Exploring The Mechanism Of Curcumin In The Treatment Of Ulcerative Colitis Based On Ferroptosis-related Signaling Pathway

Objective:1.The effect and mechanism of curcumin in the treatment of ulcerative colitis through ferroptosis were verified by in vivo experiments.2.Using network pharmacology and molecular docking techniques,the relevant signaling mechanism of curcumin in the treatment of ulcerative colitis through ferroptosis was systematically explored,providing a reference for further in vivo experimental studies.3.Furthermore,the role and mechanism of curcumin in the treatment of ulcerative colitis through ferroptosis via the JAK2-STAT3 signaling pathway were verified by further in vivo experiments.Methods:1.The protective effect of curcumin in ulcerative colitis and its relationship with the ferroptosis pathway were verified using a colitis animal model.The specific methods were as follows:36 adult male Balb/c mice(6-8 weeks old,18-22g)were selected and acclimated for one week before being randomly assigned to 6 groups(6 mice/group):Normal control group(Group A):normal diet and water;Model group(Group B):normal diet and free access to 5%DSS-containing water;High-dose curcumin group(Group C):free access to 5%DSS-containing water and feed containing 40g/L curcumin;Medium-dose curcumin group(Group D):free access to 5%DSS-containing water and feed containing 20g/L curcumin;Low-dose curcumin group(Group E):free access to 5%DSS-containing water and feed containing 10g/L curcumin.Positive control group(Group F):free access to 5%DSS-containing water and feed containing 5g/L sulfasalazine.All mice were administered the treatment for 7 days,and their general condition was recorded at the same time each day.The main observations were the stool consistency and frequency of mice,detection of occult blood or bloody stool characteristics(loose stools,diarrhea,fresh blood in stool),recording of weight changes,and evaluation of the disease activity index(DAI)score.After 7 days,the mice were fasted and water-deprived for 12 hours,then euthanized,and the colon was isolated for observation of gross morphology and measurement of its length.Colon tissue and serum were collected,and mucosal injury was scored.The colonic tissues of mice in different groups were evaluated by histological examination using HE staining and pathology scoring.Biochemical assay kits were used to detect the content of Fe,MDA,GSH,and SOD in mice from different groups.Immunohistochemistry was performed to detect the expression of ACSL4 protein in mice from different groups.2.Network pharmacology and molecular docking results:Through the identification of 100 targets of curcumin,a search in the GeneCards database yielded 6065 genes related to ulcerative colitis,and the FerrDB database provided 366 regulatory factors,markers,and disease information related to Ferroptosis.The intersection of these three sets of targets resulted in 15 overlapping genes.Topological analysis of the protein-protein interaction(PPI)network revealed 10 core targets.GO and KEGG pathway enrichment analysis indicated that curcumin’s action is closely related to inflammation and primarily involves human cytomegalovirus infection,hepatitis C,EGFR tyrosine kinase inhibitor resistance,Kaposi’s sarcoma-associated herpesvirus infection,IL-17 signaling pathway,prostate cancer,lipid and atherosclerosis,toxoplasmosis,JAK2-STAT3,and other signaling pathways regulating the progression of ulcerative colitis.JAK2 and STAT3 were selected for molecular docking.The results showed that the active compounds had a good binding ability with the selected targets.3.The protective effect of curcumin in the treatment of ulcerative colitis may be associated with the JAK2-STAT3 signaling pathway.Results:1.Results from animal experiments investigating the mechanism indicate that:(1)General conditions and DAI scores of mice:The normal mice had good mental state,were agile,had shiny fur,and normal feces.Compared to the normal group,the model group mice exhibited symptoms such as listlessness,sluggishness,sparse and dry fur,diarrhea,malformed feces,mucous and bloody stool,perianal redness and swelling,and significant weight loss four days after modeling.The mice treated with curcumin and positive control groups showed improvement in the aforementioned symptoms compared to the model group,and the improvement was dose-dependent.However,the recovery was slightly worse compared to the normal group.DAI score results showed that the high,medium,and low dose curcumin groups and the positive control group were all lower than the model group,and the difference was statistically significant(P<0.05).There was no statistically significant difference in pairwise comparisons between the high,medium,and low dose curcumin groups and the positive control group(P>0.05).(2)Pathological examination:The colonic mucosa of normal group mice appears smooth,without congestion or edema,with regular arrangement of glands.In the model group,the colonic mucosa shows erosion,significant congestion,edema,extensive mucosal ulcers,and infiltration of a large number of inflammatory cells.The pathological findings in the positive control group and curcumin group show significant improvement,and mice in the high,medium,and low dose groups of curcumin exhibit dose-dependent changes in the degree of colonic inflammation,infiltration of inflammatory cells,and mucosal barrier damage.(3)Detection of inflammatory markers indicates that the levels of pro-inflammatory factors TNF-α and IL-6 are elevated in the model group,while the level of anti-inflammatory factor IL-10 is decreased.The positive control group and curcumin group can reduce the levels of TNF-α and IL-6 in the serum and increase the level of IL-10,with a dose-dependent effect observed in the curcumin group.The differences are statistically significant(P<0.05).Therefore,curcumin can effectively improve the inflammatory condition in ulcerative colitis.(4)Biochemical index detection:Compared to the normal group,the model group shows increased levels of Fe and MDA,and decreased levels of GSH and SOD(P<0.05).Compared to the model group,the positive control group and curcumin group exhibit decreased levels of Fe and MDA,and increased levels of GSH and SOD(P<0.05).Among them,the high-dose curcumin group and the positive control group show better therapeutic effects(P<0.01),with no statistically significant difference between the two groups(P>0.05),followed by the medium-dose curcumin group and the low-dose curcumin group(P<0.05).(5)Immunohistochemistry results show that compared to the normal group,the expression of ACSL4 protein in the colonic tissue of the model group mice is significantly increased.Compared to the model group,both the positive control group and curcumin group exhibit varying degrees of reduction in ACSL4 protein expression,with statistically significant differences(P<0.05).Among them,the positive control group and the high-dose curcumin group show the greatest reduction in ACSL4 protein expression(P<0.01),with no statistically significant difference between the two groups(P>0.05),followed by the medium-dose and low-dose curcumin groups(P<0.05).This indicates that curcumin treatment of ulcerative colitis is associated with the Ferroptosis mechanism.2.Network pharmacology and molecular docking results reveal that through identification of 110 targets of curcumin,topological analysis of PPI network reveals 10 core targets.GO and KEGG pathway enrichment analysis indicate that the action of curcumin is mainly involved in regulating signaling pathways related to human cytomegalovirus infection,hepatitis C,EGFR tyrosine kinase inhibitor resistance,Kaposi’s sarcoma-associated herpesvirus infection,IL-17 signaling pathway,prostate cancer,lipid and atherosclerosis,and toxoplasmosis,which are associated with the progression of ulcerative colitis.Molecular docking was performed with JAK2 and STAT3.The results show that the active compound has a good binding ability with the screened targets.3.Animal in vivo experiments show that compared with the normal group,the expression of JAK2 and STAT3 proteins in the colonic tissue of the model group mice is significantly increased.Compared with the model group,the JAK2 and STAT3 protein expression in the positive control group and curcumin group were significantly decreased to varying degrees,with statistically significant differences(P<0.05).Among them,the positive control group and high-dose curcumin group showed the greatest reduction in JAK2 and STAT3 protein expression,with no statistically significant difference between the two groups(P>0.05),followed by the curcumin medium and low-dose groups.These results indicate that the JAK2-STAT3 signaling pathway is involved in the Ferroptosis process of curcumin treatment for ulcerative colitis.Conclusions:Curcumin may regulate the process of Ferroptosis through the JAK2-STAT3 signaling pathway and thus treat ulcerative colitis.