Investigation of the effect of encapsulated and unencapsulated aronia (Aronia melanocarpa) extract on PON1 gene mRNA expression, PON1 enzyme activity in liver tissues and atherosclerosis in rats

In this study, the effects of unencapsulated (E) and microencapsulated (C) forms of Aronia (Aronia melanocarpa) extract on serum and liver tissue lipid profiles, PON1 enzyme activity and gene expression in blood and liver tissues, histopathologic formation of atherosclerotic plaques in the aorta, and histopathology of liver tissue were investigated in Sprague-Dawley (SD) rats fed a high-fat diet (HFD). The study used 42 male SD rats at 26 weeks of age. The rats were divided into 6 groups of 7 rats each. The experimental diet groups were as follows: Standard diet control (SDC) High-fat diet (HFD) HFD400E; HFD + 400 mg/kg Aronia E HFD200E; HFD + 200 mg/kg Aronia E HFD400C; HFD + 400 mg/kg Aronia C HFD200C; HFD + 200 mg/kg Aronia C From the 10th week until 20 weeks of age, the rats (except the SDC group) were fed an HFD for 10 weeks. At the end of 20 weeks, in addition to the diets of the rats fed with HFD, 200 and 400 mg/kg dosages of Aronia E and C per 1 kg of SD body weight (BW) were administered to the rats by oral gavage for 6 weeks. The average body weights of the SD rats were measured before and after Aronia administration. As a result of the study, a slowdown was observed in the rate of body weight gain of adult rats with increasing age. Aronia HFD200E administration had a negative effect on the rate of weight gain (g/h) compared to the rats in the HFD group (-4.62 g; P<0.001). The differences between the mean Lee Obesity Index of the experimental group SD rats before (20 weeks of age) and after 6 weeks of Aronia administration (26 weeks of age) were not statistically significant (P>0.05). Total cholesterol levels in the blood serum of SD rats were found to be higher in the HFD400E group (167.7±0.57 mg/dL) than in the SDC (158.9±2.05 mg/dL; P<0.0070) and HFD (159.7±2.13 mg/dL; P<0.0166) groups. Triglyceride (TG) levels of rats were significantly lower in the HFD400E group (93.0±1.67 mg/dL) than in the HFD group (172.1±23.27 mg/dL; P<0.0107). HDL levels of rats in the HFD400E group (77.15 mg/dL) were higher and significantly higher than those in the SDC (63.09±2.34 mg/dL; P<0.0006) and HFD (61.08±0.97 mg/dL; P<0.0001) groups. The VLDL levels in the blood of rats in the HFD400E group (18.60±0.33 mg/dL) were significantly lower compared to those in the HFD group (34.43±4.65 mg/dL; P<0.0107). The LDL/HDL ratio in the blood of the experimental group SD rats was lowest in the HFD400E group (0.95±0.07 mg/dL). PON1 gene expression [2-(∆∆Ct) values] based on blood samples of rats was determined as 1.15±0.06 in the HFD400E group. Rats in the HFD400E group (64.59±0.4 ng/mL) had higher PON1 enzyme activity than rats in the SDC (49.65±4.68 ng/mL; P<0.0025) and HFD (45.39±1.63 ng/mL; P<0.00001) groups. Although the PON1 gene expression values between the groups were not significant, it was observed that 400 mg/kg Aronia extract increased PON1 enzyme activity in the HFD400E group compared to the other experimental groups. MDA values in the blood of rats were higher in the HFD400E group (3.01±0.04 nmol/mL) than in the SDC group (P<0.0290), but no statistically significant difference was observed between the HFD and other groups (P>0.05). The liver weights of SD rats in the HFD400E group (12.80±0.24 g) were significantly decreased compared to those in the SDC (17.43±0.79 g; P<0.0001) and HFD (18.26±0.81 g; P<0.0001) groups. There was a decreasing effect of 400 mg/kg extract or encapsulated Aronia on liver weights compared to the HFD group. The ratio of liver weight to body weight of rats in the HFD400E group was lower than that of rats in the SDC (P<0.0001) and HFD (P<0.0001) groups. The 400 mg/kg Aronia extract kept the liver weight-to-body weight ratio (3.20±0.18%) at the lowest level. ALT and AST results of SD rats in the experiment were within the normal standard range determined for SD rats, and the lowest ALT (35.10±1.98 U/L) and AST (26.68±2.58 U/L) values were determined in the HFD400E group. While the differences between the groups in ALT values were statistically insignificant, in terms of AST values, the HFD400E value was lower and significant compared to rats in the SDC (P<0.0001) and HFD (P<0.0001) groups. PON1 enzyme activity in liver tissues of rats ranged between 62.18±7.60 ng/mL (SDC) and 81.00±1.11 ng/mL (HFD400E). The highest enzyme activity was observed in the HFD400E group, whose diets were supplemented with 400 mg/kg Aronia extract, but the difference was not significant (P>0.05). In terms of PON1 gene expression [2-(∆∆Ct) value] of rats, although a positive effect of the diet was observed in the HFD400E group (0.48±0.17), the differences between the groups in terms of 2-(∆∆Ct) values were not significant. There was no significant difference between the groups in liver MDA values (P>0.005). HDL levels in the liver tissues of the HFD400E group (67.99±0.98 mg/dL) were found to be higher and significant (P<0.0001) compared to the HFD group (52.62±2.35 mg/dL). Histopathological examination of the liver tissue samples revealed that the liver tissues of the rats in the SDC group had a normal histological structure. In the liver tissues of the rats in the HFD group, a small amount of inflammation was found in the portal area. Therefore, a high level of microsteatosis and a moderate level of macrosteatosis were detected in the liver due to the effect of the HFD. When compared with the SDC group, the liver damage score was found to be higher in this group (P<0.001). In the liver findings of rats given HFD200C and HFD400C Aronia extract, similar findings were obtained as in the HFD group (P>0.988 and P>0.953, respectively). The liver scores of the HFD200E and HFD400E groups decreased statistically significantly when compared with the HFD group (P<0.001). However, only intragastrically in the HFD400E group was a morphology similar to the SDC group detected. Micro- and macrosteatosis findings decreased in the liver score of the HFD400E group. When aortic tissues were evaluated, normal tissue morphology was observed in all experimental groups, and no atherosclerotic plaques were found in the thoracic aorta or aortic arch tissues in any experimental group. Hyaline or mucoid changes or endothelial abnormalities were not detected. The atherosclerosis index of rats was slightly decreased in the HFD400E group (2.19±0.08) compared to the SDC (2.54±0.10) and HFD (2.62±0.05) groups, but not significantly (P>0.05). In conclusion, Aronia, which is rich in anthocyanins and other polyphenolic compounds, can be used to prevent and slow down atherosclerosis by increasing PON1 enzyme activity, which helps prevent lipid oxidation and is the first step in atherosclerosis. In particular, the administration of HFD400E is thought to positively affect PON1 gene expression, increase PON1 enzyme activity and HDL levels, and provide long-term protection against oxidative stress due to a decrease in MDA level (2.38 nmol/g) in liver tissues.