DSBs Repair Enzyme Ku70 Involvement In Hyperglycemia-mediated Enhancement Of Bupivacaine-induced Neurotoxicity

Part Ⅰ cell experiment in vitroObjective To investigate the effect of high glucose on the expression of Ku70,DNA damage and neurotoxicity induced by bupivacaine in SH-SY5Y cells.Method SH-SY5Y cells were cultured with 25,50,100 mM glucose for 1,3 and 7 days.The repair enzyme Ku70 and γ-H2AX protein were measured.we de-termined the time and concentration of high glucose which could induce the for-mation of double strand damage to DNA,and inhibited the expression of Ku70.Cell viability was detected under the concentration gradient of bupivacaine.The SH-SY5Y cell model of neurotoxicity induced by bupivacaine was established.The experiment was divided into four groups:control group,high glucose group,Bupi-vacaine group,high glucose+Bupivacaine group.Ku70 mRNA,repair enzyme Ku70,cleaved caspase-3,intracellular ROS,DNA damage and apoptosis were measured respectively.Construction of Ku70 overexpression in SH-SY5Y cell line.To evalu-ate whether Ku70 was a key repair enzyme when high glucose environment in-creased neurotoxicity induced by bupivacaine in SH-SY5Y cells.Statistical The measurement data were record mean ± standard deviation,SPSS 20.0 statistics software was used to analysis the date.Single factor analysis of vari-ance(one-way ANOVA)was used to compare the differences in SH-SY5Y cells between the two groups after high glucose or bupivacaine.Comparison of the effects of two factors on the effects of bupivacaine in a high glucose environment using a two-way ANOVA,two independent samples t test was compared between the two groups.P<0.05 for the difference was statistically significant.Results Compared with the control group,the expression of repair enzyme Ku70 increased significantly in SH-SY5Y cells treated with bupivacaine(P<0.05).The expression of Ku70 inhibited in the high glucose environment increase the apoptosis of neural cells induced by bupivacaine.Overexpression of repair enzyme Ku70 can effectively reduce neurotoxicity induced by bupivacaine.Conclusion High glucose could inhibit Ku70 expression in response to bupi-vacaine-induced DNA damage and simultaneously aggravate the neurotoxic effects of bupivacaine in SH-SY5Y cells.Ku70 as the key enzyme in the process of repair-ing damage.PartⅡ rat experiment in vivoObject To evaluate the effect of hyperglycemia on spinal nerve damage and Ku70 expression of diabetes rats after intrathecal injection of bupivacaine.Methods Establishment of type 2 diabetic rat model by intraperitoneal injection of streptozotocin combined with high glucose and high fat diet.The experiment was divided into four groups:control group,high glucose group,Bupivacaine group,high glucose+Bupivacaine group.2.5%hydrochloride bupivacaine was administered by intrathecal injection 30 ul in Bup and DM+Bup group.The equal saline was in-jected in other groups.The changes of plantar withdraw mechanical threshold(PWMT)and plantar withdraw thermal latency(PWTL)were measured before and after the block 6,12,24 h.Spinal intumescentia lumbalis of rats were tested after bupivacaine intrathecal injection at 24 h.MDA and SOD levels in spinal cord were measured.The expression of Ku70 and cleaved caspase-3 were measured by western blotting and immunohistochemistry.The spinal cord injury was observed by HE staining,the percentage of apoptotic cells was detected by Tunel.Statistical The measurement data were record mean ± standard deviation,SPSS 20.0 statistics software was used to analysis the date.The differences of PWMT and PWTL values at different time before and after blocking were analyzed by repeated measurement analysis of variance(ANOVA).Comparison of the MDA,SOD,Ku70,cleaved caspase-3 protein and the percentage of apoptosis in spinal nerve cells was performed by two-way ANOVA,two independent samples t test was compared with the two groups.Statistical significance was set at P<0.05.Results Comparison of bupivacaine intrathecal injection before,PWMT and PWTL decreased significantly after injection at the 24th h in DM+Bup group(P =0.000;P = 0.000).High glucose environment and bupivacaine can lead to the in-crease of MDA and the inhibition of SOD activity,and have synergistic effect.Compared with Bupivacaine group,the expression of Ku70 was inhibited in diabetic rats administered by intrathecal injection bupivacaine(t = 4.475,P = 0.000).Spinal tissue pathological injury was significantly aggravated in diabetic rats with intrathe-cal injection of bupivacaine.Compared with Bupivacaine group,the expression of cleaved caspase-3 protein and the percentage of apoptosis in spinal nerve cells were significantly increased in diabetic rats with intrathecal injection of bupivacaine(t =-4.707,P= 0.000;t ＝-20.041,P= 0.000).Conclusion Hyperglycemia could enhanced diabetes rat spinal nerve damage induced by bupivacaine and inhibit Ku70 enzyme expression.