| Objective:Pulmonary fibrosis is the final outcome of pulmonary interstitial disease.Because the etiology is complex and pathological mechanism is unclear,leading to progressive decline in lung function in patients,so serious harm to life and health.But there is no exact and effective promotion of clinicaltreatment of drugs.The study found that oxidative stress-induced cell oxidation/anti-oxidative imbalance is the key to the formation and progression of pulmonary fibrosis.The Nrf2/ARE pathway is an important endogenous antioxidant signaling pathway.Some traditional Chinese medicine prescriptions or monotherapy components have been shown to regulate the oxidative/antioxidant imbalance of pulmonary fibrosis by activating the Nrf2/ARE pathway.This study was based on the experimental results of the National Natural Science Foundation of China,"Huatan Chinese medicine has been used to study the mechanism of antioxidant mechanism of pulmonary interstitial fibrosis(No.81273704).The protective effect of seaweed polysaccharide on oxidative stress injury of MRC-5 cells was observed by the treatment of seaweed polysaccharides before and after the oxidative damage of human embryonic lung fibroblasts(MRC-5).The mechanism of seaweed polysaccharide regulation of oxidative/anti-oxidative imbalance in MRC-5 cells was observed by using si-RNA technique and plasmid overexpression technique to investigate the Nrf2/ARE pathway.Method:1.Construction of oxidative stress model of MRC-5 cells and selection of optimal concentration of seaweed polysaccharide.H202 was used to induce MRC-5 cell oxidative damage model.Different concentrations of H202 were used to study the changes of cell inhibition rate,Nrf2 mRNA and ROS in different time of MRC-5 cells.So,the optimal concentration and modeling time of H202 were selected.Select the optimum concentration of seaweed polysaccharide on MRC-5 cells.The IC10 was the highest non-toxic concentration.The MRC-5 cells were treated with four concentrations by its.The total protein and pulp and nuclear of Nrf2 were detected by RT-PCR,Western blotting and immunofluorescence.finally,select the best concentration of action.2.Protective effect of seaweed polysaccharide on oxidative damage induced by H202 in MRC-5 cells.The group consisted of blank control group,H202 model group,seaweed polysaccharide treatment group,seaweed polysaccharide was treated with H202 for 1 h,and H202 was treated with seaweed polysaccharide for 1 h.Time monitoring points are 0,1,2,6,12,24h.The levels of SOD,CAT,MDA and GSH were detected and compared at each time point.Western blotting was used to detect the expression of Nrf2 protein at each time point.The expression and correlation of Nrf2,keap1,NOQ1,CGLC and HO-1 mRNA were detected by RT-PCR at each time point.And the optimal time-point immunofluorescence method was used to observe the distribution of Nrf2.3.Protective effect of seaweed polysaccharide activated Nrf2/ARE signaling pathway on oxidative stress injury induced by H202 in MRC-5 cells.Divided into two parts:Nrf2 siRNA was used to screen out the best target sites.The groups consisted of blank control group,Nrf2 siRNA group,H202 treatment group,the optimal transfection of Nrf2 siRNA for 48h was treated with H202,and seaweed polysaccharide was treated with H202 for 1h and then to H202 treatment group.That groups were treated with H202.SOD,CAT,MDA,GSH and other biochemical markers were detected.RT-PCR and Western blotting were used to detect the mRNA and protein expression of Nrf2.(2)The recombinant plasmid pCDNA3.1-Nrf2 was constructed by commercial pCDNA3.1 expression vector.The groups included blank control group,MRC-5 + pCDNA3.1 +seaweed polysaccharide treatment group,MRC-5 + pCDNA3 was transfected into pCDNA3.1 empty plasmid group,transfected with pCDNA3.1-Nrf2 recombinant plasmid group,MRC-5 + seaweed polysaccharide treatment group,MRC-.1-Nrf2+seaweed polysaccharide treatment group.RT-PCR and Western blotting were used to detect the mRNA and protein expression of Nrf2,NOQ1,CGLC,HO-1 and other related genes.Result1.The optimum concentration of H202 and the choice of the optimum concentration of seaweed polysaccharide(1)When the concentration of H202 was 600μmol/L for 24 h,the cell inhibition rate was 33.96%and the expression of Nrf2 was enhanced.The inhibitory concentration,the time of action and the degree of inhibition to the cells were in accordance with the optimal modeling requirements.The model of MRC-5 oxidative injury induced by 24 h was pre-stimulated with 600 umol/L H202.(2)When the drug concentration of seaweed polysaccharide reached 0.3125mg/ml,the cell inhibition rate reached 10.39%.The dose of the drug was lower,the damage rate of MRC-5 cells was small,the drug concentration was relatively safe,Nrf2 appeared Nuclear transfer,you can consider the concentration of 0.3125mg/ml as the maximum drug concentration of the drug.2.Protective effect of seaweed polysaccharide on oxidative damage induced by H202 in MRC-5 cells(1)The activity of SOD,CAT and GSH in MRC-5 cells can be significantly increased by seaweed polysaccharide,and the expression level of MDA in the oxidative damage of the organism can be significantly reduced,which is time-dependent and statistically significant(P<0.05).(2)The expression of CGLC,HO-1,NQO1 and Nrf2 in MRC-5 cells was significantly increased by seaweed polysaccharide,and the expression of Keapl was significantly decreased(P<0.05).(3)The seaweed polysaccharide can significantly improve the expression of Nrf2 in the nucleus of MRC-5 cells,and can promote the expression of Nrf2 from cytoplasmic nucleus to nucleus and improve the antioxidant capacity.3.Protective effect of seaweed polysaccharide activated Nrf2/ARE signaling pathway on oxidative stress injury induced by H202 in MRC-5 cells(1)The expression of SOD,CAT,GSH,CGLC,HO-1 and NQO1 in the MRC-5 cells was also down-regulated after Nrf2 was effectively silenced by siRNA.And It was found that the protective effect of seaweed polysaccharide on MRC-5 oxidative stress was decreased.It was suggested that the Nrf2/ARE signal pathway was activated by up-regulating the expression of Nrf2 to improve the oxidative stress of the cells and affected the antioxidant indexes such as SOD,CAT,GSH,CGLC,HO-1,NQO1 and the antioxidant genes expression.The ability of seaweed polysaccharide to increase the stress level was stronger than that of anti-stress injury,which was statistically significant(P<0.05)(2)The expression of Nrf2 gene in MRC-5 cells was increased by using pCDNA3.1-Nrf2 plasmid in vitro.The results showed that Nrf2 gene could promote the synthesis of Nrf2 protein in the absence of seaweed polysaccharide stimulation.The expression of Nrf2,NQO1,CGLC and HO-1 was significantly increased after stimulation with seaweed polysaccharide,and the expression of Nrf2 protein was increased significantly,especially after the application of seaweed polysaccharide,and it was statistically significant(P<0.05).Conclusion:1.Hydrogen peroxide can induce oxidative stress injury in MRC-5 cells,and the expression level of Nrf2 is obviously enhanced,indicating that Nrf2 is a key factor in oxidative stress2.The safe concentration of seaweed polysaccharide can promote the transfer of Nrf2 to the nucleus,suggesting that seaweed polysaccharide can upregulate the stress level of MRC-5 cells to play a protective effect on oxidative damage.3.The protective effect of seaweed polysaccharide on oxidative stress in MRC-5 cells was better than that in therapy.The expression of HO-1 and NQO1 was up-regulated by enhancing the transcriptional activation of Nrf2 and anti-oxidative stress effect.4.It was confirmed that seaweed polysaccharide could activate Nrf2/ARE signaling pathway to improve the oxidative stress of MRC-5 cells through siRNA technology and plasmid overexpression techniques about Nrf2. |
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