Asian Pacific Journal of Tropical Medicine

914-918

Authors: Wiriyaporn Sumsakul, Wanna Chaijaroenkul, Kesara Na-Bangchang

Objective To investigate the propensity of plumbagin to inhibit the three isoforms of human cytochrome P450 (CYP), i.e., CYP1A2, CYP2C19, and CYP3A4 using human liver microsomes in vitro. Methods Inhibitory effects of plumbagin on the three human CYP isoforms were investigated using pooled human liver microsomes. Phenacetin O-deethylation, omeprazole hydroxylation and nifedipine oxidation were used as selective substrates for CYP1A2, CYP2C19 and CYP3A4 activities, respectively. Concentrations of paracetamol, 5-hydroxyomeprazole, and oxidized nifedipine were determined in microsomal incubation mixture using high-performance liquid chromatography. Results Plumbagin showed significant inhibitory effects on all CYP isoforms, but with the most potent activity on CYP2C19-mediated omeprazole hydroxylation. The IC50 (concentration that inhibits enzyme activity by 50%) values of plumbagin and nootkatone (selective inhibitor) for CYP2C19 were (0.78 ± 0.01) and (27.31 ± 0.66) μM, respectively. The inhibitory activities on CYP1A2-mediated phenacetin O-deethylation and CYP3A4-mediated nifedipine oxidation were moderate. The IC50 values of plumbagin and α-naphthoflavone (selective inhibitor) for CYP1A2 were (1.39 ± 0.01) and (0.02 ± 0.36) μM, respectively. The corresponding IC50 values of plumbagin and ketoconazole (selective inhibitor) for CYP3A4 were (2.37 ± 0.10) and (0.18 ± 0.06) μM, respectively. Conclusions Clinical relevance of the interference of human drug metabolizing enzymes should be aware of for further development scheme of plumbagin as antimalarial drug when used in combination with other antimalarial drugs which are metabolized by these CYP isoforms.

989-998

Authors: Uruj Tahir, Umair Hassan Khan, Muhammad Saad Zubair, Bahar-e-Mustafa

Dengue virus syndrome is an emerging global health challenge which is endemic in tropical countries like Pakistan. In recent years dengue incidences have increased considerably in different areas of Pakistan with more sever impacts on urban and peri-urban populations. This review is an effort to highlight the changing epidemiology of dengue fever, role of Government of Pakistan in disease management and control using preventive and community based approaches in the region. Moreover, there is an emphasis on application of Wolbachia as novel, inexpensive and environmentally benign candidate for control and eradication of dengue transmitting vectors.

1006-1012

Authors: Ke-Xin Yu, Ching-Lee Wong, Rohani Ahmad, Ibrahim Jantan

Objective To investigate the larvicidal activity, inhibition effect on development, histopathological alteration and morphological aberration induced by the extracts derived from seaweeds Bryopsis pennata (B. pennata), Sargassum binderi (S. binderi) and Padina australis in Aedes aegypti (Ae. aegypti) larvae and to characterize the phytochemical components of the three seaweeds. Methods Larvicidal activity of the seaweeds towards the larvae of Ae. aegypti was determined according to WHO. The inhibition effect of seaweeds was assessed by determining the mortality, adult emergence rate, larval and pupa duration of the treated larvae. Histopathological effect on midgut epithelium of larvae and morphological aberration induced by the methanol extracts were examined. Phytochemical analysis was done to determine the presence of alkaloids, saponins, steroids and terpenoids in the seaweeds. Results Chloroform partition of B. pennata extract exhibited the strongest larvicidal activity (LC50 = 82.55 μg/mL), followed by methanol extract of B. pennata (LC50 = 160.07 μg/mL) and chloroform partition of S. binderi extract (LC50 = 192.43 μg/mL). The methanol extract of S. binderi exhibited the strongest effect on prolongation of larval period (1.5-fold longer as compared to control) and resulted in strongest inhibition effect in adult emergence (98.67%). The histopathological study showed that larvae treated with seaweed extracts had cytopathological alteration of the midgut epithelium. The morphological observation revealed that the anal papillae and terminal spiracles of larvae were the common sites of aberrations. Conclusions The study provided information on various effects of seaweed extracts on Ae. aegypti. Further investigation on identifying the active compounds and their mechanisms of action is recommended.

1022-1026

Authors: Devika Iddawela, Kiruthiha Ehambaram, Susiji Wickramasinghe

Objective To identify worms obtained from patients with eye lesions and to describe the demographic factors of patients with ocular dirofilariasis. Methods A retrospective descriptive study was conducted in 31 worm samples from 30 patients referred by consultant ophthalmologists between 2006 and February 2014. Data on age, sex and site of the lesion were ascertained from the details given in the referral letters. Morphological identification of the worm was based on the maximum width, length and appearance of the cuticle. The sex of the worm was determined by the width, length and presence or absence of vulva opening. PCR was performed using Dirofilaria repens specific primers to confirm the species of worms which couldnot be identified morphologically. Results Most of the patients belonged to the age group of 40–49 years (mean age = 42 years). Majority of them were females (70%). Subconjunctival lesions were the most frequent presentation, while the rest (n = 4) were found on eyelids. Female worms were extracted from 18 cases, and 11 had male worms. One individual had both male and female worms in a single nodule. Adults were the most commonly affected. This pattern was different from the previous studies in Sri Lanka where the most common age group affected was younger than 9 years old. Conclusions The present study showed a considerably high incidence of ocular dirofilariasis, stressing the importance of implementing preventive measures to reduce the transmission of this zoonotic filarial disease.

1033-1037

Authors: Hong-Fen Ge, Guan-Zhi Chen, Min Pan, Hui Guo

Objective To observe the effect of the cyclophosphamide (CTX) on systemic lupus erythematosus mice by fluorescent quantitative polymerase chain reaction. Methods (BALB/c × C57BL/J6) F1 hybrid mice were used as recipients of donor lymphocytes which were injected intravenously. Mice were randomly divided into control group and treatment group, serum autoantibody and urinary protein were measured, renal pathological changes and the situation of fungal infection were observed. Results The reduced urinary protein, ameliorated renal lesions were found in the CTX-treated mice. Candida albicans and Aspergillus flavus were detected respectively in respiratory tract and gastrointestinal tract. Conclusions CTX can alleviate lupus nephritis and the fluorescent quantitative PCR is a rapid and sensitive method for the detection of fungal infection.

1043-1048

Authors: Jing-Bo Ji, Xue-Fu Li, Lei Liu, Guang-Zhong Wang, Xin-Feng Yan

Objective To study the effect of low intensity pulsed ultrasound (LIPUS) on the expression of tissue inhibitor of metalloproteinase-2 (TIMP-2) in the serum and expression of matrix metallopeptidase 13 (MMP-13) in the articular cartilage cells of rabbits with knee osteoarthritis (OA). Methods Inner patellar ligament defect method was used to establish the model of knee OA. Four weeks after the modeling, the arterial blood was drawn from the ear of each rabbit, while ELISA was employed to detect the expression of TIMP-2 in the serum. The chondrocytes were separated from animals in each group and then cultured in vitro. All rabbits were divided into control group, OA model group and OA + LIPUS group. Cells in the control and OA groups were not treated, while cells in the OA + LIPUS group were treated with LIPUS (40 mW/cm2, 1 time/day). Cells were collected 7 d later and the RNA and total protein were extracted respectively. Real-time PCR and Western blotting were employed to analyze the expression of MMP-13 in chondrocytes at the mRNA and protein level, respectively. Results The success rate of establishment of OA model was 83%. The results of ELISA showed that the content of TIMP-2 in the serum of animals with OA was 22.3%, lower than the one in the control group (P < 0.05). Compared with the normal control group, the expression of TIMP-2 in the OA model group was significantly increased, while the expression of MMP-13 was significantly increased (P < 0.05). After the stimulation of LIPUS, the expression of TIMP-2 and MMP-13 was close to the one in the normal control group. Conclusions The inner patellar ligament defect method is a mature method to establish the rabbit OA model, with high success rate. The expression of serum TIMP-2 in the OA model group is significantly decreased. LIPUS can up-regulate TIMP-2 and down-regulate MMP-13.

1055-1059

Authors: Guan-Li Zhang, Ling Jiang, Qian Yan, Rong-Hui Liu, Lu Zhang

Objective To observe the anti-tumor effect of matrine combined with cisplatin on U14 rat models of cervical cancer. Methods A total of 80 female Kunming rats were used to establish U14 rat models of cervical cancer and then divided into groups I, II, III, IV, with 20 rats in each. For Group I, the control group, injection of normal saline was given around the tumors. For Group II, injection of 2 mg/kg cisplatin was given around the tumors. For Group III, injection of 75 mg/kg matrine was given around the tumors while the combined injection of matrine and cisplatin was given for Group IV with the same doses as Groups II and III. The animals were sacrificed 10 d after the injection and tumors were taken out for the comparisons of tumor weights after injection and calculation of anti-tumor rates, while thymus and spleen were taken for thymus index and spleen index. Blood in eyeball was collected for determination of changes in serum creatinine and urea nitrogen levels. Sections of tumor issue were prepared and morphological changes in tumor tissue cells were observed by using immunohistochemistry technique. Results After injection, the thymus index and spleen index in Groups III and IV were significantly higher than those in Groups I and II (P < 0.05) while the two indexes in Group II were significantly lower than Group I (P < 0.05). The tumor weights in Groups II and IV were significantly smaller than those in Groups I and III (P < 0.05) with significantly higher anti-tumor rates than Groups I and III (P < 0.05). The serum creatinine and urea nitrogen levels in Groups III and IV were significantly lower than Group II (P < 0.05) and the two indicators in Group III were significantly lower than those in Group IV (P < 0.05). The observation under the histological microscope showed densely arranged tumor cells in Group I, growing as a crumby structure and diffuse appearance, with hyperchromatic and large nuclei, and abundant cytoplasm. In the case of Group II, it showed less tumor cells, with extensive degenerative necrosis, sparse arrangement and karyopyknosis as well as karyoclasis. For Group III, necrosis of tumor cells in different sizes and heterogeneous color in nuclei were observed. For Group IV, the number of tumor cells was significantly smaller than Groups I and III and the tumor cells presented an appearance of crumby structure as cancer nests, with more proliferation of connective tissue. Conclusions The treatment of matrine combined with cisplatin can significantly improve the anti-tumor effect on U14 rats with cervical cancer, which can be a new option for the treatment for cervical cancer.

1064-1070

Authors: Yong Yang, Da-Peng Li, Na Shen, Xiao-Cheng Yu, Jie-Bao Li, Qi Song, Jia-Heng Zhang

Objective To investigate the expression of targeting protein for Xenopus kinesin-like protein 2 (TPX2) in breast cancer tissue and to explore its role in proliferation, migration and invasion of breast cancer cells. Methods The mRNA and protein expressions of TPX2 in breast cancer tissue and cell lines were assessed by quantitative RT-PCR and Western blot. The effect of TPX2 with RNA interference on proliferation, invasion and migration of breast cancer cells was observed by MTT and Transwell assays. Results Both mRNA and protein expressions of TPX2 were upregulated in breast cancer tissues compared to tumor-adjacent tissue. TPX2 expression was also upregulated in breast cancer cell lines, and the TPX2 interfered by small interfering RNA could inhibit the proliferation, invasion and migration of breast cancer cells by inhibiting matrix metalloproteinase-2 and matrix metalloproteinase-9. Conclusions Significantly upregulated TPX2 expression is observed in breast cancer tissue and cells, and contributes to promote the proliferation, migration and invasion of breast cancer cells.

1076-1078

Authors: Wen-Ning Li, Jia-Li Wei, Ming Wu, Wei Wu, Yun Huang, Mao-Wei Xie, Hui Han

Objective To prove whether astrocyte elevated gene-1 (AEG-1) plays a role in high glucose-stimulated Rho kinase activation and epithelial–mesenchymal transition (EMT) in human renal tubular epithelial (HK-2) cells. Methods The protein levels of AEG-1, alpha-smooth muscle actin, E-cadherin and MYPT1 were determined by Western blot. Results AEG-1 protein level was upregulated in HK-2 cells stimulated with high glucose. AEG-1 siRNA downregulated Rho kinase protein expression and blocked high glucose-induced EMT. Conclusions Our results show that AEG-1 acts a key role in high glucose-induced activation of Rho kinase and EMT in HK-2 cells.

Pages 919-922

Authors: Raimunda Sâmia Nogueira Brilhante, Jamille Alencar Sales, Celia Maria de Souza Sampaio, Francisco Geraldo Barbosa, Manoel de Araújo Neto Paiva, Glaucia Morgana de Melo Guedes, Lucas Pereira de Alencar, Yago Brito de Ponte, Tereza de Jesus Pinheiro Gomes Bandeira, José Luciano Bezerra Moreira, Débora de Souza Collares Maia Castelo-Branco, Waldemiro de Aquino Pereira-Neto, Rossana de Aguiar Cordeiro, José Júlio Costa Sidrim, Marcos Fábio Gadelha Rocha

Objective To investigate the in vitro antimicrobial potential of extracts of stem, leaves, flowers, pods and seeds of Moringa oleifera (M. oleifera) against Vibrio spp. from hatchery water and the prawn Macrobrachium amazonicum. Methods The ethanol extracts of stem, leaves, pods and seeds and chloroform extract of flowers of M. oleifera were tested against Vibrio cholerae (V. cholerae) serogroups non-O1/non-O139 (n = 4), Vibrio vulnificus (n = 1) and Vibrio mimicus (n = 1). Escherichia coli (E. coli) (ATCC® 25922) was used as quality control. Vibrio species were obtained from Macrobrachium amazonicum prawns and from hatchery water from prawn farming. The Minimum Inhibitory Concentration (MIC) was determined by broth microdilution method. Results The best result was obtained with the ethanol extract of pods, which inhibited three strains of the V. cholerae, Vibrio vulnificus, Vibrio mimicus and E. coli (MIC range 0.312–5.000 mg/mL). The chloroform extract of flowers was effective against all V. cholerae strains and E. coli (MIC range 0.625–1.250 mg/mL). However, the ethanol extracts of stem and seeds showed low effectiveness in inhibiting the bacterial growth. Conclusions The extracts of pods, flowers and leaves of M. oleifera have potential for the control of Vibrio spp. Further studies are necessary to isolate the bioactive compounds responsible for this antimicrobial activity.

889-897

Authors: Luís R. Silva, Rafaela Teixeira

Objective To improve the knowledge on the metabolite composition of Endopleura uchi bark, concerning phenolic compounds, and to evaluate some of its biological capacities for further possible exploitation in food and pharmaceutical industries associated to their health-promoting qualities. Methods Two different extracts (infusion and hydroethanolic) were studied concerning phenolic composition and biological potential. Results Five compounds were determined by HPLC-DAD, being bergenin the major one. In general way, infusion presents a greater richness in these metabolites. The antioxidant, acetylcholinesterase, butyrylcholinesterase, α-glucosidase and antibacterial activity were checked by in vitro assays. A dose-dependent response was noticed against DPPHradical dot, superoxide and nitric oxide radicals, acetylcholinesterase, butyrylcholinesterase and against α-glucosidase inhibitory assay. Antibacterial capacity of both extracts was investigated against Gram-positive and Gram-negative bacteria, being more effective against the first one. The concentrations of infusion extract tested here revealed that is non-toxic for intestinal (Caco-2) cells line. Conclusions The results suggest that the extracts of Endopleura uchi may be interesting for incorporating in pharmaceutical preparations for human health, since it can suppress hyperglycaemia and inhibit cholinesteases, and or as food additive due to its antiradical and antibacterial activities.

914-918

Authors: Wiriyaporn Sumsakul, Wanna Chaijaroenkul, Kesara Na-Bangchang

Objective To investigate the propensity of plumbagin to inhibit the three isoforms of human cytochrome P450 (CYP), i.e., CYP1A2, CYP2C19, and CYP3A4 using human liver microsomes in vitro. Methods Inhibitory effects of plumbagin on the three human CYP isoforms were investigated using pooled human liver microsomes. Phenacetin O-deethylation, omeprazole hydroxylation and nifedipine oxidation were used as selective substrates for CYP1A2, CYP2C19 and CYP3A4 activities, respectively. Concentrations of paracetamol, 5-hydroxyomeprazole, and oxidized nifedipine were determined in microsomal incubation mixture using high-performance liquid chromatography. Results Plumbagin showed significant inhibitory effects on all CYP isoforms, but with the most potent activity on CYP2C19-mediated omeprazole hydroxylation. The IC50 (concentration that inhibits enzyme activity by 50%) values of plumbagin and nootkatone (selective inhibitor) for CYP2C19 were (0.78 ± 0.01) and (27.31 ± 0.66) μM, respectively. The inhibitory activities on CYP1A2-mediated phenacetin O-deethylation and CYP3A4-mediated nifedipine oxidation were moderate. The IC50 values of plumbagin and α-naphthoflavone (selective inhibitor) for CYP1A2 were (1.39 ± 0.01) and (0.02 ± 0.36) μM, respectively. The corresponding IC50 values of plumbagin and ketoconazole (selective inhibitor) for CYP3A4 were (2.37 ± 0.10) and (0.18 ± 0.06) μM, respectively. Conclusions Clinical relevance of the interference of human drug metabolizing enzymes should be aware of for further development scheme of plumbagin as antimalarial drug when used in combination with other antimalarial drugs which are metabolized by these CYP isoforms.

930–936

Authors: Jing He, Yi Cai, Lei-Ming Luo, Rong Wang

Objective To study the correlation between expression of Wnt and NCX1 and cardiomyocyte apoptosis in mouse with myocardial hypertrophy. Methods C57B/16 male mice were given the subcutaneous injection of 1 mg/kg isoprenaline to build the myocardial hypertrophy model. After 14 d of model building, mice were executed by cervical vertebra luxation. The ratio of heart weight/body weight (HW/BW) and heart weight/tibia length (HW/TL) was observed and proved using HE staining that detected the size of cardiomyocytes. 40 male C57B/16 mice were randomly divided into the sham group (normal saline) and model group (isoprenaline), with 20 mice in each group. The terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling was applied to detect the cardiomyocyte apoptosis; while Western blot and immunohistochemistry were employed to detect the expression of Wnt and NCX1. Meanwhile, the correlation between these two proteins and cardiomyocyte apoptosis was explored. Results Compared with the sham group, the ratio of HW/BW and HW/TL was increased in the model group, as well as the bigger and hypertrophied cardiomyocytes, decreased number and increased apoptosis of cardiomyocytes, and increased positive expression of Wnt3a, Wnt5a and NCX1 in the cardiac muscle tissue. Besides, there was positive correlation between the expression of Wnt and NCX1 and the cardiomyocyte apoptosis. Conclusion The expression of Wnt3a, Wnt5a and NCX1 in mouse with myocardial hypertrophy is increased and positively correlated with the cardiomyocyte apoptosis.

944-951

Authors: Zhen Tang, Li-Ping Jiang, Jian-Yong Wu

Objective To explore the effect of maxillary expansion on orthodontics. Methods Eight beagle dogs were randomly divided into two groups, with 4 dogs in each group. Dogs in group 1 were executed immediately and received the direct physical measurement. The magnetic expansion appliance was used in group 2 for the maxillary expansion. After the expansion, the model was taken again and they were executed after cone beam CT (CBCT) scanning. The model measurement method was adopted in group 1 to measure the dental measurement indicators and width of base bone arch. The CBCT measurement method was employed to measure the above dental indicators and bone indicators. The difference in the indicators measured by different methods was compared and analyzed. Results Before the expansion, there was no significant difference in the bone measurement indicators between the CBCT measurement method and direct physical measurement method. After the expansion, there was no significant difference in indicators between the CBCT measurement method and direct physical measurement. But there was significant difference among the model measurement method, CBCT measurement method and direct physical measurement method. There was the significant difference in the dental indicators between the CBCT measurement method and model measurement, as well as the bone indicators of posterior marginal spacing of greater palatine foramen, posterior marginal spacing of incisive foramen, width of base bone arch and spacing of implant anchorage. Conclusions There is no significant difference between the effect of CBCT measurement method and direct physical measurement method, but CBCT is significantly better than the model measurement.

958-963

Authors: Guang Song, Hong-Xia Cao, Shao-Xin Yao, Cang-Tuo Li

Objective To discuss the abnormal expression of Wnt inhibitory factor (WIF1) in hepatocellular carcinoma cells and its regulating effect on the hepatocellular carcinoma invasion and metastasis factors of tissue inhibitor of matrix metalloproteinases-3 (TIMP-3) and caveolin-1. Methods RT-PCR and Western blot were employed to detect the expression of WIF1 in six hepatocellular carcinoma cell lines of HepG2, Hep3B, Huh7, PLC/PRF/5, SMMC-7721 and MHCC97 and the immortalized human liver cell line THLE-3. Besides, Lipofectamine 2000 was employed to transfect the eukaryotic expression vector pcDNA3.1-WIF1 and blank plasmid pcDNA3.1 into hepatocellular carcinoma cell lines. Transwell assay was used to detect the effect of WIF1 on the invasion ability of hepatocellular carcinoma cells; Western blot was used to detect the effect of WIF1 on the expression of TIMP-3 and caveolin-1 in hepatocellular carcinoma cells, it also discussed the effect on the expression of β-catenin. Results The expression of WIF1 in hepatocellular carcinoma cell lines was lower than that in the normal liver cell lines (P < 0.01); while there was basically no expression of WIF1 in the human highly metastatic cell line MHCC-97 and moderate expression in HepG2 and SMMC-7721. Therefore, HepG2 and SMMC-7721 were chosen as the further experimental cell lines. After transfecting the eukaryotic expression vector pcDNA3.1-WIF1 and blank plasmid pcDNA3.1 into hepatocellular carcinoma cell lines, compared with the blank plasmid group, the cell viability and invasion ability in the WIF1 group were all reduced (P < 0.01), the expression of TIMP-3, caveolin-1 and mRNA were all down-regulated (P < 0.01), and the expression of β-catenin was decreased (P < 0.01). Conclusions Because of down-regulation or missing of expression of WIF1 in hepatocellular carcinoma cell lines, the up-regulation of WIF1 expression can significantly inhibit the invasion and metastasis of HepG2 and SMMC-7721 of hepatocellular carcinoma cell lines, which are related to the up-regulated expression of TIMP-3 and down-regulated expression of caveolin-1 and may be realized through the Wnt/β-catenin signaling pathway.

971-975

Authors: Wen-Lei Yang, Lin Zhang

Objective To investigate the regulation effect of pigment epithelium-derived factor (PEDF) on the growth of human lens endothelial cells (LECs) and related mechanisms in vivo and in vitro. Methods In the part of in vivo study, 82 eyes of 82 patients with age-related cataract were included to collect the central lens anterior capsule (diameter at 5.0–5.5 mm) with the informed consent of surgery for patients. The selected specimens were divided into the LECs low density group and high density group with 20 specimens for each group based on hematoxylin and eosin staining results. The relative expression level of PEDF mRNA in LECs was detected by reverse transcription PCR. In the part of in vitro study, LEC line (HLE-B3) was cultured and 50 ng/mL PEDF was added in media for 72 h in PEDF culture group, while normally cultured cells were used as the control group. The percentage of LECs at G0 and S phases and apoptotic rate of cells were assayed by using flow cytometry with annexin Ⅴ-FITC/7-AAD double staining method. Intracellular expression of vascular endothelial growth factor (VEGF) mRNA was detected by real-time fluorescence quantitative PCR. Results The central anterior subcapsular LECs density and relative expression level of PEDF mRNA were lower than those of high density group. There were no significant differences between two groups (P = 0.168). The apoptotic rate in the PEDF culture group was significantly reduced in comparison with the control group (P < 0.001). In addition, the expression level of VEGF mRNA was lower in the PEDF culture group compared with the control group (P < 0.001). Conclusions In human eyes, PEDF may function as cytotropic factor to promote survival of LECs through anti-apoptosis and reducing-expression of VEGF. Decrease of PEDF content in LECs probably modulates the pathophysiological process of lens cells and further cataractogenesis.

983-988

Authors: Feng Yuan, Ying Zhou, Ying Jiang, Rui Liu, Jian-Zhe Li, You-Ke Xie, Xue-Mei Li, Fang Dai

Objective To explore the effect and specific mechanism of lung-tonifying and expectorant decoction on lung cancer rats with Qi deficiency and blood stasis, and aim to provide a new idea on treating the disease with traditional Chinese medicine based on syndrome differentiation. Methods A total of 60 C57BL/6J male rats were included in the study. The model of Qi deficiency and blood stasis was established in 60 rats by using multiple-factor stimulation. About 10 rats were randomly taken to verify whether the model establishment was successful and the rest of 50 rats were divided into 5 groups with 10 rats each: blank control group, cisplatin group, low dose group, medium dose group and high dose group. The blank control group was treated with normal saline, and cisplatin group was treated with cisplatin while the other three groups were treated with lung-tonifying and expectorant decoction at different doses. The volume change in transplanted tumor, tumor inhibition rate, apoptosis rate, and expression of Bcl-2, Bax, cleaved caspase-3 and cleaved caspase-9 in 5 groups were compared. Results The rapidest growth rate of transplanted tumor volume was observed in blank control group and the slowest in cisplatin group. The growth rate was gradually decreased with the increasing dose of lung-tonifying and expectorant decoction, and the difference in growth of tumor volume among groups was statistically significant (P < 0.05). The cisplatin group showed the highest tumor inhibition rate, with dose-dependent increase (P < 0.05). The apoptosis rate in low dose group was higher than blank control group but lower than high dose group (P < 0.05). The apoptosis rate in medium dose group was significantly higher than blank control group (P < 0.05). The apoptosis rate in high dose group was significantly higher than control group (P < 0.05). The positive expression rates of Bcl-2 and Bax in all groups showed statistically significant difference (P < 0.05), while expression of cleaved caspase-3 and cleaved caspase-9 in 5 groups was significantly different, with dose-dependent increase (P < 0.05). Conclusions The lung-tonifying and expectorant decoction inhibits the proliferation of tumor cells by inducing and activating the cell apoptosis in treatment of lung cancer with Qi deficiency and blood stasis, probably with good clinical therapeutic effect.

779-783

Authors: Supawadee Piratae

Opisthorchiasis caused by Opisthorchis viverrini (O. viverrini) remains as medically important problem in Thailand especially in the north-eastern part. Infection with this parasite can lead to cholangiocarcinoma improvement. The highest prevalence of O. viverrini infection has been found in the Northeast Thailand and is associated with the high incidence rate of cholangiocarcinoma. To complete the life cycle of O. viverrini, the freshwater snails namely Bithynia funiculata, Bithynia siamensis siamensis and Bithynia siamensis goniomphalos (B. s. goniomphalos) are required to serve as the first intermediate host. Within these snails group, B. s. goniomphalos is distributed concisely in northeast Thailand and acts as the majority snail that transmitted the opisthorchiasis in this region. This study described the information of B. s. goniomphalos which research are needed for understanding the biology, distribution, transmission and factors influencing on the infection of the snail vector of this carcinogenic parasite.

792-797

Authors: Samrat Baral, Ramesh Pariyar, Chi-Su Yoon, Dong-Cheol Kim, Jong-Min Yun, Seok O. Jang, Sung Yeon Kim, Hyuncheol Oh, Youn-Chul Kim, Jungwon Seo

Objective To investigate the effects of Gastrodiae rhizoma, a dried root of Gastrodia elata Blume, on proliferation and differentiation of human NSCs derived from embryonic stem cells. Methods A 70% ethanol extract of Gastrodiae rhizoma (EEGR) was estimated with 4-hydroxybenzyl alcohol as a representative constituent by HPLC. Results MTT assay showed that the treatment with EEGR increased the viability of NSCs in growth media. Compared to control, EEGR increased the number of dendrites and denritic spines extended from a differentiated NSC. Whereas EEGR decreased the mRNA expression of Nestin, it increased that of Tuj1 and MAP2 in NSCs grown in differentiation media. Immunocytochemical analysis using confocal microscopy also revealed the increased expression of MAP2 in dendrites of EEGR-treated NSCs. Furthermore, EEGR decreased mRNA expression of Sox2 in NSCs grown even in growth media. Conclusions In conclusion, our study demonstrates for the first time that EEGR induced proliferation and neuronal differentiation of NSCs, suggesting its potential benefits on NSC-based therapies and neuroregeneration in various neurodegenerative diseases and brain injuries.

807-812

Authors: Yu-Cheng Song, Wen-Juan Li, Liu-Zhi Li

Objective To study the expression of miRNA 320a in the brain tissue of epileptic rats and analyze its effect on the expression of aquaporin 4 (AQP4). Methods All rats were performed with the intraperitoneal injection of lithium chloride (3 mmol/kg) and then the intraperitoneal injection of pilocarpine (30 mg/kg) 24 h later (injected twice) to prepare the epileptic model of Wistar rats. Rats in the control group were injected with the equal volume of normal saline. According to the Racine scale, rats with over stage 3 of epilepsy were chosen and the brain tissue was separated quickly and then stored at −80 °C. The immunohistochemistry was used to detect the expression of aquaporin in the brain tissue of epileptic model and the Real-time PCR was employed to determine the difference in the expression of miRNA 320a and AQP4 in the brain tissue of rats between the epileptic model group and control group. Five 5-day neonatal Wistar rats were chosen to collect the cerebral cortex and their primary astrocytes were separated and cultured. They were transfected with miRNA mimic and imitated to the endogenous miRNA 320a to up-regulate the expression of miRNA 320a. Results In the model group, the expression of AQP4 was significantly higher than the control group (P < 0.01). However, the expression of miRNA 320a in the model group was lower than control group (P < 0.05), which was negatively correlated to AQP4. In the primary astrocytes, the transfection of miRNA 320a mimic could significantly reduce the expression of AQP4, while its inhibitor could up-regulate the expression of AQP4, which indicated that miRNA 320a could reduce the expression of AQP4. Conclusions In the primary astrocytes of rats, the miRNA 320a could inhibit the expression of AQP4 and after adding the inhibitor of miRNA 320a, the expression of AQP4 was up-regulated.

829-835

Authors: Ying-Chun Meng, Zhen-Ying Ding, Hai-Quan Wang, Li-Ping Ning, Chao Wang

Objective To explore the function and mechanism of microRNA-155 to regulate the angiogenesis after the cerebral infarction of rats through the angiotensin II receptor 1 (AT1R)/vascular endothelial growth factor (VEGF) signaling pathway. Methods Female SD rats were chosen for the construction of cerebral infarction model of rats using the modified right middle cerebral artery occlusion. The real-time PCR (RT-PCR) method was employed to detect the expression of microRNA-155 in each group at different time points after the cerebral infarction (1 h, l d, 3 d and 7 d). SD rats were randomly divided into four groups (n = 20 rats): sham operation group (Sham group), MACO group, MACO+microRNA-155 mimic group, and MACO+microRNA-155 inhibitor group. Sham group was given the free graft, while MACO+microRNA-155 mimic group and MACO+microRNA-155 inhibitor group were treated with microRNA-155 mimic and microRNA-155 inhibitor respectively. The Zea Longa 5-point scale was used to score the neurologic impairment of rats in each group; 2, 3, 5-triphenyl tetrazolium chloride staining to evaluate the volume of cerebral infarction of rats in each group; the immunohistochemistry to detect the expression of CD31; Western blot and RT-PCR to detect the expression of AT1R and VEGF receptor 2 (VEGFR2). Results The expression of microRNA-155 was increased in the cerebral ischemia tissue after the cerebral infarction. It was significantly increased at 1 d of ischemia and maintained at the high level for a long time. Rats in the Sham group had no symptom of neurologic impairment, while rats in the MACO group had the obvious neurologic impairment. After being treated with microRNA-155 inhibitor, the neural function of MACO rats had been improved, with the decreased area of cerebral infarction. But after being treated with microRNA-155 mimic, the neural function was further worsened, with the increased area of cerebral infarction. Results of immunohistochemical assay indicated that microRNA-155 inhibitor could up-regulate the expression of CD31, while microRNA-155 mimic could down-regulate the expression of CD31. The RT-PCR found that, after being treated with microRNA-155 inhibitor, MACO rats had the increased expression of AT1R and VEGFR2 messenger RNA (mRNA); but after being treated with microRNA-155 mimic, the expression of AT1R and VEGFR2 mRNA was decreased. Results of Western blot showed that, after being treated with microRNA-155 inhibitor, MACO rats had the increased expression of AT1R and VEGFR2 mRNA; but after being treated with microRNA-155 mimic, the expression of AT1R and VEGFR2 mRNA was decreased. Conclusions The inhibition of microRNA-155 can improve the neurologic impairment of rats with the cerebral infarction, reduce the volume of cerebral infarction and effectively promote the angiogenesis in the region of ischemia, which may be mediated through AT1R/VEGFR2 pathway.

841-847

Authors: Rong-Zhen Zhang, Hua Qiu, Na Wang, Fu-Li Long, De-Wen Mao

Objective To explore the regulation effect of Rheum palmatum (R. palmatum) L. on NF-κB signaling pathway of ALF mice. Methods The intraperitoneal injection of d-GalN/LPS was employed for the model building. Mice in the treatment group and positive control group were given the R. palmatum L. and bifendate before the model building. Mice in the normal group were given the intraperitoneal injection of equivalent normal saline for continuously 3 d. After 16 h of model building, the blood was collected from eyeballs of mice and then mice were executed. The measurement was performed on the content of ALT, AST, NO and Il-1β in the serum of mice in each group, as well as the activity of Caspase 3 and Caspase 8 in the liver tissue. HE staining was employed to detect the pathological morphology of liver; and the western blot was used to detect the expression of iNOS, COX-2, Bax, Bcl-2, PCNA, NF-κB p65 and IκBα. Results The content of ALT, AST, NO and Il-1β in the serum and the activity of Caspase 3 and Caspase 8 in the liver tissue were increased in the mice of ALF model group. Besides, the expression of iNOS, COX-2 and Bax was increased, the expression of Bcl-2 and PCNA was decreased, the phosphorylation of NF-κB p65 and IκBα was significant and the treatment group of R. palmatum L. could inhibit such change. Conclusions Through NF-κB signaling pathway, the R. palmatum L. could reduce the content of enzyme of liver function and inflammation factor in the serum of ALF mice, regulate the expression of cell apoptosis-related protein and improve the symptoms of ALF mice.

855-859

Authors: Li-Fei Xing, Dong-Tao Wang, Yu Yang, Su-Yue Pan

Objective To explore the effects of histone deacetylase 6(HDAC-6) on the PD cell model induced by proteasome inhibitor lactacystin. Methods Human neuroblastoma SK-N-SH cells were cultured. The wild type pcDNA3.1-alpha-synuclein eukaryotic expression plasmid was transferred into the cells which then were divided into control group, group L, group T and group T+L. The cells of group L were added with 5 μmol/L lactacystin dissolved indimethylsulfoxide (DMSO) to induce PD cell model with abnormal protein aggregation, the cells of control group were treated with 5 μmol/L DMSO, the cells of group T were treated with 5 μmol/L selective HDAC-6 inhibitor tubacin dissolved in DMSO, and the cells of group T+L were treated with 5 μmol/L lactacystin and 10 μmol/L tubacin dissolved in DMSO. The expression levels of alpha-synuclein oligomers, HSP-27 and HSP-70 were detected by Western blot and the cell survival rate of all the groups was detected by MTT colorimetric assay, and compared 24 h after the cells were treated. Results The expression levels of alpha-synuclein oligomers, HSP-27 and HSP-70 of the cells of group L were significantly higher than the control group, and the cell survival rate was significantly lower (P < 0.05); the expression level of alpha-synuclein oligomers of the cells of group T+L was significantly higher than group L, but the expression level of HSP-27 and HSP-70 were significantly lower, and so as the cell survival rate (P < 0.05); the differences of the expression level of alpha-synuclein oligomers, HSP-27 and HSP-70 and the cell survival rate of the cells of group T and the control group were not statistically significant (P > 0.05). Conclusions The expression level of alpha-synuclein oligomers can be improved and the cell survival rate can be reduced by the PD cell model induced by lactacystin and treated with selective HDAC-6 inhibitor tubacin, which means that alpha-synuclein oligomers of the PD cell model induced by lactacystin can be inhibited and the cell survival rate can be improved by HDAC-6, and the mechanism may be related to the increased of HSP-27 and HSP-70.

864-869

Authors: Kuai-Fa Fang, Zhu-Jun Chen, Meng Liu, Ping-Sheng Wu, Da-Zhi Yu

Objective To study the mechanism and significance of pH change in the coronary artery microthrombosis of rats. Methods After the sodium laurate-induced model of coronary artery microthrombosis of rats was constructed, the vascular endothelial cells were separated and then cultured in the mediums with different pH values for 24 h. Enzyme linked immunosorbent assay was used to detect the content of von Willebrand factor (vWF) in the medium; while the real-time PCR and western blot assay were used to detect the expression of fibrinogen-like protein 2 (FGL2) at the mRNA and protein level. The comprehensive evaluation was performed to discuss the effect of pH change on the coronary artery microthrombosis of rats. Results The expression level of vWF detected by enzyme linked immunosorbent assay was 336.67 ± 24.95, 311.33 ± 14.98, 359.67 ± 39.63, 354.67 ± 49.01 and 332.00 ± 33.42 (pg/mL) respectively; while the expression of vWF in the model group was 570.00 ± 57.94, 524.67 ± 57.94, 437.00 ± 95.38, 415.33 ± 44.38 and 444.67 ± 74.31 respectively. Being cultured under the different pH values, the relative expression level of FGL2 mRNA in the model group was 7.93 ± 0.93, 6.70 ± 0.70, 5.03 ± 0.32, 5.13 ± 0.40 and 5.57 ± 0.83 respectively. Conclusions The coronary artery microthrombosis of rats can cause the high expression and secretion of vWF. Meanwhile, FGL2 is also up-regulated in the thrombosis and such up-regulation is more significant in the condition with low pH, which indicates that the low pH condition may be one of factors that contribute to the cardiovascular diseases.

873-877

Authors: Xiao-Yu Lou, Jing-Liang Cheng, Bo Zhang

Objective To observe the protective effect of breviscapine on mice with cisplatin-induced nephrotoxicity. Methods Mice were given a single injection of cisplatin (8 mg/kg, i.p.); then, breviscapine was given to mice at 25 mg/kg and 50 mg/kg doses, respectively, once a day for seven days. Renal tissue structure was observed after animals were sacrificed. Blood urea nitrogen (BUN), serum creatinine (Scr), lipid peroxide (MDA) and superoxide dismutase (SOD) serum levels were detected; and MDA, glutathione peroxidase, and SOD levels in the renal cortex were detected. Results Compared with the blank control group (BCG), the kidney pathological damage of mice in the model control group (MCG) was more severe. After applying different doses of breviscapine, different degrees of renal injury improvement appeared. Compared with the BCG, the serum levels of Scr and BUN in the MCG increased to (89.92 ± 6.78) μmoL/Land (15.32 ± 4.53) mmoL/L. The differences were statistical significant (P < 0.01). Compared with the MCG, the serum levels of Scr and BUN in the Bre low-dose groups and Bre high-dose groups decreased significantly (P < 0.05). Compared with the BCG, the MDA levels in serum and in the renal cortex in the MCG significantly increased, while the SOD levels significantly decreased. Both the differences were statistically significant (P < 0.01). In the Bre low-dose groups and Bre high-dose groups, MDA levels in serum and in the renal cortex significantly decreased, while SOD and glutathione peroxidase levels in the renal cortex significantly increased, compared with the MCG; and the differences were statistically significant (P < 0.05). Conclusions Breviscapine can reduce cisplatin-induced renal toxicity in mice and it's possible through inhibition of renal tubule cell lipid peroxidation and reduces the nephrotoxicity of cisplatin.

6-13

Authors: Ng Shy Kai, Tee Ai Nee, Elaine Lai Chia Ling, Tan Chin Ping, Long Kamariah, Nyam Kar Lin

Objective To determine the antihypercholesterolemic effects of kenaf seed samples and compare with the commercial hypocholesterolemic drug on serum lipids profiles and malondialdehyde (MDA) level in the rat. Methods Kenaf seed oil (KSO), microencapsulated kenaf seed oil (MKSO), kenaf seed extract (KSE) and defatted kenaf seed meal (DKSM) were prepared and phytochemicals screening on these samples were done prior in vivo study. Phenolic compounds in KSE were quantified using high performance liquid chromatography. There were 40 (divided in eight diet groups of 5) male Sprague dawley rats adapted to normal standard diet or hypercholesterolemic diet (HD) with or without the treatment of these kenaf samples for 32 days. Results All the kenaf samples exhibited to contain most of the major phytochemicals. KSE possessed gallic acid, tannic acid, catechin, benzaldehyde, benzoic acid, syringic acid, sinapic acid, ferulic acid, naringin acid, and protocatechuic acid. The significant higher (P<0.05) serum total cholesterol, low density lipoprotein cholesterol and MDA levels in HD group without treatment than the normal control group suggested the hypercholesterolemia was induced by the incorporation of cholesterol into diet. KSE exhibited higher cholesterol–lowering properties due to the significant lower (P<0.05) in serum triglycerides, total cholesterol and MDA levels. KSE showed the highest efficiency of cholesterol–lowering activity, followed by KSO, MKSO and DKSM. Conclusions DKSM, MKSO, KSO and KSE appeared to have comparable anti–hypercholesterolemic effect with the commercial hypocholesterolemic drug. Hence, kenaf seed could be used as an alternative natural source to replace the synthetic hypocholesterolemic drugs.

19-23

Authors: Kandasamy Eniyan, Urmi Bajpai

Objective To clone, express and purify 2–methylcitrate synthase (Rv1131) gene of Mycobacterium tuberculosis (M. tuberculosis) and to study its structural characteristics using various bioinformatics tools. Methods Rv1131 gene was amplified by polymerase chain reaction using M. tuberculosis H37Rv genomic DNA and cloned into pGEM–T easy vector and sequenced. The gene was sub–cloned in pET28c vector, expressed in Escherichia coli BL21 (E. coli BL21) (DE3) cells and the recombinant protein was identified by Western blotting. The protein was purified using Nickel affinity chromatography and the structural characteristics like sub–cellular localization, presence of transmembrane helices and secondary structure of the protein were predicted by bioinformatics tools. Tertiary structure of the protein and phylogenetic analysis was also established by in silico analysis. Results The expression of the recombinant protein (Rv1131) was confirmed by western blotting using anti–HIS antibodies and the protein was purified from the soluble fraction. In silico analysis showed that the protein contains no signal peptide and transmembrane helices. Active site prediction showed that the protein has histidine and aspartic acid residues at 242, 281 & 332 positions respectively. Phylogenetic analysis showed 100% homology with major mycobacterial species. Secondary structure predicts 2–methylcitrate synthase contain 51.9% alpha–helix, 8.7% extended strand and 39.4% random coils. Tertiary structure of the protein was also established. Conclusions The enzyme 2–methylcitrate synthase from M. tuberculosis H37Rv has been successfully expressed and purified. The purified protein will further be utilized to develop assay methods for screening new inhibitors.

29-34

Authors: Appadurai Daniel Reegan, Arokia Valan Kinsalin, Michael Gabriel Paulraj, Savarimuthu Ignacimuthu

Objective To evaluate the larvicidal, ovicidal and repellent properties of solvent extracts of marine sponge Cliona celata (C. celata) (Grant) against the malarial vector Anopheles stephensi (An. stephensi) Liston. Methods Marine sponge C. celata was thoroughly washed with distilled water and shade dried for 48 h. Then the sponges were homogenized and extracted sequentially with hexane, ethyl acetate and methanol. Larvicidal and ovicidal activities were tested at four different concentrations viz., 62.5, 125.0, 250.0 and 500.0 ppm. For repellent study extracts were taken in three different concentrations viz., 5.0, 2.5, 1.0 mg/cm at. Results Among the three solvent extracts of C. celata, methanol extract showed the highest larvicidal activity at 500 ppm against the fourth instar larvae of An. stephensi. The LC50 and LC90 values of C. celata methanol extract were recorded as 80.61 and 220.81 ppm against An. stephensi larvae respectively. High ovicidal activity of 91.2% was recorded at 500 ppm concentration of methanol extract. The haxane extract was found to be the most effective protectant against the adult female mosquitoes of An. stephensi. The mean protection time recorded in hexane extract was up to 245 min at 5 mg/cm2 dosage against An. stephensi adults. Conclusions The screening results suggest that the hexane and methanol extracts of C. celata are promising in mosquito control. Considering these bioactivities, C. celata could be probed further to obtain some novel pesticidal molecules.

41-47

Authors: Xi Lin, Qi Lin, Jun-Jian Ye

Objective To explore the role of cytokine, interleukin–17 (IL–17) in human degenerative disc disease. Methods Through magnetic resonance imaging, human degenerative disc tissues were confirmed from the isolated nucleus pulposus cells, which were then cultured in vitro. The cells were cultured with and without different concentrations of IL–17. 2 ng/mL, 5 ng/mL, 10 ng/mL, 15 ng/mL and 20 ng/mL IL–17 concentrations were used for stimulation. After 72 hours, the inhibition rate of proliferation was measured by MTS method. For 48 and 96 hours, the nucleus pulposus cells were cultured with and without the appropriate IL–17 concentrations. The mRNA and protein expression levels of the matrix macromolecules and degrading tissue genes were measured by Real–time PCR and Western blot analysis. Results It was noted that nucleus pulposus cell proliferation was inhibited after culturing in vitro with IL–17 stimulation, and it was further observed that the inhibition effect was significantly stronger with 15 ng/mL IL–17 concentration. With the dosage of 15 ng/mL, IL–17 stimulation induced multiple cellular responses, such as the significant increase in mRNA expression for both aggrecan (ACAN) and type I collagen (COLLA1) genes (P<0.05), and the significant decrease in mRNA expression of both degrading tissue genes, MMP3 and TIMP3 (P<0.05). Western blot results also showed that the protein level of COL1A1 was significantly decreased (t=3.199, P=0.006), while the protein level of one peptidases (ADAMTS5) significantly increased (t=2.667, P=0.021). Conclusions These findings suggest that IL–17 can inhibit proliferation and affect the metabolism of the cultured nucleus pulposus cells in vitro, and these findings could possibly contribute to the degenerative changes that occur in DDD through extracellular matrix synthesis inhibition, promoting nucleus pulposus extracellular matrix degradation and disrupting the metabolic balance.

53-59

Authors: Shu–Yuan Ma, Zhi-Qiang Feng, Ren-Fa Lai, Zhong-Da Yin

Objective To investigate the synergistic effect and mechanism of the combined application of recombinant human bone morphogenetic protein–2 (rhBMP–2) and basic fibroblast growth factor (bFGF). Methods 24 KM male mice were randomly divided into 6 groups with 4 mice in each group, namely, Group A (control group), Group B (only treated with collagen), Group C (treated with 2 ng bFGF+collagen), Group D (treated with 4 μ g rhBMP–2+collagen), Group E (treated with 4 μ g rhBMP–2+2 ng bFGF+collagen) and Group F (treated with 4 μ g rhBMP–2+4 ng bFGF+collagen). The composites were implanted into the intermuscular septum of hind legs mice; whereas in control group, intermuscular septum of mice was separated and no implantation was performed. General observation, detection of concentration of calcium content, micro computed tomography (Micro–CT), three–dimensional reconstruction scan, measurement of bone mineral density (BMD), bone volume fraction (BVF) and trabecular thickness (Tb.Th), as well as histological observation with HE staining and ALP and CD34 immumohistochemical staining were performed. Results Ectopic osteogenesis was found in Groups D, E and F mice. The difference in concentration of calcium contents was statistically significant between Groups D and E (P<0.05), but insignificant between Groups E and F (P>0.05). Micro–CT and three–dimensional reconstruction revealed continuous newborn bone substance in external surface of ectopic bone formation, and the center of bone formation did not show obvious substantial filling by bone substance. The differences in BMD, BVF and Tb.Th were statistically significant between Groups D and E or F (P<0.01 or <0.05). HE staining showed that in Groups D, E and F, newborn bone substance was mainly located at the edge of ectopic bone formation, and the bone formation in Groups E and F was better than that in Group D. ALP and CD34 immumohistochemical staining revealed the positive expression mainly at the edge of ectopic bone formation, and area of positive expression in Groups E and F was larger than that in Groups D. Conclusions rhBMP–2 possesses the capacity to induce ectopic osteogenesis independently, but bFGF does not have this ability; the combined application of rhBMP–2 and bFGF can enhance the synergetic effect on inducing ectopic osteogenesis.

64-67

Authors: Tan Wang, Xue-Bin Cao, Xiao-Wu Chen, Pei-Pei Huang, Tian Zhang, Zhi-Bin Chen, Bei-Sha Tang

Objective To investigate the neuroprotective effects of simvastatin on lipopolysaccharide (LPS)–induced rat model of Parkinson's disease (PD) and the mechanisms involved. Methods Hemiparkinsonian rat models were induced by stereotaxieal injection of LPS in the right substantia nigra compacta. After 2 weeks of simvastatin treatment, rotational behavior test was performed after the intraperitoneal injection of apomorphine. Expression of tyroxine hydroxylase (TH) and glial fibrillary acidic protein were analyzed through immunohistochemical staining of substantia nigra and striatum, and the level of TNF– α was evaluated using enzyme–linked immunosorbent assay. Results Comparing with untreated group, behavioral symptoms of the rats were significantly less in the rats that received simvastatin treatment. The TH positive cell count in substantia nigra and striatum were significantly increased (P<0.05) and TNF– α expression was significantly decreased (P<0.05) in simvastatin group compared to untreated group. Conclusions Simvastatin could effectively inhibit the activation of astrocytes, reduce TNF– α expression, and exert anti–inflammatory effects, and thus protect the dopaminergic neurons in substantia nigra and striatum of the rat model of PD.

73-78

Authors: Guang-Hao Ge, Hong-Jie Dou, Shuan-Suo Yang, Jiang-Wei Ma, Wen-Bo Cheng, Zeng-Yong Qiao, Yue-Mei Hou, Wei-Yi Fang

Objective To investigate the protective effect of glucagon–like peptid–1 (GLP–1) against cardiac microvascular endothelial cell (CMECs) injured by high glucose. Methods CMECs were isolated and cultured. Superoxide assay kit and dihydroethidine (DHE) staining were used to assess oxidative stress. TUNEL staining and caspase 3 expression were used to assess the apoptosis of CMECs. H89 was used to inhibit cAMP/PKA pathway; fasudil was used to inhibit Rho/ROCK pathway. The protein expressions of Rho, ROCK were examined by Western blot analysis. Results High glucose increased the production of ROS, the activity of NADPH, the apoptosis rate and the expression level of Rho/ROCK in CMECs, while GLP–1 decreased high glucose–induced ROS production, the NADPH activity and the apoptosis rate and the expression level of Rho/ROCK in CMECs, the difference were statistically significant (P<0.05). Conclusions GLP–1 could protect the cardiac microvessels against oxidative stress and apoptosis. The protective effects of GLP–1 are dependent on downstream inhibition of Rho through a cAMP/PKA–dependent manner, resulting in a subsequent decrease in the expression of NADPH oxidase.

386-391

Authors: Ping Zhou, Li Chang, Xiao-Hong Zhang, You-Dai Chen, Xuan-Lin Feng, Lei Deng, Jian-Dong Wang

Objective To investigate the essential biochemical indices like 1 -hydroxylase and hypocalcaemia in the rats with severe acute pancreatitis and explore the correlation between them. Methods A total of 120 SPF grade Wistar male rats which were in similar physiological status were selected and randomly divided into two groups: sham group (SO group) and severe acute pancreatitis group (SAP group). Then they were divided into 1 h, 3 h, 6 h, and 12 h subgroups according to the killing time. The severe acute pancreatitis model was established by retrograde injection of 5% sodium taurocholate. Serum calcium, serum creatinine, serum urea nitrogen and serum amylase were measured at different time. Serum 1, 25 dihydroxy vitamin D3 level was determined by enzyme linked immunosorbentassay. The expression of 1-hydroxylase protein in the kidney tissue was determined with Western blotting and immunohistochemistry to observe its location. The pathologic features of the kidney tissue section was observed under light microscope and submicroscopic structure of the proximal convoluted tubule epithelial cell was observed under transmission electron microscope. Results Compared with the SO group, rats in the SAP group showed continuous pathological injury as time went by. There was significant increase in serum creatinine, serum urea nitrogen and serum amylase in SAP group compared with the SO group 1, 3, 6, 12 hours after the operation (P<0.05). There was significant decrease in serum calcium and 1, 25 dihydroxy vitamin D3 3, 6, 12 hours after the operation (P<0.05). It also showed that the expression of the 1-hydroxylase protein in kidney tissues was upregulated at 1 h, 3 h and decreased at 6 h, 12 h compared with the SO group. The serum calcium, 1, 25 dihydroxy vitamin D3 and the expression of the 1-hydroxylase protein in kidney tissues of the SAP group showed sustaining decrease. Western blotting showed positive correlation between the 1-hydroxylase expression and serum calcium at 3 h, 6 h and 12 h (r=0.976, P<0.001; r=0.948, P<0.001; r=0.742, P=0.001) and also positive correlation between the 1-hydroxylase expression and serum 1, 25 dihydroxy vitamin D3 at 1 h, 3 h, 6 h and 12 h (r=0.935, P<0.001; r=0.952, P<0.001; r=0.917, P<0.001; r=0.874, P<0.001). Conclusions At the early stage of the kidney injury, the expression of 1-hydroxylase in the kidney tissue is reduced with the progress of the disease and the decrease in its activity has a correlation with the hypocalcaemia.

694-700

Authors: Xiao-Nan Yang, Imran Khan, Sun Chul Kang

Objective To identify the chemical constituents of leaf essential oil of Forsythia koreana (F. koreana) and evaluate its effects on bacterial strains. Methods The essential oil of leaf of F. koreana was extracted by using hydrodistillation process and the volatile components investigated with the help of gas chromatography coupled with mass spectrometry. The antibacterial study was carried out with the help of agar disc diffusion method, MIC, MBC and viable count. The mode of action was determined with help of potassium ion flux, cellular material release and scanning electron microscopy. The antioxidant activity was determined with the help of 2, 3-diphenyl-2-picrylhydrazyl method, nitric oxide scavenging activity and superoxide anion radical scavenging assay. Results Total ten compounds were identified as trans-phytol (42.73%), cis-3-hexenol (12.95%), β-linalool (10.68%), trans-2-hexenal (8.86%), trans-2-hexenol (8.86%), myrcenol (3.86%), 4-vinylphenyl acetate (3.86%), (4Z)-4,6-heptadien-1-ol (3.18%), lemonol (2.73%) and benzeneacetaldehyde (2.27%) by gas chromatography coupled with mass spectrometry. The antibacterial study was demonstrated that leaf essential oil of F. koreana act against foodborne and other pathogenic bacteria. The mode of action revealed that this essential oil acted on the cytoplasmic membrane, resulting in loss of integrity and increased permeability. In addition, leaf essential oil of F. koreana was shown to be rich in linalool, which contributes to improved antioxidant activity. Conclusions These results show that leaf essential oil of F. koreana has great potential as a natural food preservative, antibacterial and antioxidant agent.

705-709

Authors: Nazeh M. Al-Abd, Zurainee Mohamed Nor, Mustafa Kassim, Marzida Mansor, Abdulelah H. Al-Adhroey, Romano Ngui, Sinnadurai Sivanandam

Objective To determine the prevalence of the filarial parasites,ie.,Brugia malayi, Brugia, Brugia pahangi(B. pahangi), Dirofilaria immitisandDirofilaria repens (D. repens) in domestic and stray cats. Methods A total of 170 blood sample were collected from domestic and stray cats and examined for filarial worm parasites in two localities, Pulau Carey and Bukit Gasing, Selangor State, Malaysia. Results The overall prevalence of infection was 23.5% (40/170; 95% CI = 17.4–30.6). Of this, 35% (14/40; 95% CI = 22.1–50.5) and 50% (20/40; 95% CI = 35.2–64.8) were positive for single B. pahangi nd D. repens, respectively. The remaining of 15% (6/40; 95% CI = 7.1–29.1) were positive for mixed B. pahangi and D. repens. In addition, 75% of the infected cats were domestic, and 25% were strays. No Brugia malayi and Dirofilaria immitis was detected. Eighty-four cats were captured at Pulau Carey, of which 35.7% (30/84) were infected. Among the cats determined to be infected, 93% (28/30; 95% CI = 78.7–98.2) were domestic, and only 6.7% (2/30; 95% CI = 19.0–21.3) were strays. Conversely, the number of infected cats was three times lower in Bukit Gasing than in Pulau Carey, and most of the cats were stray. Conclusions B. pahangi and D. repens could be the major parasites underlying filariasis in the study area. Adequate prophylactic plans should be administrated in the cat population in study area.

714-717

Authors: Valentina Virginia Ebani, Fabrizio Bertelloni, Barbara Turchi, Dario Filogari, Domenico Cerri

Objective To determine the prevalence of zoonotic tick-borne bacteria in feeding ticks removed from hunted wild animals. Methods PCR was executed on DNA extracted from 77 tick pools to detect Anaplasma phagocytophilum, Bartonella spp., Borrelia burgdorferi sensu lato, Coxiella burnetii and Rickettsia spp. Results A total of 432 ticks were collected: 30 (6.94%) Haemaphysalis punctata, 72 (16.7%) Dermacentor marginatus and 330 (76.38%) Ixodes ricinus. For each animal one or two pools of 3 ticks of the same species was constituted. Seventy-seven tick pools were examined by PCR: 58 (75.32%) resulted infected and among them 14 (18.18%) showed co-infections. In particular, 29 (37.66%) pools were positive for Bartonella spp., 23 (29.87%) for Anaplasma phagocytophilum, 16 (20.78%) for Rickettsia spp., and 5 (6.49%) for Borrelia burgdorferi s.l. All samples were negative for Coxiella burnetii. Conclusions The results demonstrate the presence of several zoonotic tick-borne pathogens in the studied area, and underline the risk of exposure to infections for hunters not only during the outdoor activity, but also when they manipulate hunted animals infested by infected ticks.

724-730

Authors: Xiu-Li Guo, Ya-Jie Wang, Pei-Lin Cui, Yan-Bin Wang, Pi-Xia Liang, Ya-Nan Zhang, You-Qing Xu

Objective To discuss the effect of BRMS1 on the proliferation, migration and adhesion of mouse forestomach carcinoma. Methods The constructed pCMV-myc-BRMS1 recombinant plasmid and blank plasmid were transfected into mouse forestomach carcinoma. MTT method was employed to measure the activity of gastric cancer cell; the scratch assay and Transwell assay to measure the migration and invasion of gastric cancer cell; the adhesion assay to measure the adhesion of gastric cancer cell; while the Western blot assay to measure the expression of The NF-κB signal pathway, downstream matrix metalloproteinase (MMP)-2, MMP-9 and osteopontin and E-cadherin in the gastric cancer cell. Besides, the transplanted animal model of gastric cancer in mice was constructed to measure the size of tumor xenograft. Results Results of MTT assay showed that, compared with the empty vector control group, the activity of gastric cancer cell was not affected in the BRMS1 transfection group. The improved expression of BRMS1 could inhibit the adhesion, migration and invasion of gastric cancer cell (P < 0.01). Besides, compared with the empty vector control group, the phosphorylation of NF-κB p65 and IκBα was reduced in the BRMS1 transfection group, with the decreased expression of MMP-2, MMP-9 and osteopontin and the increased expression of E-cadherin (P < 0.01). Results of animal experiment also showed that the expression of BRMS1 did not affect the transplanted tumor. Conclusions The expression of BRMS1 can significantly inhibit the adhesion, migration, invasion and metastasis of mouse forestomach carcinoma gastric cancer cell, which is related to The NF-κB signal pathway.

739-746

Authors: Jin-Yang Chen, Xiao-Zhou Mou, Xiao-Chun Du, Charlie Xiang

Objective To invest the differences among mesenchymal stem cells (MSCs) derived from different tissues and their impacts on clinical applications. Methods In this study, MSCs were isolated from adipose tissue (AD), umbilical cord tissue (UC), and menstrual blood (Men) and compared their biological characteristics in terms of proliferation capacity, passage capacity, colony formation, and surface markers were compared. Results The stem cells (SCs) obtained from different sources were all characterized as MSCs, but demonstrated some differences. Umbilical cord-derived MSCs (UCMSCs) were able to overcome density inhibition. The proliferation rate decreased in the order UCMSCs > MenSCs > ADSCs, while the colony-forming ability decreased in the order MenSCs > ADSCs > UCMSCs. Based on gene-expression data for MSCs from different sources within the same donor, 768 MenSC genes were found that were specifically upregulated or downregulated compared with bone marrow-derived MSCs and UCMSCs, most of which were involved in cell function-related pathways. In addition, MenSCs appeared to be superior in terms of immune inflammation, stress response, and neural differentiation potentials, but weaker in terms of osteogenic and chondrogenic differentiation capacities, compared with UCMSCs and bone marrow-derived MSCs. Conclusions MenSCs have higher extraction efficiency, colony-forming ability, and long time passage capacity. Although the proliferation capacity is inferior to UCMSCs.

752-756

Authors: Lie-Dao Yu, Ri-Long Jin, Peng-Cheng Gu, Zhi-Heng Ling, Xiang-Jin Lin, Jing-Yu Du

Objective To investigate clinical significance of microRNA-130b (miR-130b) in osteosarcoma and its role in cell growth and invasion. Methods miR-130b expression was detected in 68 samples of surgically resected osteosarcoma and matched normal tumor-adjacent tissues by qRT-PCR. The expression of miR-130b was altered by corresponding vectors in osteosarcoma cells, and then Western blot was used to detect the expression of PPARγ. BrdU cell proliferation and Transwell assays were performed to determine cell proliferation and invasion. Results The expression of miR-130b in osteosarcoma tissues was significantly higher than that in normal tumor-adjacent tissues. Its expression in patients with metastasis was significantly higher than that in those without metastases. miR-130b expression in tumor tissues was significantly associated with tumor size, clinical stage and distant metastasis. And its expression was significantly correlated with overall survival and disease free survival. miR-130b overexpression obviously repressed the expression of PPARγ, and resulted in significant increase of Saos-2 cell proliferation and invasion. On the contrast, repressing miR-130b expression with its inhibitor significantly increased PPARγ expression, and inhibited MG-63 cell proliferation and invasion. Conclusions The high-expression of miR-130b is correlated with the adverse clinicopathological features and poor prognosis in osteosarcoma. miR-130b may regulate proliferation and invasion of osteosarcoma cells by targeting PPARγ, suggesting miR-130b may play a key role in the progression of osteosarcoma.

762-765

Authors: Shao-Xin Yao, Gui-Song Zhang, Hong-Xia Cao, Guang Song, Zang-Tuo Li, Wei-Tao Zhang

Objective To study the correlation between miR-21 and Treg/Th17 ratio in the microenvironment of rats with hepatocellular carcinoma. Methods Diethylnitrosamine was used to build the hepatocellular carcinoma model of rats; the content of Treg cells and Th17 cells and the expression of miR-21 in the peripheral blood of rats with hepatocellular carcinoma were detected. The statistical analysis was performed on the correlation between miR-21 expression and Treg/Th17 ratio. Results Hepatocellular carcinoma model of rats was successfully constructed. The proportion of Th17 cells among all CD4+T cells in the peripheral blood of rats with hepatocellular carcinoma was 5.319%, which was higher than the control group; while the proportion of Treg cells was 9.472%, which was higher than the control group. Treg/Th17 ratio in the model group was 1.781, compared with 1.478 in the control group. The expression of miR-21 was increased in the peripheral blood of rats with hepatocellular carcinoma and it showed a positive correlation with the ratio of Treg/Th17. Conclusions There is a positive correlation between the expression level of miR-21 and the ratio of Treg/Th17.