Investigation Of The Effects And Mechanisms Of The Xuanfei Duyu Decoction On Acute Lung Injury Based On ACE2/Ang(1-7)/Mas Pathway

Objective:The purpose of this study is to establish an acute lung injury model in A549cells through lipopolysaccharide(LPS)intervention,by preparing Xuanfei Dayu decoction-containing serum as a means of intervention.The study investigates its effects on ACE2/Ang(1-7)/Mas-related indices in LPS-treated A549 cells,thus examining the potential role of Xuanfei Dayu decoction in acute lung injury treatment.Methods:The Xuanfei Dayu decoction-containing serum was prepared by pre-processing the required herbal materials in a 1 g/ml standard solution.Sixty SPF-grade SD rats were randomly selected,divided into five groups:blank control(distilled water),Xuanfei Dayu decoction in low,medium and high dosages(22.4 g·kg~-1,44.8 g·kg~-1,89.6 g·kg~-1,respectively),and dexamethasone(DEX)group(0.2 mg·kg~-1).After three days of oral administration,blood was collected and centrifuged to prepare drug-containing serum.A549 cells were cultured,passaged,and cryopreserved.The cells were then revived and added to F-12 medium containing fetal calf serum and double antibodies before being placed in a humidified incubator at 37°C with 5%CO2.Six experimental groups were established,and LPS-treated A549 cells were evaluated for various effects of Xuanfei Dayu decoction-containing serum using CCK-8,ELISA,immunofluorescence,flow cytometry,Western blot,and q RT-PCR to assess ACE2/Ang(1-7)/Mas signaling pathway-related indices.Results:We found that Xuanfei Dayu decoction-containing serum led to a significant improvement in the proliferation of A549 cells and the expression of ACE2,Mas protein,IL-6,and Ang(1-7)in LPS-treated cells.Flow cytometry showed the serum’s effect on apoptosis,while Western blot and real-time q PCR showcased the changes in protein and m RNA expression of ACE2,Ang(1-7),and Mas in the cells.CCK-8 assay revealed a dose-dependent increase in growth rates of A549 cells treated with Xuanfei Dayu decoction-containing serum,and immunofluorescence demonstrated increased protein expression of ACE2 and Mas.Flow cytometry using the Annexin V-FITC/PI double-stain method indicated a dose-dependent reduction in apoptotic rates.ELISA,immunofluorescence,and Western blot assays further confirmed increased expression of ACE2,Ang(1-7),and Mas,which suggested an effect on the ACE2/Ang(1-7)/Mas signaling pathway.Lastly,q RT-PCR revealed increased m RNA expression of ACE2,Ang(1-7),and Mas,with treatment dose impacting the effect.Conclusion:The results indicate that Xuanfei Dayu decoction-containing serum exerts a protective effect on the acute lung injury model of LPS-treated A549 cells.It increases cell growth rate,inhibits apoptosis,reduces IL-6 expression,elevates Ang(1-7)expression,and enhances protein and gene expression of ACE2,Ang(1-7),and Mas.The mechanism of its action may be attributed to the ACE2/Ang(1-7)/Mas signaling pathway.