Research About High Mobility Group Box1 Protein Involved In Acute Inflammation Induced By Clostridium Difficile Toxin A

Clostridium difficile(CD) is a spore-forming Gram-positive anaerobic bacillus and is the most common cause of nosocomial antibiotic-associated diarrhea and pseudomembranous colitis. When the balance of gut microbiota is disturbed, Clostridium difficile blooms, excessive growth, becomes major intestinal flora, and causes infection with Clostridium difficile(CDI), resulting in intestinal mucosal necrosis of epidermis cells, mucous membrane permeability increased, eventually leading to severe diarrhea and intestinal inflammation and even death.In recent years, due to the widespread use of antibiotics, the rate of CDI morbidity and mortality in the global has increased year by year, and China’s large population, with widespread use of antibiotic, CDI will become one of the pandemic threat to public health. To study the pathogenesis of CDI and find a potential therapeutic target for the treatment of CDI is one of most important works. Large toxin protein Tcd A and Tcd B are two principle virulence factors of C. difficile. The traditional view that Toxin A plays a more important role than toxin B in CDI, intestinal fluid and intestinal inflammation response. Toxin A causes intestinal tissue and stimulates the release of inflammatory cytokines such as IL-1β, IL-6, tumor necrosis factor(TNF), and then induces acute enteritis reaction. The study confirmed that HMGB1 as a kind of extracellular inflammatory factor in chronic inflammation. In the paper, we investigated whether HMGB1 is involved in the regulation of acute inflammatory response caused by Clostridium difficile toxin A(Tcd A) and provided insight into the molecular mechanisms underlying the pathogenesis of CDI.In the paper, we studied whether HMGB1 is involved in the regulation of intestinal inflammation induced by Clostridium difficile toxin A in vitro with cell rounding and in vivo with the ‘ileal loop’ surgical model. Fistly, Clostridium difficile toxin A was expressed in Bacillus megaterium and then purified by Ni2+ affinity chromatography. Secondly, the purified C. difficile toxin A protein’s “cytotoxic” was assayed by MTT; The secretion of HMGB1 was detected after Tcd A treatment with CT26, Vero and HCT8 cells by western blot; In the condition of HMGB1 inhibitor, Glycyrrhizin, not neutralizing Tcd A’s “cytotoxic”; Vroe cells were pretreated with Glycyrrhizin, and then exposed to Tcd A, Cell rounding was visualized by phase-contrast microscopy to observe whether glycyrrhizin can inhibit Tcd A-induced cell rounding and then determine whether HMGB1 involved in Tcd A “cytotoxicity”. Finally, in the condition of HMGB1 inhibitor, Glycyrrhizin, not neutralizing Tcd A’s “enterotoxigenesis”. The ‘ileal loop’ surgical model was used to study the effect of HMGB1 on Tcd A-induced acute inflammation, including swelling and fluid’s detection, Histopathological assays(H&E staining), intestinal tissue myeloperoxidase(MPO) activity detecting and expression of inflammatory cytokines in m RNA levels measured by RT-PCR. The results of the paper are as follows:(1) Clostridium difficile toxin A protein was inductively expressed in Bacillus megaterium successly and purified by Ni2+affinity chromatography.(2) Results of MTT assay demonstrated the toxic of the purified toxin A, western blot detection results showed that different cell lines exposured to Toxin A caused HMGB1 extracellular secretion indicating that the system is universal.(3) The experiment showed that HMGB1 inhibitors Glycyrrhizin does not neutralize the toxicity of toxin A; in this condition, Vero cells were pretreated with glycyrrhizin 30 min before Tcd A exposure, the assays of cell rounding showed that Glycyrrhizin inhibited cell rounding induced by Tcd A, indicating that HMGB1 involves in Tcd A cell toxicity.(4) The results of ‘ileal loop’ surgical model, including swelling and fluid’s detection, Histopathological assays(H&E staining), intestinal tissue myeloperoxidase(MPO) activity and RT-PCR detection of m RNA levels of inflammatory cytokines showed that Glycyrrhizin inhibited pro-inflammatory activity induced by Tcd A, which proved that HMGB1 involves in Tcd A-induced acute inflammation...