The Protective Effect And Mechanism Of Curcumin And Bisdemethoxycurcumin On Intestinal Mucosa Of Ulcerative Colitis

Ulcerative colitis(UC)is a chronic non-specific inflammatory bowel disease,often manifested as abdominal pain,diarrhea,mucopurulent bloody stool and other systemic symptoms.UC often attack colorectal mucosa and submucosa.The intestinal inflammation of active UC can cause congestion and edema of intestinal mucosa,reduce the mucosal layer of intestinal wall,and damage the function of intestinal mucosal barrier.The intestinal mucosa is composed of epithelial cells(goblet cells)and intercellular connections(tight junction).Goblet cells can secrete a variety of mucin to compose the intestinal wall mucus layer.Tight junctions include many kinds of proteins such as Occludin,Claudins,Zonula occlusions,which are the protein framework of the mechanical barrier of intestinal mucosa barrier.When UC occurs,the decrease of mucus layer makes the pathogenic microorganism directly contact the intestinal mucosa,which makes the mucosa barrier damaged.Notch signaling pathway can regulate Th17/Treg balance to differentiate into Th17 cells,increase the secretion of inflammatory factors such as interleukin-17(IL-17),tumor necrosis factor-?(TNF-?),and aggravate the injury of mucositis.The activation of Notch signaling pathway can transform intestinal epithelial cells from secretory cell lines to absorptive cell lines to reduce the number of mature goblet cells.Objective:To investigate the therapeutic effects of Curcumin(Cur)and Bisdemethoxycurcumin(BDMC)on ulcerative colitis(UC)induced by acetic acid(AA)and dextran sulfate sodium(DSS)in mice,and the effects and mechanisms of Cur and BDMC on the inflammatory response of intestinal mucosa,the mucus layer of intestinal wall and the barrier function of intestinal mucosa.Method:1.To explore the protective effect and mechanism of Cur and BDMC on intestinalmucosa injury induced by AA in UC mice 50 female BALB/c mice were randomly divided into normal group,5% AA model group,Cur group(200mg/kg),BDMC high-dose group(200mg/kg)and BDMC low-dose group(100mg/kg).Except the normal group and model group,the other groups were given respectively corresponding doses of drugs by gavage once a day for 7 consecutive days;the normal group and model group were given the same volume of 1% CMC-Na solution by gavage.24 h after the last administration,except for the normal group,the other groups were given 5% AA solution 100?L per mice by enema through the anus,and the normal group was given the same volume of saline.24 h after established the model,blood was collected,the length and weight of colon were measured and the intestinal weight index was calculated;the general condition of colon mucosa injury was observed and the colon mucosa damage index(CMDI)was evaluated.Part of the colon was fixed in 4% paraformaldehyde solution,embedded in paraffin and sliced,the pathological changes of the colon mucosa were observed by HE staining and the histopathological score(HS)of colon was evaluated.The activity of MPO,SOD and the content of MDA,NO,PGE-2 in colonic tissue were detected by biochemical analysis,and the contents of PAF and DAO in serum were determined.The contents of TNF-?,IL-17 and IL-13 were detected by ELISA.Western Blot was used to detect the expression of IL-17 A,Notch1 and DLL1 protein content in colonic tissue.2.To explore the protective effect and mechanism of Cur and BDMC on intestinal mucosal barrier of UC mice induced by DSS 60 female BALB/c mice were randomly divided into normal group,3% DSS model group,Cur high-dose group(200mg/kg),Cur low-dose group(100mg/kg),BDMC high-dose group(200mg/kg),BDMC low-dose group(100mg/kg).Except the normal group,the mice in the other groups were free to drink 3% DSS solution everyday for 10 consecutive days;the mice in the normal group were given distilled water.Except the normal and model groups,the other groups were given respectively corresponding doses of drugs by gavage once a day for 10 consecutive days;the normal group and model group were given the same volume of 1% CMC-Na solutionby gavage.During the period of modeling and administration,mice were weighed daily,their fecal properties were observed,fecal occult blood test(OBT)was conducted,and disease activity index(DAI)was calculated to evaluate the disease status of mice.24 h after the last administration,blood and colon tissues were collected from mice.The colon was weighed,the length was measured and the weight index of intestine was calculated.The spleen was weighed and the weight index of spleen was calculated.The general condition of colon mucosa injury was observed and the colon mucosa damage index(CMDI)was evaluated.Part of the colon was fixed in 4% paraformaldehyde solution,embedded in paraffin and sliced,the pathological changes of the colon mucosa were observed by HE staining and the histopathological score(HS)of colon was evaluated.The tissue sections were stained with AB-PAS to calculate the number of goblet cells and the thickness of mucus layer.The contents of DAO and D-La in serum were measured by biochemical analysis.The protein expressions of Occludin,Claudin-1,ZO-1,Hes-1,Math-1 and MUC2 were measured by Western Blot.Result:1.The effect of Cur or BDMC on intestinal mucosa damage by AA-induced UC mice(1)The results of colon length,weight and intestinal weight index Compared with normal group,the colon length and weight of model group had evidently difference(P<0.01),and the intestinal weight index was significantly increased(P<0.001).Compared with model group,Cur group or BDMC high-dose group could evidently inhibited the above changes of colon(P<0.01).BDMC low-dose group inhibited colon shortening and intestinal weight index(P<0.05).(2)The general damage of intestinal mucosal and the results of CMDI score evaluation In model group,there were hematoma,erosion and ulcer with inflammatory exudation,and the CMDI score was significantly higher than normal group(P<0.001).Compared with model group,the administration group could alleviate the colonic hematoma and ulcer,reduce the exudation of mucositis,and decrease the CMDI score.The Cur group or BDMC high-dose group could evidently reduce the CMDI score(P<0.01),and BDMC low-dose group reduced the CMDI score(P<0.05).(3)Histopathological changes and HS evaluation results of intestinal mucosa in mice The colonic epithelium and mucosa in model group were not continuously with inflammatory cell infiltration.The goblet cell was loss reduction and the crypt structure was disappeared,so the HS was significantly higher than normal group(P<0.001).Compared with model group,the colonic epithelial structure and mucosal damage in every administration group were relieved to varying degrees.The significant decrease of HS in BDMC high-dose group(P<0.001),the evidently decrease of HS in Cur group(P<0.01),and the decrease of HS in BDMC low-dose group(P<0.05).(4)The contents of MPO,SOD,MDA,NO,PGE-2 in colonic tissue and PAF,DAO in serum of mice were measured Compared with normal group,the contents of MPO,MDA,NO,PGE-2 in colon tissue of model group increased significantly,and the activity of SOD decreased significantly(P<0.001).Compared with model group,the contents of MPO,MDA,NO and PGE-2 in BDMC high-dose group decreased significantly,and the activity of SOD increased significantly(P<0.001).In Cur group,MPO content decreased and SOD content increased evidently(P<0.01),the contents of MDA,NO and PGE-2decreased significantly(P<0.001).The contents of MPO,MDA,NO and PGE-2decreased,and the activity of SOD increased in BDMC low-dose group(P<0.05).The serum PAF and DAO contents in model group were significantly increased than normal group(P<0.001).Compared with model group,the contents of PAF and DAO in BDMC high-dose group or Cur group were significantly decreased(P<0.001),while those in BDMC low-dose group were significantly lower(P<0.01).(5)The contents of TNF-?,IL-17 and IL-13 in UC mice were determined by ELISA Compared with normal group,the content of TNF-? and IL-17 in model group increased significantly(P<0.001),while the content of IL-13 decreased evidently(P<0.01).Compared with model group,the TNF-? and IL-17 contents of Cur group or BDMC high-dose group were significantly reduced(P<0.001),and the IL-13 contentswere evidently increased(P<0.01).The content of TNF-? and IL-17 in BDMC low-dose group decreased evidently(P<0.01),while the content of IL-13 increased(P<0.05).(6)The contents of IL-17 A,Notch1 and DLL1 in UC mice were determined by Western Blot Compared with normal group,the expression of IL-17 A,Notch1 and DLL1 in colon tissue of model group increased significantly(P<0.001).Compared with model group,the expression of IL-17 A and Notch1 in Cur group or BDMC high-dose group decreased significantly(P<0.001),and the expression of DLL1 decreased(P<0.05).The expression of IL-17 A and Notch1 in BDMC low-dose group were decreased(P<0.05).2.Effects of Cur or BDMC on Notch signaling pathway and intestinal mucosal barrier in DSS-induced UC mice(1)DAI evaluation results of mice Compared with normal group,the DAI of model group increased evidently from the 5th day(P<0.01),and increased significantly from the 6th day to the end of the experiment(P<0.001).Compared with model group,the DAI in BDMC high-dose group was evidently reduced(P<0.01),and that in Cur high-dose group was lower(P<0.05).(2)The results of colon length,weight,intestine weight index,spleen weight and spleen weight index in mice Compared with normal group,the colon length and weight of model group were significantly different(P<0.001).Compared with model group,Cur high-dose group or BDMC high-dose group inhibited the colonic shortening(P<0.01)and the increasing of colon weight(P<0.001).Cur low-dose group or BDMC low-dose group could inhibit the colonic shortening(P<0.05),and inhibit the colon weight growth(P<0.05).Compared with normal group,the intestinal weight index of model group increased significantly(P<0.001).Compared with model group,Cur high-dose group or BDMC high-dose group could evidently reduce the intestinal weight index(P<0.01).Cur low-dose group or BDMC low-dose group could reduce the intestinal weight index(P<0.05).The spleen weight and the spleen weight index of model group were significantly increased than normal group(P<0.001).Compared with model group,BDMC high-dose group significantly reduced the spleen weight and spleen weight index(P<0.001),Cur high-dose group evidently reduced the spleen weight and spleen weight index(P<0.01).(3)The general damage of intestinal mucosal and the results of CMDI score evaluation Compared with normal group,the CMDI score of model group was significantly higher(P<0.001),which showed the colonic mucosa had obvious hematoma and exudation with erosion and ulcer.The intestinal wall was hard and thick,and the ulcer was deep and accompanied with blood spots in some severe cases.Compared with model group,the administration group could alleviate the intestinal mucosal injury in different degrees,and the CMDI score of Cur high-dose group or BDMC high-dose group were significantly lower(P<0.001).The CMDI score of Cur low-dose group or BDMC low-dose group were evidently different(P<0.01).(4)Histopathological changes and HS evaluation results of intestinal mucosa in mice In model group,the structure of intestinal mucosa was discontinuous,submucous edema and inflammatory cell infiltration were found in all layers of intestinal wall.The structure of goblet cells were destroyed and the crypt structure disappeared.The HS was significantly higher than normal group(P<0.001).Compared with model group,the administration group could reduce the inflammatory infiltration and restore the structure of intestinal mucosa.The HS in Cur high-dose group or BDMC high-dose group decreased significantly(P<0.001).The BDMC low-dose group could reduce HS evidently(P<0.01)and Cur low-dose group could reduce it statistical differences(P<0.05).(5)The number of goblet cells and the thickness of mucus layer in intestinal mucosa of mice Compared with normal group,the number of goblet cells in colon mucosa ofmodel group decreased significantly(P<0.001).Compared with model group,Cur high-dose group or BDMC high-dose group had more staining substance in colon mucosa,and the number of goblet cells increased significantly(P<0.001).The number of goblet cells increased evidently in Cur low-dose group(P<0.01),and increased in BDMC low-dose group(P<0.05).Compared with normal group,the distribution of the mucus layer in model group was discontinuous,damaged or disappeared,and the thickness of the mucus layer was evidently thinner(P<0.01).Cur high-dose group or BDMC high-dose group evidently increased the thickness of mucus layer(P<0.01).Cur low-dose group or BDMC low-dose group could increase the thickness of mucus layer,with statistical difference(P<0.05).(6)The results of DAO and D-La in serum of mice Compared with normal group,the levels of DAO and D-La in model group were significantly higher(P<0.001).Compared with model group,the Cur high-dose group or BDMC high-dose group could evidently reduce the serum DAO and D-La contents(P<0.01).The concentration of DAO and D-La in serum of Cur low-dose group or BDMC low-dose group decreased(P<0.05).(7)The expression of Hes-1,Math-1,MUC2,Occludin,Claudin-1 and ZO-1 by Western Blot analysis in colonic tissue The Western Blot results showed that compared with normal group,the expression of Hes-1 protein in colon tissue of model group increased significantly,while the expression of Math-1 protein and MUC2 protein decreased significantly(P<0.001).Compared with model group,the expression of Hes-1 protein in Cur high-dose group or BDMC high-dose group decreased evidently(P<0.01),and the expression of Math-1 and MUC2 protein increased evidently(P<0.01).The expression of Hes-1 protein decreased in Cur low-dose group or BDMC low-dose group(P<0.05),and the expression of Math-1 and MUC2 protein increased(P<0.05).The Western Blot results showed that compared with normal group,the expression of Occludin protein and Claudin-1 protein in colon tissue of model group decreased significantly(P<0.001),and the expression of ZO-1 protein decreasedevidently(P<0.01).Compared with model group,the protein expression of Occludin,Claudin-1 and ZO-1 increased significantly in Cur high-dose group or BDMC high-dose group(P<0.001).The protein expression of Occludin,Claudin-1 and ZO-1in Cur low-dose group or BDMC low-dose group were increased(P<0.05).1.Both Cur and BDMC can restore weight loss and improve colonic symptoms and histopathological damage in mice with ulcerative colitis;2.Both Cur and BDMC can inhibit MPO,MDA,NO,PGE2,PAF content,improve SOD activity,inhibit colon inflammation and oxidative stress in UC mice;3.Cur and BDMC can effectively decrease the expression of Notch1 and DLL1 proteins in Notch signaling pathway,then down-regulated the expression of IL-17 and TNF-? proteins,then inhibit the inflammation of colon mucosa;4.Cur and BDMC can regulate the expression of Hes-1 and Math-1 proteins downstream of the Notch signaling pathway,thereby increasing the expression of MUC2 protein and increasing the thickness of intestinal mucus layer;5.Cur and BDMC can also promote the expression of Occludin,Claudin-1 and ZO-1proteins,and reconstruct the mechanical barrier of intestinal mucosa.