The Effect Of Inonotus Obliquus Polysaccharides On Cell Inflammation Model And Its Possible Mechanism Based On The Signaling Pathway Of NLRP3 Inflammasome

Background:Inonotus is a valuable medicinal fungus with the effects of antioxidant,anti-tumor,prevention of hyperglycemia and anti-inflammatory.Our laboratory has proved that IOP can reduce the expression and secretion of pro-inflammatory cytokines such as IL-6 and TNF-a in macrophages stimulated by LPS.The activation of NLRP3 inflammasome may cause over expression of pro-iflammatory fators,leaing to the development of chronic inflammation.When the activation of NLRP3 inflammasome is inhibited,it can ameliorate the inflammation-related diseases.Whether the anti-inflammatory mechanism of IOP is related to the NLRP3,inflammasome pathway has not been reported so far.Objective:To determine whether the anti-inflammatory effect of IOP was related to the signaling pathway of NLRP3 inflammasome by using the cell inflammation model induced by LPS.Methods:In this study,IOP was selected as the research object First,RAW264.7 cells were resected and divided into normal group and four concentrations of drug groups.The drug concentrations were 20,40,80 and 160 μg/mL.After 24 hours cell culture,MTT assay was used to determine whether the IOP had drug toxicity to RAW264.7 cell and determine the drug safety of IOP.Next,an in vitro cell inflammation model was established through stimulate RAW264.7 cells using LPS(1μg/mL)The cell were divided into a normal group(Normal),a model group(LPS),and an experimental group(LPS+IOP),and the following experiments were performed.In the first part,the IOP(40 μg/mL)combined with LPS was used to extract RAW264.7 cells for 6 h/24 h,then the intracellular RNA and protein were extracted,TNF-a mRNA was detected by RT-PCR as the protein of Phospho-NF-KB-p65,COX2 were detected by WB.Those were used to determine the effect of IOP on inflammatory molecules and pathways in LPS-treated inflammatory cell models.In the second part,the IOP(40 μg/mL)combined with LPS was used to extract RAW264.7 cells for 6 h/24 h,and the intracellular RNA and protein were extracted.Then detect the components of NLRP3 inflammasome and its downstream IL-18 and IL-1β mRNA by RT-PCR.The WB method was used to detect the changes in the expression levels of IL-18 and BL-1β and the NLRP3 inflammasome components.At the same time,IOP and LPS were combined to work on the superatant of RAW264.7 cells for 24 h,and the IL-18 and IL-1β protein levels in the supernatant were further verified by ELISA.Those results could use to determine the role of NLRP3 in the development and management of inflammation.Finally,the silencing effect of β-actin and NLRP3 siRNAl,NLRP3 siRNA2,and NLRP3 siRNA3 after gene transfection was detected by WB.The cell was divided into a negative control group(NC-siRNA),a model group(LPS),an experimental group(LPS+IOP),a gene silencing with LPS treatment group(LPS+siRNA),and a gene silencing with LPS+IOP treatment group(LPS+IOP+siRNA).After 24 hours of NLRP3 gene silencing,the cells were subjected to LPS for 24 h,and the protein was extracted.The protein of NLRP3 inflammasome components were detected by WB.Results:MTT result showed that there was no significant difference in the cell viability rate of RAW264.7 cell line with different concentrations of IOP,that is,no drug toxicity.The results of RT-PCR showed that the expression of TNF-α mRNA in the model group was higher than that in the normal group(P<0.05),and the expression of TNF-a mRNA in the LPS+IOP group was significantly lower than that in the LPS group(P<0.05).At the same time,the expression of NLRP3,ASC,Caspase-1,IL-18 and IL-1β mRNA in the LPS+IOP group was significantly lower than that in the LPS group(P<0.05).The WB results showed that compared with the LPS group,the protein expression of COX 2 in the IOP group was reduced(P<0.05);the phosphorylation level of NF-κB-p65 was also significantly decreased(P<0.05).In addition,compared with the Normal group,the NLRP3 inflammasome in the LPS group were significantly activated,and the expression of the downstream pro-inflammatory cytokines IL-18 and IL-1β were also significantly increased,and IOP reversed this change(P<0.05).The result of ELISA showed that the expression of pro-inflammatory cytokines IL-18 and IL-1β in the cell culture supernatant of the LPS+IOP group was significantly lower than that of the LPS group(P<0.05).The NLRP3 gene silencing experiments showed that LPS+siRNA group and LPS+IOP+siRNA group significantly inhibited the expression of NLRP3,Caspase-1 and ASC induced by LPS compared with LPS group(P<0.05),and there was not statistically significant between LPS+siRNA group and LPS+IOP+siRNA group(P>0.05).Conclusion:IOP could inhibit the cell inflammation which induced by LPS and the possible mechanism may be achieved by inhibiting the activation of NF-κB and NLRP3 inflammasome.