Hawthorn leaf flavonoids alleviate the deterioration of atherosclerosis by inhibiting SCAP-SREBP2-LDLR pathway through sPLA2-â ¡A signaling in macrophages in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Hawthorn leaves are a combination of the dried leaves of the Rosaceae plants, i.e., Crataegus pinnatifida Bge. or Crataegus pinnatifida Bge. var. major N. E. Br., is primarily cultivated in East Asia, North America, and Europe. hawthorn leaf flavonoids (HLF) are the main part of extraction. The HLF have demonstrated potential in preventing hypertension, inflammation, hyperlipidemia, and atherosclerosis. However, the potential pharmacological mechanism behind its anti-atherosclerotic effect has yet to be explored. AIM OF THE STUDY: The in vivo and in vitro effects of HLF on lipid-mediated foam cell formation were investigated, with a specific focus on the levels of secreted phospholipase A2 type IIA (sPLA2-II A) in macrophage cells. MATERIALS AND METHODS: The primary constituents of HLF were analyzed using ultra-high performance liquid chromatography and liquid chromatography-tandem mass spectrometry. In vivo, HLF, at concentrations of 5 mg/kg, 20 mg/kg, and 40 mg/kg, were administered to apolipoprotein E knockout mice (ApoE-/-) fed by high-fat diet (HFD) for 16 weeks. Aorta and serum samples were collected to identify lesion areas and lipids through mass spectrometry analysis to dissect the pathological process. RAW264.7 cells were incubated with oxidized low-density lipoprotein (ox-LDL) alone, or ox-LDL combined with different doses of HLF (100, 50, and 25 µg/ml), or ox-LDL plus 24-h sPLA2-IIA inhibitors, for cell biology analysis. Lipids and inflammatory cytokines were detected using biochemical analyzers and ELISA, while plaque size and collagen content of plaque were assessed by HE and the Masson staining of the aorta. The lipid deposition in macrophages was observed by Oil Red O staining. The expression of sPLA2-IIA and SCAP-SREBP2-LDLR was determined by RT-qPCR and Western blot analysis. RESULTS: The chemical profile of HLF was studied using UPLC-Q-TOF-MS/MS, allowing the tentative identification of 20 compounds, comprising 1 phenolic acid, 9 flavonols and 10 flavones, including isovitexin, vitexin-4″-O-glucoside, quercetin-3-O-robibioside, rutin, vitexin-2″-O-rhamnoside, quercetin, etc. HLF decreased total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), and non-high-density lipoprotein cholesterol (non-HDL-C) levels in ApoE-/- mice (P < 0.05), reduced ox-LDL uptake, inhibited level of inflammatory factors, such as IL-6, IL-8, TNF-α, and IL-1ꞵ (P < 0.001), and alleviated aortic plaques with a thicker fibrous cap. HLF effectively attenuated foam cell formation in ox-LDL-treated RAW264.7 macrophages, and reduced levels of intracellular TC, free cholesterol (FC), cholesteryl ester (CE), IL-6, TNF-α, and IL-1ß (P < 0.001). In both in vivo and in vitro experiments, HLF significantly downregulated the expression of sPLA2-IIA, SCAP, SREBP2, LDLR, HMGCR, and LOX-1 (P < 0.05). Furthermore, sPLA2-IIA inhibitor effectively mitigated inflammatory release in RAW264.7 macrophages and regulated SCAP-SREBP2-LDLR signaling pathway by inhibiting sPLA2-IIA secretion (P < 0.05). CONCLUSION: HLF exerted a protective effect against atherosclerosis through inhibiting sPLA2-IIA to diminish SCAP-SREBP2-LDLR signaling pathway, to reduce LDL uptake caused foam cell formation, and to slow down the progression of atherosclerosis in mice.

Chlorogenic acid regulates the expression of NPC1L1 and HMGCR through PXR and SREBP2 signaling pathways and their interactions with HSP90 to maintain cholesterol homeostasis.

BACKGROUND: Hypercholesterolemia is widely implicated in the etiology of coronary heart disease, stroke, and dementia. Evidence suggests that chlorogenic acid (CA) reduces the risk of cardiovascular disease. PURPOSE: The current study aims to explore the underlying molecular mechanism of CA in lowering cholesterol based on pregnane X receptor (PXR) and sterol regulatory element-binding protein 2 (SREBP2) regulatory pathways and their interactions with heat shock protein 90 (HSP90). METHODS: A hypercholesterolemic mouse model, HepG2 and Caco2 cell models, metabolomics analysis, and co-immunoprecipitation (COIP) were used to study the mechanism of CA lowering cholesterol. RESULTS: Treatment of the hypercholesterolemic mice with CA for 12 weeks significantly reduced body weight, blood lipid, hepatic lipid accumulation, and increased lipid excretion. The nuclear aggregation of PXR and SREBP2 was inhibited simultaneously. In addition, the expression of downstream target genes, including Niemann-pick C1-like 1 (NPC1L1) and 3­hydroxy-3-methylglutaryl-CoA reductase (HMGCR), was downregulated after CA administration. Furthermore, in HepG2 and Caco2 cell models, CA reduced intracellular cholesterol levels by inhibiting the nuclear translocation of PXR and SREBP2 and the expression of NPC1L1 and HMGCR. SREBP2 interacts with PXR through HSP90, and CA reduces the binding stability of SREBP2 and HSP90 and enhances the binding of PXR and HSP90, thus reducing the nuclear accumulation of SREBP2 and PXR simultaneously. Moreover, CA promoted the phosphorylation of AMP-activated protein kinase (AMPK) and its binding to SREBP2. This was not conducive to the binding of HSP90 and SREBP2 but enhanced the binding of HSP90 and PXR, thereby inhibiting the nuclear translocation of SREBP2 and PXR and reducing intracellular cholesterol levels. However, no noticeable direct binding between AMPK and PXR was observed. CONCLUSION: CA downregulates NPC1L1 and HMGCR expression by acting on the AMPK/SREBP2 direct pathway and the AMPK/SREBP2/HSP90/PXR indirect pathway, thus retaining cholesterol homeostasis.

Induced pluripotent stem cells-based disease modeling, drug screening, clinical trials, and reverse translational research for amyotrophic lateral sclerosis.

It has been more than 10 years since the hopes for disease modeling and drug discovery using induced pluripotent stem cell (iPSC) technology boomed. Recently, clinical trials have been conducted with drugs identified using this technology, and some promising results have been reported. For amyotrophic lateral sclerosis (ALS), a devastating neurodegenerative disease, several groups have identified candidate drugs, ezogabine (retigabine), bosutinib, and ropinirole, using iPSCs-based drug discovery, and clinical trials using these drugs have been conducted, yielding interesting results. In our previous study, an iPSCs-based drug repurposing approach was utilized to show the potential of ropinirole hydrochloride (ROPI) in reducing ALS-specific pathological phenotypes. Recently, a phase 1/2a trial was conducted to investigate the effects of ropinirole on ALS further. This double-blind, randomized, placebo-controlled study confirmed the safety and tolerability of and provided evidence of its ability to delay disease progression and prolong the time to respiratory failure in ALS patients. Furthermore, in the reverse translational research, in vitro characterization of patient-derived iPSCs-motor neurons (MNs) mimicked the therapeutic effects of ROPI in vivo, suggesting the potential application of this technology to the precision medicine of ALS. Interestingly, RNA-seq data showed that ROPI treatment suppressed the sterol regulatory element-binding protein 2-dependent cholesterol biosynthesis pathway. Therefore, this pathway may be involved in the therapeutic effect of ROPI on ALS. The possibility that this pathway may be involved in the therapeutic effect of ALS was demonstrated. Finally, new future strategies for ALS using iPSCs technology will be discussed in this paper.

B7H3 increases ferroptosis resistance by inhibiting cholesterol metabolism in colorectal cancer.

Ferroptosis, a newly discovered form of regulated cell death, has been reported to be associated with multiple cancers, including colorectal cancer (CRC). However, the underlying molecular mechanism is still unclear. In this study, we identified B7H3 as a potential regulator of ferroptosis resistance in CRC. B7H3 knockdown decreased but B7H3 overexpression increased the ferroptosis resistance of CRC cells, as evidenced by the expression of ferroptosis-associated genes (PTGS2, FTL, FTH, and GPX4) and the levels of important indicators of ferroptosis (malondialdehyde, iron load). Moreover, B7H3 promoted ferroptosis resistance by regulating sterol regulatory element binding protein 2 (SREBP2)-mediated cholesterol metabolism. Both exogenous cholesterol supplementation and treatment with the SREBP2 inhibitor betulin reversed the effect of B7H3 on ferroptosis in CRC cells. Furthermore, we verified that B7H3 downregulated SREBP2 expression by activating the AKT pathway. Additionally, multiplex immunohistochemistry was carried out to show the expression of B7H3, prostaglandin-endoperoxide synthase 2, and SREBP2 in CRC tumor tissues, which was associated with the prognosis of patients with CRC. In summary, our findings reveal a role for B7H3 in regulating ferroptosis by controlling cholesterol metabolism in CRC.

The IDI1/SREBP2 axis drives intrahepatic cholestasis and is a treatment target of San-Huang-Cai-Zhu formula identified by sequencing and experiments.

San-Huang-Chai-Zhu formula (SHCZF), originates from Da-Huang-Xiao-Shi decoction (DHXSD) for the treatment of jaundice as recorded in the Chinese traditional Chinese medicine book Jin Gui Yao Lue. In the clinic, SHCZF has been used to treat cholestasis-related liver disease by improving intrahepatic cholestasis, but the treatment mechanism has not been elucidated. In this study, 24 Sprague-Dawley (SD) rats were randomly assigned to the normal, acute intrahepatic cholestasis (AIC), SHCZF, and ursodeoxycholic acid (UDCA) groups. In addition, 36 SD rats were divided into dynamic groups, namely, normal 24 h, AIC 24 h, normal 48 h, AIC 48 h, normal 72 h, and AIC 72 h groups. Alpha-naphthylisothiocyanate (ANIT) was used to induce an AIC rat model. Serum biochemical indices and hepatic pathology were detected. Part of the hepatic tissues was used for sequencing, and others were used for subsequent experiments. Sequencing data combined with bioinformatics analysis were used to screen target genes and identify the mechanisms of SHCZF in treating AIC rats. Quantitative real-time PCR (qRT-PCR) and Western blotting (WB) were used to detect the RNA/Protein expression levels of screened genes. Rats in the dynamic group were used to determine the sequence of cholestasis and liver injury. High-performance liquid chromatography (HPLC) was used to determine the representative bioingredients of SHCZF. Sequencing and bioinformatics analysis suggested that IDI1 and SREBP2 are hub target genes of SHCZF to ameliorate ANTI-induced intrahepatic cholestasis in rats. The treatment mechanism is associated with the regulation of lipoprotein receptor (LDLr) to reduce cholesterol intake and 3-Hydroxy-3-Methylglutaryl-CoA reductase (HMGCR), and 3-Hydroxy-3-Methylglutaryl-CoA synthase 1 (HMGCS1) to decrease cholesterol synthesis. Animal experiments showed that SHCZF significantly reduced the expression levels of the above genes and proinflammatory cytokine lipocalin 2 (LCN2), inflammatory cytokines interleukin 1 beta (IL-1ß) and tumor necrosis factor alpha (TNF-α), thereby improving intrahepatic cholestasis and inflammation and liver injury.

Matrine suppresses NLRP3 inflammasome activation via regulating PTPN2/JNK/SREBP2 pathway in sepsis.

BACKGROUND: Sepsis is defined as life-threatening organ dysfunction caused by a dysregulated host response to infection. Abnormal activation of NOD-like receptor thermal protein domain associated protein 3 (NLRP3) inflammasome plays a vital role in the pathogenesis of sepsis. Matrine is proved to show good anti-inflammatory properties, whereas its effect and the underlying molecular machinery on sepsis remains unclear. PURPOSE: The aim of this study is to evaluate the effect and mechanism of Matrine on sepsis. STUDY DESIGN: THP-1 cells and J774A.1 cells were stimulated by lipopolysaccharide (LPS) with nigericin or adenosine triphosphate (ATP) to establish an in vitro model. Cecal ligation and puncture (CLP)-induced sepsis mouse model was used. Matrine was given by gavage. METHODS: To investigate the NLRP3 inflammasome activation, phorbol myristate acetate (PMA)-induced THP-1 cells were first primed with LPS and then stimulated by matrine, followed by treatment with nigericin or ATP. The concentration of interleukin 1ß (IL-1ß) and interleukin 18 (IL-18) in the cell culture supernatant was detected. The mechanism was explored by cell death assay, immunoblots and immunofluorescence in vitro. C57BL/6 mice were intragastrically administered with matrine for 5 days before CLP. The therapeutic effect of matrine was evaluated by symptoms, pathological analysis, ELISA and RT-qPCR. RESULTS: Our results revealed that matrine inhibited IL-1ß and IL-18 secretion, suppressed caspase-1 activation, reduced cell death, and blocked ASC speck formation upon NLRP3 inflammasome activation. Furthermore, matrine restrains NLRP3 inflammasome activation as well as pyroptosis through regulating the protein tyrosine phosphatase non-receptor type 2 (PTPN2)/JNK/SREBP2 signaling. Matrine also prominently improved the symptoms and pathological changes with reduced levels of TNF-α, IL-1ß, and IL-6 in the lung tissues and serum in a dose-dependent manner. CONCLUSION: Matrine effectively alleviates the symptoms of CLP-induced sepsis in mice, restrains NLRP3 inflammasome activation by regulating PTPN2/JNK/SREBP2 signaling pathway, and may become a promising therapeutic agent for sepsis treatment.

Hempseed (Cannabis sativa) Peptide H3 (IGFLIIWV) Exerts Cholesterol-Lowering Effects in Human Hepatic Cell Line.

Hempseed (Cannabis sativa) protein is an important source of bioactive peptides. H3 (IGFLIIWV), a transepithelial transported intestinal peptide obtained from the hydrolysis of hempseed protein with pepsin, carries out antioxidant and anti-inflammatory activities in HepG2 cells. In this study, the main aim was to assess its hypocholesterolemic effects at a cellular level and the mechanisms behind this health-promoting activity. The results showed that peptide H3 inhibited the 3-hydroxy-3-methylglutaryl co-enzyme A reductase (HMGCoAR) activity in vitro in a dose-dependent manner with an IC50 value of 59 µM. Furthermore, the activation of the sterol regulatory element binding proteins (SREBP)-2 transcription factor, followed by the increase of low-density lipoprotein (LDL) receptor (LDLR) protein levels, was observed in human hepatic HepG2 cells treated with peptide H3 at 25 µM. Meanwhile, peptide H3 regulated the intracellular HMGCoAR activity through the increase of its phosphorylation by the activation of AMP-activated protein kinase (AMPK)-pathways. Consequently, the augmentation of the LDLR localized on the cellular membranes led to the improved ability of HepG2 cells to uptake extracellular LDL with a positive effect on cholesterol levels. Unlike the complete hempseed hydrolysate (HP), peptide H3 can reduce the proprotein convertase subtilisin/kexin 9 (PCSK9) protein levels and its secretion in the extracellular environment via the decrease of hepatic nuclear factor 1-α (HNF1-α). Considering all these evidences, H3 may represent a new bioactive peptide to be used for the development of dietary supplements and/or peptidomimetics for cardiovascular disease (CVD) prevention.

Di'ao Xinxuekang Capsule Improves the Anti-Atherosclerotic Effect of Atorvastatin by Downregulating the SREBP2/PCSK9 Signalling Pathway.

Statins are the first choice for lowering low-density lipoprotein cholesterol (LDL-C) and preventing atherosclerotic cardiovascular disease (ASCVD). However, statins can also upregulate proprotein convertase subtilisin/kexin type 9 (PCSK9), which in turn might limits the cholesterol-lowering effect of statins through the degradation of LDL receptors (LDLR). Di'ao Xinxuekang (DXXK) capsule, as a well-known traditional Chinese herbal medicine for the prevention and treatment of coronary heart disease, can alleviate lipid disorders and ameliorate atherosclerosis in atherosclerosis model mice and downregulate the expression of PCSK9. In this study, we further explored whether DXXK has a synergistic effect with atorvastatin (ATO) and its underlying molecular mechanism. The results showed that both ATO monotherapy (1.3 mg/kg) and ATO combined with DXXK therapy significantly lowered serum lipid levels and reduced the formation of atherosclerotic plaques and the liver lipid accumulation. Moreover, compared with ATO monotherapy, the addition of DXXK (160 mg/kg) to the combination therapy further lowered LDL-C by 15.55% and further reduced the atherosclerotic plaque area by 25.98%. In addition, the expression of SREBP2, PCSK9 and IDOL showed a significant increase in the model group, and the expression of LDLR was significantly reduced; however, there were no significant differences between the ATO (1.3 mg/kg) and the model groups. When ATO was combined with DXXK, the expression of LDLR was significantly increased and was higher than that of the model group and the expression of SREBP2 and PCSK9 in the liver was also significantly inhibited. Moreover, it can be seen that the expression of SREBP2 and PCSK9 in the combination treatment group was significantly lower than that in the ATO monotherapy group (1.3 mg/kg). Besides, the expression of IDOL mRNA in each treatment group was not significantly different from that of the model group. Our study suggests that DXXK might have a synergistic effect on the LDL-C lowering and antiatherosclerosis effects of ATO through the SREBP2/PCSK9 pathway. This indicates that a combination of DXXK and ATO may be a new treatment for atherosclerosis.

Targeted inhibition of PPARα ameliorates CLA-induced hypercholesterolemia via hepatic cholesterol biosynthesis reprogramming.

BACKGROUND & AIMS: Disruption of lipid metabolism is largely linked to metabolic disorders, such as hypercholesterolemia (HCL) and liver steatosis. While cholesterol metabolic re-programmers can serve as targets for relevant interventions. Here we explored the dietary conjugated linoleic acids (CLA)-induced HCL in mice and the molecular regulation behind it. METHODS: A high dose of CLA supplementation in the diet was used to induce HCL in mice and was found to cause a hyper-activated cholesterol biosynthesis programme in the liver, leading to cholesterol metabolism dysregulation. The effects of a small-molecule drug targeting PPARα, i.e., GW6471 were studied in vivo in mice fed diets with CLA supplementation for 28 days, and in primary hepatocytes derived from HCL-mice in vitro. RESULTS: We demonstrate that CLA induced HCL and liver steatosis through multiple pathways. Among which was the PPARα-mediated cholesterogenesis. It was found to cooperate with SREBP2 via binding to Hmgcr and Dhcr7 (genes encoding key enzymes of the cholesterol biosynthetic pathway) and recruits the histone marks H3K27ac and H3K4me1 and cofactors. PPARα inhibition disrupts its physical association with SREBP2 by blocking cobinding of PPARα and SREBP2 to the genomic DNA response element. We showed that NR RORγ functions as an essential mediator that facilitates the interaction of PPARα and SREBP2 to modulate the cholesterol biosynthesis genes expression. CONCLUSIONS: Our study unravels that the small-molecule compound GW6471 exerts an attractive therapeutic effect for CLA-induced HCL, involving multiple pathways with the "PPARα-RORγ-SREBP2" being a potential complex player in this hepatic cholesterol biosynthesis programming.

Lecithin Inclusion by α-Cyclodextrin Activates SREBP2 Signaling in the Gut and Ameliorates Postprandial Hyperglycemia.

BACKGROUND: α-cyclodextrin (α-CD) is one of the dietary fibers that may have a beneficial effect on cholesterol and/or glucose metabolism, but its efficacy and mode of action remain unclear. METHODS: In the present study, we examined the anti-hyperglycemic effect of α-CD after oral loading of glucose and liquid meal in mice. RESULTS: Administration of 2 g/kg α-CD suppressed hyperglycemia after glucose loading, which was associated with increased glucagon-like peptide 1 (GLP-1) secretion and enhanced hepatic glucose sequestration. By contrast, 1 g/kg α-CD similarly suppressed hyperglycemia, but without increasing secretions of GLP-1 and insulin. Furthermore, oral α-CD administration disrupts lipid micelle formation through its inclusion of lecithin in the gut luminal fluid. Importantly, prior inclusion of α-CD with lecithin in vitro nullified the anti-hyperglycemic effect of α-CD in vivo, which was associated with increased intestinal mRNA expressions of SREBP2-target genes (Ldlr, Hmgcr, Pcsk9, and Srebp2). CONCLUSIONS: α-CD elicits its anti-hyperglycemic effect after glucose loading by inducing lecithin inclusion in the gut lumen and activating SREBP2, which is known to induce cholecystokinin secretion to suppress hepatic glucose production via a gut/brain/liver axis.

Berberine mitigates nonalcoholic hepatic steatosis by downregulating SIRT1-FoxO1-SREBP2 pathway for cholesterol synthesis.

OBJECTIVE: To investigate effects of berberine (BBR) on cholesterol synthesis in HepG2 cells with free fatty acid (FFA)-induced steatosis and to explore the underlying mechanisms. METHODS: A steatosis cell model was induced in HepG2 cell line fed with FFA (0.5 mmol/L, oleic acid:palmitic acid = 2:1), and then treated with three concentrations of BBR; cell viability was assessed with cell counting kit-8 assays. Lipid accumulation in cells was observed through oil red O staining and total cholesterol (TC) content was detected by TC assay. The effects of BBR on cholesterol synthesis mediators were assessed by Western blotting and quantitative polymerase chain reaction. In addition, both silent information regulator 1 (SIRT1) and forkhead box transcription factor O1 (FoxO1) inhibitors were employed for validation. RESULTS: FFA-induced steatosis was successfully established in HepG2 cells. Lipid accumulation and TC content in BBR groups were significantly lower (P < 0.05, P < 0.01), associated with significantly higher mRNA and protein levels of SIRT1(P < 0.05, P < 0.01), significantly lower sterol regulatory element-binding protein 2 (SREBP2) and 3-hydroxy 3-methylglutaryl-CoA reductase levels (P < 0.05, P < 0.01), as well as higher Acetyl-FoxO1 protein level (P < 0.05, P < 0.01) compared to the FFA only group. Both SIRT1 inhibitor SIRT1-IN-1 and FoxO1 inhibitor AS1842856 blocked the BBR-mediated therapeutic effects. Immunofluorescence showed that the increased SIRT1 expression increased FoxO1 deacetylation, and promoted its nuclear translocation. CONCLUSION: BBR can mitigate FFA-induced steatosis in HepG2 cells by activating SIRT1-FoxO1-SREBP2 signal pathway. BBR may emerge as a potential drug candidate for treating nonalcoholic hepatic steatosis.

Gypenoside LVI improves hepatic LDL uptake by decreasing PCSK9 and upregulating LDLR expression.

BACKGROUNDS: Atherosclerotic Cardiovascular Disease (ASCVD) is defined as ischemic or endothelial dysfunction-various inflammatory diseases, which is mainly caused by excessive low-density lipoprotein cholesterol (LDL-C) in circulating blood. Gynostemma pentaphyllum is a traditional Chinese medicine, and total Gypenosides are used for the treatment of hyperlipidemia and to reduce circulating proprotein convertase subtilisin/kexin type 9 (PCSK9) level. However, which gypenoside involved in the modulation of PCSK9 expression is still unknown. PURPOSE: This study aimed to discover effective PCSK9 inhibitors from Gypenosides in accordance with the 2019 ESC/EAS guidelines. METHODS: HPLC was employed to determine major six components of Gypenosides. The inhibitory activity on secreted PCSK9 in HepG2 of six major compounds from Gypenosides were screened by ELISA. The level of low-density lipoprotein (LDL) receptor (LDLR) was determined by Flow cytometry and Immunofluorescence. The expression levels of PCSK9, LDLR and Sterol-regulatory element binding proteins-2 (SREBP-2) were analyzed by qPCR and Western blot. DiI-LDL was added to evaluated LDL uptake into HepG2. RESULTS: The results suggested that the mRNA and protein levels of PCSK9 were down-regulated by Gypenoside LVI and the LDLR degradation in lysosomes system was inhibited, thereby leading to an increasing in LDL uptake into HepG2 cells. Furthermore, Gypenoside LVI decreased PCSK9 expression induced by stains. Altogether, Gypenoside LVI enhances LDL uptake into HepG2 cells by increased LDLR level on cell-surface and suppressed PCSK9 expression. CONCLUSION: This indicates that Gypenoside LVI can be used as a useful supplement for statins in the treatment of hypercholesterolemia. This is firstly reported that monomeric compound of G. pentaphyllum planted in Hunan province has the effect of increasing LDL-C uptake in hepatocytes via inhibiting PCSK9 expression.

Identification of neolignans with PCSK9 downregulatory and LDLR upregulatory activities from Penthorum chinense and the potential in cholesterol uptake by transcriptional regulation of LDLR via SREBP2.

ETHNOPHARMACOLOGICAL RELEVANCE: Penthorum chinense has been used in East Asia for the treatment of cholecystitis, infectious hepatitis, jaundice and to treat liver problems. Recent evidences provided the potential for the clinical use of P. chinense in the treatment of metabolic disease. AIM OF THE STUDY: Based on the traditional use and recent evidences, we investigated the effects of constituents from P. chinense with modulation on proprotein convertase subtilisin/kexin type 9 (PCSK9) and low-density lipoprotein receptor (LDLR) expression, and the effect of the most active substance on cholesterol uptake, and genes relevant to lipid metabolism. MATERIALS AND METHODS: The isolation of compounds from the BuOH-soluble extract of 80% methanol extract of P. chinense was conducted using chromatographic methods and the structures were established by interpreting spectroscopic data. Quantitative real time-PCR, and Western blot analysis were performed to monitor the regulatory activity on PCSK9 and LDLR expression. PCSK9-LDLR binding interaction was also tested. The cholesterol uptake in hepatocyte was measured using 1,1-dioctadecyl-3,3,3,3-tetramethylindocarbocyanine perchlorate (DiI)-labeled LDL cholesterol. Additionally, gene network analysis of LDLR and responses of its target proteins were carried out to discover genes germane to the effect of active compound on HepG2 cells. Moreover, we performed protein-protein interaction analysis via String and constructed the compound target network using Cytoscape. RESULTS: Two new neolignans and 37 known compounds were characterized from P. chinense. Of the isolated compounds, (7'E,8S)-2',4,8-trihydroxy-3-methoxy-2,4'-epoxy-8,5'-neolign-7'-en-7-one (3), penthorin A (4) and methyl gallate (25) were found to suppress PCSK9 mRNA expression with IC50 values of 5.13, 15.56 and 11.66 µM, respectively. However, all the isolated compounds were found to be inactive in PCSK9-LDLR interaction assay. Additionally, a dibenzoxepine-type lignan analog, (7'E,8S)-2',4,8-trihydroxy-3-methoxy-2,4'-epoxy-8,5'-neolign-7'-en-7-one (3) demonstrated to upregulate LDLR mRNA and protein expression via transcriptional factor sterol regulatory element-binding protein 2 (SREBP2). Furthermore, (7'E,8S)-2',4,8-trihydroxy-3-methoxy-2,4'-epoxy-8,5'-neolign-7'-en-7-one (3) increase the LDL-cholesterol uptake in DiI-LDL assay. CONCLUSION: (7'E,8S)-2',4,8-trihydroxy-3-methoxy-2,4'-epoxy-8,5'-neolign-7'-en-7-one (3) seemed to increase potentially cholesterol uptake via the downregulation of PCSK9 and the activation of LDLR in hepatocytes. Moreover, SREBP2 was found to play an important role in regulation of PCSK9 and LDLR by (7'E,8S)-2',4,8-trihydroxy-3-methoxy-2,4'-epoxy-8,5'-neolign-7'-en-7-one.

Unripe Rubus coreanus Miquel Extract Containing Ellagic Acid Regulates AMPK, SREBP-2, HMGCR, and INSIG-1 Signaling and Cholesterol Metabolism In Vitro and In Vivo.

Our previous study demonstrated that a 5% ethanol extract of unripe Rubus coreanus (5-uRCK) has hypo-cholesterolemic and anti-obesity activity. However, the molecular mechanisms of its effects are poorly characterized. We hypothesized that 5-uRCK and one of its major bioactive compounds, ellagic acid, decrease cellular and plasma cholesterol levels. Thus, we investigated the hypocholesterolemic activity and mechanism of 5-uRCK in both hepatocytes and a high-cholesterol diet (HCD)-induced rat model. Cholesterol in the liver and serum was significantly reduced by 5-uRCK and ellagic acid. The hepatic activities of HMG-CoA and CETP were reduced, and the hepatic activity of LCAT was increased by both 5-uRCK extract and ellagic acid, which also caused histological improvements. The MDA content in the aorta and serum was significantly decreased after oral administration of 5-uRCK or ellagic acid. Further immunoblotting analysis showed that AMPK phosphorylation in the liver was induced by 5-uRCK and ellagic acid, which activated AMPK, inhibiting the activity of HMGCR by inhibitory phosphorylation. In contrast, 5-uRCK and ellagic acid suppressed the nuclear translocation and activation of SREBP-2, which is a key transcription factor in cholesterol biosynthesis. In conclusion, our results suggest that 5-uRCK and its bioactive compound, ellagic acid, are useful alternative therapeutic agents to regulate blood cholesterol.

Effects of Lactobacillus plantarum PMO 08 Alone and Combined with Chia Seeds on Metabolic Syndrome and Parameters Related to Gut Health in High-Fat Diet-Induced Obese Mice.

This study researched the effects of Lactobacillus plantarum PMO 08 alone and combined with chia seeds on metabolic syndrome and parameters related to microbiota modulation and intestinal barrier integrity in obese mice fed high-fat diets (HFDs; 45% kcal fat). Male C57BL/6J mice were acclimated for a period of 2 weeks and then randomly separated into five groups depending on whether they received a normal diet (ND group), an HFD (HFD group), an HFD with L. plantarum (PMO group), an HFD with L. plantarum combined with chia seeds (PMOChia group), or an HFD with chia seeds (Chia group). Serum lipid profiles and related markers (cholesterol metabolism-related gene expression) were measured. Intestinal barrier integrity was assessed by measuring occludin mRNA expression of tight junction proteins. Mucosal bacteria were checked with quantitative reverse transcript polymerase chain reaction (qRT-PCR). After 16 weeks of feeding, the PMO group showed significantly lower serum total cholesterol, low-density lipoprotein cholesterol levels, atherogenic index, and cardiac risk factors compared to the HFD group. Moreover, the hepatic mRNA expression of SREBP2 (sterol regulatory element binding protein 2), a protein related to cholesterol metabolism, was significantly downregulated in the PMO group. We also found a positive synergistic effect in the PMOChia group, as manifested by the hepatic mRNA expression of hepatic CYP7A1 (cholesterol 7α-hydroxylase), strengthening of the gut barrier function, and the promotion of more L. plantarum in the colonic mucosa than in either the HFD or PMO group. In conclusion, our results indicate that PMO 08 may protect against metabolic syndrome by exerting effects on the regulation of lipid metabolism. Although the effects of chia seeds alone remain uncertain based on this experiment, its combination with PMO 08 was demonstrated to improve multiple beneficial effects of PMO 08 in obese mice fed HFD, which is a promising possibility for future research.

Effect of Sulgidduk containing pine needle juice on lipid metabolism in high fat-cholesterol diet induced dyslipidemic rats / 한국영양학회지

PURPOSE: Dyslipidemia is a major risk factor for cardiovascular disease. Pine needles (Pinus densiflora seib et Zucc) are a traditional medicine used to treat dyslipidemia in clinical settings. This study examined the potential effects of sulgidduk, a Korean traditional rice cake containing pine needle juice to protect against dyslipidemia induced by a high-fat/sugidduk diet in a rat model. METHODS: Twenty one male Sprague-Dawley rats were divided randomly into three groups: normal control (NC), Sulgidduk diet (SD), Sulgidduk diet containing pine needle juice (PSD). The blood lipid levels, production of lipid peroxide in the plasma and liver, total cholesterol and triglyceride in the liver and feces, antioxidant enzyme activities in plasma and erythrocytes were measured to assess the effects of PSD on dyslipidemia. RESULTS: A high-fat/Sulgidduk diet induced dyslipidemia, which was characterized by significantly altered lipid profiles in the plasma and liver. The food intake was similar in the three groups, but weight gain and food efficiency ratio (FER) were reduced significantly in the PSD group compared to those in the SD group. The level of total cholesterol, LDL-cholesterol and TBARS in the plasma showed tendencies to decrease in the PSD group compared to those in the SD group. The levels of high-fat/Sulgidduk diet-induced sterol regulatory element-binding protein 2 (SREBP2) gene expression were reduced significantly in the PSD group. The supplementation of PSD reduced the hepatic triglyceride and total cholesterol levels significantly, and enhanced the fecal excretion of triglyceride and hepatic antioxidant enzyme activities compared to the SD group. CONCLUSION: These results suggest that the addition of 0.4% pine needle juice to Sulgidduk may be an alternative snack to control dyslipidemia.

Biological-Profiling-Based Systematic Analysis of Rhizoma Coptidis from Different Growing Regions and Its Anticholesterol Biosynthesis Activity on HepG2 Cells.

Rhizoma Coptidis is a widely cultivated traditional Chinese herb. Although the chemical profiles of Rhizoma Coptidis have been established previously, the biological profiling of Rhizoma Coptidis has not been conducted yet. In this study, we collected Rhizoma Coptidis varieties from four distinct growing regions and performed genome-wide biological response fingerprinting (BioReF) on HepG2 cells using a gene expression array. Similar biological pathways were affected by extracts of all four Rhizoma Coptidis varieties but not by their analogue, Mahoniae Caulis. Among these pathways, the terpenoid backbone biosynthesis pathway was highly enriched, and six genes in the mevalonate (MVA) pathway were all down-regulated. However, the expression, maturation, as well as the specific DNA binding capacity of their coordinate transcription factor, sterol response element binding protein 2 (SREBP2), was not affected by Rhizoma Coptidis extract (RCE) or its typical active alkaloid berberine. Cellular cholesterol content tests further verified the cholesterol-lowering function of RCE in vitro, which supplements evidence for the use of Rhizoma Coptidis in hyperlipidemia treatment. This is the first described example of evaluating the quality of Rhizoma Coptidis with BioReF and a good demonstration of using BioReF to uncover the mechanisms of herbs at a systematic level.

Epigallocatechin gallate suppresses hepatic cholesterol synthesis by targeting SREBP-2 through SIRT1/FOXO1 signaling pathway.

This study aims to explore the effect of epigallocatechin gallate (EGCG) on blood lipids, liver lipids, and cholesterol synthesis in hyperlipidemic rats. SREBP-2 transgenic rats were used to investigate the transcriptional level of SREBP-2 regulated by SIRT-1/FOXO1 and the molecular mechanism of rate-limiting enzyme HMGCR that affects cholesterol synthesis. Rat models of hyperlipidemia were established and administered EGCG. Cholesterol synthesis was observed. Enzyme linked immunosorbent assay was used to determine serum triglyceride (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), free fatty acid (FFA), superoxide dismutase (SOD), malondialdehyde (MDA), and T-AOC contents. Hematoxylin-eosin staining and oil red O staining were utilized to observe the histological changes in the liver. Biochemical method was applied to measure serum ALT and AST changes. Western blot assay and qRT-PCR were employed to detect the changes in SIRT1/FOXO1 pathway-related proteins, cholesterol synthesis-related genes, and SREBP-2. EGCG 50 mg/kg could obviously decrease the liver weight and liver coefficient, reduce serum TG, TC, LDL-C, and FFA levels (P < 0.05), and increase serum HDL-C levels in hyperlipidemic rats. EGCG could diminish hyperlipidemia-induced liver injury and reduce serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels. Oil red O staining results demonstrated that the number of red lipid droplets in hepatocytes reduced to varying degrees, especially high-dose EGCG. EGCG remarkably diminished MDA content in the liver with hypercholesterolemia and increased T-AOC and SOD activity. In the model group, SIRT1 expression increased, and FOXO1 expression decreased. EGCG activated SIRT1 and increased FOXO1 expression, whose expression trend was consistent with the fenofibrate group. HMGCR, FDPS, SS, and ABCA1 expression increased, and ACAT2 expression noticeably reduced in SREBP-2+/+ transgenic rats. EGCG could reverse the expression trend of each gene. Simultaneously, EGCG increased FOXO1 expression, and decrease SREBP-2 expression; however, no significant changes in these expression were found in SREBP-2-/- rats. EGCG can alleviate liver injury and oxidative stress in hyperlipidemic rats. EGCG can activate SIRT1, activate FOXO1 protein, regulate SREBP-2 protein, and inhibit hepatic cholesterol synthesis.

Tripterygium regelii decreases the biosynthesis of triacylglycerol and cholesterol in HepG2 cells.

In the course of screening to find a plant material decreasing the activity of triacylglycerol and cholesterol, we identified Tripterygium regelii (TR). The methanol extract of TR leaves (TR-LM) was shown to reduce the intracellular lipid contents consisting of triacylglycerol (TG) and cholesterol in HepG2 cells. TR-LM also downregulated the mRNA and protein expression of the lipogenic genes such as SREBP-1 and its target enzymes. Consequently, TR-LM reduced the TG biosynthesis in HepG2 cells. In addition, TR-LM decreased SREBP2 and its target enzyme HMG-CoA reductase, which is involved in cholesterol synthesis. In this study, we evaluated that TR-LM attenuated cellular lipid contents through the suppression of de novo TG and cholesterol biosynthesis in HepG2 cells. All these taken together, TR-LM could be beneficial in regulating lipid metabolism and useful preventing the hyperlipidemia and its complications, in that liver is a crucial tissue for the secretion of serum lipids.

Cholesterol-lowering effect of Aralia elata (Miq.) Seem via the activation of SREBP-2 and the LDL receptor.

BACKGROUND: Hyperlipidemia causes arteriosclerosis, a risk factor for coronary heart disease. Prevention of hyperlipidemia by improving dietary habits has recently attracted attention. In this regard, we investigated whether Aralia elata (Miq.) Seem (AE) extract inhibits hepatic cholesterol accumulation and modulate the cellular signaling pathway. METHODS: To determine AE's cholesterol regulating mechanism, we measured cholesterol level, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity and cholesterol regulating-related gene expression in HepG2 cells and in high-fat diet (HFD)-induced mice using ELISA and RT-PCR assay. RESULTS: The AE extract reduced cholesterol levels and HMG-CoA reductase activity in hepatocellular carcinoma HepG2 cells. In addition, it also reduced the plasma cholesterol concentrations in HFD-induced mice. Furthermore, the AE extract increased the gene expression of the LDL-receptor (LDL-R); sterol-regulatory-element binding protein-2 (SREBP-2); ATP-binding cassette, sub-family A, member 1 (ABCA1); and scavenger receptor class B member 1 (SR-B1) in a dose-dependent manner. However, the AE extract did not affect the gene expression of acetyl-coenzyme A acetyltransferase (ACAT) in either the HepG2 cells or mice. CONCLUSION: We demonstrated that the AE extract activated genes related to cholesterol metabolism, such as SREBP-2 and LDL-R, which resulted in hypocholesterolemic activities.