We aimed to investigate the effect of bromelain, the extract from stems of pineapples on the high-fat diet (HFD)-induced deregulation of hepatic lipid metabolism and non-alcoholic fatty liver disease (NAFLD), and its underlying mechanism in mice. Mice were daily administrated with HFD with or without bromelain (20 mg/kg) for 12 weeks, and we found that bromelain decreased the HFD-induced increase in body weight by ~30%, organ weight by ~20% in liver weight and ~40% in white adipose tissue weight. Additionally, bromelain attenuated HFD-induced hyperlipidemia by decreasing the serum level of total cholesterol by ~15% and triglycerides level by ~25% in mice. Moreover, hepatic lipid accumulation, particularly that of total cholesterol, free cholesterol, triglycerides, fatty acids, and glycerol, was decreased by 15–30% with bromelain treatment. Mechanistically, these beneficial effects of bromelain on HFD-induced hyperlipidemia and hepatic lipid accumulation may be attributed to the decreased fatty acid uptake and cholesteryl ester synthesis and the increased lipoprotein internalization, bile acid metabolism, cholesterol clearance, the assembly and secretion of very low-density lipoprotein, and the β-oxidation of fatty acids by regulating the protein expression involved in the above mentioned hepatic metabolic pathways. Collectively, these findings suggest that bromelain has therapeutic value for treating NAFLD and metabolic diseases.

Bromelain, a cysteine protease found in pineapple, has beneficial effects in the treatment of inflammatory diseases; however, its effects in cardiovascular pathophysiology are not fully understood. We investigated the effect of bromelain on atherosclerosis and its regulatory mechanisms in hyperlipidemia and atheroprone apolipoprotein E-null (apoe^−/−) mice. Bromelain was orally administered to 16-week-old male apoe^−/− mice for four weeks. Daily bromelain administration decreased hyperlipidemia and aortic inflammation, leading to atherosclerosis retardation in apoe^−/− mice. Moreover, hepatic lipid accumulation was decreased by the promotion of cholesteryl ester hydrolysis and autophagy through the AMP-activated protein kinase (AMPK)/transcription factor EB (TFEB)-mediated upregulation of autophagy- and antioxidant-related proteins. Moreover, bromelain decreased oxidative stress by increasing the antioxidant capacity and protein expression of antioxidant proteins while downregulating the protein expression of NADPH oxidases and decreasing the production of reactive oxygen species. Therefore, AMPK/TFEB signaling may be crucial in bromelain-mediated anti-hyperlipidemia, antioxidant, and anti-inflammatory effects, effecting the amelioration of atherosclerosis.

Background and Aims: HCC incidence is increasing worldwide due to the obesity epidemic, which drives metabolic dysfunction–associated steatohepatitis (MASH) that can lead to HCC. However, the molecular pathways driving MASH-HCC are poorly understood. We have previously reported that male mice with haploinsufficiency of hypoxia-associated factor (HAF) (SART1 ^+/−) spontaneously develop MASH-HCC. However, the cell type(s) responsible for HCC associated with HAF loss are unclear. Approach and Results: We generated SART1-floxed mice, which were crossed with mice expressing Cre recombinase within hepatocytes (Alb-Cre; hepS^−/−) or myeloid cells (LysM-Cre, macS^−/−). HepS ^−/− mice (both male and female) developed HCC associated with profound inflammatory and lipid dysregulation, suggesting that HAF protects against HCC primarily within hepatocytes. HAF-deficient hepatocytes showed decreased P-p65 and P-p50 in many components of the NF-κB pathway, which was recapitulated using HAF small interfering RNA in vitro. HAF depletion also triggered apoptosis, suggesting that HAF protects against HCC by suppressing hepatocyte apoptosis. We show that HAF regulates NF-κB activity by regulating the transcription of TRADD and RIPK1. Mice fed a high-fat diet showed marked suppression of HAF, P-p65, and TRADD within their livers after 26 weeks but showed profound upregulation of these proteins after 40 weeks, implicating deregulation of the HAF-NF-κB axis in the progression to MASH. In humans, HAF was significantly decreased in livers with simple steatosis but significantly increased in HCC compared with normal liver. Conclusions: HAF is a novel transcriptional regulator of the NF-κB pathway and is a key determinant of cell fate during progression to MASH and MASH-HCC.

Bromelain, an enzyme extracted from the stems of pineapples, exerts anticoagulant effects; however, the regulatory mechanisms are not fully understood. Here, we aimed to investigate the effects of bromelain on non-alcoholic fatty liver disease (NAFLD)-induced deregulation of blood coagulation and the underlying molecular mechanisms. C57BL/6 mice were fed a high-fat diet (HFD), with or without bromelain (20 mg/kg/day) administration, for 12 weeks. Treatment with bromelain decreased thrombus formation in the liver and prolonged HFD-induced shortened prothrombin, activated partial thromboplastin, and fibrinogen times. Moreover, liquid chromatography-mass spectrometry/mass spectrometry and Western blot analysis showed that bromelain inhibited NAFLD-induced activation of the intrinsic, extrinsic, and common pathways by upregulating the protein expression of antithrombin III, serpin family G member 1, and α1-antitrypsin, and downregulating the protein expression of fibrinogen in the liver and plasma. Bromelain also upregulated the level of plasminogen and downregulating factor XIII expression in the liver and plasma. Collectively, these findings suggest that bromelain exerts anticoagulant effects on NAFLD-induced deregulation of coagulation by inhibiting the activation of the coagulation cascade, decreasing the stability of clots, and promoting fibrinolytic activity. The present study provides new insights into the potential therapeutic value of bromelain for the prevention and treatment of thrombosis-related diseases.

HLJ1 (also called DNAJB4) is a member of the DNAJ/Hsp40 family and plays an important role in regulating protein folding and activity. However, there is little information about the role of HLJ1 in the regulation of physiological function. In this study, we investigated the role of HLJ1 in blood coagulation using wild-type C57BL/6 mice and HLJ1-null (HLJ1^-/-) mice. Western blot analysis and immunohistochemistry were used to assess the expression and distribution of HLJ1 protein, respectively. The tail bleeding assay was applied to assess the bleeding time and blood loss. A coagulation test was used for measuring the activity of extrinsic, intrinsic and common coagulation pathways. Thromboelastography was used to measure the coagulation parameters in the progression of blood clot formation. The results showed that HLJ1 was detectable in plasma and bone marrow. The distribution of HLJ1 was co-localized with CD41, the marker of platelets and megakaryocytes. However, genetic deletion of HLJ1 did not alter blood loss and the activity of extrinsic and intrinsic coagulation pathways, as well as blood clot formation, compared to wild-type mice. Collectively, these findings suggest that, although HLJ1 appears in megakaryocytes and platelets, it may not play a role in the function of blood coagulation under normal physiological conditions.

Oxidative stress is a central driver of atherosclerosis progression, promoting lipid peroxidation, vascular inflammation, and hepatic metabolic dysfunction. Forchlorfenuron (KT-30), a cytokinin-like plant growth regulator widely used on fruits such as kiwifruit, grapes, and watermelon, has been reported to elevate serum cholesterol levels, suggesting potential atherogenic effects. However, whether KT-30 induces oxidative stress and accelerates atherosclerosis remains unknown. Here, apolipoprotein E-deficient (apoE^−/−) mice were orally administered KT-30 (5 mg/kg/day) for four weeks. KT-30 significantly accelerated atherosclerotic lesion formation, elevated plasma cholesterol levels, upregulated scavenger receptors SR-A and CD36, and downregulated ABCA1 and LXRα, indicating impaired reverse cholesterol transport and enhanced foam cell formation. KT-30 also increased pro-inflammatory cytokines (IL-1β, IL-6, MIP-2) and aortic expression of F4/80 and VCAM-1. Critically, KT-30 exposure was associated with elevated oxidative stress markers, as evidenced by elevated plasma MDA levels, increased aortic 4-HNE immunostaining, and upregulation of NOX-1/4. In the liver, KT-30 induced lipid accumulation, characterized by elevated cholesterol and free fatty acids, accompanied by SREBP-1/2-driven de novo lipogenesis and impaired lipoprotein uptake. Proteomic analysis revealed significant alterations in mitochondrial oxidative phosphorylation- and sirtuin signaling-related protein expression. Taken together, KT-30-associated oxidative stress, accompanied by upregulation of NOX-1/4 and alterations in mitochondrial pathway-related protein expression, may represent an important mechanistic link between lipid homeostasis disruption and accelerated atherosclerosis in apoE^−/− mice, highlighting the potential pro-atherogenic effects of KT-30 in a susceptible experimental model and providing mechanistic evidence that warrants further investigation of its possible cardiovascular implications.