Regulation of PGC1α on SiO2-induced lipid accumulation in macrophages and fibrosis in pulmonary fibroblasts
（1）通过在巨噬细胞中转染沉默PGC1α及其对照序列，并给予SiO2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）、SiO2刺激组（转染si-NC培养48 h后，50 μg·mL−1 SiO2刺激36 h）和敲低PGC1α+SiO2组（转染si-PGC1α培养48 h后，50 μg·mL−1 SiO2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（O2K）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（COL Ι）、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。
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SiO2刺激组相比，敲低PGC1α+SiO2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+SiO2组中TC、FC和CE的水平是SiO2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+SiO2组中复合体Ι的水平是SiO2刺激组的0.63倍（ P<0.05）；巨噬细胞分泌的TGF-β1增多，敲低PGC1α+SiO2组中TGF-β1的水平是SiO2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COL Ι、FN表达水平增加，Eca表达降低，敲低PGC1α+SiO2组中COL Ι、FN和Eca的蛋白水平分别是SiO2刺激组的1.39倍、1.18倍和0.82倍（ P<0.05）。 结论
The pathogenesis of silicosis is complex and treatment methods are limited. SiO2-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 receptor γ coactivator 1α (PGC1α) mediates mitochondrial dysfunction and lipid metabolism pathways in diabetic models, but its role in silicosis has not been elucidated.
To investigate the effect of PGC1α on lipid metabolism disorder of macrophages induced by SiO2 and its effect on the progression of silicosis fibrosis.
(1) Macrophages were divided into four groups by transfecting and silencing PGC1α and its control sequence in macrophages and followed by SiO2 stimulation: negative control group (transfected with si-NC for 48 h), si-PGC1α group (transfected with si-PGC1α for 48 h), SiO2 stimulation group (stimulated with 50 μg·mL−1 SiO2 for 36 h after transfection with si-NC for 48 h), and si-PGC1α+SiO2 group (stimulated with 50 μg·mL−1 SiO2 for 36 h after transfection with si-PGC1α for 48 h). Western blot and cell immunofluorescence were used to test PGC1α expression, 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 (O2K) was used to assess the effects of PGC1α on 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 collagen Ι (COL Ι), E-cadherin (Eca), and fibronectin (FN) were detected by cell immunofluorescence and Western blot to further evaluate the effect of silencing PGC1α on fibrosis.
The protein expression level of PGC1α stimulated by SiO2 was decreased, and the relative expression level of PGC1α was 0.78 times that of the control group (
P<0.05). After transfection with si-PGC1α, the expression of PGC1α was decreased, and the relative protein expression level of the si-PGC1α group was 0.86 times that of the control group ( P<0.05). Compared with the SiO2 stimulation group, the staining area of BODIPY 493/503 in the si-PGC1α+SiO2 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 SiO2 stimulation group ( P<0.05). The activity of mitochondrial complex Ι was decreased, and the level of complex Ι in the si-PGC1α+SiO2 group was 0.63 times that in the SiO2 stimulation group ( P<0.05). The secretion of TGF-β1 by macrophages increased, and the level of TGF-β1 in the si-PGC1α+SiO2 group was 1.15 times that of the SiO2 stimulation group ( P<0.05). In addition, after stimulation of primary lung fibroblasts with macrophage supernatant, silencing PGC1α increased the expression levels of COL Ι and FN, while decreased the expression of Eca. The protein levels of COL Ι, FN, and Eca in the si-PGC1α+SiO2 group were 1.39, 1.18, and 0.82 times those in the SiO2 stimulation group, respectively ( P<0.05). Conclusion
Silencing PGC1α exacerbates SiO2-induced lipid metabolism disorder, inhibits mitochondrial respiratory chain, and aggravates the fibrosis induced by SiO2, suggesting that PGC1α may participate silicosis fibrosis by regulating mitochondrial respiratory chain and lipid metabolic disorder induced by SiO2.