Validity of serum resistin level and Il-6 as prognostic biomarkers of decompensated liver cirrhosis in chronic hepatitis C virus patients

Decompensated liver cirrhosis (DLC) is now known as a chronic inflammatory process, evidenced by elevated levels of circulatory pro-inflammatory cytokines and chemokines which in turn lead to the development of more hepatic decompensation and multi-organ failure. Resistin has a pro-inflammatory effect through the production of several cytokines (e.g., IL-1, IL-6, IL-12, and TNF-α) and cell adhesion molecules. Interleukin-6 (IL-6) is a proinflammatory cytokine playing a crucial role in acute phase responses and in regulating immune reactions through activation and differentiation of T and B lymphocytes. The current study aimed to evaluate the value of serum resistin and IL-6 as biomarkers of DLC and their role as prognostic markers of complications in these patients. This study was conducted on 90 patients divided into three groups: group I—30 patients with compensated cirrhosis (CLC); group II—40 patients with DLC; and group III consisted of 20 healthy controls. Serum resistin and IL-6 levels were statistically significantly higher in patients with DLC compared to patients with CLC at baseline. A cut-off value of > 302 pg/ml for serum resistin was found to discriminate between CLC and DLC with a specificity of 73.33% and sensitivity of 92.50% and a cut-off level of > 31 pg/mL for IL-6 differentiated between the two groups with a sensitivity of 85.0% and specificity of 76.67%. Patients with DLC were followed up for 3 months, 10 patients (25%) passed away, and 19 patients out of the remaining 30 (63.3%) patients developed complications including acute kidney injury, spontaneous bacterial peritonitis, variceal hemorrhage, encephalopathy, and hepatocellular carcinoma. Serum resistin and IL-6 were found to be significantly higher at baseline in those patients who developed complications or mortality after the follow-up period. In addition, there were positive correlations between IL-6 and resistin and MELD-NA and CRP. Serum resistin and IL-6 could be used as sensitive diagnostic and prognostic biomarkers of decompensated cirrhotic patients.


Background
Liver cirrhosis (LC) is considered the final pathway of many chronic liver diseases.It is an irreversible process in its advanced stages [1].It is one of the leading causes of death in adults [2].LC is classified into compensated and decompensated based on its natural history.Compensated liver cirrhosis (CLC) is an asymptomatic phase, while decompensated liver cirrhosis (DLC) is characterized by the occurrence of clinical deterioration in the form of ascites, encephalopathy, and variceal hemorrhage [3].
Patients with DLC have a worse prognosis than CLC.The median survival was ≤ 6 months in decompensated cirrhosis patients with Child-Pugh score ≥ 12 or a Model for End-stage Liver Disease (MELD) score ≥ 21 [4].
It is now recognized that DLC is a chronic inflammatory process, evidenced by elevated levels of circulatory pro-inflammatory cytokines and chemokines [5].This is thought to be caused by increased intestinal permeability and abnormal bacterial translocation.The release of pro-inflammatory molecules leads to the development of more hepatic decompensation and multi-organ failure [6].
Resistin is a protein formed of 108 amino acids that are named according to its supposed insulin resistance effect.It belongs to a family of cysteine-rich secretory proteins known as FIZZ (found in inflammatory zone proteins) [7].Besides its metabolic role in increasing insulin resistance [8], resistin has a pro-inflammatory effect through the production of several cytokines (e.g., IL-1, IL-6, IL-12, and TNF-α) and cell adhesion molecules [9].Although resistin is known to be secreted by human adipocytes, the most significant source appears to be mononuclear blood cells [10].High resistin level is associated with chronic inflammatory diseases like rheumatoid arthritis, inflammatory bowel disease, and chronic kidney disease [11].
IL-6 is a proinflammatory cytokine playing a crucial role in acute phase responses and in regulating immune reactions through activation and differentiation of T and B lymphocytes [12].IL-6 is considerably increased in pathological conditions like trauma, inflammation, and tumors [13].
Cirrhosis-associated immune dysfunction (CAID) occurring in patients with advanced cirrhosis is characterized by high levels of proinflammatory cytokines including IL-6 [14].An association between the degree of decompensation of liver cirrhosis, IL-6, and serum resistin levels has been suggested [15,16].

Methods
The aim of the work is to study the value of serum resistin and IL-6 as biomarkers of DLC and their role as prognostic markers to predict the development of complications in patients with DLC.
The current is a case-control study conducted on 70 patients with HCV-related liver cirrhosis divided into two groups.Group I consists of 30 patients with compensated cirrhosis.Group II contains 40 patients with decompensated liver cirrhosis.Twenty healthy subjects were taken as controls and comprised group III.
All patients were recruited from Tropical Medicine Department, Faculty of Medicine, Alexandria University, in the period between March 2021 and December 2021, after approval of the local ethical committee of Alexandria University.All patients were given an informed consent.
Sample size was calculated using med cal, according to Mariadi et al. [17] who found that IL-6 and resistin differentiated between patients with CLC and DLC with a minimum sample size of 70 patients.
Diagnosis of cirrhosis was based on FIB-4 score [20] as well as radiological evidence of cirrhosis by ultrasound evident by the shrunken liver with coarse echogenicity and manifestations of portal hypertension, ascites, and splenomegaly.FIB-4 > 3.25 was the cut-off value taken to diagnose liver cirrhosis.Patients with liver cirrhosis were classified according to Child-Pugh Score (C-P score) and MELD score to assess the degree of decompensation [21].
DLC patients were subjected to follow-up after 3 months to assess mortality and development of complications namely acute kidney injury after exclusion of causes unrelated to liver cirrhosis defined by either an absolute increase in serum creatinine (SCr) of more than or equal to 0.3 mg/dl in less than 48 h or a percentage increase in SCr of more or equal to 50% (1.5-fold from baseline) in less than 7 days [22], variceal hemorrhage, spontaneous bacterial peritonitis diagnosed based on neutrophil count in ascitic fluid of > 250/ mm 3 [23], and hepatocellular carcinoma, whom diagnosis was based on imaging techniques obtained by multiphasic CT or dynamic contrast-enhanced MRI showing arterial phase hyperenhancement (APHE) with washout in the portal venous or delayed phases [24].

Exclusion criteria
Patients with diabetes mellitus (DM), chronic kidney disease (CKD), malignancy, coronary artery disease, rheumatoid arthritis (RA), inflammatory bowel disease (IBD), and those taking immunosuppressive drugs or steroids were excluded.

Statistical analysis
Data were fed to the computer and analyzed using IBM SPSS software package version 20.0.(Armonk,NY: IBM Corp).Qualitative data were described using number and percent.The Shapiro-Wilk test was used to verify the normality of distribution.Quantitative data were described using range (minimum and maximum), mean, standard deviation, median, and interquartile range (IQR).Significance of the obtained results was judged at the 5% level.

Results
This   At a cut-off value of > 302 pg/ml serum resistin was found to discriminate between compensated and decompensated cirrhosis with specificity of 73.33% and sensitivity of 92.50% as shown in Fig. 1 and Table 2. Regarding IL-6, a cut-off level of > 31 pg/mL differentiated between the two groups with a sensitivity of 85.0% and specificity of 76.67% as shown in Fig. 1 and Table 2.
Serum resistin and IL-6 showed a significant positive correlation with each other as shown in Table 3, in addition to positive correlations with CRP, MELD-NA, FIB-4, and Child-Pugh score as shown in Table 3.
After follow-up of 40 patients with DLC after 3 months, 10 patients ( 25%) passed away and out of the remaining 30 patients, 19 ( 63.3%) patients developed complications including acute kidney injury, spontaneous bacterial peritonitis, variceal hemorrhage, and encephalopathy while 11 patients did not develop any complication as shown in Table 4.As regards the 10 patients who passed away, they died due to complications related to cirrhosis namely acute on chronic liver failure, sepsis, and variceal hemorrhage (Table 5).
A cut-off value of > 480 pg/ml for serum resistin was found to predict the development of complications in DLC at 3-month follow-up period with 90.91% and 96.55% sensitivity and specificity respectively.Regarding IL-6, a cut-off level of > 37 pg/mL was found with a sensitivity of 93.10% and specificity of 90.91% as shown in Table 6.

Discussion
It is well known now that decompensated liver cirrhosis is a chronic inflammatory status [5].Resistin and IL-6 levels which are both known to have pro-inflammatory effects were studied to assess their association with decompensated liver cirrhosis and evaluate their potential value to predict the development of complications in those patients (Table 7).
In our study, there was a statistically significant higher level of serum resistin in patients with decompensated liver cirrhosis compared to compensated patients.At baseline, higher levels of resistin were observed in patients with decompensated liver cirrhosis who developed complications or died after a period of 3 months' follow-up.In addition, significant positive correlations were found between resistin level and IL-6 and both were correlating positively to MELD score, C-P score, and CRP.This was in concordance with Yagmur et al. who found that serum resistin was correlated positively with markers of inflammation such as tumor necrosis factor-alpha (TNF-α) or C-reactive protein (CRP), as well as with clinical complications, e.g., portal hypertension [25].Also, Mariadi et al. found the same positive correlation between resistin level and IL-6 and CRP levels [17].
Human resistin is among the inflammatory regulators for guiding the subsequent actions of inflammation in macrophages, peripheral blood mononuclear cells (PBMNs), and vascular cells.When these cells are stimulated with recombinant human resistin, they produce tumor necrosis factor-alpha (TNF-α), IL-6, IL-12, and monocyte chemotactic protein-1 (MCP-1) through nuclear factor kappa B (NFκB)-mediated pathway [26].Human resistin expression is elevated during the pathological conditions of inflammation.Circulatory resistin level has been positively correlated with common inflammatory and fibrinolytic biomarkers such as CRP, TNF-a, and IL-6 [27].Moreover, these findings were in concordance with what Costa et al. who studied biomarkers of systemic inflammation across different stages of advanced chronic liver disease ( ACLD) and found statistically significant higher levels of IL-6 in decompensated stages compared to compensated stages [28].
It was also noted that IL-6 levels showed a statistically significant positive correlation with CRP levels.This was in concordance with Turco et al. who demonstrated that CRP levels increased progressively across stages of compensated and decompensated liver cirrhosis patients [29].In our research, decompensated cirrhosis patients who have IL-6 levels more than 37 pg/ml at baseline showed a statistically significant higher probability to develop complications and mortality in 3-month period of follow-up.This was in concordance with Costa et al. who found that decompensated patients.withhigh IL-6 of more than 14 pg/ml showed a significantly higher probability of liver-related death/LT [28].The difference in the cut-off valve of IL-6 to predict the probability of complications may be related to the difference in the serological kits for IL-6 used.The smaller sample size in our study may be another contributing factor to this difference.
In addition, higher levels of resistin levels were found in decompensated cirrhosis patients who developed complications and mortality in 3 months of follow-up with cut-off predictive value of more than 480 pg/ml.This was in line with Yagmur et al. who showed that high resistin levels were an unfavorable prognostic indicator for overall survival with serum resistin value of more than 5 μg/L [25].The difference in the cut-off value is likely related to different serological kits used to measure serum resistin and it may be related to the differences in the studied population on which the research was conducted.
However, unlike our observation, Da Silva et al. found that resistin levels were not associated with survival in cirrhotic patients [30].This may be attributed to due to the smaller proportion of patients with more severe liver diseases, only 3 out of 122 patients were Child-Pugh C in this study.It may be also due to differences in the etiology of cirrhosis across different studies.

Limitation of study
• Small sample size • Longer period of follow-up required to determine a more precise cut-off value for the prediction of complications.

Conclusion
Serum IL-6 and resistin could be used as sensitive diagnostic and prognostic biomarkers of decompensation in cirrhotic patients.Furthermore, the significant correlation of serum resistin and IL-6 with Child-Pugh and MELD score could suggest that they can be used as a tool to follow up the clinical outcomes of patients with liver cirrhosis and prioritize listing for liver transplantation.

Fig. 1
Fig.1ROC curve for resistin level and IL-6 level to discriminate between decompensated liver cirrhosis patients (n = 40) from compensated liver cirrhosis (n = 30) (group II vs I)

Table 3
Correlation between resistin level, IL-6 level, and different parameters in group II (decompensated liver cirrhosis) (n = 40) * Statistically significant at p ≤ 0.05 AKI Acute kidney injury; SBP Spontaneous bacterial peritonitis; HCC Hepatocellular carcinoma

Table 6
Validity (AUC, sensitivity, specificity) for IL-6 level and resistin level to discriminate DLC who developed complications (n = 29) from non-complicated DLC (n = 11) patients at follow-up

Table 7
Univariate and multivariate logistic regression analysis for complicated and mortality regarding different factors (n = 29 vs. 11) OR Odds ratio; C.I Confidence interval; LL Lower limit; UL Upper limit p, p value for odds ratio for comparing the studied groups * Statistically significant at p ≤ 0.05