Astragalosides were purchased from Shanghai Yuanye Biotechnology Co., Ltd (Shanghai, China). Rat MCs (No. HBZT-1) were obtained from Wuhan Boster Biological Technology, Ltd. (Wuhan, China). 5-Bromo-2′-deoxyuridine (BrdU) kit was purchased from Beyotime Institute of Biotechnology (Shanghai, China). The TRIzol kit and cDNA reverse transcription kit were from Takara Bio, Inc. (Otsu, Japan). The rat TGF-β1, CTGF, FN and colIV enzyme-linked immunosorbent assay (ELISA) kits were purchased from R&D Systems, Inc. (Minneapolis, MN, USA).

MCs were cultured in Gibco Dulbecco's modified Eagle's medium (DMEM; Thermo Fisher Scientific, Inc., Waltham, MA, USA) that was supplemented with 100 µg/ml streptomycin, 100 U/ml penicillin (both purchased from Beyotime Institute of Biotechnology) and 10% (v/v) fetal bovine serum (Hangzhou Sijiqing Biological Engineering Materials Co., Ltd., Hangzhou, China). The cells were incubated at 37°C in a humidified atmosphere of 5% CO₂. During the experiments, the cells were first exposed to a normal concentration of glucose [5.56 mmol/l; normal glucose (NG) group] for 4 h, and then treated with high glucose (HG group; 30 mmol/l glucose), high glucose with 50 µg/ml astragalosides (ATSL group), high glucose with 100 µg/ml astragalosides (ATSM group), and high glucose with 200 µg/ml astragalosides (ATSH group). Mannitol (MA group) was used as a control to rule out the effect of osmotic pressure. The cells were harvested for analysis after 24 and 48 h of treatment.

Cell proliferation was examined by BrdU assay. MCs were seeded into 96-well plates at a density of 1.0×10⁶ per well. After 24 and 48 h, BrdU solution was added and the MCs continued to be cultured for another 2 h. Absorbance was read at 450 nm by visible spectrometry (CliniBio 128C; Biochrom, Ltd., Cambridge, UK).

An RT-PCR procedure was performed to determine the relative mRNA quantities of TGF-β1, CTGF, ColIV and FN in MCs. Total RNA was extracted from the MCs with TRIzol reagent according to the manufacturer's protocol. The total RNA (0.5 µg) obtained was converted into cDNA using the RevertAid™ First Strand cDNA Synthesis kit with Random Hexamer primers (Thermo Fisher Scientific, Inc.). The upstream and downstream primers (Shanghai Sangon Biotech Co., Ltd., China) of these genes are shown in Table I. The reaction mixture was incubated at 48°C for 45 min for reverse transcription, and then underwent cycling. The cycling conditions of these genes were: Initial denaturation for 5 min at 94°C, 30 cycles at 94°C for 30 sec, 60°C for 30 sec, 72°C for 1 min, and final elongation at 72°C for 7 min. The RT-PCR products were separated by 2% agarose electrophoresis, and the band densities were analyzed using laser densitometry (Tanon-1600R; Tanon Science & Technology Co., Ltd., Shanghai, China). The relative quantities of mRNA of these four genes in the MCs were represented by the ratio of the band density of objective gene relative to that of β-actin.

Levels of TGF-β1, CTGF, ColIV and FN in the supernatants of the MCs were determined by ELISA. The MC culture medium was collected and centrifuged at 13,000 × g for 15 min to pelletize the debris. The levels of these four proteins were determined according to the kit manufacturer's protocol. The colorimetric reaction was measured at 450 nm.

Data are expressed as the mean ± standard deviation. Statistical analyses were performed using the paired t-test for the comparison of two groups and one-way analysis of variance with Dunnett's test for the comparison of multiple groups. A value of P<0.05 was considered statistically significant.

A BrdU assay was employed to evaluate the impact of AST on MC proliferation. As shown in Fig. 1, the proliferation of MCs was increased after 24 and 48 h in the presence of a high concentration of glucose (HG group vs. NG group, P<0.01). The cell proliferation rates in the ASTL, ASTM and ASTH groups were significantly lower than those in the HG group (P<0.05 for ASTL; P<0.01 for ASTM and ASTH). Cell viability in the MA group was almost identical to that in the NG group, indicating no evident impact of the osmotic pressure generated by high glucose. The above results indicate that the administration of AST significantly suppressed high glucose induced-MC proliferation.

The RT-PCR detection results showed that the mRNA levels of TGF-β1, CTGF, ColIV and FN in the HG group were elevated compared with those of the NG group (P<0.01). At 24 h (representative results, Fig. 2A), the TGF-β1, CTGF and FN mRNA levels in the groups treated with 100 or 200 µg/ml AST were all lower than those of the HG group (Fig. 2B, C and E). The ColIV mRNA levels in the group treated with 50 µg/ml AST were also decreased compared with those in the HG group (Fig. 2D). At 48 h, the four types of mRNA in the ASTL, ASTM and ASTH groups were notably downregulated compared with those in the HG group (Fig. 3). No significant changes in the levels of TGF-β1, CTGF, ColIV and FN mRNA were observed between the MA group and the NG group (Figs. 2 and 3). These results indicate that the administration of AST significantly decreased the high glucose-induced increases of TGF-β1, CTGF, ColIV and FN mRNA levels.

The levels of TGF-β1, CTGF, ColIV and FN in the cell culture supernatants of the MCs in the HG group were markedly upregulated compared with those of the NG group (P<0.01, Figs. 4 and 5). No significant difference between the NG group and the MA group was observed with regard to the secretion of these four proteins (Figs. 4 and 5). These results indicate that the administration of AST significantly reduced the high glucose-induced secretion of TGF-β1, CTGF, ColIV and FN proteins.