| Ulcerative colitis(UC)is a chronic inflammatory bowel disease in which the location of the disease is limited to the colonic mucosa and submucosa with an increasing global incidence.Studies have found that its occurrence and development are closely related to inflammation and oxidative stress.Dictyophora indusiata polysaccharides(DIP)is the main active substance of Dictyophora indusiata,with biological activities such as anti-tumor,antioxidant,and immune regulation.Based on the multiple biological properties of DIP,this project first studied the anti-inflammatory activity of DIP and its mechanism at the in vitro cell level,and further evaluated the effect of DIP on UC in mice through animal experiments,and explored its mechanism.The main research works are described as follows:(1)Effect of DIP on the immune activity of RAW264.7 macrophages.Flow cytometry was used to detect the expression of TLR4 and ROS secretion,qRT-PCR was used to detect the expression of ? interleukin 1(IL-1?)and IL-6 m RNA,western blotting was used to detect the expression of NLRP3 inflammasome associated proteins,the secretion of related cytokines were detected by ELISA,and the nuclear translocation of NF-?B-p65 was detected by laser confocal microscopy.The results showed that DIP activated priming of the NLRP3 inflammasome activation via enhancing TLR4 expression,phosphorylation of I?B-?,the nuclear translocation of NF-?B p65 subunit,the expression of NLRP3 protein and mRNA of inflammatory cytokines,and IL-6 secretion.However,DIP did not regulate the activation phase of NLRP3 inflammasome and had no effect on the secretion of ROS,caspase-1,IL-1? and IL-18.Therefore,DIP cannot exert an immune-enhancing effect via regulating NLRP3 inflammasome.(2)Study on the anti-inflammatory activity and mechanism of DIP in RAW264.7 macrophages.Through the use of western blotting,flow cytometry,ELISA and other related experimental techniques,the study found that in the RAW264.7 macrophage inflammation model stimulated by LPS and ATP,DIP inhibited initial and activation phases of NLRP3 inflammasome activation via inhibiting TLR4 expression,I?B? phosphorylation,nuclear translocation of NF-?B-p65,NLRP3 protein expression,ROS secretion,self-assembly of NLRP3 inflammasome,and release of mature caspase-1,IL-1?,and IL-18,thereby exerting its anti-inflammatory activity.(3)Study on protective effect and mechanism of DIP on dextran sodium sulfate-induced colitis in C57BL/6 mice.The transcription and secretion levels of inflammatory cytokines in intestinal tissues were detected using qRT-PCR and ELISA methods,western blotting detected intestinal inflammation-related signaling pathway proteins and apoptosis-related proteins expression levels,immumohistochemical staining method detected intestinal inflammation and macrophage polarization-related protein expression,and immunofluorescence method detected intestinal tight junction protein(TJP1)expression and flow cytometry detected subtypes of spleen macrophages.The results showed that DIP dose-dependently inhibited the level of oxidative stress in DSS-induced colitis,reduced the transcription and secretion levels of inflammatory cytokines,inhibited inflammation-related signaling pathways(NF-?B,STAT3 and NLRP3),increased the expression of intestinal TJP1 protein,inhibited the apoptosis of colon tissue,and down-regulated the polarization level of M1 macrophages in spleen and colon tissue.Therefore,DIP relieved DSS-induced ulcerative colitis via regulating the inflammatory response.To sum up,DIP cannot play an immune-enhancing role by activating NLRP3 inflammasome in RAW264.7 macrophages,but DIP can inhibit NLRP3 inflammasome activation to exert anti-inflammatory effects in LPS and ATP co-stimulated RAW264.7 macrophages.In addition,DIP can alleviate DSS-induced colitis.Our research provides some scientific evidences for that DIP will be developed as an effective and safe medication for colitis in the future. |
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