| High mobility group protein B1 (HMGB1) is one of members of the high mobility protein family, which was found in the 1970s. As an abandant non-histone nuclear protein, HMGB1 regulates gene transcription and stabilizes chromosomal architecture through binds DNA. As HMGB1 does not bind tightly to DNA, it is actively secreted by immunostimulated macrophages and dendritic cells or passively released by damaged or necrotic cells. Extracellular HMGB1 has been implicated as a late mediator of lethality in sepsis, ischemia-reperfusion injury, tumors, acute necrotizing pancreatitis and so on. Apart from in severe diseases, HMGB1 was also involved in the pathogenesis of autoimmune diseases, such as rheumatoid arthritis, lupus nephritis, inflammatory bowel disease. Therefore, the role of extracellular HMGB1 in inflammatory or immune diseases received extensive attention of researchers, and it will become a therapeutic targets of these diseases.Inflammatory bowel disease(IBD), including ulcerative colitis (UC) and Crohn’s disease (CD), is a kind of unknown etiology and pathogenesis unclear of chronic intestinal inflammatory diseases. Its characteristics are chronic repeated abdominal pain, diarrhea and protracted, hardly cured stool with pus, blood and mucous. The incidence of IBD is always higher in developed countries, but in recent years the rate showed a rapid rise among adult patients in our country. And it has now been proved easy to induce colorectal cancer. As a gastrointestinal disease, it has a serious harm on people’s health. As a result, the pathogenesis and treatment has been the hot topics in the study of IBD. Although the etiology and pathogenesis of IBD has not yet clear, but the immune disorder is regarded to be one of the important factors leading to the IBD. Th17 cells as a new type differs from that of Th1 and Th2 CD4+T cell subsets, mainly involved in the pathogenesis of inflammatory and autoimmune diseases. Recent studies show that the excessive activation of Th17 cells as a key role in the pathogenesis of IBD, which is expected to become the IBD new therapeutic target. Now that HMGB1 and Thl7 cells are involved in the pathogenesis of IBD, so how is the relationship between them? HMGB1 implicated in the pathogenesis of IBD whether throhgh regulating the Th17 cells activity? Therefore, we aimed to clarify whether HMGB1 cntributes to Thl7 cells activity in IBD.At present, the IBD treatments is very limited, mainly 5-aminosalicylic acid preparations, corticosteroids, immunosuppressive agents and the recent use of biological agents, but there are different limitations and side effects and some patients are still unable to maintain clinical remission after long-term administration of these drugs. Therefore, the search for new and effective drugs is the most urgent task of IBD now.Aloe is a kind of high medical value plant, has been used in the treatment of diseases such as dermatitis, ulcer for thousands of years. It has been reported that fresh aloe juice enema has better curative effect for patients with chronic ulcerative colitis. Aloe emodin (AE) is the major active ingredient of aloe, Studies have shown that AE has a certain immune regulation, inflammation suppression function. However, there is no research report on the exact mechanism and therapeutic effects of AE on colitis, that is one of scientific problems focusing on in our research.Objective1.1 Studies have shown that HMGB1 may be an endogenous immune adjuvant to activate antigen-presenting cell (APC), participated in the Th1, Treg CD4+T cell differentiation regulation. And it has been confirmed that HMGB1 promotes the proliferation and differentiation of Th17 cells involved in autoimmune myocarditis and acute allograft rejection. Then, in IBD, how is the relationship between HMGB1 and Th17 cells? So, we aimed to clarify the HMGB1 effetes on Th17 cells and the pathogenesis on IBD.1.2 To investigate the immune therapeutic effect of aloe emodin on experimental colitis Based on HMGB1-Th17 pathways, and further to explore the mechanism of aloe emodin treating IBD.Methods1.1 HMGB1 regulates Th17 cell response in the pathogenic role of experimental colitis in mice1.1.1 Establish experimental colitis and drug interventionA total of 30 femal BALB/c mice were randomly divided into 3 groups (n=10, each group), ethanol control group, model group, ethyl pyruvate (EP) group. Colitis model was induced in mice by rectal administration of trinitro-benzene sulfonic acid (TNBS) dissolved in ethanol.8 hours after TNBS administration, EP group was treated by intraperitoneal injection with 100 mg/Kg EP solution per day, control group and model group instead of with 0.2ml/10g lactic acid slinger liquid every day, each group was administrated for 6 consecutive days.1.1.2 Evaluation of therapeutic effects①The weight and the stool situation of each animal were recorded daily and on the basis of these manifestation disease activity index (DAI) was evaluated.②Colon intestinal samples was collected after 6 days, the colonic macroscopic damage index (CMDI) and tissue damage index (TDI) were evaluated.③The serum levels of HMGB1, IL-17 and IFN-y were determined by ELISA.④The colonic infiltration of Th17 cells was determined by immunofluorescence method.⑤The percentages of Th1、Th17、Tcl cells in spleen as well as in mesenteric lymph nodes(MLN) of mice were analyzied by flow cytometry(FCM).⑥The colonic expression of HMGB1 was evaluated by Western Blot.1.2 Pharmacodynamics study of aloe emodin (AE) on TNBS-induced experimental colitis in mice1.2.1 Establish experimental colitis and drug interventionA total of 40 femal BALB/c mice were randomly divided into 5 groups (n=8, each group), ethanol control group, model group, high dose of aloe emodin group, middle dose of aloe emodin group, low dose of aloe emodin group. Colitis model was induced in mice by rectal administration of trinitro-benzene sulfonic acid (TNBS) dissolved in ethanol.8 hours after TNBS administration, the three dose of AE group was treated by intragastric administration with 75mg/Kg, 100mg/Kg,150mg/Kg AE respectively per day, control group and model group instead of with 0.2ml/10g lactic acid slinger liquid every day, all the groups were administrated drugs for 6 consecutive days.1.2.2 Evaluation of therapeutic effects① The weight and the stool situation of each animal were recorded daily and on the basis of these manifestation disease activity index (DAI) was evaluated.②Colon intestinal samples was collected after 6 days, the colonic macroscopic damage index (CMDI) and tissue damage index (TDI) were evaluated.③weight animals wet spleen and wet thymus, then according to the formula to calculate organ index.1.3 Inhibitory effects of aloe-emodin on proliferation, phagocytosis and translocation of HMGB1 in mouse peritoneal macrophage induced by LPSDifferent concentrations of aloe-emodin (10μmol/L,20μmol/L,40μmol/L,80μmol/L) were added to the normal or LPS-activated mouse peritoneal macrophages. Metabolic activity of mouse peritoneal macrophages was measured by MTT assay at 12h,24h and 48h after aloe-emodin treatment. Phagocytosis and the translocation of HMGB1 in mouse peritoneal macrophages were analyzed by neutral red phagocytosis and immunofluorescence test respectively after 24h treatment by aloe-emodin.1.4 Effect of aloe-emodin on TNBS-induced experimental colitis in mice based on HMGB1-Th17 regulation pathways1.4.1 Establish experimental colitis and drug interventionA total of 40 femal BALB/c mice were randomly divided into 4 groups (n=10, each group), ethanol control group, model group, AE group and Sulfasalazine (SASP) group. Colitis model was induced in mice by rectal administration of trinitro-benzene sulfonic acid (TNBS) dissolved in ethanol.8 hours after TNBS administration, AE group and SASP group were treated by gavage with 150mg/Kg AE and 260mg/Kg SASP respectively per day, control group and model group instead of with 0.2ml/10g lactic acid slinger liquid every day, each group was administrated for 6 consecutive days.1.4.2 Evaluation of therapeutic effects① The weight and the stool situation of each animal were recorded daily and on the basis of these manifestation disease activity index (DAI) was evaluated.②Colon intestinal samples was collected after 6 days, the colonic macroscopic damage index (CMDI) and tissue damage index (TDI) were evaluated.③ The serum levels of HMGB1, IL-17 and IFN-y were determined by ELISA.④ The colonic infiltration of Th17 cells was determined by immunofluorescence method.⑤ The percentages of Th1、Th17、Tcl cells in spleen as well as in mesenteric lymph nodes of mice were analyzied by flow cytometry (FCM).⑥ The colonic expression of HMGB1 was evaluated by Western Blot.Results1.1 HMGB1 regulates Th17 cell response in the pathogenic role of experimental colitis in mice1.1.1 Compared with ethanol control group, the model group had significantly higher DAI, CMDI and TDI scores(P<0.05-P<0.001); Using EP to inhibit HMGB1, it resulted in a dramatically decreased scores on DAI, CMDI and TDI (P<0.01-.P<0.001);1.1.2 In model group, serum HMGB1, IL-17 and IFN-ylevels by ELISA were significantly increased as well as the Th17 cell infiltration and HMGB1 expression in the colon compared with controls(P<0.05 or P<0.001); Meanwhile, the percentages of Th17, Thl and Tcl cells in the spleen and MLN had a remarkably higher than control group (p<0.001). After HMGB1 inhibition with EP, the levels of HMGB1, IL-17 and IFN-γ in serum (P<0.01) and the Th17 cell infiltration and HMGB1 expression in the colon were dropped significantly compared with control group. Furthermore, the percents of Th17, Th1 and Tcl cells were also decreased in the spleen and MLN (P<0.05 or P<0.01).1.2 Pharmacodynamics study of aloe emodin(AE) on TNBS-induced experimental colitis in miceCompared with ethanol control group, the model group had significantly higher DAI, CMDI and TDI scores (P<0.001); The middle and high-dose AE can significantly reduce the DAI, CMDI and TDI scores, especially the high-dose AE (P<0.05). The organ index results demonstrated that the thymus index was lower whereas spleen index was higher in model group than control group (P<0.001). Howere, the middle and high-dose AE treatment had the effect of promoting thymus index and inhibiting spleen index (p<0.05-P<0.001).1.3 Inhibitory effects of aloe-emodin on proliferation, phagocytosis and translocation of HMGBl in mouse peritoneal macrophage induced by LPSThe MTT assay showed aloe-emodin had no significant effect on the activity of nomal mouse macrophages at the dose of (10～80) μmol/L. However, aloe-emodin at the dose of (20～80) μmol/L markedly inhibited LPS-activated mouse macrophages proliferation in a dose dependent manner (P<0.05 or P<0.01) and this inhibitory effect reached the peak at 24h after aloe-emodin treatment. Meanwhile, aloe-emodin at the dose of (10～80)μmol/L exhibited the inhibitory effect on LPS-activated mouse macrophages phagocytosis (P<0.01). Immunofluorescence test showed that with aloe emodin concentration increased, the green fluorescence, of HMGB1 was gradually strengthened in cell nucleus, while in cytoplasm, the green fluorescence of HMGB1 was gradually weakened 24h after stimulated by LPS. Furthermore, aloe emodin at the dose of (20～80) μmol/L in a dose dependent manner reduced HMGB1 gray value in cytoplasm compared to LPS group (P<0.01).1.4 Effect of aloe-emodin on TNBS-induced experimental colitis in mice based on HMGB1-Th17 regulation pathways1.4.1 Compared with ethanol control group, the model group had significantly higher DAI, CMDI and TDI scores (P<0.05-P<0.001); AE or SASP treatment can reduce dramatically the scores of DAI, CMDI and TDI (P<0.01 or P<0.001).1.4.2 In model group, serum HMGB1, IL-17 and IFN-γ levels by ELISA were significantly increased as well as the Thl7 cell infiltration and HMGBl expression in the colon compared with controls (P<0.05 or P<0.001); Meanwhile, the percentages of Th17, Thl and Tcl cells in the spleen and MLN had a remarkably higher than control group (p<0.001). After treatment with AE or SASP, the levels of HMGB1, IL-17 and IFN-γ in serum and the Th17 cell infiltration and HMGB1 expression in the colon were all dropped significantly compared with control group(P<0.05-P<0.01). Furthermore, the percents of Thl7, Thl and Tcl cells were also decreased in the spleen and MLN (P<0.05 or P<0.01).Conclusion1.1 HMGB1 play an important role in pathogenesis of experimental colitis in mice by promoting Th17 cells and Thl/Tcl type response;1.2 150 mg/Kg dose of aloe emodin has better treatment effect for experimental colitis in mice;1.3 Aloe emodin in a dose dependent manner inhibited the proliferation, phagocytosis and translocation of HMGB1 in mouse peritoneal macrophages induced by LPS.1.4 AE may attenuate TNBS-induced colitis in mice via inhibiting HMGB1 mediated the Th17 cells and Th1/Tc1 cell response, thus to play a role of treatment and protection. |
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