Intrahepatic Levels Of CXCL5,CXCL8,CXCL13,CXCL16,CX₃CL1 In Healthy Individuals And Patients With Hepatic Fibosis And Cirrhosis

Background and ObjectiveThe end-stage liver diseases which are usually caused by the hepatitis B virus threaten human health seriously. The damaged liver cells can not do normal function properly. Orthotopic liver transplantation is the most effective treatmeat for end-stage liver diseases. However, extensive application of orthotopic liver transplantation is restricted severely in clinic work. Scientists try their best to find a replacement for the treatment of liver transplantation, so that the damaged liver cells to self-repair and regenerate, to restore its normal function.The exact mechanism of liver regeneration has not yet been clear, focus of the study is to find the factors that start-up and regulate regeneration. Such factors may be of some chemical substances or biological factors which can cause or promote the proliferation of residual liver cells; may also be a decline in the concentration of some inhibitory factors start-up the division of liver cell.In recent years, the application of bone marrow-derived liver stem cells (BMDLSC) to treat end-stage liver diseases is the research focus of scholars at home and abroad, and also shows a certain application prospects. BMDLSC can resume the number of liver cells and repair the organizational structure, so that the liver can regenerate.BMDLSC settled in the bone marrow in physiological state, the mechanism of its homing to liver is not clear. In theory, the mechanism should be similar with white blood cells to the inflammatory lesions. In other words, chemokines and their receptors should play an important role in the process of BMDLSC homing to the liver.So far, all discovered chemokines can be divided into four sub-tribes (C-, CC-, CXC-, and CX3C-) according to the location and number of their N terminal cysteine residues. CXC-chemokine subfamily includes CXCL1-CXCL16 total of 16 members; Fractalkine is only one member of CX3C chemokine subfamily, the systematic name is CX3CL1. Some chemokines and their receptors play a role in the process of directional migration of bone marrow mesenchymal stem cells (BMMSC), these studies have been made by many scholars at home and abroad. Currently, many studies suggest that stromal cell-derived factor-1 (SDF-1) can attact CXCR4 positive BMMSC. However, we know little about the repair role of other chemokines in liver diseases, especially how they attact BMMSC migrate to the damaged liver. Throughout the existing reports, we deserve to pay more attention to the role of some chemokines, such as CXCL5, CXCL8, CXCL13, CXCL16 and Fractalkine, because these chemokines could drive BMMSC directional migration with the increased chemokines concentration in vitro experiments. However, in human body, whether these chemokines express in the liver tissue or not? How these chemokines express in different pathological states of liver? What role can these chemokines play in the process of BMDLSC migrate to the damaged liver? We do not know these issues, and there are almost no reports in this area. In this study, we investigate the intrahepatic levels of chemokine CXCL5, CXCL8, CXCL13, CXCL16 and Fractalkine in healthy individuals and patients with hepatic fibrosis and cirrhosis. To provide a theoretical basis for these chemokines promote BMDLSC migration to the liver to repair damaged liver from a new perspective.Materials and Methods1.Study objectThere were 30 cases in the study,17 cases were male and 13 female, with a mean age of 50 (range:19 to 77). Liver tissues were obtained in surgery from the patients who were treated in Nanfan Hospital from May.2008 to May.2009. The specimens were divided into two parts immediately after the liver tissue were cut down. One was formalin-fixed and done pathological examination, the other part was put into 1.5 mL EP tube and saved in-80℃refrigerator.2. Specimens were divided into three groups①Normal control group:The donors must be healthy with normal liver function, negative of hepatitis B surface antigen and HCV antibody. The most important thing was that their liver tissues were normal.②Liver fibrosis group:All of the patients the samples from had history of hepatitis B, positive of hepatitis B surface antigen, HCV antibody negative, fibrotic liver structure but no "false lobule" that were confirmed by pathological examination.③Group of cirrhosis:All of the patients the samples from had history of hepatitis B, positive of hepatitis B surface antigen, HCV antibody negative. Their liver tissues were abnormal remarkably, with increased intrahepatic tissue fibrosis, damaged structure of hepatic lobules and formed "false lobules" and regenerative nodules.3. Relevant indicators3.1 Symptoms and clinical signsDo physical examination to confirm whether the patients with typical symptoms and signs or not, such as jaundice, liver palms and spider nevi, ascites, hepatic encephalopathy. Then, we appraised the severity of these symptoms and signs.3.2 B-ultrasound and CT examination.3.3 Biochemical and Immunological testsThe patients must be accepted serological test of Hepatitis B and hepatitis C after admission. And it is necessary to test the value of ALT, AST, ALB, TBIL and PT in venous blood of patients in the day before operation.3.4 Pathological examination for tissueLiver tissues were observed by optical microscopy, after fixed in 10% formaldehyde, paraffin-embedded, dehydrated and finally stained by hematoxylin-eosin (HE).4. Detection of chemokine CXCL5, CXCL8, CXCL13, CXCL16, and Fractalkine content in liver tissues4.1 Homogenate and CentrifugalizationWe grinded the liver tissues to homogenate, and then diluted the homogenate with a certain proportion of PBS buffer. The homogenate was centrifugal at 4℃, 1000·g and 10 minutes, then the other homogenate was centrifugal at 4℃,25000rpm and 20 minutes. Supernatant was obtained to assayed by ELISA.4.2 The content of chemokines were assayed by ELISAWe used the way of double-antibody sandwich ELISA. Put standard and sample into micro-ELISA-plate for testing, incubate chemokines and biotin-labeled antibodies at the same time, after washing, add HRP, after another incubation and washing to remove unbound enzyme conjugate, and then add substrate A and B, react with enzyme conjugate, resulting in colors. Read the OD value of each hole at a wavelength of 450nm in microplate reader. Curve estimation was made by SPSS statistical software acoording to concentration and the OD values of standard. Finally calculate the centrifugal fluid specimens chemokine concentration.5. Statistical AnalysisThe final data are represented by x±S. The significance test of mean between multiple groups was used the One-way ANOVA and LSD-t ways by SPSS 13.0 statistical analysis software. But we used the Welch's approximate ANOVA and Dunnett's T3 to test the data when the equal variances were not assumed. We adopted Pearson Correlation or Spearman Correlation to analyze the relevance between the concentration of CXCL5, CXCL8, CXCL13, CXCL16, and Fractalkine in liver and liver function indicators and used Curve Estimation to compute standard curve equation. We usedχ2 test to test the data in R-C tables. Theα's value is equal to 0.05 in all statistical analysis.Results1. Intrahepatic levels of CXCL5, CXCL8, CXCL13, CXCL16, and FractalkineThe intrahepatic levels of CXCL5 in patients with hepatic cirrhosis were remarkably higher than that in healthy individuals and hepatic fibrosis patients (17.05±4.78 ng/g vs 0.80±0.69 ng/g and 2.03±2.04 ng/g, respectively; both P< 0.05). No significant difference was noted in intrahepatic level of CXCL5 between healthy individuals and hepatic fibrosis patients.The intrahepatic levels of CXCL8 in patients with hepatic fibrosis and cirrhosis were remarkably higher than that in healthy individuals (11.62±3.52 ng/g and 12.33±3.89 ng/g vs 6.24±3.68 ng/g, respectively; both P< 0.05). No significant difference was noted in intrahepatic level of CXCL8 between patients with hepatic fibrosis and cirrhosis.The intrahepatic levels of CXCL13 in patients with hepatic cirrhosis were remarkably higher than that in healthy individuals and hepatic fibrosis patients (12.10±10.38 ng/g vs 2.34±2.05 ng/g and 6.04±2.97 ng/g, respectively; both P< 0.05). No significant difference was noted in intrahepatic level of CXCL13 between healthy individuals and hepatic fibrosis patients.The intrahepatic levels of CXCL16 in patients with hepatic fibrosis and cirrhosis were remarkably higher than that in healthy individuals (3.34±1.81 ng/g and 3.49±1.90 ng/g vs 1.32±0.91 ng/g, respectively; both P< 0.05). No significant difference was noted in intrahepatic level of CXCL16 between patients with hepatic fibrosis and cirrhosis.The intrahepatic levels of Fractalkine in patients with hepatic fibrosis and cirrhosis were remarkably higher than that in healthy individuals (13.72±5.59 ng/g and 14.70±3.52 ng/g vs 4.84±3.72 ng/g, respectively; both P< 0.05). No significant difference was noted in intrahepatic level of Fractalkine between patients with hepatic fibrosis and cirrhosis.2. The Correlation Analysis between chemokine levels with liver function indicatorsCXCL5 with ALT, AST, PT were significantly positively correlated; CXCL8 with ALT, AST, TBIL, PT were significantly positive correlation; CXCL13 with ALT, AST were significantly positive correlation; CXCL16 with ALB was significantly negatively correlated; Fractalkine with ALB was significantly negatively correlated, but with ALT, AST, TBIL were significantly positive correlation.Conclusion1. Chemokine CXCL5, CXCL8, CXCL13, CXCL16 and Fractalkine are expressed in normal liver tissue.2. With the aggravation of hepatic fibrosis, the contents of CXCL5, CXCL8, CXCL13, CXCL16 and Fractalkine are increased with different pattents. Intrahepatic levels of CXCL5 and CXCL13 began to rise in the stage of liver fibrosis and had significant increase in cirrhosis stage. Intrahepatic level of CXCL8, CXCL16 and Fractalkine had already increased to significant level in the stage of liver fibrosis, and it remianed in high state in liver cirrhosis stage. These data suggest that CXCL5, CXCL8, CXCL13, CXCL16 and Fractalkine are likely to play a role in BMMSC migration to the liver.3. Intrahepatic levels of chemokine CXCL5, CXCL8, CXCL13, CXCL16 and Fractalkine are significantly correlated with some liver function indicators, but the correlation coefficients are lower than 0.7. The changes of CXCL5, CXCL8, CXCL13, CXCL16 and Fractalkine are related with the injury of liver, but the levels of these chemokines are not correspond with the degree of the injury of liver.