| With a deeper understanding of Type 2 diabetes mellitus(T2DM)pathogenesis,improving the toxicity of glycolipids and controlling the chronic inflammatory injury resulted from it have now become the main focus of T2DM treatment.The skeletal muscle AMPK?2 has,in recent years,become a popular target in the academia for its powerful regulation of glucose and lipid metabolism and inflammatory response.The activation of AMPK?2 not only inhibits chronic inflammation of the body mediated by NF-?B signaling pathway,its linkage with PPAR-y signals also further improves metabolism of glucose and lipids,alleviating the damage of glucolipotoxicity on the body.The "Yiqi Jiangzhuo-Tuina" method used in this study is based on a set of clinical treatment modalities proposed by Prof.Meng Feng-Xian on the core syndromes of Qi-Yin deficiency and turbidity in the pathogenesis of T2DM.Spleen and phlegm,down turbidity and stomach,the effect of cultivating vitality can improve the patient's glucose and lipid metabolism disorders and clinical symptoms,but the mechanism of the effects of the technique is not yet clear.In this study,under the support of National Natural Science Foundation of China(No.81574092)and young teachers project of Beijing University of Chinese Medicine(2016-JYB-JSPY-041,2017-JYB-JS-104),the effect mechanism of Yiqi Jiangzhuo massage therapy in T2DM treatment was explored.At the same time,through clinical studies,the clinical efficacy and advantages of phlegm-dropping and turbid massage are preliminarily clarified.1 Clinical researchPurposeTo observe the effects of Yiqi Jiangzhuo on blood glucose,blood lipids,TCM symptoms and quality of life in patients with sputum fever and turbid type T2DM,and investigate its efficacy and safety in clinical intervention of T2DM.MethodA prospective non-randomized controlled study was carried out at the Beijing University of Chinese Medicine Second Affliated Hospital Dong Fang Hospital from February 2017 to December 2017 to recruit sputum fever and turbid type T2DM patients.The patients were then chose to join either the control group or the treatment group.Patients in control group were treated with conventional hypoglycemic western medicine,while patients in the treatment group were treated with a combination of Yiqi Jiangzhuo and conventional hypoglycemic western medicine.The treatment duration lasted 12 weeks.Patient safety was observed during the treatment and treatment efficacy was evaluated after 12 weeks of treatment.(1)Main evaluating factor:HbAlc(2)1 Secondary evaluating factors:1FPG,2hPG,TC,TG,LDL-c,HDL-c;2 TCM symptoms survey and quality of life surveys(DMQLS).The safety indicators included adverse reactions during treatment,liver and kidney function,blood routine and electrocardiogram.Result54 of subjects screened,6 dropped out of the study while 48 subjects completed the study;18 of which belonged to the control group and 30 subjects in the treatment group.Baseline data that included gender,age,disease duration,BMI,HbAlc,FPG,and 2hPG were consistent and comparable in both groups(P>0.05).(1)Main evaluating factor:After treatment,FPG levels in subjects of control group and treatment group decreased significantly at 8th and 12th weeks(P<0.05,P<0.01).Comparing with subjects in control group,the decreasing trend of FPG levels on the 4th,8th,and 12th weeks was more obvious in subjects from treatment group.After treatment,2hPG levels in subjects of control group decreased significantly at 8th and 12th weeks(P<0.05)while 2hPG levels of subjects in treatment group decreased significantly at 4th,8th,and 12th weeks(P<0.01).Comparing with subjects in control group,subjects in treatment group had lower 2hPG values at the 4th and 12th weeks(P<0.05).After treatment,the HbAlc levels in subjects of both control group and treatment group decreased significantly(P<0.05,P<0.01).The decreasing trend of HbAlc in subjects of the treatment group was more significant than that in control group(P>0.05).The total effectiveness rate was 77.78%in the control group and 93.33%in the treatment group.The total effectiveness rate in the treatment group was significantly higher than that in the control group(P<0.05).(2)Secondary evaluating factors:1 After treatment,the TG levels subjects of treatment group were significantly lower than before treatment(P<0.05).There was no significant change in levels of TC,LDL-c,and HDL-c in each group.2 Symptoms and signs surveys:After treatment,the TCM symptom scores in subjects of control group decreased significantly at the 8th and 12th weeks(P<0.05)while the scores in that of treatment group decreased significantly at the 4th,8th,and 12th weeks(P<0.05,P).<0.01);TCM symptom scores were significantly lower in subjects from treatment group than in subjects from control group at week 12(P<0.05).3 DMQLS survey:After treatment,the DMQLS scores in subjects of control group decreased significantly at the 12th week(P<0.05)while DMQLS scores in subjects of treatment group were significantly lower at the 8th and 12th weeks(P<0.05,P<0.01).The scores of DMQLS at the 8th and 12th week were significantly lower in the treatment group(P<0.05,P<0.01).Subgroup analysis of DMQLS found that the scores of subjects in control group decreased significantly in two dimensions after treatment,namely disease and physiology(P<0.05,P<0.01).For subjects in treatment group,the scores decreased significantly in four dimensions,namely disease,psychology,physiology and satisfaction.(P<0.05,P<0.01).The degree satisfaction in subjects of treatment group was significantly higher than that of the control group(P<0.05).During the 12 weeks of treatment,there was no obvious abnormality in the safety indicators all subjects and no adverse reactions was reported.ConclusionThe complementation of Yiqi Jiangzhuo with conventional hypoglycemic drugs can further improve levels of HbAlc,FPG,and TG and clinical symptoms of TCM in patients with sputum fever and turbid type of T2DM,improving quality of life from a multi-dimensional approach.2 Experimental studyPurposeTo clarify whether Yiqi Jiangzhuo can inhibit NF-?B by activating AMPKa2 and PPAR-y to improve the regulation of glucose and lipid metabolism in obese T2DM mice,reducing the chronic inflammatory injury caused by glucolipotoxicity.This will be a preliminary study to reveal the effector mechanisms of tuina in T2DM intervention,providing experimental basis and research ideas for future studies to investigate the mechanisms of tuina.MethodFour-week-old healthy male SPF Wistar mice were treated with 35 mg·kg-1 intra-peritoneal injection of 1%concentration of STZ solution after 4 weeks of high-fat diet.After 72 hours,fasting plasma glucose(FPG)of ?11.1 mmol/L indicated a successful establishement of T2DM murine model.32 T2DM mice were then randomly divided into model group(MOD),western medicine group(MET)(Metformin 250 mg·kg-1·d-1),tuina group(TNA)and western medicine tuina group(MIX)with 8 normal mice in control group(CON).The mice were sacrificed after 8 weeks of intervention and their blood,rectus abdominis and abdominal adipose tissue specimens were collected.The levels of serum FPG,TC and TG were measured by oxidase method.The serum FFA levels were measured ACS-ACOD.The transcription levels of AMPK?2,GLUT4,NF-?B and abdominal adipose tissue PPAR-? and IL-6 were detected and quantified with real time-PCR.Immunohistochemical techniques were used to detect and quantify the protein expression levels of AMPK?2,GLUT4,NF-?B and abdominal adipose IL-6 in skeletal muscle,while protein expression levels of AMPK?2 and NF-?B in skeletal muscle were quantified with Western blot and GLUT4 in skeletal muscle,abdominal fat PPAR-y and IL-6 protein expression levels were quantified with ELISA.Result1.Serum biochemistry:Comparing with mice in MOD,FPG levels in mice of MET decreased significantly on the 2nd,4th,and 8th weeks(P<0.05,P<0.01)while the levels of FPG in mice from TNA decreased significantly on the 4th and 8th weeks(P<0.05)and FPG levels in mice of MET decreased significantly on 2nd,4th,and 8th weeks(P<0.05,P<0.01).Comparing with mice in MET,the mice in MIX significantly on 4th and 8th weeks(P<0.05).FPG levels in mice of MET decreased significantly on 2nd,4th,and 8th weeks(P<0.05,P<0.01).Comparing with mice in MET,the mice in MIX had more significant FPG regulating effect on the 4th week(P<0.05).Comparing with mice in TNA,the FPG regulating effect was more significant on 4th and 8th week in mice of MET(P<0.05).Comparing with all mice on 0 week,the FPG regulating effect significantly decreased in all mice on the 2nd,4thth and 8th weeks(P<0.05,P<0.01).Comparing with the mice in CON,the levels of TC,TG and FFA in mice of MOD increased significantly(P<0.01).Comparing with the mice in MOD,the levels of TG and FFA in mice of MET decreased significantly(P<0.05).Comparing with the mice in MOD,the mice in TNA showed significant decrease(P<0.05)in the levels of TG and FFA.Comparing with mice in MOD,the levels of TG and FFA in mice of MET decreased significantly(P<0.05,P<0.01).2.HE staining(skeletal muscles and adipose tissues):Comparing with mice in MOD,the amount of connective tissues between muscles was observed to have slightly increased in mice of MOD,also with presence of dilated and congested blood vessels and diffused infiltration of inflammatory cells(P<0.01).Comparing with mice in MOD,the degree of edema in every other intervention groups were lower at varying degrees,with less number of dilated and congested blood vessels.Among them,the distribution of muscle fibers in mice of MIX was more regular and compact than that of other groups,while having less atrophic muscle bundles,with presence of lamellar corpuscle,and least extent of edema around cells(P<0.05).The adipose tissues of the mice in MOD were diffused growth of white fat was observed with rare sightings of glandular lobular brown fat.A portion of these fat cells increased in cell volume with cell diameter about 3 to 4 times of normal cells.The amount of fat also increased compared with that of CON with capillary hyperplasia,dilation and congestion of blood vessels observed in the surrounding tissues together with a scattered infiltration of inflammatory cells around the peritubular(P<0.01).The number of inflammatory cells in all intervention groups were lower than that in the MOD,with less dilated and congested blood vessels and decrease in the number and volume of adipocytes(P<0.05).3.Real time-PCR(abdominal rectus AMPK?2,GLUT4,NF-?B,adipose PPAR-?,IL-6):Comparing with mice of CON,the transcription levels of AMPKa2,GLUT4 and PPAR-ygenes in mice of MOD decreased(P<0.05,P<0.01)while the levels of NF-?B and IL-6 genes transcription increased(P<0.01,P<0.05).Comparing with mice of MOD,the transcription levels of AMPK?2 and GLUT4 genes in mice of MET,TNA,and MIX all increased(P<0.05)while the transcription levels of NF-?B gene decreased(P<0.05),and a downward trend was shown in the levels of IL-6 gene transcription(P>0.05).4.Immunohistochemistry(AMPK?2,GLUT4,NF-?B,and fat IL-6 in rectus abdominis):Comparing with mice in CON,the expression of AMP?2 and GLUT4 protein in mice of MOD was significantly decreased(P<0.01)while the expression of NF-?B and IL-6 protein were significantly decreased.Comparing with mice in MOD,the protein expressions of AMPK?2 and GLUT4 with mice in MET,TNA and MIX increased(P<0.05,P<0.01)while their NF-?B and IL-6 protein expressions decreased significantly(P<0.01).Comparing with mice in TNA,GLUT4 protein expression was significantly increased in mice of MET(P<0.05),and IL-6 protein expression was significantly decreased in mice of MET(P<0.05).5.Western blot(AMPK?2 and NF-?B in the rectus abdominis muscle with with,with mice in CON,the expression of AMPK?2 protein in mice of MOD decreased significantly(P<0.05)and expression of NF-?B protein increased significantly(P<0.05),while the expression of AMPK?2 protein in mice of MET,TNA and MIX increased(P<0.05).The expression of NF-?B protein in mice of TNA and MIX decreased(P<0.05).6.ELISA(rectal GLUT4,adipose PPAR-?,IL-6):GLUT4 protein expression in mice of MOD decreased compared with mice of CON(P<0.05).Comparing with mice in MOD,mice in MET,TNA and MIX showed an increase in GLUT4 protein expression(P<0.05).Comparing with mice in CON,the expression of PPAR-y protein in mice of MOD decreased(P<0.01)while the expression of IL-6 protein increased(P<0.05).Comparing with mice in MOD,the PPAR-y protein expression in mice of MET and MIX showed an increasing trend(P>0.05).Comparing with mice in MOD,the expression of IL-6 protein in mice of MET,TNA and MIX significantly decreased(P<0.01).ConclusionYiqi Jiangzhuo tuina method can restore the glucose and lipid metabolism of obese T2DM rats by activating the skeletal muscle AMPKa2/GluT4 signal chain.At the same time,by reducing the expression of NF-?B,a key factor of inflammatory pathway,it can reduce the downstream IL-6 and other proinflammatory factors.The accumulation of factors eventually exerts a positive effect of improvement of metabolic inflammatory injury. |
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