Mechanism Of Vitamin D Receptor Regulating TLR4/NF-?? Signaling Pathway To Promote Intestinal Adaptation In Short Bowel Syndrome

Short bowel syndrome?SBS?is a syndrome of extensive resection of the small intestine or reduced absorption area after intestinal fistula surgery for various reasons.The remaining intestines can not meet the nutritional needs of the patients,resulting in water and electrolyte imbalance and nutritional malabsorption.Intestinal adaptation is a self-generated compensation phenomenon after extensive small bowel resection.It has very important clinical significance for SBS patients.Accelerating intestinal adaptation is the key to intestinal rehabilitation.Toll like receptors?TLRs?are specific receptors for lipopolysaccharides?LPS?.Many Gram-negative bacilli in the intestine are capable of producing LPS.When stress,such as surgery and trauma,cause gastrointestinal mucosal ischemia,necrosis,and barrier function damage,a large amount of endotoxin is released into the blood,causing endotoxemia,leading to activation of monocyte macrophages,endothelial cells,triggering the body's inflammatory response.Vitamin D receptor?VDR?is widely distributed in various organs of the human body and has anti-inflammatory effects.VDR can down-regulate the expression of TLR4,thereby inhibiting the TLR4/nuclear factor kappa?NF-???signaling pathway and exerting anti-inflammatory effects.Animal studies have found that intestinal administration of vitamin D₃ can promote intestinal adaptation in rats with short bowel syndrome,and the mechanism is not clear.Thus,this study hypothesized that the mechanism by which VDR promotes intestinal adaptation is achieved by inhibiting the TLR4/NF-??signaling pathway.In order to verify this hypothesis,this study will first establish a rat model of short bowel syndrome,up-regulate VDR and observe intestinal adaptation,intestinal barrier and inflammation,and detect the expression of TLR4/NF-??signaling pathway in intestinal tissue.Then,LPS-induced IEC-6 inflammatory model of rat intestinal crypt epithelial cells was established in vitro to observe the effect of VDR receptor changes on inflammation.This study will lay a foundation for improving the cause of intestinal adaptation from the new perspective of anti-inflammatory,and provide new ideas for clinical treatment of short bowel syndrome.The full text is divided into two parts.Part?:Mechanism of vitamin D receptor promoting intestinal adaptation through TLR4/NF-?B signaling pathway in a rat model of short bowel syndromeObjective:To explore the mechanism of vitamin D receptor regulating TLR4/NF-?B signaling pathway in promoting intestinal adaptation in rats with short bowel syndrome.Methods:Eighteen Sprague-Dawley?SD?male rats of 210g-230g were randomly divided into three groups:Sham-operated?Sham?group,SBS group and 1,25D₃?1,25-dihydroxy-vitamin D₃?group.After the establishment of the rat SBS model,the 1,25D₃group was intragastrically administered daily with 1,25D₃ on the third day after surgery.Two weeks later,the jejunal tissue from 1 cm-6 cm proximal to the anastomosis and blood sample were taken.The changes of serum inflammatory factors tumor necrosis factor-??TNF-??,Interferon-??IFN-??,Interleukin-2?IL-2?,Interleukin-6?IL-6?and serum calcium were detected.The expressions of VDR,MyD88 and NF-??in residual small intestine were observed.The villus height and crypt depth of residual small intestine was observed by histopathology.The expression of ki-67 in intestinal epithelial cells was detected by immunohistochemistry.The apoptosis of intestinal epithelial cells was detected by TUNEL method.The expression of Claudin-1 in intestinal epithelial cells was detected by Western blotting.Results:Compared with the Sham group,the villus height?P<0.001?and crypt depth?P<0.01?increased in the SBS group after 75%of the small intestines were removed.The expression of tight junction protein Claudin-1 was down-regulated.The expression of NF-??and MyD88 protein was up-regulated.The proliferation of ki-67 was active and apoptosis was alleviated.The serum inflammatory factors TNF-??P<0.01?,IFN-??P<0.001?,IL-2?P<0.001?,IL-6?P<0.001?increased.Intestinal tissue TNF-?,IFN-?,IL-2,IL-6 protein expression was down-regulated.There was no difference in blood calcium concentrations between the three groups?P>0.05?.Conclusion:Vitamin D receptors can promote intestinal adaptation in rats with short bowel syndrome by regulating TLR4/NF-??signaling pathway.Part ?: Protective mechanism of vitamin D receptor on LPSinduced IEC-6 inflammatory model in rat intestinal crypt epithelial cellsObjective: Vitamin D receptors have protective effects in some inflammatory cells,but there are few studies in intestinal cells.The protective effect of vitamin D receptor on LPS-induced IEC-6 inflammatory model of rat intestinal crypt epithelial cells is discussed.Methods: IEC-6 cells were cultured in vitro and divided into blank control group?conventional culture cells?,LPS group,1 n M-1,25D₃ + LPS group,10 n M-1,25D₃ + LPS group,100 n M-1,25D₃ + LPS group,200 n M-1,25D₃ + LPS group,500 n M-1,25D₃ + LPS group?add 1 n M,10 n M,100 n M,200 n M,500 n M 1,25D₃ respectively and 10 ?g/ml LPS?.The growth of cells was detected by MTT assay.The expressions of VDR,TLR4,NF-?B and My D88 were detected by real-time quantitative PCR.The expression of Claudin-1 and Occludin was detected by Western blot.The inflammatory factor IL-6 and IFN-? expression was detected by ELISA.Results: In IEC-6 cells,after treatment with a total concentration of 10 ?g/ml LPS for 24 hours,the cell viability of the LPS group was significantly decreased?P<0.05?.And with different concentrations of 1,25D₃ treatment,the cell survival rate increases as the concentration increases.Compared with the blank control group,the content of inflammatory factors in the supernatant of LPS group increased,and according to the pretreatment 1,25D₃ concentration,the other groups of inflammatory factors IFN-? and IL-6 have different degrees of decline.After pretreatment with 1,25D₃,the expression increased?P< 0.05?with VDR m RNA,TLR4,NF-?B,My D88 m RNA expression gradually reduce.Compared with the blank control group,LPS destroyed the tight junction proteins Occludin and Claudin-1,and after pretreatment with 1,25D₃,it protected the tight junction proteins Occludin and Claudin-1.Conclusion: Vitamin D receptors attenuate the inflammatory effects of LPS-induced inflammatory models of IEC-6 cells by down-regulating the TLR4/NF-?B signaling pathway.