Effect Of Inonotus Obliquus Polysaccharide On Colorectal Cancer Cell Lines Via NLRP3 Inflammasome And Its Mechanism

Background: Inonotus Obliquus Polysaccharide(IOP)is one of the effective components of Inonotus Obliquus,which has anti-inflammatory,hypoglycemic,anti-tumor,anti-oxidation and other biological characteristics.Colorectal cancer(CRC)is a common malignant tumor in the intestinal system.It is well known that inflammation is closely related to the occurrence and development of cancer,and the two promote each other.Inflammasome plays a key role in the inflammatory process,among which NLRP3 inflammasome is the most widely studied,but its role in the development of colorectal cancer is still unclear.Objective: To investigate the effect of IOP on the biological characteristics of colorectal cancer cell lines and determine whether its anti-tumor mechanism is related to NLRP3 inflammasome.Methods:1.SW620 cells were divided into normal control group and an experimental group(50,100,150 μg/m L IOP groups).After 48 hours of culture,the effect of IOP on the growth was detected by the CCK-8 method;the cell colony formation experiment was used to detect the effect of IOP on cell colony formation;cell scratch test to detect the effect of IOP on the migration ability of cells;Western blot test to detect the expression level of apoptosis-related proteins in cells.2.After IOP was applied to SW620 cells for 48 hours,real-time quantitative PCR was used to detect the expression level of ASC,caspase-1,NLRP3,IL-1β and IL-18 mRNA;Western blot was used to detect the expression of NLRP3 inflammasome protein;the ELISA method was used to detect the protein levels of IL-1β and IL-18 in the cell supernatant.3.After treating SW620 cells with NLRP3 specifically inhibiting MCC950,Western blot experiments were used to detect the expression level of ASC,caspase-1,NLRP3 after IOP treatment of SW620 cells;the protein level of IL-1β and IL-18 in the cell supernatant was detected by ELISA.4.After the overexpression of NLRP3 in SW620 cells,Western blot experiment to detect the expression of ASC,caspase-1,NLRP3 after IOP treatment of SW620 cells;ELISA method to detect the protein levels of IL-1β and IL-18 in the cell supernatant.Results:1.CCK-8 results showed that IOP inhibited SW620 cell proliferation in a dosedependent manner;cell colony formation experiments showed that IOP treatment dosedependently reduced the number of colonies formed after 7 days in culture;scratch experiments showed that IOP treatment also inhibited the migration of SW620 cells;Western blot results showed that IOP can promote SW620 cell apoptosis.2.Real-time quantitative PCR results showed that compared with the control group,IOP could significantly increase the expression of NLRP3,ASC,caspase-1,IL-1β and IL-18 mRNA levels;Western blot results showed that IOP dramatically enhanced NLRP3 inflammasome protein levels;ELISA results showed that IOP can enhanced tumor cell secretion of the cytokines IL-1β and IL-18.3.Western blot and ELISA results showed that the expression of ASC,caspase-1,NLRP3 and cytokines IL-1β and IL-18 were significantly increased in the IOP+MCC950group compared with that in the MCC950 group;the expression of ASC,caspase-1,NLRP3,IL-1β and IL-18 were also significantly increased in the IOP group compared with the IOP+MCC950 group.4.After transfection of NLRP3 overexpression,Western blot and ELISA results showed that the expressions of ASC,caspase-1,NLRP3,IL-1β and IL-18 were significantly increased in the NLRP3 overexpression group compared with the control group;and the expressions of ASC,caspase-1,NLRP3,IL-1β and IL-18 were significantly increased in the IOP+NLRP3 overexpression group compared with the NLRP3 overexpression group.Conclusion: IOP can inhibit the growth of SW620 cells,and its mechanism may be related to the activation of inflammasome NLRP3 and the up-regulation of the expression of related cytokines.