a-simple-protocol-for-isolation-culture-hepatocytes-from-masld

A Simple Protocol for the Isolation and Culture of Hepatocytes from MASLD Mice

Metabolic dysfunction-associated steatotic liver disease (MASLD), characterized by pathological lipid accumulation (steatosis) within hepatocytes, ...

University of Science and Technology of China

Natural products possess pleiotropic cardiovascular protective effects owing to their anti-oxidation, anti-inflammation and anti-thrombotic properties. Kaempferol, (3,5,7-trihydroxy-2-(4-hydroxyphenyl)-4H-1-benzopyran-4-one), is a kind of naturally occurring flavonoid existing in many common fruits and vegetables (e.g., onions, broccoli, strawberries and grapes) and particularly in traditional Chinese medicine as exemplified by . Epidemiological, preclinical and clinical studies have revealed an inverse association between the consumption of kaempferol-containing foods and medicines and the risk of developing cardiovascular diseases. Numerous translational studies in experimental animal models and cultured cells have demonstrated a wide range of pharmacological activities of kaempferol. In this article, we reviewed the antioxidant, anti-inflammatory and cardio-protective activities of kaempferol and elucidated the potential molecular basis of the therapeutic capacity of kaempferol by focusing on its anti-atherosclerotic effects. Overall, the review presents the health benefits of kaempferol-containing plants and medicines and reflects on the potential of kaempferol as a possible drug candidate to prevent and treat atherosclerosis, the underlying pathology of most cardiovascular diseases.

Kaempferol and atherosclerosis: From mechanism to medicine.

Aortic dissection (AD) is a fatal cardiovascular disease with limited pharmacotherapies. To discover novel therapeutic targets for AD, the present study was conducted on ascending aorta samples from AD patients versus those from control subjects using proteomic analysis. Integrated proteomic data analysis identified S100 calcium-binding proteins A8 and A9 (S100A8/A9) as new therapeutic targets for AD. As assessed by ELISA, the circulating levels of S100A8/A9 were elevated in AD patients. In addition, we validated the upregulation of S100A8/A9 in a mouse model of AD. In vitro and in vivo studies substantiated that S100A8/A9, as danger-associated molecular pattern molecules, promotes the smooth muscle cells phenotypic switch by inhibiting serum response factor (SRF) activity but elevating NF-κB dependent inflammatory response. Depletion of S100A8/A9 attenuates the occurrence and development of AD. As a proof of concept, we tested the safety and efficacy of pharmacological inhibition of S100A8/A9 by ABR-25757 (paquinimod) in a mouse model of AD. We observed that ABR-25757 ameliorated the incidence of rupture and improved elastin morphology associated with AD. Further single-cell RNA sequencing disclosed that the phenotypic switch of vascular smooth muscle cells (VSMCs) and inflammatory response pathways were responsible for ABR-25757-mediated protection against AD. Thus, this study reveals the regulatory mechanism of S100A8/A9 in AD and offers a potential therapeutic avenue to treat AD by targeting S100A8/A9.

A proteome-wide screen identifies the calcium binding proteins, S100A8/S100A9, as clinically relevant therapeutic targets in aortic dissection.

Nonalcoholic fatty liver disease (NAFLD) encompasses a disease continuum from simple steatosis to nonalcoholic steatohepatitis (NASH). However, there are currently no approved pharmacotherapies for NAFLD, although several drugs are in advanced stages of clinical development. Because of the complex pathophysiology and heterogeneity of NAFLD, the identification of potential therapeutic targets is clinically important. Here, we demonstrated that tripartite motif 56 (TRIM56) protein abundance was markedly downregulated in the livers of individuals with NAFLD and of mice fed a high-fat diet. Hepatocyte-specific ablation of TRIM56 exacerbated the progression of NAFLD, while hepatic TRIM56 overexpression suppressed it. Integrative analyses of interactome and transcriptome profiling revealed a pivotal role of TRIM56 in lipid metabolism and identified the lipogenesis factor fatty acid synthase (FASN) as a direct binding partner of TRIM56. TRIM56 directly interacted with FASN and triggered its K48-linked ubiquitination-dependent degradation. Finally, using artificial intelligence-based virtual screening, we discovered an orally bioavailable small-molecule inhibitor of FASN (named FASstatin) that potentiates TRIM56-mediated FASN ubiquitination. Therapeutic administration of FASstatin improved NAFLD and NASH pathologies in mice with an optimal safety, tolerability, and pharmacokinetics profile. Our findings provide proof of concept that targeting the TRIM56/FASN axis in hepatocytes may offer potential therapeutic avenues to treat NAFLD.

TRIM56 protects against nonalcoholic fatty liver disease by promoting the degradation of fatty acid synthase.

Vascular calcification, a devastating vascular complication accompanying atherosclerotic cardiovascular disease and chronic kidney disease, increases the incidence of adverse cardiovascular events and compromises the efficacy of vascular interventions. However, effective therapeutic drugs and treatments to delay or prevent vascular calcification are lacking.

Moscatilin inhibits vascular calcification by activating IL13RA2-dependent inhibition of STAT3 and attenuating the WNT3/β-catenin signalling pathway.

Within the context of residual cardiovascular risk in post-statin era, emerging evidence from epidemiologic and human genetic studies have demonstrated that triglyceride (TG)-rich lipoproteins and their remnants are causally related to cardiovascular risk. While, carriers of loss-of-function mutations of ApoC3 have low TG levels and are protected from cardiovascular disease (CVD). Of translational significance, siRNAs/antisense oligonucleotide (ASO) targeting ApoC3 is beneficial for patients with atherosclerotic CVD. Therefore, animal models of atherosclerosis with both hypercholesterolemia and hypertriglyceridemia are important for the discovery of novel therapeutic strategies targeting TG-lowering on top of traditional cholesterol-lowering. In this study, we constructed a novel mouse model of familial combined hyperlipidemia through inserting a human ApoC3 transgene (hApoC3-Tg) into C57BL/6 J mice and injecting a gain-of-function variant of adeno-associated virus-proprotein convertase subtilisin/kexin type 9 (AAV-PCSK9)-D377Y concurrently with high cholesterol diet (HCD) feeding for 16 weeks. In the last 10 weeks, hApoC3-Tg mice were orally treated with a combination of atorvastatin (10 mg·kg·d) and fenofibrate (100 mg·kg·d). HCD-treated hApoC3-Tg mice demonstrated elevated levels of serum TG, total cholesterol (TC) and low density lipoprotein-cholesterol (LDL-C). Oral administration of atorvastatin and fenofibrate significantly decreased the plaque sizes of en face aorta, aortic sinus and innominate artery accompanied by improved lipid profile and distribution. In summary, this novel mouse model is of considerable clinical relevance for evaluation of anti-atherosclerotic drugs by targeting both hypercholesterolemia and hypertriglyceridemia.

A novel mouse model of familial combined hyperlipidemia and atherosclerosis.

Heart failure with preserved ejection fraction (HFpEF) is a complex clinical syndrome with cardiac dysfunction, fluid retention and reduced exercise tolerance as the main manifestations. Current treatment of HFpEF is using combined medications of related comorbidities, there is an urgent need for a modest drug to treat HFpEF. Geniposide (GE), an iridoid glycoside extracted from Gardenia Jasminoides, has shown significant efficacy in the treatment of cardiovascular, digestive and central nervous system disorders. In this study we investigated the therapeutic effects of GE on HFpEF experimental models in vivo and in vitro. HFpEF was induced in mice by feeding with HFD and L-NAME (0.5 g/L) in drinking water for 8 weeks, meanwhile the mice were treated with GE (25, 50 mg/kg) every other day. Cardiac echocardiography and exhaustive exercise were performed, blood pressure was measured at the end of treatment, and heart tissue specimens were collected after the mice were euthanized. We showed that GE administration significantly ameliorated cardiac oxidative stress, inflammation, apoptosis, fibrosis and metabolic disturbances in the hearts of HFpEF mice. We demonstrated that GE promoted the transcriptional activation of Nrf2 by targeting MMP2 to affect upstream SIRT1 and downstream GSK3β, which in turn alleviated the oxidative stress in the hearts of HFpEF mice. In H9c2 cells and HL-1 cells, we showed that treatment with GE (1 μM) significantly alleviated HO-induced oxidative stress through the MMP2/SIRT1/GSK3β pathway. In summary, GE regulates cardiac oxidative stress via MMP2/SIRT1/GSK3β pathway and reduces cardiac inflammation, apoptosis, fibrosis and metabolic disorders as well as cardiac dysfunction in HFpEF. GE exerts anti-oxidative stress properties by binding to MMP2, inhibiting ROS generation in HFpEF through the SIRT1/Nrf2 signaling pathway. In addition, GE can also affect the inhibition of the downstream MMP2 target GSK3β, thereby suppressing the inflammatory and apoptotic responses in HFpEF. Taken together, GE alleviates oxidative stress/apoptosis/fibrosis and metabolic disorders as well as HFpEF through the MMP2/SIRT1/GSK3β signaling pathway.

Geniposide alleviates heart failure with preserved ejection fraction in mice by regulating cardiac oxidative stress via MMP2/SIRT1/GSK3β pathway.

Aclysiran, an RNAi therapeutic agent targeting ACLY, treats hypercholesterolemia and atherosclerosis in   mice.

Obesity is a debilitating global pandemic with a huge cost on health care due to it being a major underlying risk factor for several diseases. Therefore, there is an unmet medical need for pharmacological interventions to curb obesity. Here, we report that halofuginone, a Food and Drug Administration-approved anti-scleroderma and antiprotozoal drug, is a promising anti-obesity agent in preclinical mouse and pig models. Halofuginone suppressed food intake, increased energy expenditure, and resulted in weight loss in diet-induced obese mice while also alleviating insulin resistance and hepatic steatosis. Using molecular and pharmacological tools with transcriptomics, we identified that halofuginone increases fibroblast growth factor 21 (FGF21) and growth differentiation factor 15 (GDF15) levels via activating integrated stress response. Using  and  knockout mice, we show that both hormones are necessary to elicit anti-obesity changes. Together, our study reports the beneficial metabolic effects of halofuginone and underscores its utility in treating obesity and its associated metabolic complications, which merits clinical assessment.

The clinical antiprotozoal drug halofuginone promotes weight loss by elevating GDF15 and FGF21.

 Atherosclerotic cardiovascular disease (ASCVD) represents the predominant cause of mortality and morbidity globally. Given the established role of hypercholesterolemia as a significant risk factor for ASCVD, the discovery of new lipid-lowering medications is of paramount importance. ATP citrate lyase (ACLY) is a crucial enzyme in cellular metabolism, providing acetyl-CoA as the building block for the biosynthesis of fatty acids and cholesterol. Consequently, it has emerged as a promising drug target for innovative treatments of lipid metabolic disorders.  Virtual screening of a natural product library was performed to identify small-molecule ACLY inhibitors, leading to the discovery of isoginkgetin (ISOGK). The lipid-lowering and anti-atherosclerotic effects of ISOGK were validated in hypercholesterolemic diet-induced animal models (mice and hamsters). The inhibitory effects of ISOGK on ACLY enzymatic activity were measured using commercial assay kits. The direct interaction between ISOGK and ACLY was confirmed by surface plasmon resonance (SPR) and cellular thermal shift assays (CETSA). Liver-specific ACLY knockdown mice were generated using GalNAc-conjugated siRNA (GalNAc-siAcly).  ISOGK directly bind to ACLY and inhibit its enzymatic activity  and . By inhibiting ACLY, ISOGK treatment thus alleviates hypercholesterolemia and atherosclerosis in mice and hamsters. However, ISOGK fails to attenuate lipid accumulation and the expression of lipid-metabolism related genes in Acly knockout or depleted hepatocytes. , the lipid-lowering and anti-atherosclerotic effects of ISOGK were reversed by hepatic knockdown of  via treatment with GalNAc-siAcly in mice.  Taken together, the present study identifies ISOGK as an effective and naturally-occurring small-molecule inhibitor of ACLY that limits hypercholesterolemia and atherosclerosis. ISOGK thus serves as a promising drug lead in cardiovascular therapeutics.

Article	Year
Kaempferol and atherosclerosis: From mechanism to medicine. Critical reviews in food science and nutrition\| PubMed ID: 36099317	2024
A proteome-wide screen identifies the calcium binding proteins, S100A8/S100A9, as clinically relevant therapeutic targets in aortic dissection. Pharmacological research\| PubMed ID: 38056513	2024
TRIM56 protects against nonalcoholic fatty liver disease by promoting the degradation of fatty acid synthase. The Journal of clinical investigation\| PubMed ID: 38206764	2024
Moscatilin inhibits vascular calcification by activating IL13RA2-dependent inhibition of STAT3 and attenuating the WNT3/β-catenin signalling pathway. Journal of advanced research\| PubMed ID: 38432393	2025
A novel mouse model of familial combined hyperlipidemia and atherosclerosis. Acta pharmacologica Sinica\| PubMed ID: 38459255	2024
Geniposide alleviates heart failure with preserved ejection fraction in mice by regulating cardiac oxidative stress via MMP2/SIRT1/GSK3β pathway. Acta pharmacologica Sinica\| PubMed ID: 39060523	2024
Aclysiran, an RNAi therapeutic agent targeting ACLY, treats hypercholesterolemia and atherosclerosis in mice. Acta pharmaceutica Sinica. B\| PubMed ID: 39807322	2024
The clinical antiprotozoal drug halofuginone promotes weight loss by elevating GDF15 and FGF21. Science advances\| PubMed ID: 40138418	2025
A natural small molecule isoginkgetin alleviates hypercholesterolemia and atherosclerosis by targeting ACLY. Theranostics\| PubMed ID: 40225566	2025
Isoginkgetin, a natural biflavonoid from Ginkgo biloba, inhibits inflammatory response in endothelial cells via suppressing NF-κB activation. Acta pharmacologica Sinica\| PubMed ID: 40670675	2025

Meijie Chen

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