参竹心康汤通过miRNA-21调控EndMT影响心肌纤维化的实验研究

Experimental Study on the Effect of Shenzhu Xinkang Decoction(参竹心康汤)on Myocardial Fibrosis by Regulating EndMT through miRNA-21

目的:探讨参竹心康汤对心肌纤维化的作用及其机制,为参竹心康汤临床应用提供实验依据。方法:采用血清药理学方法,将20只SD大鼠随机分为中药组、空白组,每组10只。中药组大鼠灌胃给予参竹心康汤;空白组大鼠灌胃给予等量生理盐水,1次/d,连续7 d。腹主动脉采血制备大鼠含药血清和空白血清。将HUVEC细胞随机分为对照组、模型组、阳性对照组、参竹心康汤组。对照组不做特殊处理,其他组细胞以10 ng/mL TGF-β1诱导HUVEC细胞制作纤维化细胞模型,分别加入相应药物或血清处理,收集上述各组细胞。采用细胞免疫荧光法检测CD31、α-SMA、FSP1蛋白表达;采用ELISA法检测collagenⅠ含量;采用RT-qPCR法检测miRNA-21 mRNA、PTEN mRNA、PI3K mRNA、Akt mRNA表达;采用Western blotting法检测TIMP-1、MMP-9、PTEN、PI3K、Akt、pAkt蛋白表达。结果:模型组HUVEC细胞CD31荧光强度,PTEN mRNA相对表达量,TIMP-1、PTEN蛋白相对表达量均明显低于对照组(P<0.05);模型组α-SMA、FSP1荧光强度,细胞培养液上清collagenⅠ含量,miRNA-21 mRNA、PI3K mRNA、Akt mRNA相对表达量,MMP-9、PI3K蛋白相对表达量及pAkt/Ak1值均明显高于对照组(P<0.05)。阳性对照组和参竹心康汤组HUVEC细胞CD31荧光强度,PTEN mRNA相对表达量,TIMP-1、PTEN蛋白相对表达量均明显高于模型组(P<0.05),α-SMA、FSP1荧光强度,细胞培养液上清collagenⅠ含量,miRNA-21 mRNA、PI3K mRNA、Akt mRNA相对表达量,MMP-9、PI3K蛋白相对表达量及pAkt/Akt值均低于模型组(P<0.05)。参竹心康汤组HUVEC细胞CD31荧光强度,PTEN mRNA相对表达量,TIMP-1、PTEN蛋白相对表达量均明显高于阳性对照组(P<0.05);参竹心康汤组α-SMA、FSP1荧光强度,细胞培养液上清collagenⅠ含量,PI3K mRNA、Akt mRNA相对表达量,TIMP-9、PI3K、蛋白相对表达量及pAkt/Akt值均明显低于阳性对照组(P<0.05)。参竹心康汤组HUVEC细胞miRNA-21相对表达量与阳性对照组比较,差异无统计学意义(P>0.05)。结论:参竹心康汤可以抑制EndMT,从而起到抗心肌纤维化作用,其可能机制与该方下调miRNA-21水平,抑制PTEN/PI3K/AKT通路有关。

Objective:To explore the effect and mechanism of Shenzhu Xinkang Decoction against myocardial fibrosis and provide experimental basis for the clinical use of Shenzhu Xinkang Decoction.Methods:20 SD rats were randomly divided into traditional Chinese medicine group and blank group by serum pharmacology method,with 10 rats in each group.Rats in the traditional Chinese medicine group were given Shenzhu Xinkang Decoction by gavage.Rats in the blank group were given the same amount of normal saline by gavage,once a day for 7 consecutive days.Blood was collected from abdominal aorta to prepare drug containing serum and blank serum of rats.HUVEC cells were randomly divided into control group,model group,positive control group and Shenzhu Xinkang Decoction group.The control group did not receive special treatment,and other groups of cells induced HUVEC cells with 10 ng/mL TGF-β1 to make fibrosis cell models.Respectively add corresponding drugs or serum for treatment,and collect cells of the above groups.The expression of CD31,α-SMA and FSP1 were detected by immunofluorescence assay.Type I collagen was detected by ELISA.The expression of miRNA-21,PTEN,PI3K and Akt mRNA were detected by RT-qPCR.The protein expression of TIMP-1,MMP-9,PTEN,PI3K,Akt,pAkt were detected by Western blotting.Results:The fluorescence intensity of CD31,the relative mRNA expression of PTEN,the relative protein expression of TIMP-1 and PTEN in HUVEC cells in the model group were significantly lower than the control group(P<0.05).The fluorescence intensity ofα-SMA,FSP1,the content of type I collagen in the cell culture supernatant,the relative mRNA expression of miRNA-21,PI3K and Akt,the relative protein expression of MMP-9,PI3K,pAkt/Akt in the model group were significantly higher than the control group(P<0.05).The fluorescence intensity of CD31,the relative mRNA expression of PTEN,the relative protein expression of TIMP-1 and PTEN in both positive control group and Shenzhu Xinkang Decoction group were significantly higher than the model group(P<0.05).The fluorescence intensity ofα-SMA,FSP1,the content of type I collagenin the cell culture supernatant,the relative mRNA expression of miRNA-21,PI3K and Akt,the relative protein expression of MMP-9,PI3K,pAkt/Akt in both positive control group and Shenzhu Xinkang Decoction group were lower than the model group(P<0.05).The fluorescence intensity of CD31,the mRNA expression of PTEN,the protein expression of TIMP-1 and PTEN in the Shenzhu Xinkang Decoction group were significantly higher than the positive control group(P<0.05).The fluorescence intensity ofα-SMA,FSP1,the content of type I collagenin the cell culture supernatant,the relative mRNA expression of miRNA-21,PI3K and Akt,the relative protein expression of MMP-9,PI3K,pAkt/Akt in the Shenzhu Xinkang Decoction group were significantly lower than the positive control group(P<0.05).The relative expression of miRNA-21 in HUVEC cells in Shenzhu Xinkang Decoction group was not significantly different from that in the positive control group(P>0.05).Conclusions:Shenzhu Xinkang Decoction can inhibit endmt,thus playing an anti myocardial fibrosis role.The possible mechanism is related to the down-regulation of miRNA-21 level and inhibition of PTEN/PI3K/AKT pathway.