Department of Respiratory Disease, Baoshan Branch, Shuguang Hospital, Shanghai University of Traditional Chinese Medicine, China; Department of Respiratory Disease, Baoshan District Hospital of Integrated Traditional Chinese and Western Medicine, Shanghai, China.
Department of Traditional Chinese and Western Medicine, Shanghai Pulmonary Hospital, Tongji University School of Medicine, Shanghai, China.
Life Sci. 2020 Sep 1;256:117893. doi: 10.1016/j.lfs.2020.117893. Epub 2020 Jun 2.
To investigate the effect and underlying mechanism of melittin and tripartite motif (TRIM) family in human embryonic lung fibroblast (HELF).
Lentiviral RNA interference vector and lentiviral overexpression vector were constructed and packaged by transfecting 293T cells; the proliferation of HELF was examined using Cell Counting Kit 8; Western blot and qRT-PCR were performed to examine protein and mRNA expression; the interaction with protein phosphatase magnesium-dependent 1A (PPM1A) was examined by Co-immunoprecipitation.
Compared with the control group, the mRNA expression of the TRIM6, TRIM8 and TRIM47 in the IPF group significantly increased. Melittin inhibited the mRNA expression and protein expression levels of TRIM47, the HELF proliferation, the hydroxyproline levels, and the phosphorylation of Smad2/3; the interference of TRIM47 inhibited the protein expression of Vimentin, α-SMA, CTGF, the phosphorylation of Smad2/3 and the synthesis of hydroxyproline; TRIM47 overexpression elevated the phosphorylation of Smad2/3, induced ubiquitination of PPM1A and decreased the expression level of PPM1A, while TRIM47 RNA interference reversed this result.
Melittin has anti-fibrotic effect in HELF by directly reducing the phosphorylation of Smad2/3 or indirectly reducing the phosphorylation of Smad2/3 by decreasing the expression levels of TRIM47 whose overexpression induces ubiquitination of PPM1A.
研究蜂毒素和三联基序（TRIM）家族对人胚肺成纤维细胞（HELF）的作用及其潜在机制。
构建慢病毒 RNA 干扰载体和慢病毒过表达载体，通过转染 293T 细胞进行包装；使用细胞计数试剂盒 8 检测 HELF 的增殖；通过 Western blot 和 qRT-PCR 检测蛋白和 mRNA 表达；通过免疫共沉淀检测与蛋白磷酸酶镁依赖性 1A（PPM1A）的相互作用。
与对照组相比，特发性肺纤维化（IPF）组中 TRIM6、TRIM8 和 TRIM47 的 mRNA 表达明显增加。蜂毒素抑制 TRIM47、HELF 增殖、羟脯氨酸水平、Smad2/3 磷酸化的 mRNA 表达和蛋白表达水平；TRIM47 干扰抑制波形蛋白、α-SMA、CTGF、Smad2/3 磷酸化和羟脯氨酸合成的蛋白表达；TRIM47 过表达升高 Smad2/3 的磷酸化，诱导 PPM1A 的泛素化并降低 PPM1A 的表达水平，而 TRIM47 RNA 干扰则逆转了这一结果。
蜂毒素通过直接降低 Smad2/3 的磷酸化或间接通过降低 TRIM47 的表达水平（TRIM47 的过表达诱导 PPM1A 的泛素化）来降低 Smad2/3 的磷酸化，从而对 HELF 具有抗纤维化作用。
