Study On The Mechanism Of Fritillaria Components Intervention In TGF-?1 Induced MRC-5 Oxidative Damage By Notch Signaling Pathway

Objective:Pulmonary fibrosis is a chronic diffuse interstitial lung disease,which is characterized by large deposits of extracellular matrix proteins in the basement membrane and interstitial tissue,as well as damage to the epithelial cells and myofibroblasts proliferation.The clinical manifestations of patients with pulmonary fibrosis are progressive dyspnea,restrictive ventilation dysfunction,and hypoxemia,etc.,which will eventually lead to dyspnea that is difficult to cure,and most patients die of respiratory failure.Notch signaling pathway is a highly conserved signaling pathway that regulates cell proliferation,differentiation,and apoptosis,and is also an intercellular regulatory signal necessary for the early development of a variety of tissues and organs.In recent years,more and more studies have found that Notch signaling pathway is closely related to pulmonary fibrosis.This research is based on the experimental results of the National Natural Science Foundation of China "Study on Phlegm-Traditional Chinese Medicines to Regulate the Antioxidant Mechanism of Pulmonary Interstitial Fibrosis(No.81273704)",and used TGF-?1 to construct MRC-5 cells.Model,given different Fritillaria components(Peimine,Peiminine,Peimisine and Imperaline)to observe the protective effect of different Fritillaria components on oxidative damage of MRC-5 cells after modeling.At the same time,DAPT was used to block the Notch signal pathway and observe the specific mechanism of action of Fritillaria component in protecting MRC-5cells against oxidative damage.Method:1.Construction of MRC-5 oxidative damage model and selection of maximum nontoxic concentration of Fritillaria component.According to the different action time of 5ng / ml TGF-?1 on MRC-5 cells,MTT method was used to detect the cell proliferation inhibition rate,and Real-time PCR and Western Blotting method were used to detect the effects of TGF-?1 on MRC-5 cells.The expression of the concentration of 8-OHdG protein in the cells was detected by ELISA,and the optimal time for TGF-?1 modeling was selected.The optimal non-toxic concentration of Fritillaria component to MRC-5,IC10 was selected as the optimal nontoxic concentration for subsequent experiments.2.To observe the protective effect of Fritillaria component on TGF-?1-induced MRC-5 cell oxidative damage model.Divided into blank group,model group(TGF-?1 group),Peimine group(Peimine+TGF-?1),Peiminine group(Peiminine + TGF-?1),Peimisine group(Peimisine + TGF-?1)and Imperaline group(Imperaline + TGF-?1),a total of 6 groups.RT-PCR was used to detect and compare the expression of collagen?and collagen? mRNA in each group of cells;Western Blotting was used to detect and compare the expression of collagen ?,collagen ?,and ?-SMA proteins in each group of cells;Immunofluorescence was used to observe the number of ?-SMA positive myofibroblasts and the intensity of fluorescence expression in each group of cells.3.Peimine improves fibrosis by regulating TGF-?1 / Notch-mediated oxidative damageIt was divided into blank group,model group(TGF-?1 group),Peimine group(Peimine+ TGF-?1),TGF-?1 + DAPT group and Peimine group+ DAPT,a total of 5groups.Western Blotting and RT-PCR were used to detect the protein and mRNA expressions of collagen?,collagen ?,Notch1-4,Jag1-2,Hes1 and DLL1,3,4 in each group and compare within groups;ELISA method was used to detect 8-OHdG in each group Expression level;Biochemically detect and compare the expressions of SOD and GSH in each group;Lentivirus transfected the Notch1 target gene into MRC-5 cells,and detect the protein expression of collagen?,collagen ?,?-SMA and Notch1 of Peimine after treatment.Result:1.Construction of MRC-5 oxidative damage model and selection of maximum nontoxic concentration of Fritillaria component.1.1 Selected 5 ng / ml TGF-?1 pre-stimulated MRC-5 for 48 hours for oxidative damage modeling.1.2 TGF-?1 can not only induce oxidative damage to MRC-5 cells,but also cause fibrotic damage.1.3 Peimine 3.125 ?g/ml,Peiminine 6.25 ?g / ml,Peimisine 6.25 ?g / ml,and Imperaline 6.25 ?g / ml were selected as the maximum non-toxic concentrations of the experimental drug.Subsequent experiments were performed.2.To observe the protective effect of Fritillaria component on TGF-?1-induced MRC-5 cell oxidative damage model.2.1 Peimine,Peiminine,Peimisine and Imperaline all can protect MRC-5 cells induced by TGF-?1 to varying degrees.Compared to other Fritillaria components Peiminine and Peimisine decrease the mRNA and protein expression of collagen?,collagen ?,and ?-SMA fibrosis factor,which indicates that these two components of Fritillaria are resistant to TGF-?1 induced MRC-5 cells Fibrosis is stronger.2.2 The number of ?-SMA positive myofibroblasts and the fluorescence expression of the Peimine group were more obvious than those of the Fritillin group,indicating that Peimine has a stronger protective effect on MRC-5 cells and an anti-fibrotic effect.More obviously,Peimine was selected as the subsequent experimental drug.3.Peimine improves fibrosis by regulating TGF-?1 / Notch-mediated oxidative damage3.1 There was no significant difference in the mRNA and protein expressions of Notch1,Jag1,Hes1 between the Peimine+DAPT and TGF-?1+DAPT groups,indicating that Peimine may reduce the signal by inhibiting the expression of Notch1,Jag1,Hes1.The release of downstream factors of the pathway plays a role in protecting cells and reducing fibrotic damage;however,it is not expressed or meaningless in Notch2-4,Jag2,DLL1,DLL3 and DLL4.3.2 Peimine can significantly improve the expression of GSH and SOD,and reduce the expression of 8-OHdG has anti-oxidant effect;and the DAPT+TGF-?1 group shows an up-regulation of GSH and SOD,which is not significant compared with Peimine group.The difference indicates that Peimine may play an antioxidant role by blocking the Notch signaling pathway.At the same time,there is no significant difference in the expression of collagen?and collagen ? in the Peimine group compared with DAPT +TGF-?1 group.And compared with the TGF-?1 group,it was significantly reduced,indicating that Peimine may be oxidized through the Notch signaling pathway to reduce fibrotic damage.3.3 The protein expressions of collagen?,collagen?,?-SMA,and Notch1 in MRC-5 cells transfected with Notch1 virus were significantly increased,and the protein expressions of collagen?,collagen ?,?-SMA,and Notch1 in MRC-5 cells of the Peimine group were significantly reduced.This shows that Peimine can inhibit the Notch signaling pathway and reduce the expression of downstream products of the pathway,which in turn can protect cells,slow down the conversion of FB to MFB,and have a therapeutic effect on fibrosis.Conclusion:1.TGF-?1 can induce oxidative damage in MRC-5 cells,leading to fibrosis.2.The maximum non-toxic concentration of Fritillaria component can reduce the expression of fibrosis factors in MRC-5 cells induced by TGF-?1,indicating that Fritillaria component at a safe concentration can damage oxidatively damaged MRC-5cells.It plays a protective role and reduces fibrotic damage.3.Peimine can play an antioxidant and reduce the production of fibrotic factors by up-regulating the expression of SOD and GSH,and its mechanism may be related to the Notch1/Jag1/Hes1 signaling pathway.4.Peimine can inhibit the Notch signal pathway and reduce the expression of downstream products,thereby protecting cells,reducing oxidative damage,slowing the conversion process of FB to MFB,and has a therapeutic effect on fibrosis.