| Background: Inflammatory bowel disease(IBD)is a type of chronic and relapsing Inflammatory disease of intestinal tract caused by multiple etiology.IBD has become a huge burden of human life because of its repeated course,difficult to cure,easy to worsen and cause cancer.New evidence suggests that NLRP3 inflammasome,as an important component of innate immunity,plays an important role in the development of immune response IBD.Therefore,inspired by the latest mechanism discovery,it is imperative to find innovative,efficient and less toxic anti-colitis drugs in the treatment of IBD.Natural products have the advantages of wide therapeutic range,small side effects and low price,and have always been the focus of research as anti-inflammatory drugs.Bai-tou-weng-tang(BTWT)is a famous TCM prescription for the treatment of intestinal diseases caused by inflammation.It has the effects of cooling blood,stopping dysentery,clearing heat and detoxifying.According to the indications,it is often used for the treatment of enteritis,protozoal dysentery,bacterial dysentery and other digestive system infections.The main ingredient of BTWT is Pulsatilla pulsatilla,of which Anemoside B4(AB4)is a main natural saponin component isolated from the roots of Pulsatilla pulsatilla.Previous studies had shown that AB4 alleviated inflammatory injury of IBD by inhibiting NF-κB expression and decreasing the levels of inflammatory cytokines IL-1β,IL-6 and TNF-α,but whether it affects IBD inflammation by regulating NLRP3 inflammasome expression has not been reported.CD1 d has structural and functional homology with major histocompatibility complex(MHC)molecules,which can transmit mutual signals and trigger endogenous signals.Our recent work suggests that macrophage CD1 d signaling inhibits NLRP3 inflammasome activation during inflammation.Therefore,regulating the expression of inflammatory factors by regulating the CD1 d signaling pathway may be beneficial for the treatment of NLRP3 mediated IBD.Objective: From the perspective of macrophage CD1d/NLRP3 axis,the protective effect of AB4 on DSS induced IBD and its related molecular mechanism were discussed,laying a theoretical foundation for the development of novel IBD treatment drugs and treatment strategies.Methods:1.CCK-8 assay was used to detect the effects of AB4,AA3 and 23-HA(25μM、50μM、100μM、200μM)on RAW264.7 cell viability.The effects of three saponins on IL-1β m RNA and protein were detected by q RT-PCR and ELISA.2.Acute colitis model of C57BL/6 mice induced by 3.0%DSS was established,and the survival rate,body weight change,DAI score,colon length and spleen index of the mice were recorded and analyzed daily.The intestinal permeability was detected by FITC-Dextran and the expression of tight junction proteins(Occludin,Claudin-1,ZO-1)was detected by Western Blot.H&E staining was used to detect colon tissue damage.3.Compared with DSS group,AB4 significantly decreased the levels of IL-1β,IL-18,IL-6,TNF-α and i NOS in serum in mice.AB4 inhibited the activation of NLRP3 inflammasome in colon and up-regulated the expression of IL-10,IL-22 and PCNA.AB4 inhibited the m RNA expression of NLRP3,ASC,Caspase-1,IL-1β,IL-18,IL-6 and TNF-α in colon.AB4 inhibits the activation of NLRP3 inflammasome in colon macrophages,but not intestinal epithelial cells.In THE DSS induced colitis model of WT and NLRP3-/-mice,NLRP3-/-mice showed slower weight loss,lower DAI scores,and longer colons compared with WT.NLRP3-/-+DSS and NLRP3-/-+DSS+AB4 were similar to WT+DSS+AB4 without significant difference.In LPS-induced BMDMs model,AB4 significantly inhibited the m RNA expression of NLRP3,IL-1βand IL-18,but did not affect the m RNA expression of ASC and Caspase-1.AB4 significantly inhibited the protein expression of NLRP3,pro IL-1β and IL-18.AB4 inhibited the protein expression of NLRP3,Caspase-1 p20,IL-1β p17,and IL-18 in LPS+ATP or LPS+Nig induced BMDMs and THP-1 cells in vitro.4.In LPS-induced BMDMs and THP-1 cell models,AB4 significantly inhibited the expression of AKT-STAT1-PRDX1-NF-κB pathway related proteins;NF-κB inhibitor JSH-23 inhibited the protein and m RNA expression of NLRP3,pro IL-1β and IL-18,which was similar to AB4.AKT inhibitor MK2206 inhibited the expression of AKT-STAT1-PRDX1-NF-κB signaling pathway related proteins as well as NLRP3,pro IL-1β and IL-18 proteins,which was similar to AB4.SC79 reversed the inhibitory effect of AB4 on AKT-STAT1-PRDX1-NF-κB signaling pathway related proteins,NLRP3,pro IL-1β and IL-18 proteins.AB4 inhibited the expression of AKT-STAT1-PRDX1-NF-κB signaling pathway in 3.0%DSS induced enteritis in C57BL/6 mice.AB4 showed strong binding activity to CD1 d.In the 3.0%DSS induced enteritis model,AB4 significantly enhanced the expression of CD1 d protein in mouse colon tissue.In WT and CD1d-/-mice colitis models induced by 3.0%DSS,weight loss,DAI score and colon shortening were more serious in CD1d-/-mice compared with WT.AB4 administration did not alter weight loss or colon shortening in CD1d-/-mice.Western Blot and ELISA results showed that AB4 administration could not inhibit the expression of AKT-STAT1-PRDX1-NF-κB-NLRP3 signaling pathway in CD1d-/-mice.Results:1.AB4,AA3(25μM,50μM,100μM,200μM)had no toxicity to RAW264.7 cells,but 200μM23-HA had a certain toxicity to RAW264.7 cells.The inhibition rates of AB4(50μM,100μM,200μM)on IL-1β m RNA were 20%,32%,46%,respectively,while 25μM AB4 showed no significant difference in IL-1β m RNA inhibition.AA3(50μM,100μM,200μM)inhibited IL-1βm RNA by 12%,16%,26%,respectively,while 25μM AA3 did not inhibit IL-1β m RNA.The inhibition rates of IL-1β m RNA by 23-HA(25μM,50μM,100μM)were 12%,17%,26%,respectively.AB4,AA3 and 25μM 23-HA inhibited the expression of IL-1β,and AB4 had the highest inhibitory activity.2.Compared with DSS group,the survival rate of AB4 group was significantly higher;the weight loss was not obvious,DAI score was low,and colon shortening was not obvious.Spleen index is high.The content of FITC-Dextran in serum of AB4 group was low.AB4 significantly increased the protein expression of Occludin,Claudin-1 and ZO-1.H&E staining showed that AB4 reduced the number of crypts and inflammatory cell infiltration.3.Compared with DSS group,AB4 significantly decreased the levels of IL-1β,IL-18,IL-6,TNF-α and i NOS in serum in mice.AB4 inhibited the activation of NLRP3 inflammasome in colon and up-regulated the expression of IL-10,IL-22 and PCNA.AB4 inhibited the m RNA expression of NLRP3,ASC,Caspase-1,IL-1β,IL-18,IL-6 and TNF-α in colon.AB4 inhibits the activation of NLRP3 inflammasome in colon macrophages,but not intestinal epithelial cells.In the DSS induced colitis model of WT and NLRP3-/-mice,NLRP3-/-mice showed slower weight loss,lower DAI scores,and longer colons compared with WT.NLRP3-/-+DSS and NLRP3-/-+DSS+AB4 were similar to WT+DSS+AB4 without significant difference.In LPS-induced BMDMs model,AB4 significantly inhibited the m RNA expression of NLRP3,IL-1βand IL-18,but did not affect the m RNA expression of ASC and Caspase-1.AB4 significantly inhibited the protein expression of NLRP3,pro IL-1β and IL-18.AB4 inhibited the protein expression of NLRP3,Caspase-1 p20,IL-1β p17,and IL-18 in LPS+ATP or LPS+Nig induced BMDMs and THP-1 cells in vitro.4.In LPS-induced BMDMs and THP-1 cell models,AB4 significantly inhibited the expression of AKT-STAT1-PRDX1-NF-κB pathway related proteins;NF-κB inhibitor JSH-23 inhibited the protein and m RNA expression of NLRP3,pro IL-1β and IL-18.AKT inhibitor MK2206 inhibited the expression of AKT-STAT1-PRDX1-NF-κB signaling pathway related proteins as well as NLRP3,pro IL-1β and IL-18 proteins.SC79 reversed the inhibitory effect of AB4 on AKT-STAT1-PRDX1-NF-κB signaling pathway related proteins,NLRP3,pro IL-1β and IL-18 proteins.AB4 inhibited the expression of AKT-STAT1-PRDX1-NF-κB signaling pathway in3.0%DSS induced enteritis in C57BL/6 mice.AB4 showed strong binding activity to CD1 d.In the3.0%DSS induced enteritis model,AB4 significantly enhanced the expression of CD1 d protein in mouse colon tissue.In WT and CD1d-/-mice enteritis models induced by 3.0%DSS,weight loss,DAI score and colon shortening were more serious in CD1d-/-mice compared with WT.AB4 administration did not alter weight loss or colon shortening in CD1d-/-mice.Western Blot and ELISA results showed that AB4 administration could not inhibit the expression of AKT-STAT1-PRDX1-NF-κB-NLRP3 signaling pathway in CD1d-/-mice.Conclusions:1.AB4 shows the strongest anti-inflammatory effect in LPS induced macrophages in AB4,AA3 and 23-HA.2.AB4 alleviates the inflammatory injury of DSS induced inflammatory bowel disease by reducing body weight loss,DAI score,survival rate,colon length,spleen index and intestinal epithelial barrier injury in mice.3.AB4 inhibits the activation of NLRP3 inflammasome in DSS-induced mouse colon tissue and colon macrophages.In DSS-induced NLRP3-/-mice,AB4 alleviates the inflammatory injury of IBD by inhibiting NLRP3 inflammasome activation.AB4 inhibits NLRP3 inflammasome activation in vitro.4.AB4 inhibits NLRP3 inflammosome activation by targeting macrophage CD1 d by regulating AKT-STAT1-PRDX1-NF-κB signaling pathway.CD1 d macrophage specific knockdown reversed the protective effects of AB4 on DSS-induced weight loss,DAI score,colon shortening and colon injury in mice,and also reversed the inhibitory effect of AB4 on AKT-STAT1-PRDX1-NF-κB-NLRP3 signaling pathway..In conclusion,Anemoside B4 may inhibit the activation of NLRP3 inflammasome by targeting macrophage CD1 d to regulate AKT-STAT1-PRDX1-NF-κB signaling pathway,promote the balance of inflammatory factors and repair of intestinal epithelial injury,and maintain the integrity of mucosal barrier,thereby alleviating the inflammatory injury induced by DSS in mouse inflammatory bowel disease. |
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