When subclinical ketosis (SCK) occurs in dairy cows, it leads to an excessive production of β-hydroxybutyrat (BHBA), which disrupts liver lipid metabolism and triggers a series of inflammatory responses. Rutin (RT), a flavonoid extracted from plants, exhibits diverse biological activities. However, its potential to mitigate BHBA-induced liver inflammation and lipid accumulation in dairy cows remains unexplored. In this study, we investigated the effect of RT on the BHBA-induced injury of hepatocytes and the possible mechanism. First, hepatocytes were treated with BHBA (0, 0.3, 0.6, 1.2, 2.4 mM) to assess its effects on inflammation impairment and lipid accumulation. Second, hepatocytes were pretreated with RT (0, 25, 50, 100, 150 μg/mL) to evaluate its protective effects. Third, hepatocytes were divided into five treatment groups: blank control, BHBA treatment, RT + BHBA treatment, NF-κB activator (PDTC) + BHBA treatment, and RT + PDTC + BHBA treatment. This experiment further explored the underlying mechanism of RT in mitigating BHBA-induced hepatocyte injury. The results demonstrated that RT at 100 and 150 μg/mL mitigated the increases in hepatocyte interleukin-1 beta (IL-1β), IL-6, triglyceride (TG), and total cholesterol (TC) contents induced by high concentrations of BHBA (p < 0.05). Compared to the BHBA treatment, 100 μg/mL RT significantly downregulated the relative protein expression of P-NF-κB p65 and the relative mRNA expression of NF-κB p65, tumor necrosis factor-alpha (TNF-α), IL-1β, IL-6, peroxisome proliferator-activated receptor gamma (PPARγ), and microsomal triglyceride transfer protein (MTP), while upregulating the relative mRNA expression of IKBα (p < 0.05). Additionally, these effects were more pronounced with the combined pretreatment of the PDTC and RT. In conclusion, RT inhibits BHBA-triggered hepatocyte inflammation and lipid accumulation by modulating the NF-κB signaling pathway, implying that RT may be a promising target for ameliorating damage in SCK cows.

: Negative energy balance (NEB) in dairy cows induces excessive lipolysis, leading to elevated levels of β-hydroxybutyric acid (BHBA), which, when accumulated, can cause liver damage. Rutin (RT), a natural flavonoid with antioxidant and anti-inflammatory properties, has demonstrated potential hepatoprotective effects; however, its ability to mitigate BHBA-induced hepatocellular injury in calves remains unclear. This study first assessed the impact of various BHBA concentrations on oxidative stress in calf hepatocytes, then explored the protective effects and underlying mechanisms of RT, and finally employed untargeted metabolomics to further elucidate RT’s mode of action. The results showed that exposure to 1.2 mM BHBA significantly increased malondialdehyde (MDA), nitric oxide (NO) contents, and reactive oxygen species (ROS) levels, while markedly decreasing glutathione (GSH) content and catalase (CAT) activity compared with the blank control. Notably, pretreatment with 100 μg/mL RT resulted in the greatest increase in GSH contents (180%) compared to BHBA treatment alone, while 150 μg/mL RT led to the most pronounced reduction in MDA contents (220%). Furthermore, BHBA treatment significantly upregulated the expression of Kelch-like ECH-associated protein 1 (Keap1) and downregulated nuclear factor erythroid 2-related factor 2 (Nrf2), NAD(P)H quinone dehydrogenase 1 (NQO1), and heme oxygenase-1 (HO-1) at both the mRNA and protein levels. These alterations were effectively reversed by pretreatment with 100 μg/mL RT. Non-targeted metabolomics identified 1525 metabolites in total. Based on OPLS-DA, metabolites with a variable importance in projection (VIP) > 1 and p < 0.05 were considered significantly altered. Compared with the blank control, BHBA treatment upregulated 47 metabolites—including 8-hydroxy-2′-deoxyguanosine, 3-hydroxyisovaleric acid, and N-palmitoyl-sphingosine—and downregulated 58 metabolites, such as betaine, linolenic acid, and arachidonic acid. In contrast, RT pretreatment upregulated 207 metabolites relative to the BHBA treatment, including linolenic acid, taurocholic acid, and 4-hydroxybenzoic acid, and downregulated 126 metabolites, including 3-hydroxyisovaleric acid, 8-hydroxy-2′-deoxyguanosine, and pyruvaldehyde. Pathway enrichment analysis indicated that RT alleviated BHBA-induced hepatocyte injury primarily by modulating the fatty acid degradation pathway. In summary, RT mitigated BHBA-induced oxidative stress in calf hepatocytes by regulating the Keap1/Nrf2 signaling pathway and further exerted protective effects through metabolic reprogramming.