Abstract

Purpose: To investigate the importance of Toll-like receptor 4 (TLR4) expression on hepatocytes obtained from Chronic Hepatitis B patients as well as on hepatocellular carcinoma HepG2 and HepG2.2.15 cell lines.

Methods: Expression of TLR4 in liver tissues was determined by immunohistochemistry in 75 patients with CHB and in10 healthy controls. The protein and mRNA 1eve1s of TLR4 in hepatocellular carcinoma HepG2 and HepG2.2.15 cells were measured by flow cytometry (FCM) and real-time quantitative PCR (RQ-PCR), and endotoxin triggered TNF-α secretion in HepG2 and HepG2.2.15 cells was evaluated by ELISA.

Results: TLR4 expressed mainly in the cytoplasm and some on cell membrane in hepatocytes. The staining scores of TLR4 expression in the liver tissues of patients with CHB were significantly higher than that of healthy controls. The liver tissues from patients with severe CHB expressed higher level of TLR4 than those from patients with mild CHB. Furthermore, the staining scores of TLR4 expression in the liver tissues of patients with CHB were positively correlated with the grading scores. Our results also showed that the mean fluorescence intensity and TNF-α secretion induced by endotoxin as well as the protein and mRNA 1eve1s of TLR4 in HepG2.2.15 cells were all significantly higher than those in HepG2 cells.

Conclusion: TLR4 was up-regulated in the hepatocytes in patients with CHB. This indicates a potentially important interaction between TLR4 expression and the pathogenesis of CHB.

 

 

Mechanisms responsible for chronic hepatitis B remain incompletely understood, although increasing evidence points to immunological rather than direct viral effects playing a central role.^1,2 However, most studies have focused on adaptive immunity, with little research on the innate immunity. One of the key components of the innate immune response is the family of Toll-like receptors(TLRs), evolutionarily conserved pattern recognition receptors. Activation of TLRs triggered by various motifs common to microorganisms, known as pathogen-associated molecular patterns, can promote the inflammatory response, the anti-infectious response and the maturation of antigen presenting cells.^3,4 It is also believed that the innate immunity controls the adaptive immune responsies.⁵ The Toll-like receptor 4 (TLR4) is one of most important TLRs. Activation of TLR4 can cause inflammation by promoting the secretion of inflammatory cytokines such as TNF-α and interleukin-6 through the MyD88 dependent path way, and anti-virus effects by promoting the secretion of interferon β through the MyD88 independent path way.⁴ TLR4 is expressed in hepatocytes and endotoxin can induce the secretion of TNF-α and interleukin-6, ect by hepatocytes.^6-11 TLR4 is up-regulated in peripheral blood monocytes (PBMCs) and hepatocytes in patients with chronic hepatitis C(CHC) and plays an important role in the pathogenesis of CHC.^12,13 Systemic endotoxemia often occurs in patients with chronic viral hepatitis¹⁴ and the human 60 kDa heat shock proteins (hHSP 60) are markedly increased in patients with active viral hepatitis with predominant expression in areas of inflammatory infiltrates¹⁵, while endotoxin and hHSP 60 are the major ligands of TLR4, an important member of TLRs.^16,17 In preliminary studies, we found that TLR4 is up-regulated in the peripheral blood monocytes (PBMCs) in patients with chronic hepatitis B(CHB).¹⁸ However, less detailed data are available about the expression of TLR4 on hepatocytes in patients with CHB. In this study, we analyzed the importance of TLR4 expression in patients with chronic hepatitis B. Our results suggest a potentially important interaction between TLR4 expression and the pathogenesis of CHB.

 

Materials and methods

Patients

The study was approved by the ethic committee and written consent was obtained from all patients and healthy volunteers.

Patients who had been HBsAg and hepatitis B virus (HBV)-DNA positive for at least six months and had elevated blood alanine aminotransferase (ALT) levels were defined as CHB in this study.¹⁹ Patients with hepatitis delta virus, HIV infection, bacteria infection, concomitant diseases such as autoimmune diseases and gastrointestinal, pulmonary, cardiovascular, renal or neurological disorders or with alcohol abuse were excluded. Seventy-five patients with CHB and 10 healthy controls entered the study. The 10 healthy control liver samples were all of routine biopsies from the donor liver used in liver transplantation surgery and were all excluded of hepatitis viruses infections.

 

Blood samples

Blood samples were collected one week before the liver biopsy. Clinical routine items such as serum levels of alanine aminotransferase (ALT), serum total bilirubin(TB), serum HBeAg status and serum HBV-DNA levels were tested according to the regular procedures.

 

Histopathological examination of samples

Percutaneous liver biopsy samples were obtained by using liver biopsy needles after written informed consent was approved. All tissue samples were fixed in 10% buffered formalin solution, embedded in paraffin and stained with haematoxylin–eosin, trichrome and Van Gieson stains for routine histological examination. Each biopsy specimen was evaluated according to the modified grading and staging system described by Scheuer.²⁰ In addition, we classified histological activity of CHB as mild or severe, defining mild CHB as ≤2, and severe CHB as >2.²¹

 

Immunohistochemistry

Standard deparaffinization sections were subjected to microwave oven, immersed in 0.01M citrate buffer pH 6.0 for 20 min and, after washing in PBS, the immunohistochemical procedure was carried out as described as the instruction of the Elivision two step kit (Maxin Biotechnology, China). Polyclonal anti-TLR4 made in rabbit (sc-10741, Santa Cruz Biotechnology, USA) was used in a 1:100 dilution as the primary antibody. DAB-3,3′-diaminobenzidin was used for visulization. PBS instead of the primary antibody served as the negative control. Each section was semiquantitatively evaluated by two independent observers using a Nikon light microscope (Nikon Labophot,, Japan) without the knowledge of either clinical or histological diagnosis. Four distinct items were recorded on separate sheets as follows: 0-no staining; +1-positive staining in <30% of cells per high power field (×250); +2- positive staining in >30% but in <70% of cells per high power field; and +3-positive staining of >70% of cells per high power field, according to a previously validated scoring system. Finally, data were averaged to median values, giving a numerical score for each liver biopsy specimen, and then used for statistical analysis.^21,22

 

Cell culture of HepG2 and HepG2.2.15

HepG2 and HepG2.2.15 were provided by the laboratory of infectious disease, Third Affiliated Hospital, Sun Yat-Sen University. HepG2 was cultured in six-well plates with DMEM medium supplemented with 10% fetal calf serum, 100 U/ml penicillin and l00 μg/ml streptomycin, in a humidified 5% CO₂ incubator at 37^oC. The culture medium for HepG2.2.15 was added 600μg/mL G418 to that for HepG2. Cells were passaged twice weekly and routinely examined for mycoplasma contamination. When the monolayers of cells reached about 90% confluency, then cells were harvested by trypsin EDTA treatment.

 

Flow Cytometry for Determination of TLR4 Expression on HepG2 and HepG2.2.15

A total of 1×10⁶ single cell suspension were incubated with phycoerythrin-conjugated anti-human TLR4 monoantibody (sc-13593,eBioscience, USA) for 30 min in the dark and washed twice with PBS. Cells incubated with phycoerythrin-conjugated isotype mouse IgG2a were used as negative controls. Analysis was performed on a FACS Calibur flow cytometry (Becton Dickinson, USA). A minimum of 10,000 cells were analyzed in all specimens. The mean fluorescence intensity and the percentage of positive cells were determined using the CellQuest software program (Becton Dickinson,USA) and expressed as the results.

 

Real-Time RT-PCR for Determination of mRNA level of TLR4 in HepG2 and HepG2.2.15

Oligonucleotide primers were designed and synthesized by Takara (Japan) as follows: 5′-AGGATGATGCCAGGATGATGTC-3′and 5′-TCAGGTCCAGGTTCTTGGTTGAG-3′(198bp) for TLR4, 5′-TGG CAC CCA GCA CAA TGA A-3′and 5′-CTA AGT CAT AGT CCG CCT AGA AGC A-3′(186bp) for β-actin. The plasmid pBluescript-HBV (constructed by the laboratory of infectious disease, Third affiliated hospital, Sun yat-sen university) coding hepatitis B virus DNA was used to make standard curves. Total RNA was isolated from the cells by Trizol（Invitrogen, USA. One step real-time RT-PCR was performed on ABI Prism 7000 Sequence Detection System (Applied Biosystems, Foster City, Calif. USA), using the SuperScript™ III Platinum^® SYBR^® Green One-Step qRT-PCR Kit (Takara, Japan). The 50µL reaction mixture contained: 4μl forward primer(0.2μM), 4μl reverse primer(0.2μM) and 4μl total RNA or plasmid pBluescript-HBV of different dilutions. The PCR schedule consisted of the following stages: 15 minutes at 42 ^oC for 1 cycle, 2 minutes at 95 ^oC for 1 cycle, 5 seconds at 95 ^oC and then 31 seconds at 60 ^oC for a total 40 cycles, 15 seconds at 95 ^oC, 31 seconds at 60 ^oC and then 15 seconds at 95 ^oC for 1cycle. The ratio between the mRNA level of TLR4 to β-actin was termed relative TLR4 mRNA level and used for quantitative evaluation.

 

ELISA for Determination of TNF-α Secreted by HepG2 and HepG2.2.15

HepG2 and HepG2.2.15 cells were seeded at a density of 2.5×10⁴ cells/cm². All cells were seeded at the same time. Their media were changed every 24 h. Cells were seeded and, on the third change of medium (72 hr after seed), media with or without 1μg/ml endotoxin from Salmonella typhimurium (Sigma, St. Louis, MO) were added.²³ After 24 hr, the medium was collected and centrifuged during 5 min at 30000 rpm, to immediately evaluation of TNF-α. TNF-α was determined by the quantikine human TNF-alpha immunoassay (R&D Systems). The TNF-α secretion ability of HepG2 and HepG2.2.15 cells was presented as the ratio of the TNF-α concentration in the culture supernatant of endotoxin stimulated cells to that of unstimulated cells as control.

 

Statistical Analysis

Statistical analyses were performed using the Mann-Whitney rank sum test, student-t test and Spearman’s rank correlation test as appropriate (SPSS for Windows, version 11.0; SPSS Inc, USA). P < 0.05 was considered significant.

 

Results

There were no differences in age or sex distribution between the patient and control groups. Demographic parameters and laboratory findings (ALT, serum total bilirubin, HBeAg status and lg[serum HBV-DNA] ) of the patients are shown in Table 1.

Few TLR4-positive hepatocytes could be traced in the liver tissues of normal controls whereas, they existed discreetly or diffusely in the liver tissues of the patients with CHB. Positive staining of TLR4 was located mainly in the cytoplasm with some on the cell membrane of hepatocytes. Other cells in the liver were not stained (Figure 1). TLR4 expression scores in CHB liver tissue were higher than that of healthy controls (P<0.001, table 2). Staining scores of TLR4 expression in the severe CHB group were much higher than in the mild CHB group (P<0. 0.001,Table 2). There were positive correlations between staining scores of TLR4 expression and the grading scores in CHB (γ=0.579, P<0.001). There were no correlations between the staining scores of TLR4 expression and the levels of serum ALT, lg[serum HBV-DNA] and the staging scores. No difference was found between the HBeAg positive group and the HBeAg negative group for expression of TLR4 in hepatocytes. The level of serum ALT did not correlate with the histopathological grading or staging score.

The mean fluorescence intensity of TLR4, percentage of TLR4 positive cells and relative TLR4 mRNA 1eve1 in HepG2.2.15 were all higher than those in HepG2 (Table 3). After stimulation with endotoxin for 24 hr, secretion of TNF-α by HepG2.2.15 increased much more than that by HepG2 (3.3±0.2 vs 2.6±0.3, n=18, P<0.001).

 

Discussion

The expression of both mRNA and protein levels of TLR4 were much higher in HepG2.2.15 cells than in HepG2 cells and that secretion of TNF-α by HepG2.2.15 stimulated by endotoxin increased much more than by HepG2. Also, levels of TLR4 in CHB patients were higher than in healthy controls, and TLR4 expression was much more remarkable in the severe than in the mild CHB group. There was a positive correlation between the staining scores of TLR4 expression and the grading scores in CHB. However, there was no correlation between the staining scores of TLR4 expression and the levels of serum ALT and or between scores of TLR4 and levels lg[serum HBV-DNA].

TLR4, barely detected in the normal hepatocytes, can be up-regulated by NS5A protein of hepatitis c virus and beta-glucan of Aspergillus fumigatus, etc.^24,25 Our results agree with those of Mozer-Lisewska¹³, but differ from those reports that HBV virus has no effect on the expression of TLR4 on hepatocyts²⁶ We found that HBV virus could up-regulate the expression of TLR4 in hepatocyts.

These results may be explained as follows:

1. TLR4 exists not only on the cell membrane but also in the cytoplasm.^27,28 The level of TLR4 on the cell membrane can be altered by exocytosis and endocytosis. Visvanathan studied only the expression of TLR4 on cell membrane of hepatocytes in patients with CHB, while we studied the total expression of TLR4 in both cell membrane and cytoplasma.

2. Visvanathan reported that the change of expression of TLR4 on the cell membrane of HepG2 was caused by HBV by acute transduction. This should be a model of acute HBV infection, while HepG2.2.215 is a model of persistent stable chronic HBV infection of HepG2. The differences may indicate that HBV has different effects on the expression of TLR4 on hepatocytes during the course of chronic and acute infection. We found that there was no correlation between the staining scores of TLR4 expression and the levels of serum ALT. This agreed with the observation that there was no correlation between histopathological grading scores and levels of serum ALT. In fact, the levels of serum ALT may often not reflect the severity of CHB.²⁹

HepG2.2.15 is a cell line of HepG2 with stable chronic HBV infection. We concluded that up-regulation of TLR4 expression in HepG2.2.15 cells might be caused by HBV itself. Systemic endotoxemia often occurs in patients with chronic viral hepatitis¹⁶ and human 60 kDa heat shock proteins (hHSP 60) are increased in patients with active viral hepatitis with predominant expression in areas of inflammatory infiltrates.¹⁷ Both endotoxin and hHSP 60 are major ligands of TLR4. ^14,15 Elevated expression of TLR4 increases sensitivity to the ligands and leads to enhanced production of pro-inflammation cytokines.^24,25 We found that secretion of TNF-α by HepG2.2.15 stimulated by endotoxin increased more than that by HepG2. Thus, hepatocytes could produce high levels of TNF-α in chronic hepatitis. Also, TNF-α plays an important role in the pathogenesis of CHB.^30,31 In this study, there was a positive correlation between the staining scores of TLR4 expression and the grading scores in CHB. Taken together, the elevated TLR4 expression in hepatocytes may play a role in the pathogenesis of CHB. In animal models, activation of TLR4 caused inhibition of replication of HBV.^32,33 However, there was no correlation between the scores of TLR4 and the levels of lg[serum HBV-DNA] in this study. There may be no role of elevated expression of TLR4 in hepatocytes for inhibition of replication of HBV in CHB. Activation of TLR4 can cause liver injury in several pathological processes such as hemorrhagic shock, hepatic ischemia/reperfusion injury, alcohol-induced liver injury and hepatic failure.^33-37 Inhibition of TLR4 with a TLR4 antagonist or recombinant soluble forms of extracellular TLR4 domain and MD-2 can attenuate myocardial ischemia-reperfusion injury, dampen lipopoly-saccharide-induced pulmonary inflammation in mice and have anti-inflammatory effects in murine models of inflammatory bowel disease.^38-40 Our finding of elevated expression of TLR4 in hepatocytes may have a role in the pathogenesis of CHB while little effect on the inhibition of replication of HBV, TLR4 antagonist may be helpful in the treatment of CHB.

In conclusion, we have demonstrated for the first time that TLR4 expression may play an important role in the pathogenesis of CHB. This indicates that TLR4 may be a useful therapeutic target for the treatment of CHB.

 

References

1.   Nishimura T, Ohta A. A critical role for antigen-specific Th1 cells in acute liver injury in mice. J Immunol, 1999,162:6503-9.

2.   Ichiki Y, He XS, Shimoda S, et al. T cell immunity in hepatitis B and hepatitis C virus infection: implications for autoimmunity. Autoimmun Rev 2005;4:82-95.

3.   Medzhitov R. Toll-like receptors and innate immunity. Nat Rev Immunol 2001;1:135–45.

4.   Andreakos E, Foxwell B, Feldmann M. Is targeting Toll-like receptors and their signaling pathway a useful therapeutic approach to modulating cytokine-driven inflammation? Immunol Rev 2004;202:250-65.

5.   Iwasaki A, Medzhitov R. Toll-like receptor control of the adaptive immune responsies. Nat Imunnol 2004;5:987-95.

6.   Migita K, Abiru S, Nakamura M, et al. Lipopolysaccharide signaling induces serum amyloid A (SAA) synthesis in human hepatocytes in vitro. FEBS Lett 2004;569:235-9.

7.   Kobayashi N, Takesue M, Kobayashi N, et al. Evaluation of toll-like receptor 4 gene expression of immortalized human liver cell lines. Transplant Proc 2003;35:431-2.

8.   Liu S, Gallo DJ, Green AM, et al. Role of toll-like receptors in changes in gene expression and NF-kappa B activation in mouse hepatocytes stimulated with lipopolysaccharide. Infect Immun 2002;70:3433-42.

9.   Vodovotz Y, Liu S, McCloskey C, et al. The hepatocyte as a microbial product-responsive cell. J Endotoxin Res 2001;7:365-73.

10.Panesar N, Tolman K, Mazuski JE. Endotoxin stimulates hepatocyte interleukin-6 production. J Surg Res 1999;85:251-28.

11.Saad B, Frei K, Scholl FA, et al. Hepatocyte-derived interleukin-6 and tumor-necrosis factor alpha mediate the lipopolysaccharide-induced acute-phase response and nitric oxide release by cultured rat hepatocytes. Eur J Biochem 1995;229:349-55.

12.Riordan SM, Skinner NA, Kurtovic J, et al. Toll-like receptor expression in chronic hepatitis C: correlation with pro-inflammatory cytokine levels and liver injury. Inflamm Res 2006;55:279-85.

13.Mozer-Lisewska I, Sluzewski W, Kackzmarek M, et al. Tissue localization of Toll-like receptors in biopsy specimens of liver from children infected with hepatitis C virus. Scand J Immunol 2005;62:407-12.

14.Ohashi K, Burkart V, Flohé S, et al. Cutting edge: heat shock protein 60 is a putative endogenous ligand of the toll-like receptor-4 complex. J Immunol 2000;164:558-61.

15.de Graaf R, Kloppenburg G, Kitslaar PJ, et al. Human heat shock protein 60 stimulates vascular smooth muscle cell proliferation through Toll-like receptors 2 and 4. Microbes Infect 2006;8:1859-65.

16.Sozinov AS. Systemic endotoxemia during chronic viral hepatitis. Bull Exp Biol Med 2002;133:153-5.

17.Lohse AW, Dienes HP, Herkel J, et al. Expression of the 60 kDa heat shock protein in normal and inflamed liver. J Hepatol 1993;19:159-66.

18.Wei XQ, Wen ZF, Zheng FP, et al. Changes of Toll-like receptor (TLR) 2 and TLR4 on the peripheral blood mononuclear cells in patients with chronic hepatitis B and chronic severe hepatitis B. Zhonghua Gan Zang Bing Za Zhi 2007;15:354-7.

19.Society of Infectious Diseases, Parasitology and Hepatology, Chinese Medical Association. The project of prevention and treatment for viral hepatitis. Zhonghua Gan Zang Bing Za Zhi 2000;8: 342-29.

20.Scheuer PJ. Classification of chronic viral hepatitis: A need for reassessment. J Hepatol 1991;13:372–4.

21.Apolinario A, Majano PL, Alvarez-Pérez E, et al. Increased expression of T cell chemokines and their receptors in chronic hepatitis C: relationship with the histological activity of liver disease. Am J Gastroenterol 2002;97:2861-70.

22.García-Monzón C, Martín-Pérez E, Iacono OL, et al. Characterization of pathogenic and prognostic factors of nonalcoholic steatohepatitis associated with obesity. J Hepatol 2000;33:716–24.

23.Gutiérrez-Ruiz MC, Quiroz SC, Souza V, et al. Cytokines, growth factors, and oxidative stress in HepG2 cells treated with ethanol, acetaldehyde, and LPS. Toxicology 1999;134:197-207.

24.Machida K, Cheng KT, Sung VM, et al. Hepatitis C virus induces toll-like receptor 4 expression, leading to enhanced production of beta interferon and interleukin-6. J Virol 2006;80:866-74.

25.Wright MS, Clausen HK and Abrahamsen TG. Liver cells respond to Aspergillus fumigatus with an increase in C3 secretion and C3 gene expression as well as an expression increase in TLR2 and TLR4. Immunol Lett 2004;95:25-30.

26.Visvanathan K, Skinner NA, Thompson AJ, et al. Regulation of Toll-like receptor-2 expression in chronic hepatitis B by the precore protein. Hepatology 2007;45:102-10.

27.Powers KA, Szászi K, Khadaroo RG, et al. Oxidative stress generated by hemorrhagic shock recruits Toll-like receptor 4 to the plasma membrane in macrophages. J Exp Med 2006;203:1951-61.

28.Hassan F, Islam S, Tumurkhuu G, et al. Intracellular expression of toll-like receptor 4 in neuroblastoma cells and their unresponsiveness to lipopolysaccharide. BMC Cancer 2006;6:281.

29.Zavaglia C, Mondazzi L, Maggi G, et al. Are alanine aminotransferase, hepatitis B virus DNA or IgM antibody to hepatitis B core antigen serum levels predictors of histological grading in chronic hepatitis B? Liver 1997;17:83-7.

30.Ding W, Yin X. Dissection of the multiple mechanisms of TNF-α induced apoptosis in liver injury. J Cell Mol Med 2004;8:445-54.

31.Wang JY, Wang XL, Liu P. Detection of serum TNF-alpha,IFN-beta,IL-6 and IL-8 in patients with hepatitis B. World J Gastroenterol 1999;5:38-40.

32.Isogawa M, Robek MD, Furuichi Y, et al. Toll-like receptor signaling inhibits hepatitis B virus replication in vivo. J Virol 2005;79:7269-72.

33.Prince JM, Levy RM, Yang R, et al. Toll-like receptor-4 signaling mediates hepatic injury and systemic inflammation in hemorrhagic shock. J Am Coll Surg 2006;202:407-17.

34.Wang H, Li ZY, Wu HS, et al. Endogenous danger signals trigger hepatic ischemia/reperfusion injury through toll-like receptor 4/nuclear factor-kappa B pathway. Chin Med J (Engl) 2007;120:509-14.

35.Uesugi T, Froh M, Arteel GE, et al. Toll-like receptor 4 is involved in the mechanism of early alcohol-induced liver injury in mice. Hepatology 2001;34:101-8.

36.Yohe HC, O'Hara KA, Hunt JA, et al. Involvement of Toll-like receptor 4 in acetaminophen hepatotoxicity. Am J Physiol Gastrointest Liver Physiol 2006;290:G1269-79.

37.Takayashiki T, Yoshidome H, Kimura F, et al. Increased expression of toll-like receptor 4 enhances endotoxin-induced hepatic failure in partially hepatectomized mice. J Hepatol 2004;41:621-8.

38.Shimamoto A, Chong AJ, Yada M, et al. Inhibition of Toll-like receptor 4 with eritoran attenuates myocardial ischemia-reperfusion injury. Circulation 2006;114:I270-4.

39.Mitsuzawa H, Nishitani C, Hyakushima N, et al. Recombinant soluble forms of extracellular TLR4 domain and MD-2 inhibit lipopolysaccharide binding on cell surface and dampen lipopolysaccharide-induced pulmonary inflammation in mice. J Immunol 2006;177:8133-9.

40.Fort MM, Mozaffarian A, Stöver AG, et al. A synthetic TLR4 antagonist has anti-inflammatory effects in two murine models of inflammatory bowel disease. J Immunol 2005;174:6416-23.

 

 

 

Correspondence to:

 

Dr. Zhuo-Fu Wen

Department of Gastroenterology,

Third Affiliated Hospital of Zhong Shan (Sun Yat-sen)  University, 510630 Guangzhou, P.R.China

Email: wenzfu@163.com

 or weixiuqing1975@yahoo.com.cn

 

FIGURE 1. 1 Toll-like receptor 4 (TLR4) expression. A, B: Healthy control, no staining for TLR4 expression (x100, x400). C, D: Liver biopsy from a patient with chronic hepatitis B (x200), TLR4 expressed on hepatocytes, positive staining mainly located in the cytoplasm but not the nuclei (x100, x400).

 

FIGURE 2. Typical TLR4 in HepG2 (A) and HepG2.2.15 (B). Cells was stained with directly conjugated antibodies to TLR4. Diagrams represent 10,000 gated cells.

 

TABLE 1. Demographic and laboratory variables
	Chronic hepatitis B	Healthy Control
No. of patients	75	10
Sex（male/female）	11 1/2	9
Age (yr)	35±11	36±6
ALT (U/l)	125±76	25±11
Total bilirubin (µmol/l)	63.7±53.5	N/A
Numbers of HBeAg（+）/（-）	2 1/8	N/A
Lg [serum HBV-DNA]	6.19±1.64	N/A
G0/G1/G2/G3/G4 score	0/16/20/18/21	10/0/0/0/0
S0/S1/S2/S3/S4 score	0/14/21/26/14	10/0/0/0/0
TLR4 score 0/1/2/3	2/13/22/38	8/2/0/0
N/A: not applicable

 

TABLE 2. TLR4 Scores
	No. of patients	Score of Expression of TLR4
Normal Control	10	0.20±0.42
CHB	75	2.17±0.88 *
Mild CHB	36	1.75±0.87*
Severe CHB	39	2.56±0.68 *,#
Notes: CHB, chronic hepatitis B; *, P<0.05 vs normal control; #, P<0.05 vs mild CHB

 

TABLE 3. Expression of TLR4 in HepG2and HepG2.2.15
	n	MFI	TLR4 positive cells(%)	Relative TLR4 mRNA
HepG2	12	1.03±0.34	0.39±0.12	0.13±0.03
HepG2.2.15	12	10.72±2.79*	16.34±6.97*	0.60±0.09*
Notes: MFI, Mean fluorescence intensity ; *, P<0.05 vs HepG2