PPARγ Activation Research Articles

Kaempferol improves depression-like behaviors through shifting microglia polarization and suppressing NLRP3 via tilting the balance of PPARγ and STAT1 signaling.

The pathogenesis of depression is largely influenced by dyshomeostasis of neuroinflammation regulated by microglia M1/M2 polarization, and NLRP3 inflammasome acts critical roles in shifting microglia polarization. Kaempferol (Kae), a major flavonoid in edible plants, possesses anti-inflammation and anti-depression capacity, but its underlying cellular and molecular mechanisms of antidepressive effect have not yet fully explored. In vivo studies with lipopolysaccharide (LPS)-induced depressive mice were carried out to evaluate antidepressant effect of Kae. In vitro, BV2 microglia cell line stimulated by LPS along with IFN-γ to detect pharmacological effects of Kae on microglia polarization and NLRP3. Based on two depression-related GEO datasets (GSE54570 and GSE54568) and the potential targets of Kae obtained from GeneCards database, enrichment analysis and protein-protein interaction (PPI) network construction reveal potential therapeutic targets of Kae for depression. Then the precise antidepressant mechanisms of Kae were verified by western blot and immunofluorescent staining in vivo and vitro. Our results showed that Kae significantly improves LPS-induced depressive behaviors and alleviates neuroinflammation in prefrontal cortex. Moreover, Kae obviously shifted microglia polarization to M2 phenotype, and also suppressed NLRP3 in prefrontal cortex and BV2. Enrichment analysis and PPI network construction suggested PPARγ and STAT1 signaling are related to regulation of NLRP3 in depression. Furtherly, Kae remarkably enhanced PPARγ activation and inhibited nuclear translocation of p-STAT1 in microglia of prefrontal cortex and BV2. Importantly, pre-incubation with PPARγ antagonist T0070907 or overexpression with CASTAT1 (constitutively active STAT1) both prevented pharmacologic effects of Kae on shifting microglia polarization and suppressing NLRP3 in BV2. Noteworthily, T0070907 significantly blocked the inhibitory effect of Kae on STAT1 while overexpression with CASTAT1 abolished the effect of Kae on PPARγ activation in BV2. Above results suggested that pharmacologic effects of Kae on microglia polarization and NLRP3 are dependent on the balance of counter-regulatory PPARγ and STAT1 signaling. Our results indicated that the shifting microglia polarization and suppression of NLRP3 via tilting the balance of PPARγ and STAT1 signaling may be the antidepressant mechanism of Kae, which provides a novel perspective for elucidating antidepressive effect of Kae.

RBIO-02. DEVELOPMENT OF FLASH-CAR RADIOIMMUNOTHERAPY FOR PEDIATRIC BRAIN TUMORS

Abstract Pediatric brain tumors including medulloblastoma and high-grade glioma are generally refractory to T cell-based immunotherapy, largely due to an immune-hostile microenvironment infiltrated extensively with immunosuppressive macrophages. Ultra-high dose rate (FLASH) radiotherapy holds promise for treating solid tumors, given the potential lower toxicity in normal tissues and possible favorable impact on tumor immunity. Using a genetically engineered mouse model of medulloblastoma and glioma, we show that FLASH radiation stimulates pro-inflammatory polarization in tumor macrophages. Single-cell transcriptome analysis shows that FLASH proton beam radiation skews macrophages towards proinflammatory phenotypes and increases T cell infiltration in the medulloblastoma model. Further bulk transcriptome analyses reveal that FLASH radiation reduces peroxisome proliferator-activated receptor (PPAR)-γ and arginase-1 expression and inhibits immunosuppressive macrophage polarization under stimulus-inducible conditions. Mechanistically, FLASH radiation abrogates lipid oxidase expression and oxidized low-density lipid (oxLDL) generation to reduce PPARγ activity, while standard radiation induces reactive oxygen species (ROS)-dependent PPARγ activation in macrophages. Notably, FLASH radiotherapy improves infiltration and activation of chimeric antigen receptor (CAR) T cells and sensitizes tumors to GD2 CAR T immunotherapy in the autochthonous medulloblastoma and syngeneic glioma models. These findings suggest that FLASH radiotherapy may reprogram macrophage lipid metabolism to reverse tumor immunosuppression and that combination FLASH-CAR radioimmunotherapy may offer exciting opportunities for treatment of pediatric brain tumor.

Genomic and Bioinformatics Analysis of Familial Partial Lipodystrophy Type 3 Identified in a Patient with Novel PPARγ Mutation and Robust Response to Pioglitazone

Familial partial lipodystrophies (FPLDs) are very rare inherited disorders characterized by partial loss of adipose tissue from the upper and lower extremities. At least seven subtypes of FPLD have been identified and are mostly dominantly inherited. FPLD type 3 is caused by mutations in the PPARγ gene, which encodes for the protein peroxisome proliferator-activated receptor gamma (PPARγ). We identified a Saudi female with PFLD3 presented with partial lipoatrophy, uncontrolled diabetes, severe hypertriglyceridemia, and recurrent pancreatitis. The clinical and biochemical findings in this proband were described before and after treatment with Pioglitazone in addition to the conventional treatment. DNA extraction and whole exome sequencing (WES) were performed to detect the variant. The mutant gene was subjected to Sanger analysis to confirm the results. We applied five specific computational prediction tools to assess the pathogenicity of variation, namely the MT, DANN, CADD, BayesDel, and fitCons tools. We assessed protein modeling and stability with the AlphaFold-generated structures for both wild-type and mutant proteins. Finally, we conducted molecular docking using the AutoDock Vina virtual docking. Upon whole exome sequencing, a c.1024C>T p.(Gln342Ter) missense mutation was detected in the PPARγ gene associated with FPLD3. This variant is a novel mutation that has not been described in all genome databases. Sanger analysis confirmed the heterogenicity and pathogenicity of this variant. All five computational prediction tools indicate that this variant is considered highly pathogenic. Our patient showed a dramatic response to Pioglitazone, a synthetic PPARγ agonist. From structural modeling, we found that the enhanced binding affinity of the mutant PPARγ protein to Pioglitazone likely improves the activation of PPARγ, enhancing its transcriptional activity and resulting in better clinical outcomes. These findings extend the spectrum of PPARγ mutations responsible for FPLD3 and highlight the potential for personalized treatment strategies based on genetic mutations.

The Neuroprotective Mechanisms of PPAR-γ: Inhibition of Microglia-Mediated Neuroinflammation and Oxidative Stress in a Neonatal Mouse Model of Hypoxic-Ischemic White Matter Injury.

Neuroinflammation and oxidative stress, mediated by microglial activation, hinder the development of oligodendrocytes (OLs) and delay myelination in preterm infants, leading to white matter injury (WMI) and long-term neurodevelopmental sequelae. Peroxisome proliferator-activated receptor gamma (PPAR-γ) has been reported to inhibit inflammation and oxidative stress via modulating microglial polarization in various central nervous system diseases. However, the relationship between PPAR-γ and microglial polarization in neonatal WMI is not well understood. Therefore, this study aimed to elucidate the role and mechanisms of PPAR-γ in preterm infants affected by WMI. In this study, an invivo hypoxia-ischemia (HI) induced brain WMI neonatal mouse model was established. The mice were administered intraperitoneally with either RSGI or GW9662 to activate or inhibit PPAR-γ, respectively. Additionally, an invitro oxygen-glucose deprivation (OGD) cell model was established and pretreated with pcDNA 3.1-PPAR-γ or si-PPAR-γ to overexpress or silence PPAR-γ, respectively. The neuroprotective effects of PPAR-γ were investigated invivo. Firstly, open field test, novel object recognization test, and beam-walking test were employed to assess the effects of PPAR-γ on neurobehavioral recovery. Furthermore, assessment of OLs loss and OL-maturation disorder, the number of myelinated axons, myelin thickness, synaptic deficit, activation of microglia and astrocyte, and blood-brain barrier (BBB) were used to evaluate the effects of PPAR-γ on pathological repair. The mechanisms of PPAR-γ were explored both invivo and invitro. Assessment of microglia polarization, inflammatory mediators, reactive oxygen species (ROS), MDA, and antioxidant enzymes was used to evaluate the anti-inflammatory and antioxidative effects of PPAR-γ activation. An assessment of HMGB1/NF-κB and NRF2/KEAP1 signaling pathway was conducted to clarify the mechanisms by which PPAR-γ influences HI-induced WMI in neonatal mice. Activation of PPAR-γ using RSGI significantly mitigated BBB disruption, promoted M2 polarization of microglia, inhibited activation of microglia and astrocytes, promoted OLs development, and enhanced myelination in HI-induced WMI. Conversely, inhibition of PPAR-γ using GW9662 further exacerbated the pathologic hallmark of WMI. Neurobehavioral tests revealed that neurological deficits were ameliorated by RSGI, while further aggravated by GW91662. In addition, activation of PPAR-γ significantly alleviated neuroinflammation and oxidative stress by suppressing HMGB1/NF-κB signaling pathway and activating NRF2 signaling pathway both invivo and invitro. Conversely, inhibition of PPAR-γ further exacerbated HI or OGD-induced neuroinflammation, oxidative stress via modulation of the same signaling pathway. Our findings suggest that PPAR-γ regulates microglial activation/polarization as well as subsequent neuroinflammation/oxidative stress via the HMGB1/NF-κB and NRF2/KEAP1 signaling pathway, thereby contributing to neuroprotection and amelioration of HI-induced WMI in neonatal mice.

Magnolol preserves the integrity of the intestinal epithelial barrier and mitigates intestinal injury through activation of PPAR γ in COPD rat

Ethnopharmacological relevanceMagnolia officinalis Rehder & E.H. Wilson is traditionally used in the treatment of gastrointestinal disorders, diarrhea, and cough. Its main active ingredient, magnolol, exhibits protective effects on the lungs and gastrointestinal tract, including the inhibition of inflammation in these organs. Aim of the studyThis work aims to explore the molecular mechanism by which magnolol suppressed Chronic obstructive pulmonary disease (COPD) intestinal damage by improving the intestinal epithelial barrier. Materials and methodsThe study focused on investigating the mitigation effect of magnolol on intestinal injury and epithelial barrier in a COPD rat. Caco-2 cells were induced with TNF-α or IL-1β to establish the barrier injury model in order to explore the direct protective effect of magnolol on the intestinal barrier and elucidate the molecular mechanism by which it activates peroxisome proliferators-activated receptors-γ (PPARγ). ResultsMagnolol significantly improves pulmonary function and tissue damage in COPD rats by inhibiting inflammation, protease imbalance, and oxidative stress. It also suppresses colon tissue damage and inflammation, and protects colon epithelial barrier function by suppressing the decline of tight junction proteins, reducing colon epithelial permeability. In Caco-2 cells, magnolol directly reduces monolayer permeability, increases TEER, and upregulates tight junction protein expression induced by TNF-α or IL-1β. Drug Affinity Responsive Target Stability (DARTS) and thermal shift assays show that magnolol effectively binds to SRC, activating PPARγ signaling in Caco-2 cells and colon tissues of COPD rats. Furthermore, magnolol enhances the binding of PPARγ and RXRα, promoting their activation and entry into the nucleus. The PPARγ inhibitor GW9662 can reverse the effects of magnolol on PPARγ activation and tight junction protein upregulation in IL-1β or TNF-α induced Caco-2 cells. ConclusionsThis work demonstrates that magnolol enhances lung and intestinal functions in COPD rats, and elucidates its mechanism of action in protecting the intestinal epithelial barrier by activating PPARγ.

Potential involvement of PPARs in the inhibition of mammary lipid synthesis during diet-induced milk fat depression

The objective of this study was to evaluate the possible role of the peroxisome proliferator-activated receptors (PPARs: PPAR-α, PPAR-β/δ, and PPAR-γ) in diet and conjugated linoleic acid (CLA)-induced milk fat depression (MFD) in dairy cows. We hypothesized that the expression of PPARs, which regulate lipid metabolism and bind to polyunsaturated fatty acids, could be modulated by biohydrogenation intermediates that induce MFD, thereby interfering with milk fat synthesis. First, tissue profiling revealed that PPAR-α and PPAR-β/δ had low expression in mammary tissue compared with the liver. A comparison of lactating and nonlactating tissue from the same cows showed that expression of the PPARs did increase during lactation. Mammary expression of the PPARs during MFD was then observed in 9 mid-lactation cows in a 3x3 Latin square design with MFD induced by a 3-d intravenous infusion of trans-10,cis-12 CLA or feeding a high-oil/low-forage diet. The expression of the PPARs remained largely unaltered during CLA and diet-induced MFD, except for an increase in PPAR-α target genes CPT1A and ACADVL that are involved in β-oxidation. The interaction of PPAR-γ chemical agonist Troglitazone and antagonist T0070907 and CLA was then investigated in bovine mammary epithelial cells (MAC-T). The activation and inhibition of PPAR-γ did not overcome trans-10,cis-12 CLA inhibition of lipogenesis despite the agonist stimulating PPAR-γ expression. Furthermore, PPAR-γ activation did not modify the expression of lipogenic genes. Overall, the results fail to support a functional role of the PPARs in the inhibition of lipogenesis during MFD in dairy cows.

In-vitro modulation of glucose and lipid metabolism by Lentinula edodes extracts in obesity and type 2 diabetes models

IntroductionLentinula edodes (shiitake mushroom) has been integral to Traditional Chinese Medicine (TCM) for centuries, with its cultivation dating back to the Song dynasty (960–1127) in China. Traditionally valued for its immune-boosting properties, L. edodes is now being studied for its potential in managing metabolic disorders like obesity and type 2 diabetes. This study examines the effects of L. edodes fruiting body extract on key metabolic pathways related to glucose metabolism and lipid regulation. MethodsFruiting body extracts of Lentinula edodes including water, ethanolic, methanolic, and hydroalcoholic (50:50, 75:25, 25:75) were tested on L6 myoblasts. The study evaluated glucose uptake, GLUT4 expression, p-AMPK activation, and gene expression levels of PPARγ, AMPK, UCP-1, and FASN. Comparisons were made with control groups and standards like berberine and insulin. ResultsThe hydroalcoholic extracts (50:50 and 75:25) significantly enhanced glucose uptake (p < 0.001) and GLUT4 translocation, similar to the effects of insulin (p < 0.0001) and berberine (p < 0.0001). These extracts also upregulated p-AMPK (p < 0.0001) and UCP-1 expression (7.621), indicating improved insulin sensitivity and thermogenic activity. Additionally, these extracts differentially regulated PPARγ and FASN, suggesting a complex interaction with lipid metabolism. Notably, FASN expression was highly upregulated in water extract (37.792), indicating potential implications for fatty acid synthesis and storage. DiscussionLentinula edodes shows significant potential as a functional food for managing obesity and diabetes, supporting traditional TCM practices with modern scientific evidence. The study suggests that L. edodes can modulate key metabolic pathways such as glucose uptake, AMPK signalling, PPARγ regulation, and upregulation of FASN and UCP-1, making it a promising natural therapeutic agent. Further research, including clinical trials, is necessary to confirm these benefits in humans and explore the underlying mechanisms.

Cell-state dependent regulation of PPARγ signaling by the transcription factor ZBTB9 in adipocytes.

Peroxisome proliferator-activated receptor-γ (PPARγ) is a nuclear hormone receptor that is a master regulator of adipocyte differentiation and function. ZBTB9 is a widely expressed but poorly studied transcription factor that was predicted to interact with PPARγ based on large-scale protein-protein interaction experiments. In addition, genome-wide association studies (GWAS) revealed associations between ZBTB9 and BMI, T2D risk, and HbA1c levels. Here we show that Zbtb9 deficiency in mature adipocytes decreased PPARγ activity and protein level, and thus acts as a positive regulator of PPARγ signaling. In contrast, Zbtb9 deficiency in 3T3-L1 and human preadipocytes increased PPARγ levels and enhanced adipogenesis. Transcriptomic and transcription factor binding site analyses of Zbtb9 deficient preadipocytes revealed that the E2F pathway, controlled by the E2F family of transcription factors that are classically associated with cell cycle regulation, was among the most upregulated pathways. E2F1 positively regulates adipogenesis by promoting Pparg expression, independent of its cell cycle role, via direct binding to the Pparg promoter early during adipogenesis. RB phosphorylation (pRB), which regulates E2F activity, was also upregulated in Zbtb9 deficient preadipocytes. Critically, an E2F1 inhibitor blocked the effects of Zbtb9 deficiency on adipogenesis. Collectively, these results demonstrate that Zbtb9 inhibits adipogenesis as a negative regulator of Pparg expression via pRB-E2F signaling. Our findings reveal cell-state dependent roles of ZBTB9 in adipocytes, identifying a new molecule that regulates adipocyte biology as both a positive and negative regulator of PPARγ signaling depending on the cellular context, and thus may be important in the pathogenesis of obesity and T2D.

Odoratin balances ROS/NO through EZH2/PPARγ signalling to improve myocardial fibrosis.

Myocardial fibrosis is a critical concern in clinical medicine. This study explores the potential of odoratin as a treatment for myocardial fibrosis and investigates its underlying mechanisms. In vitro experiments involved stimulating primary mouse cardiomyocytes with TGF-β1, followed by odoratin treatment, to assess levels of reactive oxygen species (ROS) and nitric oxide (NO). In vivo, a mouse model of myocardial fibrosis was established using abdominal aortic constriction (AAC) and treated with odoratin. ROS and NO levels in myocardial tissue were then evaluated. Immunofluorescence and Western blotting analysis showed that odoratin reduced excess ROS, enhanced NO production and decreased fibrosis-related protein expression in vitro. In vivo, odoratin significantly improved cardiac function, reduced ROS, increased NO levels and mitigated fibrosis in AAC-induced mice. Both in vitro and in vivo, odoratin inhibited the expression of NADPH oxidase 4 and EZH2, while promoting the expression of phosphorylated endothelial nitric oxide synthase (p-eNOS) and PPARγ. The anti-fibrotic effects of odoratin were reversed by PPARγ antagonism, and EZH2 overexpression diminished PPARγ activation by odoratin. These findings suggest that odoratin may combat myocardial fibrosis by balancing ROS and NO through PPARγ activation, with EZH2 inhibition likely playing a key regulatory role.

Exploring the molecular pathways of the activation process in PPARγ recurrent bladder cancer mutants.

The intricate involvement of Peroxisome Proliferator-Activated Receptor Gamma (PPARγ) in glucose homeostasis and adipogenesis is well-established. However, its role in cancer, particularly luminal bladder cancer, remains debated. The overexpression and activation of PPARγ are implicated in tumorigenesis. Specific gain-of-function mutations (M280I, I290M, and T475M) within the ligand-binding domain of PPARγ are associated with bladder cancer and receptor activation. The underlying molecular pathways prompted by these mutations remain unclear. We employed a dual-basin structure-based model (db-SBM) to explore the conformational dynamics between the inactive and active states of PPARγ and examined the effects of the M280I, I290M, and T475M mutations. Our findings, consistent with the existing literature, reveal heightened ligand-independent transcriptional activity in the I290M and T475M mutants. Both mutants showed enhanced stabilization of the active state compared to the wild-type receptor, with the I290M mutation promoting a specific transition route, making it a prime candidate for further study. Electrostatic analysis identified residues K303 and E488 as pivotal in the I290M activation cascade. Biophysical assays confirmed that disrupting the K303-E488 interaction reduced the thermal stabilization characteristic of the I290M mutation. Our study demonstrates the predictive capabilities of combining simulation and cheminformatics methods, validated by biochemical experiments, to gain insights into molecular activation mechanisms and identify target residues for protein modulation.

Regulation of renal ischemia-reperfusion injury and tubular epithelial cell ferroptosis by pparγ m6a methylation: mechanisms and therapeutic implications

This study aimed to elucidate the role and underlying mechanisms of Peroxisome proliferator-activated receptor gamma (PPARγ) and its m6A methylation in renal ischemia-reperfusion (I/R) injury and ferroptosis of tubular epithelial cells (TECs). High-throughput transcriptome sequencing was performed on renal tissue samples from I/R injury models and sham-operated mice, complemented by in vivo and in vitro experiments focusing on the PPARγ activator Rosiglitazone and the manipulation of METTL14 and IGF2BP2 expression. Key evaluations included renal injury assessment, ferroptosis indicator measurement, and m6A methylation analysis of PPARγ. Our findings highlight the critical role of the PPARγ pathway and ferroptosis in renal I/R injury, with Rosiglitazone ameliorating renal damage and TEC ferroptosis. METTL14-mediated m6A methylation of PPARγ, dependent on IGF2BP2, emerged as a pivotal regulator of PPARγ expression, renal injury, and ferroptosis. This study reveals that PPARγ m6A methylation, orchestrated by METTL14 through an IGF2BP2-dependent mechanism, plays a crucial role in mitigating renal I/R injury and TEC ferroptosis. These insights offer promising avenues for therapeutic strategies targeting acute kidney injury.

PPAR-γ Signaling and Common Protective Pathways against Obesity and Alzheimer's Disease

Transcription factor PPAR-γ is predominantly found in adipose tissue, liver, and brain. PPARs form heterodimers, interact with ligands, and regulate the expression of the genes of the PPAR-γ downstream regulatory pathways. PPAR-γ is critical in regulating many physiological processes, including adipogenesis, glucose metabolism, fatty acid metabolism, energy homeostasis, and inflammation. This review is on the functions of PPAR-γ and how dysregulation of activity or expression of PPAR-γ can lead to obesity and Alzheimer's disease (AD). The PPAR- γ agonist inhibited the downregulated pathways, such as Wnt/β-Catenin and JAK-STAT pathways, both involved in activating NF-kB. PPAR-γ has a significant role in the APOE (Apolipoprotein E) gene expression, which reduces reducing obesity, inhibits amyloid aggregation, prevents hyperphosphorylation of tau, and inhibits dysregulation of autophagy. This review provides a perspective on the intricate interplay between PPAR-γ, obesity, and AD, focusing on the molecular mechanisms and potential therapeutic implications.

FPR1 signaling aberrantly regulates S100A8/A9 production by CD14+FCN1hi macrophages and aggravates pulmonary pathology in severe COVID-19

Excessive alarmins S100A8/A9 escalate the inflammation and even exacerbate immune-driven thrombosis and multi-organ damage. However, the regulatory mechanisms of S100A8/A9 expression in infectious diseases remain unclear. In this study, high-dimensional transcriptomic data analyses revealed a high proportion of CD14+FCN1hi macrophages within the pulmonary niche post-severe SARS-CoV-2 infection. By constructing the S100-coexpression gene list and supervised module scoring, we found that CD14+FCN1hi macrophages presented the highest scores of alarmin S100, and possibly served as the trigger and amplifier of inflammation in severe COVID-19. These CD14+FCN1hi cells lacked the positive regulatory activity of transcription factor PPARγ, and lost their differentiation ability towards mature macrophages. Ex vivo experiments further validated that the epithelial cells with high ORF-3a expression promoted the expression and secretion of S100A8/A9 through ANXA1/SAA1-FPR1 signaling. S100A8/A9 heterodimers, as well as the co-localization of S100A8/A9 with microtubules, were both diminished by the FPR1 inhibitor. Phospho-kinase protein array indicated that STAT3 promoted transcription, and PLC-γ and ERK1/2 pathways were involved in the hetero-dimerization and unconventional secretion of S100A8/A9. Our study highlights the pivotal role of FPR1 signaling in the excessive production of S100A8/A9 and provides a promising target for the prevention and control of severe COVID-19 and post-acute COVID-19 sequelae.

Role of PNPLA3 in Hepatic Stellate Cells and Hepatic Cellular Crosstalk.

Since its discovery, the patatin-like phospholipase domain containing 3 (PNPLA3) (rs738409 C>G p.I148M) variant has been studied extensively to unravel its molecular function. Although several studies proved a causal relationship between the PNPLA3 I148M variant and MASLD development and particularly fibrosis, the pathological mechanisms promoting this phenotype have not yet been fully clarified. We summarise the latest data regarding the PNPLA3 I148M variant in hepatic stellate cells (HSCs) activation and macrophage biology or the path to inflammation-induced fibrosis. Elegant but contradictory studies have ascribed PNPLA3 a hydrolase or an acyltransferase function. The PNPLA3 I148M results in hepatic lipid accumulation, which predisposes the hepatocyte to lipotoxicity and lipo-apoptosis, producing DAMPs, cytokines and chemokines leading to recruitment and activation of macrophages and HSCs, propagating fibrosis. Recent studies showed that the PNPLA3 I148M variant alters HSCs biology via attenuation of PPARγ, AP-1, LXRα and TGFβ activity and signalling. The advent of refined techniques in isolating HSCs has made PNPLA3's direct role in HSCs for liver fibrosis development more apparent. However, many other mechanisms still need detailed investigations.

Exploring the Role of Secondary Metabolites from Plants and Microbes as Modulators of Macrophage Differentiation.

Recent research has uncovered that secondary metabolites-biologically active compounds produced by plants, microbes, and other organisms-play a significant role in regulating the differentiation and function of macrophages. Macrophages, key components of the innate immune system, are crucial for a wide range of physiological processes, including immune response modulation, tissue homeostasis, and host defense against pathogens. This research delves into the mechanisms by which secondary metabolites influence macrophage differentiation signaling pathways, with a focus on how specific compounds affect macrophage polarization and functional phenotypes. Understanding these effects can open new avenues for developing therapeutic strategies that target macrophage-mediated immune responses. Secondary metabolites, such as nitrogen (N) and sulfur (S) containing compounds, terpenoids, and phenolic compounds from plants and microbes, can modulate macrophage differentiation by influencing cytokine production and activity. The activation of signaling pathways in macrophages involves multiple receptors and transcription factors, including IFN-γ receptor activation leading to STAT1 activation, TLR4 triggering IRF5, NFκB, and AP1, IL-4 receptor activation leading to STAT6 and IRF4 activation, PPARγ activation via the fatty acid receptor, TLR4 increasing CREB and C/EBP levels. The complex interplay between transcription factors and cytokines is crucial for maintaining the balance between the M1 and M2 states of macrophages. Despite these insights, further research is needed to unravel the specific molecular mechanisms involved and to identify promising secondary metabolites that could be translated into clinical applications.

Structural characteristics of a purified Evodiae fructus polysaccharide and its gastroprotection and relevant mechanism against alcohol-induced gastric lesions in rats

Evodiae fructus polysaccharide (EFP) has been previously shown to protect against alcohol-induced gastric lesions. However, which and how active fractions in EFP exert gastroprotection remains unclear. This study aimed to characterize the structure of the purified fraction (EFP-2-1) of EFP, and investigate its gastroprotection and underlying mechanisms. EFP-2-1 was obtained through column chromatography, and was characterized using instrumental analytical techniques. Gastroprotective effect of EFP-2-1 was evaluated using alcohol-induced gastric lesions in rats, and its mechanism was explored through proteomics, metabolomics and diversity sequencing. Results showed that EFP-2-1 had a molecular weight of 7.3 kDa, and consisted mainly of rhamnose, galacturonic acid, galactose and arabinose. Its backbone contained HG and RG-I domains, and branched with →5)-α-l-Araf-(1→, α-l-Araf-(1→ and →4)-β-d-Galp-(1→ residues. EFP-2-1 reduced gastric lesions and the levels of MDA, TNF-α and IL-6, activated PPARγ, primarily altered protein digestion and absorption and bile secretion metabolic pathways, regulated gut microbiota like Faecalibaculum and Lachnoclostridium, and increased short-chain fatty acids production. Correlations were observed among the gut microbiota, metabolites and biochemical indexes influenced by EFP-2-1. These findings suggest that EFP-2-1 is an active fraction of EFP for protecting against alcohol-induced gastric lesions, which may be linked to PPARγ activation, gut microbiota and serum metabolism.

8638 NEGR1 is a Mediator of Exercise-Induced Neuritogenesis in Subcutaneous White Adipose Tissue, Implications for Improved Systemic Metabolism

Abstract Disclosure: P. Nigro: None. N.P. Carbone: None. T. Caputo: None. M. Vamvini: None. M.M. Columbus: None. M.F. Hirshman: None. R. Middelbeek: None. L.J. Goodyear: None. Emerging research shows that exercise training results in robust and beneficial adaptions to subcutaneous white adipose tissue (scWAT), leading to improved systemic metabolism. The mechanisms for these benefits of exercise on are not fully understood, but we have recently demonstrated that training in mouse scWAT refines nerve connections in scWAT promoting neurite outgrowth (neuritogenesis). Our multi-omics and advanced microscopy data identifies Neuronal Growth Regulator 1 (NEGR1) as a potential player in this process. Here, we determine the role of NEGR1 as a mediator of scWAT neuritogenesis and regulator of systemic metabolism. Exercise training in mice for 11 days significantly increased NEGR1 protein expression by 35% (p&amp;lt;0.05). Next, we directly injected NEGR1 AAV into the inguinal scWAT of untrained male mice for three weeks resulting in a 43% increase in NEGR1 protein (p&amp;lt;0.05). Overexpression of NEGR1 led to an increase in the innervation markers tyrosine hydroxylase (+38%, p&amp;lt;0.01) and synaptophysin (+39%, p&amp;lt;0.01), and remarkably, a 16% decrease in adipocyte cell size (p&amp;lt;0.05), and a 15% reduction in fasting blood glucose levels (p&amp;lt;0.05). This suggests that NEGR1 plays a fundamental role in regulating scWAT morphology and function, adaptations that improve systemic glucose homeostasis. To determine mechanisms for exercise training-induced increases in NEGR1 expression, we used in silico and cell culture experiments and found that NEGR1 is under the regulation of the Peroxisome Proliferator-Activated Receptor gamma (PPARg) transcription factor. Given that numerous natural and synthetic PPARg ligands are lipids, we used targeted lipidomics to identify exercise-induced lipids capable of binding and inducing PPARg expression in inguinal scWAT. We identified 13-oxo-octadecadienoic acid (13-oxo-ODE), an oxylipin derived from linoleic acid, as a potent agonist and inducer of PPARg expression. Exercise training increased 13-oxo-ODE by 3-fold (p&amp;lt;0.05), and 13-hydroxyoctadecadienoic acid (13-HODE), the 13-oxo-ODE precursor was also increased by 3-fold (p&amp;lt;0.01). To validate that 13-oxo-ODE increases NEGR1, adipocytes were incubated with 2μM of 13-oxo-ODE for 48 hours. 13-oxo-ODE increased NEGR1 expression by 27-fold (p&amp;lt;0.0001) and NEGR1 protein by 2-fold (p&amp;lt;0.01). In summary, NEGR1 is a novel exercise-induced molecule that regulates neuronal refinement in scWAT. Our data also suggest that the synthesis of 13-oxo-ODE leads to PPARγ activation that in turn promotes NEGR1 expression in scWAT. NEGR1 has beneficial effects on scWAT that are associated with improved systemic metabolism, making NEGR1 a novel target for treatment of obesity and cardiometabolic diseases. Presentation: 6/1/2024

Isoliquiritigenin mitigates intervertebral disc degeneration induced by oxidative stress and mitochondrial impairment through a PPARγ-dependent pathway

ObjectivesOxidative stress, mitochondrial dysfunction, and apoptosis play significant roles in the degradation of extracellular matrix (ECM) in nucleus pulposus cells (NPCs), ultimately contributing to intervertebral disc degeneration (IVDD). This study investigates the potential of isoliquiritigenin (ISL), a natural extract known for its antioxidant, anti-inflammatory, and anti-atherosclerotic properties, to alleviate IVDD. MethodsThe viability of NPCs treated with ISL and tert-butyl hydroperoxide (TBHP) was assessed using the CCK-8 assay. Various techniques, including Western blot, qRT-PCR, immunofluorescence (IF), and immunohistochemistry, were employed to measure the expression of ECM components, oxidative stress markers, and apoptosis-related proteins. Mitochondrial function was evaluated through Western blot and IF analyses. Network pharmacology predicted ISL targets, and the expression levels of PPARγ were assessed using the aforementioned methods. The role of PPARγ in the therapeutic effects of ISL on IVDD was examined through siRNA knockdown. The therapeutic impact of ISL on puncture-induced IVDD in rats was evaluated using X-ray, MRI, and histological staining techniques. ResultsIn vitro, ISL reduced oxidative stress in NPCs, restored mitochondrial function, inhibited apoptosis, and improved the ECM phenotype. In vivo, ISL slowed the progression of IVDD in a rat model. Further analysis revealed that ISL enhances PPARγ activity and promotes its expression by direct binding, contributing to the delay of IVDD progression. ConclusionThis study demonstrates that ISL effectively treats puncture-induced IVDD in rats by inhibiting oxidative stress, restoring mitochondrial function, and reducing NPC apoptosis through a PPARγ-dependent mechanism. By balancing ECM synthesis and degradation, ISL presents a novel therapeutic approach for IVDD and identifies a promising target for treatment.

Chemical Investigation and Regulation of Adipogenic Differentiation of Cultivated Moringa oleifera.

Background/Objectives: Moringa oleifera is a matrix plant with the high potential to cure several diseases with its medicinal and ethnopharmacological value and nutraceutical properties. In this study, we investigated the chemical and biological properties of this plant cultivated in our local region. Methods: Leaves, roots, seeds, stem bark, and twigs of oleifera were extracted and evaluated bioactivities targeting intracellular lipid accumulation and adipocyte differentiation in 3T3-L1 preadipocytes, and UHPLC-ESI-Orbitrap-MS/MS-Based molecular networking guided isolation and dereplication of metabolites from these extracts. Results: Five extracts of different organs of M. oleifera significantly stimulated intracellular lipid accumulation and adipocyte differentiation in 3T3-L1 preadipocytes in a concentration-dependent manner. These extracts markedly increased the expression of genes related to adipogenesis and lipogenesis. Notably, these extracts promoted peroxisome proliferator-activated receptor γ (PPARγ) activity and the expression of its target genes, including phosphoenolpyruvate carboxykinase, fatty acid-binding protein 4, and perilipin-2. These adipogenic and lipogenic effects of Moringa extracts through the regulation of PPARγ activity suggests their potential efficacy in preventing or treating type 2 diabetes. Furthermore, chemical investigation revealed high contents of phytonutrients as rich sources of secondary metabolites including glycosides, flavones, fatty acids, phenolics, and other compounds. In addition, in silico studies on major components of these extracts revealed the bioavailability of major components through their binding affinity to respective proteins targeting adipocyte differentiation.

It takes two peroxisome proliferator-activated receptors (PPAR-β/δ and PPAR-γ) to tango idiopathic pulmonary fibrosis

BackgroundIdiopathic pulmonary fibrosis (IPF) is characterized by aberrant lung epithelial phenotypes, fibroblast activation, and increased extracellular matrix deposition. Transforming growth factor-beta (TGF-β)1-induced Smad signaling and downregulation of peroxisomal genes are involved in the pathogenesis and can be inhibited by peroxisome proliferator-activated receptor (PPAR)-α activation. However, the three PPARs, that is PPAR-α, PPAR-β/δ, and PPAR-γ, are known to interact in a complex crosstalk.MethodsTo mimic the pathogenesis of lung fibrosis, primary lung fibroblasts from control and IPF patients with comparable levels of all three PPARs were treated with TGF-β1 for 24 h, followed by the addition of PPAR ligands either alone or in combination for another 24 h. Fibrosis markers (intra- and extracellular collagen levels, expression and activity of matrix metalloproteinases) and peroxisomal biogenesis and metabolism (gene expression of peroxisomal biogenesis and matrix proteins, protein levels of PEX13 and catalase, targeted and untargeted lipidomic profiles) were analyzed after TGF-β1 treatment and the effects of the PPAR ligands were investigated.ResultsTGF-β1 induced the expected phenotype; e.g. it increased the intra- and extracellular collagen levels and decreased peroxisomal biogenesis and metabolism. Agonists of different PPARs reversed TGF-β1-induced fibrosis even when given 24 h after TGF-β1. The effects included the reversals of (1) the increase in collagen production by repressing COL1A2 promoter activity (through PPAR-β/δ activation); (2) the reduced activity of matrix metalloproteinases (through PPAR-β/δ activation); (3) the decrease in peroxisomal biogenesis and lipid metabolism (through PPAR-γ activation); and (4) the decrease in catalase protein levels in control (through PPAR-γ activation) and IPF (through a combined activation of PPAR-β/δ and PPAR-γ) fibroblasts. Further experiments to explore the role of catalase showed that an overexpression of catalase protein reduced collagen production. Additionally, the beneficial effect of PPAR-γ but not of PPAR-β/δ activation on collagen synthesis depended on catalase activity and was thus redox-sensitive.ConclusionOur data provide evidence that IPF patients may benefit from a combined activation of PPAR-β/δ and PPAR-γ.