| Objective: Detection the expression of HLA-DR and TLR4on peripheralblood monocytes in patients with HBV-related liver cirrhosis and therelationship with its clinical features and prognosis, and further explore thepotential mechanism. To investigate the efficacy of prophylactic antibioticstherapy in HBV-related liver cirrhosis patients, and detect monocytephenotype, the expression level of HLA-DR and TLR4before and aftertreatment, which can further our understanding the mechanism of posthepatitisliver cirrhosis and also suggest a effective therapeutic role of prophylacticantibiotics for HBV-related liver cirrhosis patients with potential clinicalapplications.Methods:1Detection of peripheral blood monocyte phenotype:54cases of HBV-relatedliver cirrhosis were selected randomly, and divided into3groups according toChild-Pugh.12cases of healthy people were chosen as control group. Wholeblood cells were seperated and then stained with fluorescent labelinganti-CD14, anti-CD16, anti-HLA-DR, and anti-TLR4monoclonal antibodies.All samples were measured by three-color flow cytometric analyses usingFACSCalibur and CELLQuest software (Becton Dickinson, San Jose, CA).2Detection of plasma LPS levels: The plasma LPS levels of patients andhealthy donors were testing by tachypleus amebocyte lysate.3Study objectsPlasma samples were obtained from54HBV infected patients hospitalized inBethune International Peace Hospital and12gender-and age-matched healthydonors. Clinical indexes such as ALT, AST, TBIL and ALB were analyzed byautomatic biochemical analyzer. The characteristics of the patients are shown in Table1. None of the patients received anti-HBV agents or steroids in6months before sampling. This study was carried out in accordance with theWorld Medical Association Helsinki Declaration and was approved by theEthics Committee of Bethune International Peace Hospital and theInstitutional Review Board of Institute of Biophysics, Chinese Academy ofSciences. Informed consent was obtained from all patients.Results:1The frequencies of monocytes and CD16~+monocytes in patients withHBV-related liver cirrhosis, the expression of HLA-DR and TLR4onperipheral blood monocytes, and the relationship with clinical features.1.1The frequencies of peripheral blood monocytes in patients with livercirrhosis: The frequencies of peripheral blood monocytes for healthy controls,patients with liver cirrhosis of Child A, Child B, Child C were (6.78±2.47)%,(9.08±3.45)%,(12.93±2.05)%,(16.51±6.01)%, respectively. The frequenciesof peripheral blood monocytes in the CHB patients were significantly higherthan that in the HCs (P<0.05), However, no significant correlation wasobserved between the frequencies of monocytes and clinical features in ChildA and Child B liver cirrhosis groups (P>0.05). but positive correlations wereobserved between the frequency of monocytes and AST, TBIL in Child C livercirrhosis group (P<0.05).1.2The frequencies of peripheral blood CD16~+monocytes in patients withliver cirrhosis: The frequencies of peripheral blood CD16~+monocytes forhealthy controls, patients with liver cirrhosis of Child A, Child B, Child Cwere (10.13±4.69)%,(12.63±5.07)%,(16.08±5.47)%,(23.86±8.11)%,respectively. There were statistically significant (P<0.05) within three livercirrhosis groups. The frequencies of peripheral blood CD16~+monocytes in thepatients Child B and Child C were significantly higher than that in the HCs(P<0.05), A positive correlation was observed between the frequency ofCD16~+monocyte in Child A liver cirrhosis group and ALT, between thefrequency of CD16~+monocyte in Child A liver cirrhosis group and AST(P<0.05), but no significant correlation was observed between the frequencies of CD16~+monocyte in Child B and Child C liver cirrhosis groups and clinicalfeatures (P>0.05).1.3The expression of peripheral blood monocytes HLA-DR in patients withliver cirrhosis: The expression of peripheral blood monocytes HLA-DR forhealthy controls, patients with liver cirrhosis of Child A, Child B, Child Cwere (84.35±6.86)%,(88.90±6.24)%,(85.47±7.54)%,(66.38±13.43)%,respectively. There were no statistically significant (P>0.05) difference amongall the tested groups. Compared with the other groups, the level of bloodmonocytes HLA-DR was significant lower in patients with Child C livercirrhosis (P<0.05).The level of HLA-DR in patients with Child A liver cirrhosis was found to benegatively correlated with the level of ALB (P<0.05). While the level ofHLA-DR in patients with Child C liver cirrhosis was found to be positivelycorrelated with the level of ALB (P<0.05). As for the level of ALT, AST andTBIL, no significant correlation was observed in HLA-DR (P>0.05).1.4The mean fluorescence intensity (MFI) of peripheral blood monocytesTLR4expression in patients with liver cirrhosis: The mean fluorescenceintensity (MFI) of peripheral blood monocytes TLR4expression for healthycontrols, patients with liver cirrhosis of Child A, Child B, Child C were(6.03±1.85),(5.55±2.13),(4.01±2.18),(2.92±1.69), respectively. There werestatistically significant (P<0.05) between the groups of Child B and Child Aliver cirrhosis. There were statistically significant (P<0.05) between thegroups of Child C and Child A liver cirrhosis. The MFI of peripheral bloodmonocytes TLR4expression was no significant correlation with clinicalfeatures (P>0.05).2The LPS levels in plasma of patients with liver cirrhosis and the relationshipwith the expression of HLA-DR and TLR4in peripheral blood monocyte.The LPS levels in plasma for healthy controls, patients with liver cirrhosis ofChild A, Child B, Child C were (3.70±2.15)pg/ml,(65.15±65.10)pg/ml,(143.37±149.16)pg/ml,(403.54±347.11)pg/ml, respectively. There werestatistically significant among four groups (P<0.05). No significant correlation was found between the LPS levels and the expression of HLA-DR and TLR4in patients with Child A and Child C liver cirrhosis (P>0.05). The LPS levelswas negatively correlated with TLR4expression, but not associated with theexpression of HLA-DR in plasma of Child B liver cirrhosis group (P<0.05).3The frequencies, phenotype of monocytes and the expression of HLA-DRand TLR4changes before and after antibiotic treatment.3.1The frequencies of monocytes in patients with Child B liver cirrhosisbefore and after antibiotic treatment were (12.49±1.56)%,(11.69±2.14)%,respectively. The frequencies of monocytes in patients with Child C livercirrhosis before and after antibiotic treatment were (12.56±2.25)%,(13.01±1.52)%, respectively. No significant associations of monocytes frequencieswere observed between Child B and Child C groups before and after antibiotictreatment.3.2The frequencies of CD16~+monocytes in patients with Child B livercirrhosis before and after antibiotic treatment were (14.76±5.61)%,(12.81±3.76)%, respectively. The frequencies of CD16~+monocytes in patients withChild C liver cirrhosis before and after antibiotic treatment were (18.58±6.50)%,(16.02±3.61)%, respectively. After treatment, the frequencies ofmonocytes were significantly lower in patients with Child B liver cirrhosis(P<0.05). But there was no statistically significant difference in Child Cpatients before and after treatment (P>0.05).3.3The expression of HLA-DR in peripheral blood monocytes in patientswith Child B liver cirrhosis before and after antibiotic treatment were (85.78±7.19)%,(86.37±5.25)%, respectively. The expression of HLA-DR inperipheral blood monocytes in patients with Child C liver cirrhosis before andafter antibiotic treatment were (71.59±14.10)%,(78.40±11.27)%,respectively. After treatment, the level of HLA-DR was significantly higher inpatients with Child B liver cirrhosis (P<0.05). But there was no statisticallysignificant difference in Child C patients before and after treatment (P>0.05).3.4The MFI of TLR4expression in peripheral blood monocytes in patientswith Child B liver cirrhosis before and after antibiotic treatment were (3.61± 1.72),(4.03±1.68), respectively. The MFI of TLR4expression in peripheralblood monocytes in patients with Child C liver cirrhosis before and afterantibiotic treatment were (4.66±1.48),(4.93±1.03), respectively. Theexpression of TLR4in patients with Child B before and after treatment werestatistically significant different (P<0.05). But there was no statisticallysignificant difference in Child C patients before and after treatment (P>0.05).Conclusions:1During the process of HBV-ralated liver cirrhosis, the frequencies of bothperipheral blood monocytes and CD16~+monocytes were markedly increasedin patients, compared with healthy controls. While the expression of HLA-DRwas found to increase at the initial stage of liver cirrhosis, but decrease in asignificant manner at the late-stage. As for the expression of TLR4, itcontinued to decline accompany with disease progression, and its changes isassociated with the severity and prognosis of disease.2Oral antibiotic by reducing endotoxin levels can decrease the frequency ofCD16~+monocytes, increase the expression of HLA-DR and TLR4, whichin part regulates the function of monocytes and improve the prognosis ofpatients with cirrhosis. |
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