缺氧诱导线粒体DNA释放后激活cGAS-STING通路促进肺动脉平滑肌细胞增殖

Hypoxia-induced mitochondrial DNA release promotes proliferation of pulmonary arterial smooth muscle cells via activating cGAS-STING pathway

目的探究线粒体DNA(mitochondrial DNA,mtDNA)释放在缺氧诱导大鼠肺动脉平滑肌细胞(rat pulmonary arterial smooth muscle cells,RPASMCs)增殖中的作用及可能机制。方法①1%O_(2)缺氧处理RPASMCs分3组(n=3):常氧组、缺氧24 h组、缺氧48 h组。②环孢素A(CsA)抑制线粒体通透性转换孔(mitochondrial permeability transition pore,MPTP)分为3组(n=3):常氧组、缺氧48 h组、缺氧48 h+CsA处理组。③siRNA敲低干扰素基因刺激因子(stimulator of interferon genes,STING)表达分为3组(n=3):常氧+NC组、缺氧48 h+NC组、缺氧48 h+si-STING组。④采用CCK-8检测细胞增殖,RT-qPCR、Western blot检测环状GMP-AMP合酶(cyclic GMP-AMP synthase,cGAS)、STING、增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)的基因与蛋白表达,qPCR检测细胞质mtDNA释放,ELISA检测细胞培养上清中CXC趋化因子配体10(C-X-C motif chemokine ligand 10,CXCL10)水平。结果缺氧可诱导RPASMCs释放mtDNA(P<0.01),显著上调cGAS、STING、PCNA的表达(P<0.05),并增强CXCL10的分泌(P<0.01),进而促进RPASMCs增殖(0.44±0.02 vs 0.72±0.03)(P<0.05)。抑制MPTP可阻断mtDNA释放(P<0.01),显著下调cGAS、STING、PCNA的表达(P<0.01),降低CXCL10的分泌水平(P<0.01),并显著抑制缺氧诱导的RPASMCs增殖(0.74±0.12 vs 0.43±0.06)(P<0.01)。敲低STING可显著下调PCNA的表达(P<0.05),降低CXCL10的分泌水平(P<0.01),并显著抑制缺氧诱导的RPASMCs增殖(0.68±0.03 vs 0.58±0.01)(P<0.01)。结论缺氧可诱导平滑肌细胞释放mtDNA,介导缺氧诱导的平滑肌细胞增殖,其机制可能与cGAS-STING通路的激活有关。

Objective To investigate the role and possible mechanism of mitochondrial DNA(mtDNA)release in the proliferation of rat pulmonary arterial smooth muscle cells(RPASMCs)induced by hypoxia.Methods①RPASMCs were divided into normoxia group,hypoxia 24-hour group and hypoxia 48-hour group(n=3 for each groups),and the latter 2 groups were exposed to 1% O_(2) for 24 or 48 h respectively.②Cyclosporine A(CsA)was used to inhibit the mitochondrial permeability transition pore(MPTP),and then the RPASMCs were divided into normoxia,simple hypoxia 48-hour and hypoxia 48-hour+CsA groups(n=3 for each).③SiRNA was adopted to knockdown the expression of stimulator of interferon genes(STING),and the cells were subsequently divided into normoxia+NC,hypoxia 48-hour+NC and hypoxia 48-hour+si-STING(n=3 for each).④The proliferation of the cells was measured by CCK-8 assay.The expression of cyclic GMP-AMP synthase(cGAS),STING and proliferating cell nuclear antigen(PCNA)at mRNA and protein levels was detected by RT-qPCR and Western blotting,respectively.Cytosolic mtDNA release was determined by qPCR,and the level of C-X-C motif chemokine ligand 10(CXCL10)in the culture supernatant was tested by ELISA.Results Hypoxia induced RPASMCs to release mtDNA(P<0.01),significantly up-regulated the expression of cGAS,STING and PCNA(P<0.05),enhanced the secretion of CXCL10(P<0.01),and thus promoted the proliferation of RPASMCs(0.44±0.02 vs 0.72±0.03,P<0.05).However,inhibition of MPTP blocked mtDNA release(P<0.01),down-regulated cGAS,STING and PCNA(P<0.01),and decreased CXCL10 level(P<0.01)and hypoxia-induced proliferation of RPASMCs(0.74±0.12 vs 0.43±0.06,P<0.01).The knockdown of STING also suppressed PCNA expression(P<0.05),reduced CXCL10 secretion(P<0.01),and inhibited the proliferation of RPASMCs(0.68±0.03 vs 0.58±0.01,P<0.01).Conclusion Hypoxia can induce smooth muscle cells to release mtDNA and promote the proliferation of RPASMCs,which may be related to the activation of the cGAS-STING pathway.