| 1 ObjectiveWe compared the effects of moxibustion on cardiac function and structure in CHF rats,observed the effects of moxibustion on the expression of inflammatory related factors and autophagy related molecules in myocardial cells of CHF rats,and further explored the mechanism of moxibustion in intervening with CHF through mTOR signaling pathway.2 MethodExperiment 1 10 of the 30 SD rats were randomly divided into the normal group and 20 into the model group.The model was established by intraperitoneal injection of2 mg/kg ADR twice a week for four consecutive weeks.After echocardiography evaluation,they were randomly and equally divided into normal group and model group without intervention,and moxibustion group with mild moxibustion at bilateral Xinshu and Feishu once a day,20 min each time,and continuous intervention for three weeks.After the intervention,the physical signs of the rats were recorded.The hearts of the rats(LA,LV,LVPW,IVS,EF and FS)were evaluated by echocardiography.The serum contents of BNP,ANP and c Tn I were detected by ELISA,and the myocardial histopathological changes were observed by HE and MASSON staining.Experiment 2 Rats were modeled in the same way.After model evaluation,sixty male SD rats were randomly divided into the normal group,model group,moxibustion group,moxibustion plus RAPA group and RAPA group(12 rats in each group).The former two groups were not intervened.In the moxibustion group,mild moxibustion was applied to bilateral Xinshu and Feishu acupoints once a day,for 20 min,for three weeks.In the moxibustion pius RAPA group,the RAPA at the dose of 1 mg/kg was injected intraperitoneally once after moxibustion for three weeks.The rats in the RAPA group were intraperitoneally injected with 1 mg/kg RAPA only once a day for three weeks.On the first day of dry expectation,three rats randomly selected from the five groups were injected with 250?L AAV2/9 through caudal vein,labeled,and then put back to continue the intervention with other rats.At the end of the intervention,the expressions of inflammatory related factors ICAM-1,VCAM-1,MMP-2 and MMP-9 in unlabeled rats were detected by immunohistochemistry,the myocardial autophagy was detected by tandem adeno-associated virus LC3 double-labeling fluorescence assay,and the expression of LC3 protein in the myocardium of unlabeled rats was detected by WB.The 13 mRNA expression levels of autophagy-related genes Vps34,ATG3,5,7,12 and13 in the unlabeled rat myocardium were detected by RT-PCR,and the correlation between inflammatory factors and autophagy-related genes was analyzed by Person correlation.Experiment 3 By the same modeling method,after model evaluation,45 male SD rats were randomly divided into the normal group,model group,moxibustion group,moxibustion plus RAPA group and RAPA group(9 rats in each group).The former two groups were intervened.In the moxibustion group,mild moxibustion was applied to bilateral Xinshu and Feishu acupoints once a day,for 20 min,for three weeks.In the moxibustion plus RAPA group,the RAPA at the dose of 1 mg/kg was injected intraperitoneally once after moxibustion for three weeks.The rats in the RAPA group were intraperitoneally injected with 1 mg/kg RAPA only once a day for three weeks.At the end of the intervention,WB detected the protein expression of PI3 K,p-PI3 K,AKT,p-AKT,mTOR,p-mTOR,P70S6 K,p-P70S6 K,ULK1,p-ULK1 in rat myocardium,and mRNA expression of PI3 K,AKT,mTOR,P70S6 K,and ULK1 in rat myocardium by RT-PCR.3 Results Experiment 11.Changes of cardiac function in each group: Compared with the normal group,rats in the model group had been thickened LV and LA(P < 0.01),weakened LVPW,IVS,EF and FS(P < 0.01),and increased serum BNP,ANP and c Tn I contents(P <0.01).Compared with the model group,LV and LA contents in the moxibustion group were decreased(P < 0.05),while LVPW and IVS were increased(P < 0.05),EF and FS were significantly increased(P < 0.01).Serum BNP,ANP and c Tn I contents were significantly decreased(P < 0.01).2.Effects of different groups on cardiac structure: Compared with the normal group,the left ventricular myocytes in the model group were loosely organized,with the appearance of vacuoles in the cytoplasm and significantly increased collagen fiber area(P < 0.01).Compared with the model group,the left ventricular myocytes in the moxibustion group were less damaged,and the collagen fiber area was significantly reduced(P < 0.01).Experiment 21.Influence of immunohistochemistry on the levels of myocarditis-related factors in unlabeled rats: Compared with the normal group,the expression level of myocarditis-related factors in the model group was increased(P < 0.01);Compared with the model group,the expression levels of ICAM-1,VCAM-1 and MMP-9 in the moxibustion +RAPA group were significantly decreased(P < 0.01),while the expression of MMP-2 was decreased(P < 0.05).Compared with the moxibustion group,the expressions of ICAM-1 and VCAM-1 in the moxibustion plus RAPA group were increased(P < 0.01),and the expressions of MMP-2 and MMP-9 were increased(P <0.05).Compared with the moxibustion plus RAPA group,the expression levels of inflammatory related factors in the RAPA group were significantly increased(P < 0.01).2.LC3 fluorescence detection and labeling of rat myocardial autophagic flow: The LC3 fluorescence relative intensity analysis of rat cardiomyocytes in model group and RAPA group revealed stronger autophagic flow,while the LC3 fluorescence relative intensity analysis of rat cardiomyocytes in moxibustion group and moxibustion plus RAPA group revealed weaker autophagic flow.3.LC3 protein expression in unlabeled rat myocardial tissue detected by WB:Compared with the normal group,the model histones LC3 I and II were highly expressed(P < 0.01).Compared with the model group,the expression levels of histone LC3 I and II induced by moxibustion were lower(P < 0.01).The expression levels of LC3 I and II in the moxibustion +RAPA group were lower(P < 0.05).Compared with the moxibustion group,the expressions of LC3?and II in the moxibustion plus RAPA group were significantly increased(P < 0.01).Compared with the moxibustion plus RAPA group,the expressions of LC3?and II in the RAPA group were significantly increased(P < 0.01).4.Detection of ATG expression in myocardial tissue by RT-PCR: Compared with the normal group,ATG expressions of autophagy-related genes in the model group were all higher(P < 0.01).Compared with the model group,ATGs expression levels in the moxibustion group were low(P < 0.01),while ATG7 and ATG13 expression levels in the moxibustion plus RAPA group were decreased(P < 0.01),and the expression levels of Vp34,ATG3,ATG5 and ATG12 were decreased(P < 0.05).Compared with the moxibustion group,the expression levels of ATG3 in the moxibustion plus RAPA group were significantly increased(P < 0.01),and the expression levels of Vp34,ATG5,ATG7,ATG12 and ATG13 were increased(P < 0.05).Compared with the moxibustion plus RAPA group,the ATG expression level of the RAPA group was significantly increased(P < 0.01).5.Person correlation analysis showed that the inflammatory factors positively correlated with autophagy genes(P<0.001).Experiment 31.Expression levels of proteins PI3 K,AKT,mTOR and their phosphorylation:Compared with the normal group,the expression levels in the model group were decreased(P < 0.01);Compared with the model group,the expression levels of PI3 K and its phosphorylation,AKT and mTOR in the moxibustion group and moxibustion plus RAPA group were increased(P < 0.01).The expression levels of p-mTOR in the moxibustion group were increased(P < 0.01).The expression levels of p-AKT in the moxibustion and moxibustion plus RAPA group were increased(P < 0.05),and p-PI3 K,AKT,and p-mTOR were lower in the moxibustion plus RAPA group than in the moxibustion group(P < 0.01).Compared with the moxibustion plus RAPA group,the PI3 K,AKT,and mTOR phosphorylation levels in the RAPA group were lower(P <0.01).2.mRNA levels of PI3 K,AKT,and mTOR detected by RT-PCR: Compared with the normal group,the mRNA expressions of PI3 K,AKT,and mTOR in the myocardium of the model group were decreased(P < 0.01).Compared with the model group,the mRNA expressions of PI3 K and AKT in the moxibustion group and moxibustion plus RAPA group were high(P < 0.01),the mRNA expression of mTOR in the moxibustion group was high(P < 0.01),and the mRNA expression of mTOR in the moxibustion plus RAPA group was high(P < 0.05).Compared with the moxibustion group,the mRNA expressions of PI3 K,AKT,and mTOR in the moxibustion plus RAPA group were lower(P < 0.05).Compared with the moxibustion plus RAPA group,the expression of RAPA was lower(P < 0.01).3.Expression levels of WB detection proteins P70S6 K,ULK1 and their phosphorylation: Compared with the normal group,the model group showed low expression levels of P70S6 K and p-p70s6k(P < 0.01),while the high expression levels of ULK1 and p-u Lk1(P < 0.01).Compared with the model group,the P70S6 K and its phosphorylation levels in the moxibustion group and the moxibustion plus RAPA group were increased(P < 0.01),while the ULK1 and its phosphorylation levels in the moxibustion group were decreased(P < 0.01).The ULK1 and its phosphorylation levels in the moxibustion plus RAPA group were decreased(P < 0.05).Compared with the moxibustion group,the expression levels of p70s6k(P < 0.01)and p-p70s6k(P< 0.05)were decreased in the moxibustion +RAPA group,while those of ULK1 and its phosphorylation were increased(P < 0.01).Compared with the moxibustion plus RAPA group,the P70S6 K and its phosphorylation levels in the RAPA group were decreased(P< 0.01),while the ULK1 and its phosphorylation levels were increased(P < 0.01).4.mRNA levels of P70S6 K and ULK1 detected by RT-PCR: Compared with the normal group,the mRNA level of P70S6 K was expressed less in the model group(P <0.01),while that of ULK1 was up-regulated(P < 0.01).Compared with the model group,the mRNA expression of P70S6 K in the moxibustion group was increased(P < 0.01),while that of ULK1 was decreased(P < 0.01).The mRNA relative expression of P70S6 K in the moxibustion plus RAPA group was increased(P < 0.05).Compared with the moxibustion plus RAPA group,the mRNA expression of P70S6 K in the RAPA group was significantly decreased(P < 0.01),while the mRNA expression of ULK1 was increased(P < 0.01).4 Conclusion1.Moxibustion can reduce the content of BNP,ANP and c Tn I in LA and LV and serum of CHF rats,and increase LVPW,IVS,EF and FS,thereby improving cardiac function and structure;2.Moxibustion can down-regulate the levels of ICAM-1,VCAM-1,MMP-2 and MMP-9 in the myocardium of CHF rats,thereby inhibiting the expression of inflammatory related factors.3.Moxibustion can down-regulate the levels of Vps34,ATG3,ATG5,ATG7,ATG12 and ATG13 in the myocardium of CHF rats,and reduce the expression of myocardial autophagy flow and LC3 protein,thereby inhibiting the expression of autophagy-related molecules.4.Moxibustion can regulate the expressions of proteins PI3 K,AKT,mTOR,P70S6 K,ULK1 and their phosphorylation,indicating that moxibustion might activate the upstream signal regulators PI3 K and AKT of mTOR pathway,activate the downstream signal molecule P70S6 K and inhibit ULK1. |
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