| PartⅠ: BMSCs injection alleviate LPS-induced acute liver injury in mouseObjective: To observer whether injection of exogenous BMSCs can protect mice from LPS induced acute liver injury,inhibit activation of NLRP3 inflammasome in KCs in vivo and reduce levels of inflammatory cytokine in serum,and get a deeper understanding about the protective role of BMSCs in sepsis and its underlying mechanism.Methods: Mouse BMSCs were isolated from the femurs and tibias of Balb/C mice.The transfection of the ptges gene or ptges sh RNA lentivirus into BMSCs was conducted using the Lentivirus Transfection Reagent.Thirty mice were randomly divided into five groups.The mice in LPS group,LPS+BMSC group,LPS+BMSC-PGE2(+)group and LPS+BMSC-PGE(-)group were given an intraperitoneal injection of 0.25 ml of LPS(10 mg/kg(mpk))for 12 h to induce acute liver injury,and this was followed by an injection of 0.4 ml of sterile PBS or 1×106 BMSCs/BMSC-PGE2(+)/ BMSC-PGE2(-)in 0.4 ml of sterile PBS.The mice were sacrificed 12 h after the caudal vein injection,and a blood sample and the liver were harvested.Paraffin sections were dewaxed and stained with hematoxylin and eosin(H&E).The morphologic changes in the tissue were assessed by calculating the infiltration of inflammatory cells and the degree of cellular swelling of the hepatocytes at a magnification of ×400 in 5 randomly chosen fields per sections.The apoptosis of the hepatocytes was evaluated using a TUNEL staining kit according to the manufacturer’s instructions.The expression levels of NLRP3,ASC,Pro-casp-1,caspase-1 and Pro-IL-1β in the total protein samples of KCs were measured using Western blot assays.Real-time fluorescent quantitation polymerase chain reaction(RT-q PCR)were used to measure gene expression of NLRP3,ASC and caspase-1 in KCs.The levels of alanine aminotransferase(ALT),aspartate aminotransferase(AST),IL-10,IL-1β and PGE2 in the sera were detected using commercial ELISA kits.Results:(1)A significant reduction in the number of inflammatory cells in mice receiving BMSC and BMSC-PGE2(+)compared to the LPS group and the LPS+BMSC-PGE2(-)group(P<0.05).While treatment with BMSC-PGE2(-)were have no effect on infiltration of inflammatory cells in liver as compared with LPS group(P>0.05).(2)The number of apoptotic cells were decreased significantly by treatment with BMSC-PGE(+)than with BMSC(P<0.05),while treatment with BMSC-PGE(-)did not decreased the number of apoptotic cells as compared with LPS(P>0.05).(3)LPS treatment resulted in abundant expression of NLRP3,ASC,Pro-Casp1,Caspase-1 and Pro-IL-1β in the LPS group.Expression of NLRP3,ASC,Pro-Casp1,Caspase-1 and Pro-IL-1β protein were reduced in the LPS+BMSC and decreased significantly in the LPS+BMSC-PGE2(+)group than that in the LPS group(P<0.05),while did not decreased markedly in the LPS+BMSC-PGE2(-)group(P>0.05).(4)The relative m RNA expression of NLRP3,ASC and caspase-1 in KCs were increased by LPS treatment in the LPS group,but decreased in the LPS+BMSC group and further reduced in the LPS+BMSC-PGE2(+)group as compared with the LPS group(P<0.05).While in the LPS+BMSC-PGE2(-)group,there were no obvious decrease of relative m RNA expression of NLRP3,ASC and caspase-1 than in the LPS group(P>0.05).(5)Serum PGE2 were increased in LPS+BMSC group and further elevated in LPS+BMSC-PGE(+)group(P<0.05),while not increased in LPS+BMSC-PGE(-)group as compared with LPS group(P>0.05).(6)Serum ALT,AST and IL-1β levels were enhanced in the LPS group and significantly decreased by treatment with BMSC and further decreased by BMSC-PGE2(+)(P<0.05),but did not decreased markedly by BMSC-PGE2(-)(P>0.05).The level of IL-10 in the serum was increased slightly in the LPS group and enhanced dramatically in the LPS+BMSC-PGE2(+)group(P<0.05),while not increased in LPS+BMSC-PGE(-)group than in LPS group(P>0.05).Conclusion: Injection of exogenous BMSCs via the caudal vein attenuate acute liver injury induced by intraperitoneal injection of LPS in mice,and inhibits activation of NLRP3 inflammasome in vivo.Overexpression of ptges raised secretion of PGE2 in BMSCs and strengthened the protective effect of BMSCs on liver and inhibition effect of BMSCs on activation of NLRP3 inflammasome.Part Ⅱ: Observe the effect of BMSCs on the activation of NLRP3 inflammasome in Kupffer cellsObjective: To further prove that whether BMSCs directly inhibit activation of NLRP3 inflammasome in KCs and to observe whether it is depend on BMSCs secreting of PGE2.Methods: KCs were plated in six-well plates at a density of 2×106 cells per well and randomly divided into five groups: control group,LPS group,LPS+BMSC group,LPS+BMSC-PGE2(+)group and LPS+BMSC-PGE2(-)group.For the co-culture experiments,BMSC/BMSC-PGE2(+)/BMSC-PGE2(-)were cultured in the lower compartment of transwell system at a density of 1×10 BMSC cells per well.Implant the KCs and BMSCs /BMSC-PGE2(+)/BMSC-PGE2(-)into co-culture system.All group except of control were treated with LPS(10 ng/ml)for 5 h and ATP(5 m M)for the final 1 h.6h later,KCs and supernatants of cell culture were harvested for detection.Results:(1)The relative m RNA expression of NLRP3,ASC and caspase-1 in KCs were increased in the LPS group than in the Control group,but decreased in the LPS+BMSC group and further reduced in the LPS+BMSC-PGE2(+)group than in the LPS group.While in the LPS+BMSC-PGE2(-)group,Relative m RNA expression of NLRP3,ASC and caspase-1 were not decreased obviously than in the LPS group.(2)Expression of protein of NLRP3,ASC,Pro-Casp1,Caspase-1 and Pro-IL-1β were increase significantly in the LPS group than the control group,and reduced in the LPS+BMSC and decreased significantly in the LPS+BMSC-PGE2(+)group than in the LPS group,while did not decreased markedly in the LPS+BMSC-PGE2(-)group.(3)Supernatant PGE2 levels were increased slightly in LPS+BMSC group,and further elevated in LPS+BMSC-PGE(+)group but not increased in LPS-BMSC-PGE(-)group than LPS group.(4)Levels of TNF-α,IL-18,IL-1β in the supernatant were enhanced dramatically in the LPS group than other three groups and significantly decreased by treatment with BMSC and further decreased by BMSC-PGE2(+),but did not decreased markedly by BMSC-PGE2(-).(5)The level of IL-10 in the supernatant was increased slightly in the LPS group and dramatically enhanced in the LPS+BMSC-PGE2(+)group,while not increased markedly in LPS+BMSC-PGE(-)group compared with LPS group.Conclusion: Co-culture experiment illustrated that activation of NLRP3 inflammasome in KCs directly inhibited by BMSCs,and the inhibitive effect of BMSCs on activation of NLRP3 infammasome in KCs were depended on BMSCs secreting of PGE2.Part Ⅲ: Mechnism of PGE2 repress NLRP3 inflammasome activation in Kupffer cellsObjective: To further demonstrate the underlying molecular mechanisms by which PGE2 mediates the BMSC-inhibited activation of the NLRP3 inflammasome.Methods: KCs were treated with/without LPS and ATP for the first 5 hours,followed by treatment with/without PGE2(0.1 m M)for the last 30 min of their incubation.For the group using inhibitors,the cells were treated with an extracellular signal regulated kinase 1(ERK1)inhibitor(FR180204,10 m M)for 30 min before PGE2 stimulation.The inflammatory cytokine in supernatants were analyzed by ELISA.KC lysates were harvested for RT-q PCR and Western blotting.Expression of NLRP3,ASC,Caspase-1 and p-ERK1 protein in KCs cells were detected by Western blotting.Levels of NLRP3,ASC and Caspase-1 m RNA were detected by RT-q PCR.Results:(1)Protein expression levels of NLRP3,ASC and caspase-1 in group B were increased significantly than that in group A and group C,while there were not significantly difference between group B and group D.The protein expression levels of p-ERK1 were dramatically increased in group C as compared with that in other three groups and there were no difference between group B and group D.(2)The expression trends of relative m RNA of NLRP3,ASC and caspase-1 in KCs were similarly to the trend of protein expression.(3)Levels of TNF-α,IL-18 and IL-1β in group B were increased significantly than in group A and group C,while there is no signicantly difference between group D and group B.(4)The level of IL-10 in the supernatant was increased slightly in group B and group D and dramatically enhanced in group C as compared with group A.Conclusion: ERK1-mediated IL-10 secretion in KCs could be the key pathway involved in PGE2-induced suppression of activation of NLRP3 inflammation. |
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