PGC1α对SiO_(2)诱导巨噬细胞脂质蓄积及肺成纤维细胞纤维化的调控作用

Regulation of PGC1αon SiO_(2)-induced lipid accumulation in macrophages and fibrosis in pulmonary fibroblasts

[背景]矽肺的发病机制复杂,且治疗方法有限。二氧化硅(SiO_(2))诱导的转化生长因子-β1(TGF-β1)的增加能够激活成纤维细胞以促进胶原蛋白沉积,最终导致纤维化。已有研究证实脂质代谢在矽肺的进展中发挥着重要作用。过氧化物酶体增殖物激活受体γ共激活因子1α(PGC1α)在糖尿病模型中可介导线粒体功能障碍和脂质代谢等通路,但在矽肺中的作用尚未阐明。[目的]探讨PGC1α在SiO_(2)诱导的巨噬细胞脂质代谢紊乱中的作用及对矽肺纤维化进展的影响。[方法](1)通过在巨噬细胞中转染沉默PGC1α及其对照序列,并给予SiO_(2)刺激,将巨噬细胞分为四组:阴性对照组(转染si-NC培养48 h)、敲低PGC1α组(转染si-PGC1α培养48 h)、SiO_(2)刺激组(转染si-NC培养48 h后,50μg·mL^(–1)SiO_(2)刺激36 h)和敲低PGC1α+SiO_(2)组(转染si-PGC1α培养48 h后,50μg·mL^(–1)SiO_(2)刺激36 h)。Western blot和细胞免疫荧光检测PGC1α的表达,4,4-二氟-1,3,5,7,8-五甲基-4-硼杂-3a,4a-二氮杂-s-引达省(BODIPY 493/503)和总胆固醇(TC)、游离胆固醇(FC)试剂盒检测脂质蓄积情况,Oroboros2k-Oxygraph呼吸检测系统(O_(2)K)评估PGC1α对线粒体呼吸链的影响。ELISA试剂盒检测巨噬细胞上清液中TGF-β1的表达。(2)将肺成纤维细胞分为上述相同的四组,将上述各组巨噬细胞的上清液刺激肺成纤维细胞,细胞免疫荧光和Western blot检测Ι型胶原(COLΙ)、E-钙粘蛋白(Eca)和纤连蛋白(FN)的表达以进一步评估沉默PGC1α对纤维化的影响。[结果]SiO_(2)刺激导致PGC1α蛋白表达水平降低,蛋白的相对表达水平是对照组的0.78倍(P<0.05)。转染si-PGC1α序列后,PGC1α表达降低,敲低PGC1α组的蛋白相对表达水平是对照组的0.86倍(P<0.05)。与SiO_(2)刺激组相比,敲低PGC1α+SiO_(2)组中BODIPY 493/503着色面积增强,胆固醇相关指标TC、FC和胆固醇酯(CE)增多,敲低PGC1α+SiO_(2)组中TC、FC和CE的水平是SiO_(2)刺激组的1.38倍、1.10倍和2.26倍(P<0.05);线粒体复合体Ι活性降低,敲低PGC1α+SiO_(2)组中复合体Ι的水平是SiO_(2)刺激组的0.63倍(P<0.05);巨噬细胞分泌的TGF-β1增多,敲低PGC1α+SiO_(2)组中TGF-β1的水平是SiO_(2)刺激组的1.15倍(P<0.05)。此外,将各组巨噬细胞的上清液刺激原代肺成纤维细胞后,沉默PGC1α导致COLΙ、FN表达水平增加,Eca表达降低,敲低PGC1α+SiO_(2)组中COLΙ、FN和Eca的蛋白水平分别是SiO_(2)刺激组的1.39倍、1.18倍和0.82倍(P<0.05)。[结论]沉默PGC1α加剧了SiO_(2)诱导的脂质代谢紊乱,抑制了线粒体呼吸链,并加剧了SiO_(2)诱导的纤维化。提示PGC1α可能通过调控线粒体呼吸链调节SiO_(2)诱导的脂质代谢紊乱参与矽肺纤维化的进展。

[Background]The pathogenesis of silicosis is complex and treatment methods are limited.SiO_(2)-induced increase of transforming growth factor-β1(TGF-β1)can activate fibroblasts to promote collagen deposition,ultimately leading to fibrosis.Previous studies have confirmed that lipid metabolism plays an important role in the progression of silicosis.Peroxisome proliferator-activated receptorγcoactivator 1α(PGC1α)mediates mitochondrial dysfunction and lipid metabolism pathways in diabetic models,but its role in silicosis has not been elucidated.[Objective]To investigate the effect of PGC1αon lipid metabolism disorder of macrophages induced by SiO_(2) and its effect on the progression of silicosis fibrosis.[Methods](1)Macrophages were divided into four groups by transfecting and silencing PGC1αand its control sequence in macrophages and followed by SiO_(2) stimulation:negative control group(transfected with si-NC for 48 h),si-PGC1αgroup(transfected with si-PGC1αfor 48 h),SiO_(2) stimulation group(stimulated with 50μg·mL^(−1) SiO_(2) for 36 h after transfection with si-NC for 48 h),and si-PGC1α+SiO_(2) group(stimulated with 50μg·mL^(−1) SiO_(2) for 36 h after transfection with si-PGC1αfor 48 h).Western blot and cell immunofluorescence were used to test PGC1αexpression,4,4-difluoro-1,3,5,7,8-pentamethyl-4-bora-3a,4a-diaza-s-indacene(BODIPY 493/503)and total cholesterol(TC)and free cholesterol(FC)kits were used to test lipid accumulation,and the Oroboros2k-Oxygraph respiratory test system(O_(2)K)was used to assess the effects of PGC1αon mitochondrial respiratory chain.ELISA kits were used to test TGF-β1 expressed in the macrophage supernatant.(2)Lung fibroblasts were divided into the same four groups as above,and stimulated with the supernatant of macrophages in the above groups.The expression of collagenΙ(COLΙ),E-cadherin(Eca),and fibronectin(FN)were detected by cell immunofluorescence and Western blot to further evaluate the effect of silencing PGC1αon fibrosis.[Results]The protein expression level of PGC1αstimulated by SiO_(2) was decreased,and the relative expression level of PGC1αwas 0.78 times that of the control group(P<0.05).After transfection with si-PGC1α,the expression of PGC1αwas decreased,and the relative protein expression level of the si-PGC1αgroup was 0.86 times that of the control group(P<0.05).Compared with the SiO_(2) stimulation group,the staining area of BODIPY 493/503 in the si-PGC1α+SiO_(2) group was enhanced,and the cholesterol-related indexes[TC,FC and cholesterol ester(CE)]were increased to 1.38,1.10,and 2.26 times those in the SiO_(2) stimulation group(P<0.05).The activity of mitochondrial complexΙwas decreased,and the level of complexΙin the si-PGC1α+SiO_(2) group was 0.63 times that in the SiO_(2) stimulation group(P<0.05).The secretion of TGF-β1 by macrophages increased,and the level of TGF-β1 in the si-PGC1α+SiO_(2) group was 1.15 times that of the SiO_(2) stimulation group(P<0.05).In addition,after stimulation of primary lung fibroblasts with macrophage supernatant,silencing PGC1αincreased the expression levels of COLΙand FN,while decreased the expression of Eca.The protein levels of COLΙ,FN,and Eca in the si-PGC1α+SiO_(2) group were 1.39,1.18,and 0.82 times those in the SiO_(2) stimulation group,respectively(P<0.05).[Conclusion]Silencing PGC1αexacerbates SiO_(2)-induced lipid metabolism disorder,inhibits mitochondrial respiratory chain,and aggravates the fibrosis induced by SiO_(2),suggesting that PGC1αmay participate silicosis fibrosis by regulating mitochondrial respiratory chain and lipid metabolic disorder induced by SiO_(2).