Identification of Senkyunolide I as a novel modulator of hepatic steatosis and PPARα signaling in zebrafish and hamster models.

ETHNOPHARMACOLOGICAL RELEVANCE: Non-alcoholic fatty liver disease (NAFLD) is the leading cause of liver-related morbidity and mortality, with hepatic steatosis being the hallmark symptom. Salvia miltiorrhiza Bunge (Smil, Dan-Shen) and Ligusticum striatum DC (Lstr, Chuan-Xiong) are commonly used to treat cardiovascular diseases and have the potential to regulate lipid metabolism. However, whether Smil/Lstr combo can be used to treat NAFLD and the mechanisms underlying its lipid-regulating properties remain unclear. PURPOSE: To assess the feasibility and reliability of a short-term high-fat diet (HFD) induced zebrafish model for evaluating hepatic steatosis phenotype and to investigate the liver lipid-lowering effects of Smil/Lstr, as well as its active components. METHODS: The phenotypic alterations of liver and multiple other organ systems were examined in the HFD zebrafish model using fluorescence imaging and histochemistry. The liver-specific lipid-lowering effects of Smil/Lstr combo were evaluated endogenously. The active molecules and functional mechanisms were further explored in zebrafish, human hepatocytes, and hamster models. RESULTS: In 5-day HFD zebrafish, significant lipid accumulation was detected in the blood vessels and the liver, as evidenced by increased staining with Oil Red O and fluorescent lipid probes. Hepatic hypertrophy was observed in the model, along with macrovesicular steatosis. Smil/Lstr combo administration effectively restored the lipid profile and alleviated hepatic hypertrophy in the HFD zebrafish. In oleic-acid stimulated hepatocytes, Smil/Lstr combo markedly reduced lipid accumulation and cell damage. Subsequently, based on zebrafish phenotypic screening, the natural phthalide senkyunolide I (SEI) was identified as a major molecule mediating the lipid-lowering activities of Smil/Lstr combo in the liver. Moreover, SEI upregulated the expression of the lipid metabolism regulator PPARα and downregulated fatty acid translocase CD36, while a PPARα antagonist sufficiently blocked the regulatory effect of SEI on hepatic steatosis. Finally, the roles of SEI on hepatic lipid accumulation and PPARα signaling were further verified in the hamster model. CONCLUSIONS: We proposed a zebrafish-based screening strategy for modulators of hepatic steatosis and discovered the regulatory roles of Smil/Lstr combo and its component SEI on liver lipid accumulation and PPARα signaling, suggesting their potential value as novel candidates for NAFLD treatment.

Inhibition of ATGL alleviates MASH via impaired PPARα signalling that favours hydrophilic bile acid composition in mice.

BACKGROUND AND AIMS: Adipose triglyceride lipase (ATGL) is an attractive therapeutic target in insulin resistance and metabolic dysfunction-associated steatotic liver disease (MASLD). This study investigated the effects of pharmacological ATGL inhibition on the development of metabolic dysfunction-associated steatohepatitis (MASH) and fibrosis in mice. METHODS: Streptozotocin-injected male mice were fed an HFD to induce MASH. Mice receiving the ATGL inhibitor, Atglistatin (ATGLi), were compared to controls using liver histology, lipidomics, metabolomics, 16s rRNA, and RNA sequencing. Human ileal organoids, HepG2 cells, and Caco2 cells treated with the human ATGL inhibitor NG-497, HepG2 ATGL knockdown cells, gel-shift, and luciferase assays were analysed for mechanistic insights. We validated its benefits on steatohepatitis and fibrosis in a low-methionine choline-deficient mouse model. RESULTS: ATGLi improved serum liver enzymes, hepatic lipid content, and histological liver injury. Mechanistically, ATGLi attenuated PPARα signalling, favouring hydrophilic bile acid (BA) synthesis with increased Cyp7a1, Cyp27a1, Cyp2c70, and reduced Cyp8b1 expression. Additionally, reduced intestinal Cd36 and Abca1, along with increased Abcg5 expression, were consistent with reduced levels of hepatic TAG-species containing PUFAs like linoleic acids as well as reduced cholesterol levels in the liver and plasma. Similar changes in gene expression associated with PPARα signaling and intestinal lipid transport were observed in ileal organoids treated with NG-497. Furthermore, HepG2 ATGL knockdown cells revealed reduced expression of PPARα target genes and upregulation of genes involved in hydrophilic BA synthesis, consistent with reduced PPARα binding and luciferase activity in the presence of the ATGL inhibitors. CONCLUSIONS: Inhibition of ATGL attenuates PPARα signalling, translating into hydrophilic BAs, interfering with dietary lipid absorption, and improving metabolic disturbances. The validation with NG-497 opens a new therapeutic perspective for MASLD. IMPACT AND IMPLICATIONS: The global prevalence of metabolic dysfunction-associated steatotic liver disease (MASLD) is a crucial public health concern. Since adherence to behavioural interventions is limited, pharmacological strategies are necessary, as highlighted by the recent FDA approval of resmetirom. However, since our current mechanistic understanding and pathophysiology-oriented therapeutic options for MASLD are still limited, novel mechanistic insights are urgently needed. Our present work uncovers that pharmacological inhibition of ATGL, the key enzyme in lipid hydrolysis using Atglistatin (ATGLi), improves metabolic dysfunction-associated steatohepatitis (MASH), fibrosis, and associated key features of metabolic dysfunction in a mouse model of MASH and MCD-induced liver fibrosis. Mechanistically, we demonstrated that attenuation of PPARα signalling in the liver and gut favours hydrophilic bile acid composition, ultimately interfering with dietary lipid absorption. One of the drawbacks of ATGLi is its lack of efficacy against human ATGL, thus limiting its clinical applicability. Against this backdrop, we could show that ATGL inhibition using the human inhibitor NG-497 in human primary ileum-derived organoids, Caco2 cells, and HepG2 cells translated into therapeutic mechanisms similar to ATGLi. Collectively, these findings open a new avenue for MASLD treatment development by inhibiting human ATGL activity.

Bioactive proteins and antioxidant peptides from Litsea cubeba fruit meal: Preparation, characterization and ameliorating function on high-fat diet-induced NAFLD through regulating lipid metabolism, oxidative stress and inflammatory response.

Non-alcoholic fatty liver disease (NAFLD) plays an increasingly significant threat to human health. In this study, the processing by-products of Litsea cubeba fruit meal were defatted by ultrasound-assisted methods, then the acetone-precipitated protein of L. cubeba (LCP) was obtained and structural analysis was performed. LCP was hydrolyzed by a two-step sequential hydrolysis method using alcalase and papain. Subsequently, antioxidant peptide fraction (IV2) was isolated and identified from the resultant hydrolysate through membrane ultrafiltration, Sephadex G-15 chromatography, and liquid chromatograph mass spectrometer (LC-MS). Animal experimentation indicated the potential of IV2 to mitigate hepatic steatosis. Moreover, IV2 could effectively reduce oxidative stress-induced damage by modulating the Keap1-Nrf2 pathway to activate downstream heme oxygenase-1 (HO-1) and NAD(P) H quinone oxidoreductase 1 (NQO1). Integrating metabolomics and transcriptomics revealed enrichment in pathways associated with glycerolipid metabolism and fatty acid ß-oxidation, suggesting the principal mechanisms underlying IV2's ameliorative effects on NAFLD. Transcriptome sequencing identified 3092 up-regulated and 3010 down-regulated genes following IV2 treatment. Interaction analyses based on different lipid compositions (DELs) and differentially expressed genes (DEGs) indicated that IV2 primarily alleviated hepatic steatosis by modulating peroxisome proliferator-activated receptor α (PPAR-α) related pathways, thereby augmenting fatty acid ß-oxidation within liver cells. These results indicate that IV2 shows potential in improving high-fat diet (HFD)-induced NAFLD, with improved fatty acid ß-oxidation and reduced triglyceride biosynthesis emerging as underlying mechanisms.

Ozone-Induced Lung Injury are Mediated Via PPAR-Mediated Ferroptosis in Mice.

In recent years, the concentration of PM2.5 in China has decreased, while the concentration of ozone remains rising. Exposure to ozone contributes to respiratory illnesses; however, little is known about the underlying molecular mechanisms. The present study established an ozone-induced lung injury mice model to investigate potential molecular biomarkers and toxic mechanisms. Collected and analyzed the ozone pollution data in Xinxiang city from 2015 to 2022. At the same time, 24 male C57BL/6 mice were randomly assigned to control group and ozone exposure group. The ozone exposure concentration is 1 ppm, with 4 h of daily exposure for 33 consecutive days. HE staining was used to assess lung histological alterations and lung injury. High-throughput sequencing performed on the lung tissues of mice was used to analyze the differential expressed genes and signal transduction pathways. Xinxiang city is suffering from ozone pollution, especially in summer. HE staining showed that the ozone exposure could induce obvious inflammatory cell infiltration, alveolar wall thickening, or fracture. Transcriptome data revealed that there is a 145 differentially expressed genes between two groups and the genes enriched in PPAR signaling pathway, ferroptosis. The pivotal genes in the PPAR pathway including Adipoq, Lpl, Pck1, and Plin1 expression were significantly reduced. Additionally, the expression of Acsl6 and Scl7a11, which are close to PPAR pathway and ferroptosis has decreased. Ozone exposure could disrupt the lipid metabolism balance via downregulating lipid peroxidation-related genes through the PPAR signaling pathway, which further induced lung cell ferroptosis and aggravated lung injury in mice.

The effect of Oleoylethanolamide supplementation on lipid profile, fasting blood sugar and dietary habits in obese people: a randomized double-blind placebo-control trial.

BACKGROUND: Abnormalities in biochemical parameters and changes in eating habits are considered complications of obesity. Oleoylethanolamide (OEA), an endocannabinoid-like compound, has been shown to have protective effects on many metabolic disorders. Given this evidence, the present study aimed to assess the effects of OEA on lipid profile parameters, fasting blood sugar (FBS), and dietary habits in healthy obese people. METHODS: In this randomized, double-blind, placebo-controlled clinical trial, which was carried out in 2016 in Tabriz, Iran, 60 obese people were enrolled in the study based on inclusion criteria. The intervention group consumed 125 mg of OEA capsules, and the placebo group received the same amount of starch twice for 8 weeks. Blood samples (5 mL) were taken at baseline and the end of the study in a fasting state. Serum concentrations of FBS, triglycerides (TGs), high-density lipoprotein cholesterol (HDL-C), and total cholesterol (TC) were measured by enzymatic methods using commercial kits. The low-density lipoprotein cholesterol (LDL-C) concentration was obtained using the Friede-Wald formula. To assess dietary habits, a food frequency questionnaire (147 items) was used at baseline and the end of the study. A value less than < 0.05 was considered to indicate statistical significance. RESULTS: The TG concentration decreased significantly in the intervention group (mean (SD): 166.29 (70.01) mg/dL to 142.22 (48.05) mg/dL, p = 0.047). Changes in the placebo group were not significant (p > 0.05). After adjusting for baseline values and demographic characteristics, the difference in TG between groups remained significant (p = 0.044). Changes in other biochemical parameters were not significant. There was no significant difference between or within groups in terms of food groups. CONCLUSION: OEA, as a complementary agent, plays a protective role in TG regulation. However, future studies with longer durations are needed to explore the impact of OEA on regulating dietary habits and to identify the mechanisms related to metabolic abnormalities in obese people. TRIAL REGISTRATION: The study was registered in the Iranian Registry of Clinical Trials (IRCT) center as IRCT201607132017N30 with URL. www.IRCT.IR in date 03/10/2016.

Peroxisomes and PPARs: Emerging role as master regulators of cancer metabolism.

Cancer is a disease characterized by the acquisition of a multitude of unique traits. It has long been understood that cancer cells divert significantly from normal cell metabolism. The most obvious of metabolic changes is that cancer cells strongly rely on glucose conversion by aerobic glycolysis. In addition, they also regularly develop mechanisms to use lipids and fatty acids for their energy needs. Peroxisomes lie central to these adaptive changes of lipid metabolism. Peroxisomes are metabolic organelles that take part in over 50 enzymatic reactions crucial for cellular functioning. Thus, they are essential for an effective and comprehensive use of lipids' energy supplied to cells. Cancer cells display a substantial increase in the biogenesis of peroxisomes and an increased expression of proteins necessary for the enzymatic functions provided by peroxisomes. Moreover, the enzymatic conversion of FAs in peroxisomes is a significant source of reactive oxygen and nitrogen species (ROS/RNS) that strongly impact cancer malignancy. Important regulators in peroxisomal FA oxidation and ROS/RNS generation are the transcription factors of the peroxisome proliferator-activated receptor (PPAR) family. This review describes the metabolic changes in tumorigenesis and cancer progression influenced by peroxisomes. We will highlight the ambivalent role that peroxisomes and PPARs play in the different stages of tumor development and summarize our current understanding of how to capitalize on the comprehension of peroxisomal biology for cancer treatment.

Selective activation of PPARα by pemafibrate mitigates peritoneal inflammation and fibrosis through suppression of NLRP3 inflammasome and modulation of inflammation.

Peritoneal inflammation and fibrosis remain major challenges to the long-term maintenance of peritoneal dialysis. Pemafibrate, a selective peroxisome proliferator-activated receptor α (PPARα) modulator, has been implicated in the management of fibrosis-related disorders. We investigated whether pemafibrate ameliorates peritoneal inflammation and fibrosis and explored the underlying mechanisms in mice with methylglyoxal (MGO)-induced peritoneal fibrosis (MGO mice). MGO mice exhibited peritoneal fibrosis with increased expression of mesenchymal markers, transforming growth factor-ß1 (TGF-ß1), and substantial deposition of extracellular matrix (ECM) proteins. Additionally, MGO mice exhibited peritoneal inflammation as indicated by elevated tumor necrosis factor-α expression and macrophage infiltration in peritoneal tissue. These effects were mitigated by pemafibrate treatment, which also restored peritoneal membrane function. Furthermore, pemafibrate promoted anti-inflammatory macrophage polarization in both mice and THP-1 cells. In human peritoneal mesothelial cells (HPMCs), pemafibrate effectively inhibited interferon-γ-induced production of TGF-ß1 and ECM while suppressing the proinflammatory cytokines nuclear factor-κB (NF-κB) and activator protein 1. The NF-κB inhibitory effect of pemafibrate involved stabilization of the NF-κB inhibitory protein IkBα. Notably, pemafibrate hindered activation of the NLR family pyrin domain containing 3/caspase-1 axis in interferon-γ-stimulated THP-1 cells. These findings suggest that pemafibrate ameliorates peritoneal inflammation and fibrosis, making it a promising candidate for peritoneal fibrosis therapy.

Mechanistic insights into antidiabetic potential of Ficus viren against multi organ specific diabetic targets: molecular docking, MDS, MM-GBSA analysis.

Ficus viren has been traditionally used to treat diabetes, and its extract inhibits carbohydrate/lipid metabolism and possesses anti-hyperglycemic potential. However, there is conflicting investigation related to F. viren extract effect on carbohydrate metabolism. Thus, bioactive and mechanism behind its antidiabetic potential is still scanty. This study explored F. viren's anti-diabetic property by identifying potential phytoconstituents and mechanism. A sequential in-silico approach was used i.e., druglikeness, molecular docking, post-docking MM-GBSA, ADMET studies, molecular dynamic simulation (MDS), and post-MDS MM-GBSA. We screened ∼32 phytoconstituents and twelve potential organ-specific diabetic targets (O.S.D.Ts i.e., IR, DPP-4, ppar-γ, ppar-α, ppar-δ, GLP-1R, SIRT-1, AMPK, GSK-3ß, RAGE, and AR). Drug likeness study identified 18 druggable candidates among 32 phytoconstituents. K3A, quercetin, scutellarein, sorbifolin, and vogeline J identified as potential ligands from druggable ligands, using IR as the standard target. Subsequently, potential ligands docked with remaining O.S.D.Ts. and data showed that K3A binds strongly with AMPK, ppar-δ, DPP-4, and GSK-3ß, while scutellarein binds with AR and ppar-α. Sorbifolin, quercetin, and vogeline J binds with ppar-α, ppar-γ, and RAGE, respectively. Post-docking MM-GBSA data (∆GBind) also depicted potential ligand's strong binding affinities with their corresponding targets. Thereafter, simulation data revealed that only scutellarein and sorbifolin showed dynamic stability with their respective targets, i.e., AR/ppar-α and ppar-α, respectively. Interestingly, post-MDS MM-GBSA revealed that only scutellarein exhibited strong ∆GBind of -55.08 kcal/mol and -75.48 kcal/mol with AR and ppar-α, respectively. Though, collective computational analysis supports antidiabetic potential of F. viren through AR and ppar-α modulation by scutellarein.

Asiatic acid mitigates testosterone-induced benign prostatic hyperplasia in rats via activation of PPAR-γ.

Prostate enlargement due to benign prostate hyperplasia (BPH) is a common, progressive disorder in elderly males with increasing prevalence. It causes devastating lower urinary tract symptoms with no satisfactory medication. Asiatic acid (AA), a natural pentacyclic triterpenoid, is known to have antiproliferative, antioxidant, and anti-inflammatory activities. The aim of this study was to evaluate the possible preventive activities of AA against BPH induced by testosterone in rats. Finasteride (0.5 mg/kg) was used as a reference drug. AA (10 or 20 mg/kg) administration inhibited the rise in prostatic weight and index induced by testosterone. Histopathological staining proved that AA mitigated the pathological features of BPH induced by testosterone, which was reflected as lower glandular epithelial in AA-treated groups. Also, the administration of AA along with testosterone restored the redox valance by inhibiting lipid peroxidation, and MDA production, and restoring the activities of superoxide dismutase (SOD) and catalase (CAT) activities. Also, AA reduced prostate interleukin-6 (IL-6), cyclooxygenase-2 (COX-2), tumor necrosis factor-alpha (TNF-α), and nuclear factor-kappa B (NF-κB) protein expression. In addition, AA modulated mRNA expression of Bax and Bcl-2 in favor of apoptosis. The effects of AA (20 mg/kg) were comparable to those of finasteride. Further, AA ameliorated the rise in insulin-like growth factor 1 receptor (IGF-1R) mRNA expression. This was associated with the enhancement of the prostatic content of PPAR-γ. It can be concluded that AA mitigated the features of BPH induced by testosterone in rats. This involves antioxidant, anti-inflammatory and pro-apototic activities of AA as well as its ability to down-regulate IGF-1R expression and enhance PPAR-γ concentration in prostatic tissues.

cRGD-platelet@MnO/MSN@PPARα/LXRα Nanoparticles Improve Atherosclerosis in Rats by Inhibiting Inflammation and Reducing Blood Lipid.

OBJECTIVE: Atherosclerosis (AS) is an inflammatory disease of arterial intima driven by lipids. Liver X receptor alpha (LXRα) and peroxisome proliferator-activated receptor alpha (PPARα) agonists are limited in the treatment of AS due to their off-target effects and serious side effects. Therefore, this study was designed to construct a novel nanoparticle (NP) and evaluate its mechanism of action on inflammation inhibition and lipid reduction in AS. METHODS: We synthesized cRGD-platelet@MnO/MSN@PPARα/LXRα NPs (cRGD-platelet- NPs) and confirmed their size, safety, and targeting ability through various tests, including dynamic light scattering and immunofluorescence. In vivo and in vitro experiments assessed cell proliferation, apoptosis, inflammation, and plaque formation. Finally, the NF-κB signaling pathway expression in rat aorta was determined using a western blot. RESULTS: The synthesis of cRGD-platelet-NPs was successful; the particle size was approximately 150 nm, and the PDI was below 0.3. They could be successfully absorbed by cells, exhibiting high safety in vivo and in vitro. The cRGD-platelet-NPs successfully reduced plaque formation, improved lipid profiles by lowering LDL-cholesterol, total cholesterol, and triglycerides, and raised HDL-cholesterol levels. Additionally, they decreased inflammatory markers in the serum and aortic tissue, suggesting reduced inflammation. Immunohistochemistry and western blot analyses indicated that these NPs could not only promote M2 macrophage polarization but also suppress the NF-κB signaling pathway. CONCLUSION: The newly developed cRGD-platelet-NPs with high safety are a promising approach to AS treatment, which can regulate ABCA1, reduce the formation of AS plaques, and enhance cholesterol efflux. The mechanism may involve the suppression of the NF-κB signaling pathway.

Inhibition of the RXRA-PPARα-FABP4 signaling pathway alleviates vascular cellular aging by an SGLT2 inhibitor in an atherosclerotic mice model.

Atherosclerosis is the pathological cause of atherosclerotic cardiovascular disease (ASCVD), which rapidly progresses during the cellular senescence. Sodium-glucose cotransporter 2 inhibitors (SGLT2is) reduce major cardiovascular events in patients with ASCVD and have potential antisenescence effects. Here, we investigate the effects of the SGLT2 inhibitor dapagliflozin on cellular senescence in atherosclerotic mice. Compared with ApoE-/- control mice treated with normal saline, those in the ApoE-/- dapagliflozin group, receiving intragastric dapagliflozin (0.1 mg kg-1 d-1) for 14 weeks, exhibited the reduction in the total aortic plaque area (48.8%±6.6% vs. 74.6%±8.0%, P<0.05), the decrease in the lipid core area ((0.019±0.0037) mm2vs. (0.032±0.0062) mm2, P<0.05) and in the percentage of senescent cells within the plaques (16.4%±3.7% vs. 30.7%±2.0%, P<0.01), while the increase in the thickness of the fibrous cap ((21.6±2.1) µm vs. (14.6±1.5) µm, P<0.01). Transcriptome sequencing of the aortic arch in the mice revealed the involvement of the PPARα and the fatty acid metabolic signaling pathways in dapagliflozin's mechanism of ameliorating cellular aging and plaque progression. In vitro, dapagliflozin inhibited the expression of PPARα and its downstream signal FABP4, by which the accumulation of senescent cells in human aortic smooth muscle cells (HASMCs) was reduced under high-fat conditions. This effect was accompanied by a reduction in the intracellular lipid content and alleviation of oxidative stress. However, these beneficial effects of dapagliflozin could be reversed by the PPARα overexpression. Bioinformatics analysis and molecular docking simulations revealed that dapagliflozin might exert its effects by directly interacting with the RXRA protein, thereby influencing the expression of the PPARα signaling pathway. In conclusion, the cellular senescence of aortic smooth muscle cells is potentially altered by dapagliflozin through the suppression of the RXRA-PPARα-FABP4 signaling pathway, resulting in a deceleration of atherosclerotic progression.

The dual-edged role of SIRT1 in energy metabolism and cardiovascular disease.

Regulation of energy metabolism is pivotal in the development of cardiovascular diseases. Dysregulation in mitochondrial fatty acid oxidation (FAO) has been linked to cardiac lipid accumulation and diabetic cardiomyopathy. Sirtuin 1 (SIRT1) is a deacetylase that regulates the acetylation of various proteins involved in mitochondrial energy metabolism. SIRT1 mediates energy metabolism by directly and indirectly affecting multiple aspects of mitochondrial processes, such as mitochondrial biogenesis. SIRT1 interacts with essential mitochondrial energy regulators such as Peroxisome Proliferator-Activated Receptor α (PPARα), PPARgcoactivator-1 (PGC1α), Estrogen-Related Receptor α (ERRα), and their downstream targets. Apart from that, SIRT1 regulates additional proteins, including Forkhead Box Protein O1 (FOXO1) and AMP-Activated Protein Kinase (AMPK) in cardiac disease. Interestingly, studies have also shown that the expression of SIRT1 plays a dual-edged role in energy metabolism. Depending on the physiological state, SIRT1 expression can be detrimental or protective. This review focuses on the molecular pathways through which SIRT1 regulates energy metabolism in cardiovascular diseases. We will review SIRT1 and discuss its role in cardiac energy metabolism and its benefits and detrimental effects in heart disease.

Multi-omics analysis provides new insights into mechanism of didymin on non-alcoholic fatty liver disease in rats.

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) is one of the most common liver diseases accompanied by lipid and glucose metabolism disorder. Didymin has been reported to have various hepatoprotective effects, however, its potential effects and mechanisms on NAFLD remain unclear from the perspective of the whole. PURPOSE: To investigate the underlying mechanism of didymin against NAFLD using multi-omics technologies. METHODS: Rats were fed with a high-fat diet (HFD) for 8 weeks to induce NAFLD, followed by didymin treatment for 8 weeks. Next, biochemical analysis and histopathological examinations were performed to evaluate the effects of didymin. The key regulating pathways were predicted using transcriptomics, metabolomics and proteomics, and the target pathways were then verified by detecting the key genes/proteins using various experiments. RESULTS: Didymin markedly mitigated liver injury and excessive lipid droplet accretion. An integrative multi-omics analysis suggested that the PPAR signaling cascade and insulin signaling pathway might serve as pivotal mechanisms underlying the modulation of lipid and glucose homeostasis by didymin. Further dissection identified five pivotal genes (PPARα, PPARß, FABP4, ANGPTL4, and PLIN2) and four genes (HK1, HK3, GCK, and PTPN1) as potential hubs within these pathways. Subsequent validation experiments, including qPCR and Western blot, demonstrated upregulated expression of PPARα and PPARß, indicating the activation of the PPAR pathway by didymin. Concurrently, didymin appeared to modulate the insulin signaling pathway, as evidenced by the upregulated expression of HK1 and downregulated expression of PTPN1. Notably, the manipulation of PPARα, PPARß, and PTPN1 expression in LO2 cells through silence or overexpression confirmed that didymin significantly reduced lipid accumulation, with its molecular targets likely being the PPAR and insulin pathways. CONCLUSIONS: Our findings demonstrate that didymin has a protective effect on NAFLD, and its underlying mechanism may be associated with the regulation of the PPAR and insulin signaling pathways.

Turkish coffee has an antitumor effect on breast cancer cells in vitro and in vivo.

BACKGROUND: Breast cancer is the most diagnosed cancer in women. Its pathogenesis includes several pathways in cancer proliferation, apoptosis, and metastasis. Some clinical data have indicated the association between coffee consumption and decreased cancer risk. However, little data is available on the effect of coffee on breast cancer cells in vitro and in vivo. METHODS: In our study, we assessed the effect of Turkish coffee and Fridamycin-H on different pathways in breast cancer, including apoptosis, proliferation, and oxidative stress. A human breast cancer cell line (MCF-7) was treated for 48 h with either coffee extract (5% or 10 v/v) or Fridamycin-H (10 ng/ml). Ehrlich solid tumors were induced in mice for in vivo modeling of breast cancer. Mice with Ehrlich solid tumors were treated orally with coffee extract in drinking water at a final concentration (v/v) of either 3%, 5%, or 10% daily for 21 days. Protein expression levels of Caspase-8 were determined in both in vitro and in vivo models using ELISA assay. Moreover, P-glycoprotein and peroxisome proliferator-activated receptor gamma (PPAR-γ) protein expression levels were analyzed in the in vitro model. ß-catenin protein expression was analyzed in tumor sections using immunohistochemical analysis. In addition, malondialdehyde (MDA) serum levels were analyzed using colorimetry. RESULTS: Both coffee extract and Fridamycin-H significantly increased Caspase-8, P-glycoprotein, and PPAR-γ protein levels in MCF-7 cells. Consistently, all doses of in vivo coffee treatment induced a significant increase in Caspase-8 and necrotic zones and a significant decrease in ß- catenin, MDA, tumor volume, tumor weight, and viable tumor cell density. CONCLUSION: These findings suggest that coffee extract and Fridamycin-H warrant further exploration as potential therapies for breast cancer.

PPAR-α Insufficiency Enhances Doxorubicin-Induced Nephropathy in PPAR-α Knockout Mice and a Murine Podocyte Cell Line.

Peroxisome proliferator-activated receptor-alpha (PPAR-α) and its exogenous activators (fibrates) promote autophagy. However, whether the deleterious effects of PPAR-α deficiency on doxorubicin (DOX)-induced podocytopathy are associated with reduced autophagy remains to be clarified. We investigated the mechanisms of PPAR-α in DOX-induced podocytopathy and tubular injury in PPAR-α knockout (PAKO) mice and in a murine podocyte cell line. DOX-treated PAKO mice showed higher serum levels of triglycerides and non-esterified fatty acids and more severe podocytopathy than DOX-treated wild-type mice, as evidenced by higher urinary levels of proteins and podocalyxin at 3 days to 2 weeks and higher blood urea nitrogen and serum creatinine levels at 4 weeks. Additionally, there was an increased accumulation of p62, a negative autophagy marker, in the glomerular and tubular regions in DOX-treated PAKO mice at Day 9. Moreover, DOX-treated PAKO mice showed more severe glomerulosclerosis and tubular damage and lower podocalyxin expression in the kidneys than DOX-treated control mice at 4 weeks. Furthermore, DOX treatment increased p-p53, an apoptosis marker, and cleaved the caspase-3 levels and induced apoptosis, which was ameliorated by fenofibrate, a PPAR-α activator. Fenofibrate further enhanced AMPK activation and autophagy under fed and fasting conditions. Conclusively, PPAR-α deficiency enhances DOX-induced podocytopathy, glomerulosclerosis, and tubular injury, possibly by reducing autophagic activity in mouse kidneys.

Pleiotropic Effects of Peroxisome Proliferator-Activated Receptor Alpha and Gamma Agonists on Myocardial Damage: Molecular Mechanisms and Clinical Evidence-A Narrative Review.

Cardiovascular diseases remain the leading cause of death in the world, and that is why finding an effective and multi-functional treatment alternative to combat these diseases has become more important. Fibrates and thiazolidinediones, peroxisome proliferator-activated receptors alpha and gamma are the pharmacological therapies used to treat dyslipidemia and type 2 diabetes, respectively. New mechanisms of action of these drugs have been found, demonstrating their pleiotropic effects, which contribute to preserving the heart by reducing or even preventing myocardial damage. Here, we review the mechanisms underlying the cardioprotective effects of PPAR agonists and regulating morphological and physiological heart alterations (metabolic flexibility, mitochondrial damage, apoptosis, structural remodeling, and inflammation). Moreover, clinical evidence regarding the cardioprotective effect of PPAR agonists is also addressed.

Enhanced fatty acid oxidation by selective activation of PPARα alleviates autoimmunity through metabolic transformation in T-cells.

While fatty acid oxidation (FAO) in mitochondria is a primary energy source for quiescent lymphocytes, the impact of promoting FAO in activated lymphocytes undergoing metabolic reprogramming remains unclear. Here, we demonstrate that pemafibrate, a selective PPARα modulator used clinically for the treatment of hypertriglyceridemia, transforms metabolic system of T-cells and alleviates several autoimmune diseases. Pemafibrate suppresses Th17 cells but not Th1 cells, through the inhibition of glutaminolysis and glycolysis initiated by enhanced FAO. In contrast, a conventional PPARα agonist fenofibrate significantly inhibits cell growth by restraining overall metabolisms even at a dose insufficient to induce fatty acid oxidation. Clinically, patients receiving pemafibrate showed a significant decrease of Th17/Treg ratio in peripheral blood. Our results suggest that augmented FAO by pemafibrate-mediated selective activation of PPARα restrains metabolic programs of Th17 cells and could be a viable option for the treatment of autoimmune diseases.

Enhancing Drug Discovery and Development through the Integration of Medicinal Chemistry, Chemical Biology, and Academia-Industry Partnerships: Insights from Roche's Endocannabinoid System Projects.

The endocannabinoid system (ECS) is a critical regulatory network composed of endogenous cannabinoids (eCBs), their synthesizing and degrading enzymes, and associated receptors. It is integral to maintaining homeostasis and orchestrating key functions within the central nervous and immune systems. Given its therapeutic significance, we have launched a series of drug discovery endeavors aimed at ECS targets, including peroxisome proliferator-activated receptors (PPARs), cannabinoid receptors types 1 (CB1R) and 2 (CB2R), and monoacylglycerol lipase (MAGL), addressing a wide array of medical needs. The pursuit of new therapeutic agents has been enhanced by the creation of specialized labeled chemical probes, which aid in target localization, mechanistic studies, assay development, and the establishment of biomarkers for target engagement. By fusing medicinal chemistry with chemical biology in a comprehensive, translational end-to-end drug discovery strategy, we have expedited the development of novel therapeutics. Additionally, this strategy promises to foster highly productive partnerships between industry and academia, as will be illustrated through various examples.

A two-step strategy to expand primary human hepatocytes in vitro with efficient metabolic and regenerative capacities.

BACKGROUND: Primary human hepatocytes (PHHs) are highly valuable for drug-metabolism evaluation, liver disease modeling and hepatocyte transplantation. However, their availability is significantly restricted due to limited donor sources, alongside their constrained proliferation capabilities and reduced functionality when cultured in vitro. To address this challenge, we aimed to develop a novel method to efficiently expand PHHs in vitro without a loss of function. METHODS: By mimicking the in vivo liver regeneration route, we developed a two-step strategy involving the de-differentiation/expansion and subsequent maturation of PHHs to generate abundant functional hepatocytes in vitro. Initially, we applied SiPer, a prediction algorithm, to identify candidate small molecules capable of activating liver regenerative transcription factors, thereby formulating a novel hepatic expansion medium to de-differentiate PHHs into proliferative human hepatic progenitor-like cells (ProHPLCs). These ProHPLCs were then re-differentiated into functionally mature hepatocytes using a new hepatocyte maturation condition. Additionally, we investigated the underlying mechanism of PHHs expansion under our new conditions. RESULTS: The novel hepatic expansion medium containing hydrocortisone facilitated the de-differentiation of PHHs into ProHPLCs, which exhibited key hepatic progenitor characteristics and demonstrated a marked increase in proliferation capacity compared to cells cultivated in previously established expansion conditions. Remarkably, these subsequent matured hepatocytes rivaled PHHs in terms of transcriptome profiles, drug metabolizing activities and in vivo engraftment capabilities. Importantly, our findings suggest that the enhanced expansion of PHHs by hydrocortisone may be mediated through the PPARα signaling pathway and regenerative transcription factors. CONCLUSIONS: This study presents a two-step strategy that initially induces PHHs into a proliferative state (ProHPLCs) to ensure sufficient cell quantity, followed by the maturation of ProHPLCs into fully functional hepatocytes to guarantee optimal cell quality. This approach offers a promising means of producing large numbers of seeding cells for hepatocyte-based applications.

Sexual dimorphism in hepatic PPAR alpha and CYP4a12a expression is associated with reduced development of drug-induced non-alcoholic steatohepatitis in female IL-33-/- mice.

Males are at higher risk for developing metabolic dysfunction-associated steatohepatitis (MASH) than females; however, mechanisms mediating sexual dimorphism in MASH development are not completely understood. Nutrition-based mouse models suggest that dysregulated fatty acid biosynthesis promotes MASH. Drugs recapitulate MASH without diet variabilities. This brief report investigates associations of sexual dimorphism with male susceptibility to MASH utilizing a drug-induced MASH model and focuses on very-long-chain fatty acid biosynthesis pathways. We assessed male and female mouse livers at 5 and 15 weeks following MASH induction by immunizations and age-matched un-immunized controls utilizing Western blot. Our results suggest that PPAR alpha and CYP4a12a protect females, while CYP4v2 does not protect males from MASH development. Our results have important implications for understanding sexual dimorphism in the pathogenesis of MASH.