Fatty liver hemorrhagic syndrome (FLHS) is a chronic hepatic disease caused by a disorder of lipid metabolism, which usually presents as steatosis, cirrhosis, liver fibrosis, and NAFLD [53, 54]. FLHS occurs in cage laying hens with high frequency and is characterized by decreased egg production and unexplained death of laying hens. FLHS accounts for 74% of the total mortality of cage laying hens in Queensland, Australia . In addition, FLHS is the most common cause of non-communicable chicken deaths in Northern California . FLHS caused huge losses to the poultry industry.
SDS is a herbal drug which grows at high altitude areas  and have numerous pharmacological effects, e.g. protective effects on mitochondrial function , anti-apoptotic and anti-inflammatory effects , and antioxidant effects [59, 60]. Multiple studies suggested that SDS could reduce the liver lipid accumulation in both the type 2 diabetic  and NAFLD mice . In the current study, we found that SDS inhibited OA-induced lipid accumulation in primary chicken hepatocytes. Moreover, SDS promoted hepatocyte proliferation and inhibited its apoptosis in an OA-induced fatty liver model. SDS increased the hepatocyte activity and the mRNA expression of proliferation related genes PCNA, CDK2, and cyclinD1, and the protein expression levels of PCNA and CDK2. Moreover, SDS decreased the cleavage levels of Caspase-9, Caspase-8, and Caspase-3, and also the hepatocytes apoptosis. These results were consistent with the previous studies which indicated that SDS increased the protein expression of cyclin-dependent kinases (CDKs)  and Cyclin D1 , and suppressed cell apoptosis by inhibiting the pro-apoptotic protein expression of cleaved-Caspase-3/9 . Our study showed that SDS significantly attenuated OA-induced ROS generation, which was consistent with the finding that SDS attenuated high-fat diet-induced ROS generation in NAFLD mice .
PI3K/AKT/Gsk3-β is a critical anti-apoptotic signaling pathway which activates a series of growth signaling pathways and blocks apoptotic signaling pathways, thereby promoting cell survival and proliferation [51, 52]. Zhang et al. suggested that SDS protected against 1-methyl-4-phenylpyridine-induced cell apoptosis in part by regulating the PI3K/AKT/Gsk3-β pathway. SDS increased the phosphorylation levels of AKT and Gsk3-β, and inhibited the activation of caspase-3, caspase-6, and caspase-9 . SDS dose-dependently increased the phosphorylation of the mitochondria-associated PI3K/AKT/Gsk3-β pathway in hepatocytes , and alleviated sepsis induced myocarditis in rats by regulating the PI3K/AKT/Gsk3-β signaling pathway . In the present study, we found that SDS increased the phosphorylation levels of PDK1, AKT, and Gsk3-β but decreased the PI3K inhibitor. We determined that SDS alleviated lipid accumulation, hepatocyte apoptosis, and promoted hepatocyte proliferation in the OA-induced fatty liver model by targeting the PI3K/AKT/Gsk3-β pathway.
Furthermore, we investigated the effects of SDS on HFD-induced FLHS in laying hens in vivo. Results indicated that the layer’s body weight, liver weight, and abdominal fat weight were significantly increased in the Model group which having severe steatosis, whereas all those traits were decreased and improved in SDS feeding groups. Additionally, high-fat diet-induced FLHS of laying hens showed the high levels of TC, TG, ALT, and AST in serum, while SDS improved all those indexes and increased SOD activity of high-fat diet-fed birds. Zheng et al. detected that SDS alleviated hepatic steatosis, oxidative stress, and inflammatory reactions in the liver of NAFLD mice, characterized by the decreased levels of TC, TG, ALT, and AST in serum, inhibition of hepatic lipid deposition and the gene expression of FAS, and suppression of the gene expression of TNF-α and IL-1β in the liver . Moreover, Amevor et al.  reported there were decreased levels of AST and ALT in serum, and no liver steatosis in chickens fed with the diets containing both dietary quercetin and vitamin E (dietary antioxidants). The increased expression of lipogenesis gene FAS accelerated the ectopic deposition of TG . In the current study, we found that SDS decreased the liver mRNA expression of PPARγ, SCD, and FAS, whereas increased the mRNA expression of PPARα and MTTP in high-fat diet-induced FLHS of laying hens in vivo. Inflammatory cytokine TNF-α, IL-6, IL-8, and IL-1β played important roles in the inflammatory response . Previous studies reported that SDS alleviated cell injury and lipid accumulation, and inhibited the mRNA expression of IL-1β and IL-6 in human NAFLD , alleviated LPS-induced injury in humans by decreasing inflammatory chemokines IL-6 and TNF-α , and improved the survival rate of endotoxemia mice by blocking the activation of NF-κB and inhibiting the expression and release of inflammatory cytokines TNF-α, IL-6, and IL-1β . We found that SDS decreased the mRNA expression of TNF-α, IL-1β, IL-6, and IL-8. These results indicate that SDS alleviates HFD-induced hepatic steatosis, oxidative stress, and the inflammatory response of laying hens in vivo.