Role of serum Nogo-B as a biomarker for diagnosis of chronic liver diseases and its severity

Nogo-B is one of the members of the reticulon family. Nogo-B influences the proliferation of the hepatic stellate cells inducing liver fibrotic changes. We aimed at measuring the serum levels of Nogo-B in patients with chronic liver disease (CLD) with different etiologies. Ninety subjects were included, 18 of them were normal healthy individuals and 72 had liver disease (fibrosis/cirrhosis) with different etiologies: post-hepatitis C infection, post-hepatitis B infection, NASH, and autoimmune hepatitis. Serum Nogo-B was assessed using ELISA. Patients were subdivided according to the Child-Pugh score into 3 groups: group 1—Child A (24 patients); group 2—Child B (24 patients); and group 3—Child C (24 patients). Serum Nogo-B levels were found to be significantly higher in patients (1477.92 ± 1113.50) when compared with healthy control (301.28 ± 180.87) (p < 0.001). There was a statistically significant difference in serum Nogo-B level between the three sub-groups of patients (p < 0.001). A positive correlation was found between serum Nogo-B and MELD score (r = 0.46, p-value < 0.001). However, there was no correlation found between Nogo-B and FIB-4 index or APRI score. There was a significant positive correlation between serum Nogo-B level and coagulation profile and serum bilirubin. An inverse correlation was found between serum Nogo-B with serum albumin. A ROC curve was done to examine the validity of Nogo-B in the diagnosis of liver cirrhosis, and the area under the curve was found to be 0.979, a cutoff value of 600 with a sensitivity of 97.2% and a specificity of 94.4% (p-value < 0.001). Nogo-B had a high value in the identification of patients with any severity of CLD. There is a highly significant correlation between Nogo-B and the synthetic function of the liver; it could be used as a measure of hepatic functional reserve.


Background
Liver cirrhosis is a major health problem in Egypt and worldwide. Liver diseases are on the top of the etiologies of mortality in Egypt, and it is predicted that there will be an upsurge in liver cirrhosis and hepatocellular carcinoma in the future [1]. The liver has an exceptional capacity to regenerate through various signaling pathways [2]. The regeneration process involves mechanisms which are still obscure and not fully understood.
Nogo-B is one of the members of the reticulon family. The reticulon family of proteins consists of 4 types of proteins which are primarily localized in the endoplasmic reticulum [3]. These proteins are highly expressed in the central nervous system and skeletal muscles [4]. They regulate the ER functions and structure and have an influence on protein trafficking and cell signaling regulation [5,6]. There are four reticulon genes and each gene encodes multiple isoforms. There are 3 gene products for reticulon 4 which are isoform A, B, and C [7]. Reticulon-4B or Nogo-B (Rtn-4B), 55 kDa protein, is found in many tissues, and one of its most important functions is the regulation of vascular remodeling and wound healing [8].
Nogo-B has a role in pathological vascular conditions through its ability to inhibit the migration and proliferation of smooth muscle cells while it enhances the endothelial cell migration [4]. Nogo-B influences proliferation and regeneration of the hepatic stellate cells [9] and induces liver fibrotic changes through stimulation of transforming growth factor β and its TGF β/Smad2 signaling pathway in fibroblasts [10].
Some studies revealed that Nogo-B levels were elevated in liver cirrhosis when compared to healthy controls [11]. Other studies examined the relationship between the levels of Nogo-B and the different clinical characteristics of liver cirrhosis, but the results were still controversial. The expression of Nogo-B in nonparenchymal liver cells was examined, and the levels of tissue Nogo-B were found to be elevated in the patients with liver cirrhosis and also correlated significantly with Child-Pugh scores [12].
In this study, the levels of serum Nogo-B in patients with hepatic dysfunction due to different etiologies were assessed.

Methods
This study was conducted in the Internal Medicine Department, Kasr El-Aini Hospital. A written informed consent was obtained from each participant or a responsible family member after explaining the possible complications of the diagnostic procedures. This study was submitted to and approved by the ethics committee of Kasr Alainy Hospital, Faculty of Medicine, Cairo University, with approval reference number [I-010316].
This observational case-control study included 90 subjects, 18 subjects were normal healthy individuals and 72 patients with liver disease (fibrosis/cirrhosis) due to the different etiologies such as post-hepatitis C infection, post-hepatitis B infection, NASH, and autoimmune hepatitis.
Patients were subjected to full clinical history and examination. They were classified into 4 groups: control group-composed of 18 normal healthy persons; group І-24 patients with chronic liver disease, Child A; group II-24 patients with chronic liver disease, Child B; and group III-24 patients with chronic liver disease, Child C. Liver function tests and liver enzymes were also done: serum albumin, prothrombin time, concentration, INR, serum bilirubin, ALT, and AST, and renal function tests: serum urea, creatinine, and complete blood count.
Abdominal ultrasound and upper gastrointestinal endoscopy were done to all patients. Patients with other etiologies of liver affection rather than hepatitis C (HBV, AIH, NASH) were included in the study according to their past documented investigations.
The exclusion criteria included the following: patients have diseases which might affect plasma Nogo-B levels as cardiovascular disease (including hypertension), central nervous system disorders, chronic obstructive pulmonary diseases (COPD), and pulmonary artery hypertension.
Samples were withdrawn, left to clot at room temperature for 10 min, and then centrifuged at the speed of 2000-3000 rpm for 5 min, and the supernatant was collected and stored at − 20°C till the time of analysis.

Statistical methods
Data were coded and entered using the statistical package SPSS (Statistical Package for the Social Sciences) version 24. Data was summarized using mean, standard deviation, median, minimum and maximum in quantitative data, and frequency (count) and relative frequency (percentage) for categorical data. Comparisons between quantitative variables were done using the nonparametric Kruskal-Wallis and Mann-Whitney tests [13]. For comparing categorical data, the chi-square (χ 2 ) test was performed. The exact test was used instead when the expected frequency is less than 5. Correlations between quantitative variables were done using the Spearman correlation coefficient. ROC curve was constructed with the area under curve analysis performed to detect the best cutoff value of Nogo-B, MELD, APRI, and FIB-4 for the detection of patients [14].

Results
Ninety participants were recruited in this study. They were divided into patients' group (72 patients with liver diseases of different etiologies) and control group (18 normal healthy individuals, age-and sex-matched). The mean age of the patients was 53.72 ± 12.46 years, 47 males (65.3%) and 25 females (34.7%), while the mean age of the control subjects was 36.00 ± 9.94 years and divided into 9 males (50%) and 9 females (50%).
Patients were further classified according to the Child-Pugh score into 3 groups: group 1-Child A (24 patients); group 2-Child B (24 patients); and group 3-Child C (24 patients). As for the liver disease etiologies, 54 patients had HCV infection (75%), 6 patients had HBV infection (8.3%), 3 patients had autoimmune hepatitis (4.1%), and 9 patients had non-alcoholic steatohepatitis (12.5%). Clinical and laboratory data of the patients and the healthy control groups are listed in Table 1.
Serum Nogo-B levels were found to be significantly higher in patients (1477.92 ± 1113.50) when compared with healthy control (301.28 ± 180.87) (p < 0.001) as shown in Fig. 1.
There was a statistically significant difference in the serum Nogo-B level between the three sub-groups of patients (p < 0.001). A high significant difference was found between the three sub-groups of patients as regards FIB-4 score (p < 0.001), APRI score (p < 0.05), and MELD score (p < 0.001) ( Table 2).
It was found that there was a positive correlation between serum Nogo-B and MELD score (r = 0.46, p-value < 0.001). However, there was no correlation found between Nogo-B and FIB-4 index or APRI score. There was a significant positive correlation between serum Nogo-B level and coagulation profile and serum bilirubin. An inverse correlation was found between serum Nogo-B and serum albumin (Table 3). HB hemoglobin, TLC total leukocytic count, PLT platelets, Na sodium, K potassium, ALT alanine transaminase, AST aspartate transaminase, Alb albumin, Bil bilirubin, PC prothrombin concentration, PT prothrombin time, P 1 between all patients and controls, P 2 between the different groups of patients A ROC curve was done to examine the validity of Nogo-B in the diagnosis of liver cirrhosis, and the area under the curve was found to be 0.979, a cutoff value of 600 with a sensitivity of 97.2% and a specificity of 94.4% (p-value < 0.001; Table 4 and Fig. 3). It has also a sensitivity of 100% in the detection of Child A and B patients (AUC 0.954 and 0.984 at a cutoff value of 540 and 600, respectively); it decreased to 95.8% in Child C patients (AUC 1.000 at a cutoff value of 927.5) with a specificity of 88.9%, 94.4%, and 100%, respectively. Nogo-B has a very high significance in the differentiation between Child A and B groups and Child A and C groups (pvalue < 0.001).

Discussion
Liver cirrhosis is a major cause of mortality around the world; the development of cirrhosis has been considered to be an irreversible event [15].
The prognosis of patients with liver cirrhosis often depends on their hepatic functional reserve. Clinical classification systems, such as MELD and Child-Pugh scoring systems, are widely used. Radiographic examination of the remnant liver volumes is also helpful [16].
In this study, we evaluated the relationship between serum Nogo-B and chronic liver disease in Egyptian patients. This study revealed that the serum Nogo-B level was very high in patients with liver diseases compared to the control group. The serum level of Nogo-B was significantly higher in patients with Child B and Child C compared with those patients with Child A.
Our findings signified the high sensitivity of serum Nogo-B as a biomarker that can be used for the evaluation of patients with chronic liver disease. Using the ROC curve for predicting significant fibrosis and cirrhosis, the sensitivity of Nogo-B was high reaching up to 97.2% and with a specificity of 94.4%.  Nogo-B was evaluated in many studies in plasma and liver tissues of patients with liver cirrhosis, and these studies showed that plasma Nogo-B levels were significantly higher in patients with liver cirrhosis than in healthy controls and were the highest in Child-Pugh class C patients. Plasma Nogo-B levels were positively correlated with Child-Pugh classes. There was no relationship between plasma Nogo-B levels and the etiology of liver diseases [12].
In 2017, Jin-Kyu et al. proved that Nogo-B is permissive of M1 (classically activated macrophages) polarization of Kupffer cells and accentuating liver injury in acute liver disease (ALD). They found a significant positive correlation between Nogo-B-positive Kupffer cells and disease severity in ALD patients [17].
The expression of Nogo-B in hepatocytes was firstly studied in a prior study in 2011 in rats and humans by Dahai et al. who stated that Nogo-B was highly expressed in non-parenchymal cells and minimally in hepatocytes in human and rat liver; Nogo-B protein levels were found to be significantly elevated in fibrotic/ cirrhotic liver. They found also that Nogo-B gene deletion reduced hepatic fibrosis and may block the development of portal hypertension [9].
We found a strong positive correlation between Nogo-B and MELD in the patient groups (p-value < 0.001) denoting the ability of Nogo-B to predict mortality in a cirrhotic patient with the same sensitivity of MELD score.
There was also a highly significant correlation between Nogo-B and synthetic function of the liver; serum albumin, coagulation profile with p-value <0.001, significantly correlated with serum total bilirubin with p-value < 0.05. This suggested that plasma Nogo-B might be a good surrogate marker for the evaluation of liver functional reserve in patients with liver fibrosis/cirrhosis.
No correlation was found regarding some other parameters like platelet count, ALT, AST, and ferritin. As Nogo-B is not produced by hepatocytes, this miscorrelation between plasma Nogo-B and ALT and AST was  expected, suggesting that serum Nogo-B level does not reflect the inflammatory status of the liver. Nogo-B and platelet miscorrelation negates the ability of Nogo-B to detect portal hypertension. These findings were also proved by Maoyao et al. [12]. Maoyao et al. found no correlation between Nogo-B and the presence of varices in contrast to our study as most of our patients had varices. This could be related to the high prevalence of bilharziasis in Egypt which led to the early development of vascular decompensation and portal hypertension without evident cellular decompensation or in Child A group. Repeated attacks of hematemesis with blood transfusion and the use of iron supplements may explain the miscorrelation between Nogo-B and hemoglobin and ferritin [12].
Thus, the serum Nogo-B may be considered a good simple noninvasive test for evaluating the degree of liver fibrosis and hepatic functional reserve after exclusion of other conditions affecting its level. Serum Nogo-B level was positively correlated with the synthetic function of the liver (albumin, coagulation profile, and serum bilirubin) in contrast to the FIB-4 index and APRI score. APRI score and FIB-4 index can reflect the degree of inflammation of hepatocytes taking into consideration decreased or normal AST and ALT levels in advanced cirrhosis.

Conclusion
Nogo-B has a high value in the identification of patients with any degree of liver fibrosis with a high positive correlation with different child classes. Nogo-B had the same significance of MELD score in the prediction of mortality in cirrhotic patients. There is a highly significant correlation between Nogo-B and the synthetic function of the liver; it could be used as a measure of hepatic functional reserve. Serum Nogo-B level does not reflect inflammation of the liver or portal hypertension.  Fig. 3 Roc curve shows the sensitivity and specificity of serum Nogo-B, APRI, and FIB-4 in the child A group