Discussion On The Analgesic Mechanism Of Massage On SNL Rats From The Regulation Of Microglial Polarization By Apoptosis In Spinal Dorsal Horn Neurons

Objective:To explore the mechanism of massage therapy by inhibiting neuronal apoptosis to promote the polarization of microglia into anti-inflammatory M2,inhibit inflammatory reaction and play an analgesic effect.Methods: 30 SD rats were randomly divided into 5 groups: spinal nerve ligation group(SNL group),normal group,massage group,sham massage group and sham operation group.Except for the normal group and the sham operation group,the other three groups all constructed SNL model.On the first day after operation,the massage group began to receive treatment,the sham massage group intervened for 14 days,and the other three groups did not intervene.The thermal pain response threshold(PWTL)and mechanical stimulation response threshold(PWMT)were measured before operation,1day,3 days,7 days and 14 days after operation,and the number of Iba1,CD86,CD206 and Nue N fluorescence positive cells in spinal dorsal horn were detected by immunofluorescence on the 14 th day after operation.Western-blot electrophoresis was used to detect the expression of apoptosis-related proteins Caspae-3 and Bcl-2.Apoptosis was observed by TUNEL fluorescence staining.The levels of serum inflammatory cytokines IL-10 and TNF-αwere measured by ELISA serum detection kit.Results:(1)Results of the study on mechanical pain thresholds of rats:the comparison of mechanical pain thresholds of rats in each group before the establishment of SNL model was statistically determined to be non-significant(P > 0.05);the mechanical pain thresholds of rats in the normal group and the sham-operated group were statistically determined to be non-significant(P >0.05)on days 1,3,7 and 14 after the successful establishment of the model and the implementation of the intervention;the mechanical pain thresholds of rats in the SNL group were statistically determined to be non-significant(P >0.05)compared with those in the sham-pushing group.The mechanical pain thresholds of rats in the SNL group were statistically determined to be insignificant(P > 0.05);the mechanical pain thresholds of rats in the normal,sham-operated,SNL,sham-operated,and pusher groups were statistically determined to be significantly different(P < 0.05)on days 1,3,and 7 after modeling;the mechanical pain thresholds of rats in the pusher,SNL,and sham-pusher groups were statistically determined to be insignificant(P > 0.05)on the first day after modeling On the third,seventh and fourteenth days after modeling,the mechanical pain thresholds of the pusher,SNL and sham pusher groups were statistically determined to be significantly different(P<0.05).(2)Results of thermal pain thresholds in rats: the comparison of thermal pain thresholds of rats in each group before the establishment of SNL model was statistically determined to be not significant(P>0.05);the thermal pain thresholds of normal and sham-operated groups were statistically determined to be not significant(P>0.05)on days 1,3,7,and 14 after modeling;the thermal pain thresholds of rats in SNL group and sham-pushing group were statistically determined to be not significant(P>0.05);at the time points of day 1,3 and 7 after modeling,the thermal pain thresholds of the normal group,sham-operated group and SNL group,sham-pushing group and pushing group were statistically determined to be significantly different(P<0.05);at day 1after modeling,the differences in thermal pain thresholds of the rats in the pushing group,SNL group and sham-pushing group were statistically determined to be not significant(P>0.05);at days 3,7 and 14 after modeling,the thermal pain thresholds of the rats in the pushing group,SNL group and sham-pushing group were statistically determined to be not significant(P>0.05).The differences in thermal pain thresholds of the pushing group were statistically determined to be significant(P<0.05).(3)Results of expression levels of apoptosis-related proteins Caspass-3and Bcl-2 in rats: When compared 14 days after modeling,no statistically significant difference was found between the normal and sham-operated groups(P > 0.05);when the normal and sham-operated groups were compared with the SNL group,sham-operated group and the Pushing group The difference was determined to be statistically significant when comparing the normal and sham surgery groups to the SNL group,the sham pussing group,and the pussing group(P<0.05);the difference was not determined to be statistically significant when comparing the pussing group to the sham pussing group(P>0.05);the difference was not determined to be statistically significant when comparing the pushing group and the sham pushing group.At day 14 of plastic surgery,the anti-apoptotic protein Bcl-2 was statistically significant(P<0.05)in the normal,sham surgery,and SNL groups compared to the sham push and push-and-take groups;in the push-and-take group,the difference was statistically significant(P<0.05)compared to the SNL,sham push,and sham surgery groups statistically significant(P<0.05).(4)Results of the expression level of activated Iba-1 in rat microglia:When comparing the normal and sham surgery groups,the difference was determined to be not statistically significant(P>0.05);when comparing the SNL and sham surgery groups,the difference was not statistically significant(P>0.05);the SNL group,sham pushing group and the sham surgery group,the difference was determined to be statistically significant(P<0.05);when comparing the rats in the pushing group to the rats in the SNL and sham pushing groups,the difference was significant(P<0.05).(5)Results of CD86 and CD206 expression of microglial polarity in rats:on day 14 of nerve injury,the expression of M1 marker CD86 in the lumbar segment of the spinal cord was higher in the SNL model rats compared to rats in the normal and sham-operated groups,and the difference is statistically significant(P<0.05);Conversely,the expression of M2 marker CD206 in the lumbar spinal cord of rats in the SNL model was decreased compared with rats in the normal and sham surgery groups,and the difference was statistically significant(P<0.05);After tui na intervention,the expression of M1 marker CD86 in the lumbar spinal cord of rats in the tui na group was showed a decreasing trend compared with the sham tui na group(P<0.05);conversely,the expression of M2 marker CD206 in the lumbar spinal cord of rats in the tui na group increased compared with the SNL group and the sham tui na group(P<0.05).(6)Results of TUNEL apoptosis staining in rats: there was no statistically significant difference between the normal and sham-operated groups(P>0.05);the difference between the SNL and sham-operated groups was determined to be statistically insignificant(P>0.05);there was basically no green fluorescence in the normal and sham-operated groups,and the SNL group,sham Pushing group,green fluorescence was significantly enhanced in the spinal cord tissue of the Pushing group(P<0.05);fluorescence intensity was significantly decreased in the Pushing group compared to the SNL and sham Pushing groups(P<0.05).(7)Results of Neu N expression in rat neurons: when comparing the normal and sham surgery groups,the difference was determined to be not statistically significant(P>0.05);when comparing the SNL and sham push-up groups,the difference was determined to be not statistically significant(P>0.05);when comparing the normal and sham surgery groups and SNL group,sham push-up group and push-up group,the difference was determined to be statistically significant(P<0.05);when comparing the pushing group with the SNL group and sham pushing group,the difference was determined to be statistically significant(P<0.05).(8)Results of serum inflammatory factor IL-10 and TNF-α expression in rats: when the serum inflammatory factor TNF-α was compared between the normal and sham surgery groups,the difference was not statistically significant(P>0.05);when the difference was compared between the normal,sham surgery and SNL and sham surgery groups,the difference was judged significant(P<0.05);when compared between the SNL group and the sham When compared between the SNL group,sham surgery group,and sham pusher group,the difference was statistically determined to be not significant(P>0.05);the difference was statistically determined to be significant(P<0.05).05),when compared between the SNL,sham-operated,and sham-pushing groups;also,the serum anti-inflammatory factor IL-10 of each rat group was not statistically significant(P>0.05)when compared between the normal,sham-operated,and SNL groups,respectively;the difference was not statistically significant(P>0.05);the normal,sham surgery group,SNL group,compared to the sham push and push-pull groups,the difference was determined to be statistically significant(P<0.05);compared to the sham push and push-pull groups,the difference was statistically significant(P<0.05);compared to each other.Conclusion: Massage can alleviate the pain hypersensitivity reaction of neuropathic pain model rats,which may be by inhibiting neuronal apoptosis,thereby promoting the transformation of microglial cell polarization from pro-inflammatory M1 type to anti-inflammatory M2 type,and reducing the inflammatory response.So as to achieve the effect of pain relief.