Anti‑fibrotic effect of Sedum sarmentosum Bunge extract in kidneys via the hedgehog signaling pathway

Abstract

Sedum sarmentosum Bunge (SSBE) is a perennial plant widely distributed in Asian countries, and its extract is traditionally used for the treatment of certain inflammatory diseases. Our previous studies demonstrated that SSBE has marked renal anti‑fibrotic effects. However, the underlying molecular mechanisms remain to be fully elucidated. The present study identified that SSBE exerts its inhibitory effect on the myofibroblast phenotype and renal fibrosis via the hedgehog signaling pathway in vivo and in vitro. In rats with unilateral ureteral obstruction (UUO), SSBE administration reduced kidney injury and alleviated interstitial fibrosis by decreasing the levels of transforming growth factor (TGF)‑β1 and its receptor, and inhibiting excessive accumulation of extracellular matrix (ECM) components, including type I and III collagens. In addition, SSBE suppressed the expression of proliferating cell nuclear antigen, and this anti‑proliferative activity was associated with downregulation of hedgehog signaling activity in SSBE‑treated UUO kidneys. In cultured renal tubular epithelial cells (RTECs), recombinant TGF‑β1 activated hedgehog signaling, and resulted in induction of the myofibroblast phenotype. SSBE treatment inhibited the activation of hedgehog signaling and partially reversed the fibrotic phenotype in TGF‑β1‑treated RTECs. Similarly, aristolochic acid‑mediated upregulated activity of hedgehog signaling was reduced by SSBE treatment, and thereby led to the abolishment of excessive ECM accumulation. Therefore, these findings suggested that SSBE attenuates the myofibroblast phenotype and renal fibrosis via suppressing the hedgehog signaling pathway, and may facilitate the development of treatments for kidney fibrosis.

Figure 1.

SSBE inhibits hedgehog signaling activity and alleviates interstitial fibrosis in UUO kidneys. (A) H&E and Masson trichrome staining indicated marked kidney injury and excessive accumulation of total collagen in UUO kidneys, but SSBE administration alleviated this effect. Scale bar, 100 µm. (B) Enhanced mRNA expression levels of Col1α1, Col3α1, TGF-β1 and TGF-β1R in UUO kidneys, determined by reverse transcription quantitative polymerase chain reaction, were inhibited by SSBE treatment. (C) Immunochemical staining indicated upregulated expression of Col3α1 and TGF-β1 in UUO kidneys, which were alleviated following SSBE administration. Scale bar, 50 µm. (D) SSBE decreased PCNA expression in kidney tissues of UUO rats. Scale bar, 50 µm. (E) SSBE administration inhibited UUO-induced downregulated protein expression levels of Ptch1, and upregulated expression of Smo. Scale bar, 50 µm. (F) UUO decreased mRNA expression levels of Ptch1 and increased expression of Smo, but were inhibited by SSBE treatment. Data are presented as the mean ± standard error. *P<0.05, **P<0.01 vs. sham; ^#P<0.05, ^##P<0.01 vs. vehicle. H&E, hematoxylin and eosin; UUO, unilateral ureteral obstruction; Col1α1, type I collagen; Col3α1, type III collagen; TGF-β1, transforming growth factor-β1; TGF-β1R, transforming growth factor β1 receptor; SSBE, Sedum sarmentosum Bunge; PCNA, proliferating cell nuclear antigen; Ptch1, protein patched homolog 1; Smo, smoothened.

Figure 2.

SSBE inhibits extracellular matrix accumulation in TGF-β1-treated renal tubular epithelial cells. (A) Immunofluorescence staining indicated that SSBE treatment decreased TGF-β1-mediated downregulated expression of E-cadherin, and upregulated expression of α-SMA and Col3α1 in NRK-52E cells. Scale bar, 50 µm. (B) Reverse transcription quantitative polymerase chain reaction analysis demonstrated that mRNA expression levels of α-SMA, Col1α1, and Col3α1 were increased, and the expression of ZO-1 was decreased in TGF-β1-treated cells; however, this effect was ameliorated following SSBE treatment. TGF-β1, 5 ng/ml; SSBE10, 10 µg/ml; SSBE100, 100 µg/ml. Data are presented as the mean ± standard error. *P<0.05 vs. control; ^#P<0.05 vs. TGF-β1. SSBE, Sedum sarmentosum Bunge; Col1α1, type I collagen; Col3α1, type III collagen; TGF-β1, transforming growth factor-β1; α-SMA, α-smooth muscle actin; ZO-1, tight junction protein 1.

Figure 3.

SSBE inhibits TGF-β1-induced activation of hedgehog signaling in renal tubular epithelial cells. (A) SSBE inhibits TGF-β1-induced PCNA expression in NRK-52E cells. Scale bar, 50 µm. (B) SSBE inhibits TGF-β1-induced upregulated expression of Smo and Gli1 in NRK-52E cells, and downregulated expression of Ptch1. Scale bar, 50 µm. (C) The mRNA expression levels of Shh, Smo and Gli1 were increased, and the expression levels of Ptch1 were decreased, in TGF-β1-treated cells. This effect was inhibited following SSBE treatment. Data are presented as the mean ± standard error. TGF-β1, 5 ng/ml; SSBE10, 10 µg/ml; SSBE100, 100 µg/ml. *P<0.05 vs. control; ^#P<0.05, ^##P<0.01 vs. TGF-β1. SSBE, Sedum sarmentosum Bunge; TGF-β1, transforming growth factor-β1; Smo, smoothened; Shh, sonic hedgehog; Ptch1, protein patched homolog 1; Gli1, Gli family zinc finger 1; PCNA, proliferating cell nuclear antigen.

Figure 4.

SSBE inhibits AA-mediated activation of hedgehog signaling and extracellular matrix deposition. (A) SSBE inhibits AA-induced overexpression of Smo, α-SMA and Col3α1 protein in NRK-52E cells, as assessed by western blot analysis. GAPDH served as an internal control. (B) Quantification of TGF-β1, Col1α1 and α-SMA mRNA expression levels, as assessed by reverse transcription-quantitative polymerase chain reaction. (C) Quantification of Ptch1 and Smo mRNA expression levels. AA10, 10 µg/ml; SSBE10, 10 µg/ml; SSBE100, 100 µg/ml. Data are presented as the mean ± standard error. *P<0.05, **P<0.01 vs. control; ^#P<0.05 vs. AA10. SSBE, Sedum sarmentosum Bunge; TGF-β1, transforming growth factor-β1; Smo, smoothened; Ptch1, protein patched homolog 1; Col1α1, type I collagen; α-SMA, α-smooth muscle actin; AA, aristolochic acid.