Asian Pacific Journal of Tropical Medicine

176-184

Authors: Watcharee Attatippaholkun, Panyupa Pankhong, Ananda Nisalak, Siripen Kalayanarooj

Objective To study evolutionary relationship of the 5'untranslated regions (5'UTRs) in low passage dengue3 viruses (DEN3) isolated from hospitalized children with different clinical manifestations in Bangkok during 24 year–evolution (1977–2000) comparing to the DEN3 prototype (H87). Methods The 5'UTRs of these Thai DEN3 and the H87 prototype were amplified by RT–PCR and sequenced. Their multiple sequence alignments were done by Codon Code Aligner v 4.0.4 software and their RNA secondary structures were predicted by MFOLD software. Replication of five Thai DEN3 candidates comparing to the H87 prototype were done in human (HepG2) and the mosquito (C6/36) cell lines. Results Among these Thai DEN3, the completely identical sequences of their first 89 nucleotides, their high–order secondary structure of 5'UTRs and three SNPs including the predominant C90T, and two minor SNPs including A109G and A112G were found. The C90T of Thai DEN3, Bangkok isolates was shown predominantly before 1977. Five Thai DEN3 candidates with the predominant C90T were shown to replicate in human (HepG2) and the mosquito (C6/36) cell lines better than the H87 prototype. However, their highly conserved sequences as well as SNPs of the 5'UTR did not appear to correlate with their disease severity in human. Conclusions Our findings highlighted evolutionary relationship of the completely identical 89 nucleotide sequence, the high–order secondary structure and the predominant C90T of the 5'UTR of these Thai DEN3 during 24 year–evolution further suggesting to be their genetic markers and magic targets for future research on antiviral therapy as well as vaccine approaches of Thai DEN3.

191-196

Authors: Sung-Suk Suh, So Jung Kim, Jinik Hwang, Mirye Park, Taek-Kyun Lee, Eui-Joon Kil, Sukchan Lee

Objective To screen the fatty acid (FA) composition of 20 marine microalgae species, including seven Diophyceae, six Bacillariophyceae, four Chlorophyceae, two Haptophyceae and one Raphidophyceae species. Methods Microalgal cells cultured at the Korea Institute of Ocean Science & Technology were harvested during the late exponential growth phase and the FA composition analyzed. Results The FA composition of microalgae was species–specific. For example, seven different species of Dinophyceae were composed primarily of C14:0, C16:0, C18:0, C20:4n–6, C20:5n–3 and C22:6n–3, while C14:0, C16:0, C16:1, C18:0, C20:5n–3 and C22:6n–3 were abundant FAs in six species of Bacillariophyceae. In addition, four Chlorophyceae, two Haptophyceae and one Raphidophyceae species all contained a high degree of C16:1n–7 [(9.28–34.91)% and (34.48–35.04)%], C14:0 [(13.34–25.96)%] and [(26.69–28.24)%], and C16:0 [(5.89–29.15)%] and [(5.70–16.81)%]. Several factors contribute to the nutritional value of microalgae, including the polyunsaturated FA content and n–3 to n–6 FA ratio, which could be used to assess the nutritional quality of microalgae. Conclusions This study is the first comprehensive assessment of the FA composition and nutritional value of microalgae species in South Korea, and identifies the potential utility of FAs as species–specific biomarkers.

203-205

Authors: Zahra Meshkat, Sadegh Chinikar, MohammadTaghi Shakeri, Lida Manavifar, Maryam Moradi, Hessam Mirshahabi, Tahmineh Jalali, Sahar Khakifirouz, Nariman

Objective To evaluate the prevalence of West Nile virus seropositivity in the general population of Mashhad, Northeast of Iran. Methods One hundred and eighty two individuals living in the city of Mashhad were studied using cluster sampling method. Both IgM and IgG antibodies against WNV were detected by ELISA method. Results In this study, the overall IgG seroprevalence of positive West Nile virus was 11%; however, IgM antibody was not found in the participants. Conclusions Our study suggested that the prevalence rate of West virus is considerable in Mashhad city. It seems necessary for clinicians and health care workers to be aware of WNV infection in the Northeast Iran.

209-213

Authors: Hong-Juan Chang, Tian-Fa Li, Jun-Li Guo, You-Ling Lan, Yue-Qiong Kong, Xin Meng, Xian-Ji Ma, Xiao-Ling Lu, Wei-Ying Lu, Shao-Jiang Zheng

Objective To explore effect of high glucose on expression of osteoprotegerin (OPG) and receptor activator of NF– κ B ligand (RANKE) in rat aortic vascular smooth muscle cells. Methods SD rats were intraperitoneally injected with streptozotocin, OPG and RANKL expression in rat thoracic aortas were detected by immunohistochemical staining. In cultured vascular smooth muscle cells (VSMCs) (A7r5), qRT–PCR and Western blot analysis were used to examine the mRNA and protein levels of OPG and RANKL. Results Our results demonstrated that OPG expression was increased in hyperglycemic rat aortic VSMCs, while RANKL expression was decreased. Besides, in vitro experiments high glucose induced OPG expression, but depressed RANKL expression by dose– and time–dependent manner in cultured A7r5. Conclusions Our findings suggested that high glucose could promote the expression of OPG, and inhibit the expression of RANKL in VSMCs, which may be partly be the molecular mechanism of diabetic vascular calcification.

220-224

Authors: Ping Gao, Ya-Jie Gao, Hong-Lu Liang

Objective To investigate the effects of NF– κ B inhibitor pyrrolidine dithiocarbamate hydrochloride (PDTC) on vascular endothelial growth factor (VEGF) and endostatin expression in mice with Lewis lung cance; and its mechanism. Methods Mice survival rate and anti–tumor effects were observed in different concentrations of NF– κ B inhibitor PDTC after the Lewis lung cancer mice model was established. VEGF and endostatin expressions were detected by immunohistochemical assay. Results Lewis lung cancer was be inhibited by 0.5 mg/kg, 1.5 mg/kg and 3.0 mg/kg of NF– κ B inhibitor PDTC (P<0.05). Microvessel density (MVD) in 0.5 mg/kg, 1.5 mg/kg and 3.0 mg/kg NF– κ B inhibitor PDTC groups were significantly lower than the control group (P<0.05). Immunohistochemical assay results showed that VEGF and endostatin expressions in the 0.5 mg/kg, 1.5 mg/kg and 3.0 mg/kg NF–κ B inhibitor PDTC groups were significantly lower than the control group (P<0.05). Western blot results also showed that NF– κ B inhibitor PDTC could inhibit VEGF and endostatin expressions in tumor tissues. Conclusions NF– κ B inhibitor PDTC can inhibit tumor formation and reduce tumor angiogenesis in mice with Lewis lung cancer; and its mechanism maybe associated to VEGF and endostatin down–regulation.

229-233

Authors: Bing Sun, Jin-Fa Jiang, Cui-Mei Zhao, Chao-Hui Hu

Objective To observe the effects antiarrhythmic peptide 10 (AAP10) aon acute ventricular arrhythmia and the phosphorylation state of ischemic myocardium connexin. Methods Acute total ischemia and partial ischemia models were established by ceasing perfusion and ligating the left anterior descending coronary artery in SD rats. The effects of AAP10 (1 mg/L) on the incidence rate of ischemia–induced ventricular arrhythmia were observed. The ischemic myocardium was sampled to detect total–Cx43 and NP–Cx43 by immunofluorescent staining and western blotting. the total–Cx43 expression was detected through image analysis system by semi–quantitative analysis. Results AAP10 could significantly decrease the incidence of ischemia–induced ventricular tachycardia and ventricular fibrillation. During ischemic stage, total ischemia (TI) and AAP10 total ischemia (ATI) groups were compared with partial ischemia (PI) and AAP10 partial ischemia (API) groups. The rates of incidence for arrhythmia in the ATI and API groups (10% and 0%) were lower than those in the TI and PI groups (60% and 45%). The difference between the two groups was statistically significant (P=0.019, P=0.020). The semi–quantitative analysis results of the ischemic myocardium showed that the total–Cx43 protein expression distribution areas for TI, ATI, PI and API groups were significantly decreased compared with the control group. On the other hand, the NP–Cx43 distribution areas of TI, ATI, PI and API groups were significantly increased compared with the control group (P>0.05). AAP10 could increase the total–Cx43 expression in the ischemic area and decrease the NP–Cx43 expression. Western blot results were consistent with the results of immunofluorescence staining. Conclusions AAP10 can significantly decrease the rate of incidence of acute ischemia–induced ventricular tachycardia and ventricular fibrillation. Acute ischemic ventricular arrhythmias may have a relationship with the decreased phosphorylation of Cx43 induced by ischemia. AAP10 may stimulate the phosphorylation of Cx43 by increasing the total–Cx43 expression and decreasing the NP–Cx43 expression in the ischemic area, so as to decrease ventricular arrhythmia.

239-242

Authors: Xun Yang, Bei-Bei Chen, Ming-Hua Zhang, Xin-Rong Wang

Objective To study the role of microRNA–126 in the development of lung cancer. Methods The biological function of microRNA–126 was detected using EdU assay and CCK–8 assay; the target gene of microRNA–126 was analyzed using real time RT–PCR and Western blot assay. Results In A549 cell line, overexpression of microRNA–126 inhibits the proliferation rate; VEGF is the target gene of microRNA–126; microRNA–126 exerts its function via regulating VEGF protein level. Conclusions microRNA–126 inhibits the proliferation in A549 cell line.

249-252

Authors: Cheng-De Wang, Xin-Rong Wang, Chao-Yang Wang, Yi-Jun Tang, Ming-Wen Hao

Objective To study the relevance of EGFR gene mutation with pathological features and prognosis in patients with non–small–cell lung carcinoma. Methods A total of 297 patients from July 2009 to May 2013 were chosen as objects. EGFR gene mutation were detected with fluorescence quantitative PCR. Relevance of EGFR gene mutation with clinical and pathological features was analyzed, and the prognosis of EGFR– mutant–patients and that of EGFR– wide type–patients was compared. Results In 297 patients, 136 (45.79%) showed EGFR gene mutation. EGFR gene mutation had no significant relevance with age, gender, smoking history, family history of cancer and clinical stage (P>0.05); there was significant relevance between EGFR gene mutation and blood type, pathologic types, differentiation and diameter of cancer (P<0.05). The difference between prognosis of EGFR– mutant–patients and that of EGFR– wide type–patients was statistical significance (P<0.05). Conclusions EGFR gene mutation has significant relevance with pathological features, the prognosis of EGFR–mutant–patients is better than that of EGFR– wide type–patients.

90-93

Authors: EI Sergeeva, VA Ternovoi, EV Chausov, SA Berillo, OK Demina, AN Shikov, IV Plasunova, M Ju Kartashov, AP Agafonov

Objective To confirm dengue infection among Russian tourists returned from Southeast and Mexico in 2010–2013 with clinical signs of infection. Methods Blood and serum samples from patients were collected. NS1 antigen and human IgM/IgG antibodies to dengue virus were identified using commercial tests manufactured by “Standard Diagnostics, INC.”, Korea. ELISA test was used for the quantitative analyses of human IgM/IgG antibodies to dengue virus (“Orgenics Ltd.”, Israel). Viral RNA was detected using commercial real–time PCR tests manufactured by “Genome Diagnostics Pvt. Ltd.”, India and “Vector”, Russia. Genotypes of revealed dengue viruses were determined employing nucleotide sequencing and phylogenetic analysis of 5′–UTR of the viral genome. Results A total of 98 collected blood samples were analyzed. Fifty samples were positive for at least one of four markers of dengue infection. IgM to dengue virus were revealed in 38 samples, in 25 samples IgM were combined with IgG. NS1 antigen was detected in 43 samples. 22 serum samples were positive for dengue virus RNA. The majority of samples (12 patients) from tourists returned from Thailand were positive for genotype 1 of dengue virus, 2nd and 4th genotype were identified each in 1 patient. Conclusions Due to laboratory confirmed cases of imported dengue fever in Russia, the differential diagnosis of dengue is strictly recommended for tourists returning from endemic areas.

98-103

Authors: Ben Slimane Badreddine, Ezzine Olfa, Dhahri Samir, Chograni Hnia, Ben Jamaa Mohamed Lahbib

Objective To evaluate toxic activities of essential oils obtained from Rosmarinus officinalis and Lavandula stoechas against the fourth larval instars of Orgyia trigotephras. Methods A total of 1 200 larvae were divided into three groups I II III. Group I was to investigate the effect of extracted essential oils from these aromatic plants as gastric disturbance. Bacillus thuringiensis and ethanol were used as control group. Group II was used as contact action and Group III was used as fumigant action. Decis and ethanol were used as control group. During the three experiments, the effect of essential oils on larvae was assessed. Results The chemical composition of essential oils from two medicinal plants was determined and, their insecticidal effects on the fourth larval state of Orgyia trigotephras were assessed. The two simples presented an insecticidal activity, nevertheless Rosmarinus officinalis essential oil was less efficient compared to Lavandula stoechas one are discussed. Conclusions The relationship between the chemical composition and the biological activities is confirmed by the present findings. Therefore the potential uses of these essential oils as bioinsecticides can be considered as an alternative to the use of synthetic products.

112-119

Authors: Arunagirinathan Koodalingam, Ramar Manikandan, Munisamy Indhumathi, Ethala Subramani Kaviya

Objective To investigate mechanism of anti–inflammatory activity of Adenanthera pavonina (A. pavonina) extracts. Methods Rat peritoneal macrophages were treated with different concentrations of lipopolysaccharide and H2O2 in the presence and absence of kernel extract from A. pavonina. Nitric oxide, Superoxide anion generation, cell viability and nuclear fragmentation were investigated. Results The pre–treatment of kernel extract from A. pavonina suppressed nitric oxide, superoxide anion, cell death, nuclear fragmentation in lipopolysaccharide and H2O2 stimulated or induced macrophages, respectively. Conclusions These results suggest that A. pavonina extract suppresses the intra cellular peroxide production.

127-131

Authors: Zelalem Addis, Emebet Adem, Abebe Alemu, Wubet Birhan, Biniam Mathewos, Belaynesh Tachebele, Yegnasew Takele

Objective To assess the prevalence and risk factors of smear positive pulmonary tuberculosis among Gondar town prisoners, North West Ethiopia. Methods A cross sectional study was conducted from February to July, 2008 in Gondar Prison. Prisoners with cough duration of more than two weeks were involved in the study by giving three sputum samples and filling the questionnaires prepared for risk factor assessment. Acid fast staining technique was employed to detect the presence of the Mycobacterium tuberculosis bacilli in the sputum samples. Data was analyzed using SPSS version13 computer software and presented in table. Chi–square test was used to assess associations and a P–value less than 0.05 was taken as significant. Results A total of 384 prisoners, 349 male and 35 females, with a mean age of 33.3 years were involved in the study. The prevalence of smear positive pulmonary tuberculosis among those prisoners with cough duration of more than two weeks was 8.59%. Only the length of imprisonment had a significant association (χ 2= 18.82, P–value<0.0001) with the prevalence of tuberculosis. Conclusions This study indicated that tuberculosis among prisoners with cough duration of more than two weeks in Gondar prison is very high. Therefore Periodic screening of the prisoners and screening of newly introduced prisoners should be practiced so as to minimize the burden of tuberculosis in prisoners.

137-141

Authors: Xiang-Yang Gao, Xian-Jie Geng, Wen-Long Zhai, Xian-Wei Zhang, Yuan Wei, Guang-Jun Hou

Objective To evaluate the efficacy of allicin combined with cyclophosphamide on neuroblastoma (NB)–bearing mice and explore the immunological mechanism in it. Methods A total of 30 NB–bearing mice were equally randomized into model group, cyclophosphamide group and combined therapy group, 10 nudemice were set as normal saline (NS) group. Cyclophosphamide group and combined therapy group were weekly injected with 60 mg/kg cyclophosphamide for four weeks; besides, combined therapy group was given with allicin (10 mg/kg/d) by gastric perfusion for 4 weeks; model group and NS group were given with the same volume of NS. Serum VEGF content was detected by ELISA pre–treating (0 d) and on the 3rd d, 14th d and 28th d; on 29th d, all mice were sacrificed and the tumor, liver, spleen and thymic tissues were weighted. Tumors were made into paraffin section for detecting tumor cell apoptosis and proliferation by TUNEL and BrdU method, respectively. Survival curves were drawn by Kaplan–Meier method. Results After treatment, both treatment groups relieved on viscera indexes, VEGF level, T cell subsets distribution and tumor growth and each index of combined therapy group was better than cyclophosphamide group (P<0.05 or 0.01); only combined therapy group could significantly increase the lifetime of NB–bearing mice (μ 2=5.667, P=0.017). Conclusions Allicin can improve T cell subsets distribution and inhibit VEGF expression through its immunomodulatory activity, thereby improve the efficiency on NB in coordination with cyclophosphamide.

147-152

Authors: Xin-Pu Miao, Xiao-Ning Sun, Lu-Jia Cui, Qin-Fang Cao, Gui-Feng Zhuang, Tao-Zhi Deng, Dong-Yan Zhang

Objective To investigate the effects of pectic polysaccharides extracted from Rauwolfia verticillata (Lour.) Baill.var.hainanensis Tsiang on an experimental murine colitis model. Methods Experimental colitis was induced by dextran sulfate sodium (DSS), and mice were divided into 4 groups: control, DSS alone, DSS plus SASP, DSS plus pectic polysaccharides. The disease activity index (DAI) and histological score were observed. The tumor necrosis factor (TNF)– α and interleukin (IL)–17 levels were measured by enzyme–linked immunosorbent assay. I κ B and NF– κ B p65 expression were assessed by western blot analysis. Myeloperoxidase (MPO) activity was determined by using MPO assay kit. Results Administration of pectic polysaccharides significantly reduced the severity of DSS–induced colitis as assessed by DAI and histological score, and resulted in down regulation of MPO activity and NF– κ B p65 expression and subsequent degradation of I κ B protein, strikingly reduced the production of TNF– a and IL–17. Conclusions Pectic polysaccharides extracted from Rauvolfia verticillata (Lour.)Baill.var. hainanensis Tsiang exerts beneficial effects in experimental colitis and may therefore provide a useful therapeutic approach for the treatment of UC.

157-161

Authors: Huan-Yu Zheng, Hong-Yu Zheng, Yun-Tao Zhou, En-Ling Liu, Jie Li, Yan-Mei Zhang

Objective To study the change of TIZ expression in epithelial ovarian cancer cells. Methods HO8910 cells were transinfected with siRNA to inhibit the expression of TIZ. pcDNA3.1–TIZ vectors were combined to increase the TIZ expression level. The cell viability, colony forming efficiency and cycle distribution of HO8910, HO8910/NC, HO8910/pcDNA3.1–NC, HO8910/TIZ–573 and H08910/pcDNA3.1–TIZ were compared, and the invasion rate, migration rate and adhesion rate between 5 groups of cells were compared. Results Compared with those of HO8910, HO8910/NC and HO8910/pcDNA3.1–NC, the cell viability, colony forming efficiency and cell cycle distribution of HO8910/TIZ–573 were increased, while the indexes of H08910/pcDNA3.1–NC were decreased with statistical significant difference (P<0.05). There was no statistical significant difference in the invasion rate, migration rate and adhesion rate between 5 groups of cells (P>0.05). Conclusions The expression of TIZ can inhibit the proliferation of epithelial ovarian cancer cells.

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.