الفهرس 
INTRODUCTIONOnly 14 pages are availabe for public view
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Abstract
Hepatitis C virus (HCV) infection is a major cause of chronic liver disease worldwide. The long-term natural history of HCV infection is highly variable. Hepatic injury can range from minimal histological changes to extensive fibrosis and cirrhosis with or without hepatocellular carcinoma (HCC). Moreover, HCV infection is associated with multiple metabolic derangements including dyslipidemia, insulin resistance (IR), and type 2 diabetes mellitus, which af¬fect the progression and prognosis of liver disease and the development of HCC. Numerous signaling pathways have been described to be dysregulated during HCV infection and may be involved in hepatocarcinogenesis, however, the exact mechanism has not been elucidated. The circadian rhythms orchestrate a wide array of crucial behavioral and biological processes involved in maintaining homeostasis and environmental adaptation. These rhythmic oscillations are the result of an endogenous autonomous, intrinsic timekeeping system called the circadian clock (CC), which consists of a set of genes including positive elements brain and muscle Arnt-like protein 1 (BMAL1) and circadian locomotor output cycles kaput (CLOCK) and negative elements Period (PER1, PER2, and PER3) and cryptochrome (CRY1 and CRY2). Disruption of the circadian rhythms (chronodisruption) has direct consequences for human health leading to a wide variety of diseases including viral infection, metabolic diseases such as diabetes, obesity, and dyslipidemia, and cancer. Emerging evidence indicates an important role of the circadian rhythm in orchestrating liver metabolism and physiology and circadian disruption has emerged as major contributor to liver diseases. However, the role of the circadian rhythms in the pathogenesis of HCV-related liver disease has not been fully elucidated.
Therefore, the present work was conducted to assess the clinical significance of circulating circadian proteins BMAL1 and PER2, positive and negative regulators of the CC, in HCV-related liver disease.
The present study included 60 treatment-naïve patients with HCV-related liver disease (20 patients with chronic hepatitis C (CHC), 20 cirrhotic patients without HCC, and 20 cirrhotic patients with HCC) who have been referred to the Hepatobiliary Unit, Department of Internal Medicine, Main Alexandria University Hospital. They were 39 males and 21 females and their ages ranged between 35-71 years (mean SD, 53.62 9.75 years). All patients had seropositivity of circulating HCV antibodies and detectable serum HCV RNA. The presence of cirrhosis was determined by clinical and radiological examination and/or liver stiffness measurement using acoustic radiation force impulse (ARFI) elastography. The diagnosis of HCC was based on typical vascular characteristics in multiphase computed tomography and/or magnetic resonance imaging. Also, 20 age- and sex-matched healthy subjects with no evidence of liver disease were included in the study as control group. They were 14 males and 6 females and their ages ranged between 38-71 years (mean SD = 56.70 9.43 years). Patients with HCV-related liver disease were excluded from the study if they have seropositivity for HBV infection; history of alcohol consumption; other known causes of chronic liver disease; concomitant schistosomiasis; autoimmune and connective tissue diseases; other infections or inflammatory disorders; malignancy other than HCC; cardiac, respiratory or renal disease. Also, patients who had received previous antiviral treatment or therapy for HCC, locoregional or systemic, were not included in the study. The study was conducted in accordance with the provisions of the Declaration of Helsinki 1979 and Good Clinical Practice guidelines. An informed consent was obtained from all participants included in the study.
All patients with HCV-related disease were evaluated clinically as regards the possible risk factors of HCV infection, symptoms and signs of chronic liver disease, liver and spleen sizes, presence of palpable focal hepatic lesions and anthropometric measurements including body mass index (BMI) and waist-to-hip ratio (WHR). Radiological examination was performed for the assessment of liver echo pattern; the presence of cirrhosis, ascites, splenomegaly, and HCC and tumor characteristics. Laboratory investigations included complete blood picture, serum creatinine, serum sodium, liver test profile [serum aspartate and alanine aminotransferases (AST and ALT respectively), serum gamma glutamyl transpeptidase (GGT), serum albumin, serum bilirubin, prothrombin time and international normalized ratio], serum HCV RNA using real time polymerase chain reaction assay, serum -fetoprotein (AFP), lipid profile [total cholesterol (TC), high density lipoprotein-cholesterol (HDL-C), low density lipoprotein-cholesterol (LDL-C) and triglycerides (TGs)], fasting plasma glucose (FPG), fasting serum insulin (FSI) and the homeostatic model assessment of IR (HOMA-IR) for estimation of IR. Serum levels of BMAL1 and PER2 levels were quantified using commercially-available enzyme-linked immunosorbent assay kits. The severity of liver disease in patients with HCV-related cirrhosis was graded according to Child-Pugh classification and the Model for End Stage Liver Disease Sodium (MELDNa) score. The staging of HCC was determined according to the Barcelona Clinic Liver Cancer (BCLC) staging system. The noninvasive liver fibrosis scores aspartate aminotransferase-to-platelet ratio index (APRI) and fibrosis index based on 4 factors index (FIB-4) were calculated. Liver stiffness was assessed using ARFI elastography and the liver shear wave velocity (LSWV) was expressed as meters per second (m/s).
Data were analyzed using IBM SPSS software package version 21.0. (Armonk, NY: IBM Corporation) and showed the following results:
The serum HCV-RNA levels ranged between 5.4-698.0 x103 IU/ml (mean ± SD = 125.15 ± 180.60 x103 IU/ml) in patients with CHC, between 13.0-945.0 x103 IU/ml (mean ± SD = 197.88 ± 245.85 x103IU/ml) in cirrhotic patients without HCC and between 14.7-568.0 x103 IU/ml (mean ± SD = 204.56 ± 185.06 x103 IU/ml) in cirrhotic patients with HCC with no statistically significant differences between patient groups
According to Child-Pugh classification, cirrhotic patients without HCC were classified as class A in 17 (85.0%) patients and class B in 3 (15.0%) patients and the Child-Pugh score ranged between 5-8 (mean ± SD = 6.05 ± 0.95) while cirrhotic patients with HCC were classified as class A in 13 (65.0%) patients, class B in 5 (25.0%) patients, and class C in 2 (10.0%) patients and the Child-Pugh score ranged between 6-10 (mean ± SD = 6.85 ± 1.66). The MELDNa score ranged between 6-16 (mean ± SD = 9.55 ± 2.42) in cirrhotic patients without HCC and between 6-17 (mean ± SD = 10.40 ± 3.50) in cirrhotic patients with HCC.
The maximum diameter of HCC ranged between 2-12 cm with a mean value of 6.88 ± 3.29 cm. The tumor size was 5 cm in 8 (40.0%) patients and > 5 cm in 12 (60.0%) patients. The number of nodules was a single nodule in 7 (35.0%) patients, two nodules in 6 (30.0%) patients and three nodules in 7 (35.0%) patients.
The HCC stage was classified as BCLC stage A in 9 (45.0%) patients, BCLC stage B in 6 (30.0%) patients, BCLC stage C in 3 (15.0%) patients and BCLC stage D in 2 (10.0%) patients.
Serum AFP levels showed significant increases in cirrhotic patients with and without HCC compared with patients with CHC and healthy subjects and in cirrhotic patients with HCC compared with cirrhotic patients without HCC (P < 0.001).
Serum TC, LDL-C, and TGs levels were significantly higher in cirrhotic patients with and without HCC than in healthy subjects (P < 0.05) while serum HDL-C levels showed a significant decrease in patients with CHC and cirrhotic patients with and without HCC compared with healthy subjects (P < 0.001) while there were no statistically significant differences between patient groups (P > 0.05).
The FPG levels showed significant increases in cirrhotic patients with and without HCC compared with patients with CHC and healthy subjects while levels of FSI and HOMA-IR were significantly higher in patients with CHC and cirrhotic patients with and without HCC than in healthy subjects and in cirrhotic patients with and without HCC than in patients with CHC (P < 0.01).
The APRI and FIB-4 were significantly higher in cirrhotic patients with and without HCC than in patients with CHC (P < 0.001), while there was no statistically significant difference between cirrhotic patients with and without HCC (P > 0.05).
The LSWV showed significant increases in cirrhotic patients without HCC, the HCC and the surrounding non-neoplastic liver compared with patients with CHC and healthy subjects and in cirrhotic patients without HCC and the surrounding non-neoplastic liver compared with the HCC (P < 0.001).
Serum BMAL1 levels were significantly lower in patients with CHC, cirrhotic patients without HCC and cirrhotic patients with HCC than in healthy subjects (2323.13 ± 493.33 pg/ml, 1799.23 ± 82.89 pg/ml and 1536.68 ± 129.91 pg/ml vs 10446.35 ± 3850.13 pg/ml respectively), in cirrhotic patients with HCC than in patients with CHC and cirrhotic patients without HCC, and in cirrhotic patients without HCC than in patients with CHC (P < 0.001).
Serum PER2 levels showed also significant decreases in patients with CHC, cirrhotic patients without HCC and cirrhotic patients with HCC compared with healthy subjects (1235.10 ± 71.33 pg/ml, 1084.40 ± 45.96 pg/ml and 978.00 ± 34.83 pg/ml vs 2642.25 ± 674.52 pg/ml respectively), in cirrhotic patients with HCC compared with patients with CHC and cirrhotic patients without HCC, and in cirrhotic patients without HCC compared with patients with CHC (P < 0.001).
By plotting receiver operating characteristic (ROC) curve, the sensitivity, specificity and predictive values of serum BMAL1 and PER2 were higher than those of APRI, and FIB-4 in discriminating cirrhotic patients without HCC from patients with CHC (AUC = 0.915 and 0.986, 0.823, and 0.870 respectively, P < 0.001 for all) and were superior to serum AFP in discriminating cirrhotic patients with HCC from cirrhotic patients without HCC (AUC = 0.963, P < 0.001; 0.985, P < 0.001; 0.783, P = 0.002 respectively).
Statistical correlations between serum BMAL1 and PER2 levels and other parameters in patients with HCV-related liver disease showed the following results:
- No statistically significant correlations were found between serum BMAL1 and PER2 levels on one hand and age and serum GGT levels on the other hand (P > 0.05).
- There was a positive correlation between serum levels of BMAL1 and PER2 (P < 0.001).
- Serum BMAL1 and PER2 levels showed inverse correlations with serum levels of AST, ALT, and HCV RNA, Child-Pugh score, MELDNa score, APRI, FIB-4, and LSWV (P < 0.05).
- Serum BMAL1 and PER2 levels were inversely correlated with metabolic parameters including BMI, WHR, serum levels of TC, LDL-C, TGs, FPG, FSI and HOMA-IR and were positively correlated with serum HDL-C levels (P < 0.05).
Statistical correlations between serum BMAL1 and PER2 levels and tumor progression parameters in cirrhotic patients with HCC showed that serum BMAL1 and PER2 levels were inversely correlated with serum AFP levels, HCC maximum diameter, number of HCC nodules, BCLC stage, and HCC LSWV (P < 0.05).