Mechanism Of Qingfei Oral Liquid Flavonoids In Treating RSV Pneumonia By Regulating Necroptosis

Objective:RSV pneumonia mice model was established and the effects of flavonoids in Qingfei Oral Liquid on epithelial barrier,mitochondrial function,necroptosis pathway and lipid metabolism in RSV-infected mice were studied by molecular biology and metabolomics,thereby revealing the mechanism of flavonoids in Qingfei Oral Liquid.Methods:1.The effect of flavonoids in Qingfei Oral Liquid on epithelial barrier damage in RSVinfected mice:BALB/c mice were randomly divided into control group,model group,flavonoids low-,high-dose(30,60 mg/kg)groups and ribavirin(46mg/kg)group.RSV-infected mice model was established.After continuous administration for four days,lung inflammation was assessed by HE staining and ELISA.RSV-F mRNA and protein expression in lung tissue was detected by immunohistochemistry and RT-qPCR.BALF protein concentration and BALF/serum fluorescence ratio were used to evaluate lung epithelial permeability.Western blot was used to detect the expression of tight junction proteins Claudin-1,Occludin and ZO-1 in lung tissue.2.The effect of flavonoids in Qingfei Oral Liquid on mitochondrial function in RSVinfected mice:ROS level in lung tissue was detected by fluorescence staining,mitochondrial membrane potential was detected by flow cytometry,and ultrastructure of lung tissue was observed by transmission electron microscope.3.The effect of flavonoids in Qingfei Oral Liquid on the necroptosis pathway in RSVinfected mice:flow cytometry was used to detect the necroptosis rate in lung tissue cell.ELISA was used to detect LDH and HMGB1 content in BALF.Western blot was used to detect the protein expression of RIP1,RIP3,MLKL,PGAM5,DRP1 and p-p38 MAPK.Immunofluorescence was used to detect the co-localization of RIP1 and RIP3,as well as the co-localization of p-MLKL and epithelial cells.Molecular docking was used to evaluate the binding ability of flavonoids in Qingfei Oral Liquid to p38 MAPK.Finally,RSV-infected mice were treated with p38 MAPK inhibitor SB 203580 to observe the effect of p38 MAPK inhibition on necroptosis pathway.4.Effects of flavonoids in Qingfei Oral Liquid on lipid metabolism in RSV-infected mice:LC-MS was used to detect and analyze the lipid metabolism profile of flavonoids in Qingfei Oral Liquid in RSV-infected mice.Then differential lipids were screened out.The relationship between ceramide and necroptosis and tight junction verified by using the ceramide inhibitor FBI,thereby exploring the lipid metabolism mechanism of flavonoids in Qingfei Oral Liquid.Results:1.Flavonoids in Qingfei Oral Liquid could reduce the pathological damage of lung tissue in RSV-infected mice and the levels of IL-6 and TNF-α in BALF,inhibit the nucleic acid transcription and protein expression of RSV-F in lung tissue,reduce the protein concentration in BALF and the BALF/serum fluorescence ratio,as well as increasing the expression of tight junction proteins Claudin-1,Occludin and ZO-1.2.Flavonoids in Qingfei Oral Liquid could reduce the levels of ROS and MDA in the lung tissue of RSV-infected mice,increase the level of SOD,improve the mitochondrial membrane potential,and restore the structure of mitochondria.3.Flavonoids in Qingfei Oral Liquid could reduce the necroptosis in lung tissue after RSV infection.The expression of HMGB1 and LDH in BALF,the protein expression of RIP 1,RIP3,MLKL,PGAM5,DRP1,p-p38 MAPK,the formation of the RIP1/RIP3 necrotic complex and the fluorescent expression of p-MLKL in lung epithelial cells were inhibited,compared to the RSV group.The p38 MAPK inhibitor SB 203580 could also inhibit the expression of RIP1,RIP3 and MLKL,which was similar to the flavonoids in Qingfei Oral Liquid.In addition,the results of molecular docking showed that luteoloside,swertisin and hesperidin in Qingfei Oral Liquid had good potential binding ability to p38 MAPK.4.Metabolism results showed that the flavonoids in Qingfei Oral Liquid had obvious regulatory effect on lipid metabolism disorder in the serum of RSV-infected mice.A total of 292 differential lipid metabolites were identified in this study,mainly enriched in phospholipids,sphingolipids and fatty acids.Among them,ceramide is an important lipid involved in regulating necroptosis and epithelial barrier.Ceramide inhibitor FBI could inhibit the expression of p-p38 MAPK,RIP1,RIP3,MLKL in lung tissue,and increase the levels of tight junction proteins Claudin-1,Occludin and ZO-1.Conclusion:1.The flavonoids in Qingfei Oral Liquid alleviate RSV-induced necroptosis,improve mitochondrial function,and reduce epithelial barrier damage by inhibiting p38 MAPK/RIP1/RIP3/MLKL/PGAM5/DRP1.2.The flavonoids in Qingfei Oral Liquid have obvious regulatory effect on lipid metabolism disorder in the serum of RSV-infected mice,which may inhibit necroptosis and protect lung epithelial barrier by regulating ceramide metabolism.