Introduction

The endoplasmic reticulum (ER) is a membranous organelle required for folding and processing of nascent proteins. Physiological, pathological or pharmacological insults that disrupt ER function induce accumulation of unfolded or misfolded proteins in the ER lumen. This condition is defined as ER stress and triggers a conserved cellular response called unfolded protein response (UPR) (1). UPR aims to relieve ER stress via different mechanisms, including enhancement of protein folding and degradation processes as well as specific inhibition of protein synthesis in the cell. UPR-related mechanisms are mediated by three different signaling pathways: protein kinase R-like ER kinase (PERK), activated transcription factor 6 (ATF6), and inositol-requiring enzyme 1 (IRE1) (1,2). Although UPR provides a defense mechanism for cells, ER stress can trigger apoptosis depending on the severity and duration of stress (2,3,4). It has been reported that a number of pathophysiological conditions, such as diabetes mellitus, cardiovascular diseases, obesity, cancer, neurodegenerative diseases and hepatic steatosis are associated with excessive or persistent ER stress (5,6,7,8,9,10).

Hepatocytes possess quite well-developed ER and high capacity for protein synthesis similar to other secretory cells. Therefore, ER stress and UPR play an important role in various liver diseases such as non-alcoholic steatohepatitis, alcoholic liver disease, and ischemic reperfusion injury as well as toxic liver damage (11,12,13,14,15). Moreover, different studies conducted on mammalian cells, including hepatocytes, have reported that viruses were also able to induce ER stress. Increased viral protein synthesis in the infected cells induces ER stress by disturbing protein folding mechanisms and enhancing protein aggregates in the ER lumen (16). It is known that hepatitis B virus (HBV) induces ER stress and UPR activation, which contributes to liver pathogenesis during HBV infection (16,17,18,19).

Glucose-regulated protein 78 (GRP78) is an ER resident chaperone protein that monitors ER stress and regulates UPR-induced survival or apoptosis. During ER stress, expression of GRP78 significantly increases; hence, increased expression of GRP78 indicates UPR induction (1,2,3,4). Khadir et al. (20) reported that obesity, which induces hepatic ER stress, caused an elevation in GRP78 levels both in plasma and liver. These results suggest that GRP78 can pass into blood from tissue during hepatic ER stress (20,21). In HBV-infected patients, activation of UPR and its contribution to HBV pathogenesis have been shown previously, however, no circulating marker has been reported. Therefore, alterationsin circulating GRP78 level in patients with chronic HBV infection is evaluated in the present study.

Materials and Methods

In the current study, samples collected from 60 patients with chronic HBV infection whose serum specimens were processed for HBV-DNA quantification in the molecular microbiology laboratory were evaluated for GRP78 analysis. Control serums were obtained from 50 hepatitis B surface antigen (HBsAg)-, anti-hepatitis C virus- and anti-human immunodeficiency virus-negative subjects who had no chronic disease including chronic liver disease. According to HBV-DNA levels, HBV-infected patients were subdivided into low (20-1x10² IU/mL, n=20), mild (1x10³-1x10⁵ IU/mL, n=20), and high HBV-DNA (1x10⁶- 1.7x10⁸ IU/mL, n=20). All the samples were stored at -40 °C until assayed throughan ELISA. This study was approved by the Clinical Research Ethics Committee in Ordu University (2017-157).

Real-time polymerase chain reaction: Quantification of HBV-DNA was performed via a real-time polymerase chain reactionmethod using the COBAS AmpliPrep/COBAS Taqman 48 system (Roche, Branchburg, NJ, USA). Viral nucleic acids were extracted from serum (500 μl) using Cobas AmpliPrep automatic extractor system according to the manufacturer’s instructions. The assay range for HBV-DNA was 20-1.7x10⁸ IU/mL.

Detection of GRP78: Levels of circulating GRP78 were determined in serum using the human GRP78 ELISA kit (Elabscience; E-EL-H5586) with a detection range of 0.63-40 ng/mL. Samples and standards were added to the appropriate micro ELISA plate wells pre-coated with an antibody specific to human GRP78 and then the manufacturer’s instructions were followed. The optical density was measured spectrophotometrically at the wavelength of 450 nm (BioTek, ELx800 brand REF ELX508 SN1310149). The level of GRP78 in the samples was calculated by comparing the optical density of the samples with the standard curve.

Statistical Analysis

All data are given as mean ± standard deviation. Statistical analysis was performed with One-Way ANOVA and Tukey’s test.A p value of less than 0.05 was considered statistically significant.

Results

Sixty patients with chronic HBV infection (42.1±16.0 years; 28 female, 32 male) and 50 control subjects (53.9±19.1; 21 female, 29 male) were included in this study. HBV-DNA content in the serum samples of the patients with chronic HBV infection was in three different ranges of low (20 - 1x10² IU/mL, n=20), mild (1x10³ - 1x10⁵ IU/mL, n=20), and high (1x10⁶-1.7x10⁸ IU/mL, n=20). Serum HBsAg, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in all patients are presented in Table 1.

The serum GRP78 concentration was found to be 14.36±10.22 ng/mL in controls. As seen in Figure 1, there was no significant difference in GRP78 concentration between the HBV subgroups (12.97±8.5, 15.01±10.7 and 9.18±6.02 ng/mL, respectively). In the control group, no correlation was found between GRP78 and serum ALT or AST levels. In each HBV subgroup, changes in serum GRP78 did not show a correlation with the serum variables such as ALT, AST and HBsAg or HBV-DNA content. When all the HBV-infected patients were included in the analysis, we did not determine a correlation between GRP78 and the other variables (Figure 2).

Discussion

Accumulation of unfolded or misfolded proteins in the ER lumen causes a condition termed ER stress (1,2,3,4). During ER stress, UPR is activated to restore cellular homeostasis, however, delayed or insufficient responses to ER stress are implicated with pathological consequences, including fat accumulation, insulin resistance, inflammation, and apoptosis, all of which play important roles in liver pathologies (11,12,13,14,15).

HBV infection is a serious health problem affecting approximately 260 million people worldwide and causing acute and chronic hepatitis, liver cirrhosis, and even hepatocellular carcinoma (22). To date, many studies have reported on the molecular mechanisms for the relationship between HBV infection and pathogenesis of hepatic diseases, but the mechanisms are still not fully understood (23-25). Recent studies indicate that ER stress may play a role in the pathogenesis of HBV infection (16,17,18,19). It has been reported that some products of HBV genome might be involved in the activation of UPR in hepatocytes. For instance, Li et al. (26) reported that regulatory X protein (HBx), which is a product of HBV genome, mediated UPR activation in Hep3B and HepG2.2.15 cells via ATF6 and IRE1-XBP1 pathways. Further studies confirmed HBx-induced ER stress in different cell lines (27,28). Additionally, Wang et al. (29,30) reported that the pre-S mutant large HBsAgs were retained in the ER lumen and induced UPR signals, leading to the increased expression of ER chaperones such as GRP78 and GRP94.

In the present study, serum GRP78 levels were evaluated as an ER stress marker in patients with chronic HBV infection whose HBV-DNA levelranged between 80 and 1.7x10⁸ IU/mL. As expected, these patients displayed higher levels of serum HBsAg compared to controls. Since the measurement of serum ALT and AST levels is a common laboratory assay to monitor liver functions, we evaluated these parameters in the subjects. In the control group, the ALT and AST levels were within the normal range. In the HBV subgroups, as the HBV-DNA content raised, serum ALT and AST levels gradually increased. However,a statistically significant elevation was observed only in the HBV subgroup with the highest HBV-DNA content. These results are consistent with the previous reports and suggest that hepatic functions declined during high viral replication (31,32,33).

GRP78 is an ER-resident molecular chaperone which also regulates ER stress and is upregulated during UPR (1,2,3,4). Since HBV induces hepatic ER stress (16,17,19,26,27,28,29,30), we hypothesized that circulating GRP78 increased in HBV-infected patients and then, measured serum GRP78 levels in controls and in patients with chronic HBV infection.Contrary to our expectation, present results showed that during chronic HBV infection, circulating GRP78 levels remained unchanged without any relationship with serum variables. These results might be attributed to a mechanism that prevents GRP78 release from hepatocytes. Li et al. (26) showed that HBx protein and GRP78 were co-localized in ER lumen and displayed a direct interaction which may results in increased GRP78 retention in ER lumen. The aforementioned HBx-GRP78 interaction or another unknown mechanism may prevent GRP78 release to the blood and be responsible for the unchanged serum GRP78 levels in HBV-infected patients. Nevertheless, GRP78 measurements are needed both in liver biopsy and serum samples to confirm this suggestion.

Study Limitations

The most important limitations of the current study are small sample size, which included only Turkish subjects, and absence of liver biopsies. It would be valuable data if we had shown the presence of ER stress in liver biopsies obtained from a larger and heterogenous subject population, despite unchanged serum GRP78 concentration.

Conclusion

Although the involvement of ER stress in HBV-induced liver damage is well-documented, the present results show that serum GRP78 remained unchanged during chronic HBV infection and there was no relationship between serum level of GRP78 and the parameters of HBV infection. Since ER stress is an important mechanism in HBV-related liver injury, other ER stress markers, such as GRP94, might be examined with liver biopsies in future studies conducted with larger patient group.

Ethics

Ethics Committee Approval: This study was approved by the Clinical Research Ethics Committee in Ordu University (2017-157).

Informed Consent: Retrospective study.

Peer-review: Externally and internally peer-reviewed.

Concept: S.C., Design: S.C., Y.Ç., Data Collection or Processing: S.C., Y.Ç., M.K.Ç., Analysis or Interpretation: S.C., A.A.Y., T.N., Literature Search: S.C., A.A.Y., Writing: S.C., Y.Ç.

Conflict of Interest: No conflict of interest was declared by the authors.

Financial Disclosure: The authors declared that this study received no financial support. This study was financially supported by Ordu University Research Funds (BAP; HD-1722).