Asian Pacific Journal of Tropical Medicine

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.