| Background Bupivacaine(Bup)is a kind of local anesthetics that widely used in clinical anesthesia and analgesia which plays a very important role in clinic anesthesia.But the neurotoxic effect of local anesthetics might bring patients mental and physical problems,which could seriously restrict the quality of patients’ life.So it is extremely worth anesthesiologists and related person attach more attention to it.Transcription factor p53 owns the function of inducing cell cycle arrest,promoting DNA repair and apoptosis,which aims to repair or remove the damage of DNA.It is very important in the process of the lives’ emergence,development and many diseases in eukaryotes.LncRNA TUG1(taurine upregulated gene 1)participates in the development of the nervous system,and have close relationship with many central nervous system diseases.But the evidence of it in peripheral nervous diseases has not been found.There has study shows that TUG1 has strong correlationship with p53 in many diseases.And TUG1 is a downstream target gene of protein p53 in certain cases.In current time,the research of p53 in nerve injury or repair mainly concentrated in the central nervous system,and very few reported in peripheral nerves.The number of studies of TUG1 in the function of DNA injury or repair are also limited and mostly concentrated in the field of tumor.So in intraspinal anesthesia or peripheral nerve block,if bupivacaine causes damage to mouse DRG cells,it may also cause DNA damage,and even cause DNA damage response(DDR)to reduced the DNA damage in p53－dependent pathway.This study intends to explore the role of p53/TUG1 pathway in the process of DRG injury induced by bupivacaine in mice at the genetic level,so as to provide reference information for the prevention and treatment of neurotoxicity of local anesthetics.Objects:1.To detect the DNA damage effect of bupivacaine on mice DRG cells2.To explore the function of p53/TUG1 pathway in DNA damage in DRG cells caused by bupivacaine.3.To explore the mechanism of p53/TUGl in bupivacaine induced DNA damage in DRG cellsMethods1.DRG cells of 4 weeks old of female C57/BL6J mouse were isolated and cultured.CCK-8 assay was used to detect cell activity after treatment with different concentrations of bupivacaine for 3 hours.IC50 value was calculated and as a reference for the optimal concentration of bupivacaine in later experiments.Annexin V-FITC assay was used to detect apoptosis.Comet assay was used to detect DNA damage.Western blot was used to detect the expression of DNA damage related protein p-h2ax to verify the damage of bupivacaine on DNA and cells.The cells were treated with DNA repair key enzyme DNA-pk inhibitor nu7441 and then treated with bupivacaine.The change of cells’ viability was detected with CCK-8 test again while the comet assay was used to detect DNA damage which were used to judge that whether bupivacaine cause cells injury by causing DNA damage.2.Protein and RNA were extracted from bupivacaine treated cells to detect the expression of p53.p53 was knocked down with siRNA and then treated with bupivacaine.Comet assay was used to detect DNA damage and cck-8 was used to detect changes of cell viability,which were used to detected the relationship between p53 and DNA as well as cell damage.The expression level of lncRNA TUG1 was detected to research the effect of p53 on TUG1.3.The interaction between p53 and TUG1 was researched with information retrieval,and then experiment of combined 1ncRNA TUG1 fluorescence in situ hybridization(FISH)with p53 protein Immunofluorescence(IF)was used to verify the information retrieval result,both of which were aimed to confirm the positional relationship between p53 and TUG1 expression in mice’s DRG cells after treated with bupivacaine.4.The expression of TUG1 was detected in DRG cells treated with bupivacaine.After TUG1 was knocked down with siRNA,treated the cells with bupivacaine followed.Comet experiment was used to detect DNA damage when cck-8 was used to detect cells vitality of different groups.All of which aimed to research the the function of TUG 1 in the relationship between DNA damage and cells damage.5.DRGs of lumbosacral segment of mice were isolated after 30 minutes of intrathecal injection of bupivacaine or normal saline,and then extract the protein and RNA,western blot detected p53 protein and DNA damage signature protein expression,and qPCR detect the expression of TUG1,which aimed from another aspect to verify the function of p53 and TUG1 in the progress of bupivacaine caused cells and DNA damage.Results:1.Bupivacaine can reduce cell viability in a concentration-dependent mannier,aggravate apoptosis.and aggravate DNA damage.2.When DNA-PK,a key enzyme for DNA repair,is inhib ited.DNA damage and cell damage caused by bupivacaine will be aggravated.3.Bupivacaine can cause up-regulation expression of p53 gene.When p53 is kn ocked down by siRNA,DNA dam age and cell damage caused by bupivacain e will be aggravated.4.When p53 is knocked down,TUG1 expression also decreases.Information retrieval shows where p53 is likely to bind to the promoter of TUG 1.P53 and TUG1 have common expression sites in mouse cells.5 Bupivacaine also causes down-regulation of lncRNA TUG1.After IncRNA is knocked down by siRNA,DNA damage caused by bupivacaine and c.ell damage will also be aggravated.6.Vivo experiments showed that intrath ecal injection of bupivacaine in mice also caused increased expression of p53 and lncRNA TUG1.Conclusions:1.Bupivacaine is neurotoxic which could cause DRG cells and DNA damage in mice.2.The DNA damage induced by bupivacaine in mice can activate the DNA damage response of the p53-dependent pathway and carry out DNA repair.3.In the DNA damage response of mouse DRG cells,p53 may take lncRNA TUG1 as the downstream target.participate in DNA repair,and reduce D NA damage to reduce cell damage. |
