Here we demonstrate that, in spite of of WD-induced fatty liver development, AF supplementation: 1) reduces Tnf-α mRNA; 2) tended to reduce liver protein carbonyl levels and did decrease 8-isoprostane levels when these animals were compared to CTL rats, albeit the latter marker was also reduced in WD rats as well; 3) tended to increase liver SOD2 mRNA, which encodes the mitochondrial superoxide dismutase antioxidant, when these animals were compared to CTL rats; and 4) prevented WD-induced alterations in select genes related to the transport and metabolism of carbohydrates in favor of select genes related to lipid transport and metabolism. However, AF supplementation did not affect fatty liver development in the presence of WD feeding. Finally, serum clinical chemistry markers and liver histopathology demonstrated that sub-chronic (30 days), twice daily AF supplementation was well-tolerated, and that the significant between-group effects were observed up to 24 hours after receiving a final treatment dose. More in depth discussion of these findings are presented herein.
WD + AF rats experienced a significant reduction in liver Tnf-α mRNA compared to WD and CTL rats. Chicanine, a major lignan present within Schisandra chinensis, an active ingredient in AF, was recently reported by Chen et al. to significantly down-regulate LPS-induced expression of Tnf-α mRNA, as well as other pro-inflammatory cytokines in murine macrophages. Similarly, Dushkin et al. recently showed that eight weeks of daily oral Rhaponticum carthamoides extract, an active present in AF, significantly lowered serum Tnf-α levels in high-fat diet fed six-month old male Wistar rats. Green tea extract, another active ingredient in AF, has been shown to protect against NASH in ob/ob mice by decreasing the expression of adipose tissue lipogenic genes, improving hepatic antioxidant defenses and decreasing liver Tnf-α[21, 22]. Likewise, Li et al. demonstrated that supplementing a high fat diet with resveratrol, an active ingredient in AF, amerliorated fibrosis, insulin resistance, glucose tolerance, dysregulated lipid metabolism, oxidative stress, and pro-inflammatory responses. Rivera et al. similarly revealed that elevated liver biomarkers of NASH were significanlty lower in obese Zucker rats following 8-week treatment of resveratrol. The authors further explained that resveratrol was able to increase adiponectin protein content and lower Tnf-α protein content in the visceral adipose tissue of rats. Therefore, a decrease in liver Tnf-α mRNA may have been due to schisandra, rhaponticum, resveratrol, green tea extract, and/or other ingredients present in AF.
Serum and liver TAC were significantly lower in WD + AF versus WD and CTL rats. We posit that the latter findings could be a result of the exogenous antioxidant ingredients being provided through AF which, in turn, decreases the expression of total endogenous antioxidant capacity across multiple tissues. In this regard, Valtuena et al. reported that diets high in total antioxidant capacity significantly improved markers of inflammation, but did not appreciably affect antioxidant status in healthy adults. Foods high in antioxidants have also been shown to prevent carotenoid secretion into circulation up to 24 hours following feeding. Likewise, while a controversial topic, the possibility exists that exogenously provided dietary antioxidants hamper endogenous antioxidant systems. However, the decrease in serum 8-isoprostane levels in the WD + AF rats versus CTL rats does provide a line of evidence suggesting that systemic oxidative stress was still lower in the former group while serum TAC was also reduced.
Liver protein carbonyl concentration within the mitochondria and nuclear compartments, but not within the soluble cellular compartments such as the cytosol have been linked to mammalian aging; specifically, short-lived animals were shown to possess higher concentrations of liver protein carbonyls within the insoluble cellular components such as the mitochondria, and long-living animals were shown to possess reduced protein carbonyls within these cellular compartments. Interestingly, within the present investigation, total liver protein carbonyls tended to be lower in WD + AF versus CTL rats, which is suggestive of AF possessing potentially anti-oxidant properties in the liver. Though AF signficantly reduced serum and liver TAC, our study findings would seem to suggest that AF possesses antioxidant properties. This is of no surprise given that AF contains green tea extract and resveratrol. Chung et al. reported that green tea extract inhibits ROS/RNS-mediated damage while reducing serum alanine aminotransferase (ALT) and decreasing lipid peroxidation and protein nitration. Rubiolo et al. similarly revealed that resveratrol protects isolated primary rat hepatocytes from necrosis induced by oxidative stress. Gómez-Zorita et al. demonstrated that a 6-week treatment of resveratrol significantly decreased lipid peroxidation, suggesting a protective antioxidant effect against obesity and steatosis-induced oxidative stress. It is noteworthy to mention, however, that WD feeding in the current study did not alter markers of oxidative stress when comparing WD versus CTL rats. This finding was likely due to the relatively sub-chronic WD feeding schedule. However, NASH development appears to have been occurring, as is evident from the observed liver fat deposition, though no overt signs of oxidative stress became apparent. Therefore, this finding suggests that, AF supplementation reduces liver protein carbonyl formation and may reduce systemic oxidative stress as evidenced by only 25% of the animals presenting detectable levels of serum 8-isoprostane compared to 50–66% of the rats within the other groups presenting detectable levels of this marker.
While liver TNF-α mRNA and oxidative stress markers were favorably altered with AF supplementation, liver lipid deposition remained unaltered. Pan et al. reported similar findings when high-fat fed mice were provided either a water- or ethanol-extracted schisandra fruit extract for 13 days. Specifically, whereas hepatic steatosis was ameliorated and liver injury reduced by the schisandra treatment, significant increases in high and low density lipoproteins occurred without any observable changes occuring within measures of total cholesterol and triglycerides. Jwa et al., however, demonstrated that supplementing a high fat diet with piperine, another active ingredient in AF, markedly decreased hepatic lipid desposition in rodents. Nagao et al. similarly reported that dietary resveratrol has an anti-obesity effect in obese rats. While it is difficult to reconcile our findings with the aforementioned two investigations, this discrepancy may be due to dosaging differences and/or the relatively short length of the current study.
A unique aspect of this study was using RNA-seq as an unbiased approach of examining liver transcriptome-wide differences in WD + AF versus WD animals when both groups were compared to CTL rats. Bioinformatic analyses suggest that AF potentially prevented WD-induced alterations in select genes that lead to the transport and metabolism of carbohydrates in favor of lipid transport and metabolism. Interestingly, phosphoenolpyruvate carboxykinase (Pck1) was down-regulated with AF supplementation. Gomez-Valades et al. have demonstrated that RNA interference against the Pck1 gene improves glycemic control, insulinemia and blood lipid levels in db/db mice. Therefore, the AF-induced downregulation of Pck1 may potentially lead to a longer-term stability in liver carbohydrate and fatty acid metabolism in the midst of WD feeding. Glut2 (Slc2a2) was also down-regulated with AF supplementation. Okamato et al. have illustrated in vitro that hepatocytes presenting steatosis show an enhanced expression of Glut2 mRNA; this being an effect which the authors suggest may be associated with liver gluconeogenesis and insulin resistance. Therefore, while the WD-fed rats supplemented with or without AF presented high fasting glucose levels suggestive of impaired glucose tolerance, the AF-induced decrease in liver Glut2 mRNA may again be potentially beneficial with regard to longer-term effects on liver metabolism. This is in agreement with Dushkin et al. that reported an improvement in glucose sensitivity and increased liver PPAR-α DNA-binding activity in male rats treated with rhaponticum extract for eight weeks. Cd36 was up-regulated with AF supplementation and functions in the uptake of long chain fatty acids along with oxidizing low-density lipoproteins. A plethora of evidence suggests that a lowered Cd36 expression is metabolically protective while overexpression is likely to result in metabolic complications[47, 48]. However, evidence also exists suggesting that Cd36 may be integral in glucose metabolism and insulin control[49–51].