| Ectopic fat deposition was one of the main causes of diabetic cardiomyopathy(DCM).DM led to varieties of complications,among which DCM always presented in the cardiovascular system,which was one of the key factors causing the death of DM patients.At present,the mechanism of DCM has not been fully studied.Some studies have shown that lipid toxicity,glucose metabolism disorder,OS,myocardial calcification,mitochondrial dysfunction and changes in myocardial function are all factors leading to the occurrence of DCM.Lipotoxic myocardial injury often causes severe myocardial dysfunction,influences the normal systolic functions of myocardium,and severely endangers the lives and health of patients.Keeping the balance of lipid metabolism and enhancing cardiac function have become an important means of prevention and treatment of DCM.Lipotoxicity is due to the increase of FFA content in human peripheral serum,which overflows the normal use range of the body,which causes the excessive accumulation of FFA in liver,muscle and other tissues,which damages the tissue and organs.In the longterm high fat environment,the accumulation of lipid and intermediate products is excessive,which will lead to cell dysfunction,metabolic abnormality and related pathological changes.Excessive lipid accumulation in the heart muscle stimulates ers,causes OS,and causes vast number of apoptosis of cardiomyocytes,which exerts a major influence on the activity of myocardium.Lipid metabolism disorder can stimulate the abnormal function of OS and mitochondria,which can cause abnormal cardiac structure and function,such as cardiac hypertrophy,myocardial infarction,heart failure and death.Lipotoxic myocardial injury plays an important role on the progress of DCM.Artemisinin and its derivatives have good therapeutic effects on arrhythmia,myocardial ischemia,atherosclerosis and plaque stability caused by various reasons.Activated NF-kB pathway can increase the expression of inflammation factors,and then promote inflammatory response and fibrosis,and eventually lead to left ventricular remodeling;studies have found that Artemisinin can inhibit NF-kB pathway to reduce myocardial remodeling.The antiarrhythmic mechanism of artemisinins is achieved by blocking ion channels and promoting the expression of connexin in myocardial cells.The antiarrhythmic effect of artemisinins is mainly reflected in anti inflammation and anti-oxidation,inhibition of vascular endothelial cell division and inhibition of macrophage phenotype transformation.Artemisinin is also effective in preventing the complications of heart,kidney and liver caused by lipotoxicity.It can inhibit inflammatory reaction,protect islet β cells,improve their survival rate,improve insulin resistance,induce islet α cells to transform into β cells,reduce the process of renal and cardiac fibrosis,and reduce urinary protein excretion.miRNAs are a sort of non-coding RNA with hairpin structure and always play certain roles in regulatory function,which is uniquely found in the eukaryotes.It distributes widely in many species and piles of organs,and functions by regulating many target genes.Some studies confirmed that miRNA not only participated in embryo heart development,but also exerted important roles in the progress of cardiovascular diseases.Along with the increasing incidence rate and mortality,miRNAs became a hot spot in the pathophysiology of cardiovascular diseases.miRNA-133b was involved in the regulation of plenty of physiological bioprocesses,including the sheltering of cardiomyocytes confronted with hypoxia and infarction situation.It was reported that miRNA-13 3b hindered the activation and proliferation of cardiac fibroblasts,and the expression level of miRNA-13 3b in peripheral plasma of patients suffered with acute myocardial infarction was increased significantly.What’s more,the role of miR-133b in myocardial lipotoxicity has not been reported.In this study,myocardial cell HHHM2 was used as the research object,the myocardial lipotoxic injury cell model was induced by PAL,and the miR-133b inhibitor was transfected to analyze the role of miR-133b induced myocardial lipotoxic injury in PAL by MTT analysis and real-time fluorescent quantitative PCR.Through the double fluorescein report experiment,it was revealed that whether Sirtl was the target gene of miR-133b,and the effect of miR-133b on the expression of Sirtl in HHHM2 cells was detected by immunoblot.Construct Sirtl overexpression plasmid(pcDNA3.1-Sirt1),and transfect cardiomyocytes with the overexpression plasmid and miR-133b mimic or inhibitor alone or at the same time.Through rescue experiments,it was verified whether miR-133b played a role in cardiomyocyte damage after PAL treatment by regulating Sirt1.Use DHA to rescue cardiomyocytes after PAL treatment,MTT was used to determine cell viability,qRT-PCR to check the expression of miR-133b,qRT-PCR to examine DHA to reduce the transcription level of cardiomyocyte apoptosis factors after PAL treatment,using luciferase system to detect the role of microRNA-133b(miR-133b)in the lipotoxic environment of cardiomyocytes,and to explore its downstream molecular mechanisms,as well as the fine mechanism of dihydroartemisinin in protecting cardiomyocytes via regulating MiR-133b,The diagnosis and treatment of hyperlipidemia and DCM provide new strategies.Part1 Role of miRNA-133b in PAL induced myocardial lipotoxicityObjective:To explore the effect of palmitic acid(PAL)on HHHM2 cardiomyocytes,establish a lipotoxic injury model of HHHM2 cardiomyocytes,analyze the mechanism of PAL-induced myocardial cell toxicity,and clarify the role of miRNA-133b in PAL-induced myocardial lipotoxic injury.Methods:1.Culture cardiomyocytes HHHM2 in vitro,and take palmitate to mimic-lipid toxicity,MTT(3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide)analysis was taken to check the effect of myocardial cells on cell viability after PAL treatment before and after miR-133b inhibition2.qRT-PCR detects the difference in expression of Caspase3,Bax,Bcl-2 and miR133b in normal cardiomyocytes and after PAL treatment3.Western blot was carried to detect the differences in the expression of Caspase3,Bax and Bcl-2 in cardiomyocytes treated with PAL in normal cardiomyocytes and in cardiomyocytes treated with PAL.4.Real-time fluorescence quantitative PCR was used to analyze the differences in the expression of miR-133b in the high-fat stimulation model cells before and after miR-133b inhibitor transfection.Results:1.Cardiomyocytes HHHM2 can be successfully cultured in vitro.The results of MTT show that after PAL treatment for 24 hours,the viability of cardiomyocytes was significantly lower than that of normal cells(p<0.05),and after PAL treatment for 72 hours,the viability of cardiomyocytes decreased significantly(p<0.01).2.The qRT-PCR test showed that after PAL treatment for 12h,the expression level of Caspase 3 was significantly higher than that of the normal control group(p<0.05),and after PAL treatment for 48h,the expression level of Caspase 3 increased more significantly(p<0.01);Bcl-2 No significant difference(p>0.05);Bax expression increased significantly after PAL treatment for 12h(p<0.05),and increased more significantly at 48h(p<0.01).3.The Western blot method showed that the expression of Caspase3 increased slightly at 12h after PAL treatment(p<0.05),and began to rise sharply at 24h(p<0.001),and continued to increase to 72h(p<0.001).The expression of Bax is similar to Caspase3,and also increases with the increase of PAL treatment time.The expression level of Bcl-2 increased significantly at the beginning of PAL treatment(12h),but gradually decreased with the passage of time.4.The expression of miR-133b in cardiomyocytes treated with PAL was analyzed by real-time fluorescent quantitative PCR.The results showed that the expression of miR-133b in cardiomyocytes increased significantly after PAL treatment.Compared with the control group,the expression level of miR-13 3b increased slightly after PAL treatment for 6h(p>0.05),and after PAL treatment for 12h and 24h,the expression level of miR-133b increased significantly by 60%and 80%,respectively.(P<0.05,p<0.01);it was significantly increased at 48h after PAL treatment(p<0.01).After 72h,the expression of miR-133b began to decrease,but it was still significantly higher than that of the control group(p<0.01).5.After myocardial cells were transfected with miR-133b inhibitor,they were treated with PAL at the same time.The qRT-PCR results showed that the expression level of miR133b in the miR-133b inhibitor transfected and PAL treated group was significantly lower than that of the control group(p<0.001),The expression level of miR-133b decreased by 50%;the expression level of miR-133b in the miR-133b inhibitor group was significantly lower than that of the Scramble group and Blank group(p<0.001).6.The results of MTT test showed that the cell viability of the Blank group and the Scramble group was significantly lower than that of the control group(p<0.01),and the cell viability of the miR-133b inhibitor transfection group was also significantly lower than that of the control group(p<0.05);Compared with the Scramble group,the cell viability of the miR-133b inhibitor transfection group was significantly increased(p<0.05).Conclusion:The proliferation of cardiomyocyte HHHM2 that can be inhibited by PAL treatment leads to the increase of apoptosis-related genes and protein expression,thereby promoting cell apoptosis.PAL stimulation can induce an increase in the expression of miR133b in cardiomyocytes,and inhibition of miR-133b expression can reduce the lipotoxic injury of cardiomyocytes induced by PAL.Part2 Study on the mechanism of miR-133b regulating myocardial lipotoxic injuryObjective:To explore the mechanism of miR-133b regulating myocardial lipotoxic injury.Methods:1.Online software(http://www.targetscan.org/)analyzes the potential target genes of miR-133b.2.The dual luciferase reporter gene system verifies that the target gene regulated by miR-133b is Sirtl(Sirtuin 1).3.Western blot method to detect the effect of miR-133b on Sirtl protein expression in cardiomyocytes.4.Construct Sirtl overexpression plasmid(pcDNA3.1-Sirt1),and transfect cardiomyocytes with miR-133b mimic or inhibitor alone or at the same time,and observe whether miR-133b regulates Sirtl after PAL treatment through rescue experiment Protect the biological role of cardiomyocytes.Results:1.Online software(http://www.targetscan.org/)analyzes Sirtl as a target gene regulated by miR-133b.2.Detection of dual luciferase activity results showed that the relative luciferase activity of the miR-133b mimic transfected and the plasmid group containing Sirt1 3’_UTR(wildtype)luciferase was significantly reduced(p<0.01),indicating that miR-133b acts directly on the predicted target site of Sirt1 3’-UTR.3.Western blot results showed that compared with the control group(Scramble group),the Sirtl protein expression level was significantly lower in the miR-133b mimic transfection group(p<0.01),indicating that overexpression of miR-133b inhibited the Sirtl protein expression.4.qRT-PCR detects the expression level of Sirtl in each group of cells after transfection of the plasmid.The results show that compared with the backbone vector transfection control group(pcDNA3.1 group),the expression of Sirtl in cardiomyocytes transfected with pcDNA3.1-Sirtl is significant Increased(p<0.01);while the expression of Sirtl in the pcDNA3.1-Sirtl+miR-133b mimic transfection group was significantly lower than that in the pcDNA3.1-Sirt1 transfection group(p<0.01),but not compared with the backbone plasmid transfection group Significant difference(p>0.01).Similarly,the expression of Sirt1 was significantly increased after pcDNA3.1-Sirt1 was transfected,while its expression decreased after miR-133b was added at the same time.5.The change of cell viability after PAL induction was detected by MTT method.The results showed that the cell viability in the pcDNA3.1-Sirt1 transfection group was significantly higher than the empty vector control group(pcDNA3.1 group)(p<0.01),while in miR-133b The mimic+pcDNA3.1-Sirt1 transfection group was significantly lower than the pcDNA3.1-Sirt1 transfection group(p<0.01),and there was no significant difference compared with the empty vector control group(pcDNA3.1 group)(p>0.05).Conclusion:The protective effect of Sirtl on cardiomyocytes is inhibited by miR-133b,which is a target gene of miR-133b.miR-133b plays a role in cardiomyocyte damage after PAL treatment by regulating Sirt1.Part3 Study on the Mechanism of DHA Regulating MiR-133b to Reduce Cardiomyocyte InjuryObjective:To explore the mechanism of DHA regulating MiR-133b to reduce myocardial cell injury.Methods:1.Use MTT method to observe the effect of DHA on myocardial cell viability after PAL treatment,and qRT-PCR to detect the expression changes of miR-133b.2.Use qRT-PCR to detect the effect of DHA on the transcription level of cardiomyocyte apoptosis factors after PAL treatment.3.The luciferase reporter gene system was used to detect that DHA regulates SIRT1 transcription by affecting miR-133b.Results:1.After the cardiomyocytes are inoculated,when their confluence rate reaches 80%,add PAL at a concentration of 0.5mM as a control group.The experimental group was treated with PAL while adding DHA at a final concentration of 10μM,and then tested at 12h,24h,48h and 72h.The cell survival rate was not detected at 12h,while the DHA group increased slightly at 24h.,But failed to detect a significant difference.The cell survival rate of DHA group was significantly higher than that of PAL group at 48h(p<0.05),and the significance of this difference increased further at 72h(p<0.01).2.Compared with the model group,DHA significantly improved the survival rate of cardiomyocytes,the difference was significant at 48h(p<0.05),and the difference was more significant at 72h(p<0.01).3.The qRT-PCR test showed that the expression of Caspase3 was significantly higher than that of the control group after 48h of PAL treatment(p<0.001),while DHA treatment significantly reduced the expression of Caspase3(p<0.01).After DHA treatment,the expression of Caspase3 was still significantly higher than that of the control group(p<0.05).Compared with the control group and the PAL group,the expression of Bcl-2 was not significantly changed after 48h after DHA treatment(p>0.05).Bax expression was significantly increased 48h after PAL treatment compared with the control group(p<0.001);Bax expression was significantly decreased compared with PAL group after DHA treatment(p<0.01).However,the expression of Bax did not return to the normal level after DHA treatment(p<0.05).4.Western blot detection showed that after 48 hours of PAL treatment,the protein content of Caspase3 increased sharply compared with the control group(p<0.001),while the expression of DHA decreased significantly compared with the PAL group(p<0.001).Compared with the control group,the difference was not significant(p<0.05);the Bax expression pattern was similar;the Bcl-2 expression increased significantly after PAL stimulation(p<0.05),and the DHA treatment increased significantly compared with the PAL group(p<0.01),significantly higher than the control group(p<0.001).5.Cardiomyocytes were treated with PAL and PAL and DHA at the same time.The cells were collected at 48h,and the expression of Caspase3 was detected by qRT-PCR.The results showed that the expression of Caspase3 after PAL treatment for 48h was significantly higher than that of the control group(p<0.001),while DHA treatment significantly reduced the expression of Sirt1(p<0.01).After DHA treatment,the expression of Caspase3 was still significantly higher than that of the control group(p<0.05).6.Cardiomyocytes were treated with PAL and DHA at the same time.The cells were collected at 48h,and the expression of miR-133b was detected by qRT-PCR.The results showed that the expression of miR-133b was significantly higher than that of the control group after PAL treatment for 48h(p<0.001),while DHA treatment significantly reduced the expression of miR-133b(p<0.01).7.To further explore whether DHA directly regulates the transcription of Sirt1 through miR-133b,a luciferase reporter gene system was used for detection.The Scramble sequence was used as a negative control group,and the wild type and mutant 3’-UTR of Sirt1 were treated with miR-133b and/or DHA,and the Luciferase intensity was observed.The results show that in the presence of Scramble,Sirt1 wild-type 3’-UTR can be transcribed normally,and the addition of DHA does not affect the effect of Scramble on the transcription activity of Sirt1 wild-type 3’-UTR(p>0.05).In the presence of DHA,Scramble’s regulation of Sirt1 mutant 3’-UTR(MUT)transcriptional activity did not change(p>0.05);in the presence of miR-133b,Sirtl wild-type 3’-UTR transcriptional activity Compared with the control group,it was significantly reduced(p<0.001).After the application of DHA,the inhibition of the transcription activity of Sirtl wild-type 3’-UTR by miR-133b was significantly reduced(p<0.001),but it did not affect the Sirtl mutant 3’-UTR.No effect(p>0.05).Conclusion:DHA can effectively antagonize the injury and apoptosis of cardiomyocytes induced by pal,and increase the expression of anti apoptotic factors.DHA can change the expression of SIRT1 by regulating miR-13 3b,and then play a protective role in cardiomyocytes. |
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