Ampelopsin, Total Glucosides Of Peony Attenuates2,4,6-trinitrobenzene Sulfonic Acid/Ethanol-induced Colitis In Rats

Introduction Inflammatory bowel disease (IBD), including ulcerative colitis (UC) and Crohn’s disease (CD), is an idiopathic, non-specific inflammatory disorder of the intestinal tract with unknown causes. It is currently believed that genetic susceptibility, dysfunction of immune regulation in intestinal mucosa and intestinal bacteria are involved in the pathogenesis of IBD. Immune regulation dysfunction might be the direct cause for onset of IBD. It is now well accepted that abnormal immune response mediated by proinflammatory cytokines such as TNF-α,IL-1β, IL-6and IL-8, and antiinflammatory cytokines IL-10plays an important role in balance of Thl and Th2as well as inflammatory severity and activity in IBD. Currently, there is a lack of an effective therapy to cure the diseases since causes and mechanisms of IBD are not totally understood. Aminosalicylates, corticosteroids, immunomodulating and immunosuppressive drugs are used for treatment of IBD by controlling active inflammation and regulating abnormal immune response. However, such treatments have multiple adverse effects, particularly for long-term administration, and relapse is high upon drug discontinuance. Biological agents such as anti-TNF-a monoclonal antibody can induce the alleviation of IBD, but at a high cost and put the patients at risk to develop treatment-related cancers, and surgical resection of the colon and ileostomy is the ultimate alternative in many cases. Therefore, there is an urgent for the development of new and specific therapies for IBD with few side effects.Part I:Ampelopsin attenuates2,4,6-trinitrobenzene sulfonic acid/ethanol-induced colitis in ratsAmpelopsin is a flavonoids extracted from Rattan tea which is a traditional Chinese herbal remedy prepared from the stems and leaves of the plant ampelopsis grossedentata. Ampelopsin was reported as a major bioactive component in ampelopsis grossedentata and first isolated from ampelopsis meliaefolia by Kotake and Kubota in1940.Recent studies have shown that ampelopsin has a broad range of biological functions including inhibition of apoptosis, hypoglycemic, anti-hypertension, anti-bacterial, anti-inflammatory, antioxidant, anti-tumor, hepatoprotective, and neuroprotective effects. These findings support the hypothesis that ampelopsin is also effective against IBD. Therefore, ampelopsin may be a bright potential candidate Chinese drug for treatment of IBD. Objective The present study is to investigate therapeutic effects of ampelopsin on2,4,6-trinitrobenzene sulfonic acid (TNBS)/ethanol-induced colitis in rats and to explore potential clinical use of ampelopsin for treatment of inflammatory bowel disease (IBD).Methods Sixty Sprague-Dawley (SD) rats were randomly grouped into normal controls, model controls, sulfasalazine (SASP) controls (100mg/kg/day), and low, medium and high-dose ampelopsin groups (125,250and500mg/kg/day, respectively).24hours following colonic instillation of TNBS, ampelopsin and SASP were given by gastric gavage three times a day for7days.Disease activity index (DAI), colon macroscopic damage index (CMDI), histopathological score (HPS) and myeloperoxidase (MPO) activity were evaluated. Levels of serum and colon TNF-α,IL-1β and IL-10were measured by enzyme-linked immunosorbent assay (ELISA), expression of TNF-α,IL-1β and IL-10mRNA in colonic tissues was detected by reverse transcription-polymerase chain reaction (RT-PCR), and expression of NF-κB p65protein in colonic tissues was detected by immunohistochemical methods, respectively.Results①Normal control rats treated with ethanol enema gained body weight and defecated normally. In the model control group, pasty to liquid gross bloody stool and weight loss were observed in all rats. When ampelopsin (250or500mg/kg/day) or SASP (100mg//kg/day) was administered on day7, bloody stools were not evident, stools were better formed, and weight loss was lessened. The colons from rats in the model control group were seriously adherent to small intestines and spleen, and showed marked hyperemia, inflammation, necrosis, ulcer, whereas the colons from rats in the normal control group showed no or a slight inflammation. Treatment with ampelopsin (250or500mg/kg/day) or SASP markedly decreased both hyperemia and inflammation in the colons. Administration of ampelopsin (250or500mg/kg/day) significantly improved the DAI and CMDI8days after TNBS instillation. And there was no significant difference compared with administration of SASP (P>0.05).②Ulcerations, massive transmural infiltration of inflammatory cells, thickening of the colon wall, goblet cell depletion, and extensive fibrosis found throughout colons in the model control group. Administration of ampelopsin (250or500mg/kg/day) improved these signs and the HPS significantly. And there was no significant difference compared with administration of SASP (P>0.05).③Rectal instillation of TNBS led to the marked enhancement of NF-κB p65protein expression in colonic tissues. Compared with the normal group, administration of ampelopsin (250or500mg/kg/day) significantly reduced the enhancement of NF-κB p65protein expression in colonic tissues. The difference was not significant when compared with administration of SASP (P>0.05).④MPO activity was very low in the normal control group, and increased significantly nearly6-fold in rats after TNBS enema in the model control group. In contrast, this increase was significantly blunted by about50%by administration of ampelopsin (250or500mg/kg/day). And there was no significant difference compared with administration of SASP (P>0.05).⑤ectal instillation of TNBS led to the marked enhancement of levels of serum TNF-α and IL-1β, and the marked decrease of levels of serum IL-10. Administration of ampelopsin (250or500mg/kg/day) significantly reduced the enhancement of levels of serum TNF-α and IL-1β and at the same time increased the decrease of levels of serum IL-10. And there was no significant difference when compared with administration of SASP(P>0.05).⑥Rectal instillation of TNBS led to the marked enhancement of mRNA expression of TNF-α and IL-1β, and the marked decrease of that of IL-10in colonic tissues. Administration of ampelopsin (250or500mg/kg/day) significantly reduced the enhancement of mRNA expression of TNF-α and IL-1β and at the same time significantly increased the decrease of that of IL-10. And there was no significant difference when compared with administration of SASP (P>0.05).⑦Protein expression of TNF-α and IL-1β was markedly elevated and that of IL-10was markedly decreased in the colons after rectal TNBS instillation, which was consistent with results of the mRNA expression.250or500mg/kg/day ampelopsin treatment significantly abrogated the elevation in protein expression of TNF-α,IL-1β and at the same time significantly increased the decrease of protein expression of IL-1O.The difference was not significant when compared with administration of SASP (P>0.05). On the other hand, administration of ampelopsin at the dose of125mg/kg/day showed modest improvement on colonic inflammation and regulatory effects on expression of TNF-α,IL-1β and IL-10, but the values were lower than administration of SASP (P<0.01).Conclusion Ampelopsin attenuates TNBS/ethanol-induced colitis in rats and its efficacy is similar to SASP, the potential mechanism might be related to the down-regulation the enhancement of NF-κB p65protein expression in colonic tissues and adjustment of Thl/Th2cytokines polarization by decreasing pro-inflammatory cytokine TNF-α and IL-1β, and increasing anti-inflammatory cytokine IL-10. Part II:Total glucosides of peony attenuates2,4,6-trinitrobenzene sulfonic acid/ethanol-induced colitis in ratsTotal glucosides of peony (TGP) is a group of glucosides extracted from peony, including paeoniflorin, hydroxy-paeoniflorin, paeonin, albiflorin and benzoylpaeoniflorin. Paeoniflorin is a monoterpene glucoside, the major active ingredient of TGP, accounting for over90%of total glucosides. Now TGP has been a common prescription drug used for the treatment of autoimmune diseases including rheumatoid arthritis, ankylosing spondylitis and systemic lupus erythematosus and shown significant efficacy in the clinic. Recent studies have shown that TGP has anti-inflammatory and immunoregulatory functions, by inhibiting Th1cytokines and enhancing Th2cytokines. These findings support the hypothesis that TGP is also effective against IBD. Therefore, TGP may be a bright potential candidate Chinese drug for treatment of IBD.Objective The present study is to investigate therapeutic effects of total glucosides of peony (TGP) on2,4,6-trinitrobenzene sulfonic acid (TNBS)/ethanol-induced colitis in rats and to explore potential clinical use of TGP for treatment of inflammatory bowel disease (IBD).Methods Sixty Sprague-Dawley (SD) rats were randomly grouped into normal controls, model controls, sulfasalazine (SASP) controls (100mg/kg/day), and low, medium and high-dose TGP groups (25,50and100mg/kg/day, respectively).24hours following colonic instillation of TNBS, TGP and SASP were given by gastric gavage three times a day for7days.Disease activity index (DAI), colon macroscopic damage index (CMDI), histopathological score (HPS) and myeloperoxidase (MPO) activity were evaluated. Levels of serum TNF-α,IL-1β and IL-10were measured by ELISA, and expression of TNF-α,IL-1β and IL-10mRNA and protein in colonic tissues was detected by RT-PCR and Western blot, respectively.Results①Normal control rats treated with ethanol enema gained body weight and defecated normally. In the model control group, pasty to liquid gross bloody stool and weight loss were observed in all rats. When TGP (50or100mg/kg/day) or SASP was administered on day7, bloody stools were not evident, stools were better formed, and weight loss was lessened. The colons from rats in the model control group were seriously adherent to small intestines and spleen, and showed marked hyperemia, inflammation, necrosis, ulcer, whereas the colons from rats in the normal control group showed no or a slight inflammation. Treatment with TGP (50or100mg/kg/day) or SASP markedly decreased both hyperemia and inflammation in the colons. Administration of TGP (50or100mg/kg/day) significantly improved the DAI and CMDI8days after TNBS instillation. And there was no significant difference compared with administration of SASP (P>0.05).②Ulcerations, massive transmural infiltration of inflammatory cells, thickening of the colon wall, goblet cell depletion, and extensive fibrosis found throughout colons in the model control group. Administration of TGP (50or100mg/kg/day) improved these signs and the HPS significantly.③MPO activity was very low in the normal control group, and increased significantly nearly5-fold in rats after TNBS enema in the model control group. In contrast, this increase was significantly blunted by about50%by administration of TGP (50or100mg/kg/day).④Rectal instillation of TNBS led to the marked enhancement of levels of serum TNF-α and IL-1β and the marked decrease of levels of serum IL-10. Administration of TGP (50or100mg/kg/day) significantly reduced the enhancement of levels of serum TNF-α and IL-1β and at the same time increased the decrease of levels of serum IL-10. And there was no significant difference when compared with administration of SASP (P>0.05).⑤Rectal instillation of TNBS led to the marked enhancement of mRNA expression of TNF-α and IL-1β, and the marked decrease of that of IL-10in colonic tissues. Administration of TGP (50or100mg/kg/day) significantly reduced the enhancement of mRNA expression of TNF-α and IL-1β and at the same time significantly increased the decrease of that of IL-10.⑥Protein expression of TNF-α and IL-1β was markedly elevated and that of IL-10was markedly decreased in the colons after rectal TNBS instillation, which was consistent with results of the mRNA expression.50or100mg/kg/day TGP treatment significantly abrogated the elevation in protein expression of TNF-α,IL-1β and at the same time significantly increased the decrease of protein expression of IL-10. The difference was not significant when compared with administration of SASP (P>0.05). On the other hand, administration of TGP at the dose of25mg/kg/day showed modest improvement on colonic inflammation and regulatory effects on expression of TNF-α,IL-1β and IL-10, but the values were lower than administration of SASP (P<0.01).Conclusion TGP attenuates TNBS/ethanol-induced colitis in rats and its efficacy is similar to SASP, the potential mechanism might be related to the adjustment of Thl/Th2cytokines polarization by decreasing pro-inflammatory cytokine TNF-α and IL-1β, and increasing anti-inflammatory cytokine IL-10.