Intervention Mechanism of Compound Tinglizi Decoction(复方葶苈子汤)on COPD-PH Based on HMGB1-Mediated Pulmonary Artery Smooth Muscle Cell Pyroptosis
Objective To investigate the effects of high mobility group box-1 protein(HMGB1)-mediated pulmonary artery smooth muscle cell pyroptosis,and intervention mechanism of Compound Tinglizi Decoction(复方葶苈子汤)on chronic obstructive pulmonary disease-associated pulmonary hypertension(COPD-PH)disease.Methods CCK-8 was used to detect the activity of pulmonary arterial smooth muscle cells(PASMCs),and the appropriate intervention concentration and time of cigarette smoke extract(CSE)and lipopolysaccharide(LPS)were selected for the establishment of cell damage model.The cells in logarithmic growth stage were divided into control group,CSE group,LPS group and CSE+LPS group.The control group was added with cell culture.The CSE group was added with cell culture containing 10％CSE,LPS group was added with cell culture containing 1 µ g/mL LPS,and CSE+LPS group was added with cell culture containing 10％CSE and 1 μg/mL LPS.After 24 hours,CCK-8 was used to detect the cell activity,real-time fluorescent quantitative PCR(RT-PCR)was used to detect the expression of mRNA,Western blot(WB)was used to detect the expression of proteins,enzyme linked immunosorbent assay(ELISA)was used to detect the content of inflammatory factors in cell supernatant,and flow cytometry was used to detect pyroptosis.The ultrastructure of cells was observed by transmission electron microscope.CCK-8 was used to detect the activity of PASMCs,and the optimal intervention concentration and time of Compound Tinglizi Decoction drug serum and glycyrrhizic acid were selected for cells.The cells in logarithmic growth stage were divided into normal group,model group,drug serum group and inhibitor group.The control group was added with cell culture.The model,drug serum and inhibitor group were added with cell culture containing 10％CSE and 1 μg/mL LPS.The culture medium was removed after 24 hours.The normal and model group were added with cell culture with 10％blank serum,drug serum group was added with cell culture containing 10％Compound Tinglizi Decoction drug serum,and inhibitor group was added with cell culture containing 150 μg/mL glycyrrhizic acid and 10％blank serum.After 24 hours,CCK-8 was used to detect the cell activity,RT-PCR was used to detect the expression of mRNA,WB was used to detect the expression of proteins,ELISA was used to detect the content of inflammatory factors in cell supernatant,and flow cytometry was used to detect pyroptosis.The ultrastructure of cells was observed by transmission electron microscope.Results 10％CSE and 1 μg/mL LPS were selected for the establishment of cell damage model,and the intervention time was 24 hours.The activity of PASMCs was reduced by CSE,LPS and CSE combined with LPS.The mRNA expression of HMGB1,RAGE,Caspase-8 and GSDMD,protein expression of HMGB1,RAGE,pro Caspase-8,cleaved Caspase-8 and GSDMD,secretion of interleukin-1β(IL-1β)and interleukin-18(IL-18),and pyroptosis were promoted by CSE,LPS and CSE combined with LPS in PASMCs.10％Compound Tinglizi Decoction drug serum and 150 μg/mL glycyrrhizic acid were selected for cellular intervention,and the intervention time was 24 hours.The activity of damaged PASMCs was increased by Compound Tinglizi Decoction drug serum.The mRNA expression of HMGB1,RAGE,Caspase-8 and GSDMD,protein expression of HMGB1,RAGE,pro Caspase-8,cleaved Caspase-8 and GSDMD,secretion of IL-1β and IL-18,and pyroptosis were inhibited by Compound Tinglizi Decoction drug serum in damaged PASMCs.Conclusion COPD-PH could be improved by Compound Tinglizi Decoction,through inhibiting the expression of HMGB 1,Caspase-8-mediated pulmonary artery smooth muscle cell pyroptosis,and inflammatory cascade.
Compound Tinglizi Decoction(复方葶苈子汤)chronic obstructive pulmonary disease-associated pulmonary hypertensionhigh mobility group protein B1pyroptosispulmonary artery smooth muscle cells
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