Lignans from the bark of Eucommia ulmoides inhibited Ang II-stimulated extracellular matrix biosynthesis in mesangial cells

RPMI-1640 medium, newborn calf serum (NCS), RT-qPCR kits with Platinum® SYBR® Green qPCR Super Mix-UDG, and primers for collagen I (Col I), Col III, collagen type IV (Col IV), fibronectin and AR were purchased from Invitrogen (Carlsbad, CA, USA). Antibodies for Col I, Col III, Col IV, fibronectin and AR for western blotting were supplied by Abcam (Cambridge, England) and Santa Cruz Biotechnology Inc. (CA, USA). The Cell Titer 96® Aqueous One Solution Proliferation Assay for the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) method was provided by Promega (Madison, WI, USA). A Revert Aid First Strand cDNA Synthesis Kit was purchased from Thermo Scientific (Austin, TX, USA). Losartan was obtained commercially from the National Institute for Food and Drug Control (Beijing, China). Human Angiotensin II and other analytical grade reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Eucommia lignans were extracted at our own laboratory as described previously [12, 19, 20]. E. ulmoides were obtained from Changsha Medical Company (Hunan, China) in July 2009, and authenticated by Dr. Dong-Sheng Ouyang, one of the authors according to the methods described in the literature [21]. A voucher specimen of Eucommia ulmoides Oliv. (IBSC_0345347) was deposited at South China Botanical Garden Herbarium, Guangdong, China. Briefly, fresh Eucommia ulmoides Oliv. bark was cut into pieces and extracted with 60% ethanol purchased from Changsha Tianshun Chemical Co., Ltd (Hunan, China) at 70°C for 2 h (twice). The extract was subjected to macroporous resin supplied by Haiguang Chemical Industrial Company (Tianjin, China) and eluted with 80% ethanol after treatment with pure water as the eluent. The eluent was freeze-dried to powder and stored at 4°C. The lignans content in Eucommia lignans was 71%, as determined by spectrophotometry on a Beckman Coulter DU 640 spectrophotometer (Beckman Coulter, Inc. USA) at 277 nm, with pinoresinoldiglucoside used as the control which was supplied by college of chemistry and chemical engineering in Central South University (Hunan, China).

RMCs (HBZY-1 cells) were purchased from China Center for Type Culture Collection (Wuhan, China). After recovery, RMCs were cultured in RPMI-1640 medium supplemented with 10% NCS at 37°C in a humidified atmosphere of 5% CO₂ in air.

RMCs were added into the wells of a 96-well plate at a density of 3000 cells per well and cultured in RPMI-1640 medium containing 10% NCS. All incubations were performed in RPMI-1640 containing 1% NCS when they grew to 60% confluence. The study included two parts: (1) Control group, Eucommia lignans groups (10, 20, 30, 40, 50, 60, 70, 80, or 90 mg/L Eucommia lignans); and (2) Control group, Ang II group (10 nM Ang II), Losartan group (10 nM Ang II with 20 μM Losartan), Eucommia lignans groups (10 nM Ang II with 20, 40 and 80 mg/L Eucommia lignans). After 48 h, the viability of RMCs was measuredby MTT method. Then, 20 μL cell Titer 96® Aqueous One Solution Reagent was added to the medium in each well, and the absorbance of solubilized blue formazan was recorded by a microplate reader (Molecular Devices, Spectra MAX. 250, USA) at 490 nm after 1 h at 37°C in a humidified 5% CO₂ atmosphere.

RMCs were assigned to six groups: Control group, Ang II group (10 nM Ang II), Losartan group (10 nM Ang II with 20 μM Losartan), and Eucommia lignans groups (10 nM Ang II with 20, 40 or 80 mg/L Eucommia lignans), in a 6-well plate, and cultured in RPMI-1640 medium containing 10% NCS for 48 h. Total RNA from RMCs was extracted by Trizol® reagent (Invitrogen, Carlsbad, CA, USA) and the concentration was determined by spectrophotometry at 260 and 280 nm. A reverted aid cDNA synthesis kit was used to perform the synthesis of first strand cDNA from total RNA templates. Real-time qPCR was performed by Platinum® SYBR® Green qPCR Super Mix-UDG following the manufacturer’s instructions. The gene-specific primers are listed in Table 1. The data were quantitatively analyzed by Stratagene Mx3000p Real-time PCR (Santa Clara, CA, USA). The glyceraldehyde phosphate dehydrogenase (GAPDH) gene was used as the internal control.

Total protein was extracted from RMCs with radio immunoprecipitation assay lysis buffer consisting of 10 mM sodium phosphate (pH 7–8), 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate and 1% Triton X 100 after a 48-h culture under the conditions described above, and the protein concentration was determined using a bicinchoninic acid assay kit (Sigma-Aldrich, USA). A total of 40 μg of total protein was separated on a 10% sodium dodecyl sulfate polyacrylamide gel (Invitrogen, Carlsbad, CA, USA) and transferred onto a polyvinylidenefluoride membrane (Millipore, USA). The membrane was blocked with 5% skim milk solution in 0.1% tris-buffered saline Tween-20 (TBST) (Shanghai Bioscience Co. Ltd, China) over night. Subsequently, one of the primary antibodies (rabbit polyclonal for Col I, mouse monoclonal [FH-7A] for Col III, rabbit polyclonal for Col IV, mouse monoclonal [IST-9] for fibronectin, goat polyclonal for AR, and rabbit polyclonal for GAPDH) was added for hybridization before being incubated with the specific secondary antibody after washing membranes with TBST (Shanghai Bioscience Co. Ltd, China) three times. Protein bands were determined by the enhanced chemiluminescence western blotting detection system. GAPDH was used as an internal standard for data normalization.

Data were shown as mean ± standard deviation (SD) and were analyzed with SPSS 17.0 software (SPSS Inc., USA). A P value less than 0.05 was considered statistically significant. Significant differences between multiple groups were analyzed by one-way analysis of variance (ANOVA) followed by a Dunnett’s post-hoc test.

In comparison with the control, there was no significant change in the number of cells treated with Eucommia lignans in the 10, 20, 30, 40, 50, 60, 70 and 80 mg/L groups (P = 0.983, 0.986, 1.000, 0.900, 0.920, 0.515, 0.515, and 0.249, respectively). However, cellular viability decreased markedly in the group incubated with 90 mg/L Eucommia lignans (P = 0.001) (Figure 1). Therefore, the incubated concentrations of Eucommia lignans for the following experiments were 20, 40 and 80 mg/L.

The Ang II receptor blocker, losartan (20 μM), significantly decreased the proliferation of RMCs induced by Ang II (P = 0.004). The inhibitory effects were also observed in the different Eucommia lignans-treated groups (P = 0.034, P < 0.001, and P < 0.001) (Figure 2).

The changes in Col I, Col III, Col IV and fibronectin production are shown in Figure 3. mRNA and protein expression increased with Ang II stimulation. All of the increased expression levels induced by Ang II could be attenuated by losartan treatment (Col IV mRNA, P = 0.007; others, P < 0.001). Furthermore, Eucommia lignans also significantly diminished their ascended expression (Col I protein of 20 mg/L, P = 0.001; Col I protein of 40 mg/L, P = 0.002; Col IV mRNA of 80 mg/L, P = 0.031; others, P < 0.001), although decreases of the Col IV mRNA level of the low and middle concentration lignans groups did not reach a statistically significant difference (P = 0.084 and P = 0.134). Eucommia lignans could suppress Ang II-stimulated biosynthesis of ECM in RMCs (Figure 3B).

The mechanisms of Eucommia lignans inhibitory effects were tentatively elucidated from data of our previous animal experiments [22]. Both mRNA and protein expression of AR were effectively enhanced by Ang II (P < 0.001) (Figure 4). Losartan and Eucommia lignans clearly attenuated all expression stimulated by Ang II (Losartan, P = 0.045; 20 mg/L Lignans, P = 0.01; others, P < 0.001). The experiment demonstrated that Eucommia lignans could suppress Ang II-induced AR expression in RMCs.