N^(6)-甲基腺嘌呤甲基转移酶3介导Bax/Bcl-2抑制慢性肾脏病血管钙化的机制

Study on mechanism of inhibition effect of N6-methyladenosine methyltransferase-like 3 on vascular calcification in chronic kidney disease through Bax/Bcl-2

目的探讨N^(6)-甲基腺嘌呤(N6-methyladenosine,m^(6)A)甲基转移酶3(methyltransferaselike 3,METTL3)调控凋亡相关蛋白在慢性肾脏病(chronic kidney disease,CKD)血管钙化中的作用机制。方法(1)临床试验:实时荧光定量PCR法检测维持性血液透析(maintenance hemodialysis,MHD)患者血清METTL3 mRNA水平。(2)细胞实验:Western印迹法检测高磷刺激的血管平滑肌细胞(vascular smooth muscle cells,VSMCs)中METTL3蛋白表达,免疫荧光双染法观察METTL3与Runt相关转录因子2(Runt-related transcription factor 2,Runx2)的分布。构建METTL3过表达和敲低质粒,分别转染VSMCs,茜素红染色检测钙化情况,Western印迹法检测成骨标志物Runx2、骨形态发生蛋白2(bone morphogenetic protein-2,BMP-2)、Ⅰ型胶原(collagenⅠ)和凋亡相关蛋白Bax、Bcl-2表达。(3)动物实验:30只SD大鼠被随机分为对照组、CKD血管钙化组、S-腺苷高半胱氨酸(S-adenosylhomocysteine,SAH)干预组,硝酸银染色评估胸主动脉钙化情况,免疫组化及Western印迹法检测Runx2、Bax、Bcl-2蛋白表达。结果(1)MHD血管钙化患者血清中METTL3 mRNA水平明显低于无钙化患者(P<0.05),且与冠状动脉钙化积分呈负相关(r=-0.65,P<0.001)。(2)高磷刺激的VSMCs中METTL3蛋白表达降低,并呈现时间依赖性。免疫荧光双染发现METTL3与Runx2共表达于细胞核。在高磷处理的VSMCs中过表达METTL3后Runx2、BMP-2、Collagen I表达明显降低,并伴有钙化结节减少,且凋亡蛋白Bax/Bcl-2比值降低(均P<0.05)。反之,敲低METTL3通过诱导凋亡加重VSMCs钙化。(3)进一步在体内模型中给予METTL3抑制剂SAH,发现抑制METTL3表达可显著增加大鼠胸主动脉钙化,且Bax/Bcl-2比值、Runx2表达上调。结论MHD血管钙化患者血清METTL3水平降低,体内外实验证实METTL3可通过介导凋亡相关蛋白Bax/Bcl-2抑制CKD血管钙化。

Objective To investigate the role and mechanism of N6-methyladenosine(m6 A)methyltransferase-like 3(METTL3)in vascular calcification(VC)of chronic kidney disease(CKD)through apoptosis-associated protein.Methods(1)Real-time fluorescence quantitative PCR was used to test METTL3 mRNA in serum of maintenance hemodialysis(MHD)patients.(2)Western blotting was used to detect the expression of METTL3 protein in high-phosphorus stimulated vascular smooth muscle cells(VSMCs),and immunofluorescence double lable was used to observe the distribution of METTL3 and Runt-related transcription factor 2(Runx2).The METTL3 overexpressed and knockdown plasmids were constructed and transfected into VSMCs.Alizarin red staining was used to detect calcification degree.Western blotting was used to detect the expressions of osteogenic markers[Runx2,bone morphogenetic protein-2(BMP-2),collagenⅠ]and apoptosisrelated proteins Bax and Bcl-2.(3)SD rats were randomly divided into control group,CKD-VC group and S-adenosylhomocysteine(SAH)intervention group.The calcification of thoracic aorta was evaluated by von Kossa staining,and the protein expressions of Runx2,Bax and Bcl-2 were detected by immunohistochemistry and Western blotting.Results(1)METTL3 mRNA expression in MHD patients with VC was significantly lower than that in non-VC patients(P<0.05),and was negatively correlated with coronary artery calcium score(r=-0.65,P<0.001).(2)The expression of METTL3 in VSMCs stimulated by high phosphorus was decreased and showed a time dependence.Immunofluorescence double label showed that METTL3 and Runx2 were co-expressed in the nucleus.METTL3 was overexpressed in high-phosphorus induced VSMCs,and the expressions of Runx2,collagen I and BMP-2 were significantly decreased,accompanied by the decrease of calcified nodules and Bax/Bcl-2 ratio(all P<0.05).Conversely,METTL3 knockdown aggravated VSMCs calcification by inducing apoptosis.(3)Furthermore,METTL3 inhibitor SAH was administered in vivo,and it was found that inhibition of METTL3 expression significantly increased the calcification of rat thoracic aorta,and the Bax/Bcl-2 ratio and Runx2 expression were up-regulated.Conclusions Serum METTL3 level is reduced in MHD patients with VC.In vivo and in vitro studies demonstrate that METTL3 inhibits VC in CKD by mediating the apoptosis-related protein Bax/Bcl-2.