【摘要】 目的：脓毒症相关急性肾损伤(sepsis-associated acute kidney injury,SA-AKI)是重症患者的重要致死原因。巨噬细胞极化失衡在SA-AKI进展中发挥关键作用，其中经典活化的M1型巨噬细胞通过释放促炎细胞因子加剧肾损伤，而线粒体稳态失衡是驱动巨噬细胞炎症表型转换的关键。中药单体大黄酚(chrysophanol,CHR)可通过调控巨噬细胞极化方向改善SA-AKI。本研究探讨CHR在SA-AKI微环境中是否通过维持巨噬细胞线粒体稳态来抑制M1型极化，旨在阐明其发挥抗炎及肾脏保护作用的具体机制。方法：采用盲肠结扎穿孔法(cecal ligation and puncture,CLP)构建C57BL/6小鼠SA-AKI模型，并设立假手术(Sham)组、模型(CLP)组及CHR治疗(CLP+CHR)组。体外实验使用人单核巨噬细胞(human monocytic-leukemia cells,THP-1)和人肾小管上皮细胞(human renal tubular epithelial cells,HK-2)构建2种细胞模型：1)建立M1型巨噬细胞与脂多糖(lipopolysaccharide,LPS)刺激的HK-2细胞的Transwell共培养体系并给予CHR干预，评估CHR在SA-AKI中的整体保护效应；2)对单层M1型巨噬细胞给予CHR处理，聚焦CHR对线粒体稳态的保护作用。采用苏木精-伊红(hematoxylin and eosin,HE)染色观察肾脏病理变化；检测血尿素氮(blood urea nitrogen,BUN)和肌酐(creatinine,Cr)水平以评价小鼠肾功能情况；采用酶联免疫吸附试验(enzyme linked immunosorbent assay, ELISA)测定血清及共培养上清中白细胞介素(interleukin, IL)-6和肿瘤坏死因子α(tumor necrosis factor alpha,TNF-α)水平；采用细胞计数试剂盒8(cell counting kit-8,CCK-8)法评估HK-2细胞活力；采用TUNEL检测评估凋亡情况；采用蛋白质印迹法检测凋亡相关蛋白表达。采用透射电镜观察线粒体超微结构；检测三磷酸腺苷(adenosine triphosphate,ATP)水平、线粒体膜电位(mitochondrial membrane potential,MMP)及烟酰胺腺嘌呤二核苷酸磷酸氧化型/还原型(nicotinamide adenine dinucleotide phosphate oxidized/reduced,NADP~+/NADPH)。采用实时荧光定量聚合酶链反应(real time fluorescent quantitative polymerase chain reaction,qPCR)检测线粒体生物合成关键基因过氧化物酶体增殖物激活受体γ辅激活因子1α(peroxisome proliferator-activated receptor gamma coactivator 1alpha,PGC-1α)、线粒体转录因子A(mitochondrial transcription factor A,TFAM)、核呼吸因子1(nuclear respiratory factor 1,NRF1)及M1型极化标志物白细胞分化抗原86(cluster differentiation 86,CD86)的mRNA表达，并采用蛋白质印迹法验证其蛋白表达；采用细胞免疫荧光染色观察CD86的表达强度。结果：与Sham组相比，CLP组模型小鼠肾组织出现明显的肾小管扩张、上皮细胞坏死脱落及管型形成，损伤评分显著升高(P<0.05)；经CHR干预可显著减轻肾组织病理损害。在体外模型中，CHR干预可显著逆转LPS刺激导致的HK-2细胞活力下降(P<0.01)。与Sham组相比，CLP组模型小鼠血清BUN、Cr、IL-6、TNF-α水平均升高，差异均有统计学意义(均P<0.01);CHR干预能显著改善小鼠肾功能，降低血清及共培养上清中促炎细胞因子水平(均P<0.05)，抑制组织与细胞中的细胞凋亡。透射电镜结果表明，CLP组线粒体嵴断裂，膜结构模糊；经CHR干预后，线粒体超微结构得到明显修复。CHR能显著提升SA-AKI模型中降低的ATP水平(P<0.001)，改善M1型巨噬细胞线粒体膜电位，降低NADP~+/NADPH，恢复线粒体抗氧化平衡(P<0.05)。蛋白质印迹法和qPCR证实：CHR能显著上调受损模型中线粒体相关基因的mRNA和蛋白表达水平(均P<0.05)，显著下调M1型巨噬细胞标志物的蛋白与mRNA表达(均P<0.05)。免疫荧光结果显示M1组CD86荧光强度高，CHR组荧光强度减弱，差异均有统计学意义(均P<0.05)。结论：CHR可显著减轻SA-AKI模型小鼠肾脏病理损伤并改善肾功能，其发挥抗炎及肾脏保护作用的机制可能与维持巨噬细胞线粒体能量与氧化还原稳态、抑制巨噬细胞向M1型极化相关。更多还原

【Abstract】 Objective: Sepsis-associated acute kidney injury(SA-AKI) is a major cause of mortality in critically ill patients. Imbalanced macrophage polarization plays a crucial role in the progression of SA-AKI, in which classically activated M1 macrophages aggravate renal injury by releasing pro-inflammatory cytokines, whereas mitochondrial homeostasis disruption is a key driver of macrophage inflammatory phenotypic switching.Chrysophanol(CHR), a monomeric active component derived from traditional Chinese medicine, has been shown to ameliorate SA-AKI by regulating macrophage polarization.This study aimed to investigate whether CHR suppresses M1 macrophage polarization by maintaining mitochondrial homeostasis in the SA-AKI microenvironment, thereby elucidating its anti-inflammatory and renoprotective mechanisms.Methods: A SA-AKI model was established in C57BL/6 mice using cecal ligation and puncture(CLP), and mice were assigned to sham, CLP, and CHR treatment(CLP+CHR)groups. In vitro experiments were performed using human monocytic leukemia cells(THP-1) and human renal tubular epithelial cells(HK-2). Two cell models were established: 1) A Transwell co-culture system of M1 macrophages and lipopolysaccharide(LPS)-stimulated HK-2 cells treated with CHR to evaluate the overall protective effects of CHR in SA-AKI;and 2) a monoculture model of CHR-treated M1 macrophages to specifically assess its effects on mitochondrial homeostasis. Renal pathological alterations were examined by hematoxylin and eosin(HE) staining. Blood urea nitrogen(BUN) and serum creatinine(Cr)levels were measured to evaluate renal function. Enzyme-linked immunosorbent assay(ELISA) was used to quantify interleukin(IL)-6 and tumor necrosis factor-alpha(TNF-α)levels in serum and co-culture supernatants. HK-2 cell viability was assessed using cell counting kit-8(CCK-8), and apoptosis was evaluated by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay and Western blotting.Mitochondrial ultrastructure was observed by transmission electron microscopy. Adenosine triphosphate(ATP) levels, mitochondrial membrane potential(MMP), and nicotinamide adenine dinucleotide phosphate oxidized/reduced(NADP+/NADPH) ratios were measured.Quantitative polymerase chain reaction(qPCR) was performed to determine the mRNA expression of mitochondrial biogenesis-related genes, including peroxisome proliferatoractivated receptor gamma coactivator 1alpha(PGC-1α), mitochondrial transcription factor A(TFAM), nuclear respiratory factor 1(NRF1), and the M1 polarization marker cluster differentiation 86(CD86), with protein expression validated by Western blotting. CD86expression was further evaluated by immunofluorescence staining.Results: Compared with the sham group, mice in the CLP group exhibited marked renal tubular dilation, epithelial necrosis and detachment, tubular cast formation, and significantly increased renal injury scores(P<0.05), whereas CHR treatment markedly alleviated these pathological changes. In vitro, CHR significantly reversed the LPS-induced reduction in HK-2 cell viability(P<0.01). Serum levels of BUN, Cr, IL-6, and TNF-α were significantly elevated in the CLP group compared with the sham group(all P<0.01). CHR treatment significantly improved renal function, reduced pro-inflammatory cytokine levels in serum and co-culture supernatants(all P<0.05), and suppressed apoptosis in both tissues and cells. Transmission electron microscopy revealed disrupted mitochondrial cristae and blurred membrane structures in the CLP group, which were markedly restored following CHR treatment. CHR significantly increased the reduced ATP levels in the SA-AKI model(P<0.001), improved mitochondrial membrane potential in M1 macrophages, decreased NADP+/NADPH ratios, and restored mitochondrial redox balance(P<0.05). Western blotting and qPCR demonstrated that CHR significantly upregulated mitochondrial-related gene and protein expression in the injured model(all P<0.05), while significantly downregulating M1 macrophage marker expression at both the mRNA and protein levels(all P<0.05). Immunofluorescence analysis showed strong CD86 fluorescence intensity in the M1 group, which was significantly attenuated after CHR treatment(both P<0.05).Conclusion: CHR significantly alleviates renal pathological injury and improves renal function in SA-AKI model mice. Its anti-inflammatory and renoprotective effects may be associated with maintaining mitochondrial energy and redox homeostasis and suppressing macrophage M1 polarization.更多还原