薤白超细粉调控上皮细胞铁死亡对肺纤维化的影响及其机制OA

Objective To investigate the inhibitory effect of ultrafine Xiebai powder(UXP)on pulmonary fibrosis and its regulatory mechanisms associated with epithelial cell ferroptosis.Methods(1)Network pharmacology analysis was performed to predict potential targets and signaling pathways of four major active components of UXP.(2)Human bronchial epithelial cells(Beas-2b)were divided into control,bleomycin(BLM),BLM+1 mg/ml UXP(UXP-L),BLM+5 mg/ml UXP(UXP-H),and BLM+1 μmol/L nintedanib(NIN)groups.Except for control group,cells in all groups were treated with BLM(10 μg/ml)to establish a pulmonary fibrosis model.Cell viability,apoptosis level,reactive oxygen species(ROS),mitochondrial function,lipid peroxidation,and ferroptosis indicators were detected in each group.(3)Co-cultured Beas-2b cells and human lung fibroblasts(HLF-1)were randomly divided into control,BLM,BLM+UXP-L,BLM+UXP+H and BLM+NIN groups.Except for control group,cells in all groups were treated with BLM(10 μg/ml)to establish a pulmonary fibrosis cell model.Immunofluorescence staining was used to assess the expression levels of collagen type I alpha 1 chain(Col1a1)and α-smooth muscle actin(Acta2)in HLF-1 cells.(4)C57BL/6 mice were randomly divided into control,BLM,BLM+UXP-L,BLM+UXP-H,and BLM+NIN groups(n=8 per group).Except for control group,mice in all groups were intratracheally instilled with BLM(0.5 mg/ml)to establish a pulmonary fibrosis model.From day 7 to day 20 after modeling,mice were intragastrically administered UXP-L(100 mg/kg),UXP-H(500 mg/kg),or NIN(50 mg/kg)daily respectively;control and BLM groups were given an equal volume of normal saline by gavage.Lung index,lung wet/dry weight ratio(W/D),Masson staining and Sirius Red staining were measured to assess pulmonary edema and pathological changes.The contents of superoxide dismutase(SOD),malondialdehyde(MDA),glutathione(GSH),and hydroxyproline were detected.RT-qPCR was used to detect mRNA expression of fibrosis-related factors,and Western blotting was performed to analyze the expression levels of related proteins.Transmission electron microscopy was employed to observe changes in mitochondrial ultrastructure.Results(1)A total of 240 overlapping target genes were identified between the main active components of UXP and pulmonary fibrosis.(2)Compared with control group,BLM group exhibited significantly decreased cell viability and mitochondrial membrane potential(P<0.05),along with markedly increased apoptosis rate,ROS levels,lipid peroxidation content,and Fe2+content(P<0.001).Compared with BLM group,BLM+UXP-L,BLM+UXP-H,and BLM+NIN groups exhibited significantly increased cell viability and mitochondrial membrane potential(P<0.001),and significantly decreased cell apoptosis rate,ROS levels,lipid peroxidation content,and Fe2+content(P<0.001).(3)Compared with control group,the levels of Acta2 and Col1a1 in HLF-1 cells co-cultured with Beas-2b were significantly higher in BLM group(P<0.001).Compared with BLM group,BLM+UXP-L,BLM+UXP-H,and BLM+NIN groups showed significantly lower levels of Acta2 and Col1a1 in HLF-1 cells(P<0.001).(4)Compared with control group,BLM group showed significant increased lung index,W/D ratio,hydroxyproline and MDA contents,mRNA expression levels of fibrosis-related and pro-ferroptotic genes(Acta2,Col1a1,Col1a2,Fn,Acsl4,Ncoa4)and protein expression levels of Acta2,Col3a1 and p-ERK(P<0.05,P<0.01,or P<0.001),while the activities of SOD and GSH and expression levels of anti-ferroptotic gene Gpx4,Slc7a11 were significantly decreased(P<0.001).Significant collagen deposition,iron deposition,and mitochondrial ultrastructural damage were observed in lung tissues.Compared with BLM group,BLM+UXP-L,BLM+UXP-H,and BLM+NIN groups showed significantly decreased lung index,W/D ratio,hydroxyproline and MDA contents,mRNA expression levels of Acta2,Col1a1,Col1a2,Fn,Acsl4,Ncoa4,and expression levels of Acta2,Col3a1 and p-ERK protein(P<0.01 or P<0.001),while SOD and GSH activities and expression levels of Gpx4 and Slc7a11 were significantly increased(P<0.05 or P<0.001).Collagen deposition,iron deposition,and mitochondrial ultrastructural damage in lung tissues were alleviated.Conclusion UXP may inhibit pulmonary fibrosis by regulating oxidative stress,lipid peroxidation,and the ferroptosis pathway.