Fluorofenidone Inhibits NLRP3 Inflammasome Activation Via The Lysosomal Pathway

Objective:Due to the critical role of NLRP3 inflammasome-mediated inflammatory response in the progression of various renal diseases,targeting NLRP3inflammasomes would be an effective way to attenuate the renal inflammatory response,where lysosomal damage is the classical pathway that triggers NLRP3inflammasome activation.Fluorofenidone（AKFPD）,a novel small molecule drug,has a wide range of well-defined anti-inflammatory effects,however,its mechanism is still unclear.In this study,we constructed a model of inflammation in mouse peritoneal-derived macrophages（PDM）and human renal cortical proximal tubule epithelial cells（HK-2）,we observed the expression of NLRP3,ASC,pro-caspase-1,caspase-1,pro-IL-1β,IL-1β,CTSB and the number of lysosomes in PDM and HK-2cells before and after treatment with Fluorofenidone（AKFPD）.To investigate the mechanism by which AKFPD inhibits NLRP3 inflammasome activation and thus exerts its anti-inflammatory effects through the lysosomal pathway.Methods:1.Cellular inflammation model construction and grouping（1）LPS+ATP stimulation model:Primary peritoneal-derived macrophages（PDM）extracted from BALB/c mice were used as the study subjects.LPS（500 ng/ml,180 min）+ATP（5 m M,30 min）were used to induce the inflammation model（LPS was added to act for 2.5 h and then ATP was added to co-incubate for 30 min）.The experiment was divided into three groups:normal group（N）,model group（LPS+ATP）,and fluorofenidone treatment group（AKFPD）.The treatment group was pretreated with AKFPD（400μg/ml）for 24h before modeling.（2）Hypoxia-induced injury model:Human renal cortical proximal tubule epithelial cells（HK-2）were used as the study target.Cells were cultured under hypoxic conditions（1%O₂,94%N₂and 5%CO₂）in nutrient-free medium for 12 h to induce hypoxic injury,and then cells were transferred back to conventional medium with oxygen for 6 h to promote reoxygenation to construct a hypoxia/reoxygenation（H/R）injury model.The experiment was divided into three groups:normal group（N）,model group（H/R）,and fluorofenidone treated group（AKFPD）.The treatment group was pretreated with AKFPD（400μg/ml）for 24 h before modeling.2.The expression of IL-1βin mouse peritoneal-derived macrophages were detected by q PCR,and the expression of NLRP3,ASC,pro-caspase-1,pro-IL-1β,caspase-1（p10）and IL-1β（p17）were detected by Western-blot.The co-localization of NLRP3 and ASC was detected by immunofluorescence co-localization.3.The expression of lysosomes in PDM and HK-2 cells were observed by Lyso-Tracker Red tracer labeling.4.Western-blot was used to detect the level of lysosomal cathepsin B（CTSB）in primary peritoneal-derived macrophages and HK-2 cells,specific activity assay kit was used to detect CTSB protein activity,and immunofluorescence confocal was used to detect the co-localization of cathepsin B and NLRP3.Results:1.The q PCR results showed that elevated levels of IL-1βexpression were seen in LPS+ATP-stimulated PDM compared to the normal group,with statistically significant differences（P<0.05）;IL-1βexpression in macrophages decreased after pretreatment with AKFPD compared to model group（LPS+ATP）,with statistically significant differences（P<0.05）.2.WB results showed that the expression of NLRP3,ASC,pro-caspase-1,caspase-1,pro-IL-1βand IL-1βwas significantly enhanced in macrophages an d HK-2 cells in the model group compared with the normal group;compared with the model group,the expression of the above proteins declined significant ly after pretreatment with AKFPD,with statistically significant differences（P<0.05）;Immunofluorescence co-localization showed that ASC and NLRP3 were co-localized in the cytoplasm,and the expression of ASC and NLRP3 was increa sed in macrophages and HK-2 cells in the model group compared with the no rmal one,with statistically significant differences（P<0.05）;the expression of A SC and NLRP3 was decreased in macrophages and HK-2 cells in the pretreat ment with AKFPD group,with statistically significant differences（P<0.05）.3.According to the immunofluorescence results,it was statistically significant（P<0.05）in the differences of lysosomes levels in macrophages and HK-2 cells compared with the normal group,the level of lysosomes was higher in model group（LPS+ATP、H/R）;and compared to the model group,the expression of lysosomes in macrophages and HK-2 cells was decreased after pretreatment with AKFPD,with statistically significant differences（P<0.05）.4.WB and immunofluorescence results showed that the levels and activiti es of CTSB in macrophages and HK-2 cells were significantly increased in the model group compared with the normal one,with statistically significant diffe rences（P<0.05）;compared with the model group（LPS+ATP、H/R）,the levels and activities of CTSB in macrophages and HK-2 cells were significantly decr eased in the treatment group,with statistically significant differences（P<0.05）.Immunofluorescence co-localization showed that CTSB and NLRP3 were co-loc alized in the cytoplasm,and the expression of CTSB and NLRP3 in macropha ges and HK-2 cells was elevated in the model group compared with the norm al one,with statistically significant differences（P<0.05）;compared with the mo del group（LPS+ATP、H/R）,the expression of CTSB and NLRP3 in macropha ges and HK-2 cells was decreased in the treatment group,with statistically sig nificant differences（P<0.05）.Conclusion:1.Fluorofenidone inhibited NLRP3 inflammasome activation,reduced the release of inflammatory cytokine IL-1βand down-regulated the inflammatory response.2.Fluorofenidone significantly reduced the activity and level of CTSB,moreover,the co-localized expression of CTSB and NLRP3 was reduced in macrophages and HK-2 cells after treatment with AKFPD,indicating that fluorofenidone may inhibit NLRP3 inflammasome activation through the lysosomal cathepsin pathway.