[Chrysophanol alleviates sepsis-associated acute kidney injury by maintaining mitochondrial homeostasis and inhibiting M1 macrophage polarization].

OBJECTIVES: 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 proliferator-activated 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. CD86 expression 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).

CONCLUSIONS: 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.