Effect Of Macrophages Depletion On CCL₄-Induced Acute Liver Injury And Liver Fibrosis Recovery Phases In Mice

Objective: to study the effect of macrophages depletion on CCL₄-induced Acute Liver Injury and Liver Fibrosis Recovery Phases in mice.Methods:1 The effect of macrophages depletion on CCL₄-induced Acute Liver Injury: Fifteen male BALB/c mice?23.70±2.43?g were divided into three groups at random namely control group?ip Oil,n=5?,model group?no depletion macrophages,iv PBS,ip CCL₄,n=5?,experiment group?depletion macrophage,iv Clodronate-Liposomal,CL,ip CCL₄,n=5?.Our experiment was based on Van Rooi jen report for Clodronate –Liposomal.As the report,intravenous injections of Clodronate –Liposomal can be selectively removing the macrophages.Macrophage uptake of liptosomes by phagocytosis.Phospholipase destroy lipid bilayer of leptosomes.This drug is taken up by macrophages and rapidly causes apoptosis of these cells.The model group was injected intravenously with PBS 150 ?l.At the same time experiment group were injected intravenously with Clodronate-Liposomal 150 ?l,but empty treatments were done in the control group.During macrophage depletion,12 hours after Clodronate-Liposomal,mice were injected intraperitoneally with CCl₄ or Oil.The model group and experiment group were injected intraperitoneally with 5%CCl₄?0.6 ul/g body weight?,control group were injected intraperitoneally with Oil?0.6 ul/g body weight?.Mice were sacrificed 24 hours after the last injection.The serum were stored-80 ?until alanine aminotransferase?ALT?? Aspartate transaminase,AST and total bilirubin?TBIL?assay.Part of the left hepatic lobe were cut and fixed in 4% paraformaldehyde.Tissue section were stained with hematoxylin-eosin?HE?,other tissue section were stained immunohistochemistrically to detect macrophages?F4/80+?and integral optical density?IOD?were measured by Image-Pro Plus.The rest of tissue were stored at-80?to detect the expression of iNos?CXCL16 mRNA by Real-Time PCR.2 The effect of macrophages depletion on CCL₄-induced liver fibrosis recovery phases: nine male BALB/c mice?26.19±4.23?g were divided into three groups at random namely control group?ip Oil,n=3?,model group?no depletion macrophages,iv PBS,ip CCL₄,n=3?,experiment group?depletion macrophage,iv CL,ip CCL₄,n=3?.The model group and experiment group were injected intraperitoneally with 5%CCl₄?0.6 ul/g body weight?,control group were injected intraperitoneally with Oil?0.6 ul/g body weight?twice weekly.Continued for 4 weeks.4 weeks after CCl₄,the model group was injected intravenously with PBS 150 ?l and experiment group were injected intravenously with Clodronate-Liposomal 150 ?l twice weekly.Empty treatments were done in the control group.Mice were sacrificed 7 days after the last injection.The serum were stored-80 ?until ALT?AST and TBIL assay.Part of the left hepatic lobe were cut and fixed in 4% paraformaldehyde.Tissue sections were stained with hem-atoxylin-eosin?HE?and Sirius red staining.Other tissue section were stained immunohistochemistrically to detect macrophages?F4/80+???-SMA ?collagen-?and TGF-?1.The IOD were measured by Image-Pro Plus.The rest of tissue were stored at-80?to detect the expression of ?-SMA?TGF-??CXCL16 protein by Western-bolt.Results:1 The effect of macrophages depletion on CCL₄-induced Acute Liver Injury: experiment group,about 80% of the macrophages were depleted by Immunohistochemical staining.Macrophages of liver could be seen by F4/80 staining.Model group show that large F4/80 positive expression of macrophage in liver tissue necrosis area.The IOD were?21496.63±5907.46?in model group?no depletion macrophages?.F4/80 immunoreactivity was almost no detected in experiment group?depletion macrophages?.The IOD were?4404.33±311.28?in experiment group.Control group show that moderate amount F4/80 positive expression of macrophage in liver tissue.The IOD were?12491.26±3958.61?in control group.The IOD were higher in model group than that in experiment group and the results were significant?P<0.01?.The IOD in control group were also higher than IOD in experiment group.The differences between control group and experiment group were significant?P<0.01?.Histological observations revealed that CCl₄ treatment induce extensive necrosis in the hepatocytes surrounding the central veins of the liver in model group.The areas of hepatocytes necrosis were reduced in experiment group.Hepatocytes morphology was normal in control group.Serum ALT levels were dramatically increased in model group?202.32±3.20?U/L compared with levels in control group?16.70±5.11?U/L.The differences between model group and control group were significant?P<0.01?.Serum ALT levels in model group compared with levels in experiment group?200.60±6.34?U/L were not reach statistical significance?P> 0.05?.Serum AST levels were?292.90±8.64?U/L in model group,?278.54±8.42?U/L in experiment group,?64.64±30.18?U/L in control group.Serum AST levels were higher in model group than that in control group.The differences between model group and control group were significant?P<0.01?.Differences between results for model group and experiment group were significant?P<0.05?.Serum TBIL levels were?19.49±1.16??mol/L in model group,?14.64±0.89??mol/L in experiment group,?6.68±0.17??mol/L in control group.Serum TBIL levels were higher in model group than that in control group and the results were significant?P<0.01?.The differences between model group and experiment group were significant?P<0.05?.RT-PCR: the expression of iNos mRNA in model group was higher than in experiment group.The differences were significant?P<0.05?.The expression of iNos mRNA in model group was also higher than in control group.The differences were significant?P<0.01?.The expression of CXCL16 mRNA in model group was higher than in experiment group.The differences were significant?P<0.05?.The expression of CXCL16 mRNA in model group was also higher than in control group.The differences were significant?P<0.05?.2 The effect of macrophages depletion on liver fibrosis recovery phases: experiment group,about 62% of the macrophages were depleted by Immunohistochemical staining.Macrophages of liver could be seen by F4/80 staining.Model group and control group show that F4/80 positive expression of macrophage in liver tissue.The IOD were?45858.33±383.15?in model group?no depletion macrophages?.The IOD were?45209.00±7466.85?in control group.F4/80 immunoreactivity was almost no detected in experiment group?depletion macrophages?.The IOD were?17810.00±7663.55?in experiment group.The IOD were higher in model group than that in experiment group and the results were significant?P<0.01?.The IOD in control group were also higher than IOD in experiment group.The differences between control group and experiment group were significant?P<0.01?.But Between the model group and control group had no statistical significance.Histological observations revealed that CCl₄ treatment induce extensive necrosis in the hepatocytes surrounding the central veins of the liver in model group and experiment group.Hepatocytes morphology was normal in control group.Sirius red: Sirius red staining was observed in model group and experiment group.Model group markedly reduced Sirius red than experiment group.The staining regions of control group were detected mainly in portal area.Serum ALT levels were a bit much in control group?17.73±4.49?U/L compared with levels in model group?13.97±6.34?U/L.But the differences were not reach statistical significance?P>0.05?.Serum ALT levels in model group compared with levels in experiment group?98.70±30.10?U/L were significant?P<0.05?.Serum AST levels were?101.17±35.10?U/L in model group,?202.20±35.60?U/L in experiment group,?75.45±4.34?U/L in control group.Compared with the control group of serum AST level model group increased serum level can be neglected.The differences were not reach statistical significance?P>0.05?.Serum AST levels in model group compared with levels in experiment group were significant?P<0.05?.TBIL levels were?6.49±0.19??mol/L in model group,?8.73±1.71??mol/L in experiment group,?7.29±1.16??mol/L in control group.But no significant difference was observed among three groups.IOD of ?-SMA in model group was?7850.83±1022.74?.?-SMA in model group was expressed most.The IOD were?13815.0±3051.69?in experiment group.The IOD were?5756.67.83±1218.78?in control group.?-SMA immunoreactivity was indentified in the smooth muscle cells of hepatic arteries and in the walls of portal and hepatic vein branches in control.Between the model group and control group had no statistical significance.The differences between control group and model group had no statistical significance.The differences between model group and experiment group were significant?P<0.05?.The differences between control group and experiment group were significant?P<0.05?.Collagen-?was expressed little in model group.IOD in model group were?9517.67±2664.57?.The IOD were?25747.00±7363.58?in experiment group.Collagen-immunoreactivity was almost no detected in control group.?The IOD were?11009.67±3272.93?in control group.The differences between control group and experiment group were significant?P<0.05?.The differences between model group and experiment group were significant?P<0.05?.TGF-?1 was expressed little in model group.IOD in model group were?9523.67±3266.61?.The IOD were?20536.67±5196.5?in experiment group.TGF-?1 immunoreactivity was almost no detected in control group.The IOD were?8846.33±2677.30?in control group.The differences between experiment group and control group were significant?P<0.05?.The differences between model group and experiment group were significant?P<0.05?.Western blot: The expressions of ?-SMA protein were detected in different chronic groups.The expressions of ?-SMA protein were?0.237±0.017?in model group,?0.490±0.016?in experiment group,?0.126±0.009?in control group.The results showed that the expressions of ?-SMA protein in model group were lower than that in experiment group.There is significance statistical difference between the two groups?P<0.05?.The expressions of protein in model group were higher than that in control group.The differences between two groups were significant?P<0.05?.The expressions of TGF-?1 protein were detected in different chronic groups.The expressions of protein were?0.174±0.023?in model group,?0.318±0.006?in experiment group,?0.087±0.012?in control group.The results showed that the expressions of protein in model group were higher than that in control group.There is significance statistical difference between the two groups?P<0.05?.The expressions of TGF-?1 protein in model group were lower than that in experiment group.The differences between two groups were significant?P<0.05?.The expressions of CXCL16 protein were detected in different chronic groups.The expressions of protein were?0.236±0.044?in model group,?0.393±0.015?in experiment group,?0.117±0.013?in control group.The results showed that the expressions of CXCL16 protein in model group were lower than that in experiment group.There is significance statistical difference between the two groups?P<0.05?.The expressions of protein in model group were higher than that in control group.The differences between two groups were significant?P<0.05?.Conclusion:1 Macrophages depletion on Acute Liver Injury can improve liver function,reduce the secretion of inflammatory cytokines and relieve liver damage,so as to protect the liver.2 Macrophages depletion on liver fibrosis recovery phases can aggravating liver damage,increase the secretion of inflammatory cytokines,attenuated matrix degradation,Slow down the recovery of liver fibrosis.