Effects Of Astaxanthin On Cardiac Fibrosis And Its Mechanism

Myocardial fibrosis(MF)is defined as the increase of collagen in myocardial tissue or changes in collagen composition.It refers to the proliferation and transformation of cardiac fibroblasts in myocardial tissue and the increase of collagen content.The degree of myocardial fibrosis is closely related to the prognosis of cardiovascular disease.However,there is still no effective method to treat myocardial fibrosis.Astaxanthin is a carotenoid with strong ability to eliminate oxygen free radicals.It has the effects of anti-inflammatory,anti-oxidaive stress,anti-cancer,anti-ag-ing and anti-organ fibrosis.However,there are few studies on the effects of astaxanthin on myocardial fibrosis.In the study,myocardial fibrosis model was constructed by isoproterenol(ISO),and the effects of different doses of astaxanthin on myocardial tissue fibrosis in rats and its mechanism were studied;moreover,cardiac fibroblasts(CFs)were induced by Ang Ⅱ and the effects of different concentrations of astaxanthin on CFs proliferation,transformation and collagen synthesis were observed.Objective:In this study,the cardiac fibrosis model induced by ISO was used to explore the effects of astaxanthin on cardiac fibrosis and its underlying mechanism through evaluation of cardiac function,the pathological changes of myocardial tissue,the antioxidative ability,the levels of inflammatory factors;moreover,CFs induced by Ang II were used to discuss the effects of astaxanthin on CFs proliferation,transformation and collagen synthesis through detection of the cell proliferation by CCK8,the level of α-SMA by immunofluorescence assay,the level of collagen protein by WB and the level of oxidative stress by DHE.Methods:Animal experiment section:The male SD rats were randomly divided into 4 groups(5 in each group),namely normal control group(Con),ISO group(ISO),low-dose astaxanthin group(ISO+LD)and high-dose astaxanthin group(ISO+HD).Rats in ISO group were continuously treated with ISO(5 mg/kg)for 14 d by intraperitoneal injection to establish the model of myocardial fibrosis,while rats in astaxanthin group were treated with astaxanthin(5 mg/kg or 10 mg/kg)on the second day of modeling for 21 d by gavage.In the 22 day of experiment,rats were sacrificed after fasting 12 h,and hearts were taken out.The hearts were washed with cold saline and fat,vascular,and atrial tissue were cut off.The tip of the heart was placed in 4%paraformaldehyde for at least 24 h for Masson staining,and the rest of the myocardial tissue was put in the-80 ℃ refrigerator for molecular biological examination.Cell experiment section:CFs were isolated by the method of enzymatic digestion,and cultured in cell incubator and passed on.The CFs of 3-5 generation were divided into 5 groups,namely normal control group,Ang Ⅱ group,low-dose astaxanthin group[Ang Ⅱ+AST(20 uM)],middle-dose astaxanthin group[Ang Ⅱ+AST(40 uM)],high-dose astaxanthin group[AngⅡ+AST(80 uM)].CFs in the model group were given Ang Ⅱ(10-7 mol/L)to induce cardiac fibrosis,while CFs in intervention group were given astaxanthin(20 uM,40 uM,80 uM,respectively).The changes of each index were assessed after 24 h.Results:Animal experiment section:1.The effect of astaxanthin on myocardial fibrosis in myocardial tissue assessed by Masson’s trichrome stainingThe myocardial tissue of ISO group showed obvious fibrosis.Compared with the ISO group,rats in both the low-dose astaxanthin group and the high-dose astaxanthin group showed decreased fibrosis.2.The effect of astaxanthin on collagen synthesisCompared with the control group,the expression of collagen Ⅲ and connective tissue factor(CTGF)in ISO group increased,on the other hand,low-dose astaxanthin group and high-dose astaxanthin group showed lower levels of collagen Ⅱ and CTGF.3.The effect of astaxanthin on inflammatory factorsRats in ISO group showed increased interleukin-1(IL-1),interleukin-6(IL-6)and monocyte chemotactic protein-1(MCP-1)mRNA in the myocardial tissue compared to control group,whereas the increased of IL-1、IL-6 and MCP-1 were markedly decreased after addition of low-dose and high-dose astaxanthin.4.The effect of astaxanthin on oxidative stress in myocardial tissueCompared with control group,the expression of SOD-1 and SOD-2 protein in myocardial tissues in ISO group decreased,while the decreased of SOD-1 and SOD-2 protein can be improved by low-dose and high-dose astaxanthin.5.The effect of astaxanthin on TGF-β31、p-Smad3Compared with control group,the expression of TGF-β1、p-Smad3 were increased in ISO group,whereas the increased of TGF-β1 and p-Smad3 can be eliminated by low-dose and high-dose astaxanthin.Cell experiment section:1.The effect of astaxanthin on CFs proliferation induced by Ang IIAng Ⅱ can induce CFs proliferation,and the speed of CFs proliferation can be repressed by different dose of astaxanthin.2.The effect of astaxanthin on CFs transformation induced by Ang IICompared with control group,the expression of alpha smooth muscle actin(a-SMA)protein and mRNA were increased,while compared with Ang II group,the expression of a-SMA protein and mRNA were decreased in astaxanthin intervention group.3.The effect of astaxanthin on CFs collagen synthesis induced by Ang IICompared with the control group,the expression of collagen I protein and mRNA were upgraded,while compared with Ang II group,the collagen I protein and mRNA were decreased in astaxanthin intervention group.4.The effect of astaxanthin on CFs oxidative stress induced by Ang IICompared with the control group,Ang II group has the increased oxidative stress,and the upgraded oxidative stress can be suppressed by different dose of astaxanthin.Conclusion:Astaxanthin can inhibit myocardial fibrosis of rats induced by ISO and improve cardiac function,which may be related to inhibition of TGF-β1/Smad3 signaling and suppression of oxidative stress.Moreover,astaxanthin can inhibit Ang Ⅱ-induced CFs proliferation,transformation and collagen synthesis and its mechanism may be associated with the effect of anti-oxidative stress.