The Impact of Combined Cranberry Supplementation and Weight Loss Diet on Inammatory, Antioxidant and Apoptosis Biomarkers in Patients with Non-Alcoholic Fatty Liver Disease

Non-alcoholic fatty liver disease (NAFLD) is a prevalent chronic liver diseases. The aim of this study was to evaluate the effect of combined weight loss diet and cranberry supplementation on inammatory, antioxidant and apoptosis biomarkers in patients with NAFLD. In this randomized, double-blinded, controlled clinical trial, 41 NAFLD patients were supplemented with either a 288-mg cranberry tablet or a placebo tablet for 12 weeks. Both groups followed a diet of 500– 1000 calories less than the estimated energy requirements. Serum levels of Total antioxidant capacity (TAC), Malondialdehyde (MDA), Cytokeratin 18-M30 (CK-18 M30), Chemokine C-C motif ligand 2 (CCL2) and Tumour necrosis factor alpha (TNF-α) were measured at both baseline and the end of the study.


Background
Non-alcoholic fatty liver disease (NAFLD) is a prevalent chronic liver diseases. The aim of this study was to evaluate the effect of combined weight loss diet and cranberry supplementation on in ammatory, antioxidant and apoptosis biomarkers in patients with NAFLD.

Methods
In this randomized, double-blinded, controlled clinical trial, 41 NAFLD patients were supplemented with either a 288-mg cranberry tablet or a placebo tablet for 12 weeks. Both groups followed a diet of 500-1000 calories less than the estimated energy requirements. Serum levels of Total antioxidant capacity (TAC), Malondialdehyde (MDA), Cytokeratin 18-M30 (CK-18 M30), Chemokine C-C motif ligand 2 (CCL2) and Tumour necrosis factor alpha (TNF-α) were measured at both baseline and the end of the study.

Results
Signi cant improvements in TAC were observed in the cranberry group and between the two groups (p = 0.006 and p = 0.011 respectively), but the changes in the placebo group were not signi cant (p = 0.325).
There were no statistically signi cant differences in the serum levels of MDA, CK-18 M30, CCL2 and TNFα between the cranberry and the placebo groups (p > 0.05).

Conclusions
It seems that daily consumption of cranberry supplement would be bene cial in increasing serum levels

Background
Non-alcoholic fatty liver disease (NAFLD) is caused by an increase in the cellular content of free fatty acids; FFAs), resulting in excessive accumulation of triglycerides (TG) in the cytoplasm of hepatocytes (> 0.5%) without the over-consumption of alcohol [1]. NAFLD that embrace a broad spectrum of physiopathological conditions from simple steatosis to non-alcoholic steatohepatitis (NASH), is highly related to obesity, diabetes, insulin resistance, hypertension, hyperlipidemia, and metabolic syndrome [2]. The prevalence of NAFLD is high worldwide and estimated to be about 24% [3]. The pathophysiology of NAFLD is complex and it has multiple manifestations/complications [4]. In terms of pathogenesis of NAFLD, it has been shown in both "two-hit" model and "multi-parallel hit" hypothesis that hepatic in ammation, oxidative stress, necrosis, apoptosis, and nally, brosis are caused by excessive fat accumulation in the hepatocytes [5]. A fundamental intervention for the management of NAFLD is Lifestyle modi cation such as having healthy eating patterns and regular exercise [6]. Many studies have been shown that in these patients the serum and liver contents of free radical oxidation products increase and the total antioxidant capacity decreases [5]. Therefore, following healthy eating patterns [7] along with antioxidant and anti-in ammatory supplements [8] could be helpful in better improvement to NAFLD, given that oxidative stress and in ammation play a role in its pathogenesis [9]. Available evidence show that food ingredients such as phytochemicals, vitamins and minerals exhibit anti-in ammatory, antioxidant and immune-regulating activity in the body [10][11][12][13][14][15]. One of the classes of phytochemicals are phenolics, which include avonoids and polyphenol [16]. Researchers have been shown that these phytochemicals have ability to scavenge free radicals, reducing in ammation, modifying the lipid pro le, lowering blood pressure, and reducing platelet aggregation [17][18][19][20]. Cranberries (Vaccinium Macrocarpon) are rich in polyphenols such as avonols, catechins, anthocyanins, resveratrol, organic acids, B-type proanthocyanidins (PACs) and a high amount of rare A-type PACs [21] and according to the United States Department of Agriculture (USDA), they have the highest free radical scavenging ability [5]. Previous studies showed some effects of cranberry bio-actives include reducing in ammation in humans and in vitro, reducing blood markers of oxidative stress in humans [22] and abating of hepatic in ammation and steatosis in mice fed a high-fat diet [23]. Impact of consumption low-calorie cranberry juice on decreasing cardiometabolic risk in overweight middle-aged population was investigated by a clinical trial. There was a signi cant improvement in C-reactive protein (CRP) for intervention group after 8 weeks of evaluation [24].
This placebo-controlled, double-blind, randomized clinical trial study was designed to explore the possible role of cranberry supplementation on in ammatory and oxidative markers in patients with NAFLD because of the anti-in ammatory and antioxidant properties of cranberry and role of in ammation and oxidative stress in pathogenesis of NAFLD.

Samples and study design
In this randomized double-blind and placebo-controlled clinical trial, the patients with NAFLD were recruited from Ahvaz Golestan hospital. Totally, 50 eligible patients were recruited. A signed consent form was collected from all subjects. The recruited subjects were randomly allocated into control (n = 25) and intervention (n = 25) groups based on the block design. Age 18 years or older, BMI of 25-5 kg/m2, patients with con rmed NAFLD (the grade of steatosis higher or equal to 2 at ultrasonography) were the inclusion criteria and history of signi cant alcohol intake (more than 10 mL/day for women and 20 ml/d for men); smoking habits; subjects affected by other liver diseases, cardiovascular, respiratory, kidney disorders, malignancies, and diabetes mellitus,, pregnancy or breastfeeding, medication in the previous 6 months, supplementation with antioxidants or vitamins, weight loss over the past 3 months, metabolism and endocrine disorders pregnancy or breastfeeding, medication in the previous 6 months, supplementation with antioxidants or vitamins, weight loss over the past 3 months, metabolism and endocrine disorders were the exclusion criteria. Subjects in the intervention and control groups received either cranberry or placebo tablets for 12 weeks. The color, size and weight of cranberry and placebo tablets were similar. All subject received two tablets; one tablet after lunch and another one after dinner.
Cranberry tablets were purchased from Shari Nutraceutical Co., Tehran, Iran. Each tablet contained 144 mg of Vaccinium macrocarpon extract with at least 36 mg proanthocyanidine (equal to 13 g dried cranberry fruit), while composition of the remaining 144 mg was unknown. Placebo tablets contained 288 mg of starch. Both groups followed a diet of 500-1000 calories less than the estimated energy requirement. Dietary intakes were obtained from three 24 h dietary recalls (1 weekend day and 2 weekdays). Physical activity levels were assessed by the metabolic equivalent of task (MET) questionnaire.
This clinical trial study was registered in the "Iranian Registry of Clinical Trials" with the IRCT number of IRCT20150124020765N2.

Biochemical And Anthropometric Measurements
At baseline, 6 weeks and after 12 weeks, all subjects underwent anthropometric measurements: Height was measured to the nearest 0.1 cm using a non-stretched tape measure. Weight, body fat (BF), and body mass index (BMI) were measured using a bioelectrical impedance analysis (OMRON device BF-511). Waist circumference (WC) (the widest area between the lower rib and the superior iliac crest) was also measured to the nearest 0.1 cm. A fasting blood sample (12 ml) was collected from subjects at baseline and the end of intervention. Blood samples were centrifuged at 3500 rpm for 10 min and then the supernates were stored at -70°C until analysis. The serum samples were used to analysis TAC, MDA, TNFα, CK-18 M30 and CCL2. Serum levels of CCL2, CK-18 M30, TNF-α and TAC were measured using ELISA method by laboratory kits (Biotech Day Crystal for CCL2, CK-18 M30, TNF-α; and LDN, PLabor Diagnostika Nord GmbH, Germany for TAC). Serum MDA levels were assessed utilizing the thiobarbituric acid reactive substances (TBARS).

Statistical analysis
Considering 95% con dence with an estimated standard deviation and difference of the fasting blood glucose (16) the sample size was calculated and 25 subjects in each group were determined. The statistical analyses were performed using SPSS (version 19). The results presented as mean ± SD. Pvalue lower than 0.05 was considered as signi cant. The Kolmogorov-Smirnov test was used to assess the normal distribution of variables. Independent samples comparisons in terms of quantitative variables were performed using two independent samples t tests and repeated measures ANOVA, as appropriate. In order to analyze nutrient intakes, "Nutritionist IV" software was applied.

Results
In this study, 9 out of 50 patients were excluded due to non-adherence to diet and medication. Therefore, 21 subjects in the group receiving cranberry supplements and 20 in the placebo group remained in the study. Patient compliance in this randomized clinical trial study was 82%. The characteristics of subjects are shown in Table 1. No statistically signi cant difference were seen between two groups in terms of their demographic characteristics or their baseline biomedical, and anthropometric measurements (p > 0.05). Table 2 shows the dietary intake and anthropometric indices of the two groups. Statistical analysis showed that after 12 weeks, the weight, BF, BMI, and WC signi cantly decreased in both groups (p < 0.05).
There was no signi cant difference in weight, BMI, BF, and WC between the two groups (p > 0.05). No signi cant differences were also observed between two groups for dietary data including the intakes of energy, and macronutrients at the end of the study.   Table 3 shows the in ammatory, antioxidant and apoptosis biomarkers at baseline and after 12 weeks for the two groups. Signi cant improvements in TAC were observed in the cranberry group and between the two groups (p = 0.006 and p = 0.011 respectively), but the changes in the placebo group were not signi cant (p = 0.325). The mean MDA was reduced (but not signi cantly) in the cranberry group after intervention, moreover no signi cant differences were seen in the serum levels of MDA between the cranberry and the placebo groups (p > 0.05). Also both within cranberry and placebo groups, there were no signi cant changes in the mean levels of CK-18 M30 post intervention compared with baseline (p > 0.05).
There were no statistically signi cant differences in the serum levels of CCL2 and TNF-α between the cranberry and the placebo groups (p > 0.05).

Discussion
The present study was conducted to evaluate effects of cranberry supplementation and weight loss diet on markers of in ammation (TNF-α and CCL-2), oxidative stress (MDA and TAC) and hepatic cell apoptosis (CK-18 M30) in NAFLD patients. Supplementing a weight loss diet with cranberry for a period of 12 weeks could signi cantly increase TAC. However, changes in other variables remained insigni cant. Different studies have been conducted on the effects of cranberry with or without dietary interventions on these markers with different intervention types (extract, juice, tablet, etc), variable doses and different ndings.
Cytokine imbalances occur in the "second hit" of NAFLD. Therefore the issue has gained a considerable attention as target for therapeutic interventions [25]. Glisan et al evaluated effects of polyphenol enriched cranberry extract (CBE) on markers of hepatic in ammation in HFD-fed obese rats and found that CBE can decrease hepatic protein levels of TNF-α and CCL-2, as well as hepatic mRNA levels of toll like receptor-4 (TLR-4) and nuclear factor κB (NFκB) [22]. Another study was performed to evaluate possible anti-brotic effects of cranberry nutraceuticals in high fat cholesterol diet induced (HFCD)-NAFLD rats.
The results showed that cranberry could alleviate markers of oxidative stress (MDA, glutathione, catalase and superoxide dismutase), in ammation (TNF-α, IL-6 and NFκB) and improved markers of insulin resistance [26]. Apoptosis is a key mechanism in the progression of steatosis to NASH and apoptosis markers are related with histologic severity of NAFLD [27]. CK-18 M30 is a well-known substrate of caspase activity during apoptotic hepatocyte death [28] and has been shown to have a high accuracy in differentiating NAFLD from control subjects [29]. Accordingly, in the current study, CK-18 M30 was assessed as a marker of apoptosis which did not signi cantly change during intervention. Studies regarding anti-apoptotic effects of cranberries are few. However, some studies have been conducted on other polyphenol rich compounds in this area. In a randomized clinical trial, 44 participants were given either 250 ml bayberry juice or placebo, twice a day for 4 wks. Bayberry consumption could signi cantly improve markers of in ammation and apoptosis including polypeptide speci c antigen and CK-18 M30 [30]. In another study, 14 days of dark chocolate consumption, as a source of polyphenols, led to a signi cant reduction in CK-18 M30 in NAFLD patients [31]. According to ndings, multistage processing of fruit extraction leads to a considerable loss in phytochemical content through thermal degradation and polyphenol oxidation which could have been considered as a reason for the null ndings. Therefore, future research should focus on comparisons between different forms of cranberry supplements.
On the other hand, a signi cant improvement in TAC was observed in intervention group which indicates possible anti-oxidative effects of cranberry supplements in NAFLD patients. Oxidative stress is a result of an imbalance between pro-oxidants and anti-oxidants and plays a crucial role in the pathogenesis of NAFLD [32]. A considerable amount of research has been conducted regarding anti-oxidative effects of berries. In one study mulberry treatment in HFD rats signi cantly suppressed hepatic reactive oxygen species (ROS) overproduction and mitochondrial oxidative stress [33]. Another research team investigated effects of raspberry on obese diabetic (db/db) mice for 8 weeks. The ndings showed that raspberry intake could improve antioxidant status and lessen IL-6 in treatment group [34]. Results of a double blind randomized trial showed that 4 weeks supplementation with maqui berry (delphinol) signi cantly reduced markers of oxidative stress (ox-LDL and urinary F2-isoprostane) in intervention group [35]. Wild blueberry consumption signi cantly improved postprandial oxidative stress in male subjects. Oxygen radical absorbance capacity (ORAC) assay and the total antioxidant status (TAS) were evaluated as markers of oxidative stress in the study [36]. On the other hand, in a randomized controlled trial, 40 post-menopausal women consumed either 22 grams of blueberry or placebo for 8 weeks. Blood markers of oxidative stress, in ammation and antioxidant defense did not change in blueberry group after 8 weeks [37].
Health bene ts of fruits and vegetables have been demonstrated in nutrition not only for their vitamins and minerals, but also their phytochemical components [38]. The American cranberry (species Vaccinium macrocarpon) has been particularly considered a health fruit for centuries [39]. Cranberries as a uniquely rich source of phytochemicals, contain over 150 phytochemicals with avonoids as the most predominant component. Some cranberry avonoids include anthocyanins, proanthrocyanidins, catechins, organic acids, resveratrol and avonols which are responsible for the fruit's color and sour astringent avor [40]. Several in vivo animal models have con rmed anticarcinogenic, antiyumorogenic, antiangiogenic, anti-in ammatory and antioxidant properties of cranberry polyphenols [41]. NAFLD has been linked to gut dysbiosis and metabolic endotoxemia which are the initial triggers of in ammatory cascade [42]. NF-kβ is a key regulator in this cascade and has the potential to control the production of pro-in ammatory cytokines including TNF-α and IL-6 [43,44]. Cranberries as a great source of polyphenols might exert as prebiotics which can have immunomodulatory and anti-in ammatory effects by interacting with gut microbiota [45]. In one study, dietary cranberry supplementation in a mouse model of IBD, not only suppressed colonic levels of pro-in ammatory cytokines (IL-1β, IL-6 and TNF-α) but also increased the abundance of bene cial gut bacteria including lactobacillus and bi dobacterium [46]. It has also been shown that phenolic compounds can suppress IL-1β secretion and exert anti-in ammatory effects through inhibition of cyclo-oxygenase and lipoxygenase activity [47]. Antioxidant properties of cranberry are attributed to free radical scavenging properties of polyphenols against ROSs as well as inhibition of lipid and protein oxidation [48]. According to studies, cranberry supplementation has also the potential to decrease NO synthase activity, improve homocysteine levels, and endothelial function, thus suppressing oxidative stress [49].

Conclusion
In conclusion, this randomized, double-blind, placebo controlled trial indicate that 288 mg/d of cranberry Availability of data and materials The data gathered and analyzed during the current study are available from the corresponding author on reasonable request.

Ethics approval and consent to participate
This study was approved by the ethics committee of the Research Deputy of Ahvaz Jundishapur University of Medical Sciences (IR.AJUMS.REC.1397.678).

Consent for publication
No personal data is noted herein.