Effect Of Heat-reinforcing Needling On Serum Metabolite Profiles In Rheumatoid Arthritis Rabbits Cold Syndrome

Objective: To explore the metabolite profiling changes in serum of rheumatoid arthritis rabbits with heat-reinforcing needling based on liquid chromatography quadrupole time-of-flight mass spectrometry(LC-Q/TOF-MS)technology,and to investigate the specificity mechanisms of heat-reinforcing needling for RA.Method: A total of 40 healthy purple blue rabbits were randomly allocated to normal control(NC),model control(MC),reinforcing-reducing needling group(RRN),twirling-reinforcing needling group(TRN),and heat-reinforcing needling group(HRN)(n=8/group).Arthritis rabbits cold model was made with ovalbumin and freezing.Except NC and MC,RRN was given acupuncture of reinforcing-reducing needling at Zusanli(ST36),TRN was given acupuncture of twirling-reinforcing needling at Zusanli(ST36),and HRN was administrated acupuncture of heat-reinforcing needling at Zusanli(ST36),once a day and retaining 30 min,a total of seven days.After the intervention,the general condition and synovial pathology of each group were observed,the pathological score were counted,and pain threshold,joint swelling of each group were observed.We collected fresh serum after the intervention and derivatized sample.Serum metabonomic profiles were detected using liquid chromatography quadrupole time-of-flight mass spectrometry(LC-Q/TOF-MS)technology after the intervention.All the datas were analyzed by principal component analysis(PCA)and partial least squares discriminant analysis(PLS-DA)by using the SIMCA-P(13.0)software for windows,and to explore its possible pathomenesis.Several metabolites based on the variable impoetance in the projection values(VIP>1),P value(P<0.05)and screening in databases were identified.Result:1 The changes in the general condition of rabbit in each groupThe diet,drinking water,activities,etc.were not any abnormal changes in NC,The rabbits in MC were curled up,the mental state were poor,the body was dirty,the dullness,the activity decreased,the diet and the drinking water decreased,the knee swells,touched the avoidance behavior,the local skin was cold.Compared with MC,The levels of mental state,activity,joint swelling and local skin temperature were significantly improved in theacupuncture groups.2 The changes of rabbit pain threshold in each groupThe pain threshold in MC was lower than the NC(P<0.05),the pain threshold in the acupuncture groups were higher than that in MC after intervention(all P<0.05),while which in HRN were obviously higher to that of RRN and TRN(all P<0.05).3 The changes of rabbit knee joint swelling in each groupThe knee joint swelling in MC was significantly higher than the NC(P<0.05),the knee joint swelling in the acupuncture groups were lower than that in MC after intervention(all P<0.05).4 The changes of rabbit synovial pathology and pathological score in each groupThe synovial linin cell layer in NC arranged in the rules,no hyperplasia and enlargement,no inflammatory cell infiltration,no activation of synovial stroma;the synovial linin cell layer in MC were strong enlargement(more than six cell layers),giant cells were frequent,strong inflammatory cell infiltration,severe synovial stroma activation;the synovial linin cell layer in RRN and TRN were moderate enlargement(four to five cell layers),some giant cells or lymphocytes,moderate inflammatory infiltration,moderate synovial stroma activation;the synovial linin cell layer in HRN were slight enlargemnet(two to three cell layers),slight inflammatory infiltration,slight synovial stroma activation.The pathological score in MC was significantly higher than the NC(P<0.05),the pathological score in the acupuncture groups were lower than that in MC after intervention(all P<0.05),while which in HRN were obviously superior to that of RRN and TRN(all P<0.05).5 The principal component analysis(PCA)resultsFrom the PCA score,it could be seen that the clustering of each group was obvious,indicating that there were significant differences in the five groups of samples,the acupuncture group along the t[1] axis return to the normal group trend,and in HRN more obvious.6 The partial least squares-discriminant analysis(PLS-DA)resultsThe serum metabolites of carnitine,LysoPC(14:0),LysoPC(18:3),LysoPE(0:0/20:5),LysoPE(0:0/22:1),decylic acid,stearic acid and lactic acid in MC mainly were increased(P<0.05),but the metabolites of Leucine,valine,glutamic acid,focal glutamine,α-ketoglutaric acid,succinic acid,fumaric acid,malic acid,galactose,mannose and glucuronic acid mainly were decreased compared with NC,these metabolites are correlated with metabolic pathway fatty acid,amino acid,the Krebs cycle,and glucose metabolism.The metabolites that various kinds above-mentioned were regulated compared with MC in all intervention of acupuncture group(P<0.05),but the metabolites concerning the Krebs cycle,and glucose metabolism in HRN were obviously superior to that of RRN and TRN(P<0.05).Conclusion:(1)The treatment of RA with heat-reinforcing needling is relatively specific;(2)Heat-reinforcing needling can significantly improve the pathological changes of RA,reduce synovial tissue pathology score relative specificity;(3)Heat-reinforcing needling can regulate the Krebs cycle and glucose metabolism of RA relative specificity;(4)Regulation of energy metabolism is one of the relative specific mechanism of heat-reinforcing needling in the treatment of RA.