Hydroxysafflor Yellow A Ameliorates Renal Fibrosis by Suppressing TGF-β1-Induced Epithelial-to-Mesenchymal Transition

HSYA (98%, C₂₇H₃₂O₁₆, m.w. 612.53) was purchased from the Wuben Biological Technology Co (Xi’an, China). Recombinant Human transforming growth factor beta1 (TGF-β1) was purchased from Peprotech (USA); Anti-TGF-β1 antibody was purchased from Cell Signaling Technology (Beverly, MA, USA); Antibodies against smad7, a-SMA, fibronectin, and collagen-I were purchased from the Boster Co (Wuhan, China); Anti-phospho-smad3, anti-smad3,E-cadherin and anti-β-actin antibody were purchased from Biosynthesis Biotechnology Co (Beijing, China); SIS3 was purchased from Santa Cruz Biotechnology (USA).

Prior to the experiments, 30 health male mice were acclimatized for 1 week. They were housed in temperature-controlled conditions, humidity, lighting (12 h light/12 h dark cycle), with free access to food and water. UUO or sham surgery was performed under 5% chloralic hydrasanesthesia, and all effortsin a humane manner as literature described [19]: the right ureter was ligated with 4–0 silk at uretero pelvic junction through a right flank incision, Sham group mice had their ureters exposed and manipulated without ligation, and then the wound was closed in layers. The mice underwent surgery were randomly divided into five groups (six rats per group): sham group, UUO groups, HSYA (10, 50 and 100 mg/kg) treatment group. Mice were given HSYA as the corresponding dose by daily gastric gavage beginning the first day after surgical procedure. Sham group and UUO group were given identical voluminal saline. Both obstructed and contralateral kidneys were harvested 14 days after surgery and either stored at -80°Cor fixed with 4% paraformaldehyde.

The blood samples, obtained from the eyeball after14-day treatment, were centrifuged at 4000 rpm for 10 min then the separated serum was stored in Eppendorf tubes stored at -80°C. Serum creatinine (Scr) and blood urea nitrogen (BUN) levels were measured with7160 automatic biochemical detector (Hitachi Co., Tokyo, Japan).

The fixed renal tissues were dehydrated in graded alcohol and then embedded in paraffin. Sections were stained with hematoxylin & eosin (HE) to assess renal injury and Masson staining to assess the level of collagen deposition. Semi-quantitative evaluation was performed at ten non-overlapping vision fields that randomly selected under Olympus microscope (Olympus IX71, Japan)and photographed in each group.

Paraffin-embedded sections(5μm) were deparaffinized in xylene and hydrated in graded ethanol, washed with PBS, treated with 3% (v/v) H₂O₂ for 15 min, blocked with 10% (w/v) normal goat serum for 1 h, then sections were incubated at 4°C overnight with primary antibodies against a-SMA, and collagen-I, respectively. After washed with PBS, co-incubated with horseradish peroxides (HRP)-labeled goat anti-rabbit/mouse secondary antibody at 37°C for 30 min. DBA colorized for 15min, hematoxylin staining for 3 min, flush 2 min. After conventional dehydration, clearing and mounting with neutral gum, slices were observed under microscope. The Image Pro-Plus Software (Media Cybernetics, Rockville, MD) was employed to calculate the expression of a-SMA and collagen-I.

In vitro experiments were performed on HK-2 cells, which cultured in DMEM/F12 (1:1) with 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100μg/ml streptomycin in a cell incubator with 5% CO₂ at 37°C. Fresh growth medium was added to cells every 2 to 3d until confluent.

After digested with 0.25% trypsin, 5×10⁴cells were seeded into 96-well plates with complete medium and incubated at 37°Cin 5% CO₂ overnight. Then HK-2 divided into seven groups:Control group, TGF-β1 (5ng/ml) group, HSYA (1, 10, 50, 100 and 200μg/ml)group. Three time points12h, 24h and 48h were applied to value the viability. MTT (1 mg/ml) solution 100ul was added into each well and then incubated for 4 h in an incubator. Thereafter, the medium was removed from each well before added with 150μl of DMSO, and then shaken for l0 min to dissolve the resulting blue crystals. The absorbance was measured at 570 nm using Microplate Reader (Thermo Fisher Scientific, Shanghai, China). Cell viability was expressed as a percentage of the control (S4 Table).

HK-2 cells cultured on coverslips were washed with sterile cold PBS three times and then fixed in 4% paraformaldehyde for 15 min and permeabilized with 0.5% TritonX-100 in PBS for 30 min at room temperature. After fixation, slides were blocked with 1% bovine serum albumin (BSA) for 1 h before washing with PBS. Subsequently slides were incubated with the primary antibody, diluted in 1%BSA overnight at 4°C. After washing, cells were incubated with the appropriate FITC-conjugated secondary and should be lucifugal. Finally, slides were mounted and analyzed by fluorescence microscopy.

HK-2 cells were lysed by RIPA lysis buffer containing protease inhibitor PMSF on ice. Then the lysates was collected after centrifugation at 10000 rpm at 4°C for 20 min. Protein concentration were tested with a Coomassie brilliant blue protein quantitative kit (Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer’s instructions, and then heated with sample buffer at 100°C for 5 min before loading and separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) on 12% polyacrylamide gels. The proteins were electro transferred to a nitro-cellulose (NC) membrane in transfer buffer for 30min. In order to prevent nonspecific background binding, the membraneswere incubated with 5% non-fat milk in Tris-buffered saline with 0.1% Tween 20 (TBST) at room temperature for 2 h. The immunoblots incubated with various primary antibodies in blocking buffer (containing 5% bovine serum albumin, 0.1% Tween 20 in 0.1 TBS, pH 7.4) at the dilutions specified by the manufacturers overnight at 4°C, following three washes with TBST, the membranes were incubated with horseradish peroxidase-conjugated secondary anti-body for 1 h at 37°C. And then an ECL detection kit was used to detect the bound antibodies. Results were normalized to β-actin and quantified with image analysis systems (Bio-Rad, USA).

All experiments were repeated as mean ± standard deviation (SD). Multiple comparisons were examined for significant differences using ANOVA, followed by individual comparison with the Tukey’ s posthoctest, with P<0.05 indicating statistical significant difference.

Scr and BUN are the classical indicators of renal function. The data (S1 Table) showed that Scr and BUN were markedly elevated in the UUO group compared with the sham group and reduced by HSYA. Indicating that UUO model had been successfully established and HSYA exerted a protective effect on renal injury and dysfunction (Fig 1).

From HE staining, we observed the morphological changes in the UUO model, including tubular atrophy, inflammatory cell infiltration, segmental thickening of glomerular basement membranes, excessively deposited mesangial matrix, and renal interstitial fibrosis remarkable. However, treatment with HSYA attenuated these ultrastructural abnormalities in a dose-dependent manner. Masson staining showed that collagen deposition and fibrosis area were significantly decreased in HSYA treatment groups than in UUO group (Fig 2).

It has been acknowledged that a-SMA and collagen-I are hallmarks of myofibroblasts and excessive extracellular matrix accumulation[20]. Immunohistochemistry and semi-quantitative data (S2 Table) demonstrated that a-SMA and collagen-I were increased in the UUO group and partly reversed after treatment with HSYA. Furthermore, the changes were more obvious in the high-dose group (Fig 3).

To investigate whether HSYA inhibits TGF-β1-induced EMT in renal tubule epithelial cells, we firstly observed the toxicity of HSYA at different concentrations (Fig 4A).Then HK-2 cells were stimulated by human recombinant TGF-β1 (5ng/ml), and treated with different concentrations of HSYA. Cell proliferation was significantly inhibited by HSYA in a dose-dependent manner, and the phenomenon was more prominent at 200μg/ml after 24 hours. Then 200μg/ml was applied in the following experiment (Fig 4B).

Due to the fact that the progression of EMT paves the way for extracellular matrix (ECM) deposition and finally deteriorate renal fibrosis, we detected the expression of a-SMA, collagen-I and fibronectin by Immunofluorescence. Results showed that the level of a-SMA, collagen-I and fibronectin were notable with TGF-β1 stimulation. However, HSYA treatment obviously down regulated a-SMA and collagen-I protein expression, and slight down regulation of fibronectin (Fig 5A–5C).We further evaluated the expression of fibronectin and collagen-I via Western blot. Results showed that both fibronectin and collagen-I protein were higher in TGF-β1-stimulated group than in control group. HSYA treatment significantly ameliorated TGF-β1-induced increase of fibronectin and collagen-I expression (Fig 5D and 5E). These results indicated that HSYA could prevent the expression of the fibroblast marker and ECM in HK-2 cells, thus slow down the progression of TGF-β1-induced EMT.

It is well-known that cell apoptosis involved in the injury and repair progress of multiple kidney disease. Growing evidence showed that TGF-β1/Smads signaling could mediate cell apoptosis by stimulating expression of pro-apoptotic members and by suppressing expression of anti-apoptotic members, respectively [21]. Here, we test the expression of pro-apoptotic Bax, anti-apoptotic Bcl-2, and cleaved caspase3 by Western blotting in HK-2 cells. Data (S5 Table) indicated that Bax and caspase3 protein exhibited higher expression in TGF-β1-stimulated group. While, Bcl-2 expression was lower in TGF-β1-stimulated group than in control group. The expression of Bax, Bcl-2 and caspase3 were all reversed in HSYA group. These phenomena suggested that HSYA could reduce cell apoptosis via regulating the ratio of Bax and Bcl-2, down-regulating the expression of cleaved caspase3. (Fig 6)

We then test the effect of HSYA on TGF-β1/Smad3 signaling, which have been confirmed a central role to regulate interstitial fibrosis [22, 23].The level of TGF-β1 was down-regulated compare with the model group, and the ratio of p-Smad3 to Smad3 was decreased in the HSYA-treated groups relative to the model group in a dose-dependent manner (Fig 7 and S3 Table). The semblable effect was identified in HK-2cells. Administration with HSYA notably reduced the high expression of TGF-β1 and p-Smad3 stimulated by TGF-β1. In addition, the negative regulator, Smad7 was up-regulated by HSYA (Fig 8). These results suggested that activation of the TGF-β1/Smads signaling pathway plays a prominent role in the progress of renal fibrosis and it was meaningful that HSYA could rebalance the TGF-β1/Smad ssignaling to exhibiting antifibrotic effect.

E-cadherin (the epithelial cell marker) and α-SMA (a specific myofibroblast marker) are usual biomarker of EMT. To further demonstrate the role of HSYA in the EMT process through TGF-β1/smads pathway, we treated HK-2withHSYA and SIS3 (5μM) after stimulated by human recombinant TGF-β1 (5ng/ml)and then to measure the expression of a-SMA and E-cadherin (Fig 9). The results showed that the expression level of a-SMA was reduced and level of E-cadherin was increased in treatment group of HSYA and SIS3, indicating that TGF-β1–induced EMT was reversed by inhibiting the smad3 signaling pathway (S6 Table). This suggested that activation of the TGF-β1/smad3 pathway appears to play a prominent role in the antifibrotic effect of HSYA.

All relevant data are within the paper and its Supporting Information files.