Study On The Anti Fibrosis Mechanism Of SAA On Human Embryonic Lung Fibroblast MRC-5 Based On Notch1 Signal Pathway

Objective:Pulmonary fibrosis?PF?is a common pathological process of interstitial lung disease.It is a group of non tumor and non infectious diseases mainly involving alveolar wall lesions,involving peripheral tissues and adjacent structures.The morbidity is high,the mortality is great,the course of disease is generally progressive,and there is no effective and effective clinical medicine at present.It is found that oxidative stress induced oxidative/antioxidant imbalance is the key to the formation and progression of pulmonary fibrosis.Recent studies have found that Notch1 signal pathway is involved in the occurrence and development of pulmonary fibrosis.Blocking this pathway can reduce the production of myofibroblast,inhibit epithelial mesenchymal transition?EMT?,and delay the progression of pulmonary fibrosis.Through the experiment of human embryonic lung fibroblasts?MRC-5?oxidative injury model with different concentrations of salvianolic acid A intervention,to observe the protective effect of SAA on oxidative stress damage in MRC-5 cells;the intervention of Notch1 signal pathway,observe the regulation of SAA on MRC-5 cells and mechanism of oxidant/antioxidant imbalance.Methods:The first use of H₂O₂ induced oxidative damage in MRC-5 cells were established,respectively at the concentration of 200 mol/L and 400 mol/L,600 mol/L,800mol/L,1000 mol/L H2O2,respectively 6h,12h and 24h were incubated with different concentrations of H2O2,MTT assay in MRC-5 cells at different detection the cell inhibition rate,H₂O₂ concentration and the best selection model of modeling time for subsequent test.The concentrations of SAAof 5mg/ml,2.5mg/ml,1.25mg/ml,0.625mg/ml,0.3125mg/ml and 0.1562mg/ml were selected respectively.The inhibitory effect of different concentrations of SAA on MRC-5 cell proliferation was determined by CCK-8 method,and the maximum non-toxic concentration of the experiment was selected.Theblank group?group A?,the experimental setup of H₂O₂ group?group B?and SAA in high concentration group?group C?,H₂O₂+SAA in high concentration group?group D?,H2O2+SAA in medium concentration group?group E?,H2O2+SAA in low concentration group?group F?.6h,12h and 24h were used as time monitoring points,and the expressions of SOD,CAT,MDA and GSH in each time point were measured and compared.Based on Notch1 signaling pathway,the protective mechanism of SAA on oxidative stress damage in MRC-5 cells induced by H₂O₂ was investigated.Western blotting was used to detect the protein expression of 24h Notch1,JAG1 and Hes1.RT-PCR was used to detect 24h Notch1mRNA,JAG1mRNA and Hes1 mRNAexpression,and the expression of collagen I and collagen III was observed by immunofluorescence.Results:1.Optimum concentration of H₂O₂ and the selection of optimal concentration of SAA.1.1 When the concentration of H₂O₂ 600?mol/L were incubated for 24h,the cell inhibition rate reached 33.96%,the concentration and action time and the degree of inhibition for cell injury in line with best modeling needs,so as to 600?mol/L H₂O₂ 24hours pre stimulation induced MRC-5 oxidative damage model for subsequent test.1.2.When the concentration of SAA reached 2.5mg/ml,the cell inhibition rate reached10.94%.Under the dosage of the drug,the cell inhibition rate was low,MRC-5 cells were less damaged,and the drug concentration was relatively safe.2.5mg/ml could be considered as the largest non-toxic concentration of the drug.2.Observe the protective effect of SAA on oxidative damage induced by H₂O₂ in MRC-5 cells.SAA can significantly increase the activities of antioxidant SOD,CAT and GSH in MRC-5 cells,and significantly reduce the expression of oxidative damage.The expression level of MDA shows a concentration dependent manner,with statistical significance?P<0.05?.3.The protective mechanism of SAA on oxidative stress injury induced by H₂O₂ in MRC-5 cells was investigated based on Notch1 signal pathway.SAA can significantly reduce the expression level of Notch1,JAG1,Hes1 in MRC-5cells,with significant statistical significance?P<0.05?.4.The expression of SAA on the expression of 24h collagen I and collagen III in MRC-5 cells.SAA can obviously inhibit the expression of collagen in MRC-5 cells and improve the antioxidant capacity of the body.Conclusion:1.A certain concentration of SAA can increase the stress level of MRC-5 cells to play a role in the treatment of oxidative damage.2.H₂O₂ upregulated the expression of Notch1,JAG1 and Hes1,suggesting that Notch1 signaling pathway plays an important role in oxidative stress injury.A certain concentration of SAA can significantly reduce the expression of Notch1,JAG1 and Hes1,suggesting that SAA can effectively antagonize oxidative stress damage in MRC-5 cells,and regulate the Notch1 signal pathway to exert the protective effect of antioxidative stress.3.SAA can inhibit the expression of collagen I and collagen III,suggesting that a certain concentration of SAA can increase the anti oxidative damage of the body.