| BackgroundIn recent years,the morbidity and mortality rates of heart failure have remained high worldwide.As the terminal outcome of many cardiovascular diseases,heart failure reduces the quality of life of patients,increases the socio-economic burden,and poses a serious threat to human health.Hence,preventing and treating heart failure is of great importance.Current research suggests that cardiac remodeling due to pathological myocardial fibrosis plays an important role in heart failure development and evolution.The pathogenesis of myocardial fibrosis mainly includes replacement fibrosis and reactive fibrosis.Acute myocardial infarction results in massive myocardial cell death followed by replacement fibrosis in the infarcted area.The scar tissue that forms maintains the structural integrity of the heart to prevent serious complications such as cardiac rupture.However,excessive fibrosis can lead to localized abnormalities in myocardial activity.In contrast,diffuse or focal reactive myocardial fibrosis is the result of pressure or volume overload caused by various etiologies such as persistent hypertension,metabolic disorders,valvular heart disease,ischaemic injury away from the infarcted region,or diffuse cardiomyopathy.Excessive replacement fibrosis and reactive fibrosis will eventually lead to a decrease in diastolic and systolic function of the heart and induce heart failure.Therefore,to prevent and treat heart failure,it is important to investigate the mechanisms of pathological myocardial fibrosis and explore the effects of drugs on fibrosis.The transforming growth factor-beta(TGFβ)family is an activating mediator of fibroblasts and is the central mediator of the fibrotic response,while TGFβ1 plays the largest role in pathological fibrosis.TGFβ1 regulates gene transcription by activating Smad-dependent classical and non-Smad-dependent non-classical pathways,transforming fibroblasts into myofibroblasts with secretory activity,expressing α-smooth muscle actin(α-SMA)and secreting extracellular matrix proteins such as Collagen Ⅰ(Col Ⅰ),ultimately causing pathological fibrosis.Galectin-3(Gal-3)is a β-galactoside-binding protein that belongs to the lectin family and has various regulatory activities such as promoting cell growth,proliferation,differentiation,cell adhesion,and tissue fibrosis.Studies have shown that there is a regulatory relationship between Gal-3 and TGFβ1,and we hypothesize that Gal-3 can promote the development of pathological fibrosis in heart failure patients by regulating TGFβ1,while inhibiting Gal-3 and TGFβ1 expression,thereby improving pathological myocardial fibrosis.Gal-3can potentially control or even reverse myocardial remodeling in heart failure patients,improving their clinical symptoms and long-term prognosis.Rosuvastatin is a selective hydroxymethylglutaryl coenzyme A reductase inhibitor,which can lower blood lipids by increasing the number of LDL receptors on the surface of hepatocytes and promoting LDL absorption and catabolism and is now widely used in treating coronary heart disease and hypercholesterolemia.Besides their lipidlowering function,statins can also have a positive effect on patients with heart failure,possibly by inhibiting myocardial fibrosis and improving myocardial remodeling.Rosuvastatin has been shown to downregulate serum Gal-3 levels in patients,but it is not known whether it can improve pathological myocardial fibrosis by modulating Gal-3 and the TGFβ1/Smad pathway.In this study,the expression levels of Procollagen type I carboxy-terminal propeptide(PⅠCP)and other related proteins in the serum of heart failure patients will be analyzed,and a pathological myocardial fibrosis cell model using Angiotensin II(Ang II)will be constructed in vitro to investigate the protective effect of rosuvastatin on pathological myocardial fibrosis and its mechanism.ObjectivesThe differences in Gal-3,PⅠCP and TGFβ1expression levels in the serum of heart failure patients are analyzed to clarify their relevance in heart failure.A pathological myocardial fibrosis cell model is constructed in vitro using Ang II to clarify the expression of fibrosis phenotypic proteins and Gal-3 and TGFβ1,and the Gal-3,TGFβ1,Smad2,p-Smad2,Smad3,and p-Smad3 expression levels are further examined after the intervention of rosuvastatin to investigate the protective effect of rosuvastatin on pathological myocardial fibrosis and its mechanism.Methods1.A total of 125 patients who were hospitalized in the Department of Cardiovascular Medicine,the Second Norman Bethune Hospital of Jilin University from March 1,2021 to August 25,2022 were selected for the cross-sectional study,among which patients with a clear heart failure diagnosis were included in the heart failure group,a total of 63 patients;patients with other cardiovascular diseases such as ischemic heart disease,hypertension,and arrhythmia diagnosed at the Department of Cardiovascular Medicine,the Second Norman Bethune Hospital of Jilin University during the same period without the diagnosis of heart failure were included in the control group.Fasting venous blood was collected from all patients in the group,centrifuged and the serum was transferred to ep tubes and frozen in a-20 °C refrigerator.After collecting all the samples,the expression of serum PⅠCP,TGFβ1,and Gal-3 was detected by enzyme-linked immunosorbent assay,and the results were statistically analyzed.The Shapiro-Wilk test for normality and the Levene test for homogeneity of variance were used.The t-test was applied to analyze the differences between groups for measurements that met the normal distribution and homogeneity of variance test;the rank sum test was applied to analyze the differences between groups for measurements that did not meet the normal distribution and/or homogeneity of variance test;the chi-square test was applied to compare groups for counting data.Spearman’s rank correlation analysis was applied to test the correlation between Gal-3,PⅠCP,and TGFβ1,respectively.2.A pathological cellular fibrosis model was constructed using Ang II stimulation of rat H9c2 cells.The expression of Col Ⅰ,α-SMA,Gal-3,and TGFβ1 under different AngⅡ stimulation concentrations was examined by western blot.Subsequently determine whether the modeling was successful and the optimal stimulation concentration for subsequent experiments and investigate the relationship between Gal-3 and TGFβ1 in the myocardial fibrosis model.3.In the myocardial fibrosis cell model,different concentrations of rosuvastatin were administered and the Col Ⅰ,α-SMA,Gal-3,TGFβ1,Smad2,p-Smad2,Smad3,and p-Smad3 expression levels were measured by western blot to determine the protective effect of rosuvastatin on myocardial fibrosis and to investigate its molecular mechanism.Results1.Serum Gal-3 expression level was significantly upregulated in the heart failure group compared to the control group(11.20(8.71,12.96)versus 6.01(4.75,7.11),p <0.01);serum TGFβ1 expression level was significantly upregulated in the heart failure group compared to the control group(21.20(15.74,24.64)versus 10.90(7.66,14.00),p < 0.01);serum PⅠCP expression level was significantly upregulated in the heart failure group compared to the control group(143.05(112.19,172.30)versus 78.44(67.76,98.61),p <0.01),all these results were statistically significant.The correlation between Gal-3,PⅠCP,and TGFβ1 was detected using the Spearman correlation analysis,and the results suggested that the serum TGFβ1 and PⅠCP expression levels were positively correlated in the heart failure group patients(r=0.3679;p<0.01).Moreover,the serum Gal-3 and PⅠCP expression levels were positively correlated in the heart failure group patients(r=0.5102;p<0.01).Finally,the serum Gal-3 and TGFβ1 expression levels in the heart failure group were positively correlated(r=0.5741;p<0.01).2.In the Ang II-induced cardiac fibrosis cell model,the Col Ⅰ and α-SMA expression levels started to increase at 1 μM Ang II concentration and gradually increased with the increase of Ang II concentration,reaching the highest level at 2 μM Ang II.The TGFβ1 and Gal-3 expression levels did not differ significantly from the control group at 1 μM Ang II stimulation,but started to increase at 1.5 μM Ang II stimulation and reached the highest level at 2 μM Ang II stimulation.3.The expression of Col I and α-SMA was not significantly different in the highdose Rosuvastatin(RVS-H)group compared to the control group but was significantly increased after 24 h of 2 μM Ang II stimulation.Compared with the Ang II group,the expression of Col Ⅰ was not significantly different in the Ang II+ low-dose rosuvastatin(Ang II+ RVS-L)and Ang II+ medium-dose rosuvastatin(Ang II+ RVS-M)groups,and was significantly lower in the Ang II+ RVS-H group.The expression of α-SMA was not significantly different in the Ang II+ RVS-L group compared with the Ang II group and was significantly lower in the Ang II+ RVS-M and Ang II+ RVS-H groups.The expression of Gal-3,TGFβ1,p-Smad2/Smad2,and p-Smad3/Smad3 was not significantly different in the RVS-H group compared to the control group but was significantly increased after 24 h of 2 μM Ang II stimulation.Compared with the Ang II group,the expression of Gal-3 was not significantly different in the Ang II+RVS-L and Ang II+ RVS-M groups but was significantly decreased in the Ang II+ RVS-H group;the expression of TGFβ1 was not significantly different in the Ang II+RVS-L group but was significantly decreased in the Ang II+ RVS-M and Ang II+ RVS-H groups;p-Smad2 p-Smad2/Smad2 expression was not significantly different in the Ang II+ RVS-L group but was significantly lower in the Ang II+ RVS-M and Ang II+ RVSH groups;p-Smad3/Smad3 expression was not significantly different in the Ang II+RVS-L and Ang II+ RVS-M groups compared to the Ang II group but was significantly lower in the Ang II+ RVS-H group.Conclusions1.Serum PⅠCP,TGFβ1,and Gal-3 expression levels were increased in heart failure patients,and Gal-3 expression levels were positively correlated with PⅠCP and TGFβ1,suggesting that Gal-3 may mediate pathological myocardial fibrosis in heart failure patients by interacting with TGFβ1.2.Rosuvastatin exerts a protective effect against pathological myocardial fibrosis by down-regulating the expression of Gal-3 and TGFβ1 and inhibiting the Smaddependent pathway.Innovation and Research Significance1.In this study,the role of Gal-3 and TGFβ1 in pathological myocardial fibrosis was clarified through the collection of clinical blood samples and in vitro cellular assays,and the relationship between Gal-3 and TGFβ1 was discussed in the context of previous studies,providing a theoretical basis for the study of the mechanism of pathological myocardial fibrosis in patients with heart failure.2.By constructing a myocardial fibrosis cell model,the protective effect of rosuvastatin on pathological myocardial fibrosis and the possible molecular mechanism were investigated,which provided a new idea for the clinical treatment of heart failure patients and a theoretical basis for further investigation of the molecular mechanism of the anti-fibrotic effect of rosuvastatin. |
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