| ObjectiveBy establishing the rat model of chronic heart failure(CHF)with qi-deficiency-blood-stasis syndrome,this paper discusses the molecular mechanism of moxibustion through regulating mitochondrial quality control,including the effects of oxidative stress response in myocardium,mitochondrial function,mitochondrial dynamics and mitophagy mediated by FUNDC1 signal pathway on rat cardiac function and cardiomyocytes.The clinical research is to evaluate the therapeutic effect and safety of moxibustion by observing the clinical effect of moxibustion on CHF patients with qi-deficiency-blood-stasis syndrome.Experimental research MethodsExperiment 1: 50 rats were randomly divided into Cont group(n = 10)and Model group(n = 40).The rats in the Cont group did not intervene.The CHF model of rats in the Model group was built by intraperitoneal injection of DOX 2.5 mg / kg once a week for 6 weeks with a cumulative dose of 15 mg / kg.Then the model of qi-deficiency-blood-stasis syndrome was built by exhaustive swimming.The successful construction of the model was judged by echocardiography.Then survival of the model rats were randomly divided into the DOX group(n = 9),the MOX group(n = 9),the Ben group(n = 8)and the MOX + Ben group(n = 8).The Cont group and DOX group were perfused with equal volume of normal saline every day in the intervention stage.The MOX group was treated with moxibustion both sides of Xinshu and Feishu points every day for 20 min.The Ben group was perfused with benazepril hydrochloride of0.86mg/kg every day,and the MOX + Ben group was treated with moxibustion both sides of Xinshu and Feishu and intragastric administration of benazepril hydrochloride0.86mg/kg every day for 3 weeks.After the intervention,the general conditions of rats were evaluated.LVIDs,EF and FS were detected by echocardiography,serum BNP level was detected by ELISA,cardiac weight index was calculated,and pathological changes of cardiomyocytes were observed by HE and Masson pathological staining.Experiment 2: 60 rats were randomly divided into the Cont group(n = 12)and the Model group(n = 48).The rats in the Cont group did not intervene.The rats in the Model group were built the CHF model with qi-deficiency-blood-stasis syndrome by the method of Experiment 1.After successful model construction,the survival of model rats were randomly divided into the DOX group(n = 12),the MOX group(n = 10),the Mdivi-1 group(n = 10),the MOX + Mdivi-1 group(n = 10).During intervention,the Cont group and DOX group were injected with equal volume DMSO solution intraperitoneally every day.The MOX group was treated with moxibustion both sides of Xinshu and Feishu points every day for 20 min.The Mdivi-1 group was intraperitoneally injected with mitochondrial fission / autophagy inhibitor Mdivi-1solution at the dose of 1.2 mg / kg every day.The MOX + Mdivi-1 group was treated with moxibustion and intraperitoneally injected with Mdivi-1 solution at the dose of 1.2mg / kg for 3 weeks.After the intervention,the general condition of rats was evaluated,the damage of myocardial mitochondria was observed by electron microscope,the content of ATP and MDA in myocardium of CHF rats were detected by detection kit,the content of SOD2 was detected by ELISA,and DRP1,FIS1,OPA1,Mfn1,Mfn2 and SIRT1 and SIRT2,PCG-1α protein expression were detected by WB.Experiment 3: 45 male SD rats were randomly divided into the Cont group(n = 9)and the Model group(n = 36).After the successful replication of the model,the Model group rats were randomly divided into the DOX group(n = 8),the MOX group(n = 7),the Mdivi-1 group(n = 7)and the MOX + Mdivi-1 group(n = 8).The intervention method was the same as that in Experiment 2.After the intervention,the autophagosome of myocardial mitochondria was observed by electron microscope,the expression of LC3Ⅱ/LC3 Ⅰ,p62,ULK1,p-ULK1,PGAM5,FUNDC1,p-FUNDC1 ser17 protein was detected by WB,and the expression of ULK1 mRNA,PGAM5 mRNA,FUNDC1 mRNA and LC3 mRNA was detected by RT-PCR.ResultsExperiment 1:(1)Model determination: six weeks after modeling,it was found that the mental and activity status in the Model group rats were worse than that in the Cont group,and the values of EF and FS detected by cardiac ultrasound were decreased(P < 0.01).(2)Changes of cardiac function: after three weeks of intervention,the treatment results showed that compared with Cont group,LVIDs and BNP in the DOX group were increased(P < 0.01),EF and FS in the DOX group were decreased(P <0.01).Compared with DOX group,LVIDs and BNP were decreased(P < 0.01),EF and FS were increased(P < 0.01)in the MOX group,Ben group and MOX + Ben group.Compared with the MOX group and Ben group,LVIDs and BNP were decreased further(P < 0.05)in the MOX + Ben group,EF and FS were increased further(P <0.05)in the MOX + Ben group.(3)Changes of cardiac mass index: compared with the Cont group,cardiac mass index was increased(P < 0.01)in the DOX group,the MOX group and Ben group.Compared with the DOX group,cardiac mass index were decreased(P < 0.01)in the MOX group,Ben group and MOX + Ben group.Compared with the MOX group and Ben group,the cardiac mass index were decreased(P < 0.05)in the MOX + Ben group.(3)Changes of cardiomyocytes: the results of pathological staining showed that compared with the Cont group cardiomyocytes in DOX group were disordered,the nuclei decreased and collagen fiber area increased(P < 0.01).Compared with the DOX group,myocardial cell damage and collagen fiber area were decreased(P < 0.05)in the MOX group,Ben group and MOX + Ben group.Experiment 2:(1)The damage of myocardial mitochondria was observed by electron microscope: compared with the Cont group,mitochondria in the DOX group were broken,vacuolated and the damage was serious.Compared with the DOX group,the mitochondrial damage was reduced in the MOX group,Mdivi-1 group and MOX +Mdivi-1 group.(2)Compared with the Cont group,the rat myocardium content of MDA was increased significantly(P < 0.01)while the contents of SOD2 and ATP were decreased significantly(P < 0.01)in the DOX group.Compared with the DOX group,the rat myocardium contents of MDA was decreased(P < 0.05)while the contents of SOD2 and ATP were increased significantly(P < 0.01)in the MOX group,Mdivi-1group and MOX + Mdivi-1 group.Compared with the MOX group and Mdivi-1 group,the rat myocardium content of MDA in the MOX + Mdivi-1 group was decreased further(P < 0.01)while the contents of SOD2 and ATP were increased further(P <0.01).(3)WB detection of myocardial mitochondrial dynamics protein: compared with the Cont group,the rat myocardium mitochondrial fusion proteins OPA1,Mfn1,Mfn2 and mitochondrial synthetic protein PCG-1α,SIRT1 and SIRT2 were decreased significantly(P < 0.01)while the expression of mitochondrial fission proteins DRP1 and FIS1 increased significantly(P < 0.01)in the DOX group.Compared with DOX group,the OPA1,Mfn1,Mfn2 and PCG-1α,SIRT1,SIRT2 were increased significantly(P < 0.01)while the expression of DRP1 and FIS1 were decreased(P < 0.05)in the MOX group,Mdivi-1 group,MOX + Mdivi-1 group.Compared with the MOX group and Mdivi-1 group,the OPA1,Mfn1,Mfn2 and PCG-1α,SIRT1,SIRT2 in the MOX +Mdivi-1 group were increased further(P < 0.05),and the expression of DRP1 and FIS1 protein decreased further(P < 0.01).Experiment 3:(1)Electron microscopic observation of myocardial mitochondrial autophagosomes: compared with the Cont group,the number of autophagosomes in the DOX group were significantly increased(P < 0.01).Compared with the DOX group,the number of autophagosomes in the MOX group,Mdivi-1 group and MOX + Mdivi-1 group were decreased(P < 0.05).(2)WB detected the expression of mitophagy protein and FUNDC1 signal pathway related molecules: compared with the Cont group,the expression of the LC3Ⅱ/LC3Ⅰ,ULK1,p-ULK1,PGAM5,FUNDC1,p-FUNDC1 ser17 protein were increased significantly(P < 0.01)while the expression of p62 protein were decreased significantly(P < 0.01)in the DOX group. Compared with the DOX group,the expression of the LC3Ⅱ/LC3Ⅰ,ULK1,p-ULK1, PGAM5,FUNDC1,p-FUNDC1 ser17 protein were decreased(P < 0.05)while p62 protein were increased(P < 0.05)in MOX group,Mdivi-1 group and MOX + Mdivi-1 group.Compared with the MOX group and the Mdivi-1 group,the expression of LC3Ⅱ/LC3Ⅰ,ULK1,p-ULK1,PGAM5,FUNDC1 and p-FUNDC1 ser17 protein were decreased(P < 0.05)while the expression of p62 protein were increased(P < 0.05)in the MOX + Mdivi-1 group.(3)RT-PCR was used to detect the expression of FUNDC1 signal pathway related molecules: compared with the Cont group,the expression of ULK1 mRNA,PGAM5 mRNA,FUNDC1 mRNA and LC3 mRNA were increased(P < 0.01)in the DOX group.Compared with the DOX group,the expressions of ULK1 mRNA,PGAM5 mRNA,FUNDC1 mRNA and LC3 mRNA were decreased(P < 0.05)in the MOX group,Mdivi-1 group and MOX + Mdivi-1 group.Compared with the MOX group and Mdivi-1 group,the expressions of ULK1 mRNA,PGAM5 mRNA,FUNDC1 mRNA and LC3 mRNA were decreased(P < 0.05)in the MOX + Mdivi-1 group.Clinical research MethodsPatients who met the inclusion criteria were randomly divided into the control group and the treatment group,with 30 cases in each group.The two groups were treated with conventional drugs against chronic heart failure according to the condition.The treatment group was treated with conventional drugs and moxibustion both sides of Xinshu and Feishu points every day for 20 min,once a day for three weeks.The basic information and medical history of patients were collected in detail before treatment.The quantitative score of TCM symptoms,the six minute walking test distance and the changes of serum NT-pro BNP,hs-CRP and related safety indexes detected by clinical laboratory were recorded before and after treatment.LVEDD and EF were measured by cardiac ultrasound before and after treatment;serum MR-pro ANP and TNF-α、IL-6content were detected by ELISA before and after treatment.Results(1)General data: the basic information and medical history of the two groups were compared,and there was no significant difference(P > 0.05),indicating that the data of the two groups were comparable.(2)Quantitative scoring table of TCM symptoms:after treatment,the TCM syndrome scores of the control group and the treatment group were lower than those before treatment,and the difference was statistically significant(P < 0.05);after treatment,the score of TCM syndrome in the treatment group was further reduced compared with that in the control group(P < 0.05).(3)Cardiac ultrasound: LVEDD value was decreased while EF value was increased after treatment compared with that before treatment(P < 0.05);after treatment,compared with the control group,LVEDD value in the treatment group decreased further and EF value increased further(P < 0.05).(4)Serum markers: the content of NT-pro BNP,Mr-pro ANP,hs-CRP and TNF-α in the two groups after treatment were lower than that before treatment(P < 0.05).Compared with the control group after treatment,the serum NT-pro BNP,Mr-pro ANP,hs-CRP and TNF-α,IL-6 in the treatment group were decreased further(P < 0.05).(5)The walking tolerance of patients in both groups increased after six minutes of treatment(P < 0.05);after treatment,the exercise tolerance of the treatment group was further increased compared with that of the control group(P < 0.05).(6)Degree of vascular sclerosis: after treatment,CRI-Ⅰ,CRI-Ⅱ and AC were lower than those before treatment(P < 0.05);there was no significant difference between the treatment group and the control group(P > 0.05).(7)Curative effect of TCM syndrome: the total effective rate of TCM syndrome treatment in the control group was 77% and that in the treatment group was 97%,the difference was statistically significant(P < 0.05).(8)Effect of improving cardiac function: the total effective rate of improving cardiac function in the control group was 73% and that in the treatment group was 93%,the difference was statistically significant(P < 0.05).(9)Safety: during the treatment,the patients had no obvious adverse reactions,and the safety indexes had no significant changes during the treatment(P > 0.05).Conclusions1 Moxibustion can improve the cardiac function of CHF rats with qi-deficiencyblood-stasis syndrome,and reduce the damage of myocardial cells.Moxibustion combined with medicine is better than single moxibustion or medicine.2 Moxibustion can reduce the oxidative stress reaction in myocardium of CHF rats with qi-deficiency-blood-stasis syndrome and improve the damage of myocardial mitochondria..3 Moxibustion can inhibit mitochondrial excessive fission in myocardium of CHF rats with qi-deficiency-blood-stasis syndrome,and promote mitochondrial fusion and synthesis.The specific mechanism may be related to the targeted inhibition of DRP1 protein expression4 Moxibustion may inhibit myocardial mitochondrial autophagy in CHF rats of qi-deficiency-blood-stasis syndrome through FUNDC1 signal pathway.5 Moxibustion can improve the TCM symptoms and cardiac function of CHF patients with qi-deficiency-blood-stasis syndrome,which proves that moxibustion has a definite clinical effect and meets the safety standard. |
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