The Clinical Effect Of Acupuncture On CFS Immunological Abnormality And The Mechanism Of MiR-184/NFAT2/NKCC Signaling Pathway

Purpose:1.The NKCC and NFAT2 protein activation levels and serum IFN-γexpression levels of CFS patients before and after acupuncture treatment were analyzed,and the clinical effects of acupuncture on the immune function of CFS patients were preliminarily discussed.2.To explore whether acupuncture can change NKCC through miR-184 / NFAT2/ NKCC signaling pathway to exert its immunoregulatory effect.Methods:1.Clinical trial: The clinical effect of acupuncture on the immune function in patients with CFS.A total of 30 healthy subjects and CFS patients were recruited through strict inclusion criteria.(1)With healthy subjects as controls and CFS patients as subjects,the activity of peripheral blood NK cells was detected by LDH method,the expression of NFAT2 and p-NFAT2 protein in NK cells was detected by WB method,and the serum cytokine IFN-γ was detected by ELISA method,and analyze its characteristics.(2)Taking acupuncture as an intervention method,CFS patients were randomly divided into true acupuncture group(group A,n=14)and placebo acupuncture group(group B,n=16)for 6 weeks.The NK cell activity,NFAT2,pNFAT2 and IFN-γ level was analyzed by comparing the difference between A and B groups,and before and after comparison.2.Animal experiment: The mechanism of acupuncture regulating miR-184/NFAT2/NKCC signal pathway in CFS model rats.Forty SD male rats were randomly divided into group A(blank group),group B(model group),group C(model+acupuncture group),and group D(model+acupuncture+miR184 antagomirgroup),with 10 rats in each group.The three groups B,C and D adopted the compound stress method of "chronic confinement + exhaustion swimming" to build the model,and judged whether the model was successful through exhaustion swimming,tail suspension experiment and open field experiment.During the treatment period,the two groups A and B were fixed for 15 minutes a day without acupuncture intervention;groups C and D were treated with acupuncture,and the group D was injected with miR184 antagomir on the first day before weekly treatment.After 3 weeks,RT-q PCR was used to detect the expression of miR-184 in NK cells in each group;the detection methods of NK cell activity,NFAT2 protein and serum IFN-γ expression in each group were the same as in clinical trials.Since there is no rat p-NFAT2 antibody,the degree of dephosphorylation and activation of NFAT2 can only be reflected by the expression level of NFAT2 protein in the cytoplasm and nucleus of NK cells.Outcome:1.Clinical trial(1)Compared with healthy subjects,the expression of NKCC and serum IFN-γin CFS patients were significantly reduced(P=0.013,P<0.01),and the expression of cytoplasmic NFAT2 and p-NFAT2 protein were significantly reduced(P<0.01),the expression of NFAT2 protein in the nucleus was significantly increased(P<0.01).(2)Compared with before treatment,NKCC in group A was significantly increased(P<0.05),serum IFN-γ was not significantly different(P>0.05),and cytoplasmic NFAT2 and p-NFAT2 protein expression were significantly increased(P<0.05,P<0.01),nuclear NFAT2 protein expression was significantly reduced after treatment(P<0.01);group B NKCC and serum IFN-γ expression were notsignificantly different(both P>0.05),cytoplasmic NFAT2 and p-NFAT2 protein expression were both Significantly increased(both P<0.01),and nuclear NFAT2 protein expression was significantly reduced after treatment(P<0.01).(3)Compared with group B,NKCC in group A was significantly improved(P<0.01),and there was no significant difference in serum IFN-γ,nuclear NFAT2,cytoplasmic NFAT2 and p-NFAT2 after treatment(all P> 0.05).2.Animal experiment(1)miR-184 expression: after acupuncture treatment,Compared with group A,the expression of miR-184 in group B rats was significantly decreased(P<0.01).Compared with group B,the expression of miR-184 in group C rats was increased(P<0.01).Compared with group C,the expression of miR-184 in group D decreased(P<0.01).NFAT2 protein expression: Compared with group A,the expression of NFAT2 in the nucleus of rats in group B was significantly increased,and the expression of NFAT2 in the cytoplasm was significantly decreased,the differences were statistically significant(P<0.01);compared with group B,group C the expression of NFAT2 in the nucleus of rats was significantly decreased,and the expression of NFAT2 in the cytoplasm was significantly increased,and the differences were statistically significant(P<0.01);compared with group C,group D the expression of NFAT2 in the nucleus was significantly increased,and the expression of NFAT2 in the cytoplasm was significant The difference was statistically significant(P<0.05,P<0.01).(3)NKCC: After acupuncture treatment,Compared with group A,NKCC of group B rats decreased significantly(P<0.01).Compared with group B,NKCC ofgroup C rats was significantly increased(P<0.01).Compared with group C,NKCC in group D decreased significantly(P<0.01).(4)Serum IFN-γ expression: after acupuncture treatment,compared with group A,the serum IFN-γ expression of group B rats decreased(P<0.01).Compared with group B,serum IFN-γ expression was increased in group C(P<0.01).Compared with group C,serum IFN-γ expression decreased in group D(P<0.01).Conclusion:(1)Compared with healthy subjects,CFS patients have reduced NKCC and decreased serum IFN-γ expression,and NK cell NFAT2 activation.(2)Compared with placebo acupuncture,acupuncture can effectively improve NKCC and serum IFN-γ in CFS patients,,and its clinical effect may be related to the induction of miR-184 expression,inhibition of intracellular NFAT2 activation,and thus play a role in regulating downstream pathways.(3)The miR-184/NFAT2/NKCC signaling pathway may be one of the possible mechanisms for regulating CFS immune dysfunction,and whether NFAT2 activation is specific remains to be further studied.