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Role of Galectins in the Liver Diseases: A Systematic Review and Meta-Analysis

Background: Galectins, a family of β-galactoside-binding proteins, are related to the development and progression of various human diseases such as cancer, heart failure, and chronic kidney disease. However, its role in liver diseases is unclear. Methods: The PubMed, Embase, and Cochrane Library databases were searched. Hazard ratios (HRs), odds ratios (ORs), and mean differences (MDs) with 95% CIs were pooled to evaluate the association of the galectins with the outcomes and risk of liver diseases by a random effects model. Results: Thirty three studies involving 43 cohorts and 4,168 patients with liver diseases were included. In the patients with hepatocellular carcinoma (HCC), high expression of galectin-1 and -3 in the tissues was significantly associated with worse overall survival (galectin-1: HR = 1.94, 95% CI = 1.61–2.34, p < 0.001; galectin-3: HR = 3.29, 95% CI = 1.62–6.68, p < 0.001) and positive vascular invasion (galectin-1: OR = 1.74, 95% CI = 1.18–2.58, p = 0.005; galectin-3: OR = 2.98, 95% CI = 1.58–5.60, p = 0.001); but, high expression of galectin-4 and −9 in the tissues was significantly associated with better overall survival (galectin-4: HR = 0.53, 95% CI = 0.36–0.79, p = 0.002; galectin-9: HR = 0.56, 95% CI = 0.44–0.71, p < 0.001) and negative vascular invasion (galectin-4: OR = 0.36, 95% CI = 0.19–0.72, p = 0.003; galectin-9: OR = 0.60, 95% CI = 0.37–0.97, p = 0.037). Serum galectin-3 level was significantly higher in HCC (MD = 3.06, 95% CI = 1.79–4.32, p < 0.001), liver failure (MD = 0.44, 95% CI = 0.23–0.66, p < 0.001), liver cirrhosis (MD = 1.83, 95% CI = 1.15–2.51, p < 0.001), and chronic active hepatitis B (MD = 18.95, 95% CI = 10.91–27.00, p < 0.001); serum galectin-9 level was significantly higher in HCC (MD = 3.74, 95% CI = 2.57–4.91, p < 0.001) and autoimmune hepatitis (MD = 8.80, 95% CI = 7.61–9.99, p < 0.001). Conclusion: High galectin-1 and -3 and low galectin-4 and -9 expression indicate worse outcomes of patients with HCC. Serum galectin-3 and -9 levels are positively associated with the risk of chronic liver diseases.


INTRODUCTION
Liver diseases, including chronic hepatitis, liver fibrosis or cirrhosis, acute liver injury or liver failure, and hepatocellular carcinoma (HCC), are a major global health burden. They are often subtle, but potentially lethal (1). According to the report of the Global Burden of Disease Study 2019, there are 79,200 deaths from acute hepatitis (2), 1,470,000 deaths from liver cirrhosis and other chronic liver diseases (3), and 485,000 deaths from HCC (4) in the world. Early assessment and identification of liver diseases by molecular biomarkers are clinically important.
Galectins play a regulatory role in liver diseases by binding their CRDs to the glycoconjugates expressed in the hepatocytes (9). Abnormal expression of the galectins may be related to the development of hepatitis and liver fibrosis/cirrhosis and the progression of HCC (10). In this study, we conducted a systematic review and meta-analysis to evaluate the role of galectins in various liver diseases.

METHODS
This meta-analysis was performed in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement (11).

Registration
The registration number was CRD42020210038 in the PROSPERO.

Selection Criteria
The inclusion criteria were as follows: (1) study population should be the patients diagnosed with liver diseases and (2) galectin expression or level was detected in patients with liver diseases. The exclusion criteria were as follows: (1) duplicate papers; (2) reviews, meta-analyses, or case reports; (3) notes, conferences, corrections, editorials, comments, or letters; (4) experimental or animal studies; and (5) studies which were lacking of detailed data regarding galectin expression or level.

Data Extraction
We extracted the following data from each study, including first author, publication year, country, study design, enrollment period, sample size, subtypes of the galectins, and methods to detect the galectins. As for the studies regarding the clinicopathological features and the outcomes of HCC, we specifically extracted the data as follows: galectin expression and its grouping; clinicopathological features including tumor size, tumor-node-metastasis (TNM) stage, differentiation grade, and vascular invasion; and outcomes, which include overall survival (OS), disease-free survival (DFS), and relapse-free survival (RFS). As for the studies regarding the risk of liver diseases, we specifically extracted the data regarding the type of liver diseases, the Child-Pugh class, and the level of serum galectins.
As for the survival data, we directly extracted or indirectly estimated the hazard ratio (HR) and 95% CI. If a study did not give the HR and 95% CI, but only reported the Kaplan-Meier curves, we would employ the Engauge Digitizer 4.1 software (Linux, Mac OSX, and Windows Slashdot Media, CA, USA) to extract the survival rate at the different time points from the Kaplan-Meier curves and then utilize Tierney's table (12) to estimate its correlative HR with 95% CI.

Study Quality Assessment
Quality of the case-control and cohort studies were evaluated by the Newcastle-Ottawa Scale (NOS), which included the three parts (i.e., selection, comparability, and outcomes) and eight questions (13). The highest NOS score was nine points. High quality was considered if the NOS score was more than six points.

Statistical Analysis
The Stata version 12.0 (Stata Corporation, College Station, Texas, USA) was employed for the statistical analysis. Only a random effects model was implemented. HRs, odds ratios (ORs), and mean differences (MDs) with 95% CIs were pooled. A two-sided p < 0.05 was considered as statistically significant. If the data were expressed as median with range, mean with SD would be estimated (14). Heterogeneity was evaluated by the I² statistics and the Cochran's Q test. I 2 > 50% or p < 0.1 was considered as a statistically significant heterogeneity. Sensitivity analysis was performed after omitting one study at a time in order to check the consistency to estimate the overall effect. Publication bias was assessed by Egger's test (15) and p < 0.1 was considered to imply a significant publication bias.

Relapse-Free Survival
The relationship between the galectins and RFS was explored in three study cohorts (20,30,32).

Hepatocellular Carcinoma
The relationship between the galectins and the risk of HCC was explored in six study cohorts (25,30,36,(41)(42)(43). Among them, five study cohorts selected the healthy volunteers as the control TABLE 4 | Galectins with the risk of different liver diseases: results of the meta-analyses.

Heterogeneity Groups
No. studies Pooled proportion using random-effects mode P-value I 2 P-value subjects, and one study cohort selected the patients with chronic hepatitis as the control subjects. Serum galectin-3 level was significantly higher in the patients with HCC compared to the healthy volunteers or the patients with chronic hepatitis (MD = 2.71, 95% CI = 1.56-3.85, p < 0.001) with a significant heterogeneity (I 2 = 86.9%, p < 0.001). Sensitivity analysis illustrated that the study by Akyuz et al. (42) displayed an apparent influence on the overall result of the meta-analysis (Supplementary Figure 5). After the exclusion of this study, the pooled MD was similar (MD = 2.28, 95% CI = 2.07-2.50, p < 0.001), but the heterogeneity was statistically insignificant (I 2 = 0.6%, p = 0.389).

Liver Failure
The relationship between galectin-3 and the risk of liver failure was explored in two study cohorts, both of which selected the healthy volunteers as the control subjects (33,35).

Liver Cirrhosis
The relationship between galectin-3 and the risk of liver cirrhosis was explored in six study cohorts, all of which selected healthy volunteers as the control subjects (25,36,38,40,48).
Serum galectin-3 level was significantly higher in the patients with liver cirrhosis compared to the healthy volunteers (MD = 1.83, 95% CI = 1.15-2.51, p < 0.001) with a significant heterogeneity (I 2 = 98.3%, p < 0.001). Sensitivity analysis did not find any source of heterogeneity.

Other Chronic Liver Diseases
The relationship between the galectins and the risk of other chronic liver diseases, including inactive hepatitis B, chronic active hepatitis B, non-alcoholic steatohepatitis, hepatitis C, autoimmune hepatitis, non-alcoholic fatty liver disease, and biliary atresia, was explored in 10 study cohorts. All of them selected healthy volunteers as the control subjects (25,34,37,39,(44)(45)(46)(47).
T cells and promote tumor growth in the mice receiving the tumor-reactive CD8 + T cells (54). Silencing of galectin-3 can significantly reduce the mRNA and protein levels of urokinase-type plasminogen activator receptor (uPAR) and downstream signaling transduction pathway of uPARs in the HCC cells by inhibiting the MEK/ERK signaling pathway, further influencing the proliferation, migration, and invasion of the HCC cells (55). Third, galectin-9 can inhibit the growth of the HCC cell lines by inducing cell apoptosis (56). Galectin-9 also increases the number of Tim-3 + dendritic cells and CD8 + T cells and enhances antitumor immunity through the interaction of galectin-9 with Tim-3 (57). By comparison, blockade of the Tim-3/galectin-9 signaling pathway importantly increases the functionality of tumor-infiltrating Tim-3 + T cells and is negatively associated with the survival of patients with HCC (58).
Another major finding of this study was that higher serum galectin-3 level was associated with an increased risk of liver failure, liver cirrhosis, and chronic active hepatitis B. Other evidence was also in favor of the importance of galectin-3 in these liver diseases. First, if the patients with acute-on-chronic liver failure related to hepatitis B had galectin-3 methylated promoter, they would have shorter survival time, higher 3-month mortality, and higher model for end-stage liver disease (MELD) score (59). Second, galectin-3 modulates the phagocytosis-induced hepatic stellate cell activation and liver fibrosis in vivo (60). Galectin-3 level is significantly higher in the Child-Pugh class C and positively correlates with the MELD score, suggesting the association of galectin-3 level with hepatic decompensation (61). By comparison, the galectin-3 inhibitor can reduce the hepatic venous pressure gradient in patients with esophageal varices (62). Third, galectin-3 deficiency can lead to a significant reduction in the incidence of concanavalin A-induced hepatitis in mice by inhibiting inflammation (63). This study did not find any significant association of serum galectin-3 level with inactive hepatitis B, non-alcoholic steatohepatitis, or non-alcoholic fatty liver disease. This illustrated that the impact of galectin-3 level on chronic liver diseases might be dependent upon the severity and stage of liver damage (40). Indeed, the evidence regarding the role of galectin-3 in non-alcoholic fatty liver disease and non-alcoholic steatohepatitis is also controversial. Some studies have shown that galectin-3 deficiency in male mice can spontaneously develop non-alcoholic fatty liver disease and more severe hepatic injury (64,65). In contrast, other studies have reported that galectin-3 ablation protected the mice from the diet-induced non-alcoholic steatohepatitis (66).
There were several limitations in this study. First, this meta-analysis contained a relatively small number of studies, which might lead to insufficient statistical power. Second, the cutoff values of high galectin expression were heterogeneous among the studies. Third, HR values were not directly reported in the six included studies, where their survival data were extracted from the Kaplan-Meier curves by the Engauge Digitizer 4.1 software. Fourth, most of the included studies were from Asia. Our findings are not a global representation.
In conclusion, based on this systematic review and metaanalysis, both high galectin-1 and -3 and low galectin-4 and -9 expression in the tissues were significantly related to worse prognosis and positive vascular invasion in patients with HCC and serum galectin-3 level was associated with the risk of HCC, liver failure, liver cirrhosis, and chronic active hepatitis B (Figure 3). Further studies are needed to explore the role of galectins as a potential therapeutic target and biomarker for liver diseases.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author/s.

AUTHOR CONTRIBUTIONS
XQ contributed to the conceptualization, supervision, and project administration. YA, SX, YL, XX, and XQ contributed to the methodology, formal analysis, data curation, and writing the original draft. YA, SX, YL, XX, CP, JC, NM-S, XG, and XQ contributed to the validation, writing, review, and editing. All authors contributed to the article and approved the submitted version.