Emodin promotes fibroblast apoptosis and prevents epidural fibrosis through PERK pathway in rats

Emodin (molecular formula: C₁₅H₁₀O₅ EMO) [9] was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. The purity of EMO is 95%.

Human fibroblasts were sourced from ScienCell Research Laboratories (Shanghai, China). Then, the cells were cultured in DMEM (Invitrogen, CA, USA) which contains 15% fetal bovine serum (FBS, Gibco, USA) and 1% penicillin/streptomycin (Gibco, CA, USA) under 5% CO₂ at 37 °C. Fibroblasts between passages 4 and 6 were involved in all experiments. The cells were transferred into various dishes overnight. After reaching 60–70% density, the cells were washed with phosphate-buffered saline. Subsequently, various concentrations (0, 5, 10, 20 μg/ml) of EMO were applied to treating the fibroblast.

The cell viability of fibroblasts treated with EMO was detected by Cell Counting Kit-8 (CCK-8) (Dojindo, Tokyo, Japan). The cells at exponential stage were transferred into 96-well plates. When the cell density reached 60–70%, EMO of varying concentrations was added to each well for a period of 24 h. Then, the cells were treated with 10 μl CCK-8 solution well for another 2 h. The absorbance at 450 nm was measured by microplate absorbance reader (TECAN). In the same way, the cells with EMO (10 μg/ml) were treated for differing lengths of time (0–72 h). Cell survival rate was calculated according to the instruction book.

Fibroblasts were cultured into six-well plates, before being incubated for 24 h. Three wells were assigned as the control group, and another three wells were set as the EMO-treated group. After the EMO-treated group was treated with 10 μg/ml EMO for 24 h, all cells were collected, prior to being washed with 4 °C PBS buffer for three times. The cells with 1 ml 1× binding buffer were resuspended, and 100 μl cell suspension was transferred to the tube. Then, 5 μl PI and 5 μl FITC Annexin V were added to the tubes. After the addition of 400 μl 1× binding buffer and incubation by FITC Annexin V and PI for 15 min at room temperature away from light, the mixture was detected by flow cytometry.

After the EMO of varying concentrations was treated, all cells were collected. Resuspended fibroblasts were lysed by RIPA buffer (Beyotime, Shanghai, China) for 15 min. The protein concentration was measured by using BCA Protein Assay Kit (Beyotime, Shanghai, China). Forty micrograms of protein per well was used for western blot. After being soaked in the blocking buffer at room temperature for a 2-h spell, the PVDF membranes (Millipore, Bedford, MA) were incubated with primary and secondary antibodies successively in line with the instructions. The primary antibodies used were anti-cleaved-poly ADP-ribose polymerase (cleaved PARP), anti-Bax, anti-Bcl-2, anti-78-kDa glucose-regulated protein (GRP78), anti-CHOP, anti-PERK, anti-phospho-PERK (P-PERK), anti-eukaryotic translation initiation factor 2α (eIF2α), anti-phospho-eIF2α(P-eIF2α), and anti-GAPDH antibodies (CST, Beverly, MA, USA). The secondary antibodies involved were the anti-mouse or anti-rabbit IgG (CST, Beverly, MA, USA).

Fibroblasts were seeded into six-well plates, with a glass slide contained in each well for 24 h. After treatment of 10 μg/ml EMO for 24 h, the fibroblasts were fixed by 4% paraformaldehyde at room temperature for 15 min. The TUNEL staining (KeyGEN, Nanjing, China) procedures were developed based on the manufacturer’s instructions. After brief steps of staining, fluorescence microscopy was employed for detection of the apoptotic fibroblasts.

The study was granted approval from the Animal Ethics Committee of Yangzhou University. Twelve male 250–280 g SD male rats were split into three groups on a random basis, namely saline group, 50 mg/ml emo group, and 100 mg/ml emo group. Based on previous studies [15, 16], rats were anesthetized by intraperitoneal injection of 1% pentobarbital sodium (40 mg/kg), and we performed laminectomy model to remove L1-L2. Subsequently, topical 1 × 1 cm gauze containing the corresponding (0/50/100 mg/kg) concentration of EMO covered the wound for 5 min, prior to the wounds being rinsed with saline and sutured in layers carefully.

Four weeks later, after anesthesia, all rats were perfused with 4% paraformaldehyde. The spine of rats after laminectomy was collected by groups, and the specimen was immersed in formalin for 3 days before decalcification with ethylenediamine tetraacetic acid (EDTA) for 1 month. Successive 4-μm sections were obtained through the surgical vertebra. Hematoxylin-eosin (HE) and Masson’s trichrome staining were used to evaluate the degree of epidural fibrosis. The images of epidural fibrosis, scar adhesion, and collagen synthesis were observed by optical microscopy at the magnification of × 40. The fibroblast counting was calculated by three fields (100 × 100 mm each) in the sites of epidural defect at the magnification of × 100.

After denitrification and rehydration, these sections were subjected to pretreatment with sodium citrate to activate their antigenicity. Endogenous peroxidase is blocked by 3% hydrogen peroxide. The sections of epidural fibrosis tissue were incubated with anti-GRP78 at room temperature for 1 h and then incubated with anti-rabbit IgG at room temperature for 2 h. Then, the sections were stained by using DAB kit and counterstained by using hematoxylin. Finally, sections were observed under a light microscope.

The data was analyzed with SPSS statistical 19.0 software. All of our data were presented as mean ± standard deviation. Independent Student’s t test was conducted to draw comparison between groups. P value < 0.05 was introduced to define statistical significance.

To ascertain whether EMO could induce fibroblast apoptosis, the cells were treated with varying concentrations of EMO for 24 h. Besides, the fibroblasts with EMO (10 μg/ml) were treated for different lengths of time (0–72 h). Then, the CCK-8 assay was performed to detect the effect of EMO on the cell viability. As revealed in Fig. 1a, b, EMO plays a role in inhibiting fibroblast viability in a dose- and time-dependent manner. In order to validate the effect of EMO on fibroblast apoptosis, we performed morphological examinations (TUNEL assay). As shown in Fig. 1c, d, the control group was found to have few TUNEL-positive cells; however, the TUNEL-positive cells were observed to have a significant increase in the EMO-treated group. Besides, as EMO concentration was on the rise; western blot (Fig. 1e) analysis showed that the expression of pro-apoptotic markers was upregulated, such as cleaved PARP and Bax. By contrast, the expression of anti-apoptotic marker Bcl-2 was in decline. As revealed by the Annexin V-FITC/PI double labeling (Fig. 1f, g), the apoptosis rate of fibroblast was increased after EMO treatment. In summary, the above results evidenced that EMO is effective in inducing fibroblast apoptosis significantly.

In order to figure out the molecular mechanism of EMO on fibroblast apoptosis, the cells with four concentrations of EMO were treated, and then, the expression of ER stress pathway-related proteins was detected by performing western blot analysis. Figure 2a demonstrates that EMO promoted the expression of GRP78, p-PERK, and p-eIF2α. Then, the expression of two ER stress pathway proteins (GRP78 and CHOP) (Fig. 2b) was analyzed, which led to the discovery that the protein expression was upregulated when the EMO concentrations were increased. Then, detection was made of a classic pathway of ER stress relative protein (PERK, elF2α, P-PERK, P-elF2α) (Fig. 2c), and the ratios of P-PERK/PERK and P-elF2α/elF2α showed a noticeable upsurge with the concentration of EMO on the increase. All the results indicated that EMO induces fibroblast apoptosis via the upregulation of PERK signal pathway.

Figure 3a reveals that the control group epidural fibrosis was more severe as compared to the EMO-treated group. In addition, with the increase in EMO concentration, the degree of epidural fibrosis was in decline incrementally. The HE staining images (× 100) show that the number of fibroblasts in the EMO-treated groups was less than that in the control group (Fig. 3b, c). The Masson staining images (Fig. 3d) show that the density of collagen was higher in the control group than in the EMO-treated group. Moreover, the density of collagen decreased as its concentration rose.

Figure 4a, b demonstrates that the expression of GRP78 was enhanced with the rising level of EMO concentrations in the epidural scar tissue. GRP78 expression in the 100 mg/kg EMO-treated group was noticeably increased as compared with the control group. In previous studies [15], it was demonstrated that GRP78 increased notably after ER stress activation. All these results suggested that ER stress signaling plays an essential role in EMO-induced reduction of epidural fibrosis.