| [Objective]To study the effects of Jinmaitong Capsule (JMT) on cell apoptosis and oxidative stress in sciatic nerves of STZ-DM rats, as well as to study the effects of medicated serum of JMT on apoptosis and oxidative stress of Schwann Cells cultured in high glucose from the aspects of integral level, cellular level and molecular level.[Methods]1. In vivo experimentAll Sprague Dawley (SD) rats were randomly divided into normal group and "diabetic" group. "Diabetic" rats were induced by STZ which were randomly divided into model group, low-dose JMT group (treated with JMT similar to the quintupling dose of adult recommended dosage), middle-dose JMT group (similar to the decuple dose of adult recommended dosage), high-dose JMT group (similar to the twenty-fold dose of adult recommended dosage) and Vitamin C group (similar to the decuple dose of adult recommended dosage). Normal group included ten rats, other group included fourteen diabetic rats. All rats were given intragastric administration for 16 weeks (the normal and model groups were treated with distilled water) and then killed. Body weight and blood glucose were detected before and at the 4th,8th,12th,16th week after treatment. The pain threshold to mechanical stimulation with Von Frey filament were carried out before death. The expression of NADPH oxidase phox p22 subunit, iNOS, Bcl-2, Caspase-3 and their mRNAs in sciatic nerve were detected by immunohistochemical method and real-time fluorogenetic quantitative PCR respectively.2. In vitro experiment Schwann cells were cultivated and identified with S-100 protein antibody. The 3rd passage schwann cells were cultured respectively in following conditions including DMEM, high glucose(50mmol) media supplemented with 20% rat serum,50mmol glucose media containing medicated serum of JMT and Vitamin C. DMEM served as negative control. Schwann cell apoptosis is detected by TUNEL kit. the expression of NADPH oxidase phox p22 subunit, iNOS, Bcl-2 and Caspase-3 in SC were detected by real-time fluorogenetic quantitative PCR and confocal laser scanning microscope respectively.[Results]1. In vivo experiment(1) Blood glucose and body weightThe blood glucose levels of STZ-DM rats were much higher than those of normal rats (P<0.01). In all the treated groups, there were no significant differences among them compared each other or compared with model group (P>0.05). And it got the same result when concerning about body weight no matter how the rats were dealt with (P>0.05).(2) Pain threshold to mechanical stimulation with Von Frey filament: Compared with normal group, the pain thresholds of model group, high-dose and low-dose JMT group and Vitamin C group decreased extremely (P<0.01) while middle-dose JMT group didn't lower much (P>0.05). Compared with model group, the threshold values of low-dose, middle-dose, high-dose JMT group and Vitamin C group raised strikingly (P<0.01). Compared with Vitamin C group, the threshold values of middle-dose raised strikingly (P<0.01). Compared among JMT groups, the threshold values of middle-dose were much higher than other JMT groups (P<0.01).(3) NADPH oxidase phox p22 subunit expression of sciatic nerveThe integrated option density of p22 subunit expression in STZ-DM rats was much higher than the normal (P<0.01). And the levels of p22 subunit in all the treated groups decreased notably compared with model group (P <0.01). The levels of p22 subunit in middle-dose JMT group were lower than low-dose JMT group and Vitamin C group (P<0.01). (4) NADPH oxidase phox p22 subunit mRNA expression of sciatic nerveThe levels of p22 subunit mRNA expression in STZ-DM rats were much higher than those of the normal rats (P<0.01). Compared with model group, p22 subunit mRNA expression of low-dose, middle-dose and high-dose JMT groups down-regulated noticeably (P<0.01, P<0.01, P<0.05); There was no significant difference between model group and Vitamin C group (P>0.05). Compared with Vitamin C group, p22 subunit mRNA expression of low-dose and middle-dose JMT treated groups down-regulated noticeably (P<0.05, P<0.01).(5) iNOS expression of sciatic nerveThe integrated option density of iNOS expression in STZ-DM rats was much higher than that of the normal (P<0.01). And the levels of iNOS in all the treated groups decreased notably compared with model group (P <0.01). The levels of iNOS in middle-dose JMT group were higher than those of low-dose JMT group and Vitamin C group (P<0.01).(6) Bcl-2 expression of sciatic nerveThe integrated option density of Bcl-2 expression in STZ-DM rats was much lower than that of the normal (P<0.01). And the levels of Bcl-2 in low-dose and middle-dose JMT groups increased notably compared with model group (P<0.01). The levels of Bcl-2 in middle-dose JMT group were higher than those of Vitamin C group (P<0.01).(7)Bcl-2 mRNA expression of sciatic nerveThe levels of Bcl-2 mRNA expression in STZ-DM rats were much lower than those of the normal rats (P<0.01). Compared with model group, Bcl-2 mRNA expression of low-dose, middle-dose and high-dose JMT groups up-regulated noticeably (P<0.01); There was no significant difference between model group and Vitamin C group (P>0.05). Compared with Vitamin C group, the Bcl-2 mRNA expression of low-dose, middle-dose and high-dose JMT treated groups up-regulated noticeably (P<0.01).(8) Caspase-3 expression of sciatic nerve The integrated option density of Caspase-3 expression in STZ-DM rats was much higher than the normal (P<0.01). And the levels of Caspase-3 in high-dose and middle-dose JMT groups decreased notably compared with model group (P<0.05, P<0.01). The levels of Caspase-3 in middle-dose JMT group were much lower than those of lower-dose JMT group and Vitamin C group (P<0.05, P<0.01).(9) Caspase-3 mRNA expression of sciatic nerveThe levels of Caspase-3 mRNA expression in STZ-DM rats were much higher than those of the normal rats (P<0.01). Compared with model group, Caspase-3 mRNA expression of low-dose, middle-dose and high-dose JMT groups down-regulated noticeably (P<0.01, P<0.01, P<0.05); Compared with Vitamin C group, Caspase-3 mRNA expression of middle-dose JMT treated groups down-regulated noticeably (P<0.05).2. In vitro experiment(1) NADPH oxidase phox p22 subunit mRNA expression of schwann cells①The expression of p22 subunit mRNA in schwann cells cultured in high glucose condition were much higher than those of normal condition (P <0.01).②p22 subunit mRNA expression in schwann cells of JMT group down-regulated compared with high glucose group (P<0.01)③p22 subunit mRNA expression in schwann cells of JMT group was lower than that of Vitamin C group (P<0.01).(2) iNOS expression of schwann cells①The fluorescence intensities of iNOS in schwann cells cultured in high glucose condition were much higher than those of normal condition (P<0.01).②The fluorescence intensities of iNOS in schwann cells in JMT and Vitamin C groups weakened remarkably compared with high glucose group (P<0.01).③The fluorescence intensities of iNOS in schwann cells in JMT group were much lower than that of Vitamin C groups (P<0.01).(3) Bcl-2 expression of schwann cells①The fluorescence intensities of Bcl-2 in schwann cells cultured in high glucose condition were much lower than those of normal condition (P <0.01).②The fluorescence intensities of Bcl-2 in schwann cells in JMT groups reinforced remarkably compared with high glucose group (P<0.01), There were no significant differences between Vitamin C group and high glucose group (P>0.05).③The fluorescence intensities of Bcl-2 in schwann cells in JMT group were much higher than that of Vitamin C groups (P <0.01).(4)Bcl-2 mRNA expression of schwann cells①The expression of Bcl-2 mRNA in schwann cells cultured in high glucose condition were much lower than those of normal condition (P< 0.01).②Bcl-2 mRNA expression in schwann cells of JMT and Vitamin C groups up-regulated compared with high glucose group (P<0.01)③Bcl-2 mRNA expression in schwann cells of JMT group was higher than that of Vitamin C group (P<0.01).(5) Caspase-3 expression of schwann cells①The fluorescence intensities of Caspase-3 in schwann cells cultured in high glucose condition were much higher than those of normal condition (P<0.01).②The fluorescence intensities of Caspase-3 in schwann cells in JMT and Vitamin C groups weakened remarkably compared with high glucose group (P<0.01).③The fluorescence intensities of Caspase-3 in schwann cells in JMT group were much lower than that of Vitamin C groups (P<0.01).(6) Caspase-3 mRNA expression of schwann cells①The expression of Caspase-3 mRNA in schwann cells cultured in high glucose condition were much higher than those of normal condition (P <0.01).②Caspase-3 mRNA expression in schwann cells of JMT group down-regulated compared with high glucose group (P<0.01)③Caspase-3 mRNA expression in schwann cells of JMT group was lower than that of Vitamin C group (P<0.01).(7) the percent of schwann cell apoptosis①There were no cell apoptosis in schwann cells cultured in normal condition.②The percent of cell apoptosis in schwann cells cultured in high glucose condition were much higher than those of normal condition (P<0.01).③the percent of schwann cells apoptosis of JMT group down-regulated compared with high glucose group (P<0.01), but there was no significant difference between high glucose group and Vitamin C group (P>0.05).④The percent of schwann cells apoptosis of JMT group was lower than that of Vitamin C group (P<0.01).[Conclusion]1. In vivo experiment①SZT-induced diabetic rats (single intraperitoneal injection, 60mg/kg) had hyperalgia at 16w, which demonstrated the sensory nerve fibers were injured and the DPN models were established. JMT can alleviate hyperalgia strikingly.②The NADPH oxidase phox p22 subunit and iNOS expression of sciatic nerve increased significantly in DPN rats.③JMT could down-regulate the expression of NADPH oxidase phox p22 subunit and iNOS in sciatic nerve in DPN rats.④The Bcl-2 expression of sciatic nerve degressed significantly in DPN rats, while the caspase-3 expression increased.⑤JMT could up-regulate the expression of Bcl-2 and down-regulate the expression of Caspase-3 in sciatic nerve in DPN rats; the therapeutic effect of JMT was much better than Vitamin C.2. In vitro experiment①The purity coefficient of Schwann cells isolated from the sciatic nerves of newborn Wistar rats, cultivated and purified by methods of repeated explanation, differential velocity adherent technique, low density trypsin digestion and application of G418 could reach more than 90%through identification of SABC immunohistochemical method with S-100 protein antibody.②High glucose promoted the expression of NADPH oxidase phox p22 subunit and iNOS in schwann cells obviously. ③The medicated serum of JMT could decrease the level of NADPH oxidase phox p22 subunit and iNOS in schwann cells cultured in high glucose effectively. The therapeutic effect of JMT was much better than Vitamin C.④High glucose depressed the expression of Bcl-2 and promoted the expression of Caspase-3 in schwann cells obviously, and High glucose increased the percent of schwann cell apoptosis.⑤The medicated serum of JMT could increase the level of Bcl-2 and decrease the level of Caspase-3 in schwann cells cultured in high glucose effectively, it could decrease the percent of schwann cell apoptosis effectively.The therapeutic effect of JMT was much better than Vitamin C.[Innovation]To study the effects of Jinmaitong Capsule on cell apoptosis and oxidative stress in sciatic nerve of STZ-DM rats, as well as to study the effects of medicated serum of JMT on the role of schwann cell apoptosis and oxidative stress cultured in high glucose from the aspects of integral level, cellular level and molecular level, which hasn't been reported home and abroad. This research can provide the experimental foundation for the application of JMT to clinical treatment of DPN.[Key words]Diabetic peripheral neuropathyoxidative stresscell apoptosisSchwann cellReal-time fluorogenetic quantitative PCRTraditional Chinese Medicine Jinmaitong Capsule...
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