The Mechanism Of LKB1 Attenuates Liver Ischemia/Reperfusion Injury Via Inhibiting NLRP3 Inflammasome

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THE EXPRESSION OF LKB1 IN MOUSE LIVER ISCHEMIA-REPERFUSION MODELS IN VIVO AND IN VITROObjective: To detect the expression level of LKB1 in mouse liver ischemia/reperfusion(I/R)models in vivo and in vitro.Methods: The mouse liver I/R model and a RAW 264.7 macrophage hypoxia-reoxygenation(H/R)models were constructed.The microplate method was used to detect the levels of serum alanine aminotransferase(s ALT)and serum aspartate transaminase(s AST).Hematoxylin and eosin(H&E)staining was used to detect the degree of liver tissue damage for each group,and Suzuki score was used to calculate the degree of liver tissue damage.WB was used to examine the protein levels of LKB1.Results: The mouse I/R model and the RAW 264.7 H/R model were successfully constructed.The s ALT and s AST levels of mice was increased with the prolonging of ischemia/reperfusion time.The pathological slices of liver tissues suggested that after treatment of ischemia/reperfusion,the liver presents different degrees of injury and changes,mainly manifested as hepatocyte necrosis,sinusoidal congestion and vacuole-like changes.The protein level of LKB1 was decreased in the I/R and H/R models.It was determined that the optimal I/R treatment time for subsequent experiments was I1R6;the optimal H/R treatment time was H6/R6.Conclusion: The expression of LKB1 is decreased in mouse liver I/R and RAW 264.7 macrophage H/R models,and the optimal I/R treatment time for subsequent experiments was I1R6;the optimal H/R treatment time was H6/R6.PART ? LKB1 ATTENUATES LIVER ISCHEMIA-REPERFUSION INJURY VIA INHIBITING THE NLRP3 INFLAMMASOMEObjective: To investigate the effect of LKB1 on liver ischemia/reperfusion injury.Methods: The mice were injected with adeno-associated virus(AAV)overexpressing LKB1 through the tail vein two weeks before liver ischemia/reperfusion surgery.Animal experiments are divided into four groups:(1)sham operation group,(2)I/R group,(3)adeno-associated virus negative control(I/R+AAV-NC)group,(4)Adeno-associated virus overexpression(I/R+AAV-LKB1)group.The expression of LKB1 in mouse liver tissue for each group was performed.H&E was carried out on the degree of liver damage for each group,and Suzuki score was presented with calculating the severity of liver damage for each group.The concentration of s ALT and s AST were determined.The serum concentration of IL-1? and TNF-? were made by the method of ELISA.The protein levels of LKB1 and NLRP3 inflammasome-related molecules in mouse liver tissues were demonstrated by WB.Results: Both of the tissue m RNA and protein levels of LKB1 in mice which transfected with adenovirus overexpressing LKB1 were remarkably higher than those in the negative control transfection group and the sham group.The liver tissue damage in the I/R+AAV-LKB1 group was distinctly decreasing than that in I/R group.The concentration of s ALT and s AST in the I/R+AAV-LKB1 group were lower than I/R group.The concentration levels of IL-1? and TNF-? in the I/R+AAV-LKB1 group were more reduced with the I/R group.WB tests were carried out the protein of NLRP3,IL-1?,IL-18,and caspase-1 in the I/R group were overtly increased,while the protein of the above molecules in the I/R+AAV-LKB1 group were decreased.Conclusion: LKB1 could alleviate liver ischemia/reperfusion injury in mice.PART ? THE MOLECULAR MECHANISM OF LKB1 AMELIORATING ISCHEMIA-REPERFUSION INJURY VIA THE NLRP3 INFLAMMASOME DEPENDING ON FOXO1Objective: To explore the molecular mechanism of LKB1 alleviating liver ischemia-reperfusion injury in mice in vitro.Methods: LKB1 overexpressed lentivirus was used to infect RAW264.7 cells and stable cell strains were isolated.Small interfering RNA(si RNA)down-regulated FoxO1 expression level.The H/R model of RAW264.7 cells was constructed.The first part of the cell experiment grouping is as follows:(1)Blank infection group(LV-NC): RAW 264.7 cells were infected with negative control(NC)lentivirus,(2)Overexpression LKB1 lentivirus infection group(LV-LKB1): RAW 264.7 cells was infected with overexpressed LKB1 lentivirus,(3)Hypoxia-reoxygenation + blank infection group(H/R+LV-NC): RAW 264.7 cells was infected with NC lentivirus in H/R model,(4)Hypoxia and reoxygenation + LKB1 overexpression lentivirus infection group(H/R+LV-LKB1): RAW 264.7cells was infected with overexpressed LKB1 lentivirus in H/R model.The second part of the cell experiment is divided into the following groups:(1)control group,(2)hypoxia-reoxygenation treatment group(H/R),(3)hypoxia-reoxygenation + overexpression LKB1 group(H/R+LV-LKB1):RAW 264.7 cells was infected with overexpressed LKB1 lentivirus in H/R model,(4)Hypoxia and reoxygenation + overexpression of LKB1+down-regulated FoxO1 group(H/R+LV-LKB1+si RNA-FoxO1): RAW264.7 cells overexpressed LKB1 and down-regulated FoxO1 with si RNA in H/R model.The protein levels of NLRP3 inflammasome-associated molecules and NF-?B signaling pathway related molecules were performed.The localization of NLRP3 in cells and the nuclear translocation of p65 were performed with immunofluorescence.The concentrateion of cytokines TNF-? and IL-1? in the cell supernatant were tested.Results: The stable cell strains overexpressed LKB1 were successfully isolated,and the most suitable si RNA sequence that down-regulates FoxO1 was selected.We constructed the H/R model of RAW 264.7 cells in vitro.The cell experiment results of the first part were as follows.The protein levels of NLRP3 inflammasome-associated molecules and NF-?B signaling pathway related proteins in the H/R+LV-NC group elevated while the above molecules in H/R+LV-LKB1 group reduced.NLRP3 was mainly located in the cytoplasm of RAW 264.7cells and the fluorescence intensity of NLRP3 in the H/R+LV-LKB1 group was apparently weaker than that in the H/R+LV-NC group;the nuclear translocation of p65 was significant in the H/R+LV-NC group increased,but weakened in the H/R+LV-LKB1 group.The data of the second part cell experiment were as follows.The protein levels of NLRP3,IL-1?,IL-18,caspase-1 and p-I?B-? and p-NF-?B p65 reduced in H/R group after overexpression of LKB1 but was raised in FoxO1-silenced group.Immunofluorescence showed that the fluorescence intensity of NLRP3 was localized in the cytoplasm.In the H/R model,the fluorescence intensity of NLRP3 decreased after the treatment of overexpression of LKB1;while at the same time,the fluorescence intensity of NLRP3 increased in FoxO1-silenced group.The concentration of TNF-? and IL-1? in H/R group elevated remarkably and which decreased obviously in LKB1-upregulated group but partly down-expressed in FoxO1-silenced group.Conclusion: LKB1 exerts a protective effect in liver IRI by inhibiting NLRP3 inflammasome through FoxO1 in vitro.