Xian Huang Fang Inhibits the PKCδ/MAPK Signaling Pathway to Attenuate Renal Tubular Epithelial Cell Senescence and Thereby Delay Renal Interstitial Fibrosis

Yanru Wang,1– 4 Minghai Shao,1– 4 Tingting Li,1– 4 Xiu Du,1– 4 Kanjun Chen,5 Lin Xu1– 4 1Department of Nephrology, Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine, Shanghai, 201203, People’s Republic of China; 2Key Laboratory of Liver and Kidney Diseases, Ministry of Education, Shanghai University of Traditional Chinese Medicine, Shanghai, 201203, People’s Republic of China; 3TCM Institute of Kidney Disease, Shanghai University of Traditional Chinese Medicine, Shanghai, 201203, People’s Republic of China; 4Shanghai Key Laboratory of Traditional Chinese Clinical Medicine, Shanghai University of Traditional Chinese Medicine, Shanghai, 201203, People’s Republic of China; 5Department of Traditional Chinese Medicine, Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine, Shanghai, 201203, People’s Republic of ChinaCorrespondence: Lin Xu, Department of Nephrology, Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine, No. 528 Zhangheng Road, Pudong District, Shanghai, 201203, People’s Republic of China, Email [email protected]: The Chinese herbal medicine compound Xian Huang Fang (XHF) protects renal function and delays the progression of chronic kidney disease (CKD). Currently, XHF has been found to alleviate renal interstitial fibrosis, but the specific mechanism is still unclear, and its underlying molecular mechanism needs to be further elucidated.Purpose: The objective of this study was to investigate the protective effects of XHF against renal fibrosis and the underlying mechanisms.Methods: Chemical profiling of the reconstituted XHF freeze-dried powder was performed using ultra-high-performance liquid chromatography coupled with quadrupole-Orbitrap high-resolution mass spectrometry (UHPLC-Q-Orbitrap HRMS). Chronic renal failure model was established via 5/6 nephrectomy (A/I), and renal tubular epithelial (HK-2) cell fibrosis was induced by 5 ng/mL Transforming growth factor β 1 (TGF-β 1). Mechanisms were evaluated using network pharmacology, histopathology, Western blotting, immunofluorescence (IF), and senescence-associated β-galactosidase (SA-β-gal) staining.Results: XHF significantly ameliorated renal injury, reduced inflammatory infiltration in the renal interstitium, and effectively improved renal interstitial fibrosis (RIF) in 5/6 nephrectomy (A/I) model rats. Network analysis revealed Protein Kinase C delta (PKCδ) as a key target; XHF inhibited the phosphorylation of PKCδ (Tyr311), subsequently downregulating phosphorylated (p)-p38, p-ERK, p-JNK, and p-NF-κB both in vivo and in vitro. Furthermore, it upregulated the expression of the cell proliferation marker Ki67, while suppressing senescence markers (SA-β-gal activity, p21) and the senescence-associated secretory phenotype (SASP) factors (eg, IL-6).Conclusion: XHF alleviates renal interstitial fibrosis by inhibiting the PKCδ/MAPK signaling pathway, thereby reducing the senescence of renal tubular epithelial cells.Keywords: XHF, CKD, PKCδ, cellular senescence, SASP, network pharmacology