Tetrandrine Modulates Rheb/mTOR Mediated Selective Autophagy And Protects Pulmonary Fibrosis

Background and PurposePulmonary fibrosis is the most common type of interstitial lung disease.It has a high mortality rate and currently lacks effective treatments.Traditional Chinese Medicine is an important part of my country’s medicine.At the same time,drug research on the effective monomer components of traditional Chinese medicine is also one of the current hot spots in the development of the new use of old drugs.Tetrandrine is an alkaloid extracted from the roots of tetrandrine.It has anti-inflammatory,immune regulation,and anti-fibrosis activities.However,the therapeutic effect of tetrandrine in pulmonary fibrosis and the mechanism needs to be further explored.Experimental Approach1.To construct a bleomycin-induced pulmonary fibrosis model in mice,starting from the first day(prevention group)or the eighth day(treatment group)after the model was established,intraperitoneal injection of tetrandrine A was carried out on the 21st day.The mice were sacrificed.Use HE staining to observe the effect of tetrandrine on the structure of fibrotic lung tissue,Masson staining and hydroxyproline kit to detect the effect on the deposition and distribution of pulmonary interstitial collagen,and immunohistochemistry and Western Blot to detect protein a-The influence of expression of SMA,vimentin,collagen-Ⅰ,fibronectin.2.Use the lung fibroblasts induced by TGF-β1 to construct an in vitro fibrosis model.After treatment with tetrandrine,the expression of fibrosis-related proteinsα-SMA,vimentin,collagen-Ⅰ and fibronectin were detected by Western Blot and immunofluorescence.3.Using Western Blot,immunofluorescence,electron microscopy,mRFP-GFP-LC3 tandem fluorescent adenovirus,small interfering RNA,co-immunoprecipitation(Co-IP)and other methods proved that tetrandrine can promote selective autophagy.4.Use immunofluorescence colocalization and immunoprecipitation(Co-IP)methods to detect the localization and interaction of collagen-Ⅰ with SQSTM1 and MAP1LC3.Chromatin immunoprecipitation(ChIP)experiment detects the binding of transcription factor NRF2 to the promoter region of SQSTM1.5.Western Blot,immunofluorescence and other methods proved that tetrandrine inhibits the Rheb/mTOR signaling pathway,and the constructed Rheb plasmid was further transferred into fibroblasts to explore the role of Rheb in tetrandrine in regulating pulmonary fibrosis.6.Statistical processing:Data is expressed as mean ± standard deviation,and GraphPad Prism software is used for statistical analysis.The comparison between two independent sample groups was performed by t test;the one-way ANOVA was used for single-way comparison between multiple groups;the Two-way ANOVA was used for two-way comparison between multiple groups,and the Dunnett or Tukey method was further used for multiple comparisons of multiple sample means.Key Results1.The effect of tetrandrine on the pathomorphology of lung tissue in mice with pulmonary fibrosis:Compared with the pulmonary fibrosis model group,tetrandrine preventive treatment group and tetrandrine fibrosis treatment group can improve lung structural disorders,reduce Ashcroft score,reduce lung tissue collagen deposition and fibroblast activation Expression of the marker α-SMA.2.The effect of tetrandrine on the proliferation,activation and extracellular matrix deposition of lung fibroblasts:①Tetrandrine can reduce the protein expression of α-SMA,vimentin,collagen-Ⅰ,fibronectin and the expression of p-smad2 and p-smad3 in fibroblasts induced by TGF-β1.②Compared with the TGF-β1 group,the tetrandrine treatment group significantly inhibited the proliferation of myofibroblasts.3.The effect of tetrandrine on the selective autophagy of fibroblasts induced by TGF-β1:①Prediction from bioinformatics websites proved that tetrandrine can improve pulmonary fibrosis by regulating apoptosis and/or autophagy signaling pathways.②With the increase of tetrandrine concentration,the expression of apoptosis marker cleaved caspase3 and the number of apoptotic cells did not change significantly,suggesting that tetrandrine does not affect the apoptotic pathway of fibroblasts.③Tetrandrine can significantly increase the expression of autophagy-related proteins MAP1LC3Ⅱ,SQSTM1 and the number of autophagosomes in lung fibroblasts induced by TGF-β1.At the same time,the use of autophagy inhibitors 3MA or siATG7 to inhibit autophagy can significantly reduce the inhibitory effect of tetrandrine on the expression of α-SMA,vimentin,collagen-Ⅰ,and fibronectin,suggesting that tetrandrine can promote autophagy Improve pulmonary fibrosis.④Compared with TGF-β1 group,tetrandrine treatment group can increase the co-localization and interaction of SQSTM1 with MAP1LC3B and ubiquitinated protein.Further experimental results confirmed that tetrandrine can significantly increase the co-localization of collagen-Ⅰ with MAP1LC3B and the lysosomal marker LAMP2,and at the same time strengthen the interaction between collagen-Ⅰ and SQSTM1,suggesting that tetrandrine can promote SQSTM1-mediated selection Sexual autophagy increases the degradation of collagen-Ⅰ.⑤Predictions from NCBI and JASPAR websites suggest that there is a sequence recognized and bound by NRF2 in the promoter region of SQSTM1,and further use ChIP-PCR experiments to confirm that tetrandrine can increase the binding capacity of NRF2 in the promoter region of SQSTM1,suggesting tetrandrine Increase the protein expression of SQSTM1 by regulating NRF2.⑥Compared with the TGF-β1 group,the tetrandrine treatment group significantly inhibited the activity of Rheb,and then promoted autophagy by inhibiting the phosphorylation of mTOR.Using plasmid overexpression of Rheb can significantly reduce the inhibitory effect of tetrandrine on the expression of α-SMA,vimentin,collagen-Ⅰ,and fibronectin,suggesting that tetrandrine activates autophagy through the Rheb/mTOR signaling pathway.ConclusionThis subject proved through in vivo and in vitro experiments that tetrandrine can alleviate pulmonary fibrosis.Tetrandrine promotes SQSTM1-mediated selective autophagy by inhibiting the Rheb/mTOR signaling pathway,thereby inhibiting fibroblast activation and promoting collagen degradation;at the same time,tetrandrine can increase the expression of SQSTM1 through NRF2 to promote selection Sexual autophagy,to achieve the effect of anti-pulmonary fibrosis.This study provides a certain experimental basis for the application of tetrandrine to the clinical prevention and treatment of pulmonary fibrosis.