Nanoparticles Encapsulated γ-Oryzanol as a Natural Prodrug of Ferulic Acid for the Treatment of Oxidative Liver Damage.

Antioxidants are promising therapeutics for treating oxidative stress-mediated liver diseases. Previously, we studied a potent natural antioxidant, ferulic acid, and developed a liposomal formulation of ferulic acid (ferulic-lipo) to improve its solubility. Ferulic-lipo significantly attenuated oxidative damage in the liver by inhibiting reactive oxygenase species (ROS). However, antioxidative liposomes must be less reactive with ROS prior to reaching the target sites to effectively neutralize existing ROS. But ferulic-lipo tends to be oxidized before reaching the liver. Besides, γ-oryzanol has been reported to decompose into ferulic acid in vivo; accordingly, we hypothesized that γ-oryzanol could be employed as a natural prodrug of ferulic acid to improve stability and antioxidative effectiveness. Therefore, in this study, we prepared a liposomal formulation of γ-oryzanol (γ-ory-lipo) and investigated its therapeutic effects in a CCl4-induced rat model of liver injury. We found that γ-ory-lipo has a higher chemical stability than does free γ-oryzanol. Although the antioxidative effect of γ-ory-lipo was lower than that of ferulic-lipo, pretreatment of the HepG2 cells with γ-ory-lipo improved the viability of CCl4-treated cells to a similar level as treatment with ferulic-lipo. γ-Oryzanol was shown to be converted into ferulic acid in vitro and in vivo. Furthermore, intravenous administration of γ-ory-lipo exhibited a similar effectiveness as ferulic-lipo against CCl4-induced hepatotoxicity, which should be the due to the conversion of γ-oryzanol into ferulic acid. These findings demonstrated that γ-ory-lipo could be a good natural prodrug of ferulic acid for eradicating its stability problem.

Effects of saroglitazar in the treatment of non-alcoholic fatty liver disease or non-alcoholic steatohepatitis: A systematic review and meta-analysis.

AIM: This systematic review and meta-analysis was conducted to evaluate the efficacy and safety of 4 mg saroglitazar treatment in patients with non-alcoholic fatty liver disease (NAFLD) or non-alcoholic steatohepatitis (NASH). METHODS: PubMed, Embase, Scopus, Cochrane CENTRAL, medRxiv (pre-print), bioRxiv (pre-print), and ClinicalTrials.gov databases were searched for relevant studies. The primary outcome was the change in the serum alanine transaminase (ALT) level. The secondary outcomes were changes in liver stiffness, liver function test parameters, and metabolic parameters. Pooled mean differences were calculated using random-effects models. RESULTS: Of 331 studies that were screened, ten were included. Treatment with adjunct saroglitazar showed a reduction in ALT [mean difference: 26.01 U/L (95% CI: 10.67 to 41.35); p = 0.009; i2: 98%; moderate GRADE evidence] and aspartate transaminase [mean difference: 19.68 U/L (95% CI: 8.93 to 30.43); p<0.001; i2: 97%; moderate GRADE evidence] levels. There was a significant improvement in liver stiffness [mean difference: 2.22 kPa (95% CI: 0.80 to 3.63); p = 0.002; i2: 99%; moderate GRADE evidence]. There were significant improvements in glycated hemoglobin [mean difference: 0.59% (95% CI: 0.32 to 0.86); p<0.001; i2: 78%; moderate GRADE evidence], total cholesterol [mean difference: 19.20 (95% CI: 1.54 to 36.87); p = 0.03; i2: 95%; moderate GRADE evidence], and triglyceride [mean difference: 105.49 mg/dL (95% CI: 11.18 to 199.80); p = 0.03; i2: 100%; moderate GRADE evidence] levels. Saroglitazar treatment was safe. CONCLUSION: Treatment with adjunct 4 mg saroglitazar could significantly improve liver enzymes, reduce liver stiffness, and improve metabolic parameters (serum glucose and lipid profile) in patients with NAFLD or NASH.

Unsaturated bond recognition leads to biased signal in a fatty acid receptor.

Individual free fatty acids (FAs) play important roles in metabolic homeostasis, many through engagement with more than 40G protein-coupled receptors. Searching for receptors to sense beneficial omega-3 FAs of fish oil enabled the identification of GPR120, which is involved in a spectrum of metabolic diseases. Here, we report six cryo-electron microscopy structures of GPR120 in complex with FA hormones or TUG891 and Gi or Giq trimers. Aromatic residues inside the GPR120 ligand pocket were responsible for recognizing different double-bond positions of these FAs and connect ligand recognition to distinct effector coupling. We also investigated synthetic ligand selectivity and the structural basis of missense single-nucleotide polymorphisms. We reveal how GPR120 differentiates rigid double bonds and flexible single bonds. The knowledge gleaned here may facilitate rational drug design targeting to GPR120.

Darobactins Exhibiting Superior Antibiotic Activity by Cryo-EM Structure Guided Biosynthetic Engineering.

Over recent decades, the pipeline of antibiotics acting against Gram-negative bacteria is running dry, as most discovered candidate antibiotics suffer from insufficient potency, pharmacokinetic properties, or toxicity. The darobactins, a promising new small peptide class of drug candidates, bind to novel antibiotic target BamA, an outer membrane protein. Previously, we reported that biosynthetic engineering in a heterologous host generated novel darobactins with enhanced antibacterial activity. Here we utilize an optimized purification method and present cryo-EM structures of the Bam complex with darobactin 9 (D9), which served as a blueprint for the biotechnological generation of twenty new darobactins including halogenated analogs. The newly engineered darobactin 22 binds more tightly to BamA and outperforms the favorable activity profile of D9 against clinically relevant pathogens such as carbapenem-resistant Acinetobacter baumannii up to 32-fold, without observing toxic effects.

Syringin exerts anti-breast cancer effects through PI3K-AKT and EGFR-RAS-RAF pathways.

BACKGROUND: Breast cancer (BC) is one of the most common malignant tumors with the highest mortality in the world. Modern pharmacological studies have shown that Syringin has an inhibitory effect on many tumors, but its anti-BC efficacy and mechanism are still unclear. METHODS: First, Syringin was isolated from Acanthopanax senticosus (Rupr. & Maxim.) Harms (ASH) by systematic solvent extraction and silica gel chromatography column. The plant name is composed of genus epithet, species additive words and the persons' name who give its name. Then, the hub targets of Syringin against BC were revealed by bioinformatics. To provide a more experimental basis for later research, the hub genes which could be candidate biomarkers of BC and a ceRNA network related to them were obtained. And the potential mechanism of Syringin against BC was proved in vitro experiments. RESULTS: Syringin was obtained by liquid chromatography-mass spectrometry (LC-MS), nuclear magnetic resonance (NMR), and high-performance liquid chromatography (HPLC). Bioinformatics results showed that MAP2K1, PIK3CA, HRAS, EGFR, Caspase3, and PTGS2 were the hub targets of Syringin against BC. And PIK3CA and HRAS were related to the survival and prognosis of BC patients, the PIK3CA-hsa-mir-139-5p-LINC01278 and PIK3CA-hsa-mir-375 pathways might be closely related to the mechanism of Syringin against BC. In vitro experiments confirmed that Syringin inhibited the proliferation and migration and promoted apoptosis of BC cells through the above hub targets. CONCLUSIONS: Syringin against BC via PI3K-AKT-PTGS2 and EGFR-RAS-RAF-MEK-ERK pathways, and PIK3CA and HRAS are hub genes for adjuvant treatment of BC.

Effectiveness of Gamma Oryzanol on prevention of surgical induced endometriosis development in rat model.

Infertility is believed to be triggered by endometriosis whose pathophysiology and the etiology is still unknown. Certain genes play pivotal roles in pathogenesis of endometriosis. Natural products and plants are used as important sources for production of new drugs. The current study assesses the effects of gamma-oryzanol (GO) in a rat model with surgically induced endometriosis. The experimental endometriosis was induced in 24 wistar rats, and the animals were subsequently subdivided into endometriosis-sole (endom group), 3000 and 6000 µg/kg GO (GO-3000 and GO-6000) and Vit C groups. The protein levels of estrogen receptor-alpha (ER-α), Steroidogenic factor 1 (SF1), Sirtuin 1 (SIRT1), heme oxygenase 1 (HO1), light chain 3 (LC3B) and Beclin1 (BECN1) were assessed. TUNEL staining was used for detecting the apoptosis rate. The results revealed that protein levels of SF1, HO1, and total LC3B significantly (P < 0.05) decreased in GO-6000-treated groups compared to endom group. Moreover, the protein level of BECN1 and SIRT-1 significantly (P < 0.05) increased in GO-6000-treated groups compared to endom group. GO treatment did not imply any significant difference (P > 0.05) concerning the protein levels of ER-α. The TUNEL staining results showed higher TUNEL-positive cells reactions in the rats treated with GO-6000 and Vit C. Thus, GO is involved in improving condition rats involved with endometriosis through modulation in the protein levels of some molecules and also induction of apoptosis.

Isolation, Characterization and In Silico Studies of Secondary Metabolites from the Whole Plant of Polygala inexpectata Pesmen & Erik.

Polygala species are frequently used worldwide in the treatment of various diseases, such as inflammatory and autoimmune disorders as well as metabolic and neurodegenerative diseases, due to the large number of secondary metabolites they contain. The present study was performed on Polygala inexpectata, which is a narrow endemic species for the flora of Turkey, and resulted in the isolation of nine known compounds, 6,3'-disinapoyl-sucrose (1), 6-O-sinapoyl,3'-O-trimethoxy-cinnamoyl-sucrose (tenuifoliside C) (2), 3'-O-(O-methyl-feruloyl)-sucrose (3), 3'-O-(sinapoyl)-sucrose (4), 3'-O-trimethoxy-cinnamoyl-sucrose (glomeratose) (5), 3'-O-feruloyl-sucrose (sibiricose A5) (6), sinapyl alcohol 4-O-glucoside (syringin or eleutheroside B) (7), liriodendrin (8), and 7,4'-di-O-methylquercetin-3-O-ß-rutinoside (ombuin 3-O-rutinoside or ombuoside) (9). The structures of the compounds were determined by the spectroscopic methods including 1D-NMR (1H NMR, 13C NMR, DEPT-135), 2D-NMR (COSY, NOESY, HSQC, HMBC), and HRMS. The isolated compounds were shown in an in silico setting to be accommodated well within the inhibitor-binding pockets of myeloperoxidase and inducible nitric oxide synthase and anchored mainly through hydrogen-bonding interactions and π-effects. It is therefore plausible to suggest that the previously established anti-inflammatory properties of some Polygala-derived phytochemicals may be due, in part, to the modulation of pro-inflammatory enzyme activities.

Gamma-Oryzanol-Rich Fraction from Purple Rice Extract Attenuates Lipopolysaccharide-Stimulated Inflammatory Responses, Migration and VEGFA Production in SW480 Cells via Modulation of TLR4 and NF-κB Pathways.

Inflammatory response facilitating colorectal cancer (CRC) progression is a serious event following operative infection, which can occur in CRC patients. This event is mainly mediated by bacterial lipopolysaccharide (LPS), via a toll like receptor 4 (TLR4) and NF-κB. Hexane soluble fraction (HSF) from purple rice extract (PRE) has been identified as a γ-oryzanol (OR)-rich fraction. Recently, HSF possessed inhibitory effect of LPS-stimulated metastasis of human colon cancer SW480 cells, however the related mechanism was unknown. Thus, this study aimed to investigate the effect of HSF on inflammatory response-associated cancer progression of LPS-stimulated SW480 cells. The various inflammatory mediators, vascular endothelial growth factor-A (VEGFA) and related pathways were evaluated by Western blot and ELISA. Furthermore, cell migration was also determined by migration assays. Of all, HSF seemed to be stronger than OR to attenuate the responsiveness of LPS on various inflammatory mediators, which was related to an obvious reduction of cancer cell migration as well as indistinct disruption on VEGFA production in SW480 cells, via downregulation of TLR4 and NF-κB. Therefore, OR-rich fraction from PRE, against the subsequent inflammatory response and CRC progression following surgery, which could be combined with conventional treatments to increase the survival rate.

Proof-of-concept study to evaluate the safety and efficacy of saroglitazar in patients with primary biliary cholangitis.

BACKGROUND & AIM: Saroglitazar is a novel peroxisome proliferator-activated receptor (PPAR) agonist with dual agonistic properties (α/γ). Due to a strong mechanistic rationale, we aimed to test the safety and efficacy of saroglitazar in patients with primary biliary cholangitis (PBC) who were either ursodeoxycholic acid (UDCA) resistant or intolerant. METHODS: In this double-blind, phase II proof-of-concept trial, 37 patients with PBC were randomized to saroglitazar 4 mg (n = 13), saroglitazar 2 mg (n = 14), or placebo (n = 10) daily for 16 weeks. The primary efficacy endpoint was the reduction in alkaline phosphatase (ALP) level at Week 16. RESULTS: A significant reduction of mean ALP levels was observed at Week 16 relative to baseline in both the saroglitazar 4 mg (least-squares [LS] mean =-163.3 U/L, SE = 25.1, p <0.001) and 2 mg (LS mean =-155.8 U/L, SE = 24.4, p <0.001) groups, compared with placebo (LS mean =-21.1 U/L, SE = 28.9). Treatment with saroglitazar resulted in a rapid reduction of ALP concentration at Week 4 that was sustained through the study duration. At least 1 treatment-emergent adverse event occurred in 11 (84.6%) patients in the saroglitazar 4 mg group, in 12 (85.7%) patients in the 2 mg group and in 8 (80%) patients in the placebo group. Study drug was discontinued in 4 patients (3 patients in the 4 mg group and 1 patient in the 2 mg group) due to aminotransferase increases that promptly returned to baseline values after drug discontinuation. CONCLUSIONS: Saroglitazar at 2 mg and 4 mg daily was tolerated and resulted in rapid and sustained improvements in ALP. Further studies are underway at a daily dose of 2 mg and 1 mg due to the higher incidence of elevated liver enzymes observed with the 4 mg dose. CLINICALTRIALS. GOV IDENTIFIER: NCT03112681 LAY SUMMARY: Saroglitazar resulted in a rapid and sustained improvement in alkaline phosphatase levels in patients with primary biliary cholangitis. The mean percentage reductions in alkaline phosphatase levels were 49% and 51% in the saroglitazar 4 mg and 2 mg groups compared to 3% in the placebo group.

Mutasynthetic Production and Antimicrobial Characterization of Darobactin Analogs.

There is great need for therapeutics against multidrug-resistant, Gram-negative bacterial pathogens. Recently, darobactin A, a novel bicyclic heptapeptide that selectively kills Gram-negative bacteria by targeting the outer membrane protein BamA, was discovered. Its efficacy was proven in animal infection models of Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa, thus promoting darobactin A as a promising lead compound. Originally discovered from members of the nematode-symbiotic genus Photorhabdus, the biosynthetic gene cluster (BGC) encoding the synthesis of darobactin A can also be found in other members of the class Gammaproteobacteria. Therein, the precursor peptides DarB to -F, which differ in their core sequence from darobactin A, were identified in silico. Even though production of these analogs was not observed in the putative producer strains, we were able to generate them by mutasynthetic derivatization of a heterologous expression system. The analogs generated were isolated and tested for their bioactivity. The most potent compound, darobactin B, was used for cocrystallization with the target BamA, revealing a binding site identical to that of darobactin A. Despite its potency, darobactin B did not exhibit cytotoxicity, and it was slightly more active against Acinetobacter baumannii isolates than darobactin A. Furthermore, we evaluated the plasma protein binding of darobactin A and B, indicating their different pharmacokinetic properties. This is the first report on new members of this new antibiotic class, which is likely to expand to several promising therapeutic candidates. IMPORTANCE Therapeutic options to combat Gram-negative bacterial pathogens are dwindling with increasing antibiotic resistance. This study presents a proof of concept for the heterologous-expression approach to expand on the novel antibiotic class of darobactins and to generate analogs with different activities and pharmacokinetic properties. In combination with the structural data of the target BamA, this approach may contribute to structure-activity relationship (SAR) data to optimize inhibitors of this essential outer membrane protein of Gram-negative pathogens.

GPR120 Inhibits RANKL-Induced Osteoclast Formation and Resorption by Attenuating Reactive Oxygen Species Production in RAW264.7 Murine Macrophages.

Osteoclasts are large, multinucleated cells that are responsible for the resorption of bone. Bone degenerative diseases, such as osteoporosis, are characterized by overactive osteoclasts. Receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL) binding to its receptor on osteoclast precursors will trigger osteoclast formation and resorption. The production of reactive oxygen species (ROS) is known to play a crucial role in RANKL-induced osteoclast formation and resorption. G-protein coupled receptor 120 (GPR120) signalling has been shown to affect osteoclast formation, but the exact mechanisms of action require further investigation. RAW264.7 murine macrophages were seeded into culture plates and exposed to the GPR120 agonist, TUG-891, at varying concentrations (20-100 µM) and RANKL to induce osteoclast formation. TUG-891 was shown to inhibit osteoclast formation and resorption without affecting cell viability in RAW264.7 macrophages. TUG-891 further decreased ROS production when compared to RANKL only cells. Antioxidant proteins, Nrf2, HO-1 and NQO1 were shown to be upregulated while the ROS inducing protein, Nox1, was downregulated by TUG-891. Gene silencing revealed that TUG-891 exerted its effects specifically through GPR120. This study reveals that GPR120 signalling may inhibit osteoclast formation and resorption through inhibition on ROS production.

High-throughput metabolomics using UPLC/Q-TOF-MS coupled with multivariate data analysis reveals the effect and mechanism of syringin against ovariectomized osteoporosis.

Osteoporosis is an increasing public health problem in the worldwide and has caused socioeconomic burden. Natural products as candidates have the potential to promote bone formation and suppress bone resorption for osteoporosis treatment. Previously, syringin has showed the potent anti-osteoporosis activity, however the detailed mechanism of syringin against osteoporosis is still unclear. This study aimed to reveal the pharmacological effect and mechanism of syringin through the high-throughput metabolomics. In this study, metabolomics techniques were used to explore the metabolic biomarkers and profiles provides deep insights into the pharmacological effects and mechanism of syringin against osteoporosis. The metabolite biomarkers were monitored based on the high-resolution mass spectrometry. By the integration analysis of metabolomics technology, a total of 23 metabolic biomarkers were discovered and we found the highly relevant pathway involved in glycine and serine metabolism, butyrate metabolism, methionine metabolism, catecholamine biosynthesis, tyrosine metabolism, etc. Interestingly, synthesis and degradation of ketone bodies, phenylalanine, tyrosine and tryptophan biosynthesis, arachidonic acid metabolism, tyrosine metabolism, glycine, serine and threonine metabolism, butanoate metabolism, was related with efficacy of syringin. The present work showed that the metabolomics technology can provide novel strategies for revealing insights into the metabolic effects and action mechanism of drug.

Saroglitazar improved hepatic steatosis and fibrosis by modulating inflammatory cytokines and adiponectin in an animal model of non-alcoholic steatohepatitis.

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) have become significant global health concerns. In the present study, we aimed to investigate the effects of saroglitazar, a dual PPARα/γ agonist, fenofibrate, a PPAR-α agonist, and pioglitazone, a PPAR-γ agonist on an animal model of NASH. METHODS: Male Wistar rats were fed a high-fat (HF) emulsion via gavage for 7 weeks to induce NASH. The HF-treated rats were grouped into four groups to receive saroglitazar, pioglitazone, fenofibrate, or vehicle. We measured body and liver weight, liver enzymes, serum levels of adiponectin and leptin. We also performed histopathological examinations and gene expression analysis of interleukin 6 (IL-6), tumor necrosis factor-alpha (TNF- α), transforming growth factor-beta (TGF-ß), and monocyte chemoattractant protein 1 (MCP-1). RESULTS: Body weight was markedly normalized by both saroglitazar and fenofibrate, while the liver index only decreased significantly with saroglitazar. Saroglitazar corrected ALT, AST, leptin, and adiponectin levels better than pioglitazone and fenofibrate. All PPAR agonists significantly attenuated the upregulation of the proinflammatory and TGF-ß genes, which correlated with the improved steatosis, inflammation of liver tissue, and fibrotic lesions. CONCLUSIONS: As documented by our results, the dual activation of PPARα/γ by saroglitazar could effectively improve steatosis, fibrosis, and aspects of necro-inflammation in the HF-induced NASH model more than fenofibrate and pioglitazone, and it can be more beneficial in the management of NASH.

Artepillin C, a major component of Brazilian green propolis, inhibits endoplasmic reticulum stress and protein aggregation.

Propolis, a compound produced by honeybees, has long been used in food and beverages to improve health and prevent diseases. We previously reported that the ethanol extracts of Brazilian green propolis and its constituents artepillin C, kaempferide, and kaempferol mitigate oxidative stress-induced cell death via oxytosis/ferroptosis. Here, we investigated the potential of Brazilian green propolis and its constituents to protect against endoplasmic reticulum stress in the mouse hippocampal cell line HT22. Ethanol extracts of Brazilian green propolis, artepillin C, and kaempferol attenuated tunicamycin-induced unfolded protein response and cell death. Interestingly, artepillin C inhibited both tunicamycin-induced protein aggregation in HT22 cells and the spontaneous protein aggregation of mutant canine superoxide dismutase 1 (E40K-SOD1-EGFP) in Neuro2a cells. These findings indicate that in addition to oxidative stress, the ethanol extracts of Brazilian green propolis help prevent endoplasmic reticulum stress-related neuronal cell death, which is proposedly involved in several neurodegenerative diseases. Moreover, artepillin C, a major constituent of Brazilian green propolis, may exhibit chemical chaperone-like properties.

Total Syntheses and Anti-inflammatory Activities of Syringin and Its Natural Analogues.

Syringin (1), a natural bioactive glucoside isolated from the root of Acanthopanax senticosus (Rupr. Maxim.) Harms, possesses significant anti-inflammatory activity. In this study, we have accomplished the total syntheses of syringin (1), along with its natural analogues 2-12, from a common starting material, syringaldehyde (13), in 4-8 steps with an overall yields of 11.8-61.3%. The anti-inflammatory activities of these compounds were determined against NO production in the LPS-stimulated RAW264.7 cells. Among them, compounds 1-5, 7, and 9 exhibited different levels of anti-inflammatory activity.

Loxoprofen enhances intestinal barrier function via generation of its active metabolite by carbonyl reductase 1 in differentiated Caco-2 cells.

Nonsteroidal anti-inflammatory drugs (NSAIDs) are used worldwide as antipyretic analgesics and agents for rheumatoid arthritis and osteoarthritis, but known to cause damage to the gastrointestinal mucosae as their serious adverse effects. Few studies showed the impairment of intestinal epithelial barrier function (EBF) by high concentrations (0.5-1 mM) of NSAIDs, but the underlying mechanism is not fully understood. This study is aimed at clarifying effects at a low concentration (50 µM) of three NSAIDs, loxoprofen (Lox), ibuprofen and indomethacin, on intestinal EBF using human intestinal epithelial-like Caco-2 cells. Among those NSAIDs, Lox increased the transepithelial electric resistance (TER) value, decreased the paracellular Lucifer yellow CH (LYCH) permeability, and upregulated claudin (CLDN)-1, -3 and -5, indicating that low doses of Lox enhanced EBF through increasing expression of CLDNs. Lox is known to be metabolized to a pharmacologically active metabolite, (2S,1'R,2'S)-loxoprofen alcohol (Lox-RS), by carbonyl reductase 1 (CBR1), which is highly expressed in human intestine. CBR1 was expressed in the Caco-2 cells, and the pretreatment with a CBR1 inhibitor suppressed both the Lox-evoked CLDN upregulation and EBF enhancement. In addition, the treatment of the cells with Lox-RS resulted in higher TER value and lower LYCH permeability than those with Lox. Thus, Lox-RS synthesized by CBR1 may greatly contribute to the improving efficacy of Lox on the barrier function. Since EBF is decreased in inflammatory bowel disease, we finally examined the effect of Lox on EBF using the Caco-2/THP-1 co-culture system, which is used as an in vitro inflammatory bowel disease model. Lox significantly recovered EBF which was impaired by inflammatory cytokines secreted from THP-1 macrophages. These in vitro observations suggest that Lox enhances intestinal EBF, for which the metabolism of Lox to Lox-RS by CBR1 has an important role.

The Anti-Atherosclerotic Action of FFAR4 Agonist TUG-891 in ApoE-Knockout Mice Is Associated with Increased Macrophage Polarization towards M2 Phenotype.

Background: Over the past few years, a better understanding of the biology of G-protein coupled receptors (GPRs) has led to the identification of several receptors as novel targets for free fatty acids (FFAs). FFAR4 has received special attention in the context of chronic inflammatory diseases, including atherosclerosis, obesity and NAFLD, through to its anti-inflammatory effect. Methods: The present study investigates the influence of prolonged treatment with TUG-891-FFAR4 agonist on the development of atherosclerosis plaque in apoE-knockout mice, using morphometric and molecular methods. Results: TUG-891 administration has led to the reduction of atherosclerotic plaque size and necrotic cores in an apoE-knockout mice model. TUG-891-treated mice were administered subcutaneously at a dose of 20 mg/kg three times a week for 4 months. The FFAR4 agonist reduced the content of pro-inflammatory M1-like macrophages content in atherosclerotic plaques, as evidenced by immunohistochemical phenotyping and molecular methods. In atherosclerotic plaque, the population of smooth muscle cells increased as evidenced by α-SMA staining. We observed changes in G-CSF and eotaxin markers in the plasma of mice; changes in the levels of these markers in the blood may be related to macrophage differentiation. Importantly, we observed a significant increase in M2-like macrophage cells in atherosclerotic plaque and peritoneum. Conclusions: Prolonged administration of TUG-891 resulted in significant amelioration of atherogenesis, providing evidence that the strategy based on macrophage phenotype switching toward an M2-like activation state via stimulation of FFAR4 receptor holds promise for a new approach in the prevention or treatment of atherosclerosis.

Structural Basis for Anti-non-alcoholic Fatty Liver Disease and Diabetic Dyslipidemia Drug Saroglitazar as a PPAR α/γ Dual Agonist.

Peroxisome proliferator-activated receptors (PPARs) are nuclear receptor-type transcription factors that consist of three subtypes (α, γ, and ß/δ) with distinct functions and PPAR dual/pan agonists are expected to be the next generation of drugs for metabolic diseases. Saroglitazar is the first clinically approved PPARα/γ dual agonist for treatment of diabetic dyslipidemia and is currently in clinical trials to treat non-alcoholic fatty liver disease (NAFLD); however, the structural information of its interaction with PPARα/γ remains unknown. We recently revealed the high-resolution co-crystal structure of saroglitazar and the PPARα-ligand binding domain (LBD) through X-ray crystallography, and in this study, we report the structure of saroglitazar and the PPARγ-LBD. Saroglitazar was located at the center of "Y"-shaped PPARγ-ligand-binding pocket (LBP), just as it was in the respective region of PPARα-LBP. Its carboxylic acid was attached to four amino acids (Ser289/His323/His449/Thr473), which contributes to the stabilization of Activating Function-2 helix 12, and its phenylpyrrole moiety was rotated 121.8 degrees in PPARγ-LBD from that in PPARα-LBD to interact with Phe264. PPARδ-LBD has the consensus four amino acids (Thr253/His287/His413/Tyr437) towards the carboxylic acids of its ligands, but it seems to lack sufficient space to accept saroglitazar because of the steric hindrance between the Trp228 or Arg248 residue of PPARδ-LBD and its methylthiophenyl moiety. Accordingly, in a coactivator recruitment assay, saroglitazar activated PPARα-LBD and PPARγ-LBD but not PPARδ-LBD, whereas glycine substitution of either Trp228, Arg248, or both of PPARδ-LBD conferred saroglitazar concentration-dependent activation. Our findings may be valuable in the molecular design of PPARα/γ dual or PPARα/γ/δ pan agonists.

The Dietary Supplement γ-Oryzanol Attenuates Hepatic Ischemia Reperfusion Injury via Inhibiting Endoplasmic Reticulum Stress and HMGB1/NLRP3 Inflammasome.

The purpose of this study is to investigate the protective effect of γ-oryzanol (ORY) against hepatic ischemia reperfusion (HIR) injury and the potential protective mechanisms of ORY. ORY is an important biologically active ingredient isolated from rice bran oil, which has anti-inflammatory and antiapoptotic effects. However, it is still unknown whether ORY can protect the liver from the HIR damage. In this study, ORY was administered orally for seven days, after which the animals were subjected to liver ischemia for 60 minutes and reperfused for 6 hours. Related indicators were analyzed. The results showed that ORY pretreatment significantly reduced the levels of AST and ALT, relieved hepatocellular damage and apoptosis, and attenuated the exhaustion of SOD and GSH and accumulation of MDA and MPO. Interestingly, ORY treatment could significantly decreased ER stress. Furthermore, ORY pretreatment remarkably reduced the protein expressions of HMGB1, NLRP3, caspase-1 (p20), and IL-1ß to protect the liver from I/R-induced inflammasome activation and apoptosis. In conclusion, we demonstrated the potential effect of ORY in modulating oxidative stress, endoplasmic reticulum stress, and inflammasome activation during HIR.