Protective Effect And Involved Mechanism Of Duloxetine On Peripheral Neuropathy Induced By Oxaliplatin

Background and purpose:Oxaliplatin(OXA)is a third-generation platinum-based chemotherapy drug widely used in the treatment of advanced metastatic colorectal cancer,ovarian cancer,breast cancer and lung cancer.However,while effective in tumors,it also produces severe peripheral neuropathy(OIPN)in approximately 90%of patients.This peripheral neuropathy is the only major dose-limiting toxicity associated with OXA.It feels dull for the periphery and feels abnormal for the limbs.In order to alleviate this side effect,various drugs such as anticonvulsants and tricyclic antidepressants have been clinically prevented.However,these drugs also cause many side effects and hinder their wide application.The US Food and Drug Administration(FDA)has approved duloxetine for the treatment of peripheral neuropathic pain caused by diabetes.In addition,in a randomized,double-blind,placebo-controlled,crossover clinical trial,compared with the placebo group,Patients in the loxetine-treated group had more reduction in CIPN induction.Duloxetine is a serotonin and norepinephrine reuptake inhibitor and is a well-known antidepressant.However,it is also considered effective against various pains such as fibromyalgia,osteoarthritis knee pain and chronic low back pain.Regarding OXA-induced neuropathic pain,the American Society of Clinical Oncology(ASCO)recommended duloxetine as a drug for the treatment of peripheral neuropathy caused by chemotherapy based on several clinical trials.However,the site and mechanism of duloxetine action are not known.Therefore,the purpose of this study is to make a preliminary discussion on the mechanism of the OIPN mechanism based on the experimental results and conjectures of the experts in this field.Research program:Chapter 1:The activity of MTX assay was used to detect the effect of different concentrations of duloxetine on OXA-induced DRG cells.The appropriate concentration of duloxetine was determined by axon staining to determine whether it has protective effect on axons.8 method to detect the effect of duloxetine on the anticancer effect of oxaliplatin.Chapter 2:Grouping cells into control group,OXA model group,duloxetine treatment group and monotherapy group,using protein immunoblotting and fluorescence quantitative polymerase chain reaction to detect oxidative stress and apoptosis-related proteins and Changes in mRNA;further study of the changes of p53 and SARM in the treatment of duloxetine in the process of cell injury by immunofluorescence;detection of NAD+/NADP content in cells by NAD kit;detection of reactive oxygen species in ROS kit and use of calcium imaging technology To determine the mechanism of oxaliplatin and duloxetine affecting DRG cell activity.Chapter 3:The mice were divided into 5 groups by pre-experiment,which were control group,OXA model group,duloxetine high-low dose group and single-agent group,10 mice in each group.Behavioral experiments related to mechanical stimulation.After four weeks,the changes of fiber density in the epidermis of each group were observed by immunohistochemistry.The effect of duloxetine on the expression of oxidative stress and apoptosis-related proteins in DRG and sciatic nerve tissue was examined by Western blotting.Time PCR was used to detect the expression of related mRNA in DRG and sciatic nerve;the changes of p53 and SARM in the treatment of DRG tissue injury were detected by immunohistochemistry.Chapter 4:Flow cytometry,cell transfection and Western blotting were used to further explore whether the mechanism of duloxetine treatment of OIPN is related to p53-caspase-3 or p53-Bax/Bcl2 signaling pathway.Research result:In the first chapter,by comparing the axon status between groups,it was found that duloxetine(1μM)can alleviate the influence of oxaliplatin damage on DRG neuron axons to some extent;duloxetine in cells Levels have no significant inhibition or synergistic effect on the anti-tumor effect of oxaliplatin.Analysis of the second chapter found that oxaliplatin-induced DRG nerve cell damage was mainly caused by apoptosis,although it may not be related to oxidative stress,but ROS detection found that the ROS content of the treatment group was significantly higher than the model group.This indicates that the synergistic effect of oxaliplatin and duloxetine may cause an increase in ROS in DRG neurons and further cause oxidative stress.This result needs further confirmation.However,the final results indicate that duloxetine can reduce the expression of P53-Bax/Bcl2 and protect DRG cell axons from damage and reduce apoptosis.The third chapter proves that the mouse model is successful by analyzing the behavioral results,and duloxetine can effectively prevent peripheral nerve pain caused by oxaliplatin.In the analysis of different groups of different methodologies,the mechanism of duloxetine treatment of OIPN was associated with signaling pathways downstream of p53(caspase family or bcl2 family)and did not appear to be associated with oxidative stress.In the last chapter,by silencing the expression of p53 protein in DRG cells,the immunoblotting results showed that the p53-Bax/Bcl2 apoptosis pathway must be one of the mechanisms of duloxetine in the treatment of OIPN.Analysis conclusion:1.The mechanism of duloxetine in relieving OIPN may not be related to oxidative stress,but the combination of oxaliplatin and duloxetine increases the reactive oxygen species in DRG cells.2.In vitro studies have shown that duloxetine has the effect of alleviating oxaliplatin-induced DRG neuronal damage,and has no effect on the anti-cancer effect of OXA.In vivo studies have shown that duloxetine has a significant preventive effect on various pains caused by OXA,and the mechanism may be to further terminate the apoptosis process in vivo by inhibiting p53 molecules and downstream Bax/Bcl2 proteins and caspase families.In summary,duloxetine is currently a more effective treatment for OIPN,and its mechanism will help to develop more effective targeted drugs for OIPN in the future.