Gallic Acid Can Promote Low-Density Lipoprotein Uptake in HepG2 Cells via Increasing Low-Density Lipoprotein Receptor Accumulation.

Gallic acid (GA) is a type of polyphenolic compound that can be found in a range of fruits, vegetables, and tea. Although it has been confirmed it improves non-alcoholic fatty liver disease (NAFLD), it is still unknown whether GA can improve the occurrence of NAFLD by increasing the low-density lipoprotein receptor (LDLR) accumulation and alleviating cholesterol metabolism disorders. Therefore, the present study explored the effect of GA on LDLR and its mechanism of action. The findings indicated that the increase in LDLR accumulation in HepG2 cells induced by GA was associated with the stimulation of the epidermal growth factor receptor-extracellular regulated protein kinase (EGFR-ERK1/2) signaling pathway. When the pathway was inhibited by EGFR mab cetuximab, it was observed that the activation of the EGFR-ERK1/2 signaling pathway induced by GA was also blocked. At the same time, the accumulation of LDLR protein and the uptake of LDL were also suppressed. Additionally, GA can also promote the accumulation of forkhead box O3 (FOXO3) and suppress the accumulation of hepatocyte nuclear factor-1α (HNF1α), leading to the inhibition of proprotein convertase subtilisin/kexin 9 (PCSK9) mRNA expression and protein accumulation. This ultimately results in increased LDLR protein accumulation and enhanced uptake of LDL in cells. In summary, the present study revealed the potential mechanism of GA's role in ameliorating NAFLD, with a view of providing a theoretical basis for the dietary supplementation of GA.

Effects of PCSK9 on thrombosis and haemostasis in a variety of metabolic states: Lipids and beyond (Review).

Proprotein convertase subtilisin kexin type 9 (PCSK9) inhibitors are widely recognised as being able to induce a potent reduction in low­density lipoprotein­cholesterol. An increasing number of studies have suggested that PCSK9 also influences the haemostatic system by altering platelet function and the coagulation cascade. These findings have significant implications for anti­PCSK9 therapy in patients with specific coagulation conditions, including expanded indications, dose adjustments and drug interactions. The present review summarises the changes in PCSK9 levels in individuals with liver diseases, chronic kidney diseases, diabetes mellitus, cancer and other disease states, and discusses their impact on thrombosis and haemostasis. Furthermore, the structure, effects and regulatory mechanisms of PCSK9 on platelets, coagulation factors, inflammatory cells and endothelial cells during coagulation and haemostasis are described.

Statin administration or blocking PCSK9 alleviates airway hyperresponsiveness and lung fibrosis in high-fat diet-induced obese mice.

BACKGROUND: Obesity is associated with airway hyperresponsiveness and lung fibrosis, which may reduce the effectiveness of standard asthma treatment in individuals suffering from both conditions. Statins and proprotein convertase subtilisin/kexin-9 inhibitors not only reduce serum cholesterol, free fatty acids but also diminish renin-angiotensin system activity and exhibit anti-inflammatory effects. These mechanisms may play a role in mitigating lung pathologies associated with obesity. METHODS: Male C57BL/6 mice were induced to develop obesity through high-fat diet for 16 weeks. Conditional TGF-ß1 transgenic mice were fed a normal diet. These mice were given either atorvastatin or proprotein convertase subtilisin/kexin-9 inhibitor (alirocumab), and the impact on airway hyperresponsiveness and lung pathologies was assessed. RESULTS: High-fat diet-induced obesity enhanced airway hyperresponsiveness, lung fibrosis, macrophages in bronchoalveolar lavage fluid, and pro-inflammatory mediators in the lung. These lipid-lowering agents attenuated airway hyperresponsiveness, macrophages in BALF, lung fibrosis, serum leptin, free fatty acids, TGF-ß1, IL-1ß, IL-6, and IL-17a in the lung. Furthermore, the increased RAS, NLRP3 inflammasome, and cholecystokinin in lung tissue of obese mice were reduced with statin or alirocumab. These agents also suppressed the pro-inflammatory immune responses and lung fibrosis in TGF-ß1 over-expressed transgenic mice with normal diet. CONCLUSIONS: Lipid-lowering treatment has the potential to alleviate obesity-induced airway hyperresponsiveness and lung fibrosis by inhibiting the NLRP3 inflammasome, RAS and cholecystokinin activity.

Targeting PCSK9 to upregulate MHC-II on the surface of tumor cells in tumor immunotherapy.

BACKGROUND: Proprotein convertase subtilisin/kexin type 9 (PCSK9), the last member of the proprotein convertase family, functions as a classic regulator of low-density lipoprotein (LDL) by interacting with low-density lipoprotein receptor (LDLR). Recent studies have shown that PCSK9 can affect the occurrence and development of tumors and can be used as a novel therapeutic target. However, a comprehensive pan-cancer analysis of PCSK9 has yet to be conducted. METHODS: The potential oncogenic effects of PCSK9 in 33 types of tumors were explored based on the datasets of The Cancer Genome Atlas (TCGA) dataset. In addition, the immune regulatory role of PCSK9 inhibition was evaluated via in vitro cell coculture and the tumor-bearing mouse model. Finally, the antitumor efficacy of targeted PCSK9 combined with OVA-II vaccines was verified. RESULTS: Our results indicated that PCSK9 was highly expressed in most tumor types and was significantly correlated with late disease stage and poor prognosis. Additionally, PCSK9 may regulate the tumor immune matrix score, immune cell infiltration, immune checkpoint expression, and major histocompatibility complex expression. Notably, we first found that dendritic cell (DC) infiltration and major histocompatibility complex-II (MHC-II) expression could be upregulated by PCSK9 inhibition and improve CD8+ T cell activation in the tumor immune microenvironment, thereby achieving potent tumor control. Combining PCSK9 inhibitors could enhance the efficacies of OVA-II tumor vaccine monotherapy. CONCLUSIONS: Conclusively, our pan-cancer analysis provided a more comprehensive understanding of the oncogenic and immunoregulatory roles of PCSK9 and demonstrated that targeting PCSK9 could increase the efficacy of long peptide vaccines by upregulating DC infiltration and MHC-II expression on the surface of tumor cells. This study reveals the critical oncogenic and immunoregulatory roles of PCSK9 in various tumors and shows the promise of PCSK9 as a potent immunotherapy target.

Inhibition of PCSK9 with evolocumab modulates lipoproteins and monocyte activation in high-risk ASCVD subjects.

BACKGROUND: Mechanistic studies suggest that proprotein convertase subtilisin/kexin type 9 inhibitors can modulate inflammation. METHODS: Double-blind, placebo-controlled trial randomized 41 ASCVD subjects with type 2 diabetes with microalbuminuria and LDL-C level >70 mg/dL on maximum tolerated statin therapy received subcutaneous evolocumab 420 mg every 4 weeks or matching placebo. The primary outcomes were change in circulating immune cell transcriptional response, lipoproteins and blood viscosity at 2 weeks and 12 weeks. Safety was assessed in all subjects who received at least one dose of assigned treatment and analyses were conducted in the intention-to-treat population. RESULTS: All 41 randomized subjects completed the 2-week visit. Six subjects did not receive study medication consistently after the 2-week visit due to COVID-19 pandemic suspension of research activities. The groups were well-matched with respect to age, comorbidities, baseline LDL-C, white blood cell counts, and markers of systemic inflammation. Evolocumab reduced LDL-C by -68.8% (p < 0.0001) and -52.8% (p < 0.0001) at 2 and 12 weeks, respectively. There were no differences in blood viscosity at baseline nor at 2 and 12 weeks. RNA-seq was performed on peripheral blood mononuclear cells with and without TLR4 stimulation ("Stress" transcriptomics). "Stress" transcriptomics unmasked immune cell phenotypic differences between evolocumab and placebo groups at 2 and 12 weeks. CONCLUSIONS: This trial is the first to demonstrate that PCSK9 mAB with evolocumab can modulate circulating immune cell properties and highlights the importance of "stress" profiling of circulating immune cells that more clearly define immune contributions to ASCVD.

Leveraging Bidirectional Nature of Allostery To Inhibit Protein-Protein Interactions (PPIs): A Case Study of PCSK9-LDLR Interaction.

The protein PCSK9 (proprotein convertase subtilisin/Kexin type 9) negatively regulates the recycling of LDLR (low-density lipoprotein receptor), leading to an elevated plasma level of LDL. Inhibition of PCSK9-LDLR interaction has emerged as a promising therapeutic strategy to manage hypercholesterolemia. However, the large interaction surface area between PCSK9 and LDLR makes it challenging to identify a small molecule competitive inhibitor. An alternative strategy would be to identify distal cryptic sites as targets for allosteric inhibitors that can remotely modulate PCSK9-LDLR interaction. Using several microseconds long molecular dynamics (MD) simulations, we demonstrate that on binding with LDLR, there is a significant conformational change (population shift) in a distal loop (residues 211-222) region of PCSK9. Consistent with the bidirectional nature of allostery, we establish a clear correlation between the loop conformation and the binding affinity with LDLR. Using a thermodynamic argument, we establish that the loop conformations predominantly present in the apo state of PCSK9 would have lower LDLR binding affinity, and they would be potential targets for designing allosteric inhibitors. We elucidate the molecular origin of the allosteric coupling between this loop and the LDLR binding interface in terms of the population shift in a set of salt bridges and hydrogen bonds. Overall, our work provides a general strategy toward identifying allosteric hotspots: compare the conformational ensemble of the receptor between the apo and bound states of the protein and identify distal conformational changes, if any. The inhibitors should be designed to bind and stabilize the apo-specific conformations.

Optimization of lipid nanoparticles for gene editing of the liver via intraduodenal delivery.

Lipid nanoparticles (LNPs) have recently emerged as successful gene delivery platforms for a diverse array of disease treatments. Efforts to optimize their design for common administration methods such as intravenous injection, intramuscular injection, or inhalation, revolve primarily around the addition of targeting ligands or the choice of ionizable lipid. Here, we employed a multi-step screening method to optimize the type of helper lipid and component ratios in a plasmid DNA (pDNA) LNP library to efficiently deliver pDNA through intraduodenal delivery as an indicative route for oral administration. By addressing different physiological barriers in a stepwise manner, we down-selected effective LNP candidates from a library of over 1000 formulations. Beyond reporter protein expression, we assessed the efficiency in non-viral gene editing in mouse liver mediated by LNPs to knockdown PCSK9 and ANGPTL3 expression, thereby lowering low-density lipoprotein (LDL) cholesterol levels. Utilizing an all-in-one pDNA construct with Strep. pyogenes Cas9 and gRNAs, our results showcased that intraduodenal administration of selected LNPs facilitated targeted gene knockdown in the liver, resulting in a 27% reduction in the serum LDL cholesterol level. This LNP-based all-in-one pDNA-mediated gene editing strategy highlights its potential as an oral therapeutic approach for hypercholesterolemia, opening up new possibilities for DNA-based gene medicine applications.

​Comprehensive mendelian randomization analysis of plasma proteomics to identify new therapeutic targets for the treatment of coronary heart disease and myocardial infarction.

BACKGROUND: Ischemic heart disease is one of the leading causes of mortality worldwide, and thus calls for development of more effective therapeutic strategies. This study aimed to identify potential therapeutic targets for coronary heart disease (CHD) and myocardial infarction (MI) by investigating the causal relationship between plasma proteins and these conditions. METHODS: A two-sample Mendelian randomization (MR) study was performed to evaluate more than 1600 plasma proteins for their causal associations with CHD and MI. The MR findings were further confirmed through Bayesian colocalization, Summary-data-based Mendelian Randomization (SMR), and Transcriptome-Wide Association Studies (TWAS) analyses. Further analyses, including enrichment analysis, single-cell analysis, MR analysis of cardiovascular risk factors, phenome-wide Mendelian Randomization (Phe-MR), and protein-protein interaction (PPI) network construction were conducted to verify the roles of selected causal proteins. RESULTS: Thirteen proteins were causally associated with CHD, seven of which were also causal for MI. Among them, FES and PCSK9 were causal proteins for both diseases as determined by several analytical methods. PCSK9 was a risk factor of CHD (OR = 1.25, 95% CI: 1.13-1.38, P = 7.47E-06) and MI (OR = 1.36, 95% CI: 1.21-1.54, P = 2.30E-07), whereas FES was protective against CHD (OR = 0.68, 95% CI: 0.59-0.79, P = 6.40E-07) and MI (OR = 0.65, 95% CI: 0.54-0.77, P = 5.38E-07). Further validation through enrichment and single-cell analysis confirmed the causal effects of these proteins. Moreover, MR analysis of cardiovascular risk factors, Phe-MR, and PPI network provided insights into the potential drug development based on the proteins. CONCLUSIONS: This study investigated the causal pathways associated with CHD and MI, highlighting the protective and risk roles of FES and PCSK9, respectively. FES. Specifically, the results showed that these proteins are promising therapeutic targets for future drug development.

Exacerbated atherosclerosis in progeria is prevented by progerin elimination in vascular smooth muscle cells but not endothelial cells.

Hutchinson-Gilford progeria syndrome (HGPS) is a rare disease caused by the expression of progerin, a mutant protein that accelerates aging and precipitates death. Given that atherosclerosis complications are the main cause of death in progeria, here, we investigated whether progerin-induced atherosclerosis is prevented in HGPSrev-Cdh5-CreERT2 and HGPSrev-SM22α-Cre mice with progerin suppression in endothelial cells (ECs) and vascular smooth muscle cells (VSMCs), respectively. HGPSrev-Cdh5-CreERT2 mice were undistinguishable from HGPSrev mice with ubiquitous progerin expression, in contrast with the ameliorated progeroid phenotype of HGPSrev-SM22α-Cre mice. To study atherosclerosis, we generated atheroprone mouse models by overexpressing a PCSK9 gain-of-function mutant. While HGPSrev-Cdh5-CreERT2 and HGPSrev mice developed a similar level of excessive atherosclerosis, plaque development in HGPSrev-SM22α-Cre mice was reduced to wild-type levels. Our studies demonstrate that progerin suppression in VSMCs, but not in ECs, prevents exacerbated atherosclerosis in progeroid mice.

Dihydrostilbenes and flavonoids from whole plants of Jacobaea vulgaris.

The isolation of previously undescribed 12 compounds from the MeOH extract of Jacobaea vulgaris whole plants is disclosed, comprising 11 dihydrostilbenes (1-11) and one flavanone (12), and eight known compounds (six flavonoids, one dihydrostilbene, and one caffeoylquinic acid). Structural elucidation employed spectroscopic methods, including 1D and 2D NMR spectroscopy, HRESIMS, and ECD calculations. Evaluation of the compounds' effects on PCSK9 and LDLR mRNA expression revealed that compounds 1 and 3 downregulated PCSK9 mRNA while increasing LDLR mRNA expression, suggesting potential cholesterol-lowering properties.

Potential use of proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibition and prevention method in viral infection.

Cellular lipid membranes serve as the primary barrier preventing viral infection of the host cell and provide viruses with a critical initial point of contact. Occasionally, viruses can utilize lipids as viral receptors. Viruses depend significantly on lipid rafts for infection at virtually every stage of their life cycle. The pivotal role that proprotein convertase subtilisin/kexin Type 9 (PCSK9) plays in cholesterol homeostasis and atherosclerosis, primarily by post-transcriptionally regulating hepatic low-density lipoprotein receptor (LDLR) and promoting its lysosomal degradation, has garnered increasing interest. Conversely, using therapeutic, fully humanized antibodies to block PCSK9 leads to a significant reduction in high LDL cholesterol (LDL-C) levels. The Food and Drug Administration (FDA) has approved PCSK9 inhibitors, including inclisiran (Leqvio®), alirocumab (Praluent), and evolocumab (Repatha). At present, active immunization strategies targeting PCSK9 present a compelling substitute for passive immunization through the administration of antibodies. In addition to the current inquiry into the potential therapeutic application of PCSK9 inhibition in human immunodeficiency virus (HIV)-infected patients for hyperlipidemia associated with HIV and antiretroviral therapy (ART), preclinical research suggests that PCSK9 may also play a role in inhibiting hepatitis C virus (HCV) replication. Furthermore, PCSK9 inhibition has been suggested to protect against dengue virus (DENV) potentially and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viruses. Recent evidence regarding the impact of PCSK9 on a variety of viral infections, including HCV, HIV, DENV, and SARS-CoV-2, is examined in this article. As a result, PCSK9 inhibitors and vaccines may serve as viable host therapies for viral infections, as our research indicates that PCSK9 is significantly involved in the pathogenesis of viral infections.

PCSK9 stimulates Syk, PKCδ, and NF-κB, leading to atherosclerosis progression independently of LDL receptor.

Proprotein convertase subtilisin/kexin type-9 (PCSK9) binds to and degrades low-density lipoprotein (LDL) receptor, leading to increase of LDL cholesterol in blood. Its blockers have emerged as promising therapeutics for cardiovascular diseases. Here we show that PCSK9 itself directly induces inflammation and aggravates atherosclerosis independently of the LDL receptor. PCSK9 exacerbates atherosclerosis in LDL receptor knockout mice. Adenylyl cyclase-associated protein 1 (CAP1) is the main binding partner of PCSK9 and indispensable for the inflammatory action of PCSK9, including induction of cytokines, Toll like receptor 4, and scavenger receptors, enhancing the uptake of oxidized LDL. We find spleen tyrosine kinase (Syk) and protein kinase C delta (PKCδ) to be the key mediators of inflammation after PCSK9-CAP1 binding. In human peripheral blood mononuclear cells, serum PCSK9 levels are positively correlated with Syk, PKCδ, and p65 phosphorylation. The CAP1-fragment crystallizable region (CAP1-Fc) mitigates PCSK9-mediated inflammatory signal transduction more than the PCSK9 blocking antibody evolocumab does.

Oral PCSK9 Inhibitors.

PURPOSE OF REVIEW: In this review, we will discuss the data from early clinical studies of MK-0616 and summarize clinical trials of other oral proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitors. RECENT FINDINGS: The success of PCSK9 inhibition with monoclonal antibody injections has fueled the development of additional therapies targeting PCSK9, including oral formulations, the most advanced of which is MK-0616. MK-0616 is a novel, orally administered macrocyclic peptide that binds to PCSK9 and inhibits binding of PCSK9 to the LDL receptor, thereby decreasing plasma levels of LDL-C. Clinical trial data on the safety and efficacy of MK-0616 are promising and report LDL-C-lowering efficacy comparable to that provided by injectable PCSK9 inhibitors. Ongoing and future studies of oral PCSK9 inhibitors in development will evaluate the safety, efficacy, and effectiveness of these agents and their potential role in preventing cardiovascular disease events.

Multifaceted roles of Meg3 in cellular senescence and atherosclerosis.

BACKGROUND AND AIMS: Long noncoding RNAs are involved in the pathogenesis of atherosclerosis. As long noncoding RNAs maternally expressed gene 3 (Meg3) prevents cellular senescence of hepatic vascular endothelium and obesity-induced insulin resistance, we decided to examine its role in cellular senescence and atherosclerosis. METHODS AND RESULTS: By analyzing our data and human and mouse data from the Gene Expression Omnibus database, we found that Meg3 expression was reduced in humans and mice with cardiovascular disease, indicating its potential role in atherosclerosis. In Ldlr-/- mice fed a Western diet for 12 weeks, Meg3 silencing by chemically modified antisense oligonucleotides attenuated the formation of atherosclerotic lesions by 34.9% and 20.1% in male and female mice, respectively, revealed by en-face Oil Red O staining, which did not correlate with changes in plasma lipid profiles. Real-time quantitative PCR analysis of cellular senescence markers p21 and p16 revealed that Meg3 deficiency aggravates hepatic cellular senescence but not cellular senescence at aortic roots. Human Meg3 transgenic mice were generated to examine the role of Meg3 gain-of-function in the development of atherosclerosis induced by PCSK9 overexpression. Meg3 overexpression promotes atherosclerotic lesion formation by 29.2% in Meg3 knock-in mice independent of its effects on lipid profiles. Meg3 overexpression inhibits hepatic cellular senescence, while it promotes aortic cellular senescence likely by impairing mitochondrial function and delaying cell cycle progression. CONCLUSIONS: Our data demonstrate that Meg3 promotes the formation of atherosclerotic lesions independent of its effects on plasma lipid profiles. In addition, Meg3 regulates cellular senescence in a tissue-specific manner during atherosclerosis. Thus, we demonstrated that Meg3 has multifaceted roles in cellular senescence and atherosclerosis.

Sesamin alleviates lipid accumulation induced by oleic acid via PINK1/Parkin-mediated mitophagy in HepG2 cells.

Sesamin, a special compound present in sesame and sesame oil, has been reported a role in regulating lipid metabolism, while the underlying mechanisms remain unclear. Autophagy has been reported associated with lipid metabolism and regarded as a key modulator in liver steatosis. The present work aimed to investigate whether sesamin could exert its protective effects against lipid accumulation via modulating autophagy in HepG2 cells stimulated with oleic acid (OA). Cell viability was evaluated using the CCK-8 method, and triglycerides (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein, cholesterol (LDL-C), alanine aminotransferase (ALT), along with aspartate aminotransferase (AST) were assessed by oil red O staining, transmission electron microscopy (TEM), and biochemical kits to investigate the lipid-lowering effects of sesamin. Differentially expressed genes were screened by RNA sequencing and validated using real-time quantitative PCR and Western blot. Autophagy and mitophagy related molecules were analyzed employing TEM, Western blot, and immunofluorescence. The data shows that in HepG2 cells stimulated by OA, sesamin reduces levels of TG, TC, LDL-C, ALT, and AST while elevating HDL-C, alleviates the lipid accumulation and improves fatty acid metabolism through modulating the levels of fat metabolism related genes including PCSK9, FABP1, CD36, and SOX4. Sesamin restores the suppressed autophagy in HepG2 cells caused by OA, which could be blocked by autophagy inhibitors. This indicates that sesamin improves fatty acid metabolism by enhancing autophagy levels, thereby mitigating the intracellular lipid accumulation. Furthermore, sesamin significantly enhances the mitophagy and improves mitochondrial homeostasis via activating the PINK/Parkin pathway. These data suggest that sesamin alleviates the excessive lipid accumulation in HepG2 caused by OA by restoring the impaired mitophagy via the PINK1/Parkin pathway, probably playing a preventive or therapeutic role in hepatic steatosis.

A promising therapy for fatty liver disease: PCSK9 inhibitors.

BACKGROUND: Fatty liver disease (FLD) poses a significant global health concern worldwide, with its classification into nonalcoholic fatty liver disease (NAFLD) and alcoholic fatty liver disease (AFLD) contingent upon the presence or absence of chronic and excessive alcohol consumption. The absence of specific therapeutic interventions tailored to FLD at various stages of the disease renders its treatment exceptionally arduous. Despite the fact that FLD and hyperlipidemia are intimately associated, there is still debate over how lipid-lowering medications affect FLD. Proprotein Convertase Subtilisin/ Kexin type 9 (PCSK9) is a serine protease predominantly synthesized in the liver, which has a crucial impact on cholesterol homeostasis. Research has confirmed that PCSK9 inhibitors have prominent lipid-lowering properties and substantial clinical effectiveness, thereby justifying the need for additional exploration of their potential role in FLD. PURPOSE: Through a comprehensive literature search, this review is to identify the relationship and related mechanisms between PCSK9, lipid metabolism and FLD. Additionally, it will assess the pharmacological mechanism and applicability of PCSK9 inhibitors (including naturally occurring PCSK9 inhibitors, such as conventional herbal medicines) for the treatment of FLD and serve as a guide for updating the treatment protocol for such conditions. METHODS: A comprehensive literature search was conducted using several electronic databases, including Pubmed, Medline, Embase, CNKI, Wanfang database and ClinicalTrials.gov, from the inception of the database to 30 Jan 2024. Key words used in the literature search were "fatty liver", "hepatic steatosis", "PCSK9", "traditional Chinese medicine", "herb medicine", "botanical medicine", "clinical trial", "vivo", "vitro", linked with AND/OR. Most of the included studies were within five years. RESULTS: PCSK9 participates in the regulation of circulating lipids via both LDLR dependent and independent pathways, and there is a potential association with de novo lipogenesis. Major clinical studies have demonstrated a positive correlation between circulating PCSK9 levels and the severity of NAFLD, with elevated levels of circulating PCSK9 observed in individuals exposed to chronic alcohol. Numerous studies have demonstrated the potential of PCSK9 inhibitors to ameliorate non-alcoholic steatohepatitis (NASH), potentially completely alleviate liver steatosis, and diminish liver impairment. In animal experiments, PCSK9 inhibitors have exhibited efficacy in alleviating alcoholic induced liver lipid accumulation and hepatitis. Traditional Chinese medicine such as berberine, curcumin, resveratrol, piceatannol, sauchinone, lupin, quercetin, salidroside, ginkgolide, tanshinone, lunasin, Capsella bursa-pastoris, gypenosides, and Morus alba leaves are the main natural PCS9 inhibitors. Excitingly, by inhibiting transcription, reducing secretion, direct targeting and other pathways, traditional Chinese medicine exert inhibitory effects on PCSK9, thereby exerting potential FLD therapeutic effects. CONCLUSION: PCSK9 plays an important role in the development of FLD, and PCSK9 inhibitors have demonstrated beneficial effects on lipid regulation and FLD in both preclinical and clinical studies. In addition, some traditional Chinese medicines have improved the disease progression of FLD by inhibiting PCSK9 and anti-inflammatory and antioxidant effects. Consequently, the inhibition of PCSK9 appears to be a promising therapeutic strategy for FLD.

Harnessing RNA Interference for Cholesterol Lowering: The Bench-to-Bedside Story of Inclisiran.

Lowering low-density lipoprotein cholesterol (LDL-C) is a cornerstone of reducing risk for atherosclerotic cardiovascular disease. Despite the approval of nonstatin therapies for LDL-C lowering over the past 2 decades, these medications are underused, and most patients are still not at guideline-recommended LDL-C goals. Barriers include poor adherence, clinical inertia, concern for side effects, cost, and complex prior authorization processes. With atherosclerotic cardiovascular disease-related mortality increasing globally, there remains a need for additional therapeutic options for lowering LDL-C as part of an atherosclerotic cardiovascular disease prevention strategy. Following the identification of PCSK9 (proprotein convertase subtilisin/kexin type 9) as a promising therapeutic target, inclisiran was developed using the natural process of RNA interference for robust, sustained prevention of hepatic PCSK9 synthesis. Twice-yearly maintenance subcutaneous inclisiran (following initial loading doses at Day 1 and Day 90) reduces circulating LDL-C levels by ≈50% versus placebo when added to maximally tolerated statins. Long-term safety and tolerability of inclisiran have been assessed, with studies underway to evaluate the effects of inclisiran on cardiovascular outcomes and to provide additional safety and effectiveness data. In 2021, <20 years after the discovery of PCSK9, inclisiran became the first RNA interference therapeutic approved in the United States for LDL-C lowering in patients with established atherosclerotic cardiovascular disease or familial hypercholesterolemia and has since been approved for use in patients with primary hyperlipidemia. This article reviews the journey of inclisiran from bench to bedside, including early development, the clinical trial program, key characteristics of inclisiran, and practical points for its use in the clinic.

An update on inclisiran for the treatment of elevated LDL cholesterol.

INTRODUCTION: The burden of atherosclerotic cardiovascular disease (ASCVD) persists globally, demanding innovative therapeutic strategies. This manuscript provides an expert opinion on the significance of managing low-density lipoprotein cholesterol in ASCVD prevention and introduces inclisiran, a novel small interfering RNA targeting proprotein convertase subtilisin/kexin type 9 (PCSK9). AREAS COVERED: This work delves into the intricate mechanism of inclisiran, highlighting its unique approach of hepatic intracellular PCSK9 inhibition, its precision and low off-target effects risk. Pharmacodynamic and pharmacokinetic distinctions from PCSK9 monoclonal antibodies are explored, underlining inclisiran's efficiency, extended duration, and clearance. Clinical trials, including pivotal phase-III placebo-controlled studies (ORION-9, -10, -11), the open-label ORION-3 and pooled safety analysis of these trails including the open-label phase of ORION-8, as well as real-word data are discussed to provide a comprehensive evaluation of inclisiran's efficacy and safety. EXPERT OPINION: Inclisiran stands as a first-in-class breakthrough in lipid-lowering therapies, showing potential in alleviating the global burden of ASCVD and is supported by multiple global regulatory approvals. To optimize inclisiran's utilization and comprehend its long-term effects, future directions include pediatric studies, cardiovascular outcome trials, and extended-duration investigations. Overall, inclisiran emerges as a precise and effective therapeutic option, offering significant promise for preserving cardiovascular health.

Arenobufagin modulation of PCSK9-mediated cholesterol metabolism induces tumor-associated macrophages polarisation to inhibit hepatocellular carcinoma progression.

BACKGROUND: The tumor microenvironment (TME) of hepatocellular carcinoma is heterogeneous enough to be prone to drug resistance and multidrug resistance during treatment, and reprogramming of cholesterol metabolism in TME mediates tumor-associated macrophages (TAMs) polarization, which has an impact on the regulation of malignant tumor progression. Arenobufagin (ARBU) was extracted and isolated from toad venom (purity ≥98 %), which is the main active ingredient of the traditional Chinese medicine Chan'su with good anti-tumor effects. PURPOSE: To investigate the regulatory effect of ARBU on lipid metabolism in tumor microenvironment, interfere with macrophage polarization, and determine its mechanism of action on liver cancer progression. METHODS: In this study, the inhibitory effect of ARBU on the proliferation of Hepa1-6 in C57 mice and the safety of administration were evaluated by establishing a transplanted tumor model of Hepa1-6 hepatocellular carcinoma mice and using 5-FU as a positive control drug. In addition, we constructed a co-culture system of Hepa1-6 cells and primary mouse macrophages to study the effects of ARBU on the polarization phenotypic transformation of macrophages and the proliferation and migration of hepatoma cells. The influence of ARBU on the metabolism of lipids in the hepatocellular carcinoma mouse model was investigated by combining it with lipidomics technology. The influence of ARBU on the PCSK9/LDL-R signaling pathway and macrophage polarization, which regulate cholesterol metabolism, was tested by using qRT-PCR, gene editing, IF, and WB. CONCLUSION: ARBU significantly inhibited the proliferation of Hepa1-6 in vivo and in vitro, regulated cholesterol metabolism, and promoted the M1-type polarization of macrophages in the tumor microenvironment. ARBU inhibits cholesterol synthesis in the TME through the PCSK9/LDL-R signaling pathway, thereby blocking macrophage M2 polarization, promoting apoptosis of the tumor cells, and inhibiting their proliferation and migration.

PCSK9 plasma concentration is associated with epicardial adipose tissue volume and metabolic control in patients with type 1 diabetes.

Patients with type 1 diabetes (T1D) have a greater risk of cardiovascular disease. Proconvertase subtilisin-kexin 9 (PCSK9) is involved in the atherosclerosis process. This study aimed to determine the relationship between PCSK9 levels and epicardial adipose tissue (EAT) volume and cardiometabolic variables in patients with T1D. This was an observational cross-sectional study including 73 patients with T1D. Clinical, biochemical and imaging data were collected. We divided the patients into two groups according to their glycemic control and the EAT index (iEAT) percentile. We performed a correlation analysis between the collected variables and PCSK9 levels; subsequently, we performed a multiple regression analysis with the significant parameters. The mean age was 47.6 ± 8.5 years, 58.9% were men, and the BMI was 26.9 ± 4.6 kg/m2. A total of 31.5%, 49.3% and 34.2% of patients had hypertension, dyslipidemia and smoking habit, respectively. The PCSK9 concentration was 0.37 ± 0.12 mg/L, which was greater in patients with worse glycemic control (HbA1c > 7.5%), dyslipidemia and high EAT volume (iEAT > 75th percentile). The PCSK9 concentration was positively correlated with age (r = 0.259; p = 0.027), HbA1c (r = 0.300; p = 0.011), insulin dose (r = 0.275; p = 0.020), VLDL-C level (r = 0.331; p = 0.004), TG level (r = 0.328; p = 0.005), and iEAT (r = 0.438; p < 0.001). Multiple regression analysis revealed that 25% of the PCSK9 variability was explained by iEAT and HbA1c (p < 0.05). The PCSK9 concentration is associated with metabolic syndrome parameters, poor glycemic control and increased EAT volume in patients with T1D.