宣肺败毒方来源sRNA靶向血管紧张素转换酶改善脂多糖诱导的小鼠急性肺损伤OA

Objective To investigate the protective effect and mechanism of small RNAs(sRNA)derived from Xuanfei Baidu Formula(宣肺败毒方,XFBD)targeting angiotensin-converting enzyme(ACE)in lipopolysaccharide(LPS)-induced acute lung injury(ALI)in mice.Methods The modified CTAB method was employed to extract sRNA from XFBD and a library was constructed,followed by prediction and screening of sRNAs targeting ACE.The targeting specificity was verified using a dual-luciferase reporter system,and sRNAs capable of suppressing ACE expression were screened in human pulmonary microvascular endothelial cells(HPMEC).The effects of sRNA on angiotensin Ⅱ(Ang II)generation,inhibitor of inhibitor of nuclear factor-κB α(IκBα)and inflammatory cytokine expressions were examined by ELISA,Western blotting and qRT-PCR.An LPS-induced ALI mice model was established,control group,model group,captopril(10 mg/kg)group,XFBD(9.2 g/kg)group,NC-sRNA(10 nmol/animal)group,ACE-sRNA-1(10 nmol/animal)group and ACE-sRNA-26(10 nmol/animal)group were set up,with six mice in each group.Hematoxylin-eosin(HE)staining and Micro CT were used to evaluate the pathological and imaging changes of lung tissue;The number of white blood cells and neutrophils in peripheral blood,as well as the total protein concentration,white blood cells,neutrophils and lymphocytes numbers in bronchoalveolar lavage fluid(BALF)were detected;Immunohistochemistry was used to detect the expressions of vascular endothelial-cadherin(VE-cadherin)and intercellular adhesion molecule-1(ICAM-1)in lung tissue;Western blotting and qRT-PCR were used to detect the expressions of ACE-Ang Ⅱ-Ang Ⅱ type 1 receptor(AT1R)pathway,IκBα and inflammatory factors in lung tissue;ELISA was used to detect the levels of Ang II and inflammatory factors in serum.Results A total of 50 potential ACE-targeting sRNAs were screened from XFBD,with 26 sRNAs validated to exhibit targeting effects,among which 12 sRNAs significantly inhibited ACE expression in HPMEC cells(P<0.05,0.01,0.001).Seven sRNAs significantly suppressed Ang II generation,with ACE-sRNA-1/26 demonstrating the most potent effect(P<0.001),inhibited IκBα protein and inflammatory factor expressions(P<0.01,0.001).In animal experiments,mice in model group had severe lung injury(P<0.001);Compared with model group,ACE-sRNA-1 and ACE-sRNA-26 significantly improved lung injury in mice(P<0.05,0.01,0.001),up-regulated VE-cadherin expression in lung tissue(P<0.001),down-regulated ICAM-1,ACE,AT1R,IκBα and inflammatory factor expressions in lung tissue(P<0.05,0.01,0.001),and reduced Ang II and inflammatory factor levels in serum(P<0.05,0.01,0.001).Conclusion ACE-sRNA-1 and ACE-sRNA-26 derived from XFBD could target the inhibition of ACE expression and activity,alleviate pulmonary endothelial inflammation and barrier damage,and improve LPS-induced ALI in mice.The mechanism may be related to the regulation of ACE-Ang Ⅱ-AT1R pathway.