| PART I:THE EFFECT OF IL-34 ON KUPFFER CELL POLARIZATION Objective:To investigate the effect of IL-34 on the polarization of Kupffer cells(KCs)in vitro.Methods:Rat KCs were obtained by differential centrifugation method which has improved by our research group.The purity of KCs was detected by flow cytometry(FCM).The phagocyte function of KCs was assessed by ink phagocytosis test.KCs were treated by LPS(1OOng/ml)and different concentrations of rat IL-34 recombinant protein.The expression levels of M1 macrophage molecular markers IL-10,TGF-? and M1 macrophage molecular marker IL-12 were detected by RT-PCR and ELISA.Western blot was used to detect the expression of Arg-1.Results:(1)The purity of KCs obtained by differential centrifugation method was more than 90%.(2)IL-12 expression level in KCs was significantly increased after treated by LPS.(3)The increase of IL-12 in LPS-treated KCs was impaired by treating with rat IL-34 recombinant protein;and the expression levels of IL-10,TGF-P and Arg-1 were increased after treated by rat IL-34 recombinant protein.(4)The expression levels of IL-10,TGF-? and IL-12 was not affected after treated by rat IL-34 recombinant protein alone.Conclusions:IL-34 could induce KCs M2 polarization in vitro.PART II:THE MOLECULAR MECHANISM OF KUPFFER CELL M2 POLARIZATION INDUCED BY IL-34 Objective:To investigate the molecular mechanism of KCs M2 polarization induced by IL-34 in vitro.Methods:The KCs were divided into four groups as following:(1)Control group:KCs cultured in normal media,(2)LPS group:KCs were treated with LPS(1OOng/ml);(3)IL-34 group:KCs were treated with LPS and rat IL-34 recombinant protein(5ug/ml)sequentially;(4)LY294002 group:KCs were pretreated with PI3K/Akt signaling pathway inhibitor LY294002(40?M)before treatmrt with rat IL-34 recombinant protein and LPS;(5)Rapamycin group:KCs were pretreated with mTOR signaling pathway inhibitor Rapamycin(lOnM)before treatment with rat IL-34 recombinant protein and LPS.Western blot was used to detect the phosphorylation status of Aktl/2?Aktl?Akt2?mTOR?4E-BP?S6K?p38 MAPK and p65 and the expression levels of I?B and Arg-1.The expressions of IL-10?TGF-? and IL-12 at serum and mRNA levels were detected by RT-PCR and ELISA,respectively.FCM was used to assess the percentage of M2 KCs.Results:(1)Phosphorylation status of Aktl/2 in LPS group was higher than that in control group.The phosphorylation status of Aktl/2 and Aktl in IL-34 group was higher than in LPS group and LY294002 group.The difference of phosphorylation status of Akt2 among IL-34 group,LPS group and LY294002 group was not obviously.The expression of IL-12 in LPS group was higher than that in control group,while the IL-12 expression in IL-34 group was significantly lower than that in LPS group and LY094002 group.The expression levels of IL-10,TGF-? and Arg-1 in IL-34 group were significantly higher than those in LPS group and LY094002 group.The percentage of M2 KCs in IL-34 group was significantly higher than that in LPS group and the LY094002 group.(2)The phosphorylation status of mTOR,S6K,and 4E-BP in IL-34 group was significantly higher than that in LPS group and LY094002 group.IL-12 expression level in IL-34 group was significantly lower than those in LPS group and Rapamycin group,and the expression levels of IL-10,TGF-?and Arg-1 in IL-34 group were significantly higher than those in LPS group and Rapamycin group.The percentage of M2 KCs in IL-34 group was also significantly higher than that in LPS group and Rapamycin group.(3)The phosphorylation status of p38 MAPK and p65 in LPS group was higher than that in control group and the phosphorylation status of p38 MAPK and p65 in IL-34 group was significantly lower than that in LPS group and LY094002 group.The expression level of Ir?B-? in LPS group was lower than that in control group,the IKB-a expression level in IL-34 group was significantly higher than that of LPS group and LY094002 group.Conclusions:(1)IL-34 induces KCs M2 polarization by activating PI3K/Akt signaling pathway;(2)Activation of the mTOR signaling pathway,downstream of PI3K/Akt signaling pathway,is involved in the M2 polarization of KCs induced by IL-34;(3)Down-regulation of NF-?B and p38 MAPK signaling pathways,downstream of PI3K/Akt signaling pathway,may also involved in M2 polarization of KCs induced by IL-34.PART III:THE M2 POLARIZATION OF KUPFFER CELL WAS INVOLVED IN THE ACUTE REJECTION INHIBITION OF RAT LIVER TRANSPLANTATION INDUCED BY IL-34 Objective:(1)To investigate the effect of IL-34 over-expression in recipient rat on acute rejection(AR)of rat liver transplantation;(2)To determine whether the effect of IL-34 on acute rejection of rat liver transplantation was mediated by M2 polarization of KCs.Methods:This part of the study contains two parts of animal experiments.The model of AR of rat orthotopic liver transplantation(LEW rat as donor,BN rat as recipient,LEW-BN)was performed by modified two-cuff technique.The first part of animal experiment was grouped as follow:(1)Control group:BN rats were injected with normal saline(NS)one month before liver transplantation;(2)AAV-GFP:BN rats were injected with adeno-associated virus which expressing green fluorescent protein(AAV-GFP)one month before liver transplantation;(3)AAV-IL-34 group:BN rats were injected with adeno-associated virus which expressing IL-34(AAV-IL-34)one month before liver transplantation.The survival of recipient rats was monitored after liver transplantation.Liver tissue was obtained for HE staining and the rejection activity index(RAI)was scored according Banff pattern on day 7 after liver transplantation.Serum ALT,AST and TBIL were detected on day 1 before liver transplantation and day 1,3,5,7 after liver transplantation.Serum IL-10 and IFN-? on day 7 after liver transplantation were detected by ELISA.Western blot was used to detect the Arg-1 expression level in liver tissues on day 7 after liver transplantation.The expression levels of IL-10.IL-12 and TGF-? in liver tissues on day 7 after liver transplantation were detected by RT-PCR.The second part of animal experiment was grouped as follow:(1)Control group:BN rats were injected with NS one month before liver transplantation;(2)AAV-IL-34 group:BN rats were injected with AAV-IL-34 one month before liver transplantation;(3)AAV-IL-34+ clodronate group:Clodronate was used to deleted the KCs of LEW rats before liver transplantation;(4)AAV-IL-34+clodronate+KCs transfer group:KCs of LEW rats were deleted by clodronate,and non-treated KCs were adoptive transferred into recipient rat through portal vein during liver transplantation;(5)AAV-IL-34+clodronate+LPS-treated KCs transfer group:KCs of LEW rats were deleted by clodronate,and LPS-treated KCs(M1 phenotype KCs)were adoptive transferred into recipient rat through portal vein during liver transplantation.The survival of recipient rats was monitored after liver transplantation.Liver tissue was obtained for HE staining and the RAI was scored on day 7 after liver transplantation.Serum ALT,AST and TBIL were detected on day 7 after liver transplantation.Results:The first part of animal experiments:(1)Survival of recipient rats in the AAV-IL-34 group was significantly longer than that in the control group and AAV-GFP group.The RAI of AAV-IL-34 group was significantly lower than that of the Control group and AAV-GFP group.Serum IL-10 of AAV-IL-34 group was higher than that in the Control group and AAV-GFP group,while the serum IFN-y of AAV-IL-34 group was lower than that in the Control group and AAV-GFP group.Serum ALT,AST and TBIL levels in AAV-IL-34 group were significant lower than those in Control group and AAV-GFP group.(2)IL-12 expression in KCs of AAV-IL-34 group was lower than that of Control group and AAV-GFP group,while expression levels of Arg-1,IL-10 and TGF-? in KCs of AAV-IL-34 group were higher than those of Control group and AAV-GFP group.(3)The survival of recipient rats in AAV-IL-34+clodronate group was significantly shorter than that in AAV-IL-34 group,AAV-IL-34+clodronate+non-treated KCs transfer group and AAV-IL-34+clodronate+LPS-treated KCs transfer group.The liver injury of AAV-IL-34+clodronate group was more severe than that in AAV-IL-34 group,AAV-IL-34+clodronate+non-treated KCs transfer group and AAV-IL-34+clodronate+LPS-treated KCs transfer group,and the RAI of AAV-IL-34+clodronate group was higher than that in AAV-IL-34 group,AAV-IL-34+clodronate+non-treated KCs transfer group and AAV-IL-34+clodronate+LPS-treated KCs transfer group.Serum ALT,AST and TBIL levels in AAV-IL-34+clodronate group were higher than that in AAV-IL-34 group,AAV-IL-34+clodronate+non-treated KCs transfer group and AAV-IL-34+clodronate+LPS-treated KCs transfer group.There was no significant difference in the degree of AR between AAV-IL-34+clodronate+non-treated KCs transfer group and AAV-IL-34+ clodronate+LPS-treated KCs transfer group.Conclusions:(1)Over-expression of IL-34 in recipient rat could inhibit the AR of rat liver transplantation;(2)IL-34 could induce M2 polarization of KCs in graft after liver transplantation;(3)The M2 polarization of KCs mediates the inhibitory effect of IL-34 on AR of rat liver transplantation. |
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