| Myocardial infarction(MI)is one of the leading causes of disability and death worldwide.The disability and mortality rate of MI patients have increased year by year,and the situation in China is also severe.Intravenous drug thrombolytic therapy or percutaneous coronary intervention is the most effective treatment for MI.These treatments can restore the blood supply of ischemic myocardial cells as soon as possible,which can effectively save the ischemic myocardium,reduce the area of myocardial infarction,improve the clinical outcome of patients,and improve patients quality of life in the future.However,the treatment window for acute MI is short,once the best treatment time for MI is missed,the efficacy of the patients would significantly reduce,and even causes disability and death.Therefore,it is of crucial importance to find targeted therapeutic agents for MI and to elucidate their possible mechanisms.Icariside Ⅱ(ICS Ⅱ),also known as Baohuoside I,is active flavonoids derived from epimedium,which have a variety of biological and pharmacological effects including anti-inflammatory,anti-osteoporosis,antioxidant,anti-aging and anti-cancer.Previous studies have demonstrated that Epimedium and its active components can combat cardiomyocyte damage by upregulating protein expression of silencing information regulator 1(SIRT1)and NF-E2-associated factor 2(Nrf2);and also reduce cardiomyocyte apoptosis through activation of the PI3K/Akt/eNOS pathway and downregulation of p-p38 and p-JNK protein levels.Icariin may inhibit the activation of NF-κB signaling molecules,thereby inhibiting the release of inflammatory factors,and alleviate myocardial injury in rats with myocardial ischemia and reperfusion.Further research found that ICS Ⅱ also has a variety of cardioprotective effects,such as reducing myocardial fibrosis and improving diabetic cardiomyopathy.However,the specific role of ICS Ⅱ in ischemic-hypoxia-induced cardiomyocytes and related molecular mechanisms remain unclear.Micrornas(MiRNA/miRs)are a group of endogenous non-coding small single-stranded RNAs,which are involved in various biological processes such as cell growth,differentiation and apoptosis.In recent years,researchers have discovered that a variety of drugs can exert pharmacological activity by regulating miRNA transcription and expression including ICS Ⅱ.MicroRNA-7-5p(miR-7-5p)is a newly discovered miRNA in the miRNAs family,which plays an important role in the development and development of a variety of cancers,such as esophageal cancer,gastric cancer,nasopharyngeal cancer,and lung cancer.In addition,studies have shown that miR-7-5p reduces myocardial cell damage induced by ischemia-reperfusion through targeting ADP-ribose polymerase 1.However,whether miR-7-5p plays a role in myocardial cell injury induced by ischemia and hypoxia has not been reported.PI3K/Akt signal is involved in a variety of cell biological processes such as cell proliferation,differentiation,glucose transport and apoptosis.It is one of the most important signal pathways in the body.A large number of studies have demonstrated that the PI3K/Akt signaling pathway plays a critical role in cardiac protection of hypoxic damage.Previous researchers have also confirmed that ICS Ⅱ can play a role by regulating the PI3K/Akt signaling pathway.However,whether ICS Ⅱ can protect cardiomyocytes from hypoxia-induced damage by regulating the PI3K/Akt signaling pathway is still unclear.Therefore,we put forward the following hypothesis:ICS Ⅱ reduces hypoxia-induced cardiomyocyte damage by regulating the expression of miR-7-5p.In order to verify our hypothesis,we will construct a model of H9C2 cardiomyocyte hypoxia injury in vitro.To clarify whether ICS Ⅱ plays a role in hypoxia-induced cardiomyocyte damage and possible mechanisms,CCK8 is used to detect cell viability,lactic dehydrogenase(LDH)release assay is used to checked cytotoxicity,flow cytometry and Western blot is used to examined apoptosis and the related protein expression,the cardiomyocyte migration and invasion capability also test,miRNA microarray analysis,bioinformatics analysis,dual luciferase reporter gene detection are performed.Part Ⅰ Icariin Ⅱ reduces hypoxia-induced cardiomyocyte damagePurpose:To explore whether ICS Ⅱ can reduce the damage of H9C2 cardiomyocytes induced by hypoxia.Methods:1)H9C2 cardiomyocytes were hypoxic for 4 h,8 h,12 h and 24 h respectively.CCK8 was then used to detect the activity of cardiomyocytes,in order to determine the best hypoxia time.Subsequently,lactate dehydrogenase(LDH)release test was used to detect cytotoxicity,flow cytometry was used to detect the level of apoptosis,and Western blot was used to detect apoptosis-related proteins Cleaved caspase-9,Procaspase-9,Cleaved caspase-3,Procaspase-3,Bax and Bcl2 expression.The myocardial cell migration and invasion ability detection were also investigated to confirm that hypoxia-induced H9C2 myocardial cell injury model was successfully constructed.2)H9C2 cardiomyocytes were pretreated with 2 μM,4 μM and 8 μM ICS Ⅱ for 24 h,and CCK8 was used to detect the activity of cardiomyocytes,in order to determine the optimal intervention concentration of ICS Ⅱ.Experimental groups were set as follow:normal control group(Ctrl),cardiomyocyte hypoxia injury group(Hypoxia),cardiomyocyte hypoxia injury+ICS Ⅱ intervention group(Hypoxia+ICS Ⅱ).CCK8 was used to detect cardiomyocytes activity,LDH release test was used to detect cytotoxicity,flow cytometry was used to detect cell apoptosis,and Western blot was used to detect apoptosis-related proteins Cleaved caspase-9,Procaspase-9,Cleaved caspase-3,Procaspase-3,Bax And Bcl2 expression,cardiomyocyte migration and invasion ability test were also investigated to confirm whether ICS Ⅱ can reduce hypoxia-induced H9C2 cardiomyocyte damage.Results:1)Compared with the normal control group,the cell viability of H9C2 cardiomyocytes decreased significantly after hypoxia for 8,12 and 24 h,among which the cell viability was the lowest for 24 h of hypoxia(*P<0.05,**P<0.01,***P<0.001),and finally we use 24 h hypoxia as the hypoxic injury model.Compared with the normal control group,the apoptosis level of H9C2 cardiomyocytes after hypoxic injury was significantly increased(***P<0.001),and the cytotoxicity was significantly increased(***P<0.001).Compared with the normal control group,the apoptosis level of H9C2 cardiomyocytes,the ratio of Cleaved caspase-9/Procaspase-9,the ratio of Cleaved caspase-3/Procaspase-3,and the expression of pro-apoptotic protein Bax increased(***P<0.001,***P<0.001,***P<0.001,***P<0.001),the expression of anti-apoptotic protein Bcl-2 decreased(***P<0.001).After 24 h of hypoxia,the migration and invasion ability of H9C2 cardiomyocytes was significantly reduced(***P<0.001,***P<0.001).2)There was no significant difference in the intervention of cardiomyocytes with different concentrations of ICS Ⅱ(P>0.05).Therefore,we chose 8 μM ICS Ⅱ for the following experiments.Compared with the Hypoxia group,the cell viability of Hypoxia+ICS Ⅱ group was significantly increased(**P<0.01),and the cytotoxicity was significantly reduced(**P<0.01).Compared with the Hypoxia group,the apoptosis level and the ratio of Cleaved caspase-9/Procaspase-9,the ratio of Cleaved caspase-3/Procaspase-3 and the expression of pro-apoptotic protein Bax in the Hypoxia+ICS Ⅱ group decreased(**P<0.01,**P<0.01,***P<0.001,**P<0.01),the expression of anti-apoptotic protein Bcl-2 increased(**P<0.01).Compared with the Hypoxia group,the myocardial cell migration and invasion ability of the Hypoxia+ICS Ⅱ group was significantly increased(***P<0.001,**P<0.01).Conclusions:1)The hypoxia-induced H9C2 cardiomyocyte injury model was successfully established.2)ICS Ⅱ could reduce hypoxia-induced H9C2 cardiomyocyte damage.Part Ⅱ ICS Ⅱ relieves hypoxia-induced cardiomyocyte damage by up-regulating the miR-7-5p/BTG2 axisPurpose:1)To clarify whether ICS Ⅱ reduces hypoxia-induced H9C2 cardiomyocyte damage by up-regulating miR-7-5p.2)Explore whether miR-7-5p could negatively regulates BTG2 expression to reduce hypoxia-induced H9C2 cardiomyocyte damage.3)Clarify the role of BTG2 in hypoxia-induced H9C2 cardiomyocyte damage.Methods:1)After ICS Ⅱ pretreated H9C2 cardiomyocytes,MiRNA microarray analysis was used to screen the downstream MiRNAs with changes in expression.Subsequently,H9C2 cardiomyocytes were pretreated with 2 μM and 8 μM ICS Ⅱ,and the expression of miR-7-5p was detected by RT-PCR to verify whether ICS Ⅱ could regulate the expression of miR-7-5p.Experimental groups were set as follow:normoxia+miR-7-5p NC group,Hypoxia+miR-7-5p NC mimics group,Hypoxia+ICSⅡ+miR-7-5p NC group,Hypoxia+ICS Ⅱ+miR-7-5p mimics group,Hypoxia+ICSⅡ+miR-7-5p inhibitors group.Then CCK8 was used to detect cardiomyocyte activity,LDH release test was used to detect cytotoxicity,flow cytometry was used to detect the level of apoptosis,and Western blot was used to detect apoptosis-related proteins Cleaved caspase-9,Procaspase-9,Cleaved caspase-3,Procaspase-3,Bax and Bcl2 expression,myocardial cell migration and invasion ability test were also investigated to confirm whether ICS Ⅱ can reduce hypoxia-induced H9C2 cardiomyocyte damage by regulating miR-7-5p.2)TargetScan bioinformatics software was used to predict the downstream target gene of miR-7-5p,and the binding site was verified by luciferase reporter gene detection.After transfection of miR-7-5p mimics and miR-7-5p inhibitors,RT-PCR and Western bolt were used to detected the expression of BTG2.The BTG2 overexpression plasmid(pcDNA3.1 BTG2)was constructed,and Western bolt was used to verify whether BTG2 was successfully overexpressed after pcDNA3.1 BTG2 was transferred into H9C2 cells.Experimental groups were set as follow:normal control group,Hypoxia group,Hypoxia+miR-7-5p mimics group,Hypoxia+pcDNA3.1+miR-7-5p mimics group,Hypoxia+pcDNA3.1 BTG2+miR-7-5p mimics group.CCK8 was used to detect cardiomyocyte activity,LDH release test was used to detect cytotoxicity,flow cytometry was used to detect cell apoptosis level,Western blot was used to detect apoptosis-related proteins Cleaved caspase-9,Procaspase-9,Cleaved caspase-3,Procaspase-3,Bax and Bcl2 expression,myocardial cell migration and invasion ability were detected to determine whether miR-7-5p can reduce hypoxia-induced cardiomyocyte damage by targeting regulation of BTG2 expression.3)Two BTG2 interference sequences(siBTG2#1 and siBTG2#2)were constructed,western blot was used to detected the BTG2 expression in H9C2 cells to confirm whether that the siBTG2 silencing vector was successfully constructed.Experimental groups were set as follow:normal control group,Hypoxia+si Ctrl group,Hypoxia+si BTG2#1 group,Hypoxia+si BTG2#2 group.CCK8 was used to detect cardiomyocyte activity,LDH release test was used to detect cytotoxicity,flow cytometry was used to detect cell apoptosis level,western blot was used to detect apoptosis-related proteins Cleaved caspase-9,Procaspase-9,Cleaved caspase-3,Procaspase-3,Bax and Bcl2 expression,myocardial cell migration and invasion ability were also detected to clear the role of BTG2 in hypoxia-induced H9C2 cell injuryResults:1)The miRN chip results showed that after treating with ICS Ⅱ,the expression of 11 of miRNAs of H9C2 cardiomyocytes was up-regulated,and the expression of 11 of miRNAs was down-regulated,of which miR-7-5p was the most up-regulated.After treating H9C2 cardiomyocytes with 2 μM and 8 μM ICS Ⅱ,the expression of miR-7-5p was detected by RT-PCR,and it was found that the expression of miR-7-5p was significantly increased after ICS Ⅱ intervention(**P<0.01,***P<0.001).After transfection of miR-7-5p mimics and miR-7-5p inhibitors,the expression of miR-7-5p was detected by RT-PCR,and it was found that the expression of miR-7-5p was significantly up-regulated after transfection of miR-7-5p mimics(***P<0.001),the expression of miR-7-5p was significantly down-regulated after transfection of miR-7-5p inhibitors(**P<0.01).CCK8 and LDH tests showed that compared with Hypoxia+miR-7-5p NC mimics group,the cell viability of Hypoxia+ICSⅡ+miR-7-5p NC group was significantly increased(**P<0.01),and cytotoxicity was reduced(***P<0.001);Compared with Hypoxia+ICS Ⅱ+miR-7-5p NC group,the cell viability of Hypoxia+ICS Ⅱ+miR-7-5p mimics group was significantly increased(***P<0.001),and cytotoxicity was reduced(*P<0.05),the cell activity of the Hypoxia+ICS Ⅱ+miR-7-5p inhibitors group was significantly reduced(**P<0.01),and the cytotoxicity was significantly increased(***P<0.001).Compared with Hypoxia+miR-7-5p-NC mimics group,the level of apoptosis in Hypoxia+ICS II+miR-7-5p NC group(***P<0.001),apoptosis protein Cleaved caspase-9/Procaspase-9 ratio(***P<0.001),Cleaved caspase-3/Procaspase-3 ratio(***P<0.001),and the expression of pro-apoptotic protein Bax were all downregulated(**P<0.01),while anti-apoptotic protein The expression of Bcl-2 was up-regulated(***P<0.001).Compared with Hypoxia+ICS Ⅱ+miR-7-5p NC group,the apoptosis level of Hypoxia+ICS Ⅱ+miR-7-5p mimics group(*P<0.05)decreased;compared with Hypoxia+ICS Ⅱ+miR-7-5p NC group,Hypoxia+ICS Ⅱ+miR-7-5p inhibitors group apoptosis level(**P<0.01),apoptotic protein Cleaved caspase-9/Procaspase-9 ratio(***P<0.001),Cleaved The ratio of caspase-3/Procaspase-3(***P<0.001),and the expression of pro-apoptotic protein Bax(**P<0.01)all decreased significantly,and the expression of anti-apoptotic protein Bcl-2 decreased(***P<0.001).2)It was predicted by TargetScan Bioinformatics that BTG2 was a potential target of miR-7-5p.Luciferase reporter gene analysis revealed that there was a binding site between miR-7-5p and BTG2.Luciferase activity of BTG2-wt was significantly inhibited by miR-7-5p(***P<0.001),while BTG2-mut had no significant change.The expression of BTG2 was detected by RT-PCR and Western Bolt,and it was found that the expression of BTG2 was significantly down-regulated after overexpression of miR-7-5p(***P<0.001,***P<0.001),while the expression of BTG2 was significantly up-regulated after inhibition of miR-7-5p(***P<0.001,***P<0.001).Western bolt detection revealed that BTG2 was overexpressed in H9C2 cells after transfection of pcDNA3.1 BTG2(***P<0.001).Cell migration and invasion experiments showed that compared with Hypoxia group,Hypoxia+miR-7-5p mimics group had enhanced myocardial cell migration and invasion ability(***P<0.001,***P<0.001);compared with Hypoxia group+pcDNA3.1+miR-7-5p mimics group,Hypoxia+pcDNA3.1 BTG2+miR-7-5p mimics group,cell migration and invasion ability was significantly reduced(***P<0.001,***P<0.001).CCK8 and LDH tests showed that compared with Hypoxia group,Hypoxia+miR-7-5p mimics group had significantly increased cardiomyocyte cell viability(***P<0.001)and decreased cytotoxicity(***P<0.001);Hypoxia+pcDNA3.1+miR-7-5p mimics group,Hypoxia+pcDNA3.1 BTG2+miR-7-5p mimics group cell viability decreased significantly(***P<0.001),cytotoxicity increased(***P<0.001).Compared with the Hypoxia group,the apoptosis level(***P<0.001)and the expression of apoptotic protein in the Hypoxia+miR-7-5p mimics group were reduced(***P<0.001,***P<0.001,**P<0.01),the anti-apoptotic protein increased(***P<0.001);compared with Hypoxia+pcDNA3.1+miR-7-5p mimics group,Hypoxia+pcDNA3.1 BTG2+miR-7-5p mimics group apoptosis increase(***P<0.001,***P<0.001,***P<0.001,**P<0.01,***P<0.001).3)Western blot detection revealed that after siBTG2#1 and siBTG2#2 were transfected into H9C2 cells for 24 h,the expression of BTG2 was significantly down-regulated compared with the normal control group(***P<0.001,***P<0.001).Compared with Hypoxia+si Ctrl,Hypoxia+si BTG2#1 group and Hypoxia+si BTG2#2 group have significantly increased cell viability(***P<0.001,***P<0.001),and reduced cytotoxicity(***P<0.001),***P<0.001).Flow cytometry and Western blot detection of cardiomyocyte apoptosis found that compared with Hypoxia+si Ctrl,Hypoxia+si BTG2#1 and Hypoxia+si BTG2#2 groups had significantly decreased apoptosis(***P<0.001,***P<0.001).Compared with Hypoxia+si Ctrl,the cell migration and invasion ability of Hypoxia+si BTG2#1 group and Hypoxia+si BTG2#2 group were significantly increased(***P<0.001,***P<0.001).Conclusions:1)ICS Ⅱ reduces hypoxia-induced H9C2 cardiomyocyte damage by up-regulating miR-7-5p.2)miR-7-5p could negatively regulate the expression of BTG2 to reduce hypoxia-induced H9C2 cardiomyocyte damage.3)Silencing BTG2 reduces hypoxia-induced H9C2 cardiomyocyte damage.Part Ⅲ ICS Ⅱ reduces hypoxia-induced cardiomyocyte damage by up-regulating miR-7-5p to activate PI3K/Akt pathwayPurpose:1)To determine whether ICS Ⅱ activates the PI3K/Akt pathway by up-regulating miR-7-5p.2)To clarify whether ICS Ⅱ can reduce hypoxia-induced cardiomyocyte damage by activating the PI3K/Akt pathway.Methods:1)Experimental groups were set as follow:normal control group,Hypoxia group,Hypoxia+ICS Ⅱ+miR-7-5p-NC mimics group,Hypoxia+ICS Ⅱ+miR-7-5p mimics group,Hypoxia+ICS Ⅱ+miR-7-5p-NC inhibitors group,Hypoxia+ICSⅡ+miR-7-5p inhibitors group.Western blot was used to detect the expression of p-PI3K,PI3K,p-Akt and Akt.2)Experimental groups were set as follow:Normal control group,Hypoxia group,Hypoxia+ICS Ⅱ group,Hypoxia+ICS Ⅱ group+LY294002 group.To determine whether ICS Ⅱ can reduce hypoxia-induced cardiomyocyte damage by activating the PI3K/Akt signaling pathway,western blot was used to detect the expression of p-PI3K,PI3K,p-Akt and Akt.CCK8 was used to detect cardiomyocyte activity,LDH release test was used to detect cytotoxicity,flow cytometry was used to detect cell apoptosis level,western blot was used to detect apoptosis-related proteins Cleaved caspase-9,Procaspase-9,Cleaved caspase-3,Procaspase-3,Bax and Bcl2 expression,myocardial cell migration and invasion ability were used.Results:1)Compared with Hypoxia group,the ratio of p-PI3K/PI3K and p-Akt/Akt in Hypoxia+ICS Ⅱ+miR-7-5p-NC mimics group increased significantly(**P<0.01,***P<0.001).Compared with the Hypoxia+ICS Ⅱ+miR-7-5p mimics group,the p-PI3K/PI3K and p-Akt/Akt ratios in the Hypoxia+ICS Ⅱ+miR-7-5p inhibitors group were significantly lower(***P<0.001,**P<0.01).2)Compared with Hypoxia group,the ratio of p-PI3K/PI3K and p-Akt/Akt in Hypoxia+ICS Ⅱ group increased significantly(***P<0.001,***P<0.001),compared with Hypoxia+ICS Ⅱ Group,Hypoxia+ICS Ⅱ group+LY294002 group p-PI3K/PI3K and p-Akt/Akt ratio(**P<0.01,***P<0.001).Compared with Hypoxia+ICS Ⅱ group,Hypoxia+ICS Ⅱ group+LY294002 group had significantly reduced cardiomyocyte cell viability and significantly increased cytotoxicity(**P<0.01,**P<0.01).Flow cytometry and Western blot experiments showed that compared with Hypoxia+ICS Ⅱgroup,Hypoxia+ICS Ⅱ group+LY294002 group had significantly increased cardiomyocyte apoptosis(**P<0.01,***P<0.001).Compared with Hypoxia+ICS Ⅱgroup,the cell migration and invasion ability of Hypoxia+ICS Ⅱ group+LY294002 group was significantly reduced(**P<0.01,*P<0.05).In conclusion:1)ICS Ⅱ activates the PI3K/Akt pathway by up-regulating miR-7-5p.2)ICS Ⅱ reduces hypoxia-induced cardiomyocyte damage by activating the PI3K/Akt pathway.ConclusionsIn this study,we confirmed that ICS Ⅱ could reduce hypoxia-induced cardiomyocyte damage by reducing cytotoxicity and apoptosis,and enhancing cell viability,migration and proliferation.Further investigating of the mechanism of ICSⅡ found that ICS Ⅱ could reduce hypoxia-induced cardiomyocyte damage by regulating the expression of miR-7-5p.miR-7-5p could negatively regulate the expression of BTG2.Silencing the expression of BTG2 may reduce hypoxia-induced H9C2 cardiomyocyte damage.All these indicated that ICS Ⅱ plays a protective role against hypoxia-induced cardiomyocyte damage by regulating the miR-7-5p/BTG2 axis.In addition,ICS Ⅱ reduced hypoxia-induced cardiomyocyte damage by regulating miR-7-5p to activate PI3K/Akt signaling pathway.In conclusion,in this study,we clarified the role of ICS Ⅱ in hypoxia-induced cardiomyocyte damage and its molecular mechanism,which would provide new ideas and new targets for the prevention and treatment of myocardial infarction. |
PDF Full Text Request