| Objectives:To evaluate the therapeutic effects of Weichangling capsule on rat ulcerative colitis(UC)induced by 2,4-dinitrochlorobenzene(DNCB)combined with acetic acid,study its effect on the colonic inflammatory response and intestinal mucosal barrier-related molecule expression in UC rats,and to explore the molecular mechanism of Weichangling capsule in the treatment of UC.Methods:1.70 male SD rats were randomly divided into normal control group,model group,low(200 mg/kg),medium(400 mg/kg),high(800 mg/kg)doses of Weichangling capsule group,420 mg/kg mesalazine as western medicine positive control group,400 mg/kg Changweining capsule as Chinese medicine positive control group,each with 10 rats.2.Rat UC model was duplicated:DNCB combined with acetic acid was used to duplicate the rat UC model.After 3 days,the symptoms such as diarrhea and mucopurulent bloody stool were observed.Colonic histopathological examination to confirm the successful replication of the model.3.After the model was successfully replicated,the related drugs were given by intragastric administration once a day according to the above groups.The normal group and the model group were given intragastric 0.5%sodium carboxymethyl cellulose.After 14 days of continuous administration,the rat samples would be collected for testing.4.During the modeling and administration period,the general conditions such as activities,diet,body weight,colour and luster of hair were observed every day,and the disease activity index(DAI)was scored.5.After the administration,the samples were collected,the colon length and colon wet weight index of animal were measured,the gross damage of the colon mucosa was observed,and the colon mucosa damage index(CMDI)score was performed.Hematoxylin-eosin(HE)staining method was used to observe the changes of colonic histopathology,and the colonic histopathological score(HS)was performed to comprehensively evaluate the therapeutic effect of Weichangling capsule on UC in rats.6.Colonic periodic acid-schiff stain(PAS)was used to observe the integrity of colonic mucosa epithelium and the changes of goblet cells.7.DNA fragmentation in situ end labeling(TUNEL)was used to detect the apoptosis of rat colon tissue cells.8.Evans blue(EB)was gavaged,and the concentration of EB in rat serum was determined to evaluate the changes in colonic mucosal permeability.9.Enzyme-linked immunosorbent assay(ELISA)was used to determine the concentrations of interleukin(IL)-8,IL-10,IL-17,IL-23,interferon-y(IFN-y)and epidermal growth factor(EGF)in rat serum,respectively.10.RT-qPCR method was used to detect the mRNA expression of TNF-α,MUC2,Occludin,Claudin-1,ZO-1 in colon tissue.11.Immunohistochemistry and western blot were used to detect the protein expression of TNF-α,MUC2,Occludin,Claudin 1,ZO-1.Results:1.Three days after modeling,the UC model rats had a significant decrease in food and water intake,lethargy,and grouping together,loose stools around the anus,and pus and blood in some rats.The body weight of the UC rats dropped significantly.The body weight still decreased to varying degrees on 1-3 days after treatment.Compared with the normal group,the body weight loss of the model group was significantly different(P<0.001),yet gradually increased from the 6th day.Compared with the model group,the body weight change of low-dose Weichangling capsule group was not significantly different(P>0.05),and the body weight retention of the middle and high-dose group was greater than that of the model group(P<0.05).Compared with mesalazine or Changweining capsule group,there was no significant difference in the body weight change of the medium-dose Weichangling capsule group(P>0.05).2.During the experiment,rats in the normal group were given 0.5%sodium carboxymethylcellulose by gavage,and individual rats showed a slight weight loss,and then all increased steadily.The stools were normal,the occult blood negative,and DAI score was near zero.Compared with the normal group,the DAI score of the model group increased significantly(P<0.001).DAI score decreased to varying degrees on 2-3 days after administration.Compared with the model group,DAI score of 200 mg/kg Weichangling capsule group had no significant change(P>0.05),while DAI scores of the other administration groups decreased(P<0.05).Compared with mesalazine group or Changweining capsule group,there was no significant difference in DAI scores of the medium and high-dose groups of Weichangling capsule(P>0.05).3.Compared with the normal group,the colon of the model group was shortened(P<0.05),and the colon wet weight index and colon CMDI score were higher(P<0.05).Compared with the model group,except 200 mg/kg Weichangling capsule group,the other treated groups could reduce colon shortened degree,colon wet weight index,and colon CMDI score(P<0.05).The high dose of Weichangling capsule obtained the similar effects to those of mesalazine and Changweining capsule(P>0.05).4.The results of HE staining and HS showed that the model group had local erosive ulcers in the colon,with a large number of exudates and necrotic tissues at the bottom,a large number of inflammatory cell infiltrations in the mucosa and submucosa,and severe gland atrophy.Compared with the normal group,colonic HS of the model group was significantly increased(P<0.01).Compared with the model group,the colonic epithelial structure and intestinal mucosal injury of rats in the each treated group was relieved to varying degrees,the crypt structure was repaired,and the inflammatory cell infiltration was reduced.HS of 400,800 mg/kg Weichangling capsule group,420 mg/kg mesalazine group,and 400 mg/kg Changweining capsule group significantly decreased(P<0.01).Compared with the Changweining capsule group,there was no significant difference in the medium and high-dose groups of Weichangling capsule(P>0.05).Compared with mesalazine group,HS scores were higher in medium and high-dose of Weichangling capsule(P<0.05).5.The results of colonic periodic acid-schiff stain(PAS)showed that the colonic mucosa epithelium of the model group was intact,the goblet cells were irregularly arranged,their morphology was incomplete,and the number of them was reduced.Some of the cells were vacuolated,and the content of glycogen and other mucous materials were severely reduced.Compared with the model group,the goblet cells in 400,800 mg/kg Weichangling capsule group,420 mg/kg mesalazine group and 400 mg/kg Changweining capsule group had regular arrangement,complete morphology,normal number and no vacuolation,and the number of PAS positive cells was significantly increased(P<0.01).Compared with Changweining capsule group there was no significant difference in the number of PAS positive cells of the medium and high-dose Weichangling capsule group(P>0.05).Compared with mesalazine group,the number of PAS positive cells in the middle and high-dose Weichangling capsule group was less(P<0.05).6.TUNEL test results showed that a large number of apoptotic cells were observed in the colon of the model group.Compared with the normal group,the number of apoptotic cells in the colon of the model group increased significantly(P<0.01).Compared with the model group,400,800 mg/kg Weichangling capsule,420 mg/kg mesalazine,and 400 mg/kg Changweining capsule decreased the number of apoptotic cells(P<0.01).Compared with Changweining capsule group,there was no significant difference in the medium and high dose groups of Weichangling capsule(P>0.05).Compared with mesalazine group,the number of colonic apoptotic cells in the medium and high-dose groups of Weichangling capsule was more(P<0.05).7.Colonic permeability test results showed that the serum EB concentration in the model group was significantly higher than the normal group(P<0.01).Compared with the model group,the serum EB concentration of UC rats decreased significantly in 400,800 mg/kg Weichangling capsule group,420 mg/kg mesalazine group,and 400 mg/kg Changweining capsule group(P<0.01).Compared with the Changweining capsule group,there was no significant difference of the medium and high dose of Weichangling capsule group(P>0.05).Compared with mesalazine group,the concentration of EB in the medium and high dose of groups Weichangling capsule was higher(P<0.05).8.ELISA results showed that compared with the normal group,the concentrations of IL-8,IL-17,IL-23,and IFN-y in the model group were significantly increased(P<0.001),while IL-10 and EGF were significantly decreased(P<0.001).Compared with the model group,the concentration of IL-8,IL-17,IL-23,and IFN-y in each treated group decreased significantly(P<0.001),and the concentrations of IL-10 and EGF increased significantly(P<0.001).9.RT-qPCR results showed that compared with the normal group,the mRNA expression of TNF-α in the model group was significantly increased(P<0.01),and the mRNA expression of MUC2,Occludin,Claudin-1,and ZO-1 were significantly reduced(P<0.01).Compared with the model group,400,800 mg/kg Weichangling capsule,420 mg/kg mesalazine,and 400 mg/kg Changweining capsule decreased the mRNA expression of TNF-α,and increased the mRNA expression of MUC2,Occludin,Claudin-1 and ZO-1(P<0.05),and the medium and high doses of Weichangling capsule obtained the similar effects to Changweining capsule.10.The results of immunohistochemistry showed that compared with the normal group,the expression of TNF-α in the model group increased,and the expression of MUC2,Occludin,Claudin-1,and ZO-1 decreased.Compared with the model group,400,800 mg/kg Weichangling capsule,420 mg/kg mesalazine,and 400 mg/kg Changweining capsule decreased the expression of TNF-α,and increased the expression of MUC2,Occludin,Claudin-1 and ZO-1(P<0.01).The medium and high doses of Weichangling capsule obtained the similar effects to Changweining capsule.11.Western blot results showed that compared with the normal group,the protein expression of TNF-α in the model group was significantly increased(P<0.01),and the protein expression of MUC2,Occludin,Claudin-1,and ZO-1 were significantly reduced(P<0.01).And compared with the model group,400,800 mg/kg Weichangling capsule,420 mg/kg mesalazine,and 400 mg/kg Changweining capsule decreased the protein expression of TNF-α,and increased the protein expression of MUC2,Occludin,Claudin-1 and ZO-1(P<0.05).The medium and high doses of Weichangling capsule obtained the similar effects to Changweining capsule.Conclusions:1.Weichangling capsule shows a protective effect against rat UC induced by 2,4-dinitrochloride benzene combined with acetic acid.2.Weichangling capsule can alleviate the inflammatory response in the colon tissues of UC rats.The mechanism may be related to the reduction of IL-8,IL-17,IL-23,IFN-y,and increasing IL-10 and EGF in the serum of UC rats.3.Weichangling capsule can reduce the inflammatory response in the colon of UC rat by inhibiting the protein and mRNA expression of TNF-α,and can restore the number of colonic goblet cells,increase the protein and mRNA expression of MUC2,Occludin,Claudin-1,ZO-1 to repair intestinal mucosal barrier. |
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