| Objective(1)To investigate the effect of Gua Sha on the inflammatory cytokines in the rat model of lumbar disk herniation(LDH)by enzyme linked immunosorbent assay(ELISA)method.(2)To examine the potential effect of Gua Sha on the serum proteome in the rat model of LDH.Methods(1)72 male SD rats were randomly divided into model,sham and Gua Sha groups(n =24/group).A rat model of noncompressive LDH induced by autologous NP was established in model and Gua Sha groups,the sham surgery was performed in the sham operation group.Gua Sha was performed on the 5th days after the surgery,once every other day,3 times for a period of treatment,and totally 3 periods.On the day before Gua Sha,the end of the first,second and third treatment circle,6 rats in each group were picked randomly and their Paw Withdrawal Thermal Latency(PWTL)was observed.Blood samples of rats were drawn to assess the expression of IL-1?,IL-4,IL-6,IFN-? and TNF-? by ELISA method.(1)Parts of the serum sample of rats in the model and Gua Sha groups were collected.They were marked as M1(model group,1 period of treatment),M2(model group,2 periods of treatment),M3(model group,3 periods of treatment),Gl(Gua Sha group,1 period of treatment),G2(Gua Sha group,2 periods of treatment)and G3(Gua Sha group,3 periods of treatment),with 6 samples in each group,totally 6 groups.Isobaric tags for relative and absolute quantitation(iTRAQ)was used to discover different protein expression profiles in different groups at 3 different periods.The molecule functions,cellular component,and biological process were analyzed via Gene Ontology(GO)analysis.Ingenuity(?)Pathway Analysis(IPA)database was used to discover the canonical pathways and networks involving these proteins.Results(1)Compared to rats in the model group,the therapy of Gua Sha could attenuate thermal hyperalgesia potently,and inhibit the expression of IL-1?,IL-6,IFN-y and TNF-a(P<0.05).There was no statistical difference for the expression of IL-4 between rats in the model and Gua Sha group(P>0.05).IFN-?/IL-4 ratio was negatively correlative with PWTL and Gua Sha could decrease the ratio.(2)? G1 vs M1:A total of 198 differential proteins were identified.Via GO analysis,A total of 10 molecule functions,13 cellular components and 19 biological processes were identified.In molecule functions,ranked the top three,respectively was binding(48%),catalytic activity(22%)and molecular function regulator(5%).In cellular components,ranked the top three were cell part(28%)and organella(18%)and organella part(14%).In biological processes,ranked the top three were cellular process(19%),biological regulation(15%)and single-organism process(15%).IPA database discovered 306 canonical pathways.According to their-Log(P-value),the top ten were FAK Signaling,Regulation of eIF4 and p70S6K Signaling,Actin Cytoskeleton Signaling,PI3K/AKT Signaling,Erythropoietin Signaling,IL-3 Signaling,LPS-stimulated MAPK Signaling,ERK/MAPK Signaling,Cancer Drug Resistance By Drug Efflux and Semaphorin Signaling in Neurons.A total of 18 networks were found to be related to these different expressing proteins.? G2 vs M2:A total of 182 differential proteins were identified.Via GO analysis,A total of 11 molecule functions,13 cellular components and 20 biological processes were identified.In molecule functions,ranked the top three,respectively was binding(44%),catalytic activity(21%)and molecular function regulator(9%).In cellular components,ranked the top three were cell part(27%)and organella(18%)and organella part(13%).In biological processes,ranked the top three were cellular process(16%),biological regulation(15%)and single-organism process(14%).IPA database discovered 306 canonical pathways.According to their-Log(P-value),the top ten were Acute Phase Response Signaling,LXR/RXR Activation,Intrinsic Prothrombin Activation Pathway,Actin Cytoskeleton Signaling,FXR/RXR Activation,Lanosterol Biosynthesis,Ubiquinol-10 Biosynthesis(Eukaryotic),Rac Signaling,Regulation of the Epithelial-Mesenchymal Transition Pathway and Clathrin-mediated Endocytosis Signaling.A total of 13 networks were found to be related to these different expressing proteins.? G3 vs M3:A total of 170 differential proteins were identified.Via GO analysis,A total of 10 molecule functions,15 cellular components and 18 biological processes were identified.In molecule functions,ranked the top three,respectively was binding(45%),catalytic activity(25%)and molecular function regulator(6%).In cellular components,ranked the top three were cell part(27%)and organella(18%)and organella part(13%).In biological processes,ranked the top three were cellular process(20%),biological regulation(16%)and single-organism process(15%).IPA database discovered 245 canonical pathways.According to their-Log(P-value),the top ten were LXR/RXR Activation,FXR/RXR Activation,5-aminoimidazole Ribonucleotide Biosynthesis I,Inhibition of Angiogenesis by TSP1,Melatonin Degradation II,tRNA Charging,Tetrahydrofolate Salvage from 5,10-methenyltetrahydrofolate,Tyrosine Degradation I,Serotonin Receptor Signaling and Ceramide Biosynthesis.A total of 13 networks were found to be related to these different expressing proteins.Conclusions(1)Gua Sha might alleviate neuropathic pain thermal hyperalgesia of LDH rats via inhibiting the expression of proinflammatory cytokines.Moreover,Gua Sha could inhibit the Thl type immunization of LDH rats,thus promotes the Thl/Th2 balance.(2)The expression of a great number of proteins in serum of LDH rats were changed.The variety of functions these proteins involved indicated that the body may undergo an extensive and complex change.Gua Sha may not only regulate the pathological change of LDH,but also treat it by stimulating the physiological responses.Different canonical pathways were activated according to priority,among which FAK Signaling,Acute Phase Response Signaling and LXR/RXR Activation were the leading pathways.Moreover,via regulating ERK1/2 and NF?B pathways,Gua Sha participated in networks involving cell morphology,organismal injury and abnormalities,nervous system development and function.The result of signal pathways and networks indicated that differentially expressed proteins mainly involved in the process of inflammation and immunity,neuroprotection,and maintaining cytoskeleton.During these processes,proteins like Racl,Orml,Hpx,Gapdh,Hsp70 and Htt played important roles and may be potential targets. |
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