Alleviative Effects Of Total Flavone Of Abelmoschus Manihot Against Ulcerative Colitis Via Inhibiting NF-κB/NLRP3 Signaling Pathway

Ulcerative colitis(UC)is one of the clinically intractable diseases.Studies have found that UC model mice have damaged intestines and release a class of cytokines such as tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β),IL-6.and IL-7,Its expression is affected by the activation of nuclear factor-kappa B(NF-κB)and nucleotide binding oligomerization domain-like receptor protein 3(NLRP3).Based on the theory of internal and external ulcers,the research group found that hollyhock flower is a potential effective traditional Chinese medicine for the treatment of inflammatory bowel disease(IBD).Total flavone of Abelmoschus Manihot(TFA),as the main active ingredient of hollyhocks,has a significant inhibitory effect on cytokines in the colon tissue of UC model mice.It is speculated that TFA may be an effective drug to resist intestinal inflammation and promote intestinal mucosal repair.This study intends to further clarify the efficacy of TFA in the treatment of UC,and interpret its therapeutic mechanism based on the NF-κB/NLRP3 signaling pathway.Objectives:Elucidation of the therapeutic mechanism of TFA in the treatment of UC based on the NF-κB/NLRP3 signaling pathway.Methods:1.2.5%dextran sulfate sodium salt(Dextran sulfate,DSS)solution established UC mouse model,The general conditions(body weight,food and water consumption,fecal status and mortality),disease activity index(DAI)scores of mice in each group were recorded,Hematoxylin-Eosin staining(HE)was used for pathological detection and scoring,and the colon corrected length and colon weight,spleen index,pathological score and other indicators were recorded.Enzyme-linked immunosorbent assay(ELISA)was used to detect the levels of myeloperoxidase(MPO),TNF-α,IL-6,IL-17,IL-18 and IL-1β in colon tissue.2.Reverse Transcription-Polymerase Chain Reaction(RT-PCR)was used to detect the mRNA level of NLRP3 inflammasome in mouse colon tissue.Western blot(WB)and immunohistochemistry were used to detect the expression of NLRP3 inflammasome protein in the intestinal tissue of mice in each group.The expressions of NF-κB p65/p-NF-κB p65 and NF-κB inhibitor of nuclear factor kappa Bα(IκBα)and p-IκBα in colon tissue were also detected.3.THP-1 cells were cultured in vitro,and the concentration of TFA was screened by cell counting kit-8(CCK-8)method,then use appropriate concentration of TFA to intervene THP-1 cells co-stimulated with lipopolysaccharide(LPS)+adenosine triphosphate(ATP),ELIS A was used to detect the contents of TNF-α,IL-1β and IL-18 in each group,and RT-PCR was used to detect the mRNA level of NLRP3 inflammasome.Western-Blot method was used to observe the protein expressions of NF-κB p65/p-NF-κB p65,IκBα/p-IκBα and NLRP3 inflammasome in the cells of each group,and the expression of NLRP3 inflammasome was observed by immunofluorescence method.Result:1.The high-dose TFA group can significantly reduce the mortality of mice,DAI score,colon histopathological macroscopic score,spleen index,histopathological score,etc.,compared with the model group,and the content of TNF-α and other pro-inflammatory cytokines is also significantly reduced.2.The high-dose TFA group could reduce the expression level of NLRP3 mRNA,and the high-dose TFA group could reduce the expression levels of caspase-1 mRNA and NLRP3 protein in colon tissue;however,there was no significant difference in the expression level of ASC protein among the experimental animals.There was no significant difference in the expression of pro-caspase-1 among the groups,but the expression of its active Caspase-1(p20)was significantly increased in the model group,and the expression level of Caspase-1 protein in the high-dose TFA group was lower than that in the model group.The results of immunohistochemistry were consistent with the above results,the immunohistochemical results of NF-κB p65 indicated that the nuclear staining rate of TFA high-dose group was low;the expression level of p-NF-κB p65 protein in the colon tissue of mice in the high-dose TFA group was significantly lower than that in the model group,and the high-dose TFA group could inhibit the phosphorylation of IκBα.3.CCK-8 method was used to evaluate the toxicity of TFA to THP-1 cells,and it was found that the toxicity of TFA to cells was most obvious at 200 μg/mL,and 25 μg/mL,50μg/mL and 100 μg/mL were selected for the experiment;after co-stimulation of THP-1 cells with LPS+ATP,cytokines such as IL-1β,IL-18,and TNF-α were significantly increased compared with the normal group,and the expression levels of these cytokines could be reduced in each dose group of TFA;the expression level of NLRP3 protein was significantly increased in LPS+ATP group and LPS+ATP+dimethyl sulfoxide(DMSO)group,after TFA intervention,the expression levels of NLRP3 protein in TFA(50 μg/mL)and TFA(100μg/mL)groups were significantly decreased;After LPS+ATP co-acted on cells,the expression of ASC and its active caspase-1(p20)protein in the model group was significantly increased,while the levels of ASC and caspase-1(p20)protein in each dose group of TFA were significantly decreased in a dose-dependent manner.The levels of p-IκBα,p-NF-κB p65/NF-κB p65 were significantly increased in LPS+ATP group and LPS+ATP+DMSO group,each dose group of TFA could inhibit the phosphorylation of IκBα and the expression level of p-NF-κB p65/F-κB p65.The results of immunofluorescence showed that LPS+ATP could stimulate the expression of NLRP3 inflammasome to increase,and TFA could inhibit the cellular inflammatory response in a dose-dependent manner.Conclusions:TFA has a certain protective effect on the intestinal tract of UC model mice,mainly in that TFA can maintain the body weight of UC model mice and protect the intestinal mucosa,etc.The mechanism of its efficacy may be related to its inhibition of the activation of the NFκB/NLRP3 inflammasome pathway and the reduction of the levels of pro-inflammatory cytokines such as TNF-α,IL-1β and IL-18 in inflammatory tissues.