| Ulcerative colitis(UC) is an inflammatory disease of unknown cause that exhibits a clinical course with remission and exacerbations,characterized by abdominal pain, rectal bleeding and diarrhea.Inflammation usually occurs in the rectum and lower part of the colon.The number of cases increased rapidly in China.With long term of bowel symptoms and systemic symptoms,the quality of life and social function of patients are also affected severely.The research of UC is a hot topic nowadays.One study showed 70.2%were proctosigmoiditis or proctitis.For the treatment of UC confined to the rectum and sigmoid,topical administration of medicines,such as glucocorticoids and 5-aminosalicylic acid(5-ASA),to the injured colonic mucosa has been shown to be more effective and less hazardous than systemic administration. However,current topical treatments for UC have their limitations.For example,the high price of 5-ASA prevents it to be wildly used in our country.When applying glucocorticoids topically,there is still a significant absorption of glucocorticoids through the inflamed mucosa that causes systemic effects.It is necessary to develop alternative therapeutic approaches that are effective,affordable and have less adverse effects. Muscovite is one kind of natural clay consisting of an insoluble double silicate of aluminium and magnesium.In traditional Chinese medicine it has been documented that muscovite has antidiarrheal activity and can protect mucosa.It's possible that rectal administration of muscovite can alleviate inflammation of UC.Adequate animal model of UC is a useful tool of research on the therapeutic agents.Several models of experimental colitis resembling ulcerative colitis have been reported previously.The most widely used models are induced by administering toxic chemical such as dextran sulfate sodium(DSS),trinitrobenzene sulfonic acid(TNBS). However,DSS-induced colitis results in inflammation mainly in the distal colonic mucosa.The histopathology of this model showed some characteristics resemblance to UC,enabling research into the pathogenesis and therapy of this disease.But an obvious obstacle with regard to the DSS model is that DSS is very expensive.Thus it is difficulty to apply this model widely in some regions.TNBS-induced colitis is a traditional model of inflammatory bowel disease and is widely used.In order to investigate whether rectal administration of muscovite can alleviate inflammation of UC,we established two animal models of UC to doublely check its effect.Besides TNBS-induced colitis,we developed an inexpensive DSS/ethanol model of UC rats.Female rats were randomized into four groups.In group A and B rats received 2%dextran sulfate sodium in the drinking water for 3 days.On day 4,rats in group A and B received 0.5ml 30%ethanol or saline intracolonically respectively.As a control,rats in group C and D only received intracolonic 30%ethanol or saline respectively.Before enema,the rats were lightly anesthetized with ether.30%ethanol or saline was instilled into the lumen of the colon through the rubber catheter.Disease Activity Index(DAI) was used to quantify the clinical evolution of every group.At various times(24h,3 th day,7th day,14 th day and 21 th day) after intracolonic administration of 30%ethanol or control saline,5 rats from each treatment group were randomly selected and killed to assess for colonic inflammatory and ulcerative responses with respect to gross morphologic damage and histopathologic scoring system.The results showed the DSS/ethanol induced significant weight loss,diarrhea and hematochezia in rats.Ulcerations and inflammation of the distal part of rat colon were developed rapidly.Histological examination showed an increased infiltration of polymorphonuclear leukocytes,lymphocytes and existence of cryptic abscesses and dysplasia.The weight of rat with colitis was decreased while the gross morphologic and histopathologic scores were increased.The successfully established DSS/ethanol model is characterized by a clinical course,localization of the lesions and histopathological features similar to human UC while it is much cheaper than DSS model.First we examined the effect of rectal administration of muscovite on DSS/ethanol induced colitis.The experimental animals were randomly divided into 4 groups to take the rectal administration of muscovite,5-ASA and the equal amount of control respectively from beginning of the induction of the colitis rats to the end of study while another group was administrated rectally with only saline served as normal control. After 10 days of treatment,the animals were assessed for colonic inflammatory and ulcerative responses with respect to change of body weight,gross morphologic damage and histopathologic scoring system.The results showed the loss of weight in the colitis groups was significantly higher than that of normal group(P＜0.01).Rectal administration of muscovite improved the loss of body weight of DSS/ethanol induced colitis.Treatment of rats with muscovite or 5-ASA decreased both morphologic and histopathologic scores of DSS/ethanol induced rats.This part showed that rectal administration of muscovite could ameliorate colonic inflammation of DSS/ethanol induced colitis.Then we examined the effect of administration of different dosage of muscovite on TNBS-induced colitis in rats.Female SD rats with TNBS-induced colitis were treated with rectal administration of different dosage of muscovite daily for 14 days.The changes in body weight,macroscopic damage and histologic scores were subsequently evaluated.The effect of muscovite was compared with that of 5-ASA.We found rectal administration of muscovite improved the loss of body weight,macroscopic and histologic scores of TNBS-induced colitis in a dose-dependent manner.This part showed that rectal administration of different dosage of muscovite could ameliorate colonic inflammation of TNBS-induced colitis.Based on these observations we proved that muscovite is useful for the treatment of these two kinds of colitis.Further experiments were also done to ascertain the possible mechanisms involved in the anti-inflammtory effect of muscovite.An exaggerated intestinal immune response in colon plays a key role in the pathophysiology of UC.Pathological processes accompanying UC are associated with aberrant expression of many proinflammatory cytokines,including tumor necrosis factor-α(TNF-α),Interleukin-1β(IL-1β) and IL-8.To ascertain the possible mechanisms involoed in the anti-inflammtory effect of muscovite,we examined the effect of muscovite on the mucosal inflammatory changes by studying the expression of IL-1βand TNF-αin the colonic tissues in rats with TNBS-induced colitis.Female SD rats with TNBS-induced colitis were treated with rectal administration of different dosage of muscovite daily for 14 days.Gene expression of TNF-α,IL-1β,mucin2 (MUC2) and intestinal trefoil factor(ITF) in the colonic tissues was assessed by semiquantitative reverse-transcription polymerase chain reaction(RT-PCR) while protein levels of TNF-αand IL-1βwere detected by enzyme-linked immunosorbent assay(ELISA).MUC2 expression in colonic mucosa was detected by immunohistochemistry and Alcian blue-periodic acid Schiff(AB-PAS).The results showed TNBS-induced expression of TNF-α,IL-1βwas reduced by administration of muscovite and 5-ASA.Reduction of MUC2 expression in colitis rats was reversed by muscovite and 5-ASA treatment.However,the expression of ITF mRNA in colonic mucosa was not affected.This part showed rectal administration of different dosage of muscovite can decrease the expression of TNF-α,IL-1β. In the end,the capacity of muscovite to adsorb cytokines in vitro was determine by measuring the residual amount of standard TNF-α,IL-1βand IL-8 after these cytokines were incubated with muscovite for 1 hours as well as the changes in the amount of TNF-α,IL-1βsecreted by lipopolysaccharide(LPS)-stimulated THP-1 cells and IL-8 secreted by LPS- stimulated HT-29 cells.Muscovite inhibited the expression of TNF-α, IL-1βsecreted by THP-1 and IL-8 secreted by HT-29 cells in a dose-dependent manner. The levels of standard TNF-α,IL-1βand IL-8 were also decreased dramatically after incubating with muscovite for 1 hour.In conclusion,we successfully established a model of chemincal-induced experimental colitis which is characterized by a clinical course,localization of the lesions and histopathological features similar to human UC.We confirmed that rectal administration of muscovite can ameliorate colonic inflammation of two different kinds of chemical-induced experimental colitis and can ameliorate colonic inflammation of TNBS-induced colitis in dose-depend manner.The mechanism of therapeutic effect of muscovite is related with the fact that rectal administration of different dosage of muscovite can decrease the mRNA and protein expression of TNF-α,IL-1β.The possible mechanisms of muscovite include a direct adsorptive action of cytokines production by mucosal cells.
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