Association of 5-Hydroxytryptamine 3 Receptor Antagonists With the Prognosis of Liver Failure

Liver failure is a severe clinical syndrome with high mortality. 5-Hydroxytryptamine 3 receptor antagonists (5-HT3RAs) can reduce liver damage in animal models. We investigated whether 5-HT3RAs may improve the prognosis of liver failure. We analyzed the 28 and 90 days mortality of liver failure patients in relation to the use of 5-HT3RAs using data from a tertiary hospital in northwest China. According to the use of 5-HT3RAs, 419 patients with liver failure (46 acute, 93 sub-acute, 44 chronic, 236 acute on chronic) were divided into 5-HT3RA group (n = 105) and control group (n = 314). 5-HT3RAs were associated with decreased 28 days (HR 0.18, 95% CI 0.10-0.34, p < 0.001) and 90 days (HR 0.21, 95% CI 0.13-0.33, p < 0.001) mortality. After propensity score matching (PSM) (n = 67 in each group), 5-HT3RAs were still significantly associated with reduced 28 days (HR 0.10, 95%CI 0.04-0.26, p < 0.001) and 90 days (HR 0.16, 95%CI 0.08-0.31, p < 0.001) mortality. 5-HT3RA group patients had significantly higher 28 and 90 days survivals than controls both before and after PSM (all p < 0.001). This study shows that 5-HT3RAs are associated with increased survival of liver failure patients and thus may be used to treat liver failure if the findings are confirmed by additional studies.


INTRODUCTION
Liver failure, a severe hepatic damage caused by various etiologies, is a devastating clinical syndrome of liver disease with many complications and high mortality. It manifests a group of clinical symptoms including jaundice, coagulation function disorders, hepatorenal syndrome, hepatic encephalopathy (HE), and ascites (Liver Failure and Artificial Liver Group, 2019). The treatment of liver failure is a challenging clinical problem due to the lack of effective approaches. Liver transplantation (LT) may improve the prognosis of liver failure (Lee et al., 2008;Artru et al., 2017). However, LT has many limitations, including the shortage of organs, surgical risks and the long-term use of immunosuppressants that may induce complications. Therefore, novel and alternative treatments are urgently needed for liver failure. 5-Hydroxytryptamine 3 receptors (5-HT3Rs), also known as the Cys-loop superfamily in eukaryotes, are cation-conducting pentameric ligand-gated ion channels found in the peripheral and central nervous system (Gaddum and Picarelli, 1997;Hargreaves et al., 1994). 5-HT3Rs are targeted by many therapeutic drugs such as the 5-HT3R antagonists (5-HT3RAs) that are widely used to prevent and treat nausea and vomiting caused by cancer chemotherapy, radiotherapy, anesthesia, and post-operation (Faerber et al., 2007). Anti-oxidative, anti-inflammatory and immunomodulatory properties have been observed for 5-HT3RAs (Fiebich et al., 2004a;Fiebich et al., 2004b;Stratz et al., 2012;Gong et al., 2019). At the same time, experiments in animals have indicated that 5-HT3RAs can alleviate liver damage induced by various causes (Fiebich et al., 2004a;Haub et al., 2011;Liu et al., 2011;Gholizadeh-Ghaleh Aziz et al., 2019;Amini et al., 2020).
In view of the anti-oxidative, anti-inflammatory and immunomodulatory properties (Fiebich et al., 2004a;Fiebich et al., 2004b;Stratz et al., 2012;Gong et al., 2019) and the protective effect on liver damage in animal models (Fiebich et al., 2004b;Haub et al., 2011;Liu et al., 2011;Gholizadeh-Ghaleh Aziz et al., 2019;Amini et al., 2020), we hypothesize that 5-HT3RAs may potentially influence the disease outcome in patients with liver failure. To this end, we performed a retrospective study to evaluate whether the use of 5-HT3RAs may affect the prognosis of patients with liver failure.

Study Population
This study included patients who were diagnosed with liver failure in the First Affiliated Hospital of Xi'an Jiaotong University from May 2013 to June 2019. The inclusion criteria in the patients were as follows: 1) age ≥18 years; and 2) clinical diseases met the diagnosis standards of "Guideline for diagnosis and treatment of liver failure (2018)" (Liver Failure and Artificial Liver Group, 2019). The exclusion criteria in the patients were as follows: 1) age <18 years; 2) pregnancy; 3) malignant tumor; 4) history of liver transplantation; 5) circulatory failure (Shock, congestive heart failure, etc.); and 6) incomplete data for analysis. The study protocol conforms to the ethical guidelines of the 1975 Declaration of Helsinki and the Ethic Committee of the First Affiliated Hospital of Xi'an Jiaotong University approved the study (XJTU1AF2020LSK-203) and waived the need of informed consent.

Definitions
The definitions of liver failure are based on the "Guideline for diagnosis and treatment of liver failure (2018)" formulated by Liver Failure and ArtificiaI Liver Group, Chinese Society of Infectious Diseases, Chinese Medical Association; Severe Liver disease and Artificial Liver Group (Liver Failure and Artificial Liver Group, 2019). This Guideline referred the definitions from the Asian Pacific Association for the Study of the Liver (Sarin et al., 2014). Briefly, the criteria for the diagnoses of liver failures were as follows: Acute liver failure (ALF): The occurrence of at least grade II HE within 2 weeks of the onset of symptoms in the absence of pre-existing liver disease with the following manifestations: 1) extreme fatigue, accompanied by severe gastrointestinal symptoms such as anorexia, abdominal distension, nausea, and vomiting; 2) progressively deepening jaundice [serum total bilirubin (TBIL) ≥10×upper limit of normal (ULN) or daily increase ≥17.1 μmol/l]; 3) hemorrhage tendency [prothrombin activity (PTA) ≤40%, or international normalized ratio (INR) ≥1.5 without other causes]; and 4) progressive liver shrinkage.
Acute-on-chronic liver failure (ACLF): On the basis of a preexisting chronic liver disease, the development of liver failure induced by various precipitating events or unidentifiable events and manifestation of acute jaundice deepening (TBIL ≥10 × ULN or daily increase ≥17.1 μmol/l), coagulation dysfunction (PTA ≤40% or INR ≥1.5), HE, ascites, electrolyte disorders, secondary infection, hepatorenal syndrome, hepatopulmonary syndrome, and the failure of extrahepatic organs.

Data Collection
Demographic, clinical, treatment, and laboratory data were collected from the electronic medical records. Demographic data included gender, age, ethnic (Han and non-Han), smoking, and alcohol drinking. Clinical data included etiology of the liver disease, classification of liver failure, hypertension, diabetes mellitus, nausea and vomiting symptoms, and complications (bleeding, ascites, infection, HE, hepatorenal syndrome, hepatopulmonary syndrome, electrolyte disturbance, and encephaledema). Treatment included artificial liver support treatment [plasma exchange (PE), plasma adsorption (PA), continuous renal replacement therapy (CRRT)], use of antivirals in hepatitis B virus (HBV) infected patients, anti-infective treatment, and the use of 5-HT3RAs. Laboratory data included liver and renal biochemistry, serum potassium, serum sodium, routine hematology, prothrombin time (PT), PTA, and INR. The model for end-stage liver disease (MELD) score was calculated in the patients based on laboratory data.
Exposure to 5-HT3RAs and Outcomes 5-HT3RAs were used in patients with liver failure who needed symptomatic treatment for nausea and/or vomiting according to Frontiers in Pharmacology | www.frontiersin.org April 2021 | Volume 12 | Article 648736 the judgment of clinicians. 5-HT3RAs used included ondansetron, granisetron, palonosetron, and tropisetron. Dosing regimes: ondansetron hydrochloride injection (4 ml: 8 mg), a single dose of 8 mg for intravenous injection (I.V.). Granisetron hydrochloride injection (3 ml: 3 mg), a single dose of 3 mg for I.V. Palonosetron hydrochloride injection (5 ml: 0.25 mg), a single dose of 0.25 mg for I.V. Tropisetron hydrochloride injection (5 ml: 5 mg), a single dose of 5 mg for I.V. The number of times of 5-HT3RA use was divided into <2 times or ≥2 times. The primary outcomes were 28 and 90 days mortality, defined as death or liver transplantation.

Statistical Analysis
Categorical variables were described as frequencies (percentage) and were compared using the Pearson chi-squared test or Fisher's exact test. Continuous variables were tested for normality. The normally distributed variables were described using mean ± standard and were compared using t-test. The non-normally distributed variables were expressed as the median (max, minimum) and were compared using Mann-Whitney U test. Patient 28 and 90 days survivals were estimated by Kaplan-Meier curves with log-rank tests for differences. In addition, collinearity between variables was detected by calculating the correlation coefficient and the p value between the variables. It was generally considered that the correlation coefficient >0.7, and p < 0.05 can be considered the existence of collinearity between the variables. In the two statistics of collinearity diagnosis of variables, namely tolerance and variance inflation factor (VIF), tolerance <0.2 or VIF >10 indicated that there was multicollinearity between variables. After calculation, the correlation coefficients between the variables in this study were all <0.7, tolerance >0.2 and VIF <10. Therefore, there was no collinearity between variables of this study. Cox proportional hazards regression model was used to assess the impact of all baseline covariates on the outcomes. In the regression model, hazard ratio (HR) and its 95% confidence intervals (CI) were calculated for each variable. Propensity score matching (PSM) was used to minimize the influence of data bias and confounding variables on the outcome. After calculating the propensity score, we used the 5-HT3RA group and the control group for patients with similar propensity scores using a 1:1 ratio matching, and the propensity score allowed the difference between the two groups to be 0.05. Inverse probability treatment weighting (IPTW) was also used. PSM and IPTW were performed used EmpowerStats 2.0 software, and the other statistical analyses used SPSS 25.0 statistical software. According to the test level of α 0.05, p < 0.05 was considered as statistically significant.

Characteristics of the Patients
During the study period, 509 patients were diagnosed with liver failure in the First Affiliated Hospital of Xi'an Jiaotong University. Of these patients, 67 patients were excluded from our study (5 aged <18 years, 1 pregnant, 54 with malignant tumors, 2 with a history of liver transplantation, 2 with circulatory failure, and 3 patients with incomplete data). There were 442 patients eligible for the study. After exclusion of 23 patients who were lost to follow-up, 419 patients were finally included in the study (Supplementary Figure S1). According to the use of 5-HT3RAs, the patients were divided into 5-HT3RA group (n 105) and control group (n 314, Table 1). PSM was performed to match all characteristics and 134 patients were selected (67 patients in each group, Table 1). The characteristics after IPTW were shown in Supplementary Table S1.
Among all the patients, males made up a higher proportion (61.9% and 70.7% in the 5-HT3RA group and the control group, respectively). The age in the 5-HT3RA group and the control group had no statistical difference. In terms of etiology, hepatitis B virus (HBV) accounted for the largest proportion (58.1% and 55.1% in 5-HT3RA group and control group, respectively). ACLF accounted for the largest proportion (59% and 55.4% in 5-HT3RA group and control group, respectively). The proportion of diabetes mellitus patients in the control group was significantly higher than that in the 5-HT3RA group (10.8% vs. 2.9%, p 0.013). The proportion of patients with nausea and vomiting symptoms (71.4% vs. 17.5%, p < 0.001), bleeding (40.0% vs. 23.6%, p 0.001), secondary infection (86.7% vs. 72.6%, p 0.003), hepatorenal syndrome (14.3% vs. 6.7%, p 0.016), PE (50.5% vs. 33.1%, p 0.001), CRRT (16.2% vs. 8.6%, p 0.028), and anti-infective therapy (96.2% vs. 80.9%, p < 0.001) in the 5-HT3R group was significantly higher than that in the control group (Table 1). After PSM, the differences between the two groups were not statistically significant ( Table 1). There was no significant difference in the baseline laboratory parameters between the two groups of patients (Supplementary Table S2).

Characteristics of Patients With the Use of 5-HT3RAs According to Classifications of Liver Failure
According to the classifications of liver failure, patients with ALF, CLF, and ACLF had more males than females, except for patients with SALF. In patients with ALF, the main causes were severe infection (40%) and unknown reason (40%). The main cause of SALF was drug-induced (45.5%). The main cause of CLF and ACLF was HBV. Ondansetron accounted for the largest proportion of 5-HT3RAs used in all the patients (>70%). Among the patients with different liver failures, gender, etiology, the use of ondansetron, PE, CRRT, and antiviral therapy were statistically different (Supplementary Table S3).
Comparison of 90 days survivors and non-survivors showed that nausea and vomiting symptoms, bleeding, ascites, secondary infection, HE, hepatorenal syndrome, electrolyte disturbances, use of 5-HT3RAs, the times of 5-HT3RAs used, CRRT, antiinfective treatment, and MELD scores were significantly different (Supplementary Table S5).
In terms of different 5-HT3RA drugs, the 28 and 90 days survivals of patients who used ondansetron were significantly higher than those who did not (28 days: 93.1% vs. 66.9%, p < 0.001; 90 days: 80.5% vs. 48.1%, p < 0.0010, respectively, Supplementary Figures S3A,B, Table 4). The subgroup analysis of patients using ondansetron according to the classifications of liver failure resulted in similar results to the subgroup analysis in all the 5-HT3RAs (Supplementary Figures  S3C-J). Subgroup analysis was not performed in other 5-HT3RAs because of the small number of patients.

DISCUSSION
This study is the first population-based study to establish an association between 5-HT3RAs and prognosis of patients with liver failure. In the patients included, there were more males than females. The etiologies of liver failure were varied and the 3 leading causes were HBV, alcohol, and drug. Drug was the main cause of SALF, accounting for 45.5%. There were 15.5% of the patients with indeterminate etiology. These results were similar to the report of southwest China (Xie et al., 2016), but different from the report of western countries (Sarin et al., 2019). ACLF was the predominant clinical type of liver failure, a finding similar to the report from other area of China (Liu et al., 2008).
This study showed that, in all patients and patients after PSM, HE, hepatopulmonary syndrome, the use of 5-HT3RAs, antiviral therapy and MELD score were independent influencing factors for 28 and 90 days outcome. It has been well known that the presence of complications such as HE and hepatopulmonary syndrome is a major risk factor for mortality in liver failure patients (Chen et al., 2019), antiviral treatment is effective in decreasing the short-term fatality of HBV-related ACLF (Zhang et al., 2014;Li et al., 2017), and the MELD score is widely used to predict the prognosis of liver failure (Katoonizadeh et al., 2007;Mishra and Rustgi, 2018). Most importantly, the use of 5-HT3RAs was associated with substantially reduced 28 and 90 days mortality in the patients with liver failure. This finding was consistently observed in unadjusted, multivariate-adjusted, propensity score-matched, and inverse probability treatment weighting analysis. The survival rate of liver failure patients with the use of 5-HT3RAs was significantly higher than that of patients without the use. Though, the difference of 28 and 90 days survivals in patients with ALF and CLF and in etiologies other than HBV was no statistically significant in subgroup analyses that may be related to the small numbers of patients in these subgroups. Our findings are consistent with previous studies in animal models showing that the 5-HT3RAs were able to reduce liver damage caused by various reasons including sepsis (Fiebich et al., 2004a), hemorrhagic shock (Liu et al., 2011), diabetes induced (Gholizadeh-Ghaleh Aziz et al., 2019Amini et al., 2020) and obesity-associated (Haub et al., 2011) liver injuries.
The mechanisms pertinent to the association of 5-HT3RAs with a significantly reduced risk of mortality in liver failure patients might be multiple and complex. First, 5-HT3RAs have anti-oxidative, anti-inflammatory and immunomodulatory properties (Fiebich et al., 2004a;Fiebich et al., 2004b;Stratz et al., 2012;Gong et al., 2019). Experiments in animals have indicated that 5-HT3RAs can alleviate liver damage induced by various causes via antioxidant and anti-inflammatory effects (Fiebich et al., 2004a;Haub et al., 2011;Liu et al., 2011;Gholizadeh-Ghaleh Aziz et al., 2019;Amini et al., 2020). Host immune dysregulations including T cell responses are considerably involved in the development of liver failure (Khamri et al., 2017) while 5-HT3RA tropisetron was found to suppress T cell activation (Vega et al., 2005). Therefore, the immunoregulatory action of 5-HT3RAs may also contribute to the effect on liver failure. Second, intestinal motility and permeability and gut microbiota play important role in hepatic disorders (Bajaj et al., 2014;Chopyk and Grakoui, 2020). 5-HT3R is a key factor in the regulation of intestinal motility and permeability. 5-HT3RAs such as tropisetron can reduce hepatic lesions of obesity-associated fatty liver disease in mice by reducing portal vein plasma endotoxin levels, attenuating the increased MyD88 and tumor necrosis factor-α mRNA expression in the liver, and increasing tight junction proteins in the duodenum (Haub et al., 2011). Gut microbiota can regulate 5-HT3R expression and modulate host secretory response to 5-HT (Bhattarai et al., 2017). In sepsis-induced liver injury mice, granisetron, a 5-HT3RA, generated by intestinal microbiota confers resistance to sepsis, protects mice against death and liver injury, and reduces proinflammatory cytokine expression by macrophages after lipopolysaccharide challenge (Fiebich et al., 2004a). In septic patients, gut microbial granisetron levels were negatively correlated with plasma transaminase levels (Fiebich et al., 2004b). Therefore, 5-HT3RAs may exert hepatoprotective effect on liver failure by the regulation of intestinal motility and permeability and the involvement of gut microbiota. Third, inflammation-associated mitochondrial dysfunction is revealed to be a potential mechanism of organ failures in ACLF (Moreau et al., 2020). 5-HT3RA tropisetron is shown to alleviate the mitochondrial dysfunction of the cerebral cortex in mice following social isolation stress (Haj-Mirzaian et al., 2016). Thus, the mitochondrial dysfunction-attenuating effect of 5-HT3RAs may potentially play a role in alleviating liver failure.
Fourth, multiple organ failure is directly related to the mortality of liver failure patients (Arroyo et al., 2015;Mahmud et al., 2020). 5-HT3RAs have multiorgan protective effects. For example, 5-HT3RA tropisetron shows renal protective effect on early diabetic nephropathy (Barzegar-Fallah et al., 2015) and improves pancreas function, increases insulin synthesis and secretion, and attenuates pancreas apoptosis in streptozotocin-induced diabetic rats (Naderi et al., 2020a;Naderi et al., 2020b). 5-HT3RAs, ondansetron, tropisetron and palonosetron, show anti-inflammatory effect, restore the delayed gastrointestinal transit and exhibit therapeutic action against post-operative ileus (Maehara et al., 2015). In mice, ondansetron shows potential therapeutic effect for Alzheimer's disease (Shinohara et al., 2019) and tropisetron shows protective action against brain aging (Mirshafa et al., 2020). These findings suggest that the multiple organ protective effect of 5-HT3RAs may potentially play a paramount role in reducing the mortality of liver failure. Fifth, thrombotic disorders including portal vein thrombosis and intrahepatic microthrombosis may be involved in the pathogenesis of liver disease including liver failure (Pieri et al., 2013). A recent study demonstrated that ondansetron considerably reduces the risk of hospital-acquired venous thromboembolism (Datta et al., 2020). Therefore, 5-HT3RAs such as ondansetron may also exert a therapeutic effect on liver failure through reduction of thrombotic disorders.
Of note, the doses of 5-HT3RAs used in the patients of this study were very small and this may call into question of the therapeutic effect on liver failure. The 5-HT3RAs used such as ondansetron are highly selective and preclinical studies in rat have shown the effects of small doses of ondansetron on cognition, behavioural sensitisation, and epilepsy (Kwan et al., 2020). In a recent study, perioperative use of 5-HT3RAs (palonosetron or ramosetron) is associated with increased recurrence-free survival in patients after thoracotomy for lung cancer although the doses used were not presented in detail and ondansetron was excluded from the 5-HT3RAs (Lee et al., 2019). It is suggested that small doses of 5-HT3RAs may also exhibit peripheral effect. Furthermore, we did not observe a dosedependent effect of the 5-HT3RAs on liver failure. Therefore, the optimal dose and timing of 5-HT3RAs need to be determined in future studies.
Lastly and importantly, there are many potential limitations or confounders that may influence the results of our study. First, this study is a retrospective study, and there may have been some confounding factors and clinical variables that we were unable to measure and control for. Though, we adopted a variety of strategies such as multivariate adjustment, PSM and IPTW to reduce the influence of data bias and confounding variables on the outcome. Second, single center and small sample size of the study may also compromise the potency of our analyses, especially the subgroup analyses. It is difficult to obtain large samples of patient data at present owing to the unconventional use of 5-HT3RAs for liver failure. Third, the etiology of liver failure in this study was mainly HBV and thus the findings might not be applicable to liver failure mainly related to other reasons although we included patients with liver failure associated with etiologies other than HBV in the analysis. Fourth, this study was Frontiers in Pharmacology | www.frontiersin.org April 2021 | Volume 12 | Article 648736 unable to include a validation analysis due to the lack of other eligible patient populations. Therefore, additional studies are definitely needed to verify the findings of this study.

CONCLUSION
This study suggests that the use of 5-HT3RAs is associated with significantly increased short-term survivals of patients with liver failure, especially SALF and ACLF. Due to the lack of effective medical treatment, the poor prognosis of liver failure and the clinical safety profile of 5-HT3RAs in humans, our findings might have considerable clinical implications for the treatment of liver failure. High quality multi-center, large-sample, prospective, randomized and double-blind controlled studies are required to confirm our findings.

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.

ETHICS STATEMENT
The studies involving human participants were reviewed and approved by the Ethic Committee of the First Affiliated Hospital of Xi'an Jiaotong University. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.

AUTHOR CONTRIBUTIONS
YC participated in the conceptualization, data curation, formal analysis and writing-original draft. JS participated in data curation, formal analysis, and writing-review and editing. XF participated in data curation, formal analysis and writing -review and editing. XW participated in data curation, formal analysis and writing-review and editing. LZ participated in data curation, formal analysis and writing -review and editing. XZ participated in data curation, formal analysis and writing-review and editing. KZ participated in data curation, formal analysis and writingreview and editing. NL participated in data curation, formal analysis, resources and writing-review and editing. QH participated in conceptualization, resources, supervision and writing-review and editing. ZL participated in conceptualization, resources, important conceptual inputs, supervision, and writing-review and editing.