基于化学蛋白质组学技术揭示雷公藤甲素抑制肺周细胞衰老的作用机制OA

Objective To identify the protein targets of triptolide(TPL)in the treatment of idiopathic pulmonary fibrosis(IPF)using chemical proteomics technology,explore the molecular mechanism of its pharmacological effects,and thereby provide experimental references for the development of new drugs and the clinical application of TPL.Methods Mouse pulmonary microvascular pericytes(SNPM-M175)were tested for the effect of pericyte proliferation in pulmonary microvessels by CCK-8.The effect of TPL on apoptosis in the help of flow cytometry.Whether TPL can occupy the protein binding site of the triptolide(TPL-p)probe in pulmonary microvascular pericytes is tested using click chemical reaction and gel electrophoresis.Using tandem mass spectrometry tag(TMT)combined with liquid chromatography-tandem mass spectrometry(LC-MS/MS)technology,the key targets of TPL anti-IPF,and the target proteins of TPL in the treatment of pulmonary fibrosis,and the bioinformatics analysis to identify the target.The technique was used to analyze the differential protein expression profiles of lung fibrosis tissues between the TPL administration group and the model group mice by LC-MS/MS.The expressions of TGF-β1 and Smad3 proteins in lung tissues was determined by Western Blot.Results The key to the inhibition of lung fibroblast proliferation is the induction of apoptosis of pulmonary microvascular peripheral cells.The results showed that the apoptosis rate of the control,TPL 20 μmol/L,TPL 10 μmol/L,and TPL 5 μmol/L groups were 5.82%,8.39%,21.46%,and 58.37%,respectively.TPL 20 μmol/L drug administration could effectively inhibit pulmonary microvascular periimeter senescence.After 12 h,ROS were tested,and the results showed that intracellular ROS levels were significantly increased in the TPL 5 μmol/L,TPL 10 μmol/L and TPL 20 μmol/L groups compared with the control group(P<0.001).The cysteine residue binding site of the protein in lung microvascular pericytes occupied by the TPL-p probe could be competed out by triptolide.Compared with the control group,the expressions of TGF-β1 and Smad3 proteins in lung tissues of the TPL 20 μmol/L group,TPL 10 μmol/L group and TPL 5 μmol/L group were significantly increased(P<0.01).Compared with the TPL 5 μmol/L group,the expression of TGF-β1 protein in the TPL 20 μmol/L group was significantly decreased(P<0.05),and the expression of Smad3 protein in lung tissues was significantly down-regulated(P<0.05).Conclusion By targeting TGF-β1 and Smad3,it inhibited pulmonary microvascular pericyte senescence and may provide a new strategy against pulmonary fibrosis.