The Role And Mechanisms Of DRP1 In The Neurotoxicity Induced By Local Anaesthetic

With the development of medicine,although general anesthesia has become the first choice of most surgical patients,but local anesthesia is still applied to date because of its many advantages,such as operating easily,keeping patients safe and awake,and fewer impacts on physiological function.In recent years,the concept of comfortable medical treatment and ERAS is gradually strengthening,as well as the ultrasound technology is more and more applied to clinical anesthesia,the safety and accuracy of regional block technology has greatly improved.Local anesthetics are more and more applied to clinical works,while the incidence of adverse reactions is also increasing.The potential neurotoxicity of local anesthetics has gradually attracted the attention of anesthesiologists.For the most of the adverse reactions of local anesthetics,such as hypersensitivity and cardiotoxicity,we all have prevention methods and treatment measures.But for the neurotoxicity,whose mechanisms are not clear now,we can only reduce the concentration of drugs without anyother effective measures.But reducing drug concentration can also reduce the effect of local anesthetics,so it is difficult to balance the risk and benefit.Even worse,the dysfunctions due to neurotoxicity are often characterized by severe symptoms and slow recovery.At present,there is no consensus on the mechanisms of neurotoxicity caused by local anesthetics,so it is very necessary to do some research.Objective:The mechanisms of neurotoxicity are not clear,at present,most of the studies show that the apoptosis is the pathological basis of neuron damage.Mitochondrial homeostasis plays an important role in endogenous mitochondrial pathway,which plays an important role in neuron apoptosis.Mitochondrial dynamic related protein 1(DRP1)is a key GTP enzyme regulating mitosis,and its abnormal activation is involved in the mitochondrial homeostasis.This study explores the role of DRP1,which is closely related to mitochondrial homeostasis,played in mechanisms of neurotoxicity caused by ropivacaine from cell level to animal level.It also reveals the regulatory mechanisms of DRP1 and its downstream signaling pathways.This study not only provides the experimental evidence for clarifying the mechanisms of neurotoxicity caused by ropivacaine,but also provides the intervention target for the clinical treatment of neurotoxicity and the development of new local anesthetics.Methods:(1)Part 1 experiment took SH-SY5 Y cells as the study object,which were divided into blank control group(Ctrl)and experimental groups.Ctrl group was a complete medium without ropivacaine,while the experimental groups all contained ropivacaine,whose concentration was set to 0.5,1,2,5 mM and stimulation time was set to 12,24,48 and 72 h.CCK-8 method was used to detect cell viability;The flow cytometry of annexin V-FITC/PI staining was used to to detect cell apoptosis rate;Mito tracker dye was used to label cell mitochondria and JC-1 dye was used to detect cell mitochondria membrane potential(MMP);The expressions of related proteins were measured by Western Blot.At last,we construct a study cell model for neurotoxicity of ropivacaine in vitro culture.(2)Part 2 experiment established a cell model by using chemically modified siRNA oligo,which could interfer the expression of target gene,to reduce the DRP1.Ctrl group was normal SH-SY5 Y cells without ropivacaine for 24h;si-DRP1 group was SH-SY5 Y cells transfected by DRP1 siRNA,induced by 2 mM ropivacaine for24h;si-NC group was SH-SY5 Y cells transfected by control siRNA,induced by 2mM ropivacaine for 24 h.CCK-8 method was used to detect cell viability;The flow cytometry of annexin V-FITC/PI staining was used to to detect cell apoptosis rate;MMP,ROS and ATP were detected to determine the changes of mitochondrial function;The expressions of mRNA and related proteins were measured by qRT-PCR and Western blot.(3)Part 3 experiment divided 40 SPF-grade SD male rats into sham operation group and ropivacaine group.The animal model of neurotoxicity induced by ropivacaine was established through directly exposing the sciatic nerve of the right hind limb and injecting the required drugs.After nerve injection for 24 hours,the sciatic nerve tissue at the injection site was taken,then TUNEL method was used to detect the apoptosis of sciatic nerve;MMP,ROS and ATP were detected to determine the changes of mitochondrial function.Western blot and immunohistochemistry were used to detect the expression level of related proteins.Results:(1)The results of CCK-8 test showed that: the relative activity of SH-SY5 Y cells decreased to(83.20±3.21)%,(71.70±5.25)%,and(53.05±3.45)%,after being stimulated by ropivacaine at concentrations of 0.1,0.5,1,2,and 5 mM for 24 hours,and the differences were all significant(P<0.05);while the relative activity of SH-SY5 Y cells decreased to(75.18±2.87)%,(63.25±2.25)%,(58.50±2.66)%and(48.15±3.65)%,after 12,24,48 and 72 hours of 2 mM ropivacaine stimulation,and the the differences were all significant(P<0.05).(2)The apoptosis rate of Ctrl group was 2.70%,but the rate of other 4 groups after being stimulated by 0.1,0.5,1,2,and 5 mM ropivacaine for 24 hours,increased to 4.02%,8.75%,9.85% and 14.24%,and the differences were all significant(P <0.05);The apoptosis rate of Ctrl group was 2.28%,while the rate of other 4experimental groups,after being stimulated by 2 mM ropivacaine for12,24 48,72 hours,increased to 4.80%,7.51%,9.10% and 13.08%,and the differences were all significant(P< 0.05).(3)After induced with 2 mM ropivacaine for 24 hours,Mito tracker test showsed that the number of normal mitochondria decreased and the accumulation of ROS in cells increased.It suggested that ropivacaine might cause the abnormal function of SH-SY5 Y cells by oxidative stress.Accordingly,JC-1 test showed that the MMP of the SH-SY5 Y cells was reduced by ropivacaine significantly.Western Blot showed that the expression of Mfn11,Mfn2,DRP1 and Caspase-3 in SH-SY5 Ycells were all significantly increased after 24 h induction of ropivacaine.(4)After interfering with the expression of DRP1 in SH-SY5 Y cells,CCK-8test showed that the relative activity of si-NC group was(61.22±5.58)%,while si-DRP group was(89.65±6.87)%,and there was statistically significant difference between groups(P<0.01).Similarly,the apoptosis rate of si-DRP group was(6.35±0.65)%,while the si-NC group was(9.13±0.64)%,and there were also statistically significant difference between groups(P<0.01).(5)After interfering with the expression of DRP1 in SH-SY5 Y cells,controlled with Ctrl group,mitochondria in si-NC group changed as follows:the relative concentration of ROS was(290.27±12.98)%,ATP was(384.28±39.18)Um/g,MMP decreased to(85.07±8.76)%;While controlled with Ctrl group,mitochondria in si-DRP1 group changed as follows:the relative concentration of ROS was(204.44±12.98)%,ATP was 453.38±28.90 Um/g,MMP was(103.25±10.02)%.There were statistically significant differences about ROS,ATP and MMP between si-NC group and si-DRP1 group(P<0.01).These results indicated that inhibition of DRP1 could restore mitochondrial dysfunction of SH-SY5 Y cells induced by ropivacaine.(6)Western Blot test showed that the expressions of DRP1,p-DRP1 and p-p38 MAPK were all decreased statistically significantly in si-DRP1 group controlled with si-NC group(P<0.01).The expression of Caspase-3 of si-DRP1 group was also significantly different from that of si-NC group(P<0.05).It suggested that the function changes of the mitochondria might be activated by the DRP1/p38 MAPK signaling pathway,which could also induce apoptosis.(7)In the animal model experiment,TUNEL test showed that the apoptotic rate of sciatic nerve cells in Sham group was(6.31± 1.23)%,while that in Ropi group was statistically significantly increased to(14.20±2.18)%(P<0.01).Compared with Sham group,the ROS content,ATP content and mitochondrial membrane potential of the sciatic nerve tissue in Ropi group were all significantly changed(P<0.001).The results of immunohistochemistry showed that the expression of DRP1 and the activation of p-P38 MAPK in the sciatic nerve tissue of the Ropi group increasedcompared with sham group;The Western Blot results further confirmed that the expression of DRP1,p-DRP1,P-P38 MAPK and Caspase-3,whose relative values were(87.23±19.20)%,(198.39±20.05)%,(310.82±25.52)% and(185.23±1.66)%,were all increased significantly in Ropi group compared with sham group(P<0.01).Conclusions:1.Ropivacaine can cause mitochondrial dysfunctions and increase the expression of DRP1 in SH-SY5 Y cells.It can also inhibit the activity and increase the apoptosis rate significantly,and the changes show concentration and time dependence.We think that ropivacaine has neurotoxic effect on SH-SY5 Y cells.2.After interfering with the expression of DRP1 in SH-SY5 Y cells,the neurotoxic effect induced by ropivacaine changes as follows:the inhibition of the mitochondrial function reduces significantly,the activity of cells increases significantly,and the rate of apoptosis decreases significantly.It suggests that DRP1 plays an important role in the neurotoxic effect of ropivacaine on SH-SY5 Y cells.While the expression of DRP1 decreases,the expression of P-DRP1,p-p38 MAPK and Caspase-3 are also significantly decreased,we can assum that p38 MAPK is involved in the apoptosis of SH-SY5 Y cell induced by ropivacaine,and DRP1/p38 MAPK signaling pathway is involved and plays a key role in the neurotoxic effect of ropivacaine on SH-SY5 Y cells.3.Animal experiment,whose results are consistent with that of cell experiment,confirms that ropivacaine has neurotoxic effect and increases apoptosis rate significantly on sciatic nerve cell through DRP1/p38 MAPK signaling pathway.It also suggests that DRP1/p38 MAPK signaling pathway plays a key role in the neurotoxic effect of ropivacaine.It also provides the intervention target for the clinical treatment of neurotoxicity and the development of new local anesthetics.