The Effects And Mechanisms Of Vitamin D Receptor On Helicobacter Pylori Infection

Background:Vitamin D is a hormone carrier with a wide range of biological effects, related to the hormone’s well known functions in calcium and phosphate homeostasis, also to maintenance of normal cellular growth in many different organs,1,25(OH)2D3, is being increasingly recognized for its potent immunomodulatory activities. Vitamin D3exerts its effect through vitamin D receptor, VDR also known as NR111(nuclear receptor subfamily1, group Ⅰ, member1), is a member of the steroid-thyroid-retinoid receptor gene superfamily. Upon activation by1,25(OH)2D3, the VDR forms a heterodimer with the retinoid-X receptor and binds to hormone response elements on DNA resulting in expression or transrepression of specific gene products.VDR involves in calcium metabolism, bone formation, cell growth and differentiation, tumor suppression, immune regulation, VDR also regulates the expression of target gene cathelicidin antimicrobial peptides (CAMP) which is closely related to pathogenic microorganisms. CAMP plays a major role in antibacterial activity, but also get involved in immunomodulatory, inhibit tissue damage and promote wound repair functions, which is an important component of the host defense system. As an important member of transcription factors, it has not been reported in the literature whether VDR took immune protective effect in H. pylori infection. According to current research, VDR plays an important role in the anti-infective immunity which is associated with to clear microbial infection and reduce tissue damage. It’s great important to further study the VDR protective immunity to bacterial infection-induced inflammation and immune signaling pathway in the anti-infective process.Method:1) Collect outpatient gastric mucosa samples:biopsy2mucosal specimens under gastroscopy, one sample sent to take pathological examination, another stored in liquid nitrogen.2)H. pylori infected gastric epithelial cells:Gastric epithelial cells (GES-1) infected with different values of the MOI (MOI of0,10,50,100) of H. pylori for24h or co-cultured at MOI of100for0,6,12,24h after GES-1cells incubated in culture plate for24h.3) Gastric epithelial cells transfected with si VDR:GES-1cells were inoculated in RPMI medium without antibiotics and the confluence of cells reached to30to50%before transfection. GES-1cells were transfected with control siRNA as VDR-negative siRNA and siVDR using the liposome LipofectamineTM2000as a transfection reagent according to the manufacturer’s instruction.4)RT-qPCR Analysis:Total RNA was isolated from GES-1cells or gastric mucosa tissues by Trizol reagent and detected of RNA concentration and purity. The first strand cDNAs were synthesized from the RNA, the real-time RT-PCR quantitation for individual target mRNAs such as VDR、CAMP、IL-6、IL-8、CYP24Al、DEFB4was performed using a TaKaRa real-time PCR kit. Gene expression levels were normalized to GAPDH and the data were analyzed using a comparative cycle threshold calculations. 5) Western Blot Analysis:The cells were scrapped into the lysis buffer, Total protein was quantified using the Bradford assay and Equal amounts of protein were separated by12%SDS-PAGE and then transferred to a polyvinylidene difluoride membrane, the membranes were blocked at room temperature for1h with5%non-fat milk in1TBST and subsequently incubated with primary antibodies at4℃overnight, then the blots were incubated with the appropriate HRP-conjugated secondary antibody at room temperature for1h and then developed with enhanced chemiluminescence according to the manufacturer’s protocol. Finally, the images of films took under the gel imaging system photos.6)ELISA Test:lessons the medium of a12-well culture plate,5000g/min After centrifugation, the supernatant obtained was diluted in accordance with a certain proportion, the concentration of the standard and the sample in accordance with serial dilutions of the reagent instructions detection of inflammatory factors.7)Bactericidal Assays:GES-1cells were infected with H. pylori SS (1108bacteria/mL) in the presence or absence of siVDR, siCAMP or1,25(OH)2D3. After incubation for2hours, the cells were lysed and diluted then plated on Columbia agar plates and counting the number of visible colonies after3-5days of incubation.8) Statistical analysis:All data are expressed as mean±standard deviation (SD). Statistical analysis was performed using t-est or one-way ANOVA or two-tailed Pearson’s correlation coefficient analysis to analyze statistically significant differences between groups by SPSS software (version16.0). Differences at P<0.05were considered significant.Results:1)The effects of H. pylori infection on mRNA expression levels in gastric mucosa tissues. VDR CAMP, IL-6and IL-8mRNA expression levels were significantly elevated in H. pylori-positive patients compared with H. pylori-negative patients. There was a significant positive correlation between VDR mRNA levels and chronic inflammation scores (correlation coefficient, r=0.536, p<0.001). The CAMP levels were significantly increased in parallel with VDR levels(r=0.814, p<0.001).2) The effects of H. pylori infection on mRNA expression levels in vitro. H. pylori infection up-regulated the VDR, CAMP expression levels depending on MOI values and incubation time by RT-qPCR or Western-Blot assay.3) The effects of transfected with si VDR on CAMP and cytokines expression after H. pylori infection:VDR downstream gene CAMP expression levels reduced and IL-6, IL-8expression levels increased after suppressed VDR expression by siRNA pools compared to the negative control siRNA treated group.4) The effects of VDR agonists1,25(OH)2D3on H. pylori infection:1,25(OH)2D3down-regulated IL-6and IL-8and up-regulated CAMP, DEFB4and CYP24A1expression.5) The effects of siVDR, siCAMP and1,25(OH)2D3on H. pylori activity:Treatment with siVDR or siCAMP increased the viability of bacteria in the siRNA transfected cells as measured by CFU, on the other hand,1,25(OH)2D3H. pylori reduced the number of viable bacteria.Conclusion:1) H. pylori infection up-regulated VDR, CAMP, IL-6and IL-8expression levels;2) VDR and1,25(OH)2D3may regulate the expression of downstream gene of antimicrobial peptides CAMP, defensin beta4DEFB4and CYP24A1; VDR and1,25(OH)2D3regulate immune response in H. pylori infection.