The Role Of Mir-489 In Regulating Myocardial Fibrosis And The Treatment Of Ginsenoside Re

Myocardial fibrosis(MF)is a common pathological change in the later stage of many cardiovascular diseases,which often causes myocardial systolic and diastolic dysfunction,and is the main reason for the continuous progress and irreversible ventricular remodeling.The pathogenesis of MF is very complex,involving interactions among neurohumoral,growth factors,pro-inflammatory cytokines and gene transcription.Cardiac fibroblasts(CFs)are the main effector cells of MF,which can be transdifferentiated into myofibroblasts with migration and secretion functions under the stimulation of ischemia,inflammatory factors and other pro-fibrotic factors.The activation of myofibroblasts secretes a large amount of extracellular matrix proteins,which leads to increased ventricular stiffness,decreased compliance and abnormal conduction of myocardial electrical signals,inducing refractory heart failure and malignant arrhythmias.At present,there are no specific drugs for the treatment of MF.Therefore,it is urgent to find the key factors of MF pathogenesis and effective therapeutic drugs for the treatment of MF.MircoRNAs(miRNAs)are a class of short non-coding RNAs that are widely expressed in eukaryotic cells.They can induce mRNAs degradation and/or inhibit mRNAs translation by binding to the 3’UTR sequence of target genes,thereby regulating a variety of biological functions.miR-489 is widely expressed in cardiomyocytes and fibroblasts.The overexpression of miR-489 in cardiomyocytes can inhibit the hypertrophic response induced by AngⅡ by inhibiting the expression of downstream myd88.Importantly,AngⅡ-induced MF was significantly reduced in miR-489 transgenic mice,indicating that miR-489 can inhibit AngⅡ-induced MF,and miR-489 may be a potential therapeutic target for the treatment of MF.However,the regulatory mechanism of miR-489 is still unclear,and whether miR-489 also plays the same role in myocardial infarct induced MF deserves further study.There is no direct discussion about MF in traditional Chinese medicine,but MF is a pathological change of many cardiovascular diseases in the late stage,which is a relatively chronic pathological change.The traditional Chinese medicine theory holds that "long-term sickness causes weakness" and "long-term sickness causes blood stasis".Therefore,treatment should follow the principle of invigorating qi and promoting blood circulation.Ginseng is a kind of valuable traditional Chinese herb for invigorating qi,which has a wide range of pharmacological effects,such as anti-myocardial ischemia,anti-arrhythmia,anti-atherosclerosis and anti-fibrosis.Ginsenoside Re is one of the main active components of ginseng.Our previous research found that ginsenoside Re has anti-fibrosis effect after myocardial infarction and can regulate the expression of Smad2/3.Previous studies have confirmed that Smad3 is a direct downstream target of miR-489.Whether ginsenoside Re can play an anti-fibrosis role by regulating miR-489 is worthy of further study.In this study,the CFs model transfected with miR-489 was constructed in vitro to confirm the regulation of miR-489 on MF and myd88/NF-κB signaling pathway.Ginsenoside Re was used as an intervention method.By constructing mice acute myocardial infarction models and AngⅡ-induced CFs models,the anti-MF effect of ginsenoside Re was observed,and the regulatory effect of ginsenoside Re on miR-489 and its downstream myd88/NF-κB was explored.It provides scientific basis for Chinese herb anti-MF.Part 1 The mechanism of miR-489 regulating myocardial fibrosisObjective:miR-489 mimic and inhibitor were transfected into normal CFs and AngⅡ-induced CFs to observe the effects of miR-489 on CFs biological behaviors such as phenotypic differentiation,migration and secretion,and to explore the mechanism of action.Methods:Primary CFs was extracted from neonatal mice,and the purity was identified by immunofluorescence with vimentin,and the groups were as follows:(1)miR-489 mimic was transfected into normal CFs to construct the miR-489 overexpression model.The CFs are divided into 3 groups:normal group(Normal),miR-489 overexpression group(mimic)and miR-489 negative control group(mimic-NC).(2)miR-489 mimic was transfected into AngⅡ-induced CFs to construct the miR-489 overexpression model.The CFs are divided into 3 groups:AngⅡ group(AngⅡ),AngⅡ+miR-489 overexpression group(AngⅡ+mimic)and AngⅡ+miR-489 negative control group(AngⅡ+mimic-NC).(3)miR-489 inhibitor was transfected into normal CFs to construct the miR-489 inhibition model.The CFs are divided into 3 groups:normal group(Normal),miR-489 inhibition group(inhibitor)and miR-489 negative control group(inhibitor-NC).(4)miR-489 inhibitor was transfected into AngⅡ-induced CFs to construct the miR-489 inhibition model.The CFs are divided into 3 groups:AngⅡ group(AngⅡ),AngⅡ+miR-489 inhibition group(AngⅡ+inhibitor)and AngⅡ+miR-489 negative control group(AngⅡ+inhibitor-NC).The expression level of miR-489 in CFs transfected with miR-489 mimic was detected by qPCR technique to verify the transfection effect.The expression ofα-SMA in each group was detected by Western blot to observe the phenotypic differentiation of CFs.The proteins expression of Collagen Ⅰ and Collagen Ⅲ in each group were measured to observe the secretion of CFs.The expression of myd88,NF-κB p-p65 and NF-κB p65 protein were detected to observe the regulatory effect of miR-489.Results:(1)Vimentin immunofluorescence detection confirmed that the purity of the primary CFs was more than 95%,which met the experimental requirements.(2)Compared with Normal group and mimic-NC group,the expression level of miR-489 was significantly increased in mimic group(P<0.01).There was no statistical difference between Normal group and mimic-NC group(P>0.05).These data indicated that the transfection of miR-489 mimic was successful.Compared with Normal group and inhibitor-NC group,the expression of myd88 in the inhibitor group was significantly up-regulated(P<0.01).There was no statistical difference between Normal group and inhibitor-NC group(P>0.05).These data indicated that the transfection of miR-489 inhibitor was successful.(3)In normal CFs and AngⅡ-treated CFs,overexpression of miR-489 could significantly inhibit the expression of myd88,α-SMA,Collagen Ⅰ and Collagen Ⅲ,and reduce the ratio of p-NF-κB p65/NF-κB p65,while inhibition of miR-489 could promote the expression of myd88,α-SMA,Collagen Ⅰ and Collagen Ⅲ,and increase the ratio of p-NF-κBp65/NF-κB p65.Conclusion:Overexpression of miR-489 in normal CFs and CFs treated by AngⅡ can inhibit the activation of downstream myd88/NF-κB signaling pathway and the fibrotic reaction of CFs,while inhibition of miR-489 produces opposite results.These results suggest that miR-489 plays an anti-MF role in CFs by regulating the myd88/NF-κB signaling pathway in physiological and pathological conditions.Part 2 The mechanism research of ginsenoside Re regulates myocardial fibrosis after acute myocardial infarctionObjective:To observe the effects of ginsenoside Re on cardiac function,serum markers,histopathological changes and related genes and proteins expression in mice with MF after acute myocardial infarction,and to explore the molecular mechanism of ginsenoside Re in improving MF after infarction.Methods:The MF model after myocardial infarction in C57BL/6 mice was established by ligation of the anterior descending branch of the left coronary artery.Mice were randomly divided into 4 groups:sham operation group(Sham),model group(Model),low-dose ginsenoside Re group(L,19.5 mg/kg/d),high-dose ginsenoside Re group(H,39 mg/kg/d),10 mice in each group.On the second day after operation,mice were given intragastric administration once a day for 4 weeks.Left ventricular ejection fraction(LVEF)and left ventricular fractional shortening(LVFS)were detected by echocardiography;The heart weight index(HWI),heart-tibial ratio(HW/TL)and the expression of ANP,BNP andβ-MHC mRNA in myocardium were measured to observe the compensatory hypertrophy of myocardium after myocardial infarction;The serum markers levels such as myocardial injury(CK-MB),heart failure(BNP),hepatic and renal function(ALT,S-Cr)and RAAS(AngⅡ)were evaluated by ELISA;HE and Masson staining were used to observe myocardial tissue and fibrotic lesions after myocardial infarction;The proteins and genes expressions of miR-489,myd88,p-NF-κB p65,NF-κB p65,α-SMA,Collagen Ⅰand Collagen Ⅲ were observed by Western blot or qPCR.Results:(1)Compared with Sham group,LVEF and LVFS were obviusly decreased(P<0.01),while LVEF and LVFS were increased in low and high dose ginsenoside Re group,when compared with Model group(P<0.01).(2)Compared with Model group,the serum levels of CK-MB,BNP and AngⅡ were significantly decreased in low and high dose ginsenoside Re group(P<0.05),while the serum levels of ALT and S-Cr have no significant change(P>0.05).(3)Compared with Sham group,HWI and HW/TL as well as myocardium mRNAs expression of ANP,BNP and β-MHC in Model group was markedly increased(P<0.01),while ginsenoside Re could reverse this tendency(P<0.05).(4)Compared with the Sham group,the structure of myocardium in the Model group was disordered,and the muscle fibers were thick and wavy,accompanied by a large number of inflammatory cells infiltration and collagen deposition.The histopathological changes were significantly improved after the intervention of ginsenoside Re.Compared with Model group,collagen volume fraction(CVF)dramatically decreased in low and high dose ginsenoside Re(P<0.05).(5)Compared with Sham group,myd88,p-NF-κB p65,α-SMA,Collagen Ⅰ and Collagen Ⅲ protein expression were up-regulated,while the expression of miR-489 was down-regulated(P<0.05).Ginsenoside Re can significantly inhibit this trend(P<0.01).In the above tests,there was no statistical difference between the low and high dose ginsenoside Re groups(P>0.05).Conclusion:In mice after myocardial infarction,Ginsenoside Re can improve the cardiac function,inhibit compensatory myocardial hypertrophy,reduce myocardial ischemia injury and collagen deposition,and improve histopathological changes.Meanwhile,there was no obvious hepatorenal toxicity.The mechanism may be related to the ginsenoside Re regulation of miR-489/myd88/NF-κB pathways in myocardial infarction mice.Part 3 The mechanism research of ginsenoside Re regulates the cellular biological behaviors in AngⅡ-induced CFsObjective:To observe the effects of ginsenoside Re on cellular biological behaviors,including differentiation,migration and secretion in AngⅡ induced neonatal mouse cardiac fibroblasts(CFs),and explore the related mechanism.Methods:The neonatal mouse CFs were extracted and the safe concentration range of ginsenoside Re was screened by CCK-8 method.CFs were randomly divided into 5 groups:normal group(Normal),model group(Model)and ginsenoside Re low-dose,medium-dose and high-dose groups.After the appropriate time of intervention,the cells were collected for related detection.Transwell test and scratch test were used to detect the migration ability of CFs.Immunofluorescence staining and Western blot were used to quantify the expression of α-SMA,Collagen Ⅰ and Collagen Ⅲ in order to observe the phenotype transformation of CFs and the production of collagen.The expression of miR-489,myd88,NF-κB p65 and p-NF-κB p65 were measured by qPCR and Western blot technology.Results:(1)By CCK-8 method,50μmol/L,100μmol/L,200μmol/L were selected as the intervention concentration of ginsenoside Re.(2)Compared with Normal group,the number of cells passing through the Transwell chamber were significantly increased and the scratch width was narrowed in the Model group(P<0.01).Compared with Model group,the number of cells migrating through the Transwell chamber were markedly reduced and the scratch width were wider in low,medium and high dose ginsenoside Re(P<0.01).(3)Compared with Model group,the fluorescence intensity and protein expression of α-SMA,Collagen Ⅰ and Collagen Ⅲwere significantly decreased in low,medium and high dose ginsenoside Re(P<0.01).(4)Compared with Normal group,miR-489 level was significantly decreased,while myd88 and p-NF-κB p65/NF-κB p65 were significantly increased in the Model group(P<0.01).Ginsenoside Re can reverse this trend(P<0.01).There was no statistical difference among low,medium,and high dose ginsenoside Re groups(P>0.05).Conclusion:Ginsenoside Re can inhibit AngⅡ-induced biological behavior changes of CFs,such as migration,phenotypic differentiation and secretion.The mechanism is related to the ginsenoside Re regulation of miR-489/myd88/NF-κB pathways in AngⅡ-induced CFs.