Baicalin ameliorates renal fibrosis via inhibition of transforming growth factor β1 production and downstream signal transduction

Abstract

Previous studies have demonstrated the potential antifibrotic effects of baicalin in vitro, via examination of 21 compounds isolated from plants. However, its biological activity and underlying mechanisms of action in vivo remain to be elucidated. The present study aimed to evaluate the effect of baicalin on renal fibrosis in vivo, and the potential signaling pathways involved. A unilateral ureteral obstruction (UUO)‑induced renal fibrosis model was established using Sprague‑Dawley rats. Baicalin was administrated intraperitoneally every 2 days for 10 days. The degree of renal damage and fibrosis was investigated by histological assessment, and detection of fibronectin and collagen I mRNA expression levels. Epithelial‑mesenchymal transition (EMT) markers, transforming growth factor-β1 (TGF-β1) levels and downstream phosphorylation of mothers against decapentaplegic 2/3 (Smad2/3) were examined in vivo and in an NRK‑52E rat renal tubular cell line in vitro. Baicalin was demonstrated to markedly ameliorate renal fibrosis and suppress EMT, as evidenced by reduced interstitial collagen accumulation, decreased fibronectin and collagen I mRNA expression levels, upregulation of N‑ and E‑cadherin expression levels, and downregulation of α‑smooth muscle actin and vimentin expression. Furthermore, baicalin decreased TGF‑β1 expression levels in serum and kidney tissue following UUO, and suppressed Smad2/3 phosphorylation in rat kidney tissue. In vitro studies identified that baicalin may inhibit the phosphorylation of Smad2/3 under the same TGF‑β1 concentration. In conclusion, baicalin may protect against renal fibrosis, potentially via inhibition of TGF‑β1 production and its downstream signal transduction.

Figure 1.

Baicalin attenuates UUO-induced renal injury and fibrosis. (A) Representative images of H&E and Sirius red staining of kidney tissue in the sham, UUO and UUO + baicalin groups. (B) Quantification of tubular injury scores and tubulointerstitial collagen areas. (C) Fold-changes of mRNA expression levels of fibronectin and collagen I in the renal tissue of all groups. Data are expressed as the mean ± standard deviation. *P<0.01; ***P<0.001. UUO, unilateral ureteral obstruction; H&E, hematoxylin and eosin.

Figure 2.

Baicalin treatment inhibits epithelial-mesenchymal transition in kidney tissue, as assessed by immunohistochemistry. (A) Representative images of N- and E-cadherin, vimentin, and α-SMA staining in kidney sections from sham, UUO and UUO + baicalin groups. (B) Percentages of positive areas of N- and E-cadherin, vimentin and α-SMA. Data are expressed as the mean ± standard deviation. *P<0.05; ***P<0.001. UUO, unilateral ureteral obstruction; N, neural; E, epithelial; α-SMA, α-smooth muscle actin.

Figure 3.

Baicalin treatment inhibits epithelial-mesenchymal transition in kidney tissue, as assessed by western blotting. (A) Representative western blot images demonstrating the expression of N- and E-cadherin, vimentin, and α-SMA in kidney tissue from the sham, UUO and UUO + baicalin groups. (B) The ratio of N- and E-cadherin, vimentin, and α-SMA expression levels to GAPDH. Data are expressed as the mean ± standard deviation. *P<0.05; **P<0.01; ***P<0.001. UUO, unilateral ureteral obstruction; α-SMA, α-smooth muscle actin; N, neural; E, epithelial.

Figure 4.

Baicalin treatment inhibits TGF-β1 expression levels and phosphorylation of Smad2/3 in vivo. (A) Quantification of serum TGF-β1 levels in the sham, UUO and UUO + baicalin groups, as measured by ELISA. (B) Fold-change of mRNA expression levels of TGF-β1 in renal tissue. (C) Representative western blot images of p-Smad2, p-Smad3 and Smad2/3 expression in sham, UUO and UUO + baicalin groups. GAPDH served as an internal control. (D) The ratio of p-Smad2/3 to Smad2/3. Data are expressed as the mean ± standard deviation. *P<0.05; **P<0.01; ***P<0.001. UUO, unilateral ureteral obstruction; TGF-β1, transforming growth factor-β1; p, phosphorylated; Smad, mothers against decapentaplegic.

Figure 5.

Baicalin treatment inhibits TGF-β1 signaling in a NRK-52E rat renal tubular epithelial cell line in an in vitro TGF-β1-induced fibrosis model. (A) Representative western blot images of p-Smad2, p-Smad3 and Smad2/3 expression in control, TGF-β1 and TGF-β1 + baicalin groups. GAPDH served as an internal control. (B) The ratio of p-Smad2/3 to Smad2/3 expression levels. (C) Representative western blot images of N- and E-cadherin, vimentin and α-SMA expression. (D) Quantification of the ratio of N and E-cadherin, vimentin, and α-SMA expression levels to GAPDH. Data are expressed as the mean ± standard deviation. *P<0.05; **P<0.01; ***P<0.001. α-SMA, α-smooth muscle actin; N, neural; E, epithelial; p, phosphorylated; Smad, mothers against decapentaplegic; α-SMA, α smooth muscle actin; TGF-β1, transforming growth factor-β1.

Figure 6.

Schematic diagram of the potential mechanisms of action of baicalin on the amelioration of renal fibrosis. The binding of TGF-β1 to its receptor triggers Smad2/3 phosphorylation. Once combined with Smad4, the Smad complex is formed, which later translocates to the nucleus to regulate target gene transcription, resulting in fibrogenesis. Baicalin ameliorates renal fibrosis via inhibition of TGF-β1 production and its downstream signal transduction. Smad, small mothers against decapentaplegic; TGF-β1, transforming growth factor β1; p, phosphorylated.