Involvement Of S1P/S1PR3 In Myocardial Fibrosis And Its Mechanism In Rats With Myocardial Infarction

Background: Myocardial infarction(MI)is a common clinical emergency.After myocardial infarction,cardiac fibroblasts(CFs)activate proliferation and transdifferentiate into myofibroblasts,secreting more collagen fibers to promote ventricular remodeling However,the continuous activation of cardiac fibroblasts will lead to fibrosis of the myocardial interstitium,severely impair myocardial systolic and diastolic function,reduce myocardial compliance,and ultimately cause serious consequences such as arrhythmia and heart failure.Sphingosine-1-phosphate(S1P)is an important biologically active lipid in mammals.Studies have shown that the S1 P receptor S1PR3 promotes fibrosis of the liver,lungs and heart,but S1PR3 and the heart The relationship of fibroblasts to myofibroblasts transdifferentiation is still unclear.The purpose of this paper is to explore whether S1P/S1PR3 is involved in the transdifferentiation of myofibroblasts to promote myocardial fibrosis,and to explore the mechanism.Methods: Animal experiments:(1)Ligation of the left anterior descending coronary artery to construct a SD rat model of myocardial infarction.The sham operation group was only threaded under LAD without ligation.(2)The SD rats were randomly divided into sham operation group(Sham),myocardial infarction group(MI)and administration group(CAY10444).The administration group was injected with S1PR3 antagonistic daily from 3 days before surgery to 4 weeks after surgery(CAY10444)1mg/kg,the Sham group and MI group were injected with the same amount of normal saline daily,and euthanized after 6 weeks of normal diet feeding.Myocardial tissue was taken for HE and Masson staining;Western blot was used to detect α-SMA,Collagen Ⅰ,and Collagen Ⅰ in each group.The expression levels of CollagenⅢ,PPARγ,and p-PPARγ were detected by immunofluorescence to detect the expression of PPARγ and α-SMA in myocardial tissue.Cell level:(1)Place rat cardiac fibroblasts(RCFs)in a hypoxia incubator(3% O2),and CCK-8will detect cells with different hypoxia time(0h,6h,12 h,24h,48h)Viability,WB detects the expression level of hypoxia-inducible factor HIF-1α at the corresponding time to determine the optimal hypoxia time;cells are treated with different concentrations of S1P(0,200,400,600,800 n M)and cultured under hypoxia to detect α-SMA level,determine the optimal S1 P concentration.(2)WB detects the expression levels of α-SMA,Collagen Ⅰ,Collagen Ⅲ,PPARγ,p-PPARγ,ERK1/2,and p-ERK1/2 of RCFs in the control group(control)and hypoxia(hypoxia)group.(3)The cells were pretreated with CAY10444(10μM)for 30 minutes and cultured for 24 hours after ischemia and hypoxia.The expression levels of α-SMA,Collagen Ⅰ,Collagen Ⅲ in each group were detected by WB.(4)The CAY10444 treatment group and the CAY10444 + PPARγ inhibitor(GW9662)group were set up.After 24 hours of hypoxia,WB was used to detect the protein expression of α-SMA,Collagen Ⅰ,Collagen Ⅲ in each group.(5)WB was used to detect the protein expression levels of PPARγ,p-PPARγ,ERK1/2,and p-ERK1/2 in the CAY10444 treatment group.(6)The cells were treated with ERK1/2 inhibitors U0126(10μM)and CAY10444 for 30 min and cultured under hypoxia for 24 h.WB was used to detect the levels of PPARγ,p-PPARγand nuclear PPARγ in each group.Results: Animal experiments:(1)Immediately after the operation,the ST segment of the MI group showed obvious ischemic changes;6 weeks after the operation,HE and Masson staining of myocardial tissue showed that compared with the Sham group,the MI group had myocardial cell damage and myocardial interstitial Fibrosis was obvious;WB showed that the expression of Collagen Ⅲ(P<0.05),Collagen Ⅰ(P<0.01)and α-SMA(P<0.05)increased in myocardial tissue.(2)HE and Masson staining showed that compared with Sham,the degree of myocardial injury and myocardial fibrosis in the MI group was significantly increased,while the degree of myocardial injury and myocardial fibrosis in the CAY10444 treatment group was significantly reduced;WB showed that the total PPARγ level between each group was not Significant difference(P>0.05).Compared with the Sham group,Collagen Ⅲ(P<0.05),Collagen Ⅰ(P<0.01),α-SMA(P<0.05)expression increased in MI group,and p-PPARγ(P<0.05)0.01).Compared with the MI group,the expression of Collagen Ⅲ,Collagen Ⅰ,and α-SMA in the CAY10444 treatment group decreased(P<0.05),and the level of p-PPARγincreased(P<0.05).Cell level:(1)The expression level of HIF-1α is the highest when the cells are hypoxic for 24 hours.At this time,the cell viability is about 75%.When the S1 P concentration is 600 n M,the α-SMA expression is the largest.Take 600 n M S1 P ischemia and hypoxia for 24 hours.Condition to construct a cell hypoxia model.(2)WB showed that the expression of Collagen Ⅲ(P<0.05),Collagen Ⅰ(P<0.01),and α-SMA(P<0.05)of RCFs in hypoxia group increased significantly,and the expression of p-PPARγ(P<0.05)The expression level was significantly reduced.In addition,the phosphorylation level of ERK1/2 was significantly increased(P<0.05).(3)CAY10444 significantly reduced the expression of Collagen Ⅲ(P<0.05),Collagen Ⅰ(P<0.01),and α-SMA(P<0.05).(4)The PPARγ inhibitor GW9662 significantly weakened the protective effect of CAY10444.(5)Compared with the hypoxia group,CAY10444 promoted the expression of p-PPARγ and reduced the expression of p-ERK1/2(P<0.05).(8)Compared with the hypoxia group,U0126 promoted the phosphorylation of PPARγ,CAY10444 had a similar effect,and both treatments increased the expression level of PPARγ in the nucleus(P<0.05).Conclusions:(1)S1P/S1PR3 promotes myocardial fibrosis in rats with myocardial infarction.(2)Under hypoxic conditions,S1 P induces the transdifferentiation of cardiac fibroblasts into myofibroblasts and the synthesis of collagen fibers through S1PR3,and participates in myocardial fibrosis.(3)S1P/S1PR3 participates in the transdifferentiation of myofibroblasts through the ERK1/2/PPARγ pathway.