Effects Of Jinmaitong Capsule On Cell Apoptosis Induced By Oxidative DNA Damage In Sciatic Nerve Of Diabetic Rats And Schwann Cells

【Objective】To study the effects of Jinmaitong Capsule on cell apoptosis induced by oxidative DNA damage in sciatic nerve of STZ-DM rats, as well as to study the effects of medicated serum of JMT on apoptosis induced by oxidative DNA damage of Schwann Cells cultured in high glucose from the aspects of integral level, cellular level and molecular level.【Methods】1. In vivo experimentSTZ-induced diabetic rats were randomly divided into 5 groups including 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). Ten normal rats matching with weight, age and sex served as normal control group. 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 8-OHdG, PARP-1 (89kDa) Actived-Caspase-3 and its mRNA in sciatic nerve were detected respectively.2. In vitro experiment Schwann cells were cultivated and were 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. After 48h, the expression of ROS-DEC and Actived-Caspase-3 in SC were detected by confocal laser scanning microscope. Enzyme-linked immunosorbent assay (ELISA) was explored to determine the concentration of 8-OHdG in the supernatant of cultured SC. The expression of PARP-1 (89kDa) was detected by Western Blot,and the level of Actived-Caspase3 mRNA in SC was detected by real-time fluorogenetic quantitative PCR.【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 filamentCompared 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). Compared with model group, the threshold values of low-dose, middle-dose,high-dose JMT group and Vitamin C group raised significantly (P<0.01). Compared with other treated groups, the threshold values of middle-dose raised significantly (P＜0.01).(3) 8-OHdG expression of sciatic nerveThe integrated option density of 8-OHdG expression in STZ-DM rats was much higher than the normal (P<0.01). And the levels of 8-OHdG in all the treated groups increased significantly compared with normal group (P< 0.01),and decreased significantly compared with model group(P<0.01). Compared with other treated groups,the level of 8-OHdG in middle-dose JMT group was lower significantly(P<0.01). There was no significant difference between Vitamin C group and high-dose JMT group(P>0.05),both of them were lower than low-dose JMT group(P<0.05).(4)PARP-1 (89kDa) expression of sciatic nerveThe level of PARP-1(89kDa)expression in high-dose JMT group, middle-dose JMT group,low-dose JMT group and Vitamin C group decreased significantly compared with model group(P<0.01,P<0.01,P<0.05,P<0.05).Compared with other treated groups,the level of PARP-1 (89kDa) in middle-dose JMT group was lower significantly(P<0.01). There was no significant difference between Vitamin C group and low-dose JMT group(P >0.05),otherwise high-dose JMT group were lower than Vitamin C group (P<0.05).(5)Actived caspase-3(17kDa) expression of sciatic nerveThe integrated option density of actived caspase-3(17kDa) expression in model group was much higher than the normal group(P< 0.01). Compared with model group, high-dose JMT group, middle-dose JMT group,low-dose JMT group and Vitamin C group decreased(P<0.05, P<0.01, P<0.05, P<0.05). Compared with other treated groups,the level of actived caspase-3(17kDa) in middle-dose JMT group was lower significantly(P<0.01). There was no significant difference between Vitamin C group and high-dose JMT group(P> 0.05),otherwise increased significantly compared with middle-dose JMT group and low-dose JMT group (P<0.01, P<0.05)(6) Actived caspase-3(17kDa) mRNA expression of sciatic nerveThe level of actived caspase-3(17kDa) mRNA expression in STZ-DM rats was much higher than those of the normal rats (P<0.01). Compared with other treated groups,the actived caspase-3(17kDa) mRNA expression in middle-dose JMT group was lower significantly(P<0.01). There was no significant difference among Vitamin C group,high-dose JMT group and low-dose JMT group(P>0.05). (7) Correlation analysis of actived caspase-3(17kDa) protein and mRNALinear correlation analysis show that the expression of actived caspase-3(17kDa) protein and mRNA in all groups was positive correlation(r=0.935, P<0.01).2. In vitro experiment(1)Level of ROS-DEC in schwann cellDetected by Confocal laser scanning microscope. The fluorescence intensities of ROS-DEC in schwann cells cultured in high glucose condition were much higher than control group(P<0.01). Compared with high glucose group,the fluorescence intensities of ROS-DEC in schwann cells cultured in JMT and VC groups were weaker significantly(P<0.01). There were no significant differences between these two treated groups(P>0.05).(2)Level of 8-OHdG in the supernatant of cultured schwann cellDetermined by Enzyme-linked immunosorbent assay (ELISA).The concentration of 8-OHdG in the supernatant in schwann cell cultured in high glucose condition were much higher than control group(P< 0.01). Compared with control group, The concentration of 8-OHdG of JMT group and VC group was much higher than control group(P<0.01) while much lower than high glucose group(P< 0.01).Compared with VC group,the concentration of 8-OHdG in the supernatant in schwann cell cultured in JMT group was significantly lower(P<0.05).(3)Expression of PARP-1(89kDa) in schwann cellDetermined by Western Blot.The level of PARP-1(89kDa) in schwann cells cultrued in high glucose,JMT and VC group were much higher than control group(P<0.01,P< 0.05,P<0.01).Compared with high glucose group,the expression of PARP-1(89kDa) in schwann cells cultrued in JMT group decreased significantly(P<0.01).The expression of JMT group was also much lower than VC group(P<0.01).(4) Expression of actived caspase-3 (17kDa) in schwann cell Detected by Confocal laser scanning microscope. The fluorescence intensities of actived caspase-3 (17kDa) in schwann cells cultured in high glucose,JMT and VC condition were much higher than control group(P<0.01,P<0.05,P<0.01). Compared with high glucose group and VC group,the fluorescence intensities of actived caspase-3 (17kDa) in schwann cells cultured in JMT group were weaker significantly(P<0.01). There was no significant difference between high glucose group and VC group(P>0.05).(5) Expression of actived caspase-3 (17kDa)mRNA in schwann cellDetermined by real-time fluorogenetic quantitative PCR.The expression of actived caspase-3 (17kDa)mRNA in schwann cells cultured in high glucose condition were much higher than control group(P<0.01). Compared with high glucose group, the expression of actived caspase-3 (17kDa)mRNA in schwann cells cultured in JMT group decreased significantly (P<0.01). There was no significant difference between high glucose group and VC group(P>0.05). The expression of actived caspase-3 (17kDa)mRNA in schwann cells cultured in JMT group was much lower than in VC group(P<0.01).【Conclusion】1. In vivo experiment(1) SZT-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 significantly.(2) JMT could down-regulate the expression of 8-OHdG of sciatic nerve and reduced the oxidative DNA damage induced by oxidative stress in DPN rats.(3)JMT could down-regulate the expression of PARP-1(89kDa) of sciatic nerve, inhibite its enzymolysis to prevent the start of programmed cell death patially.(4) JMT could down-regulate the expression of actived caspase3 protein and mRNA, and reduced the cell apoptosis of sciatic nerve in DPN rats.2. In vitro experiment (1) High glucose promoted the expression of ROS and 8-OHdG in schwann cells,it showed that there was oxidative stress in schwann cell which cultred in high glucose condition and induced oxidative DNA damage.(2) The medicated serum of JMT could could down-regulate the expression of ROS and 8-OHdG in schwann cells,it showed that JMT could act as antioxydatant and reduce the oxidative DNA damage.(3) The medicated serum of JMT could inhibite the overactivtion and enzymolysis of PARP-1,reduce the schwann cell apoptosis.The effect of the medicated serum of JMT maybe act through the inhibiton of oxidative DNA damage pathway.[Innovation]To study the effects of Jinmaitong Capsule on cell apoptosis induced by oxidative DNA damage 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 induced by oxidative DNA damage 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.