| Part 1: The role of dietary tryptophan in regulating multi-barriers in DSS-induced acute colitisObjective:To investigate the effect of dietary tryptophan on DSS-induced acute colitis in mice based on the mucus barrier,intestinal epithelial barrier and gut-vascular barrier.Methods:1.SPF male C57BL/6 mice were randomly divided into 3 groups: DSS+LTRP group,DSS+MTRP group and DSS+HTRP group.Before the administration of DSS,mice were pretreated with low concentration tryptophan(0.132%,LTRP),medium concentration tryptophan(0.2%,MTRP)and high concentration tryptophan(0.5%,HTRP)for two weeks,respectively.On the day of administration of the 2% DSS solution(day 0),the initial body weight of mice was measured,and then 2% DSS solution was freely drunk for 7 days.After administrating DSS solution for 7 days,the drinking water was changed to sterile distilled water,and mice were observed for another 5 days.The inflammatory damage of colitis model was evaluated by body weight change,disease activity index(DAI),colon length,HE staining,histological activity index(HAI),and m RNA expression of proinflammatory factor such as IL-1β,IL-6 and TNF-α.Western Blotting and RT-PCR were used to detect the expression of Ah R target gene CYP1A12.The thickness of intestinal mucus layer was detected by AB-PAS staining.The expression of mucin2 was evaluated by immunofluorescence.The trans-epithelium electrical resistant of intestine epithelial was measured by Ussing chamber technology.The intestinal permeability was assessed by concentration of serum FD4.The expression of tight junction related proteins such as occludin and ZO-1 was detected by Western Blotting and RT-PCR.The permeability of the gut-vascular barrier was measured by the concentration of FD70 in serum,spleen and liver.The expression of Plasmalemma vesicle associated Protein1(PV-1)was detected by Western Blotting,RT-PCR and immunofluorescence.Results:1.Compared with low tryptophan diet,medium tryptophan diet and high tryptophan diet improved colon length,weight loss,DAI score,colonic epithelial damage,inflammatory infiltration,and proinflammatory factors IL-1β,IL-6 and TNF-α m RNA expression,while increased the expression of Ah R target gene CYP1A1 in DSS-induced colitis.2.Compared with low tryptophan diet,medium tryptophan diet and high tryptophan diet increased the thickness of intestinal mucus and the expression of mucin2 in DSS-induced colitis.3.Compared with the low tryptophan diet,the medium tryptophan diet and the high tryptophan diet increased the trans-epithelial resistance of the intestine,reduced the permeability of FD4,and increased the expression of tight junction related proteins such as occludin and ZO-1 in DSS-induced colitis.4.Compared with low tryptophan diet,medium tryptophan diet and high tryptophan diet reduced serum FD70 fluorescence,liver and spleen FD70 density,and the expression of PV-1.Conclusions:1.Tryptophan-rich diet improves the inflammatory damage in DSS-induced acute colitis.2.Tryptophan-rich diet reduces the destruction of mucus barrier in DSS-induced acute colitis.3.Tryptophan-rich diet restores the epithelial barrier of DSS-induced colitis.4.Tryptophan-rich diet alleviates the damage of the gut-vascular barrier in DSSinduced colitis.Part 2: Tryptophan metabolism/Ah R pathway mediating the recovery of multi-barriers in improving DSS-induced acute colitisObjective:To investigate the role of Ah R pathway on multi-barriers in DSS-induced acute colitis.Methods:1.SPF male C57BL/6 mice were randomly divided into 3 groups: DSS+LTRP group,DSS+HTRP group and DSS+HTRP+CH223191 group.Before the administration of DSS,mice were pretreated with low concentration tryptophan(0.132%,LTRP),and high concentration tryptophan(0.5%,HTRP)for two weeks.On the day of administration of the 2% DSS solution(day 0),the initial body weight of mice was measured.The 2% DSS solution was administrated freely for 7 days.After administrating the DSS solution for 7 days,the drinking water was changed to sterile distilled water,and then mice were observed for another 5 days.On the day of DSS administration,mice in DSS+HTRP+CH223191 group were intraperitoneally injected with Ah R antagonist CH223191(10mg/kg),once a day,until the end of modeling.As a control,mice in DSS+HTRP and DSS+LTRP were intraperitoneally injected with DMSO.The inflammatory damage of colitis model was evaluated by body weight change,disease activity index(DAI),colon length,HE staining,histological activity index(HAI),and m RNA expression of proinflammatory factor such as IL-1β,IL-6 and TNF-α.Western Blotting and RT-PCR were used to detect the expression of Ah R target gene CYP1A12.The thickness of intestinal mucus layer was detected by AB-PAS staining.The expression of mucin2 was detected by immunofluorescence.The trans-epithelium electrical resistant of intestine tissue was measured by Ussing chamber technology.The intestinal permeability was assessed by concentration of serum FD4.The expression of tight junction related proteins such as occludin and ZO-1 was detected by Western Blotting and RT-PCR.The permeability of the gut-vascular barrier was measured by the concentration of FD70 in serum,spleen and liver.The expression of plasmalemma vesicle associated Protein1(PV-1)was detected by Western Blotting,RT-PCR and immunofluorescence.Results:1.Compared with low tryptophan diet,high tryptophan diet improved colon length,weight loss,DAI score,colonic epithelial damage,inflammatory infiltration,and proinflammatory factors IL-1β,IL-6 and TNF-α m RNA expression,while increased the expression of Ah R target gene CYP1A1 in DSS-induced colitis.CH223191 partially offset the effect of high tryptophan diet,and inhibited the expression of Ah R target gene CYP1A1.2.Compared with low tryptophan diet,high tryptophan diet increased the thickness of intestinal mucus and the expression of mucin2 in DSS-induced colitis.CH223191 partially offset the effect of high tryptophan diet.3.Compared with the low tryptophan diet,high tryptophan diet increased the trans-epithelial resistance of the intestine,reduced the permeability of FD4,and increased the expression of tight junction related proteins such as occludin and ZO-1in DSS-induced colitis.CH223191 partially offset the effect of high tryptophan diet.4.Compared with low tryptophan diet,high tryptophan diet reduced serum FD70 fluorescence,reduced liver and spleen FD70 density,and reduced the expression of PV-1.CH223191 partially offset the effect of high tryptophan diet.Conclusions:1.Tryptophan-rich diet improves the inflammatory damage of DSS-induced acute colitis by activating Ah R pathway.Inhibition of the Ah R pathway will reduce the effect of high tryptophan diet.2.Tryptophan-rich diet reduces the destruction of the mucus barrier of DSSinduced acute colitis by activating Ah R pathway.Inhibition of the Ah R pathway will reduce the effect of high tryptophan diet.3.Tryptophan-rich diet restores the epithelial barrier of DSS-induced colitis by activating Ah R pathway.Inhibition of the Ah R pathway will reduce the effect of high tryptophan diet.4.Tryptophan-rich diet alleviates the damage of the gut-vascular barrier in DSSinduced colitis by activating Ah R pathway.Inhibition of the Ah R pathway will reduce the effect of high tryptophan diet.Part 3: The role of Ah R pathway in the regulation of multi-barriers in DSS-induced acute colitisObjective: To investigate whether dietary tryptophan promoted the recovery of multi-barriers in DSS-induced acute colitis through the Ah R pathway.Methods:1.SPF male C57BL/6 mice were randomly divided into 4 groups: CON group,DSS group,DSS+FICZ group and DSS+CH223191group.Before the administration of DSS,mice were pretreated with a medium concentration of tryptophan(0.2%,MTRP)for two weeks.The drinking water of the CON group was sterile distilled water throughout the whole process.The other groups were administrated by the 2% DSS solution freely for 7 days.After administrating the DSS solution for 7 days,the drinking water was changed to sterile distilled water,and then mice were observed for another 5days.After 2 days of DSS administration,mice in DSS+FICZ group were intraperitoneally injected with Ah R agonist FICZ(1ug/mouse)once a day,until the end of modeling.On the day of DSS administration,mice in DSS+HTRP+CH223191group was intraperitoneally injected with Ah R antagonist CH223191(10mg/kg)once a day,until the end of modeling.As a control,mice in CON group and DSS group were injected intraperitoneally with DMSO.The inflammatory damage of colitis model was evaluated by body weight change,disease activity index(DAI),colon length,HE staining,histological activity index(HAI),and m RNA expression of proinflammatory factor such as IL-1β,IL-6 and TNF-α.Western Blotting and RT-PCR were used to detect the expression of Ah R target gene CYP1A1.2.The thickness of intestinal mucus layer was detected by AB-PAS staining.The expression of mucin2 was detected by immunofluorescence.The trans-epithelium electrical resistant of intestine tissue was measured by Ussing chamber technology.The intestinal permeability was assessed by concentration of serum FD4.The expression of tight junction related proteins such as occludin and ZO-1 was detected by Western Blotting and RT-PCR.The permeability of the gut-vascular barrier was measured by the concentration of FD70 in serum,spleen and liver.The expression of plasmalemma vesicle associated Protein1(PV-1)was detected by Western Blotting,RT-PCR and immunofluorescence.Results:1.FICZ improved colon length,weight loss,DAI score,colonic epithelial damage,inflammatory infiltration,and proinflammatory factors IL-1β,IL-6 and TNF-α m RNA expression,while increased the expression of Ah R target gene CYP1A1 in DSSinduced colitis.CH223191 showed the opposite trend.2.FICZ increased the thickness of intestinal mucus and the expression of mucin2 in DSS-induced colitis.CH223191 showed the opposite trend.3.FICZ increased the transepithelial resistance of the intestine,reduced the permeability of FD4,and increased the expression of tight junction related proteins such as occludin and ZO-1 in DSS-induced colitis.CH223191 showed the opposite trend.4.FICZ reduced serum FD70 fluorescence,reduced liver and spleen FD70 density,and reduced the expression of PV-1.CH223191 showed the opposite trend.Conclusions:1.FICZ improves the inflammatory damage of DSS-induced acute colitis.CH223191 has the opposite effect.2.FICZ reduces the destruction of mucus barrier in DSS-induced acute colitis.CH223191 has the opposite effect.3.FICZ restores the epithelial barrier of DSS-induced colitis.CH223191 has the opposite effect.4.FICZ alleviates the damage of the gut-vascular barrier in DSS-induced colitis.CH223191 has the opposite effect. |
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