Rhubarb was purchased from Tong Ren Tang Technologies Co., Ltd (http://www.tongrentangkj.com). Rg was extracted from rhubarb according to the method from a previous study (9).

HMCs were purchased from ScienCell Research Laboratories, Inc. and cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc.) with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.), penicillin (100 U/ml) and streptomycin (100 mg/ml). High glucose culture media was made by supplementing normal DMEM medium with additional D-glucose at a final concentration of 25 mM (HG). All of these cells were maintained at 37˚C with 5% CO₂.

Cells were seeded in the six-well plates at a density of 5x10⁵ cells per well and treated with Rg (20 or 80 µM) or transfected with lincRNA ANRIL small interfering (siRNA; 50 nM), let-7a mimics (50 nM) or negative control siRNA (50 nM) using Lipofectamine^® 3000 transfection reagent (Invitrogen; Thermo Fisher Scientific, Inc.) and cultured using HG DMEM medium according to the manufacturer's protocol at 37˚C for 48 h. The lincRNA ANRIL siRNA sequence was 5'-GGUCAUCUCAUUGCUCUAU-3', and let-7a mimics sequence was 5'-UGAGGUAGUAGGUUGUAUAGUU-3', and negative control siRNA sequence was 5'-UUCUCCGAACGUGUCACGUTT-3'. The lincRNA ANRIL siRNA, let-7a mimics and negative control were synthesized by Shanghai GenePharma Co., Ltd.

Cell proliferation was detected using a Cell Counting Kit-8 (CCK-8; Dojindo Molecular Technologies, Inc.) as previously described (10).

Apoptosis was detected using flow cytometry. In apoptosis assay, harvested cells were double-stained with Annexin V (room temperature for 15 min) and propidium iodide (PI; room temperature for 5 min) according to the protocol of a FITC-Annexin V cell apoptosis assay kit (BD Biosciences). Then the cells were analyzed using a flow cytometer (FACScan; BD Biosciences) equipped with CellQuest pro software (v5.2, BD Biosciences).

RNA of cells was extracted using a Total RNA Rapid Extraction kit (BioTeke Corporation) according to the manufacturer's protocol. After detecting the concentration, 1 µg RNA sample was reverse transcribed into cDNA with M-MLV reverse transcriptase (BioTeke Corporation) in the presence of oligo(dT) and 50 random primers (Invitrogen; Thermo Fisher Scientific, Inc.). The RT procedure is 42˚C for 60 min, and 70˚C for 10 min. The instruments used for this experiment were pre-treated using surface RNase Erase (Tiandz, Inc.) and the reagents were RNase-free. The cDNA (1 µl each reaction) was used for real-time PCR to detect the gene expressions using 2X Power Taq PCR MasterMix (BioTeke Corporation) and SYBR Green (Beijing Solarbio Science & Technology Co., Ltd.), with GAPDH as the internal control. The PCR procedure was as follows: 95˚C for 10 min, 38 cycles of 95˚C for 12 sec, 60˚C for 18 sec and 72˚C for 30 sec, and finally 4˚C for 5 min. Calculations were performed using the 2^-ΔΔCq method (11). The following primer pairs were used for amplification: ANRIL forward, 5'-GGACTACAGATGCACCACCAT-3', ANRIL reverse, 5'-TGAGCACTGTGTCCATAGCA-3'; GAPDH forward, 5'-AAATCCCATCACCATCTTCCAG-3', and GAPDH reverse, 5'-GAGTCCTTCCACGATACCAAAGTTG-3'.

Hairpin-it^™ let-7a RT-qPCR Primer Set (Shanghai GenePharma Co., Ltd.) was used for the measurement of the relative quantity of let-7a. The reaction conditions were as follows: 95˚C for 4 min, 30 cycles of 95˚C for 30 sec, 57˚C for 30 sec and 72˚C for 30 sec. The mRNA expression of let-7a was normalized to the endogenous expression of U6. The primer sequences were as follows: Let-7a forward: 5'-CACCCACCACTGGGAGATAAC-3', and let-7a reverse, 5'-TATGGTTGTTCACGACTCCTTCAC-3'; U6 forward: 5'-GCTTCGGCAGCACATATACTAAAAT-3', and U6 reverse, 5'-CGCTTC.ACGAATTTGCGTGTCAT-3'.

Protein was extracted using a whole-cell lysis kit (CWBio) from cells and the concentration of protein was measured using a BCA protein quantitative kit (Beyotime Institute of Biotechnology). After being denatured by boiling, the protein sample (40 µg for each lane) was separated by 10% SDS-PAGE and transferred to a PVDF membrane (EMD Millipore). After blocking with 5% skim milk (Inner Mongolia Yili Industrial Group Co., Ltd.) at room temperature for 1 h, the membrane was incubated with the primary antibodies at 4˚C overnight. After rinsing with TBS with Tween-20, the membrane was incubated with goat anti-rabbit Ig G labeled with horseradish peroxidase (HRP; cat. no. sc-2004; 1:5,000; Santa Cruz Biotechnologies, Inc.) or goat anti-mouse Ig G-HRP (cat. no. sc-2005; 1:5,000; Santa Cruz Biotechnologies, Inc.) at 37˚C for 45 min, and exposed with ECL reagent (Thermo Fisher Scientific, Inc.). Optical density values of bands were analyzed using a gel image processing system ImageLab software (version: 3.0, Bio Rad Laboratories, Inc.). The primary antibodies were as follows: Bcl-2 antibody (cat. no. ab185002; 1:1,000; Abcam); cleaved caspase-3 antibody (cat. no. ab2302; 1:1,000; Cell Signaling Technology, Inc.); TGF-β1 antibody (cat. no. ab92486; 1:1,000; Abcam); phosphorylated (p)-Smad2 antibody (cat. no. ab188334; 1:1,000; Abcam); Smad2 antibody (cat. no. ab33875; 1:1,000; Abcam); Smad7 antibody (cat. no. ab216428; 1:1,000; Abcam); and β-actin antibody (cat. no. ab179467; 1:1,000; Abcam).

Statistical analyses were performed using GraphPad Prism 6 (GraphPad Software, Inc.). The data in the present study are presented as the mean ± SD of three or five individual experiments, and analyzed by one-way ANOVA followed by Tukey's multiple comparison test. P<0.05 was considered to indicate a statistically significant difference.

HMCs were cultured in HG (25 mM) DMEM for 24 h, and then treated with Rg for 48 h. The results showed that HG significantly induced the apoptosis of HMCs, and 20 and 80 µM Rg could both inhibit the apoptosis induced by HG (Fig. 1A and B). Using a Cell Counting Kit-8 assay, it was also observed that HG significantly suppressed the cell proliferation of HMCs, and 20 and 80 µM Rg could both promote the cell growth, which was inhibited by HG (Fig. 1C).

A previous study reported that HG and diabetes could upregulate lincRNA ANRIL in human retinal endothelial cells and in the retina, and lincRNA ANRIL could also regulate vascular endothelial growth factor expression (12). The present study examined whether HG regulates the expression of lincRNA ANRIL in HMCs, and whether Rg inhibits the apoptosis of HMCs induced by HG through lincRNA ANRIL. The present study demonstrated that HG significantly upregulated the expression of lincRNA ANRIL, and 20 and 80 µM Rg decreased the HG-induced lincRNA ANRIL expression (Fig. 1D). HG decreased the expression of let-7a, and 20 and 80 µM Rg could both increase the HG-reduced let-7a expression (Fig. 1E).

The expressions of apoptosis-associated proteins Bcl-2 and active caspase-3 were further detected. The present results showed that HG significantly inhibited Bcl-2 expression and increased active caspase-3 expression, and Rg treatment recovered the expressions of Bcl-2 and caspase-3 affected by HG (Fig. 2A-C). The present results suggested that Rg inhibited the apoptosis of HMCs induced by HG by upregulating Bcl-2 and downregulating caspase-3.

Previous studies demonstrated that dencichine could ameliorate kidney injury in induced type II DN via the TGF-β/Smad signaling pathway and 1,25(OH)2D3/VDR could attenuate HG-induced epithelial-mesenchymal transition in human peritoneal mesothelial cells via the TGF-β/Smad3 pathway (13,14). The present study aimed to determine whether the TGF-β/Smad signaling pathway is involved in the regulation of Rg on the HG-affected cell apoptosis and proliferation. Using a western blotting assay, HG significantly upregulated TGF-β1 expression, downregulated Smad7 expression and activated the phosphorylation of Smad2. Both 20 and 80 µM Rg treatment recovered the expression levels of TGF-β1 and Smad7, and the phosphorylation status of Smad2 regulated by HG (Fig. 2D-G).

To further confirm whether lincRNA ANRIL and let-7a are involved in regulation of cell apoptosis and the TGF-β1/Smad signaling pathway by Rg treatment in the condition of HG, the present study detected whether knockdown of lincRNA ANRIL and overexpression of let-7a had the same results as Rg treatment. RT-qPCR was used to confirm the efficacy of lincRNA ANRIL knockdown and let-7a overexpression (Fig. 3A). The results showed that knockdown of lincRNA ANRIL and overexpression of let-7a significantly inhibited HG-induced apoptosis, similar to Rg treatment (Fig. 3B and C). Overexpression of let-7a had no effect on the expression of lincRNA ANRIL in an HG condition (Fig. 3D), but knockdown of lincRNA ANRIL significantly upregulated the level of let-7a compared with the HG group (Fig. 3E). The present results suggested that lincRNA ANRIL could negatively regulate let-7a in an HG condition and that lincRNA ANRIL was an upstream regulator of let-7a.

Knockdown of lincRNA ANRIL and overexpression of let-7a significantly increased the HG-suppressed Bcl-2 expression and decreased the HG-induced caspase-3 expression, similar to the Rg treatment (Fig. 4A-C). Knockdown of lincRNA ANRIL and overexpression of let-7a significantly reduced the HG-induced TGF-β1 level, inhibited the HG-activated phosphorylation of Smad2, and increased the HG-reduced Smad7 level (Fig. 4D-G). The present results suggested that Rg attenuated HG-induced apoptosis by regulating the lincRNA ANRIL/let-7a/TGF-β1/Smad signaling pathway.