Introduction
Soluble Intercellular Adhesion Molecule-1 (sICAM-1) is a cell surface glycoprotein, which belongs to the immunoglobulin superfamily (Springer Reference Springer1990). sICAM-1 expresses at a low basal level in immune, endothelial, and epithelial cells, but is upregulated in response to inflammatory stimulation (Hubbard and Rothlein Reference Hubbard and Rothlein2000). Thus, sICAM-1 has emerged as a master regulator of many essential tissue functions both at the onset and the resolution of pathologic conditions (Philpott and Miner Reference Philpott and Miner2008; Wichert et al. Reference Wichert, Juliusson, Johansson, Sonesson, Teige, Wickenberg, Frendeus, Korsgren and Hansson2017; Chu et al. Reference Chu, Chen, Chan, Lu, Huang, Mao, Sze and Sun2023). sICAM-1 is generally not expressed on the surface of normal liver cells, but only weakly expressed on liver sinusoid endothelial cells, hepatocytes, Kupffer cells, and interstitial fibroblasts (Iwasawa et al. Reference Iwasawa, Kameyama, Ishikawa and Sawa2008). Previous studies have shown us the relationship between sICAM-1 and chronic hepatitis B (CHB). When infected with hepatitis B virus (HBV), sICAM-1 is strongly expressed on sinusoidal lining cells, mediating the interaction between immunocompetent cells to clear HBV. The level of sICAM-1 expression reflects the activity of hepatitis and the extent of liver tissue damage (Horiike et al. Reference Horiike, Onji, Kumamoto and Masumoto1994; Nouri-Aria et al. Reference Nouri-Aria, Koskinas, Tibbs, Portmann and Williams2019). It is closely associated with the occurrence, development, and prognosis of liver injury.
Hepatitis B virus (HBV) infection has been a long-standing global public health issue, with approximately 257 million chronic HBV-infected individuals worldwide and approximately 887,000 deaths from HBV related diseases each year (Nelson et al. Reference Nelson, Easterbrook and McMahon2016). According to the classification criteria of the World Health Organization, China is considered a country with moderate prevalence of HBV. There are approximately 70 million cases of chronic HBV infection in China, which is the main cause of viral hepatitis, cirrhosis, liver cancer, and liver failure (Shin et al. Reference Shin, Oh, Masuyer, Curado, Bouvard, Fang, Wiangnon, Sripa and Hong2010; Honer Zu Siederdissen and Cornberg Reference Honer Zu Siederdissen and Cornberg2014; Li et al. Reference Li, Zhang and Zhu2023).
Clonorchiosis is a serious zoonotic parasitic disease caused by Clonorchis sinensis (C. sinensis) in the liver and bile ducts of humans and other mammals (Qian et al. Reference Qian, Utzinger, Keiser and Zhou2016). About 12.5 million people in China are infected with C. sinensis, accounting for 85% of the global total number of infections. In 2020, the National Health Commission investigated the status of human parasitic infections, revealing that the prevalence of C. sinensis infection has increased by 75% compared to 1990 (Keiser and Utzinger Reference Keiser and Utzinger2009; Na et al. Reference Na, Pak and Hong2020). Chronic infection of C. sinensis can cause a series of liver and gallbladder diseases, such as liver fibrosis, gallstones, and common bile duct stones, and even lead to liver cirrhosis and liver and gallbladder cancer (Pak et al. Reference Pak, Lee, Jeon, Dai, Yoo and Hong2019; Shang et al. Reference Shang, Sun, Wu, Gong, Tang, Meng, He, Yu, Huang and Li2020; Vale et al. Reference Vale, Gouveia, Gartner and Brindley2020). The repeated infection of populations in epidemic areas can continuously promote the development of liver fibrosis, bringing a devastating disease burden to patients and society (Qian et al. Reference Qian, Chen, Fang, Xu, Zhu, Tan, Zhou, Wang, Jia, Yang and Zhou2011; Yan et al. Reference Yan, Wang, Li, Zhang, Zhang, Wang, Li, Chen, Tang and Zheng2015; Machicado and Marcos Reference Machicado and Marcos2016).
Co-infection with C. sinensis and HBV is a phenomenon common in areas where C. sinensis is prevalent (Qian et al. Reference Qian, Chen, Liang, Yang and Zhou2012; Shen et al. Reference Shen, Hou, Yang and Xiao2015). Although the pathogenic mechanism of C. sinensis differs from that of HBV infection, both can cause damage to hepatocytes and speed up the process of the disease. At present, there is no clear consensus on whether C. sinensis will affect the infection of HBV. Studies have shown that the presence of C. sinensis can affect the anti-HBV treatment. In co-infected patients, the antiviral effect of simultaneous antiparasitic treatment is better than that of simple antiviral treatment (Li et al. Reference Li, Dong, Huang, Chen, Kong, Sun, Yu and Xu2016). However, in the clinical process of diagnosis and treatment, patients with C. sinensis were prone to be misdiagnosed. As mentioned earlier, the level of sICAM-1 expression can reflect the activity of hepatitis and the extent of liver tissue damage. In this study, we evaluated the level of sICAM-1 in patients co-infected with HBV and C. sinensis, and we explored whether it can indicate infection with C. sinensis, providing clues for further examination and treatment.
Materials and methods
Patients
This study included a total of 156 participants divided into six groups: patients with C. sinensis mono-infection (C. sinensis, n=27), patients with only HBV (CHB, n=32), chronic hepatitis B (CHB) patients co-infected with C. sinensis (CHB+C. sinensis, n=24), patients with single post-hepatitis B liver cirrhosis (LC, n=18), post-hepatitis B liver cirrhosis patients (LC) with C. sinensis co-infection (LC+C. sinensis, n=16), and healthy controls (HCs, n=39). The participants were selected from patients in the Third Affiliated Hospital of Sun Yat-Sen University.
The diagnosis in all subjects was based on clinical and laboratory data. The inclusion criteria for participants infected with C. sinensis were identification of eggs through microscopic exams for stool, excluding hepatitis B virus infection. As for participants infected with HBV, the inclusion criteria were HBV surface-antigen-positive and HBV DNA >10 IU/ml. The inclusion criteria for co-infected with C. sinensis and HBV were that C. sinensis eggs were found in the stool, as well as HBV infection. The inclusion criteria for chronic hepatitis B patients with liver cirrhosis was that the patients were clinically confirmed to have liver cirrhosis by liver biopsy and imaging tests after HBV infection. The inclusion criteria for patients with liver cirrhosis after co-infection were that C. sinensis eggs were found in the stool based on chronic hepatitis B with liver cirrhosis. The inclusion criteria for the healthy control group were negative for both HBV and C. sinensis as well as normal liver function indexes.
The participants were not included if they were under 18 years old or over 70 years old, pregnant, co-infected with other parasites and other types of the hepatitis virus, had liver diseases caused by metabolic disorders, drugs, and poisoning, acute infection, malignant tumor, human immunodeficiency virus (HIV) infection, thyroid dysfunction, autoimmune diseases, and so on.
The detection of sICAM-1 in serum
The serum was separated from peripheral blood of patients. The level of serum sICAM-1 was detected by enzyme-linked immunosorbent assay (ELISA) (Reagent kits purchased from Dakewe Biotech Co., Ltd.). First, 100μl diluted cytokine standard or 100μl diluted serum samples were added to each well. Second, 50μl biotinylated antibodies were added to all wells above. They were covered and incubated for one hour at room temperature. Then, the wells were washed three times by a washing buffer. Added 100μl diluted Streptavidin-HRP to each well and incubated all wells for twenty minutes at room temperature. The wells were washed again and incubated with 100μl TMB for 15 minutes before the addition of stop solution. Finally, the ELISA plate was determined at 450 nm with a correction wavelength of 630 nm.
The detection of liver function index
The level of aminotransferase (ALT) and aspartate aminotransferase (AST) in serum was detected by turbidimetric immunoassay using the Hitachi 7600 automated biochemical analyzer from Japan.
Statistical analysis
The data were analyzed by Prism 9.5.0 statistical software. The normally distributed data were presented as the mean±SD, and the skewness distributed data were presented as the median (IQR). The comparison between two groups was analyzed by the Mann-Whitney test. The comparison with more than two groups used the one-way ANOVA test or Kruskal-Wally’s test. The correlation between two indexes was analyzed by Spearman’s correlation analysis. P<0.05 was regarded as statistically significant.
Results
General information
General information of study participants is presented in Table 1, including age and levels of AST and ALT in all groups and results of both fecal C. sinensis egg count and HBV DNA copies in the patient groups. There were no significant differences in age among six groups and in fecal C. sinensis egg counts among the patient groups. All five infected groups of patients showed higher levels of AST and ALT than the healthy control subjects (P<0.05, respectively). The CHB+C. sinensis group had higher levels of AST and ALT than the CHB group (P<0.05). The LC+C. sinensis group had higher levels of AST than the LC group (P<0.05), while there was no difference in the ALT level.
Clinical statistics of the study subjects
Abbreviations: CHB: chronic hepatitis B; C. sinensis: Clonorchis sinensis; HCs: healthy controls; LC: liver cirrhosis.
a Statistically significantly different vs. healthy control subjects
b Statistically significantly different vs. CHB patients
c Statistically significantly different vs. C. sinensis patients
d Statistically significantly different vs. LC patients
The HBV DNA copies in the patient groups is shown in Figure 1. There were no significant differences between the CHB+C. sinensis and LC+C. sinensis groups, as well as the CHB and LC groups. The CHB+C. sinensis group had a significantly higher level of HBV DNA copies than the CHB group (a: P<0.05). Compared with the LC group, the LC+C. sinensis group had a significantly higher level of HBV DNA copies (b: P<0.05).
HBV DNA copies in patient groups. HBV DNA was detected by FQ-PCR and showed in log. Patients in CHB+C. sinensis group had higher HBV DNA copies than the CHB group (a: P<0.05). Patients in LC+C. sinensis group had higher HBV DNA copies than LC group (b: P<0.05). Analyzed by Mann-Whitney test.
The comparison of the levels of sICAM-1 in all groups
As shown in Figure 2, compared with the healthy control group, the level of sICAM-1 significantly elevated in all the patient groups (P<0.05, respectively). In the co-infected groups, the CHB+C. sinensis group exhibited a significant elevation in sICAM-1 level than the CHB group (P<0.05), and the LC+C. sinensis group demonstrated a significant increase in sICAM-1 level compared with the LC group (P<0.05). However, there was no significant difference in sICAM-1 level between the CHB+C. sinensis and LC+C. sinensis group. In mono-infected groups, the LC group had a higher level of sICAM-1 than the CHB group (P<0.05).
The levels of sICAM-1 in all groups. The level of sICAM-1 was detected by ELISA. All the patient groups had higher sICAM-1 levels than the healthy control group (a: P<0.05, compared with the HCs group). The CHB+C. sinensis and LC group exhibited a significant elevation in sICAM-1 level than CHB group (b: P<0.05, compared with the CHB group). The LC+C. sinensis group had higher sICAM-1 level compared with the LC group (c: P<0.05, compared with the LC group). Analyzed by Kruskal-Wally’s test.
The correlation analysis between the levels of sICAM-1 and AST, ALT in co-infected groups
The correlation analysis between the levels of sICAM-1 and AST, ALT is shown in Figure 3. Figure 3A shows a positive correlation between the level of sICAM-1 and AST in the CHB+C. sinensis group (Pearson r=0.657, p<0.0001). The correlation equation is Y = 16.31*X + 2839. Figure 3B shows a positive correlation between the level of sICAM-1 and ALT in the CHB+C. sinensis group (Pearson r=0.556, p=0.0037). The correlation equation is Y = 8.741*X + 31567. Figure 3C showed a positive correlation between the level of sICAM-1 and AST in the LC+C. sinensis group (Pearson r=0.756, p=0.0039). The correlation equation is Y = 9.624*X + 2877. Figure 3D showed a positive correlation between the level of sICAM-1 and ALT in the LC+C. sinensis group (Pearson r=0.632, p=0.0021). The correlation equation is Y = 11.31*X + 2747.
The correlation analysis between the levels of sICAM-1 and AST, ALT in CHB+C. sinensis and LC+C. sinensis group. There was a positive correlation between the levels of sICAM-1 and AST, ALT in the CHB+C. sinensis group (A and B) (r=0.657, 0.556, P<0.05) and the LC+C. sinensis group (C and D) (r=0.756, 0.632, P<0.05). Analyzed by Spearman’s correlation analysis.
Discussion
This study found that co-infection could exacerbate liver tissue inflammation and liver function damage in patients, raising the sICAM-1 level and HBV DNA copies. Our data showed that compared with the healthy control group, the sICAM-1 levels in all the experimental groups were significantly increased(P<0.05). sICAM-1 is a single chain cell surface glycoprotein, which is usually not expressed on the surface of normal liver cells but is up-regulated in response to inflammatory stimulation. When infected with HBV, sICAM-1 is strongly expressed on sinusoidal lining cells, mediating the interaction between immune active cells to clear HBV (Hubbard and Rothlein Reference Hubbard and Rothlein2000; Iwasawa et al. Reference Iwasawa, Kameyama, Ishikawa and Sawa2008; Nouri-Aria et al. Reference Nouri-Aria, Koskinas, Tibbs, Portmann and Williams2019).
Our study also showed that the levels of sICAM-1 and HBV DNA copies in co-infected groups were significantly higher compared to the mono-infected groups. We consider that co-infection could exacerbate liver tissue inflammation in patients, raising the levels of sICAM-1 and HBV DNA copies. Previous studies have found that compared to patients infected with HBV, the expression of Th1 cytokines decreased in patients with co-infection, leading to a decline in the ability to clear HBV, while the expression of Th2 cytokines increased, exacerbating the persistent infection of HBV (Leung Reference Leung1999; Henri et al. Reference Henri, Chevillard, Mergani, Paris, Gaudart, Camilla, Dessein, Montero, Elwali, Saeed, Magzoub and Dessein2002; Dong et al. Reference Dong, Zhao, Sun, Shang, Lv, Yu, Hu and Huang2022). The combined action of proteins from HBV and C. sinensis can directly promote the activation of hepatic stellate cells, leading to the release of sICAM-1 (Hellerbrand et al. Reference Hellerbrand, Tsukamoto, Brenner and Rippe1996; Li et al. Reference Li, Dong, Huang, Chen, Kong, Sun, Yu and Xu2016), which supports our findings.
Interestingly, compared with the CHB group, the levels of sICAM-1 and HBV DNA copies in the LC group increased significantly, while in the co-infected groups, there was no significant difference in the levels of sICAM-1 and HBV DNA copies. This suggests that co-infection may lead to the chronicity of HBV infection. Our previous research results from in vitro experiments showed that the levels of pro-inflammatory cytokines in the co-infection group were lower than those in the HBV group, while the levels of liver fibrosis-related molecules were the opposite (Dong et al. Reference Dong, Zhao, Sun, Shang, Lv, Yu, Hu and Huang2022). This suggests that co-infection may exacerbate the development of liver fibrosis, leading to the chronicity of HBV infection. However, compared with the CHB group, the levels of sICAM-1 and HBV DNA copies in the CHB+C. sinensis group increased significantly. This indicates that the ability to clear HBV is not determined by a single cytokine, but the combined actions of various cytokines. As mentioned above, the decreased expression of Th1 cytokines and increased expression of Th2 cytokines exacerbated the persistent infection of HBV. Other research revealed that when C. sinensis infection occurs, secretion metabolic proteins (Clonorchis sinensis excretory secretory proteins, Cs ESP) are produced, among which the Cs Severin component has been reported to enhance the ability of IFN-α to clear HBV (Ahlenstiel et al. Reference Ahlenstiel, Edlich, Hogdal, Rotman, Noureddin, Feld, Holz, Titerence, Liang and Rehermann2011; Edlich et al. Reference Edlich, Ahlenstiel, Zabaleta Azpiroz, Stoltzfus, Noureddin, Serti, Feld, Liang, Rotman and Rehermann2012; Li et al. Reference Li, Dong, Huang, Chen, Kong, Sun, Yu and Xu2016). During co-infection, there is a more disrupted alteration in cytokine levels within the body. When the expression of cytokines that promote HBV infection is dominant, it is manifested as a persistent infection of HBV (Dong et al. Reference Dong, Zhao, Sun, Shang, Lv, Yu, Hu and Huang2022; Henri et al. Reference Henri, Chevillard, Mergani, Paris, Gaudart, Camilla, Dessein, Montero, Elwali, Saeed, Magzoub and Dessein2002). Under the joint action of cytokines and macrophage inflammatory factors, sICAM-1 expression is directly or indirectly up-regulated (Li et al. Reference Li, Dong, Huang, Chen, Kong, Sun, Yu and Xu2016). When the disease progresses to a certain extent, the levels of cytokines in the body tend to stabilize. The ability to clear HBV and promote HBV infection reaches a balance, leading to the chronicity of HBV infection. Therefore, the level of sICAM-1 did not increase significantly. We believe that sICAM-1 was closely related to the degree of liver function damage in patients with co-infection.
In this study, we also detected the levels of AST and ALT in each group. The levels of AST and ALT in the mono-infected groups were significantly higher than those in the healthy control group, but still within the normal range. Compared with the mono-infected groups, the levels of AST and ALT in the co-infected groups were significantly higher. ALT and AST are important transaminases in human body and mainly exist in hepatocytes. They are mainly used in clinical practice as evaluation indicators of liver function (Pol et al. Reference Pol, Nalpas, Vassault, Bousquet-Lemercier, Franco, Lacour, Berthelot, Hanoune and Barouki1991; Li et al. Reference Li, Ye, Ran, Liu, Tang, Liu, Liao, Zhang, Xiao, Lu, Zhang, He and Hu2021). Some patients with HBV infection may be in the immune tolerance period or low replication period of the natural history of hepatitis B. There is only mild inflammation in the liver tissue, so the level of transaminase in the blood continues to be within the normal range (European Association for the Study of the Liver. Electronic address and European Association for the Study of the 2017). CHB patients with normal transaminase may also have varying degrees of inflammation, obvious fibrosis, and even cirrhosis (Tan et al. Reference Tan, Zhou, Ye, He and Ge2017). The clinical manifestations of Clonorchiasis can be acute or chronic depending on the degree of infection, duration, and immune status. Most infected individuals had normal serological indicators (Li et al. Reference Li, Dong, Huang, Chen, Kong, Sun, Yu and Xu2016). Co-infection of HBV and C. sinensis can aggravate the inflammation of liver tissue, leading to the increase of transaminase levels. Through the statistical analysis, there was a positive correlation between the levels of sICAM-1 and AST, ALT in the co-infected groups. This further confirmed that sICAM-1 could reflect the degree of liver inflammation after co-infection.
Research has demonstrated the involvement of sICAM-1 in the pathological damage mechanisms of chronic hepatitis B. It is closely related to the occurrence, progression, and prognosis of liver injury. We believe that during clinical diagnosis, a high level of sICAM-1 may alert doctors to the possibility of co-infection with C. sinensis in CHB patients, and sICAM-1 may serve as an indicator for monitoring treatment efficacy.
Abbreviations
- sICAM-1:
-
Soluble Intercellular Adhesion Molecule-1
- HBV:
-
hepatitis B virus
- C. sinensis:
-
Clonorchis sinensis
- LC:
-
liver cirrhosis
- CHB:
-
chronic hepatitis B
- HCs:
-
healthy controls
- AST:
-
Aspartate aminotransferase
- ALT:
-
Alanine aminotransferase.
Author contribution
All the authors have agreed with the publication of this paper.
Study design: Huimin Dong
Information collection: Wanshan Li
Sample assessment: Hongbin Zhang
Formal analysis: Mei Shang
Supervision: Yuan Liao
Writing: Jieru Qiu
All the authors participated in the critical revision and agreed to be responsible for all aspects of the work.
J. Qiu and M. Shang contributed equally to this work.
Data availability statement
The datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request.
Financial support
This work was supported by grants from the National Natural Science Foundation of China (81902082).
Competing interest
The authors declare that they have no competing interests.
Ethical standard
The study was approved by The Ethics Committee of the Third Affiliated Hospital of Sun Yat-sen University ([2020]02-045-01)
