The Molecular Mechanism Research Of The Activation Of Ros-NLRP3 Inflammasome Pathway In Kupffer Cell By TxnipAbstract

Part Ⅰ:The association between Kupffer cells, IL-1β and the progression of NAFLD patientsObjective:To compare the serological results and pathology results of NAFL and NASH patients, discuss the mechanism of NASH.Methods:Collecting the liver specimens of NAFLD patients according to the inclusion criteria and exclusion criteria. First, we divided the patients into normal group, NAFL group and NASH group according to the NAFLD activity score system. Second, we compared the levels of ALT, AST, triacylglycerol, FFA and IL-1β. Third, we observed the accumulation of KCs and collagen proliferation through immunohistochemistry and sirius red staining.Results:(1) We collected 8 cases of liver specimens. There were 2 cases of normal liver,3 cases of NAFL,3 cases of NASH. (2) The levels of ALT, AST, triacylglycerol, FFA and IL-1β were all normal in normal and NAFL groups. The levels of ALT, AST and triacylglycerol were all normal in NASH group. However, the levels of FFA and IL-1β in NASH group were higher than in normal and NAFL groups. (3) The accumulation of KCs was more obvious in NAFL and NASH groups than in normal group. Sirius red staining showed that there was no difference of collagen proliferation among the three groups.Conclusion:FFA, KCs and pro-inflammatory cytokine IL-1β may take part in the progression from NAFL to NASH.Part Ⅱ:The effects of suppressing the expressions of TXNIP and NLRP3 on NLRP3 inflammasome signal pathway in Kupffer cellsObjective:To discuss the mechanism of the activation of NLRP3 inflammasome by FFA in KCs through knockouting the genes of TXNIP and NLRP3.Methods:KCs were isolated by collagenase digestion in vitro and density gradient centrifugation combined selective adherence method from wild type mice, NLRP3-/- mice and TXNIP-/- mice. F4/80 immunofluorescence staining and flow cytometry were used to identify the purity of KCs. Trypan blue dye and ink swallowing experiment were used to judge the activity and phagocytic function of KCs. The dynamic morphological change of KCs was observed in electron microscope. Then, we seeded the cells cultured for 48 h in culture bottles and culture dishes, and randomly divided KCs into two groups:FFA Group in which KCs was given 0.3 mM palmitic acid stimulation for 8 h and CON Group. The cells were collected after 8 h of stimulation. The mRNA and protein levels in KCs of TXNIP, NLRP3, ASC and Caspase-1 were measured by Q-PCR, Western blotting and immunofluorescence. The levels of IL-1β in culture supernatants of KCs were detected by enzyme-linked immunosorbent assay (ELISA).Results:(1) Immunofluorescence staining result indicated that F4/80 molecules expressed highly on the cell membrane. The purity of KCs was higher than 90%. Trypan blue staining result showed the vitality of KCs was more than 96.5%. Swallowing ink test showed that KCs swallowed lots of carbon particles. (2) There was no significant difference in KCs morphology in the light microscope between FFA and CON groups, and the transmission electron microscope result showed that KCs swallowed lots of lipid droplets. (3) There were no significant differences in mRNA levels of TXNIP, ASC and Caspase-1 of WT KCs between FFA and CON groups (P>0.05), and the mRNA level of NLRP3 in FFA group was a little higher than in CON group (P<0.05). The mRNA levels of TXNIP, ASC and Caspase-1 of NLRP3-/- KCs in FFA group were higher than in CON group (P<0.05). The mRNA levels of NLRP3, ASC and Caspase-1 of TXNIP-/-KCs in FFA group were significantly higher than in CON group (P<0.05). (4) The protein levels of TXNIP, NLRP3, ASC and Caspase-1 of WT KCs in FFA group were higher than in CON group (P<0.05). There were no significant differences in protein levels of TXNIP, ASC and Caspase-1 of NLRP3-/- KCs between FFA and CON groups (P>0.05). The protein levels of NLRP3, ASC and Caspase-1 of TXNIP-/- KCs in FFA group were higher than in CON group (P<0.05). (5) The results of immunofluorescence were similar to the results of Western blotting. (6) The levels of IL-1βin culture supernatants of WT and TXNIP-/- KCs in FFA groups were higher than in CON groups (P<0.05). There were no significant differences between FFA and CON groups of NLRP3-/- KCs (P>0.05).Conclusion:The NLRP3 inflammasome in WT and TXNIP-/- KCs can be activated by FFA, accompanying by elevated secretion of IL-1β. Knockouting the gene of NLRP3 can suppress the activation of NLRP3 inflammasome and secretion of EL-1β.Part Ⅲ:Effects of suppressing the expressions of TXNIP and NLRP3 on the formation of NAFL and NASHObjective:To discuss the effect of the activation of NLRP3 inflammasome on the formation of NAFL and NASH.Methods:ND, HFD and MCD were used to feed WT mice, TXNIP-/-mice and NLRP3-/- mice to establish NAFL and NASH models. The body and liver weight data were collected. The serum levels of ALT, AST, FFA and IL-1β were detected. HE staining, Oil red staining and transmission electron microscope were used to observe the pathologic changes of hepatic tissues. The expression levels of TXNIP, NLRP3 and Caspase-1 in hepatic tissues were measured by immunofluorescence. Then the KCs in NAFL and NASH models were isolated. The protein levels of TXNIP, NLRP3, ASC and Caspase-1 in KCs were measured by Western blotting, and the activation of NLRP3 inflammasome was detected by immunoprecipitation.Results:(1) The levels of serum ALT and AST in MCD groups of WT mice, TXNIP-/- mice and NLRP3-/- mice were significantly higher than in ND groups (P<0.05), and there was no difference between HFD and ND groups (P>0.05). The levels of FFA in HFD and MCD groups of WT mice were significantly higher than ND group (P<0.05), and there was no significant difference among the three groups of TXNIP-/- mice and NLRP3-/- mice (P>0.05). The levels of serum IL-1β in MCD groups of WT mice and TXNIP-/- mice were significantly higher than in ND and HFD groups (P<0.01), and the levels of serum IL-1β in MCD groups of NLRP3-/-mice were a little higher than in ND and HFD groups (P<0.05). (2) The results of HE staining and Oil red staining showed that HFD groups of WT mice and TXNIP-/- mice formed NAFL, fatty degeneration in hepatocytes, rare inflammation and ballooning degeneration. MCD groups of WT mice and TXNIP-/- mice formed NASH, spotty necrosis of hepatocytes, and inflammatory cell infiltration. The F4/80 positive cells in MCD group significantly increased compared with ND and HFD groups. The results of transmission electron microscope showed that hepatocytes swelling, cytoplasm rarefaction, lots of lipid droplets, spotty necrosis scattering, hepatic sinusoid destruction in MCD group of WT mice. HFD group of NLRP3-/- mice also formed NAFL, fatty degeneration in hepatocytes, rare inflammation, and the extent of hepatocyte necrosis and inflammation in MCD group were moderate, which did not form typical NASH. There was no significant difference in F4/80 positive cells between HFD and MCD groups. (3) Immunofluorescence staining showed that the expression levels of NLRP3 and Caspase-1 in MCD group of hepatic tissue of WT and TXNIP-/- mice were significantly higher than in ND group. The expression levels of TXNIP and Caspase-1 in MCD group of hepatic tissue of NLRP3-/- mice were a little higher than in ND group. (4) The protein levels of NLRP3, ASC and Caspase-1 of KCs of WT and TXNIP-/- mice in MCD group were higher than in ND and HFD groups (P<0.05), and there were no significant differences between ND and HFD groups (P>0.05). There were no significant differences in protein levels of ASC and Caspase-1 between ND and MCD groups of NLRP3-/- mice (P>0.05). (5) The results of immunoprecipitation showed that the NLRP3 inflammasome in KCs of MCD group of WT mice was obviously activated compared with ND and HFD groups (P<0.05), and there were no significant differences between ND and HFD groups (P>0.05). NLRP3 inflammasome could still formed in KCs of MCD group of TXNIP-/- mice, which was much more than in ND group (P>0.05). There was still some ASC-Caspase-1 oligomer in KCs of MCD group of NLRP3-/- mice, and there were no significant differences between ND and MCD groups (P>0.05).Conclusion:FFA can induce the formation of NASH by activating NLRP3 inflammasome and promoting the secretion of IL-1β.Knockouting the TXNIP gene cannot prevent the formation of NASH. Knockouting the NLRP3 gene can prevent the formation of NASH by suppressing the activation of NLRP3 inflammasome in KCs and secretion of IL-1β.