| Objective: There are90million carriers of hepatitis B surface antigen inchina. This is the main cause of chronic hepatitis B(CHB) progresses to liverfibrosis, cirrhosis and hepatocellular carcinoma. The excessive accumulationof the extracellular matrix, which was produced by activated hepatic stellatecells (HSCs) and myofibroblas, is main pathological process of the liverfibrosis. Endocannabinoids are lipid signal molecules, its effect mainlythrough by two specific receptors Cannabinoid receptor1(CB1) andcannabinoid receptor2(CB2). A significant upregulation of CB1and CB2wascertified in mice models of liver damage induced by carbon tetrachloride(CCl4) or bile duct ligation. It was known that upregulated CB1promoted theoccurrence and development of the process of fibrosis in fatty liver andnonalcoholic fatty liver disease (NAFLD). But the studies about expressionand meaning of CB1in HSCs from liver fibrosis induced by hepatitis B wereseldom.Adipocyte Fatty Acid-Binding Protein, known as Adipocyte FABP,A-FABP or FABP4, is one of the9types of fatty acid binding protein atpresent. FABP4was discovered in adipocyte and macrophages at first, it wasnot only involves in lipid metabolism but also takes part in inflammatory andimmunoreaction. It is known that hepatocyte express FABP1, but there is littlestudy on FABP4in liver. The study about FABP4in liver disease has not beenreported.This study was divided into two sections: First,80biopsy specimenswere selected from liver fibrosis tissue induced by chronic hepatitis B. theexpression of alpha SMA, CB1, CB2and CD68in liver fibrosis was detectedby immunohistochemistry, quantitative analysis of them was measured.Second,80biopsy specimens cases were all liver biopsy specimens from all kinds of liver disease, the expression of FABP4, CB1and CD68in liver tissuewere detected by immunohistochemistry, quantitative analysis of theexpression of FABP4and investigate the relationship between CB1andFABP4, aimed to provide a evidence for the treatment of various liver diseasecaused by hepatitis B.Materials and Methods：1Distribution and quantitative analysis of α-SMA, CB1and CB2andCD68in liver fibrosis from CHB.A total of80samples (20samples per stage) with CHB-related liverfibrosis from liver biopsy were selected to detect the expression ofalpha-smooth muscle actin (α-SMA), cannabinoid receptor1(CB1),cannabinoid receptor2(CB2) and CD68by immunohistochemistry. Positivecells of α-SMA, CB1and CB2in hepatic lobule were counted. Coexpressionof α-SMA and CB1was identified by means of double immunofluorescencestaining.2The distribution and quantitative analysis of FABP4, CB1and CD68inliver fibrosis, cirrhosis and HCC caused by hepatitis B.80cases were selected from early liver fibrosis, advanced fibrosis,cirrhosis, hepatocellular carcinoma,20cases in each group. The expression ofFABP4, CB1and CD68in these liver diseases were detected byimmunohistochemistry. Quantitative analysis was measured.Result:1Expression pattern and positive cell count of α-SMA, CB1, CB2andCD68in the liver fibrosis of four stages1.1The expression of α-SMA: Positive cells mainly located in smallvascular smooth muscle of portal area in S1, occasionally in hepatic lobule; S2,faintly positive cells was observed in hepatic sinusoid besides portal area; S3,positive cells was observed in vascular smooth muscle, hepatic sinusoid,fibrotic septa, focal necrosis and angiogenesis area; the pattern of S3wassimilar to S4. The counts of positive cells in liver lobule were as follow: S1:13.84±5.88; S2:21.16±11.64; S3:26.08±11.29; S4:39.12±17.76. The number was increased significantly (P<0.05) in S2, S3, S4compared with S1. Butthere was no significant difference between S2and S3.1.2The expression of CB1: Positive cells was observed in hepatic sinusoid,fibrotic septa, portal area and focal necrosis, new-formed vessels. The countsof positive cells in liver lobule were as follow: S1:18.00,18.00; S2:25.00,27.00; S3:30.00,27.00; S4:41.00,31.25. The number of positive cells isincreased significantly (P<0.05) in S2, S3, S4compared with S1. But therewas no significant difference between S3and S4.1.3The expression of CB2: Positive cells CB2was mainly located in hepaticsinusoid, fibrotic septa, portal area and focal necrosis. Pattern of each stagewas similar but the number of positive cells was increase with the increase ofstage of fibrosis. The positive cells of each stage in liver lobule were as follow:S1:18.00,9.00; S2:30.00,15.00; S3:34.50,36.50; S4:44.00,19.25. Thenumber of positive cell was increased significantly (P<0.05) in S3, S4compared with S1. But there was no significant difference between S3and S4.1.4The expression of CD68: Positive cells were located in hepatic sinusoidand portal area. The positive cells of each stage in liver lobule were as follow:S1:13.00,13.25; S2:14.50,21.00; S3:35.50,18.50; S4:30.00,15.50. Thenumber of positive cell is increased significantly (P<0.05) in S3, S4comparedwith S1. But there were no significant difference between S1and S2, betweenS3and S4.1.5Correlation analysis between α-SMA and CB1： the expression ofα-SMA and CB1was increased with the increase of stage of fibrosis, α-SMAwas positively correlated with CB1(r=0.880, P<0.05).1.5Double immunofluorescence stainingDouble immunofluorescence staining of CB1and α-SMA: all positivecells of α-SMA was also positive expression of CB1in hepatic sinusoid.Double immunofluorescence staining of CD68and α-SMA: both of themwere positive expression in hepatic sinusoid, but no coexpression wasobserved.2The Distribution, localization and content of FABP4, CB1and CD68in liver fibrosis, cirrhosis and HCC caused by hepatitis B.2.1The expression and quantitative analysis of FABP4: In normal liver, littleexpression was observed endothelium of small artery in the portal area; liverfibrosis, expressed in endothelium of small artery of the portal area, or littlevessels near necrosis area and the edge of hepatic lobule; positive expressionin part of the vascular of mesenchyme of the HCC nests. The IOD of positiveexpression was as follow: normal liver:938.53,1629.23; early liver fibrosis:1484.45,4513.56; advanced liver fibrosis:2721.36,5396.19; liver cirrhosis:11872.68,15844.55; HCC:1472.87,4788.79. FABP4positive expression inliver fibrosis significantly increased (P<0.05), compared with normal livertissue. But no significantly difference between HCC and early liver fibrosis.2.2The expression and quantitative analysis of CB1: positive cells werevisibled in hepatic sinusoid, fibrotic septa, portal area and focal necrosis,newborn capillaries; liver cells whose cytoplasm staining near focal necrosisarea and portal area; few positive cells in mesenchyme near the edge of HCCnests. The number of positive cells: early liver fibrosis:18.00,18.25;advanced liver fibrosis:38.00,30.00; liver cirrhosis:33.30,15.75; HCC:9.00,8.25. Expression differences have statistical significance between earlyfibrosis and other disease (P<0.05). Between advanced hepatic fibrosis andcirrhosis no statistically significant.2.3The expression and quantitative analysis of CD68: positive cells werelocated at liver sinus and portal area at early fibrosis stage and significantlyincreased at advanced fibrosis and cirrhosis. In HCC, scattered among cancercells. The number of positive cells: early liver fibrosis:14.00,17.00; advancedliver fibrosis:30.00,17.75; liver cirrhosis:42.00,10.05; HCC:20.00,16.00.Expression differences have statistical significance between early fibrosis andother disease (P<0.05).Conclusion:1With gradual increasing of stages of hepatic fibrosis, positive cells ofα-SMA and CB1were both significantly increased. Coexpression of α-SMAand CB1in activated hepatic stellate cells (HSCs) was identified by means of double immunofluorescence staining. It is well accepted that activated CB1receptor promotes fibrogenesis. Our results more showed that CB1in HSCsplay an important role in the process of liver fibrosis.2The CB2positive cells are increasing in chronic hepatitis B(CHB)-related liver fibrosis, The meaning of expression and the relationshipbetween CB1and CB2were need further study.3FABP4was expressed in endothelial cells of some microvessels. Theexpression increasing in liver fibrosis, cirrhosis and HCC. There was mostexpression in cirrhosis among these diseases. Compared with advanced liverfibrosis and cirrhosis, the expression of FABP4in HCC was significantlydecreased. The result suggested that the endothelial cells was involved in thepathogenesis of inflammatory and liver fibrosis. |
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