| Objectives:Acute liver failure is due to various reasons(viruses,drug,metabolism)led to liver function damage sharp,high mortality rate.The orthotopic liver transplantation is the preferred method for treatment of acute liver failure,but because of the lack of sources,serious postoperative reaction and economic reasons so that it can not become a routine clinical treatment.Stem cell transplantation has not achieved satisfactory clinical therapeutic effect,due to the limited number of implanted stem cells into functional liver cells.The paracrine mechanism that macrophages participate in plays an important role in stem cell transplantation.Macrophages under the influence of microenvironment polarization can be expressed as M1 and M2,play a different role in remodeling process and necrosis inflammation because of its different phenotype and function.To clarify the clinical efficacy of bone marrow mesenchymal stem cells(MSCs)in the treatment of acute liver failure,comparing the survival rate,hepatocyte regeneration capability and liver pathology in acute liver failure rats after treatment of MSCs.To clarify whether the polarization of macrophages is related to the clinical efficacy of MSCs by investigating the distribution of M1 and M2 macrophages in the liver of survival and death group after bone marrow mesenchymal stem cells treatment.To make clear the source of M2 macrophages polarization-related factors IL-10 and IL-4 in survival group.Mass spectrometry was used to quantitatively compare the differences between the survival and death group after treatment,and presumed molecular pathways to activating macrophages toward M2 polarization by detecting the expression of signal and activator of transcription 6(STAT6),and GATA binding protein 3(GATA3).To investigate the role of macrophage polarization in the treatment of acute liver failure by bone marrow-derived mesenchymal stem cells and to provide a theoretical basis for improving the therapeutic effect of hepatic stem cells in patients with acute liver failure.Methods:Part Ⅰ: Evaluation of the effect of bone marrow mesenchymal stem cells in the treatment of acute hepatic failure in rats.The standard of modeling was as follows: the survival rate of rats decreased to 10-20%,serum biochemical indicators of alanine aminotransferase(ALT),aspartate aminotransferase(AST),albumin(ALB),total bilirubin(total bilirubin,TBIL)were significantly abnormal,large cell necrosis is visible in pathological structure of liver,and no other fatal diseases.Rats in acute liver failure were treated with tail vein injection of GFP-labeled rat bone The control group received intravenous injection of Dulbecco’s phosphate-buffered saline(DPBS).The survival rate,liver function and liver histopathology were used to evaluate the efficacy of bone marrow mesenchymal stem cells for treatment of acute liver failure.Part Ⅱ: To analyze the relationship between the phenotype polarization of macrophages and the function of liver regeneration.(1)The function of hepatocyte regeneration after stem cell therapy was evaluated by immunohistochemical detection of epithelial cell adhesion molecule(epithelial cell adhesion molecule,EpCAM)in liver tissue.After immunofluorescence staining,the number of Ki67 positive cells in the liver tissues of the treatment group was evaluated,and the number of hepatocytes proliferating was counted.Meanwhile,the number of TUNEL positive cells was counted to evaluate the number of apoptotic hepatocytes.(2)The polarization of macrophages in the livers of survival and death groupwere detected respectively.The markers of M1 polarization were Cluster of differentiation 68(CD68),interferon-γ(IFN)-γ),tumor necrosis factor alpha(TNF-α)and inducible nitric oxide synthase(INOS).The markers of M2 polarization are Cluster of differentiation 63,CD163,interleukin 4(IL-4),interleukin 10(IL-10)and arginase-1(Arg-1).Immunofluorescence double staining method was used to detect the distribution of CD68 and CD163 in the liver between the two groups.The concentrations of IFN-γ and IL-4 in serum of the two groups were detected by flow cytometry.The expressions of IL-10 and IL-4 in liver tissue were detected by double immunofluorescence staining to determine whether IL-10 and IL-4 originated from intrahepatic macrophages or implanted bone marrow Mesenchymal stem cells.Part Ⅲ: STAT6 and GATA3 in JAK-STAT pathway were differentially expressed in survivial and death group after treatment.Quantitative analysis protein expression differences of in survival and death group after MSCs treament,and western blot was used to examine the protein levels of STAT6 and GATA3 in the liver of the two groups.Activation through the JAK-STAT pathway.Survival curves were evaluated using Log-rank(Mantel-Cox)test and SPSS 13.0 software was used for statistical analysis of the data.Each group of samples were 4-6,each sample repeated three experiments,high magnification count each sample taken randomly when five fields(portal area 2,the central vein 2,the area between the portal area and the central vein 1).The statistics were described by means of mean ± standard deviation.Each group passed t-test with P <0.05 as statistical significance.Results:Part Ⅰ: Evaluation of clinical efficacy bone marrow mesenchymal stem cells in the treatment of acute liver failure rats.(1)The mortality rate of rats in acute liver failure group was about 80%-90%.Compared with the control group,serum ALT,AST,ALB and TBIL in rats with acute liver failure were significantly abnormal.Liver HE staining showed that liver cells Necrosis,nuclear pyknosis,fragmentation,dissolution,a large number of inflammatory cell infiltration,visible interstitial congestion.(2)The survival rate of rats in treatment group after treatment with MSCs was 37.5% higher than that in DPBS treated group(10%).At 24 hours and 48 hours after treatment,biochemical indicators were significantly improved than the DPBS treatment group.At 48 hours after treatment,no significant changes in the pathological structure of liver were observed when compared with those treated with DPBS.However,the hepatic inflammatory injury in the treated group was significantly recovered at 5 days after treatment.Part Ⅱ: To analyze the relationship between the phenotype polarization of macrophages and the function of liver regeneration.(1)The expression of EpCAM,a marker of hepatocyte regeneration,in the treatment group was significantly higher than that of the untreated group(P <0.001);in addition,expression of EpCAM in the survival group after treatment was significantly higher than that in the death group after treatment(P = 0.03).The number of Ki67-positive cells(55.5±29.5 个/HPF)in the liver of the treatment group was more than that of the DPBS-treated group(14.5±8.0 个/HPF)(P=0.003).On the contrary,the number of TUNEL-positive cells(7.5±4 个/HPF)was significantly lower than that of the untreated group(64.4±28.8 个/HPF)(P<0.001).(2)The expression level of CD68 in acute liver failure group was significantly higher than that in control group,and the expression level of CD163 was also higher than that in control group.The expression of CD68 in acute liver failure group was higher than that of CD163 by immunofluorescence double staining.The expression of CD163 in the group was higher than that of CD68.(3)After treatment,the expression of CD163 in the liver tissue of survival group was significantly higher than that of the control group(88.51 ± 24.51 pg / ml)and 34.61 ± 6.6 pg / ml(P <0.001).The gene expression levels of IL-10 and Arg-1 were higher than those of death group after treatment(P <0.001).(4)After treatment,the expression of CD68 in the liver tissue of the deceased group was significantly increased,and the concentration of IFN-γ in the serum was 542.11 ± 51.59 pg / ml higher than that of the survived group(104.07 ± 42.80 pg / ml)(P<0.001).At the same time,the gene expression levels of TNF-α and INOS were higher than that of survival after treatment(P <0.001).(5)Immunofluorescence double staining showed that both IL-4 and IL-10 in the surviving group were not colocalized with the implanted GFP-labeled MSCs after treatment.Part Ⅲ: Quantitative analysis protein expression differences of in survival and death group after MSCs treament,elucidating macrophage polarization molecular mechanism.(1)The expression of STAT protein in the survival group after MSCs treatment was significantly higher than that in the control group(P <0.05).(2)The difference protein of the survival group after treatment and the death group after treatment was analyzed to participate in the process of the immune system and the transcription activation process by GO.The differential protein STAT protein was analyzed to participate in the JAK-STAT pathway by KEGG.(3)The protein expression levels of STAT6 and GATA3 between survival group and death group were compared.After treatment,the expression of STAT6 and GATA3 in surviving group were higher than those in untreated group(P <0.001).Conclusions:1.Bone marrow-derived mesenchymal stem cells treat rats with acute liver failure can improve the survival rate of rats,improve liver function and liver tissue structure.2.Bone marrow-derived mesenchymal stem cells increase liver regeneration after treatment,and macrophage significantly upregulates liver regeneration when polarized to the M2 phenotype.3.Bone marrow mesenchymal stem cells treat acute liver failure rats by inducing M2 polarization through the JAK-STAT pathway. |
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