Chrysophanol, a main anthraquinone from Rheum palmatum L. (rhubarb), protects against renal fibrosis by suppressing NKD2/NF-κB pathway

Chronic kidney disease (CKD), characterized as renal dysfunction and multi-system damage, has become a serious public health problem with high prevalence and mortality. Rheum palmatum L. (rhubarb) is one of the most widely used Chinese herb with renal protective activity. However, the active components and underlying mechanisms of rhubarb remain unknown. In this work, we tried to explore the pharmacological mechanism of chrysophanol, a main anthraquinone from rhubarb, against CKD by in vivo and in vitro models. The therapeutic effect of chrysophanol and its underlying mechanism were investigated using CKD mouse model induced by unilateral ureteral occlusion (UUO), and human kidney 2 (HK-2) cells stimulated by TGF-β1 in vivo. The impact of chrysophanol on renal function, inflammation, fibrosis of CKD mice were evaluated. Then, the protein expressions of FN1, collagen ɑI, α-SMA, NF-κB and naked keratinocyte homolog 2 (NKD2) were investigated. In vitro studies, the inhibition on inflammation and fibrogenesis by chrysophanol was further validated in TGF-β1-stimulated HK2 cells, and the regulation of chrysophanol on NKD2/NF-κB pathway was analyzed. Moreover, NKD2 was overexpressed in HK-2 cells to confirm the role of NKD2/NF-κB pathway in chrysophanol-mediated efficacy. Finally, the binding mode of chrysophanol with NKD2 was studied using in silico molecular docking and microscale thermophoresis (MST) assay. Chrysophanol could significantly improve the kidney dysfunction, alleviate renal pathology, and reverse the elevated levels of renal fibrosis markers such as FN1, collagen ɑI and α-SMA. Furthermore, chrysophanol effectively inhibited TNF-α, IL-6, and IL-1β production, and suppressed NF-κB activation and NKD2 expression. The findings of in vitro study were consistent with those of animal expriment. Using NKD2-overexpressing HK-2 cells, we also demonstrated that overexpression of NKD2 significantly compromised the anti-fibrotic effects of chrysophanol. In addition, molecular docking and MST analysis revealed that NKD2 was a direct target of chrysophanol. Together, our work demonstrated for the first time that chrysophanol could effectively ameliorate renal fibrosis by inhibiting NKD2/NF-κB pathway. Chrysophanol can potentially prevent CKD by suppressing renal NKD2 expression directly.