Effect of Celastrus orbiculatus in inhibiting Helicobacter pylori induced inflammatory response by regulating epithelial mesenchymal transition and targeting miR-21/PDCD4 signaling pathway in gastric epithelial cells

The herbal pieces of Celastrus orbiculatus were purchased from Guangzhou Zhixin Pharmaceutical Co Ltd. (Guangzhou, China). The preparation procedure of COE has been described previously [11]. The chemical constituents from the stems of Celastrus orbiculatus were investigated by High Performance Liquid Chromatography (HPLC) assay (Additional file 1). The resultant COE micropowder was dissolved in dimethyl sulfoxide (DMSO, Sigma-Aldrich Co., St Louis, MO, USA).

The human gastric epithelial cell line GES-1 was obtained from Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China). Cells were cultured in DMEM Medium supplemented with 10% FBS at 37 °C with 5% CO₂. H. pylori, suspended in DMEM, was added to cells at a bacterium/cells ratio of 200:1. The cells were treated by COE for 24 h after H. pylori stimulation for 2 h.

GES-1 cells were planted in a 96-well plate and cultured overnight. The cells were treated with different concentrations of COE (10, 20, 40 and 80 μg/mL) for 24, 48 and 72 h. After incubation with MTT solution for 4 h, the absorbance was measured at 490 nm in a microplate reader.

The assessment of the IL-6, IL-8 and TNF-α levels in the the supernatant fluid of cells was performed by ELISA, using ELISA kits according to the manufacturer’s instructions. The absorbance at 450 nm was measured.

Cells were fixed with paraformaldehyde and then blocked with Triton X-100 and serum for 30 min. Then, the cells were incubated with primary antibodies at 4 °C overnight. After washed with PBS for 3 times, the secondary antibodies were incubated at 37 °C for 1 h. The nuclei were dyed with 4,6-diamidino-2-.

phenylindole (DAPI) (Bioworld Technology, St Louis, USA). Image were obtained under fluorescence microscope at × 400 magnification.

Proteins were separated by SDS–PAGE gel and transferred into nitrocellulose membranes (Millipore, Bedford, MA). The membrane was blocked with 5% non-fat milk for 2 h. The membranes were washed and secondary antibody was added to the system. Immunoreactive protein bands were detected using an Molecular Imager Chemi Doc XRS System (Bio-Rad). The bands from western blotting were quantified by Quantity One analysis software (Bio-Rad).

Total RNAs were isolated according to the manufacturer’s instructions. Primers were obtained from Shanghai Sangon Biological Engineering Technology and Services (Shanghai, China) and their sequences were: E-cadherin, forward primer 5′-ATG AGG TCG GTG CCC GTA TT-3′, reverse primer 5′- CGT TGGT CTT GGG GTC TGT GA-3′; N-cadherin, forward primer 5′-TCA GTG GCG GAG ATC CTA C-3′, reverse primer 5′-GTG CTG AAT TCC CTT GGC TA-3′; Vimentin, forward primer 5′-AAG CAG GAG TCA AAC GAG TA-3′, reverse primer 5′-GTT GGC AGA GGC AGA GAA AT-3′; PDCD4, forward primer 5′-AAA GGC GAC TAA GGA AAA CTC ATC-3′, reverse primer 5′-GCC TAT CCA GCA ACC TTC CCT-3′; GAPDH, forward primer 5′-CTC AAC TAC ATG GTC TAC ATG TTC CA-3′, reverse primer 5′-CTT CCC ATT CTC AGC CTT GAC T-3′. RT-PCR was conducted using an ABI 7500 Real-Time PCR System (Applied Biosystems). GAPDH was used as an internal normalization control. The results were calculated using the 2^-∆∆Ct method.

Total DNA was extracted using the Universal Genomic DNA Extraction Kit Ver.3.0 (TaKaRa). Non-methylated and methylated regions of DNA were amplified after treatment with sodium bisulfite. The PDCD4 gene methylated primer sequences used were as follows: 5′-TCG TCG TTA CGA TTG GTT AGT C -3′ (forward) and 5′-GAA AAA TCT CTA ACC CTT CTC GC -3′ (reverse). The PDCD4 gene non-methylated primer sequences used were as follows: 5′- GGT TTT GTT GTT ATG ATT GGT TAG TT -3′ (forward) and 5′-CAA AAA ATC TCT AAC CCT TCT CAC T -3′ (reverse). DNA methylation analysis was conducted following methylation specific PCR amplification. The products of PCR were detected by 1.5% agarose gel electrophoresis.

miR-21 precursor and negative control precursor miRNA were purchased from GenePharma (Shanghai, China). PDCD4 small interfering RNA (siRNA) and scrambled control siRNA were obtained from Santa Cruz Biotechnology. They were similarly transfected using Lipofectamine 2000 (Invitrogen, San Diego, CA, USA).

Date were shown as the means ± SD. Unpaired two-tailed Student’s t-test was used to compare two independent groups. One-way analysis of variance (ANOVA) was used to compare more than two groups. SPSS for Windows, version 17 (SPSS, Inc.) was used for all statistical analyses, and P < 0.05 was considered to indicate a statistically significant difference.

COE inhibited the viability of GES-1 cells in a concentration and time dependent manner. No obvious change in cell viability was observed at 10 and 20 μg/mL of COE (Fig 1a). Therefore, the non-cytotoxic concentrations (below 20 μg/mL) of COE was chosen in further experiments. The appearances of the GES-1 cells are polygonal or fusiform shape. After 24 h co-culture with H.pylori, it transformed from multiangular to round or irregular shapes of various sizes, with disrupted cell walls and cytoplasmic leakage. Interestingly, the morphology changes were noticeably suppressed by pretreatment with COE (Fig.1b).

The levels of pro-inflammatory cytokines IL-6, IL-8 and TNF-α may serve as sensitive and responsive biomarkers of change in H. pylori-induced inflammatory response [12]. As shown in Fig.1c, levels of IL-6, IL-8 and TNF-α were obviously higher in H. pylori infection group than in normal group. After treatment with different concentrations of COE, the levels were less when compared with H. pylori infection group. It significantly decreased in the 20 μg/mL COE group.

Our date showed that H. pylori infection-induced loss of epithelial marker E-cadherin, while up-regulation of mesenchymal markers N-cadherin and Vimentin at both protein and mRNA levels. However, pretreatment with COE effectively prevented these variations in a concentration-dependent manner (Fig. 2a-b). In addition, immunofluorescence staining also revealed that EMT-related gene expression were in keeping with the above research work (Fig. 3a-c). Taken together,

Recent studies have shown that the expression of PDCD4 is frequently silenced or reduced as a result of hypermethylation in tumor inflammatory microenvironment [13]. Therefore, the methylation status of the PDCD4 promoter region was investigated. The methylation level of PDCD4’s promoter was higher in H. pylori infection group than in normal group. However, after the treatment of COE, the promoter of PDCD4 had demethylation (Fig.4a). In addition, the effect of COE on PDCD4 demethylation was subjected, with western blotting and RT-PCR assay, which supported these findings (Fig.4b-c). These results demonstrated that COE significantly up-regulated the expression of PDCD4 by influencing the methylation status of its promoter.

Increasing evidence indicated that miR-21 may serve as a key mediator of the anti-inflammatory response [14]. The data from the present study revealed that COE inhibited the elevation of miR-21 after H. pylori infection in a dose-dependent manner (Fig. 5a). Moreover, as shown in Fig. 5b, cells were cotransfected reporter construct containing the wild-type miR-21 binding site within the PDCD4 3′ UTR or a mutated miR-21 binding site for luciferase reporter assay. The results indicate that PDCD4 luciferase activity increased in cells incubated with 20 μg/mL COE, suggesting that COE modulates PDCD4 expression via miR-21.

To evaluate the role of miR-21/PDCD4 signaling pathway in the anti-inflammatory effect of COE, miR-21 precursor were designed and performed. As shown in Fig. 6a, transfection with miR-21 precursor significantly increased miR-21 expression levels, comparing with control cells. Furthermore, over-expression of miR-21 significantly suppressed the COE-induced increase of PDCD4 expression levels, compared with the group treated with COE (Fig. 6b). Moreover, similar findings were observed when PDCD4 was knocked down in GES-1 cells exposure to COE (Fig. 6c). In addition, the methylation status of the PDCD4 promoter was high and the production of pro-inflammatory cytokines was significantly increased in GES-1 cells treated with COE and miR-21 precursor or PDCD4 siRNA, compared with the group treated with COE alone (Fig. 6d-e). These results suggested that miR-21 over-expression and PDCD4 silencing significantly change the methylation status of PDCD4 promoter and attenuated the anti-inflammatory effect in GES-1 cells after treatment with COE.