| Objective: This study is based on the previous proof that the "Yunfu Tongjing" massage method is clinically effective in the treatment of obesity insulin resistance.The effect of reducing fat and glucose is not as significant as the positive drug metformin hydrochloride,but the TCMSS is significantly higher than that of the drug group,especially when combined with drug treatment,the incidence of adverse events is significantly lower than that of the drug group.Therefore,this study takes OHIR as the disease carrier,and a prospective RCT will be used to evaluate the whole process of intervention in all aspects and dimensions according to the whole body and mind,and analyze the synergy and alleviating drug side effects of "Yunfu Tongjing" massage in the adjuvant treatment of OHIR,Improve the quality of life and other auxiliary clinical effects.At the same time,Seq RNA technology is used to analyze the differentially expressed m RNA,smallrna,noncoding RNA and other genes of "Yunfu Tongjing" massage in the adjuvant treatment of OHIR.It is planned to explore the distribution signal pathway of differentially expressed genes,explore the network biological regulation of "Yunfu Tongjing" massage affecting multiple genes,multiple targets and multiple pathways,and realize the auxiliary effect,To provide ideas and methods for further clarifying the clinical auxiliary effect and mechanism of "Yunfu Tongjing" massage in the treatment of OHIR,and to provide reference for reducing drug use in clinic and evaluating the whole process of patients’ body and mind.Methods:1.Clinical researchBased on the premise efficacy study,prospective RCT assistance is carried out.TCM SS of diabetes was used as the main outcome index.The SF-36,MSAS,HAMA and the incidence of adverse reactions as secondary outcome indicators,through 122 items and 22 dimensions,the whole process of "Yunfu Tongjing" massage combined drugs and drug group(non acupoint pressing and non combined metformin hydrochloride tablets)was evaluated from the whole body and mind of patients.2.Analysis of differentially expressed genes and exploration of related mechanismsAfter 6 weeks of treatment,the hepatic tissues of rats in each group were collected,and the total RNA of hepatic tissues of rats in each group was extracted.After passing the quality inspection and completing the library construction,the samples were sequenced with Illumina Hiseq Sequencing platform for High-Throughput Sequencing,and then the significantly differentially expressed m RNA,small RNA,noncoding RNA and other genes were clustered and enriched(Gene Ontology,GO)functional analysis,KEGG pathway enrichment analysis,differential m RNA,smallrna,noncoding RNA and other genes and target gene interaction analysis,and finally selected with "Yunfu Tongjing" The differential genes that may be related to the clinical effect of massage were verified by quantitative RT-PCR.At the same time,the distribution of key genes and main signal channels were analyzed.Combined with the enrichment analysis results of KEGG pathway,the possible signal pathways related to the auxiliary clinical effect of "Yunfu Tongjing" massage were explored,and the key proteins of the speculated signal pathway were detected by Western Blot and the genes were detected by RT-PCR.Results:1.Clinical researchFrom the beginning of enrollment to the end of the 10 th week of follow-up,TCMSSSevaluation:Massage has a certain dose effect accumulation,and the adverse reactions in the first two weeks are greater than the clinical effects;The TCMSSS score of massage combined drug group decreased more than that of drug group,and there was significant difference between the two groups(P<0.001).SF-36 evaluation: The massage combined drug group and the drug group improved the quality of life in nine aspects of SF-36 PF,RP,BP,GH,VT,SF,RE,MH and HT,and the score of SF-36 in the massage combined drug group decreased more,and the difference between the two groups was statistically significant(P<0.05),Especially in the two dimensions of body pain(BP)and energy(VT),there were significant differences between the two groups(P<0.001).MSAS evaluation: Massage had a certain dose effect accumulation,and the MSAS score decreased more,and the difference between the two groups was statistically significant(P<0.05).HAMA evaluation: Compared with the baseline,the difference increased gradually and showed waveform changes respectively.The massage combined with drugs group decreased gradually during the treatment,and the decline rate was faster after two weeks of treatment.It rebounded slightly during the follow-up period,and increased first and then decreased in the drug group.Comparison between groups: compared with massage combined drug group and drug group,the HAMA score decreased more,and the difference between the two groups was statistically significant(P<0.05).2.Experimental researchEffects of obesity status:Weight and Lee’s index,After 6 weeks of intervention,compared with Model group,Massage group,Drug group and Mas+Drug group all had weight loss effect on OHIR rats and improved obesity(*P<0.05),and the weight loss effect of Mas+Drug group was better than that of Drug group(#p<0.05);Compared with Blank group,the average body weight and Lee’s index of Blank+Mas Group were lower than those of Blank group(&P<0.05).Effects of hepatic enzymes:Compared with Model group,Massage group,Drug group and Mas+Drug group all had a regulatory effect on the expression of hepatic enzymes ALT and AST in OHIE rats(* P<0.05),and Mas+Drug group had a better regulatory effect on the expression of hepatic enzymes ALT and AST in OHIR rats than Drug group(#p<0.05);Compared with Blank group,the average expression of ALT and AST in Blank+Mas Group was lower than that in Blank group(&P<0.05).Effects of Blood Glucose and Blood Lipid: Compared with the Model group,Massage group,Drug group and Mas+Drug group all had a down-regulation effect on the levels of blood lipid TG and TC in OHIR rats(*P<0.05),and the down-regulation effect of Mas+Drug group on the levels of blood lipid TG and TC in OHIR rats was better than that in Drug group(#p<0.05);Compared with Blank group,the average levels of TG and TC in Blank + Mas Group were lower than those in Blank group(&P<0.05).Effects of insulin sensitivity: Compared with the Model group,the Massage group,the Drug group and the Mas+Drug group all had a down-regulation effect on the contents of FBG,FINSand C-peptide in the blood of OHIR rats.At the same time,they could improve insulin sensitivity and improve the state of obese insulin resistance(*P<0.05),and the Mas+Drug group had a better down-regulation effect on the contents of FBG,FINS and C-peptide in the blood of OHIR rats,the increase of insulin sensitivity and the improvement of insulin resistance than the Drug group(#p<0.05);Compared with Blank group,the average contents of FBG,FINS and C-peptide in blood of Blank+Mas Group were lower than those of Blank group(&P<0.05).Impact of OGTT: Under fasting condition,the blood glucose of Model group was significantly higher than that of Blank group.Compared with Model group,the area under the blood glucose curve from 0 to 120 min after the intervention of Massage,Drug and Mas+Drug decreased significantly(*P<0.01),and Mas+Drug was better than that of Drug group(#p<0.05);Compared with Blank group,the area under the blood glucose curve in Blank+Mas Group was lower than that in Blank group(&P<0.05).Influence of hepatic histomorphology: After 6 weeks of intervention,the hepatic tissue structure of Blank+Mas Group and Blank group was observed under light microscope and transmission electron microscope;There were obvious lipid deposition,vacuolar degeneration and watery degeneration of hepatocytes,inflammatory cell infiltration and a small amount of punctate necrosis of hepatocytes in the Model group.The vacuolar degeneration of hepatic in Massage group,Drug group and Mas+Drug group decreased significantly(*P<0.01),and the Mas+Drug group was better than Massage group,and the Massage group was better than Drug group(#p<0.05).3.Analysis of differentially expressed genes(1)Comparison of key differentially expressed genesThe sequencing results showed that the number of up-regulated and down-regulated differential genes of Drug vs Mas+Drug was 264,of which the most obvious up regulation was gene Nim1 k and the most obvious down regulation was gene Lcn12;Massage vs Mas+Drug up-regulated 284 differential genes and down-regulated 301 differential genes,of which the most obvious up-regulated gene is Gstt3 and the most obvious down-regulated gene is Sds;The number of differential genes upregulated by Massage vs Drug was 109,and the number of differential genes downregulated was 105,with the most upregulated being the gene Plaat1 and the most downregulated being the gene Nudt10;Model vs Mas+Drug the number of differential genes up-regulated was 320 and the number of differential genes down-regulated was 216,of which the most significantly up-regulated was gene Gck and the most significantly down-regulated was gene Cfap126;The number of Model vs Massage up-regulated differential genes was 196,and the number of down-regulated differential genes was 194,of which the most up-regulated was gene Slc25a30 and the most downregulated was gene Slc35c2;Model vs Blank up-regulated 112 differential genes and down-regulated 78 differential genes,of which the most obvious up-regulated gene is RT1-CE10 and the most obvious down-regulated gene is LOC100909524.(2)Go analysisAfter 6 weeks of treatment,the Mas+Drug group was compared with the Drug group,and 585 significantly different genes were obtained.Through the GO enrichment analysis of top GO,7031 differential GO terms were obtained,including 5521 BP,471 CC and 939 MF.In the first 20 articles,16 Biological Processes were involved in the positive regulation of Biological Process,the regulation of development process,the positive regulation of Cellular Process,the regulation of cell differentiation,oxygen transport and the positive regulation of development process,and 3 cell components such as extracellular matrix,collagen containing extracellular matrix and basement membrane,1 molecular function such as oxygen carrier activity.(3)KEGG pathway analysisAfter 6 weeks of treatment,KEGG pathway analysis showed that after the adjuvant treatment of "Yunfu Tongjing" massage,the expression of COL1 was up-regulated and IL1 was down regulated in the AGE-RAGE signal pathway related to insulin resistance in obese hepatic;P21 was up-regulated in PI3 K Akt signaling pathway;P21 was up-regulated and AOX and SOCS were down regulated in JAK STAT signaling pathway.RT-PCR was used to verify P21,COL1,IL1 A,AOX1 and SOCS1.The results showed that there were significant differences in the expression of P21,COL1,IL1 A,AOX1 and SOCS1 between the two groups before and after treatment(P<0.05).4.Real time PCR verification of differentially expressed genes and verification of network biological effectsAfter 6 weeks of intervention,real time PCR was used to detect that compared with the Model group,the expression levels of genes P21,COL1 and SOCS1 in the Mas+drug treatment group were significantly increased(*P<0.01),and the expression levels of genes IL1 A and AOX1 were lower(*P<0.05);Compared with the Drug group,the expression levels of P21,COL1 and SOCS1 in the Mas+Drug treatment group were higher(#p<0.05),and the expression level of IL1 A was lower(#p<0.05);Compared with the Drug group,the expression levels of P21,COL1 and SOCS1 in the Massage treatment group were the same(§P>0.05),and the expression level of IL1 A decreased significantly(§P<0.01).Western blot showed that compared with the Model group,the protein expression levels of PI3K、AKt、p-AKt、mTOR、p-mTOR,AEGs、RAGE、NF-κB、p-NF-κB in the Mas+Drug group have significantly decreased,and that of JAK2,STAT3,and p-STAT3 significantly increased(*P<0.01);Compared with the Blank group,the protein expression of the Mas+Drug was comparable(**P>0.01),and the protein expression of the Blank+Mas group was lower(**P<0.05);Compared with the Drug group,the protein expressions in the Mas+Drug were lower(##P<0.05),and the protein expressions in the Mas+Drug were lower(##P<0.05).Conclusion:1."Yunfu Tongjing" massage can prevent the occurrence of OIR.It can reduce weight and fat and improve insulin sensitivity in OHIR patients.The effect is not as good as metformin hydrochloride.However,combined with drugs,it can play a synergistic role and reduce the occurrence of adverse reactions.At the same time,it can protect or repair liver injury,effectively improve quality of life,regulate physical and mental state,alleviate obesity anxiety and other auxiliary clinical effects.2.Seq RNA sequencing was used to reveal the presence of aberrantly expressed RNAs in the hepatic of OHIR rats,And clinical "Yunfu Tongjing" massage might regulate the expression levels of m RNA,small RNA,noncoding RNA genes in OHIR patients and thus produce auxiliary clinical effects.3.The treatment of OHIR by clinical "Yunfu Tongjing" massage may be through regulating COL1,IL1 in AGEs-RAGE signaling pathway and P21 in PI3K-Akt signaling pathway;Expression of P21,AOX,SOCS related genes in Jak2-STAT3 signaling,In turn,the AGEs-RAGE / PI3K-Akt / Jak2-STAT3 network pathway is regulated,thus serving as an adjuvant therapeutic effect.However,further research on other relevant organizations is still needed to verify. |
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