Effect Of SIRT1 Regulating HMGB1/TLR4/NF-κB Pathway On BPDE-induced Inflammatory Injury In BEAS-2B Cells

SIRT1 is a NAD+-dependent histone deacetylase that regulates inflammatory responses.Studies have found that HMGB1/TLR4/NF-κB inflammatory pathway is involved in multiple inflammatory diseases.Benzo(a)pyrene [Benzo(a)pyrene,B(a)P]is a persistent organic pollutant that can cause pulmonary inflammatory damage and is closely related to respiratory diseases.In B(a)P metabolic end product Benzo(a)pyrene-7,8-diol-9,10-epoxide(BPDE)induced bronchial epithelial cells(BEAS-2B)injury,whether SIRT1 can participate in it through modulation of the HMGB1/TLR4/NF-κB inflammatory pathway is unclear.ObjectiveTo explore whether SIRT1 regulates HMGB1/TLR4/NF-κB pathway and participates in BPDE-induced inflammatory injury of BEAS-2B cells,which providives a theoretical basis for early prevention and treatment of acute lung injury.Methods1.BEAS-2B cells were cultured in vitro.Blank control group and DMSO solvent control group were set up,and the cells were treated with different concentrations(0.50,0.75 and 1.00 μmol/L)of BPDE for 24 h.The contents of ROS,SOD and MDA in BEAS-2B cells in each group were detected.ELISA was used to detected the expression levels of IL-1β,IL-6 and HMGB1 in the supernatant of each group of cells.Changes in the expression of SIRT1,HMGB1,TLR4,IL-1β and IL-6 were detected by q RT-PCR.Changes in the expression levels of SIRT1,HMGB1,TLR4,NF-κB p65 and p-p65 protein were detected by Western blot.2.CCK-8 method was used to detect the cell viability of BEAS-2B cells treated with different concentrations of SIRT1 activator SRT1720(1,1.5,2,2.5,3 μmol/L)and different concentrations of SIRT1 inhibitor EX527(1,5,10,15,20 μmol/L),and the appropriate concentrations were screened for subsequent experiments.DMSO group,BPDE group,DMSO+SR1720 group,BPDE+1720 group,DMSO+EX527 group and BPDE+EX527 group were established.Changes in oxidative stress indexes of cells in each group were detected.The inflammatory changes in each group were detected by ELISA.The distribution of cellular HMGB1 protein was observed by immunofluorescence and q RT-PCR was used to detect the changes of IL-1β,IL-6,SIRT1,HMGB1 and TLR4 expression.Western blot was used to detect the changes of SIRT1,HMGB1,ac-HMGB1 TLR4,NF-κB p65,and p-p65 protein expression levels.Results1.Effects of BPDE on oxidative stress and inflammatory damage in BEAS-2B cellsCompared with the control group,after treated with 0.50,0.75 and 1.00 μmol/L BPDE for 24 h,the intracellular ROS production increased,the SOD activity decreased,and the MDA activity level increased(P < 0.05,respectively).With the BPDE concentrations increasing,the relative expressions of inflammatory factors IL-1β and IL-6 m RNA were increased,and the expressions of IL-1β and IL-6 in the cell supernatant increased(P<0.05,respectively).2.Effect of BPDE on SIRT1 and HMGB1/TLR4/NF-κB pathway in BEAS-2B cellsqRT-PCR results showed that the expression of SIRT1 was decreased,while the expressions of HMGB1 and TLR4 were increased.ELISA results showed that the expression of HMGB1 increased in cell supernatant.The results of Western blot showed that after BPDE exposure to BEAS-2B cells for 24 h,the expression of SIRT1 was decreased and the expression of HMGB1,TLR4 and phosphorylated p65 were increased significantly(P<0.05,respectively).3.SIRT1 regulates HMGB1/TLR4/NF-κB pathway involved in BPDE-induced inflammatory injuryWhen the concentration of SRT1720 was 2 μmol/L and the concentration of EX527 was 20 μmol/L,the cell viability was significantly decreased(P<0.05).1.5μmol/L of SRT1720 and 15 μmol/L of EX527 were selected as the concentrations for subsequent experiments.Western blot results showed that compared with the BPDE group,the SIRT1 protein expression in the BPDE+SRT1720 group was significantly increased,while the protein expression in the BPDE+EX527 group was decreased(P<0.05).ROS expression was decreased,SOD activity was increased,and expression of inflammatory factors IL-1β and IL-6 was significantly decreased,the expression of TLR4,p-p65,and acetylated HMGB1 protein expression was decreased in the BPDE+SRT1720 group compared with the BPDE group(P<0.05,respectively).After SIRT1 expression was Inhibited by EX527,ROS expression and MDA activity increased,and the expression levels of inflammatory factor IL-1β increased(P<0.05,respectively).The immunofluorescence and ELISA results showed that the transfer and release of HMGB1 was increased,HMGB1,ac-HMGB1,TLR4 and p-p65 protein expression were increased(P<0.05,respectively).Conclusion1.BPDE induces oxidative stress in BEAS-2B cells and induces activation of HMGB1/TLR4/NF-κB inflammatory pathway in BEAS-2B cells.2.SIRT1 may be involved in BPDE-induced in flammatory injury of BEAS-2B cells by regulating the HMGB1/TLR4/NF-κB inflammatory pathway.