Study On The Effect And Mechanism Of Message On Rats With Chronic Neuropathic Pain Based On ERK Signaling Pathway

Objective: To establish a chronic constrictive injury(CCI)model in this experiment,observe the paw withdrawal latency,paw withdrawal threshold,and inclined plane test in each group after the establishment of the model,and detect the protein and gene expression of Raf-1,ERK,and CREB in the ERK signaling pathway and the expression of the inflammatory factor IL-1? in spinal cord(L4-L6)of ras.To investigate the mechanism of analgesia of massage from the molecular level.Methods: 70 SD male rats were randomly divided into 4 groups:(1)normal group,(2)sham-operated group,(3)CCI model group,(4)massage group,with 10 cases in each group.The normal group was naturally fed without any intervention.The sham group exposed the sciatic nerve without ligation.The CCI group was established according to Bennett's method.Massage therapy was respectively applied to Huantiao acupoint?Fengshi acupoint and Yanglingquan acupoint.The therapy began at day 3 after CCI operation and resumed once a day for 9 minutes until the rats were killed at day 14 after the last treatment.The latency latency(PWL)of SD rats was observed before CCI operation(0)days and at days 3,7,10 and 14 after operation.The mechanical paw withdrawal threshold(PWT)and inclined plane test(IPT)were observed before CCI operation(0)days and at days 3 and 14 after operation.On the 14 th day,the expression of Raf-1,ERK,CREB and IL-1? mRNA in spinal cord(L4-L6)in ERK signal pathway was detected by real-time fluorescence quantitative PCR.The expression of Raf-1,ERK,and CREB protein was detected by western blot.Results:(1)The thermal pain threshold measurement results showed that: 3 days after the operation,the thermal pain threshold was higher in the normal group and the sham group.The pain threshold was significantly lower in the model group and the massage group after CCI operation 3 days,and there was no significant difference between the model group and the massage group(p> 0.05);The pain threshold of the massage group was obviously increased compared with the model group after message invention at day 7(P <0.05).The pain threshold of the massage group was increasing improved at the 10 th and 14 th day after operation,which was significantly different from that of the model group(P <0.01).The threshold of thermal pain increased gradually in the sham group at day 3 after operation and there was no significant difference between the sham group and normal group after operation 14 days(p> 0.05).(2)The paw withdrawal threshold measurement results showed that: The paw withdrawal threshold in model group and CCI group decreased compared with the normal group after 3 days and 14 days of operation(p<0.05);After the intervention of the massage,the mechanical pain threshold in the massage group was significantly higher compared with the CCI group on the 14 th day after the operation(p<0.01).(3)Results of the inclined plane test: Compared with the normal group,the inclined plate angles of the CCI group and the massage group were reduced(p<0.05)at day 3 after the operation,and the difference between the CCI group and the massage group was no significantly(p> 0.05).Compared with the sham group,the inclined plate angles of the CCI group and the massage group decreased(p<0.05).At 14 days after surgery,compared with the normal group,the angles of the inclined plate test in the CCI group and the massage group were still lower(p<0.05);the difference between the massage group and the CCI model group was statistically significant(p<0.01).(4)The results of real-time fluorescence quantitative PCR showed that the mRNA expression of Raf-1,ERK,and CREB in CCI group was increased at days 3 compared with the normal group(P<0.05).Compared with sham group,the mRNA expression of Raf-1,ERK,and CREB in CCI group was also increased(P <0.05).On the 14 th day after operation,the mRNA expression of Raf-1,ERK,and CREB in the CCI group and the massage group increased compared with the normal group(P<0.05).Compared with the sham group,the CCI group and the massage group Raf-1,The mRNA expression of ERK and CREB was increased(P<0.05).Compared with CCI group,the mRNA expression of Raf-1,ERK and CREB in the massage group was decreased(P<0.05).Compared with normal group and sham operation group,the expression of IL-1? in spinal cord was increased in CCI model group(P < 0.01).After intervention by massage,the expression of IL-1? decreased in message group,and the difference was significant compared with CCI model group(P < 0.05).(5)Western blot results showed that the expression of protein Raf-1,p-ERK and p-CREB in the spinal cord of the CCI model group was higher than that of the normal group and the sham-operated group(P<0.05).Compared with the CCI model group,the expression of Raf-1,p-ERK and p-CREB protein in the spinal cord was significantly down-regulated by message in the message group on the 14 th day(P<0.05).Conclusions: 1.CCI model can cause neuropathic pain in rats with paw withdrawal latency,paw withdrawal threshold and inclined plane test reducing.Massage can improve the paw withdrawal latency,paw withdrawal threshold and inclined plane test in CCI model rats,and there were cumulative effects of time.2.ERK signaling pathway is involved in the process of chronic neuropathic pain.The mechanism of analgesia by massage may be related to its down-regulation of Raf-ERK-CREB gene and protein expression in the ERK signaling pathway,thereby inhibiting the expression of the downstream inflammatory cytokine IL-1?.