β-Mangostin Alleviates Renal Tubulointerstitial Fibrosis via the TGF-β1/JNK Signaling Pathway

Abstract

The epithelial-to-mesenchymal transition (EMT) plays a key role in the pathogenesis of kidney fibrosis, and kidney fibrosis is associated with an adverse renal prognosis. Beta-mangostin (β-Mag) is a xanthone derivative obtained from mangosteens that is involved in the generation of antifibrotic and anti-oxidation effects. The purpose of this study was to examine the effects of β-Mag on renal tubulointerstitial fibrosis both in vivo and in vitro and the corresponding mechanisms involved. As shown through an in vivo study conducted on a unilateral ureteral obstruction mouse model, oral β-Mag administration, in a dose-dependent manner, caused a lesser degree of tubulointerstitial damage, diminished collagen I fiber deposition, and the depressed expression of fibrotic markers (collagen I, α-SMA) and EMT markers (N-cadherin, Vimentin, Snail, and Slug) in the UUO kidney tissues. The in vitro part of this research revealed that β-Mag, when co-treated with transforming growth factor-β1 (TGF-β1), decreased cell motility and downregulated the EMT (in relation to Vimentin, Snail, and N-cadherin) and phosphoryl-JNK1/2/Smad2/Smad3 expression. Furthermore, β-Mag co-treated with SB (Smad2/3 kinase inhibitor) or SP600125 (JNK kinase inhibitor) significantly inhibited the TGF-β1-associated downstream phosphorylation and activation of JNK1/2-mediated Smad2 targeting the Snail/Vimentin axis. To conclude, β-Mag protects against EMT and kidney fibrotic processes by mediating the TGF-β1/JNK/Smad2 targeting Snail-mediated Vimentin expression and may have therapeutic implications for renal tubulointerstitial fibrosis.

Keywords: EMT; JNK1/2; Smad2; TGF-β1; renal tubulointerstitial fibrosis; β-mangostin.

Figure 1

Histopathological examination of kidney tissue samples from the unilateral ureteral obstruction (UUO) mice with or without being subjected to β-Mag treatment. (A) Summary of the in vivo experiment. The C57BL/6 mice underwent a UUO or sham operation on the first day. Subsequently, these mice were orally administered β-Mag (either 10 or 20 mg per kilogram of body weight daily). After drug therapy for 7 consecutive days, the kidney tissues of each mouse were harvested for histological and protein analyses. The grouping details are further described in the text. (B) The gross appearance, Hematoxylin and Eosin (H&E) staining, Masson trichrome stain for collagen I fiber, and immunohistochemical staining for α-smooth muscle actin (α-SMA), collagen I, and Vimentin in each study group. (C–E) Quantitative results of the histological findings in (B). Results are shown as % of protein expression of each experiment group. ** p < 0.01 when compared to the sham group; # p < 0.05 when compared to the UUO group.

Figure 2

The inhibitory effect of β-Mag administration on the relative protein expression of EMT-associated markers and fibrosis-related markers in the unilateral ureteral obstruction (UUO) model. Western blot analysis regarding the protein expression of collagen I, α-SMA, Vimentin, N-cadherin, Snail, and Slug, for which glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. The histograms reveal the relative protein expression. Results are shown as relative fold change in protein expression in comparison to sham, where the expression level was set as 1. ** p < 0.01 when compared to the sham group; # p < 0.05 when compared to the UUO group.

Figure 3

Effect of β-Mag treatment on TGF-β1-induced cell growth and motility in cultured human HK2 cells in vitro. (A) The results of the MTT assay, revealing the changes in cell proliferation after treatment with different concentrations of β-Mag. (B) Cell growth under the 24 h co-treatment with TGF-β1 and β-Mag at concentrations of 0, 2, and 4 µM. (C) The scratch wound healing assay demonstrated that β-Mag inhibited the cell migrating ability induced by TGF-β1. ** p < 0.01 when compared to the control group; # p < 0.05 when compared to the TGF-β1 group.

Figure 4

β-Mag inhibits TGF-β1-induced EMT-associated markers and fibrosis-related markers in HK2 cells. HK2 cells were co-treated with β-Mag (2 and 4 μM) and TGF-β1 for 24 h. (A) Western blot analysis comparing protein expression of collagen I and α-SMA between groups. (B) Western blot analysis comparing protein expression of Vimentin, N-cadherin, Snail, and Slug between groups. GAPDH was used as a loading control. (C) Immunofluorescence staining of Snail, Vimentin, and N-cadherin between treatment groups. Results are shown as relative fold change in protein expression in comparison to control, where the expression level was set as 1. ** p < 0.01 when compared to the control group; # p < 0.05 when compared to the TGF-β1 group. N.S, not significant. Scale bar: 20 μm.

Figure 5

The Smad2/Smad3 pathway acting as a major target of β-Mag in the mediation of renal EMT. (A) HK2 cells were co-treated with β-Mag (2 and 4 μM) and TGF-β1 for 24 h. Then, Western blot analysis was conducted to demonstrate the expression of p-Smad2, p-Smad3, t-Smad2, and t-Smad3. (B) HK2 cells were added with or without SB431542 (SB, 10 μM), combined with or without TGF-β1, along with β-Mag (4 μM), and then the expression of p-Smad2, t-Smad2, Vimentin, and Snail was measured after β-Mag administration. GAPDH was used as a loading control. (C) Immunofluorescence staining of Snail and Vimentin between the treatment groups. Results are shown as relative fold change in protein expression in comparison to control, where the expression level was set as 1. ** p < 0.01 when compared to the control group; # p < 0.05 when compared to the TGF-β1 group. Scale bar: 20 μm.

Figure 6

The effect of β-Mag on TGF-β1-induced JNK activation and the triggering of p-Smad2/Snail targeting Vimentin expression in HK2 cells. (A) HK2 cells were co-treated with β-Mag (2 and 4 μM) and TGF-β1 for 24 h. Then, using Western blot analysis, we determined the expression of MAPKs (p-MEK1/2, t-MEK1/2, p-ERK1/2, t-ERK1/2, p-p38, t-p38, p-JNK1/2, and t-JNK1/2). (B) HK2 cells were added with or without SP600125 (SP, 10 μM), combined with or without TGF-β1, along with β-Mag (4 μM), and the expression of p-JNK1/2, t-JNK1/2, p-Smad2, t-Smad2, p-Smad3, t-Smad3, Vimentin, N-cadherin, and Snail was measured using Western blotting. The histogram results are presented as the average fold change in the level of each protein normalized to GAPDH. (C) Immunofluorescence staining of Snail and Vimentin between the treatment groups. (D) After transfecting the JNK plasmid (Ov-JNK) in HK2 cells for 24 h, we added β-Mag for another 24 h and detected the protein expression of p-JNK1/2, Vimentin, and Snail. GAPDH was used as loading control. Results are shown as relative fold change in protein expression in comparison to control, where the expression level was set as 1. ** p < 0.01 when compared to the control group; # p < 0.05 when compared to the TGF-β1 group. N.S, not significant. Scale bar: 20 μm.

Figure 7

Summary of the proposed mechanism of action for β-Mag in suppressing renal tubulointerstitial fibrosis.