Investigation Of The Improvement Effect And Possible Mechanism Of Huatanjiangqi Formula On Glucocorticoid Resistance In Lung Bronchial Epithelial Cells Induced By Cigarette Smoke

Objective:Glucocorticoid（GC）resistance model was established by CSE-induced16HBE cells to explore the influence of Huatanjiangqi Formula-containing serum on the GC resistance of 16HBE cells stimulated by CSE,and to further study the correlation between the improvement effect of it on the GC resistance and Keap1/Nrf2/ARE signaling pathway.The aim is to clarify the role and mechanism of Huatanjiangqi Formula in reversing GC resistance under COPD,and provide new targets and research ideas for the prevention and treatment of COPD in traditional Chinese medicine.Methods:1.Twenty SD rats were randomly divided into two groups.The Huatanjiangqi Formula group was administered with Huatanjiangqi Formula twice daily,and the dose was 1.4 g/kg.The blank group was given an equal volume of normal saline.After the last gavage for 1 h,blood samples were collected from the abdominal aorta of all the rats and supernatant was centrifuged to obtain rat blank serum and Huatanjiangqi Formula-containing serum.16HBE cells were cultured in vitro,and GC resistance model was established by CSE-induced cells.Cells were incubated with low and high concentrations of Huatanjiangqi Formula-containing serum as well as combined with DEX(10^-6mol/L),respectively.The expressions of Nrf2,HO-1,MRP1 and HDAC2proteins in the cells were observed.The IL-8 expression in the cell culture supernatant and the intracellular GSH levels were determined.In addition,the IL-8 inhibition rate and the dexamethasone half inhibitory concentration(IC₅₀-DEX)of cells in each group were calculated with the change of DEX concentration in each group.2.GC resistance model was established based on CSE-induced 16HBE cells.The cells were incubated with low,medium and high concentrations of exogenous GSH to detect the IL-8 content in the cell culture supernatant and the HDAC2 protein expression.In addition,the IL-8 inhibition rate and the IC₅₀-DEX value of cells in each group were calculated.3.GC resistance model was established based on CSE-induced 16HBE cells.Low,medium and high concentrations of sulforaphane（SFN,Nrf2 pathway activator）were applied to intervene the cells.The expressions of Nrf2,HO-1,MRP1 and HDAC2proteins in the cells were observed,and the IL-8 content in the cell culture supernatant and the intracellular GSH levels were determined.In addition,the IL-8 inhibition rate and the IC₅₀-DEX value of cells in each group were calculated.Results:1.When the DEX concentrations were 10^-10～10^-6mol/L,the IL-8 inhibition rates in the CSE group were all significantly lower than those in the control group（P<0.05）;The IC₅₀-DEX values of the control group and the CSE group were（1.15±0.15）×10^-9mol/L and（1.26±0.12）×10^-6mol/L,and the difference was statistically significant（P<0.01）.2.When the DEX concentrations were 10^-10～10^-6mol/L,the IL-8 inhibition rates in the low dose group,the high dose group and the combination group of Huatanjiangqi Formula-containing serum were all higher than those in the CSE group.Among them,the inhibitory effect of IL-8 in the combination group was more obvious than that of Huatanjiangqi Formula-containing serum group（P<0.05）.The IC₅₀-DEX values of Huatanjiangqi Formula-containing serum in the low dose group,the high dose group and combination group were（4.26±0.21）×10^-7mol/L,（1.30±0.14）×10^-7mol/L and（1.22±0.17）×10^-8mol/L,which were all significantly lower than that in the CSE group.Among them,the combination group had a more significant effect than Huatanjiangqi Formula-containing serum group（P<0.01）.The IL-8 levels in the CSE group were significantly higher than those in the control group（P<0.01）.However,the IL-8 levels in the low dose group,the high dose group and the combination group of Huatanjiangqi Formula-containing serum were significantly lower than those in the CSE group.Among them,the combination group had a more significant effect（P<0.01）.The levels of intracellular GSH in the CSE group was significantly lower than that in the control group（P<0.01）.The intracellular GSH levels in the low dose group,the high dose group and the combination group of Huatanjiangqi Formula-containing serum were significantly higher than those in the CSE group.Among them,the combination group had a more prominent effect（P<0.01）.The expressions of MRP1,Nrf2,HO-1and HDAC2 proteins in the CSE group were all significantly reduced compared with the control group（P<0.01）.However,the expressions of MRP1,Nrf2,HO-1 and HDAC2 proteins in the low dose group,the high dose group and the combination group of Huatanjiangqi Formula-containing serum were all significantly higher than those in the CSE group（P<0.01）.3.When the DEX concentrations were 10^-10～10^-6mol/L,the inhibition rates of IL-8 in the low-dose,the medium-dose and the high-dose GSH groups were all higher than those in the CSE group.Among them,the inhibitory effect of IL-8 in the high-dose GSH group was more significant than that in the low-dose and the medium-dose GSH groups（P<0.05）.The IC₅₀-DEX values of exogenous GSH in the low-dose,the medium-dose and the high-dose groups were（8.09±0.23）×10^-7mol/L,（3.85±0.11）×10^-7mol/L and（2.92±0.13）×10^-8mol/L,which were all significantly lower than that in the CSE group.Among them,the high-dose GSH group was more effective than the low-dose and the medium-dose GSH groups（P<0.01）.The IL-8 levels in the CSE group were significantly higher than those in the control group（P<0.01）.However,the IL-8 levels in the low-dose,the medium-dose and the high-dose GSH groups were all significantly reduced compared with the CSE group.Among them,the high-dose GSH group had a more significant effect（P<0.01）.The expression of HDAC2 protein in the CSE group was significantly decreased compared with the control group（P<0.01）.However,the expressions of HDAC2 protein in the low-dose,the medium-dose and the high-dose GSH groups were all significantly higher than that in the CSE group.Among them,the high-dose GSH group had a more obvious effect（P<0.01）.4.When the DEX concentrations were 10^-10～10^-6mol/L,the inhibition rates of IL-8 in the low-dose,the medium-dose and the high-dose SFN groups were all higher than those in the CSE group.Among them,the inhibitory effect of IL-8 in the high-dose SFN group was more significant than that in the low-dose and the medium-dose SFN groups（P<0.05）.The IC₅₀-DEX values of SFN in the low-dose,the medium-dose and the high-dose groups were（4.60±0.29）×10^-7mol/L,（1.90±0.17）×10^-7mol/L and（2.03±0.22）×10^-8mol/L,which were all significantly lower than that in the CSE group.Among them,the high-dose SFN group was more effective than the low-dose and the medium-dose SFN groups（P<0.01）.The IL-8 levels in the CSE group were significantly higher than those in the control group（P<0.01）.However,the IL-8 levels in the low-dose,the medium-dose and the high-dose SFN groups were all significantly decreased compared with the CSE group.Among them,the high-dose SFN group had a more significant effect（P<0.01）.The amount of intracellular GSH in the CSE group was significantly lower than that in the control group（P<0.01）.However,the amount of intracellular GSH in the low-dose,the medium-dose and the high-dose SFN groups were significantly higher than that in the CSE group.Among them,the high-dose SFN group had a more prominent effect（P<0.01）.The expressions of MRP1,Nrf2,HO-1and HDAC2 proteins in the CSE group cells were significantly lower than those in the control group（P<0.01）.However,the expressions of MRP1,Nrf2,HO-1 and HDAC2proteins in the low-dose,the medium-dose and the high-dose SFN groups were all significantly higher than those in the CSE group.Among them,the high-dose SFN group had a more significant effect on the expression of these proteins than the low-dose and the medium-dose SFN groups（P<0.01）.Conclusion:1.The increase of IC₅₀-DEX in the CSE group suggested that the GC resistance model of 16HBE cells induced by CSE was successfully established.2.Inactivation of Keap1/Nrf2/ARE signaling pathway in 16HBE cells induced by CSE,the expression of HDAC2 protein was weakened,which led to a decrease in the inhibitory effect of DEX on the IL-8 secretion of LPS-induced cells under CSE stimulation.That was to say,the sensitivity of CSE-induced cells to DEX was decreased.Improving intracellular GSH levels,restoring HDAC2 protein expression and reducing cell secretion of IL-8 could reverse GC resistance of 16HBE cells under oxidative stress.Huatanjiangqi Formula-containing serum could alleviate the GC resistance of 16HBE cells induced by CSE,and the mechanism might be related to activating the Keap1/Nrf2/ARE signaling pathway,improving intracellular GSH levels and restoring HDAC2 protein expression.