Bupivacaine Aggravates Diabetic Peripheral Neuropathy Via The Sirt3 Pathway

BackgroundDiabetic Peripheral Neuropathy(DPN)is the most common chronic complication of diabetes and the most common type of diabetic neuropathy.Diabetic patients are prone to multiple organ or system diseases,and the proportion of diabetic patients requiring anesthesia or surgical treatment has also increased significantly.Spinal block and regional nerve block are commonly used clinical anesthesia methods.However,local anesthetics have been proven to have direct or indirect neurotoxic effects.ObjectiveEstablish a mouse model of type 2 diabetes,to clarify the behavioral phenotype of DPN aggravated by bupivacaine and the role of Sirt3 signaling pathway in DPN aggravated by bupivacaine;specific mechanism of action.MethodThis study is divided into two parts:animal experiment and cell experiment.Animal experiment:the first part is divided into 4 groups:NDM group,NDM+BP group,DM group and DM+BP group.,by measuring the mechanical pain withdrawal threshold(PWT)of the mouse plantar and the paw withdrawal latency(PWL)of the plantar photothermal stimulation to determine the sensitivity of the mouse to mechanical pain stimulation and thermal pain stimulation to determine the spinal cord nerve of the mouse degree of functional impairment.The second part is divided into four groups:DM group,DM+3-TYP group,DM+BP group and DM+BP+3-TYP group,the dose of 50mg/kg is given to DM+3-TYP group and DM+BUP+3 The mice in thetyp group were intraperitoneally injected with 3-TYP,once every two days,for a total of 3 injections,and continued to measure PWT and PWL.Frozen sections of mouse spinal cord tissue were used to detect ROS levels;Western blot was used to detect protein expression levels of Sirt3,SOD2,Drp 1,and Cleaved caspase-3.Cell experiments are grouped into NHG group,NHG+3-TYP group,NHG+BP group,NHG+BP+3-TYP group,HG group,HG+3-TYP group,HG+BP group and HG+BP+3-TYP group Group.CCK8 was used to detect cell viability;JC-1 was used to detect mitochondrial membrane potential;qRT-PCR was used to detect the mRNA expression levels of Opal,Mfnl,Fis1 and Drpl in ND7/23 cells.Result1.Model in vivoBehavioral results:The PWL value of the DM+BP group increased 1 hour after intrathecal injection compared with the DM group,and the PWL value of the DM group decreased compared with the NDM group,and the difference between the groups was statistically significant(P<0.05).In the second part of the behavioral experiment,the PWT value of the DM+BP+3-TYP group was lower than that of the DM+3-TYP group 3 hours after intrathecal injection,and the difference was statistically significant(P<0.01).The PWL value of the DM+BP+3-TYP group was lower than that of the DM+BP group,and the difference was statistically significant(P<0.05).The results of detecting ROS level by frozen section of mouse spinal cord:the ROS level of DM+3TYP group was significantly higher than that of DM group,and the difference was statistically significant(P<0.05);the ROS level of DM+BP+3-TYP group was significantly higher than that of DM group The difference was statistically significant(P<0.01).Western blot results showed that the expression level of Sirt3 in the DM group was significantly lower than that in the NDM group(P<0.01);the expression in the DM+BP group was significantly higher than that in the DM group(P<0.05);the expression level of DM+BP+3-TYP was significantly higher than that in the DM+group The expression level of Sirt3 in BP group was significantly decreased(P<0.01).The expression of Drpl in the NDM+BP group was significantly higher than that in the NDM group(P<0.05);the expression in the DM+BP group was significantly higher than that in the DM group(P<0.01).The expression of SOD2 in the DM+3-TYP group was significantly lower than that in the DM group(P<0.05);the expression in the DM+BP+3-TYP group was significantly lower than that in the DM+BP group(P<0.01).The expression level of Cleaved caspase-3 in the DM+3-TYP group was significantly lower than that in the DM group,and the difference was statistically significant(P<0.05);the expression level in the DM+BP+3-TYP group was significantly lower than that in the DM+BP group(P<0.0001).2.Model in vitroThe results of JC-1 showed that compared with the NHG group,the mitochondrial membrane potential of each group decreased,and it showed a stepwise decline with the increase of intervention conditions.Compared with NHG+3-TYP group,the mitochondrial membrane potential of HG+BP decreased significantly(P<0.01).The results of qRT-PCR showed that the expression level of Opal in HG+BP+3-TYP group was significantly lower than that in NHG group(P<0.05).The expression level of Mfnl mRNA in the NHG+3-TYP group was significantly lower than that in the NHG group(P<0.0001);the NHG+BP+3-TYP group was significantly lower than that in the NHG+BP group(P<0.001);the HG+3-TYP group Compared with the HG group,it was significantly lower(P<0.01);the HG+BP+3-TYP group was significantly lower than the HG+BP group(P<0.05).The expression level of Fis1 mRNA in NHG+3-TYP group was significantly higher than that in NHG group(P<0.05);NHG+BP+3-TYP group was higher than that in NHG+BP group,and the difference was statistically significant(P<0.01).The expression level of Drpl in NHG+3-TYP group was significantly higher than that in NHG group(P<0.01).Conclusion1.Bupivacaine aggravates the phenomenon of hyperalgesia in diabetic mice,and Sirt3 inhibitor 3-TYP can further reduce the pain domain of DPN mice;2.The synergistic effect of high glucose environment and bupivacaine leads to mitochondrial damage,and the excessive production of ROS by Drpl-mediated mitochondrial fragmentation;3.The high-glucose environment reduces the expression of Sirt3,inhibits the expression of SOD2 downstream of it,and makes its ability to scavenge ROS insufficient,eventually leading to cell apoptosis and nerve damage.