| Studies have shown that bupivacaine hydrochloride can cause damage to dorsal root ganglion(DRG)in diabetic mice,but there are no effective drugs or measures for this.In this study,the protective effects of astragalus polysaccharide(APS)on dorsal root ganglion neurons in diabetic mice and the related mechanisms were investigated by pretxeatment with APS in C57 BL/6J mice and ND7/23 cells.OBJECTIVE1.To explore the effects of APS pretreatment on DRG neurons damage induced by bupivacaine hydrochloride in diabetic mice by in vivo experiments;2.To explore the effects of APS pretreatment on ND7/23 cells damage induced by bupivacaine hydrochloride in high glucose culture environment in vitro experiments.METHODS1.Type 2 diabetes mellitus mice model was established,and then the mice were injected intraspinal with bupivacaine hydrochloride.To observe the effect of bupivacaine hydrochloride on DRG neurons in diabetic mice by detecting plantar pain threshold in mice,ROS staining of DRG,TUNEL assay,and detection of apoptosis-related proteins by westem blotting(WB)and immunofluorescence(IF)methods.2.Diabetic mice were pretreated with APS(0d/7d/14d/21d/28d),and then injected intraspinal with bupivacaine hydrochloride.To observe the effects of APS pretreatment on bupivacaine hydrochloride-induced DRG neurons injury in diabetic mice by plantar pain threshold test,ROS staining,TUNEL assay and apoptosis related proteins tests using WB and IF.3.ND7/23 cells were treated with APS(0μM/50μM/250μM/500μM)in high glucose culture environment,and then treated with bupivacaine hydrochloride.To observe the effects of APS on ND7/23 cells was observed by JC-1 staining,which detect mitochondrial transmembrane potential of ND7/23 cells.Meanwhile,apoptosis-related proteins were detected.4.Sirt3 gene expression and mitochondrial metabolism of ND7/23 cells were detected after treatment with bupivacaine hydrochloride.RESULTS1.After treatment with bupivacaine hydrochloride,ROS accumulation,apoptosis and apoptosis related proteins of DRG increased,with statistically significant differences.In addition,the thermal pain threshold of mice decreased slightly after treatment with bupivacaine hydrochloride and there was no statistical difference in thermal pain threshold and mechanical pain threshold among all groups.2.Compared with the mice in groups without APS pretreatment,ROS accumulation,apotosis,and apoptosis-related proteins expression of DRG neurons in the APS pretreatment groups were decreased.The longer APS administered,the more these indexes were alleviated.However,there was no statistically significant difference in behavior of mice.3.APS pretreatment could alleviate the decrease of mitochondrial transmembrane potential caused by bupivacaine hydrochloride in ND7/23 cells,but there was no statistically significant difference in the apoptosis related proteins.4.The mitochondrial transmembrane potential decreased,glutathione oxidation increased,and Sirt3 expression level increased in ND7/23 cells after treatment with bupivacaine hydrochloride.CONCLUSION1.Intraspinal injection of bupivacaine hydrochloride can lead to oxidative damage and increased apoptosis of DRG neurons in mice,which can be aggravated by diabetes.2.APS can alleviate the injury of DRG neurons induced by bupivacaine hydrochloride in diabetic mice,which may be related to the apoptosis pathway.3.In vitro experiment,APS reversed the decrease of mitochondrial transmembrane potential induced by bupivacaine hydrochloride in ND7/23 cells,but there was no statistical difference in Cleaved-caspase3,Cleaved-caspase9,Bax/Bcl-2.Therefore,we speculated that the effect of APS on ND7/23 cells was not related to apoptosis,but through other functions of mitochondria.4.Sirt3-related mitochondrial metabolic pathway may play an important role in bupivacaine hydrochloride induced DRG neurons injury in diabetes mellitus. |
