Protective Effects Of Jinmaitong On Oxidative Stress And Apoptosis In Experimental Diabetic Neuropathy Rats

[Objective]To investigate protective effects of Jinmaitong (JMT), a compound Chinese herbal medicine, on oxidative stress, mitochondrial dysfunction and apoptosis in experimental diabetic neuropathy at the animal, cellular and molecular level.[Methods]1. In vivo experiment:A single dose of streptozotocin (STZ) was used for the induction of diabetes in male Sprague-Dawley rats. These diabetic rats were randomly divided into5groups:diabetes (DM) group, low-dose JMT group (JMT at dose of.044g/kg/d), middle-dose JMT group (JMT at dose of0.88g/kg/d), high-dose JMT group (JMT at dose of1.75g/kg/d) and vitamin C group (VC at dose of0.05g/kg/d). Ten normal rats with matching weight and age were selected as control group. All rats then received intragastric administration for16weeks (the control and DM groups were treated with distilled water) and then killed. The pain threshold to mechanical stimulation were done before death. Body weight and blood glucose were detected before and at4th,8th,12th,16th week after treatment. Morphological changes in DRG was checked by Nissl staining. The expressions of NADPH oxidase p22-phox subunit, Cyt C, Bcl-2and Caspase-3were detected by immunohistochemical method and quantitative realtime polymerase chain reaction (qRT-PCR). The apoptotic cells were detected by the Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay.2. In vitro experiment①Male Sprague-Dawley rats were randomly divided into control group (distilled water), JMT group (JMT at dose of1.31g/kg/d) and vitamin C group (vitamin C at dose of0.08g/kg/d) to prepared drug-containing serum. DRG are harvested from embryonic day15SD rats, dissociated in0.25%trypsin, purified by density gradient centrifugation and differential attachment technique, then cultured in Neurobasal medium supplemented with B-27additives and nerve growth factor, and immunofluorescence method with anti-NSE was used to identify them.②DRGn were randomly divided into control group and high glucose (50,75,100mmol/L) group. After12,24and48h cultures, MTT assay was used to determine the optimal concentration of high glucose and the optimal time of drugs.③DRGn were randomly divided into control group, high glucose group (cultured with50mmol/L glucose medium), JMT1:1group, JMT1:2group, JMT1:4group (cultured with different concentration of JMT containing serum) and VC group (cultured with VC containing serum). After24cultures, MTT assay was used to determine the optimal concentration of JMT.④DRGn were randomly divided into high glucose group (cultured with50mmol/L glucose medium), JMT group (cultured with JMT containing serum) and VC group (cultured with VC containing serum). After24cultures, The levels of superoxide anion and MMP were determined by fluorescent probe. The expressions of Bcl-2and Caspase-3were detected by Western Blot analysis and qRT-PCR. The apoptotic cells were detected by the TUNEL assay.[Results]1. In vivo experiment①Blood glucose and body weight:The blood glucose levels in DM group were much higher than those in control group (P<0.01). At different time, there was no significant difference among model groups (P>0.05). The blood glucose and body weight levels had no marked changes before and after treatment (P>0.05)②Pain threshold to mechanical stimulation:The pain thresholds in DM group were much lower than those in control group (P<0.01). Compared with DM group, in treatment groups significantly increased (P<0.01). Among treatment groups, the the pain thresholds in middle-dose JMT group increased markedly (P<0.01) ③Oxidative stress:The protein levels of NADPH oxidase p22-phox subunit in DM group were much higher than those in control group(P<0.01). Compared with DM group, the levels of NADPH oxidase p22-phox subunit in treatment groups significantly decreased (P<0.05or P<0.01). Among treatment groups, large-dose JMT group showed better effect than VC group (P<0.01)④Apoptosis:The cytochrome protein levels, the mRNA and protein levels of Caspase-3and the apoptosis percentage in DM group were much higher than those in control group (P<0.01), and the mRNA and protein levels of Bcl-2were much lower (P<0.01). Compared with DM group, the cytochrome protein levels, the mRNA and protein levels of Caspase-3and the apoptosis percentage in treatment groups significantly decreased (P<0.05or P<0.01), the mRNA and protein levels of Bcl-2significantly increased (P<0.05or P<0.01). Among treatment groups, large-dose JMT group showed better effect than VC group (P<0.01)2. In vitro experiment①MTT assay:The cell viability levels in high glucose groups and control group significantly decreased at24h compared with12h (P<0.01), there was no significant difference between24h and48h (P>0.05). Compared with control group, the cell viability levels in high glucose groups markedly decreased at24h(P<0.05or P<0.01). Among high glucose groups, the cell viability levels in50mmol/LGlu group were much higher than75and100mmol/LGlu groups(P<0.01). Compared with control group, the cell viability levels in50mmol/LGlu group significantly decreased at24h (P<0.01), The cell viability levels in treatment groups were much lower than those in50mmol/LGlu group (P<0.01). Among treatment groups, there was no difference between JMT1:1group and VC group (P>0.05)②Oxidative stress:The levels of superoxide anion in high glucose group were much higher than those in control group (P<0.01). Compared with high glucose group, the levels of superoxide anion in JMT group significantly decreased (P<0.01) and JMT was superior to VC (P<0.01)③Apoptosis:The MMP levels, the mRNA and protein levels of Caspase-3and the apoptosis percentage in high glucose group were much higher than those in control group (P<0.01), and the mRNA and protein levels of Bcl-2were much lower (P<0.01) Compared with high glucose group, the MMP levels, the mRNA and protein levels of Caspase-3and the apoptosis percentage in JMT groups significantly decreased(P<0.01), and the mRNA and protein levels of Bcl-2significantly increased (P<0.01). JMT was superior to VC (P<0.01)[Conclusion]1. In vivo experiment①A single intraperitoneal STZ (60mg/kg) injection-induced diabetic rats showed significant hyperalgesia at16w, indicating development of DPN.②The levels of NADPH oxidase p22-phox subunit, Cyt C, Caspase-3and the apoptosis percentage in DPN rats significantly increased compared to control rats, and Bcl-2levels significantly reduced.③JMT treatment significantly ameliorated the alteration in hyperalgesia, NADPH oxidase p22-phox subunit, Cyt C, Bcl-2, Caspase-3and the apoptosis percentage in DPN rats. The large-dose of JMT showed the best effectiveness④These results suggest that JMT antagonized the oxidative stress, activation of the mitochondrial pathway and apoptosis in DPN rats. And its effectiveness was independent of hypoglycemia and insulin.2. In vitro experiment①The purity coefficient of DRGn which were isolated from E15SD rats, dissociated in trypsin digestion, purified by density gradient centrifugation and differential attachment technique, then cultured in Neurobasal medium supplemented with B-27additives and nerve growth factor, could reach more than90%through identification of immunofluorescence method with anti-NSE.②High glucose remarkably inhibited the cell viability levels of DRGn and JMT treatment significantly ameliorated the inhibition.③The levels of superoxide anion, Caspase-3and the apoptosis percentage of DRGn in cultured in high glucose medium significantly increased compared to control group, but MMP and Bcl-2levels significantly reduced. ④JMT treatment significantly ameliorated the alteration in superoxide anion, MMP, Bcl-2, Caspase-3and the apoptosis percentage of DRGn in cultured in high glucose medium. And JMT was superior to VC.⑤These results suggest that JMT containing serum antagonized the oxidative stress, activation of the mitochondrial pathway and apoptosis of DRGn in cultured in high glucose medium.