Proanthocyanidins Treat Ulcerative Colitis In Mice By Regulating The Balance Of Treg/Th17 Cells

ObjectiveThe aim of this study is to investigate the therapeutic effect and mechanism of proanthocyanidins on DSS-induced UC in mice,and to provide theoretical support for seeking safe and effective natural drugs for the treatment of UC.MethodsTwenty-four SPF-grade BABL/c male mice,weighing about 25 g,were selected and randomly divided into four groups.(1)Normal group(2)DSS group(3)Proanthocyanidins group(4)Mesalazine group.There were six mice in each group.Different treatments were used for a total of 14 experimental days.Days 1-7:The normal group drank water freely.The rest of three groups drank2.5% DSS solution.Days 8-14: The normal and DSS groups were fed with water;the proanthocyanidins group was gavaged with 500 mg/kg proanthocyanidins;the mesalazine group was gavaged with 100 mg/kg mesalazine.Standard diet was used for each group.The body weight,stool color and stool properties of the mice were recorded and scored at 2:00 pm every day.All the mice were executed on 15 th day and the following indexes were recorded(1)The colon was removed and photographed and the length of the colon was measured,and the mucosal tissue was observed visually for CDMI scoring.(2)H&E staining of the colon,microscopic observation for histological scoring.(3)ELISA was performed to detect the levels of IL-10,IL-6,TGF-β,IL-1β,TNF-α,IL-17 in colonic tissues;(4)RT-PCR was performed to determine the expression of Foxp3 m RNA,ROR-γt m RNA,STAT3 m RNA in colon tissues.(5)WB was performed to determine the expression of NF-κB p65,Foxp3,ROR-γt,STAT3 protein expression in colonic tissues.(6)Determination of the percentage of Treg and Th17 cells in fresh spleen of mice.Rresults1.The mice in the normal group had normal eating,normal activity,gradually increased body weight and no occult blood in the feces;the mice in the DSS group had reduced food,slow behavior,gradually decreased body weight and thin feces with blood;the mice in the proanthocyanidin group had eating better,increased activity,gradually increased body weight,more formed feces and reduced blood in the feces;the colonic length of the mice in the DSS group was shorter than that in the normal group,and the colonic mucosa CDMI score increased(P < 0.01).The colonic length of mice in the DSS group was shorter than that in the normal group,and the colonic mucosal CDMI score increased(P<0.01);the colonic length of mice in the proanthocyanidin group was not significantly different from that in the DSS group(P>0.05).The colonic mucosal sarcoid CDMI scores of mice in the proanthocyanidin group were lower than those in the DSS group(P < 0.05).On 7th day of the experiment,the body weight of mice in the DSS group was reduced compared with the normal group(P < 0.01);the DAI score of mice in the DSS group was increased compared with the normal group(P < 0.01).On the 14 th day of the experiment,the body weight of mice in the DSS group was reduced and the DAI score was increased compared with that in the proanthocyanidin group(P <0.05).2.In the DSS group,the morphology of the colonic mucosa was incomplete,small ulcers were seen,the glands were not neatly arranged,and the content of cupped cells was reduced;in the proanthocyanidin group,the colonic mucosa was more intact,the glands were more neatly arranged,and the degree of infiltration of inflammatory cells was not obvious.According to the HS score,the histological score of colonic mucosa in the DSS group was higher than that in the normal group of mice(P < 0.01);the colonic mucosa score in the proanthocyanidin group was not different from that in the mesalazine group(P > 0.05),and significantly lower than that in the DSS group,(P <0.01)3.The expression of IL-1β,TNF-ɑ,IL-17 in the colon tissue of mice in the DSS group was significantly increased compared with the normal group(P < 0.01);the expression of IL-6 in the colon tissue of mice in the DSS group was increased compared with the normal group(P < 0.05);the expression of IL-10 and TGF-β1 in the colon tissue of mice in the DSS group was decreased compared with the normal group(P <0.01).The expression of IL-1β,TNF-a,IL-17 and IL-6 in the colonic tissues of proanthocyanidin group was lower than that of DSS mice,(P < 0.05).The expression of IL-10 and TGF-β1 in the colonic tissue of mice in the proanthocyanidin group was increased compared with that in the DSS group,(P < 0.05)4.The percentage of splenic Th17 cells in the DSS group was increased compared with the normal group(P < 0.01);the percentage of splenic Th17 cells in the proanthocyanidin group was significantly decreased compared with the DSS group(P <0.05).5.The percentage of splenic Treg cells was significantly increased in the DSS group compared with the normal group of mice,(P < 0.05);the percentage of splenic Treg cells was increased in the proanthocyanidin group compared with the DSS group of mice(P < 0.05).6.The expression of Foxp3 m RNA in the colon tissue of mice in the DSS group was increased compared with that in the normal group(P < 0.05);the expression of Foxp3 m RNA in the colon tissue of mice in the proanthocyanidin group was significantly increased compared with that in the DSS group(P < 0.05);the expression of ROR-γt m RNA and STAT3 m RNA in the colon tissue of mice in the DSS group was increased compared with that in the normal group(P < 0.01);the expression of ROR-γt m RNA and STAT3 m RNA in the colon tissue of mice in the proanthocyanidin group was decreased compared with that in the DSS group(P < 0.05)7.The grayscale values of Foxp3 in the colonic tissues of mice in the DSS group were increased compared with those in the normal group(P < 0.05);the grayscale values of Foxp3 in the colonic tissues of mice in the proanthocyanidin group were increased compared with those in the DSS group(P < 0.05);the grayscale values of NF-κB p65,ROR-γt,and STAT3 in the colonic tissues of mice in the DSS group were increased compared with those in the normal group(P < 0.01);the grayscale values of NF-κB p65,ROR-γt,and STAT3 in the proanthocyanidin.The grayscale values of NF-κB p65,ROR-γt,and STAT3 in the colonic tissues of mice in the DSS group were lower than those in the DSS group(P < 0.05)ConclusionProanthocyanidin has a significant effect on the treatment of ulcerative colitis induced by DSS in mice.The mechanism is that proanthocyanidin can mobilize Th17/Treg cells in mice to relieve intestinal inflammation.The mechanism of proanthocyanidin in the treatment of UC was studied to provide a theoretical basis for the use of proanthocyanidin in the treatment of UC.