| Objective:To study the role of macrophages in CCL4 induced liver fibrosis in miceMethods: Heathly male BALB/c mouse(22.15±2.52)g were randomly divided into three groups:control group,model group,experiment group.The mouse in the model group and the experimental group were injected intraperitoneally with 5% CCl4(0.6 ul /g)for twice weekly;the mouse in the control group were injected intraperitoneally with oil(0.6 ul/g)twice weekly.Continued for four weeks.The model group was injected intravenously with PBS 150 ul and experiment group was injected intravenously with clodronate-liposomal 150 ul twice weekly.Empty treatments were done in the control group.Mouse were sacrificed 7 days after the last injection.Part of the left hepatic lobe were cut and fixed in 4% paraformaldehyde.Tissue sections were stained with hematoxylin-eosin(HE)and Sirius red staining.Other tissues section were stained immunohistochemistrically to detect macrophages(F4/80+),?-SMA,collagen-?,PDGFB,TNF-?,MMP-13,IL-10.The OD was measured by Image-pro plus.The rest of tissues were stored at-80?.Results:1 Establishment of liver fibrosis model:mouse in model group of HE staining showed that infiltrated inflammatory cells increased,portal area disorder;Sirius red staining showed fibrous connective tissue increased.2 Macrophage depletion:macrophage specific antibody F4/80 for immun ohistochemical staining of liver tissue to understand macrophage depletion,which brown granules were positive staining.Model group and control group show that F4/80 positive expression of macrophage in liver tissue,the optical density(OD)was(0.326±0.026),(0.331±0.015)respectively,only a small number of F4/80 positive macrophages were found in the liver tissue of miceinjected with CL,the OD was(0.127±0.028).Compared with the control group and the model group,the expression of F4/80 in experimental group was lower,the differences were statistically significant(P<0.01);there was no significant difference between the control group and the model group(P>0.05).The results showed that at least 62% of macrophages were successfully removed in the experimental group than in the model group.3 Histopathological observation of liverHE staining:In the control group,the morphology of hepatocytes was normal,and there was no inflammation and necrosis;in the model group,a small amount of necrosis was found in the liver tissue,which was mainly located around the central vein;the liver injury in experimental group was more serious than that in the model group,and there was a map like necrosis area around the central vein.Sirius red staining : In the control group,only a small amount of collagen fibers were found in the portal area and central venous wall.The collagen deposition in the model group was mainly located in the interlobular vein and hepatic sinusoid.In the experimental group,the deposition of collagen fibers increased significantly,mainly in the portal area and central vein.4 Effects of macrophage depletion on ?-SMA and Collagen-I:In the control group,only a small amount of ?-SMA was expressed in vascular smooth muscle cells,the OD was(0.047±0.005);in the model group?-SMA was few expressed in the portal area and hepatic sinus,the OD was(0.064±0.005);in the experimental group ?-SMA was large expressed in the portal area and hepatic sinus,the OD was(0.113±0.013).Compared with the control group,the expression of ?-SMA in experimental group was higher(P<0.01),the difference was statistically significant;compared with the model group,the expression of the experimental group was slightly higher(P<0.05),the difference was also statistically significant;There was no significant difference between the control group and the model group(P > 0.05).In the control group,only a small amount of Collagen-I was expressed in the portal area and central vein,the OD was(0.085±0.013);in the model group,there was a little expression in the interlobular vein and the hepatic sinusoid,the OD was(0.073±0.010);in the experimental group,Collagen-I was a large number of expression in hepatic sinusoid,the OD was(0.198±0.029).Compared with the control group,the expression of Collagen-I in experimental group was slightly higher(P<0.05),the difference was also statistically significant;compared with the model group,the expression of Collagen-I in experimental group was higher(P<0.01),there was significant difference;there was no significant difference between the control group and the model group(P>0.05).5 Effects of macrophage depetion on the expression of inflammatory cytokines PDGFB,TNF-? and IL-10The expression of PDGFB was mainly located in cytoplasm of positive cells,the mice in the control group were only slightly expressed in the portal vein;the model group positive expression was mainly located in the portal area and hepatic sinus;The experimental group expressed more in the portal area and perisinusoidal.The results of immunohistochemistry showed that the expression of PDGFB in the experimental group(0.133±0.006)was higher than that in the model group(0.110±0.003)and the control group(0.071±0.001),the differences were statistically significant(P < 0.01);there were differences between the control group and the model group(P<0.01).The normal control group rarely expressed in the portal area,the expression of model group in the portal area and hepatic sinusoidal small,a large number of expression in the experimental group in the portal area and hepatic sinus.The results of immunohistochemical quantitative analysis showed that the expression of TNF-? in the experimental group(0.128±0.005)was higher than that in the model group(0.106±0.005)and the control group(0.085±0.002),the differences were statistically significant(P < 0.01);there were differences between the control group and the model group(P<0.01).In normal control group,IL-10 was rarely expressed;in the model group positive cells distributed around the portal area,central vein and liver sinus;the experimental group of positive cells in the central vein and portal area expressed less.The results of immunohistochemistry showed that the expression level of IL-10 in model group was significantly higher than that innormal group(0.128±0.002 vs 0.075±0.003,P<0.01),and the difference was statistically significant;The expression of the experimental group was significantly lower than the model group(0.09±0.004 vs 0.128±0.002,P < 0.01),the difference was statistically significant;the expression of the experimental group was slightly higher than that of the normal control group(0.09±0.004 vs 0.075±0.003,P < 0.05),the difference was also statistically significant.6 Effects of macrophage depletion on the expression of MMP-13 proteinThe expression of MMP-13 was mainly located in cytoplasm of positive cells.Normal control group Rarely expressed in the portal area.In the model group,the positive staining was mainly located in the fibrous septum,the central vein and the hepatic sinusoid.The experimental group was mainly expressed in the portal area.The results of immunohistochemistry showed that the expression level of MMP-13 in model group(0.154±0.016)was higher than that in normal group(0.089±0.003)and experimental group(0.108±0.003),the differences were statistically significant(P < 0.01);there was no significant difference in the expression of MMP-13 between the experimental group and the control group(P > 0.05).Conclusion:1 Depletion of macrophages in the stage of liver fibrosis recovery,the expression of ?-SMA was up-regulated,which indicated that the activation of HSC was increased and the degree of liver fibrosis was increased.2 Depletion of macrophages in the stage of liver fibrosis recovery,up regulation of PDGFB and proinflammatory cytokines TNF-? expression,down regulation of IL-10 expression.3 Depletion of macrophages in the stage of liver fibrosis recovery,the expression of MMP-13 was decreased,the ECM degradation was blocked and recovery of liver fibrosis delayed. |
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