Based On Dectin-1/TLRs/NF-κB Pathway To Study The Mechanism By Which Paeonol Alleviates DSS-induced Ulcerative Colitis With Candida Albicans Colonization

Objective: To investigate the mechanism of paeonol against ulcerative colitis under scheduled implantation of Candida albicans based on Dectin-1/NF-κB related signaling pathway.Method: This study established a UC mice model induced by dextran sulfate sodium under scheduled implantation of Candida albicans to observe the protective mechanism of paeonol after intervention.Sixty female C57BL/6 mice were randomly divided into five groups: sham group,DSS group,model group,PAE group,Mesalazine group.The model lasted for 11 days.In the first 4 days,the suspension of C.albicans standard strain SC5314(1.0×108 CFU/ mouse/Day)was intragastrically administered.On the 5th to 11 th days,3% DSS solution was freely consumed,and the mice were sacrificed on the 12 th day.The sham group was given physiological saline intragastrically every day,and the drug intervention group was given C.albicans intragastrically on the first 4 days.Besides drinking DSS solution,PAE(400 mg/kg/d)and MES(200 mg/kg/d)were given intragastrically on the 5-11 days,respectively.The feces of mice were collected on day 1,3,5,7,9 and 11.After modeling,serum,liver,spleen and colon samples were collected.Observe the general state of the mouse,disease activity index,colon morphology and length,test the C.albicans load in feces and organs by the dilution coating method,and ELISA was used to detect the serum anti-Saccharomyces cerevisiae antibody,β-glucan,and myeloperoxidase levels in mice and the levels of cytokines in mice serum and colon.,HE staining to observe pathological changes in mouse colon;immunofluorescence method to detect colonic macrophage F4/80 expression,immunohistochemistry and Western Blot methods to detect colonic TLR 2 and TLR 4,Dectin-1,NF-κBp65 protein expression.In cell experiments,RAW264.7 macrophages were precultured exogenously with PAE/Dectin-1 receptor inhibitors(Laminarin)/ TLR2 receptor inhibitors(C29)/ TLR4 receptor inhibitors(C34),and then co-incubated with Zymosan/C.albicans.MTT assay was used to detect the effects of PAE and inhibitors on cell activity,Western Blot method to detect Dectin-1,Syk,p-Syk,Card 9,TLR2,TLR4,My D88,NF-κB p65,IκB-α protein expression,ELISA to detect cell supernatant TNF-α,IL-6 levels.Results: DSS-induced UC mice,The pre-implantation of Candida albicans aggravated the degree of damage and inflammation of colon mucosa;Increased fecal and tissue fungal burden,Serum anti-Saccharomyces cerevisiae antibody,β-glucan and myeloperoxidase levels increased significantly;At the same time,the activation degree of macrophages in colon tissue was increased,the expressions of serum and colonic pro-inflammatory factors TNF-α,IL-1β,IL-6 and IL-8 were up-regulated,and the level of anti-inflammatory factor IL-10 was down-regulated;which also stimulated the expression of Dectin-1、TLR2 and TLR4 and their downstream effector NF-κB in colon tissues;But the model mice after PAE intervention improved to some extent compared with the model group,are statistically significant;Hint the protective mechanism of PAE in model mice be related to Dectin-1/NF-κB signaling pathway.Raw264.7 cells were stimulated by Zymosan,and the levels of TNF-α,IL-6 were gradually rising,and Dectin-1,TLR2 was highly adjusted;after C.albicans stimulated,Raw264.7 cells Dectin-1,TLR2 and TLR4 proteins were increased.Zymosan / C.albicans stimulated RAW264.7 cells after PAE pretreatment,can reduce Dectin-1,TLR2,TLR4,Card 9 protein level,inhibit Syk,NF-κBp65 phosphorylation and negative regulation of TLR2 / TLR4 receptor joint protein My D88 and confrontation of IκB-α ubiquitinization.Conclusions: The mechanism of paeonol be related to macrophages and inhibition of Dectin-1/NF-κB signaling pathway.