四逆汤通过NCOR1介导线粒体自噬调控巨噬细胞极化影响动脉粥样硬化斑块形成的机制研究OA

Objective To investigate the effects of Sini Decoction(SND)on atherosclerotic(AS)plaque formation through the regulation of nuclear receptor corepressor 1(NCOR1)expression,activation of mitophagy,and modulation of macrophage polarization.Methods Twenty SD rats were randomly divided into a blank control group and a SND group.Rats in SND group were administered the SND orally at a dose of 2.43 g/kg(converted based on body surface area equivalence)twice daily for 7 consecutive days,while rats in the blank control group were gavaged with an equal volume of normal saline.Serum was isolated and used to prepare both blank control serum and SND-containing serum.The CCK-8 assay was employed to determine cell viability,based on which the optimal concentration for subsequent experiments was selected.The RAW 264.7 macrophage cell line was used.An in vitro inflammatory model of AS was established by stimulating the cells with 1 μg/mL lipopolysaccharide(LPS)for 24 hours.The experiment included seven groups:blank group,model group,low-(5%),medium-(10%),and high-dose(20%)SND-containing serum groups,the inhibitor group(with 10 μmol/L of Cyclosporine A,CsA),and the inhibitor+SND group(with 10 μmol/L of CsA+20%SND-containing serum).The expression levels of macrophage polarization markers(M1:IL-6,TNF-α,and CD16/32;M2:IL-10,Arg1,and CD206)and NCOR1 were detected by ELISA,real-time quantitative PCR(qPCR),and Western blot.The expression of key mitophagy-related proteins(PINK1,Parkin,p62,and LC3)was analyzed by Western blot.Mitochondrial ultrastructure was observed using transmission electron microscopy(TEM).Cell viability and mitochondrial membrane potential were assessed by MTS assay and flow cytometry,respectively.Results Compared with the model group,SND-containing serum dose-dependently increased cell viability(P<0.05),upregulated the expression levels of M2 markers(IL-10,Arg1,and CD206)and NCOR1(P<0.05),while downregulating the expression levels of M1 markers(IL-6,TNF-α,and CD16/32)(P<0.05).SND intervention promoted mitophagy,as evidenced by increased protein expression of PINK1 and Parkin,an elevated LC3-Ⅱ/Ⅰ ratio,and decreased p62 accumulation(P<0.05).TEM observation revealed improved mitochondrial morphology and an increased number of autophagosomes(P<0.05).In contrast,CsA inhibitor treatment exacerbated LPS-induced cell damage,manifested as a further decrease in cell viability,aggravated pro-inflammatory polarization,inhibited NCOR1 expression,and obstructed mitophagy(P<0.05).Under the background of CsA inhibition,the combined application of SND-containing serum partially reversed the above damage,significantly improving cell viability,promoting anti-inflammatory polarization,up-regulating NCOR1 expression,and restoring the mitophagic flux(P<0.05).Conclusion Sini Decoction may exert a stabilizing effect on AS plaques by upregulating NCOR1 expression,activating mitophagy,improving mitochondrial functional homeostasis,and inhibiting macrophage polarization towards the pro-inflammatory M1 phenotype.