| Object: Neuropathic pain is a kind of intractable chronic pain with high incidence and obvious clinical symptoms,which seriously threatens the life and health of patients.The pathogenesis of neuropathic pain has not been fully elucidated.At present,it is generally accepted that oxidative stress is the main cause of neuropathic pain,and a large number of studies have confirmed that inhibiting oxidative stress can greatly improve neuropathic pain.Therefore,methyl ferulic acid,a traditional Chinese medicine with strong antioxidant stress response,was selected in this study to confirm its pharmacological effect on neuropathic pain and its potential therapeutic mechanism.Methods: Neuropathic pain was induced by SNI model in male SD rats.Methyl ferulic acid(5 mg/kg,10mg/kg,20 mg/kg)was given intragastric treatment for 14 days after the establishment of SNI models.Nox4 overexpression was made by microinjection of Nox4 adeno-associated virus 4 weeks before modeling.Pain behavior tests including PMWT,PWCD and PTWL were performed 1 day before surgery and 1,3,7 and14 days after surgery to observe the effect of methyl ferulic acid on neuropathic pain.Western blotting and immunofluorescence staining were used to detect the expression of Nox4 protein and ferroptosis marker protein such as ACSL4 and GPX4 on DRG.ROS levels of DRG neurons were detected by fluorescence probe,iron content of DRG tissues was detected by tissue iron assay kit,and mitochondrial morphology of DRG neurons was observed by transmission electron microscopy.Results:(1)Compared with the Sham group,the thresholds of PMWT and PWCD in the SNI group decreased(both P<0.001).There was no significant difference in PTWL between SNI group and Sham group(P>0.05).In addition,compared with Sham group,after SNI injury,the expression of Nox4increased(P<0.001),the expression of ferroptosis marker protein ACSL4 increased(P<0.001),and the expression of GPX4 decreased gradually(P<0.001).At the same time,ROS,the content of iron and the number of damaged mitochondria also increased(all P<0.001).(2)Compared with SNI group,the thresholds of PMWT and PWCD in SNI+MFA 5mg/kg group,SNI+MFA 10mg/kg group and SNI+MFA20mg/kg group were significantly increased(all P<0.001).The effects of SNI+MFA 20mg/kg and SNI+MFA 10mg/kg groups on PMWT and PWCD were stronger than those of SNI+MFA 5mg/kg group(all P<0.001).The effects of SNI+MFA 20mg/kg and SNI+MFA 10mg/kg groups on PMWT and PWCD were not statistically significant(P>0.05).There was no significant difference in PTWL among all groups(P>0.05).Compared with SNI group,SNI+MFA 5mg/kg,SNI+MFA 10mg/kg and SNI+MFA 20mg/kg groups were found to have lower expression of Nox4,lower expression of ACSL4,and higher expression of GPX4(all P<0.001).Meanwhile,ROS expression was lower(P<0.001),the percentage of abnormal mitochondria was lower(P<0.05),and iron content was lower(P<0.05).(3)Compared with Sham group,PMWT and PWCD thresholds were lower in SNI+AAV-Nox4 group(P<0.001)and were lower than SNI and SNI+AAV-NC,but were reversed after administration of methyl ferulic acid by gavage(P>0.05).There was no significant difference in paw thermal withdrawal among all groups(P>0.05).Nox4 expression was higher in the SNI+AAV-Nox4 group compared to the SNI+AAV-NC group and the SNI group(both P<0.01),and ACSL4 was also higher(both P<0.001)and GPX4 was lower(P<0.05,P<0.01).In addition,ROS,iron content and percentage of abnormal mitochondria were higher(P<0.001,P<0.01,P<0.05).After methyl ferulic acid treatment,compared with SNI+AAV-Nox4 group,Nox4,ACSL4,ROS and iron contents were lower(all P<0.001),GPX4 was higher(P<0.001),and the number of abnormal mitochondria was lower(P<0.001).Conclusion: Methyl ferulic acid improved PMWT and PWCD of neuropathic pain in rats,which was associated with the inhibition of Nox4 and ACSL4 expression and the up-regulation of GPX4 expression. |
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