Effect Of Klotho On Lipopolysaccharide-induced Inflammatory Response In Macrophages Via NLRP3 Inflammatory Vesicles And A Preliminary Investigation Of The Mechanism

Background:Klotho is an anti-aging protein that also has anti-inflammatory,anti-tumor and antioxidant properties.Our laboratory has previously demonstrated that Klotho can regulate the infiltration of macrophages by inhibiting the activity of NF-κB and oxidative stress,and inhibiting the release of inflammatory mediators.As a downstream protein of NF-κB signaling pathway,NLRP3 inflammasome is considered to be the core of inflammatory response,and activation can lead to the occurrence of inflammatory response.Studies have confirmed that Klotho can effectively inhibit the inflammatory response of the disease,but there is no definite report on whether its anti-inflammatory mechanism is related to the NLRP3 inflammasome.Objective:To investigate the effect of Klotho on lipopolysaccharide(LPS)-induced inflammatory responses in macrophages and to explore whether its anti-inflammatory mechanism is rassociated with NLRP3 vesicles.Methods:1.After resuscitation,the mouse macrophage line RAW264.7 cells were divided into normal and drug groups,Klotho was selected as the research object,the concentrations were(200,400,600,800 pm/L),and cultured for 24h.Afterwards,the drug toxicity of Klotho to RAW264.7 cells were assayed by MTT method,and screened for appropriate drug concentrations.Subsequently,RAW264.7 cells were stimulated with LPS(lμg/mL)to establish an in vitro inflammatory model and given drug treatment.The mouse macrophage line RAW264.7 cells were divided into normal group(Normal),LPS group and LPS+Klotho(400pm/L)group.2.After co-culturing RAW264.7 cells Klotho(400pm/L)with LPS for 12h,RT-PCR was used to detect the expression levels of ASC,Caspase-1,NLRP3,TNF-α,IL-1β,and IL-18 mRNA.3.After RAW264.7 cells were co-cultured with Klotho(400pm/L)and LPS for 24h,Western blot was used to detect the intracellular levels of Phospho-NF-κB-p65,COX2,ASC,Caspase-1,NLRP3,TNF-α,IL-1β,and IL-18 protein expression levels.4.AW264.7 cells were co-cultured with Klotho(400pm/L)and LPS for 24h.The cell supernatants were collected and the levels of IL-1β and IL-18 in the supernatants were measured by ELISA.Results:1.MTT results showed that the concentration of Klotho between 200pm/L-800pm/L had no effect on the growth and viability of RAW264.7 cells,and the cell survival rate was the highest at 400 pm/L,so the experiment selected mouse macrophages were treated at 400 pm/L.2.RT-PCR results indicate that compared with the Normal group,the LPS group had significantly higher ASC,Caspase-1,NLRP3,TNF-α,IL-1β,IL-18 mRNA gene expression in RAW264.7 cells treated with LPS.Compared with the LPS group,the ASC,Caspase-1,NLRP3,TNF-α,IL-1β,IL-18 mRNA gene expressions in the LPS+Klotho group were significantly reduced.3.The results of the Western blot showed that,compared to the LPS group,Klotho in the LPS+Klotho group reduced the expression of COX-2 and significantly inhibited the phosphorylation level of NF-κB-p65 in cells.In addition,compared with the Normal group,the protein expression levels of ASC,Caspase-1,NLRP3,TNF-α,and the cytokines IL-1β and IL-18 were also increased significantly in the LPS group,while the LPS+Klotho group decreased ASC,Caspase-1,NLRP3,TNF-α,and the protein expression levels of cytokines IL-1β,IL-18.4.ELISA results showing expression of pro-inflammatory factors IL-1β and IL-18,compared to the LPS group,LPS+Klotho cell supernatant was significant reduction.Conclusion:Klotho plays an anti-inflammatory role in LPS-induced inflammatory response in mouse macrophage RAW264.7,and its mechanism may be related to the inhibition of NLRP3 inflammasome activation.