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Renal fibrosis is a crucial pathological change of chronic kidney disease (CKD) resulting in progressive loss of kidney function. Currently, therapeutic strategies for treating renal fibrosis remain limited. Jolkinolide B (JB) is a natural compound with anti-inflammatory activity from Euphorbia fischeriana Steud. We evaluated effect of JB on kidney fibrosis in mice with unilateral ureteral obstruction (UUO). JB (10 and 30 mg/kg) or vehicle was orally administered once daily for 3 days before occlusion, and then was continued for 10 days. Hematoxylin and eosin (H&E) and Masson stain showed that administration of JB (30 mg/kg) could reduce renal fibrosis and attenuate renal injuries. The results showed that JB reduced the release of inflammatory factors such as interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α, and decreased the expression of extracellular matrix proteins such as fibronectin (FN) and collagen I (Col I). Furthermore, treatment with JB down-regulated the protein levels of phosphorylated nuclear factor-kappaB p65 (p-NF-κB) p65, transforming growth factor-β1 (TGF-β1), p-Smad2/3, α-smooth muscle actin (α-SMA), vimentin and up-regulated the protein level of E-cadherin. The facts confirmed that JB might reduce renal fibrosis and protect the kidneys by inhibiting NF-κB and TGF-β1/Smad pathway coincident with epithelial-mesenchymal transition (EMT).