| [Object]To observe the effect and mechanism of Tang Bi Kang(TBK)treating in diabetic mice peripheral neuropathy,as well as TBK serum intervention of high-glucose-induced schwann cells,by regulating the sciatic nerve on oxidative damage by PTEN and apoptosis pathway.[Methods]1.In vivo experiment60 male SPF grade ICR mice were selected and given a intraperitoneal injection of 1%STZ solution.the normal control group was injected with the same amount of citrate buffer.After 72 hours of STZ injection,the tail-point blood glucose was measured.The mice with two random blood glucose levels not lower than 300 mg/dL(?16.7 mmol/L)were grouped according to the random number table of blood glucose and body weight,and were recorded as model group.Tang Bi Kang-high,medium and low group,and western medicine group,10 in each group.Continuous administration was intervened for 16 weeks.During the experiment,the general condition of the mice was recorded,and the body weights and blood glucose changes at 4w,8w,12w,and 16w before and after treatment were measured.The thermal pain threshold was measured at 16w after treatment.The sciatic nerve conduction velocity and nerve blood flow were measured under anesthesia.Serum levels of ROS and T-AOC were measured.The sciatic nerve tissue was harvested and paraffin sections were fixed with 4%paraformaldehyde for routine HE staining and pathological observation.The expression level of 8-OHdG in the sciatic nerve homogenate was determined by enzyme-linked immunosorbent assay;immunohistochemistry The expression of cleaved-PARP and PTEN in paraffin sections of sciatic nerves was detected.The mRNA expression of PTEN and caspase3 were detected by real-time qPCR.2.In vitro experimentRSC96 were cultured in vitro.SD rats were intragastrically administered for 3 days to prepare glycosylated serum and lipoic acid-containing serum.The immortal rat Schwann cell line RSC96 was routinely resuscitated,cultured and passaged to the 3rd to 5th passages.The cytokinetic effects of Tang Bi Kang drug-containing serum at 24h and 48h time points were detected by CCK-8 method.The flow cytometer was used.The level of apoptosis of FITC-Annexin V/PI dual-labeled cells was detected.The expression of ROS labeled by DCFH.DA was detected by inverted fluorescence microscope.The level of 8-OHdG secreted by cell supernatant was detected by ELISA.Nrf2 was detected in the nucleus by Western blot.Expression and transfer rate of cytoplasm/cytoplasm and expression of PTEN,p-AKT,AKT,cleaved-caspase3,cleaved-PARP,phase ? detoxification enzymes NQO1 and HO-1;real-time fluorescence quantitative PCR detection of PTEN,caspase3,NQO1,HO-1 mRNA expression.[Results]1.In vivo experimentAfter 16 weeks of drug intervention,the mice in the model group presented with multiple drinking,polyuria,weight loss,slow weight gain,and elevated blood glucose(p<0.05 or p<0.01).Compared with the time points,the high,medium,and low doses of the traditional Chinese medicine group and Western medicine group blood glucose levels were significantly lower(p<0.05 or p<0.01),and weight gain was significantly increased(p<0.05 or p<0.01).The results of behavioral tests in each group showed that 16 weeks after the stz model was established,severe thermal hyperalgesia was observed in the model group,and the thermal pain threshold in the sole and the tail was significantly increased.The TFL and PWTL of the mice in each dose group and western medicine group were reduced(p<0.05 or p<0.01),among which the improvement effect was most significant in the middle dose group of traditional Chinese medicine.Neuroelectrophysiological examination and microcirculation perfusion detection results showed that mice in the model group had slower MNCV,SNCV,and decreased CMAP,SNAP,and Flux.Chinese medicine groups and ALA interventions significantly increased the nerve conduction velocity SNCV,SNAP,and Flux(p<0.05 or p<0.01),of which the highest dose of traditional Chinese medicine has the most significant effect.Morphological observation showed that the sciatic nerve fibers were loosely arranged,disorganized,and disappearance of axons in the mice of the model group.All groups of Chinese medicine and western medicine groups showed different degrees of remission.Serum test results showed that ROS levels in the model group increased significantly and T-AOC decreased;ROS decreased and T-AOC increased in each dose group and western medicine group(p<0.05 or p<0.01).The results of 8-OHdG content in sciatic nerve tissue showed that the expression of 8-OHdG in the model group was significantly increased,and high-and middle-dose groups of TBK could effectively reduce 8-OHdG levels,which were better than that of ALA group(p<0.05).The results of immunohistochemistry showed that the protein expression of PTEN and cleaved-PARP increased significantly in the model group,and the expressions of PTEN and cleaved-PARP in the high,middle dose and ALA groups were decreased(p<0.05 or p<0.01).The effect of the group was better than that of the medium and low dose units and the western medicine group.The results of RT-PCR showed that the expression of PTEN and caspase3 mRNA expression in the model group was significantly increased,and the expression of PTEN and caspase3 mRNA in the high-and middle-dose of TBK groups,as well as those of ALA groups(p<0.05 or p<0.01).2.In vitro experimentCell viability assay results showed that with the prolonged stimulation time,high glucose can significantly reduce Schwann cell viability and inhibit cell proliferation;TBK serum and ALA drug-containing serum can relieve the inhibitory effect of high glucose on Schwann cell proliferation activity(p<0.05 or p<0.01).Apoptosis test results showed that the total apoptotic levels in the HG group and the treatment group were significantly higher(p<0.01).Compared with the HG group,the apoptotic levels in the treatment group were decreased(p<0.05 or p<0.01).The early apoptotic rate in the TBK group was significantly lower(p<0.01).The results of fluorescenceshowed that ROS in HG group was significantly higher(p<0.01),and ROS levels in TBK group and ALA group were significantly lower(p<0.01).The result of ELISA showed that the content of 8-OHdG in the supernatant of the cells was significantly increased in the HG group,and that in the TBK group and the ALA group was decreased(p<0.05 or p<0.01).The result of Western blot showed that high glucose could up-regulate the expression of PTEN,cleaved-caspase 3,cleaved-PARP,and inhibit the phosphorylation of AKT.After drug intervention,nuclear transfer rate of Nrf2 in TBK group was further enhanced.So as the p-AKT,NQO1,HO-1 expression.Meanwhile,PTEN,cleaved-caspase3 and cleaved-PARP protein expression were downregulated in TBK and ALA groups.Real-time fluorescence quantitative PCR showed that,the mRNA expression of PTEN and caspase3,compared with the normal group,were increased in the high glucose group,and there was no difference in the expression of NQO1 and HO-1 mRNA(p>0.05).TBK and ALA could down-regulate the expression of PTEN and caspase 3 mRNA and up-regulate the expression of NQO1 and HO-1 mRNA(p<0.05 or p<0.01).[Conclusions](1)In vivo experiment,TBK relieved blood glucose elevation and weight loss in DPN mice,reduce thermal pain threshold,increase nerve conduction velocity,improve nerve microcirculation blood flow,and reduce nerve fiber structural disorder.The mechanism may be to regulate the oxidative stress state of nervous tissue and reduce DNA damage and apoptosis.(2)In vitro experiment,TBK serum can reduce high glucose inhibited cell proliferation,reduce DNA damage and apoptosis.This may be achieved through the PTEN/AKT-Nrf2 pathway to enhance cellular antioxidant enzyme levels. |
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