| Background and Purpose:Ulcerative colitis, an idiopathic inflammatory bowel disease (IBD) with chronic and relapsing inflammation along the gastrointestinal tract. It belongs to the "abdominal pain", "'diarrhea" and "hemafecia" in Chinese medicine. The detailed pathological mechanism is still unclear. However, NF-?B activation and NLRP3 mediated inflammasome activation play an important role in the occurrence and development of ulcerative colitis. Now days, the conventional drug was found to induce side effects, and new therapies and alternative medicines, therefore, are urgently needed. Because of their low toxicity profiles and high patient compliance, herbal medicines have attracted lots of studies for curing IBD.Fagopyrum cymosum (Trev.) Meisn (Fag), which belongs to Polygonaceae family, has been widely used to treat inflammatory diseases, including lung injury and diarrhea. Also, some clinical or animal study revealed that Fag could ameliorate irritable bowel syndrome (IBS) partially through reducing the intestinal inflammation and enhancing the function of mucosal epithelium through regulating the structure and function of tight junctions. To the best of our knowledge, the therapeutic effect of Fag on IBD has not been explored. Thus, in this study, we would conduct our study as follows:Part 1:Investigating the tissue distribution of the main active constitutes in DSS induced colitic mice after oral administration of Fag extracts. Part 2: The potential therapeutical effects of Fag extract on TNBS induced colitis mice. Part 3:The potential therapeutical effects of Fag extract on DSS induced colitis mice. Part 4:The potential anti-inflammatory effects of Fag extract on NF-?B activation and NLRP3. Thus, we could partially explain the effectiveness of Fag on UC from an in vivo and in vitro aspect.Main Methodsand Results:Firstly, we developed a HPLC method for the simultaneously determination of the protocatechuic acid, catechin, epicatechin, procyanidins B1, and procyanidins B2 in the Fag extracts. The results showed that there were 6.98 mg protocatechuic acid,4.07 mg procyanidin B1,2.64 mg catechin,8.43 mg procyanidin B2, and 17.84 mg epicatechin in each gram of the crude 50% ethanol extracts. Based on these results, a LC-MS/MS analytical method was developed and validated to detect catechin, epicatechin, procyanidins Bl1 and procyanidins B2 in mouse biological samples. The method was validated by specificity, linearity, lower limits of quantification (LLOQ), precision, accuracy, matrix effect, recovery and stability. DSS was selected to induce colitis mice. And the plasma, liver, jejunum and colon samples were collected and analyzed after oral administration of Fag extracts (2.24g/kg). The results indicated that this method could be used for the determination of the concentration of catechin, epicatechin, procyanidins B1, and procyanidins B2 in mouse biological samples. The systemic exposure of catechin, epicatechin, procyanidins B1, and procyanidins B2 were significantly increased in the DSS induced colitis mice, similar results could be seen in the liver, jejunum and colon tissues. These results indicated that the increased systemic exposure may be beneficial for the therapeutical effects of Fag for UC.In the second part,2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced colitis mice were applied as the model of IBD. The different dose of Fag extract was orally administrated for the therapy of UC, and the conventional drug, salicylazosulfapyridine (SASP), was also used as a control. Body weight changes, colon lengths, histological analysis, immunohistochemical study of inflammatory cells (F4/80), colonic myeloperoxidase (MPO) activity, LPS and pro-inflammatory cytokines in plasma, and mRNA expression of IL-1?, TNF-? and IL-6 in colon were also studied. The results showed that Fag significantly ameliorated TNBS-induced body weight loss and colonic shortening in mice. Meanwhile, Fag suppressed tissue mRNA levels of pro-inflammatory cytokines and reduced macrophage infiltration into colonic tissues. The plasma LPS and pro-inflammatory cytokines were also inhibited by Fag treatment.In the third part, a similar animal model, DSS-induced colitis mice were applied for exploring the potential effects of Fag. The different dose of Fag extract, the conventional drug, salicylazosulfapyridine (SASP) were orally administrated, body weight changes, colon lengths, histological analysis, colonic myeloperoxidase (MPO) activity, colonic tight junction proteins (ZO-1 and occludin) expression, immunohistochemical study of NF-?B, LPS, cytokines (IL-6, TNF-? and IL-10) in plasma, and mRNA expression of IL-6, TNF-? and IL-10 in colon, and the gut permeability (FD4 as a marker) were also studied. Results indicated that in DSS-induced colitis mice, Fag significantly ameliorated TNBS-induced body weight loss and colonic shortening in mice. Meanwhile, Fag suppressed tissue mRNA levels of pro-inflammatory cytokines, increased the IL-10 mRNA expression in colonic tissue. Fag could reduced the colonic MPO activity, increased the ZO-1 and occluding expression in DSS induced colitis mice. Also the plasma LPS and pro-inflammatory cytokines were inhibited by Fag treatment, while Fag could increase the plasma IL-10 level in DSS induced colitis mice. Meanwhile the gut leakage could be restored by Fag treatment.In the last part, to further verify the anti-inflammatory effects of Fag at the molecular level, a murine macrophage cell line, Raw264.7 and THP-1 cell, a human acute monocytic leukemia cell line, were employed. The anti-inflammatory effects of Fag on LPS induced secretion of IL-6 and TNF-?, the protein level of Cox-2 and iNOS were studied. The NF-?B p65 nuclear translocation and I?B phosphorylation were determined using western blot in RAW264.7 cell. The NLRP3 pathway were also studied..The results demonstrated that Fag inhibited the production of pro-inflammatory cytokines through the inhibition of NF-?B p65 nuclear translocation and I?B phosphorylation. Moreover, Fag could inhibit the LPS induced up-regulation of Cleaved Caspase-1, IL-1?, NLRP3 and ASC in THP-1 cells.Conclusions and Significance:Our study demonstrated that Fag could significantly ameliorate the DSS or TNBS induced colonic inflammation and the intestinal barrier function. The inhibition of p65 nuclear translocation and I?B phosphorylation may partially contribute to anti-inflammatory effects of Fag. Also, our study indicated that Fag could inhibit the Cleaved Caspase-1, IL-1?, NLRP3 and ASC. Our study showed that Fag could be served as a better anti-inflammatory drug for UC. |
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