Buyang Huanwu Decoction Inhibits Mitochondrial-dependent Apoptosis Of The Dorsal Root Ganglion In Diabetic Peripheral Neuropathy Rats Through The SIRT1/p53 Pathway

Part one The effect of Buyang Huanwu decoction on sensory function and dorsal root ganglion nerve cell apoptosis in diabetic peripheral neuropathy ratsObjective: To observe the effects of Buyang Huanwu Decoction with different doses of Huangqi on sensory function and dorsal root ganglion(DRG)nerve cell apoptosis in Diabetic Peripheral Neuropathy(DPN)rats.Methods: 90 SD rats were randomly divided into control group,DPN group,DPN plus Buyang Huanwu decoction containing Astragalus 120g(DPN+BYHW120 group),DPN plus Buyang Huanwu decoction containing Astragalus 60 g group(DPN+BYHW60 group),DPN plus Buyang Huanwu decoction group containing astragalus 30g(DPN+BYHW30 group),DPN plusα-lipoic acid group(DPN+ALA group).The control group was given a standard diet.The other groups of rats developed diabetes by intraperitoneal injection of streptozotocin(STZ)and high-sugar and high-fat diet,and then continued drug intervention for 12 weeks.The DPN+BYHW120 group,DPN+BYHW60 group and DPN+BYHW30 group were intragastric given Buyang Huanwu decoction containing 120 g astragalus,60 g astragalus and 30 g astragalus respectively.DPN+ALA group rats were given α-lipoic acid intragastrically;the control group and DPN group were given equal volumes of saline.Fasting blood glucose and body weight were measured at 4 weeks,8 weeks and 12 weeks after drug intervention.Sensory nerve conduction velocity and mechanical pain threshold were measured before the rats were sacrificed at the 12 th week,and the rats with both decreased were DPN animals.Isolated the dorsal root ganglia of L4-5 on both sides of experimental rats and observed the pathological changes of each group by HE staining and toluidine blue staining.TUNEL staining was used to detect the DNA breakage of sensory nerve cells in dorsal root ganglia.The expression of cleaved caspase-3 in the dorsal root ganglia was detected by immunohistochemistry and Western blot.Result:1.Changes of fasting blood glucose and body weightThe blood glucose levels of diabetic rats continued to be maintained at a high level,and there was no significant difference in blood glucose levels between the drug intervention groups and the DPN group.There was no significant difference in body weight of experimental rats in each group.2.Sensory nerve conduction velocity The sensory nerve conduction velocity of the DPN group was significantly lower than that of the control group.The sensory nerve conduction velocity in the DPN+ALA group,DPN+BYHW120 group and DPN+BYHW60 group was higher than that in the DPN group.The sensory nerve conduction velocity of DPN+BYHW60 group and DPN+BYHW30 group was lower than that of DPN+BYHW120 group.3.Mechanical pain thresholdThe mechanical pain threshold of the DPN group was significantly lower than that of the control group.The mechanical pain threshold of DPN+ALA group,DPN+BYHW120 group and DPN+BYHW60 group was higher than that of DPN group.The mechanical pain threshold in the DPN+BYHW60 group and DPN+BYHW30 group was lower than that in the DPN+BYHW120 group.4.Pathological changes of dorsal root gangliaThe cell bodies of DRG sensory neurons in the control group contained a lot of Nissl bodies.The Nissl body in the DPN group was significantly reduced.The Nissl body of DPN+ALA group and DPN+BYHW120 group was more than that of DPN group.5.DNA damage of sensory neurons in the dorsal root ganglionNo TUNEL-positive cells were found in the control group.A large number of positive cells were seen in the DPN group.The percentage of positive cells in the DPN+ALA group,DPN+BYHW120 group and DPN+BYHW60 group were lower than that in the DPN group.6.The expression of cleaved caspase-3 in the dorsal root gangliaImmunohistochemistry showed that cleaved caspase-3 in diabetic rats were expressed to varying degrees in the cytoplasm of neurons.Western blot results showed that the relative expression of cleaved caspase-3 in the DPN group was higher than that in the control group,and the relative expression of cleaved caspase-3 in each medication intervention group was lower than that in the DPN group.The relative expression of cleaved caspase-3 in DPN+BYHW60 group and DPN+BYHW30 group was higher than that in DPN+BYHW120 group.Conclusion: Buyang Huanwu Decoction can increase the sensory nerve conduction velocity in diabetic peripheral neuropathy rats,reduce its pain sensitivity,restore the function of sensory neurons in the dorsal root ganglia,and reduce neuronal apoptosis.Its therapeutic effect on DPN does not depend on blood sugar control.The therapeutic effect is related to the dosage of Astragalus in Buyang Huanwu Decoction,and the Buyang Huanwu Decoction containing 120 g of Astragalus has a more significant therapeutic effect on diabetic peripheral neuropathy than the Buyang Huanwu Decoction containing60 g and 30 g of Astragalus.Part two The effect of Buyang Huanwu decoction on mitochondria of dorsal root ganglion in diabetic peripheral neuropathy ratsObjective: To observe the effect of different doses of Buyang Huanwu Decoction on the structure and function of dorsal root ganglion mitochondria in DPN rats.Methods: 90 SD rats were randomly divided into control group,DPN group,DPN plus Buyang Huanwu decoction containing Astragalus 120g(DPN+BYHW120 group),DPN plus Buyang Huanwu decoction containing Astragalus 60 g group(DPN+BYHW60 group),DPN plus Buyang Huanwu decoction group containing astragalus 30g(DPN+BYHW30 group),DPN plusα-lipoic acid group(DPN+ALA group).The control group was given a standard diet.The other groups of rats developed diabetes by intraperitoneal injection of streptozotocin(STZ)and high-sugar and high-fat diet,and then continued drug intervention for 12 weeks.The DPN+BYHW120 group,DPN+BYHW60 group and DPN+BYHW30 group were intragastric given Buyang Huanwu decoction containing 120 g astragalus,60 g astragalus and 30 g astragalus respectively.DPN+ALA group rats were given α-lipoic acid intragastrically;the control group and DPN group were given equal volumes of saline.The rats were sacrificed after 12 weeks of drug intervention,and bilateral dorsal root ganglia of L4-5 were obtained.The morphology and structure of mitochondria in sensory neurons of dorsal root ganglion of rats were observed by transmission electron microscopy.Colorimetric method is used to detect respiratory chain complex Ⅰ,Ⅱ,Ⅲ,Ⅳ activity of the dorsal root ganglion.The mitochondrial membrane potential of dorsal root ganglion was detected qualitatively and quantitatively by carbonylanthocyanin fluorescence staining.Result:1.Mitochondrial ultrastructural changes of sensory neurons in the dorsal root ganglionIn the DPN group,the mitochondria of dorsal root ganglion neurons were swollen obviously,and the mitochondrial crest was broken,sparse or even disappeared,and most of them were vacuolated.The mitochondria of dorsal root ganglion neurons in the DPN+BYHW120 group and DPN+ALA group were slightly swollen,the structure was relatively complete,the mitochondrial crest was clear,and the damage was significantly reduced.The mitochondria of neurons in the DPN+BYHW60 group and DPN+BYHW30 group were swollen,some of them were vacuolated,and the mitochondrial cristae were broken.2.Activity of respiratory chain complex of dorsal root gangliaThe activities of respiratory chain complex Ⅰ,Ⅱ,Ⅲ,and Ⅳ in the DPN group were significantly lower than those in the control group.Compared with the DPN group,the activity of respiratory chain complexes Ⅰ,Ⅱ and Ⅲ increased in the DPN+BYHW120 group and DPN+ALA group;the activity of respiratory chain complex Ⅳ in the DPN+ALA group,DPN+BYHW120 group and DPN+BYHW60 group was higher than that of the DPN group.3.Mitochondrial membrane potential of dorsal root ganglionThe mitochondrial membrane potential of the DPN group was significantly lower than that of the control group,and the mitochondrial membrane potential of the DPN+BYHW120 group and DPN+ALA group was higher than that of the DPN group.The mitochondrial membrane potential of DPN+BYHW30group was lower than that of DPN+BYHW120 group.Conclusion: Impairment of mitochondrial structure,decreased activity of respiratory chain complex and decreased membrane potential are the pathological basis of qi deficiency and blood stasis in DPN.Buyanghuanwu decoction can reduce the structural damage of mitochondria in sensory neurons of dorsal root ganglion of diabetic rats,increase the activity of respiratory chain complex and mitochondrial membrane potential,and reduce the damage of mitochondrial bioenergy.The protective effect of Buyang Huanwu Decoction on mitochondria has a certain relationship with the content of Astragalus in it.Buyang Huanwu Decoction containing 120 g of Astragalus has more protective effect on DPN dorsal root ganglion mitochondria than Buyang Huanwu Decoction containing 60 g and 30 g of Astragalus.Part three Buyang Huanwu decoction inhibits mitochondrialdependent apoptosis of the dorsal root ganglion in diabetic peripheral neuropathy rats through the SIRT1/p53 signaling pathwayObjective: To observe the effects of Buyang Huanwu Decoction with different doses of Huangqi on sensory function and DRG nerve cell apoptosis in DPN rats.Methods: 90 SD rats were randomly divided into control group,DPN group,DPN plus Buyang Huanwu decoction containing Astragalus 120g(DPN+BYHW120 group),DPN plus Buyang Huanwu decoction containing Astragalus 60 g group(DPN+BYHW60 group),DPN plus Buyang Huanwu decoction group containing astragalus 30g(DPN+BYHW30 group),DPN plusα-lipoic acid group(DPN+ALA group).The control group was given a standard diet.The other groups of rats developed diabetes by intraperitoneal injection of50 mg/kg streptozotocin(STZ)and high-sugar and high-fat diet,and then continued drug intervention for 12 weeks.The DPN+BYHW120 group,DPN+BYHW60 group and DPN+BYHW30 group were intragastric given Buyang Huanwu decoction containing 120 g astragalus,60 g astragalus and 30 g astragalus respectively.DPN+ALA group rats were given α-lipoic acid intragastrically;the control group and DPN group were given equal volumes of saline.The rats were sacrificed after 12 weeks of drug intervention,and bilateral dorsal root ganglia of L4-5 were obtained.The expression of SIRT1,acetylated p53,Drp1,BAX,and BCL-2 were detected by immunohistochemistry and Western blotting,and the mRNA expression of the above proteins was detected by q-PCR.Result:1.Protein expression of SIRT1/p53 pathwayThe expression of SIRT1 in DPN group was significantly lower than that in control group,and the acetyl-p53,Drp1,the ratio of BAX to BCL-2 were all higher than that in control group.Compared with the DPN group,the expression of SIRT1 in the DPN+ALA group and the DPN+BYHW120 group was upregulated,and the expression of acetyl-p53,Drp1 and the ratio of BAX to BCL-2 were down-regulated in each group of drug treatment.Compared with the DPN+BYHW120 group,the expression of SIRT1 in the DPN+BYHW60 group was down-regulated,and the expression of acetyl-p53 in the DPN+BYHW30group was up-regulated.2.mRNA expression of SIRT1/p53 pathway related proteinsCompared with the control group,the expression of SIRT1 mRNA was down-regulated in the DPN group,and the expression of p53,Drp1,BAX and BCL-2 mRNA were up-regulated.Compared with the DPN group,the expression of SIRT1 mRNA was up-regulated in each drug treatment group,and the expression of p53,Drp1,BAX mRNA was down-regulated;the expression of BCL-2 mRNA was up-regulated in the DPN+ALA group and DPN+BYHW120 group.Compared with DPN+BYHW120 group,SIRT1 mRNA expression in DPN+BYHW60 group and DPN+BYHW30 group was down-regulated,and Drp1 and BAX mRNA expression in DPN+BYHW30group were up-regulated.Conclusion: p53-mediated mitochondrial destruction induces apoptosis is an important molecular mechanism leading to DPN.Buyang Huanwu Decoction and α-lipoic acid can up-regulate the expression of SIRT1 in the dorsal root ganglion of DPN rats and inhibit p53-mediated mitochondrialdependent cell apoptosis through its deacetylation effect.The effect of Buyang Huanwu Decoction on SIRT1/p53 pathway is related to the amount of Astragalus in the prescription.Buyang Huanwu Decoction containing 120 g of Astragalus up-regulates SIRT1 and inhibiting p53-dependent cell apoptosis more significantly than Buyang Huanwu Decoction containing 60 g and 30 g of Astragalus.Part Four Effect of astragaloside Ⅳ on mitochondrial dependent apoptosis of dorsal root ganglion in rats with diabetic peripheral neuropathy through SIRT1/p53 pathwayObjective: To investigate the effect of astragaloside Ⅳ(AS-Ⅳ)on mitochondrial-dependent apoptosis in the dorsal root ganglion of Diabetic peripheral neuropathy(DPN)rats through the SIRT1/p53 pathway.Methods: Diabetic rat model was induced by high-carbohydrate/high-fat diet and intraperitoneal injection of STZ.Diabetic rats were divided into three groups(n =16 per group): DPN group,AS-Ⅳ group(60mg/kg/d)and α-lipoic acid(ALA)group(60mg/kg/d).Weight and blood sugar levels were monitored every 4 weeks for 12 weeks.DPN was evaluated using the mechanical pain threshold and nerve conduction velocity.The dorsal root ganglia of rats were isolated,and the pathological changes of mitochondria were observed by electron microscopy.The activity of mitochondrial electron transport chain complex,mitochondrial membrane potential were measured.Neural apoptosis was detected using TUNEL assay kit.The cleaved caspase-3,major proteins in the SIRT1 /p53 pathway,including SIRT1,acetyl-p53,Drp1,BAX,and BCL-2,were detected using immunohistochemistry and Western blot.Gene expression of major proteins in the SIRT1 /p53 pathway was also detected.Results: The blood glucose of rats after intraperitoneal injection of streptozotocin was significantly increased.After 12 weeks of treatment,AS-Ⅳ and ALA did not significantly affect body weight or fasting glucose levels but reduced mechanical abnormal pain in DPN and improved nerve conduction velocity.Both AS-Ⅳ and ALA can reduce mitochondrial damage,improve mitochondrial electron transport chain complex activity and mitochondrial membrane potential,and reduce the percentages of positive cells with DNA fragmentation and the expression of cleaved caspase-3 protein.AS-Ⅳ and ALA up-regulated the expression of SIRT1 and down-regulated the expression of acetyl-p53,DRP1 and the ratio of BAX to BCL-2.Changes in gene expression were similar.Conclusion: Both AS-Ⅳ and ALA can inhibit mitochondrial-dependent apoptosis by regulating SIRT1/p53,increase the nerve conduction velocity and mechanical pain threshold of DPN rats,and have similar therapeutic effects on DPN.Conclusions:1.p53-mediated mitochondrial-dependent apoptosis is an important pathogenesis of DPN.2.Damaged mitochondrial structure of dorsal root ganglion,decreased respiratory chain complex activity and mitochondrial membrane potential are the pathological basis of DPN with deficiency of qi and blood stasis.3.Buyang Huanwu Decoction can regulate SIRT1/p53 pathway to reduce mitochondrial-dependent apoptosis,improve the quality of mitochondria and protect nerve function,have a therapeutic effect on DPN.4.The therapeutic effect of Buyang Huanwu Decoction on DPN is related to the dosage of Astragalus in the prescription,and Buyang Huanwu Decoction containing 120 g of Astragalus had better neuroprotective effect on DPN rats than Buyang Huanwu Decoction containing 60 g and 30 g of Astragalus.5.Astragaloside Ⅳ,as the most important component of Buyang Huanwu Decoction,can regulate SIRT1/p53 pathway to inhibit mitochondrial-dependent cell apoptosis,and has a similar therapeutic effect on DPN as ALA.