Nobiletin restores HFD-induced enteric nerve injury by regulating enteric glial activation and the GDNF/AKT/FOXO3a/P21 pathway.

BACKGROUND: To explore whether nobiletin has a protective effect on high-fat diet (HFD)-induced enteric nerve injury and its underlying mechanism. METHODS: An obesity model was induced by a HFD. Nobiletin (100 mg/kg and 200 mg/kg) and vehicle were administered by gastric gavage for 4 weeks. Lee's index, body weight, OGTT and intestinal propulsion assays were performed before sacrifice. After sampling, lipids were detected using Bodipy 493/503; lipid peroxidation was detected using MDA and SOD kits and the expression of PGP 9.5, Trem2, GFAP, ß-tubulin 3, Bax, Bcl2, Nestin, P75 NTR, SOX10 and EDU was detected using immunofluorescence. The GDNF, p-AKT, AKT, p-FOXO3a, FOXO3a and P21 proteins were detected using western blotting. The relative mRNA expression levels of NOS2 were detected via qPCR. Primary enteric neural stem cells (ENSCs) were cultured. After ENSCs were treated with palmitic acid (PA) and nobiletin, CCK-8 and caspase-3/7 activity assays were performed to evaluate proliferation and apoptosis. RESULTS: HFD consumption caused colon lipid accumulation and peroxidation, induced enteric nerve damage and caused intestinal motor dysfunction. However, nobiletin reduced lipid accumulation and peroxidation in the colon; promoted Trem2, ß-tubulin 3, Nestin, P75NTR, SOX10 and Bcl2 expression; inhibited Bax and GFAP expression; reduced NOS2 mRNA transcription; and regulated the GDNF/AKT/FOXO3a/P21 pathway. Nobiletin also promoted PA-induced impairment of ENSCs. CONCLUSIONS: Nobiletin restored HFD-induced enteric nerve injury, which may be associated with inhibiting enteric nerve apoptosis, promoting enteric nerve survival and regulating the GDNF/AKT/FOXO3a/P21 pathway.

Nobiletin enhances mitochondrial function by regulating SIRT1/PGC-1α signaling in porcine oocytes during in vitro maturation.

Nobiletin is a natural flavonoid found in citrus fruits with beneficial effects, including anti-inflammatory, anti-cancer and anti-oxidation effects. The aim of this study was to investigate whether nobiletin improves mitochondrial function in porcine oocytes and examine the underlying mechanism. Oocytes enclosed by cumulus cells were cultured in TCM-199 for 44 h with 0.1% dimethyl sulfoxide (control), or supplemented with 5, 10, 25, and 50 µM of nobiletin (Nob5, Nob10, Nob25, and Nob50, respectively). Oocyte maturation rate was significantly enhanced in Nob10 (70.26 ± 0.45%) compared to the other groups (control: 60.12 ± 0.47%; Nob5: 59.44 ± 1.63%; Nob25: 63.15 ± 1.38%; Nob50: 46.57 ± 1.19%). The addition of nobiletin reduced the levels of reactive oxygen species and increased glutathione levels. Moreover, Nob10 promoted mitochondrial biogenesis by upregulating the protein levels of sirtuin 1 (SIRT1) and peroxisome proliferator-activated receptor-gamma coactivator 1α (PGC-1α). This resulted in an increase in the number of active mitochondria, mitochondrial DNA copy number, mitochondrial membrane potential, and ATP production, thereby enhancing mitochondrial function. The protein level of p53 decreased, followed by the phosphorylation of B-cell lymphoma 2, suggesting a reduction in mitochondria-mediated apoptosis in the Nob10 group. Additionally, the release of cytochrome c from the mitochondria was significantly diminished along with a decrease in the protein expression of caspase 3. Thus, nobiletin has a great potential to promote the in vitro maturation of porcine oocytes by suppressing oxidative stress and promoting mitochondrial function through the upregulation of the SIRT1/PGC-1α signaling pathway.

Recent advances in flavonoid compounds for the treatment of prostate cancer.

Prostate cancer is a malignant epithelial tumor of the prostate gland and is the most common malignant tumor of the male genitourinary system. Pharmacological therapies, including chemotherapy and androgen deprivation therapy, play a key role in the treatment of prostate cancer. However, drug resistance and side effects limit the use of these drugs and so there is a need for new drug therapies for prostate cancer patients. Flavonoids, with their wide range of sources and diverse biological activities, have attracted much attention in the field of anti-tumor drug screening. In 2016, at least 58 flavonoids were reported to have anti-prostate cancer activity. In recent years, six additional flavonoid compounds have been found to have anti-prostate cancer potential. In this review, we have collected a large amount of evidence on the anti-prostate cancer effects of these six flavonoids, including a large number of cellular experiments and a small number of preclinical animal experiments. In addition, we predicted their drug-forming properties using Schrödinger's QikProp software and ADMETlab due to the lack of in vivo pharmacokinetic data for the six compounds. In conclusion, this review has fully confirmed the anti-prostate cancer effects of these six flavonoids, summarized their mechanisms of action and predicted their druggability. It provides a reference for the further development of these compounds into anti-prostate cancer drugs.

Nobiletin alleviates atherosclerosis by inhibiting lipid uptake via the PPARG/CD36 pathway.

BACKGROUND: Atherosclerosis (AS) is a persistent inflammatory condition triggered and exacerbated by several factors including lipid accumulation, endothelial dysfunction and macrophages infiltration. Nobiletin (NOB) has been reported to alleviate atherosclerosis; however, the underlying mechanism remains incompletely understood. METHODS: This study involved comprehensive bioinformatic analysis, including multidatabase target prediction; GO and KEGG enrichment analyses for function and pathway exploration; DeepSite and AutoDock for drug binding site prediction; and CIBERSORT for immune cell involvement. In addition, target intervention was verified via cell scratch assays, oil red O staining, ELISA, flow cytometry, qRT‒PCR and Western blotting. In addition, by establishing a mouse model of AS, it was demonstrated that NOB attenuated lipid accumulation and the extent of atherosclerotic lesions. RESULTS: (1) Altogether, 141 potentially targetable genes were identified through which NOB could intervene in atherosclerosis. (2) Lipid and atherosclerosis, fluid shear stress and atherosclerosis may be the dominant pathways and potential mechanisms. (3) ALB, AKT1, CASP3 and 7 other genes were identified as the top 10 target genes. (4) Six genes, including PPARG, MMP9, SRC and 3 other genes, were related to the M0 fraction. (5) CD36 and PPARG were upregulated in atherosclerosis samples compared to the normal control. (6) By inhibiting lipid uptake in RAW264.7 cells, NOB prevents the formation of foam cell. (7) In RAW264.7 cells, the inhibitory effect of oxidized low-density lipoprotein on foam cells formation and lipid accumulation was closely associated with the PPARG signaling pathway. (8) In vivo validation showed that NOB significantly attenuated intra-arterial lipid accumulation and macrophage infiltration and reduced CD36 expression. CONCLUSIONS: Nobiletin alleviates atherosclerosis by inhibiting lipid uptake via the PPARG/CD36 pathway.

Screening of active components in Astragalus mongholicus Bunge and Panax notoginseng formula for anti-fibrosis in CKD: nobiletin inhibits Lgals1/PI3K/AKT signaling to improve renal fibrosis.

The Astragalus mongholicus Bunge and Panax notoginseng formula (A&P) has been clinically shown to effectively slow down the progression of chronic kidney disease (CKD) and has demonstrated significant anti-fibrosis effects in experimental CKD model. However, the specific active ingredients and underlying mechanism are still unclear. The active ingredients of A&P were analyzed by Ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-HR-MS). A mouse model of CKD was constructed by 5/6 nephrectomy. Renal function was assessed by creatinine and urea nitrogen. Real-time PCR and Western Blot were performed to detect the mRNA and protein changes in kidney and cells. An in vitro fibrotic cell model was constructed by TGF-ß induction in TCMK-1 cells. The results showed that thirteen active ingredients of A&P were identified by UPLC-HR-MS, nine of which were identified by analysis with standards, among which the relative percentage of NOB was high. We found that NOB treatment significantly improved renal function, pathological damage and reduced the expression level of fibrotic factors in CKD mice. The results also demonstrated that Lgals1 was overexpressed in the interstitial kidney of CKD mice, and NOB treatment significantly reduced its expression level, while inhibiting PI3K and AKT phosphorylation. Interestingly, overexpression of Lgals1 significantly increased fibrosis in TCMK1 cells and upregulated the activity of PI3K and AKT, which were strongly inhibited by NOB treatment. NOB is one of the main active components of A&P. The molecular mechanism by which NOB ameliorates renal fibrosis in CKD may be through the inhibition of Lgals1/PI3K/AKT signaling pathway.

Involvement of Bile Acid Metabolism and Gut Microbiota in the Amelioration of Experimental Metabolism-Associated Fatty Liver Disease by Nobiletin.

Metabolism-associated fatty liver disease (MAFLD), a growing health problem worldwide, is one of the major risks for the development of cirrhosis and liver cancer. Oral administration of nobiletin (NOB), a natural citrus flavonoid, modulates the gut microbes and their metabolites in mice. In the present study, we established a mouse model of MAFLD by subjecting mice to a high-fat diet (HFD) for 12 weeks. Throughout this timeframe, NOB was administered to investigate its potential benefits on gut microbial balance and bile acid (BA) metabolism using various techniques, including 16S rRNA sequencing, targeted metabolomics of BA, and biological assays. NOB effectively slowed the progression of MAFLD by reducing serum lipid levels, blood glucose levels, LPS levels, and hepatic IL-1ß and TNF-α levels. Furthermore, NOB reinstated diversity within the gut microbial community, increasing the population of bacteria that produce bile salt hydrolase (BSH) to enhance BA excretion. By exploring further, we found NOB downregulated hepatic expression of the farnesoid X receptor (FXR) and its associated small heterodimer partner (SHP), and it increased the expression of downstream enzymes, including cholesterol 7α-hydroxylase (CYP7A1) and cytochrome P450 27A1 (CYP27A1). This acceleration in cholesterol conversion within the liver contributes to mitigating MAFLD. The present findings underscore the significant role of NOB in regulating gut microbial balance and BA metabolism, revealing that long-term intake of NOB plays beneficial roles in the prevention or intervention of MAFLD.

The naturally occurring flavonoid nobiletin reverses methotrexate resistance via inhibition of P-glycoprotein synthesis.

Methotrexate (MTX) is the first-line treatment for rheumatoid arthritis (RA). However, after long-term treatment, some patients develop resistance. P-glycoprotein (P-gp), as an indispensable drug transporter, is essential for mediating this MTX resistance. In addition, nobiletin (NOB), a naturally occurring polymethoxylated flavonoid, has also been shown to reverse P-gp-mediated MTX resistance in RA groups; however, the precise role of NOB in this process is still unclear. Here, we administered MTX and NOB alone or in combination to collagen II-induced arthritic (CIA) mice and evaluated disease severity using the arthritis index, synovial histopathological changes, immunohistochemistry, and P-gp expression. In addition, we used conventional RNA-seq to identify targets and possible pathways through which NOB reverses MTX-induced drug resistance. We found that NOB in combination with MTX could enhance its performance in synovial tissue and decrease P-gp expression in CIA mice compared to MTX treatment alone. In vitro, in MTX-resistant fibroblast-like synoviocytes from CIA cells (CIA-FLS/MTX), we show that NOB treatment downregulated the PI3K/AKT/HIF-1α pathway, thereby reducing the synthesis of the P-gp protein. In addition, NOB significantly inhibited glycolysis and metabolic activity of CIA-FLS/MTX cells, which could reduce the production of ATP and block P-gp, ultimately decreasing the efflux of MTX and maintaining its anti-RA effects. In conclusion, this study shows that NOB overcomes MTX resistance in CIA-FLS/MTX cells through the PI3K/AKT/HIF-1α pathway, simultaneously influencing metabolic processes and inhibiting P-gp-induced drug efflux.

The clock modulator Nobiletin mitigates astrogliosis-associated neuroinflammation and disease hallmarks in an Alzheimer's disease model.

Alzheimer's disease (AD) is a devastating neurodegenerative disorder, and there is a pressing need to identify disease-modifying factors and devise interventional strategies. The circadian clock, our intrinsic biological timer, orchestrates various cellular and physiological processes including gene expression, sleep, and neuroinflammation; conversely, circadian dysfunctions are closely associated with and/or contribute to AD hallmarks. We previously reported that the natural compound Nobiletin (NOB) is a clock-enhancing modulator that promotes physiological health and healthy aging. In the current study, we treated the double transgenic AD model mice, APP/PS1, with NOB-containing diets. NOB significantly alleviated ß-amyloid burden in both the hippocampus and the cortex, and exhibited a trend to improve cognitive function in these mice. While several systemic parameters for circadian wheel-running activity, sleep, and metabolism were unchanged, NOB treatment showed a marked effect on the expression of clock and clock-controlled AD gene expression in the cortex. In accordance, cortical proteomic profiling demonstrated circadian time-dependent restoration of the protein landscape in APP/PS1 mice treated with NOB. More importantly, we found a potent efficacy of NOB to inhibit proinflammatory cytokine gene expression and inflammasome formation in the cortex, and immunostaining further revealed a specific effect to diminish astrogliosis, but not microgliosis, by NOB in APP/PS1 mice. Together, these results underscore beneficial effects of a clock modulator to mitigate pathological and cognitive hallmarks of AD, and suggest a possible mechanism via suppressing astrogliosis-associated neuroinflammation.

Nobiletin as a chemopreventive natural product against cancer, a comprehensive review.

As a leading cause of death, second only to heart disease, cancer has always been one of the burning topics in medical research. When targeting multiple signal pathways in tumorigenesis chemoprevention, using natural or synthetic anti-cancer drugs is a vital strategy to reduce cancer damage. However, toxic effects, multidrug resistance (MDR) as well as cancer stem cells (CSCs) all prominently limited the clinical application of conventional anticancer drugs. With low side effects, strong biological activity, unique mechanism, and wide range of targets, natural products derived from plants are considered significant sources for new drug development. Nobiletin is one of the most attractive compounds, a unique flavonoid primarily isolated from the peel of citrus fruits. Numerous studies in vitro and in vivo have suggested that nobiletin and its derivatives possess the eminent potential to become effective cancer chemoprevention agents through various cellular and molecular levels. This article aims to comprehensively review the anticancer efficacy and specific mechanisms of nobiletin, enhancing our understanding of its chemoprevention properties and providing the latest research findings. At the end of this review, we also give some discussion and future perspectives regarding the challenges and opportunities in nobiletin efficient exploitation.

Nobiletin and xanthohumol counteract the TNFα-mediated activation of endothelial cells through the inhibition of the NF-κB signaling pathway.

Angiogenesis, a process characterized by the formation of new blood vessels from pre-existing ones, is a crucial step in tumor growth and dissemination. Given the ability of tumors to interfere with multiple or different molecular pathways to promote angiogenesis, there is an increasing need to therapeutically block tumor progression by targeting multiple antiangiogenic pathways. Natural polyphenols present health-protective properties, which are likely attributed to their ability to activate multiple pathways involved in inflammation, carcinogenesis, and angiogenesis. Recently, increased attention has been addressed to the ability of flavonoids, the most abundant polyphenols in the diet, to prevent cancer by suppressing angiogenesis. Here we investigate the mechanisms by which xanthohumol (the major prenylated flavonoid of the hop plant Humulus lupulus L.) and nobiletin (flavonoid from red-orange Citrus sinensis) can modulate the effects of Tumor Necrosis Factor-α (TNF-α) on human umbilical vein endothelial cells (HUVEC). The results reported in this paper show that xanthohumol and nobiletin pretreatment of HUVEC inhibits the effects induced by TNF-α on cell migration, invasion capability, and colon cancer cell adhesion on the endothelial monolayer. Moreover, the pretreatment reduces metalloproteinases and adhesion molecules' expression. Finally, our results highlight that xanthohumol and nobiletin can counteract the effects of TNF-α on angiogenesis and invasiveness, mainly through Vascular Endothelial Growth Factor and NF-κB pathways. Since angiogenesis plays an important pathological role in the progression of several diseases, our findings may provide clues for developing xanthohumol and nobiletin as therapeutic agents against angiogenesis-associated diseases.

Novel Pharmaceutical Cocrystals and Solvate Crystals of Nobiletin, a Citrus Flavonoid with Potent Pharmacological Activity.

Nobiletin (NOB) is a flavonoid with attractive pharmaceutical characteristics, including anti-Alzheimer's, anti-inflammation, and anti-cancer properties, but it has low solubility in water, resulting in reduced bioavailability. Its solubility must be improved to develop NOB as a drug. Cocrystal engineering can change the physicochemical properties of an active pharmaceutical ingredient and generate remarkable drug candidates that are superior in drug formulation. In this report, extensive co-crystal screening of NOBs with 31 cocrystal formers (coformers) with various functional groups was carried out by the liquid-assisted grinding method. As a result, four cocrystals (NOB with urea (URE), oxalic acid, gallic acid and salicylic acid) and one solvate crystal (NOB with formic acid (FOR)) were found. Powder X-ray diffraction and thermal analysis revealed the unique crystal morphology of all the obtained samples. In addition, the crystal structures of two of them (NOB-URE and NOB-FOR) were determined by single crystal X-ray diffraction. The results revealed that NOB-URE and NOB-FOR are new cocrystals or solvate crystals consisting of molar ratios of 1 : 2 and 1 : 0.73, respectively. In NOB-URE, we could observe a transient increase in solubility due to supersaturation, suggesting that URE is one of the better coformers of NOB.

Nobiletin with AIEE Characteristics for Targeting Mitochondria and Real-Time Dynamic Tracking in Zebrafish.

Nobiletin is a natural product with multiple physiological activities and is the main ingredient of Pericarpium Citri Reticulatae. We successfully discovered that nobiletin exhibits aggregation induced emission enhancement (AIEE) properties and it has significant advantages such as a large Stokes shift, good stability and excellent biocompatibility. The increase in methoxy groups endows nobiletin a greater fat-solubility, bioavailability and transport rate than the corresponding unmethoxylated flavones. Ulteriorly, cells and zebrafish were used to explore the application of nobiletin in biological imaging. It emits fluorescence in cells and is specifically targeted at mitochondria. Moreover, it has a noteworthy affinity for the digestive system and liver of zebrafish. Due to the unique AIEE phenomenon and stable optical properties of nobiletin, it paves the way for discovering, modifying and synthesizing more molecules with AIEE characteristics. Furthermore, it has a great prospect with regard to imaging cells and cellular substructures, such as mitochondria, which play crucial roles in cell metabolism and death. Indeed, three-dimensional real-time imaging in zebrafish provides a dynamic and visual tool for studying the absorption, distribution, metabolism and excretion of drugs. In this article, more directions and inspiration can be presented for the exploration of non-invasive pharmacokinetic research and intuitive drug pathways or mechanisms.

The citrus flavonoid "Nobiletin" impedes STZ-induced Alzheimer's disease in a mouse model through regulating autophagy mastered by SIRT1/FoxO3a mechanism.

The prominence of autophagy in the modulation of neurodegenerative disorders has sparked interest to investigate its stimulation in Alzheimer's disease (AD). Nobiletin possesses several bioactivities such as anti-inflammation, antioxidation, and neuroprotection. Consequently, the study's aim was to inspect the possible neurotherapeutic impact of Nobiletin in damping AD through autophagy regulation. Mice were randomly assigned into: Group I which received DMSO, Groups II, III, and IV obtained STZ (3 mg/kg) intracerebroventricularly once with Nobiletin (50 mg/kg/day; i.p.) in Group III and Nobiletin with EX-527 (2 mg/kg, i.p.) in Group IV. Interestingly, Nobiletin ameliorated STZ-induced AD through enhancing the motor performance and repressing memory defects. Moreover, Nobiletin de-escalated hippocampal acetylcholinesterase (AChE) activity and enhanced acetylcholine level while halting BACE1 and amyloid-ß levels. Meanwhile, Nobiletin stimulated the autophagy process through activating the SIRT1/FoxO3a, LC3B-II, and ATG7 pathway. Additionally, Nobiletin inhibited Akt pathway and controlled the level of NF-κB and TNF-α. Nobiletin amended the oxidative stress through enhancing GSH and cutting down MDA levels. However, EX527, SIRT1 inhibitor, counteracted the neurotherapeutic effects of Nobiletin. Therefore, the present study provides a strong verification for the therapeutic influence of Nobiletin in AD. This outcome may be assigned to autophagy stimulation through SIRT1/FoxO3a, inhibiting AChE activity, reducing neuroinflammation and oxidative stress.

Suppressing NLRP3 activation and PI3K/AKT/mTOR signaling ameliorates amiodarone-induced pulmonary fibrosis in rats: a possible protective role of nobiletin.

Amiodarone (AMD), a medicine used to treat life-threatening arrhythmias, is frequently linked to pulmonary fibrosis (PF). Despite the involvement of NLRP3 inflammasome and PI3K/Akt/mTOR axis in fibrosis modulation and development, their significance in the etiology of AMD-induced PF remains uncertain. Nobiletin (NOB), a citrus flavonoid, has recently gained attention for its ability to reduce fibrotic processes in a variety of organs through inhibiting NLRP3-associated inflammation and suppressing PI3K/AKT/mTOR fibrotic pathway. Therefore, this research aimed to investigate the possible beneficial impact of NOB against AMD-induced PF, taking into account the roles of NLRP3 and PI3K/AKT/mTOR axis in its pathogenesis. Twenty-four rats were randomly specified into Vehicle; NOB 20 mg/kg; AMD 30 mg/kg, and NOB + AMD. All treatments were administered orally once a day for 4 weeks. The lung oxidant/antioxidant status, as well as the expression of inflammatory and fibrotic markers were all assessed. The results revealed that NOB, by improving Nrf2/HO-1 pathway, could reduce ROS production and NLRP3 activation, which in turn hindered IL-1ß release, prohibited TGF-ß1-related PI3K/AKT/mTOR cascade, suppressed α-SMA expression, and impeded collagen deposition. These findings point to a novel strategy by which NOB may alleviate the AMD-prompted NLRP3 inflammatory responses and associated PF through blocking PI3K/AKT/mTOR signaling.

Stabilization of high internal-phase Pickering emulsion using nobiletin crystals: assembly of supramolecular metal-polyphenolic coordination complexes for surface coating.

BACKGROUND: High internal-phase Pickering emulsions have attracted interest due to their unique properties and they have the potential for broad application in the food field, for example as fat replacers, for packaging, for delivery of nutrients or probiotics, and in the 3D printing of food. However, efficient and edible high internal-phase Pickering stabilizers are still a persistent challenge for food scientists. RESULTS: Nobiletin (NOB) was selected as a model substance. The particles' physicochemical properties (droplet size, rheological behavior, and transmission profile) showed that supramolecular metal-polyphenolic coordination networks could suppress the ripening and growth of crystals on the oil-water interface. When the ratio of tannic acid (TA) to Fe3+ was 3:1, the growth of NOB crystals could be inhibited effectively. Due to the reduction of energy steric hindrance in the adsorption process, the resulting NOB-TA3 -Fe3+ 1 (NT3 Fe1 ) nanoparticles had the greatest potential to extend emulsion storage time. CONCLUSION: The NOB-TA3 -Fe3+ 1 (NT3 Fe1 ) nanoparticles were able to stabilize a high internal-phase emulsion, of which the oil phase was 80%, for at least 30 days, eventually leading to high system viscosity. The findings in this work provide a novel selection of healthy emulsifiers and an effective emulsion delivery system for hydrophobic and crystalline nutrients. © 2023 Society of Chemical Industry.

The biological clock enhancer nobiletin ameliorates steatosis in genetically obese mice by restoring aberrant hepatic circadian rhythm.

Nonalcoholic fatty liver disease (NAFLD) is associated with disruption of homeostatic lipid metabolism, but underlying processes are poorly understood. One possible mechanism is impairment in hepatic circadian rhythm, which regulates key lipogenic mediators in the liver and whose circadian oscillation is diminished in obesity. Nobiletin enhances biological rhythms by activating RAR-related orphan receptor nuclear receptor, protecting against metabolic syndrome in a clock-dependent manner. The effect of nobiletin in NAFLD is unclear. In this study, we investigate the clock-enhancing effects of nobiletin in genetically obese (db/db) PER2::LUCIFERASE reporter mice with fatty liver. We report microarray expression data suggesting hepatic circadian signaling is impaired in db/db mice with profound hepatic steatosis. Circadian PER2 activity, as assessed by mRNA and luciferase assay, was significantly diminished in liver of db/db PER2::LUCIFERASE reporter mice. Continuous animal monitoring systems and constant dark studies suggest the primary circadian defect in db/db mice lies within peripheral hepatic oscillators and not behavioral rhythms or the master clock. In vitro, nobiletin restored PER2 amplitude in lipid-laden PER2::LUCIFERASE reporter macrophages. In vivo, nobiletin dramatically upregulated core clock gene expression, hepatic PER2 activity, and ameliorated steatosis in db/db PER2::LUCIFERASE reporter mice. Mechanistically, nobiletin reduced serum insulin levels, decreased hepatic Srebp1c, Acaca1, Tnfα, and Fgf21 expression, but did not improve Plin2, Plin5, or Cpt1, suggesting nobiletin attenuates steatosis in db/db mice via downregulation of hepatic lipid accumulation. These data suggest restoring endogenous rhythm with nobiletin resolves steatosis in obesity, proposing that hypothesis that targeting the biological clock may be an attractive therapeutic strategy for NAFLD.NEW & NOTEWORTHY NAFLD is the most common chronic liver disease, but underlying mechanisms are unclear. We show here that genetically obese (db/db) mice with fatty liver have impaired hepatic circadian rhythm. Hepatic Per2 expression and PER2 reporter activity are diminished in db/db PER2::LUCIFERASE mice. The biological clock-enhancer nobiletin restores hepatic PER2 in db/db PER2::LUCIFERASE mice, resolving steatosis via downregulation of Srebp1c. These studies suggest targeting the circadian clock may be beneficial strategy in NAFLD.

Nobiletin protects enteric nerves and ameliorates disordered bowel motility in diet-induced obese mice via increasing Trem2 expression.

Nobiletin can regulate lipid metabolism and protect the central nervous system. However, its role in the enteric nervous system (ENS) of obese subjects is still unclear. To investigate the ENS protective effects and mechanism of nobiletin in obese mice, male C57BL/6 mice were fed a chow diet and a high-fat diet (HFD) for 8 weeks. The identified obese and control mice were grouped and administered vehicle, nobiletin 40 mg/kg, 100 mg/kg or 200 mg/kg daily for 4 weeks. The major indexes of obesity, intestinal transit rate, PGP9.5, nNOS, TNF-α, IL-1ß, IL-6, IL-10, Bcl2 and Bax were measured. The full-length transcriptome was used to analyze differentially expressed genes (DEGs) in the colon. The results indicated that nobiletin effectively improved major indexes of obesity and bowel motility function, suppressed the expression of TNF-α, IL-1ß, IL-6 and Bax, and upregulated the expression of IL-10, Bcl2, PGP9.5 and nNOS. Based on full-length transcriptome sequencing, nobiletin regulated lipid metabolism and inflammation via the PPAR and NOD-like receptor signaling pathways. Trem2 expression was significantly reduced in obese mice. However, Trem2 expression was significantly increased after nobiletin treatment in obese mice. The enrichment analysis showed that Trem2 plays an important role in enteric neuroinflammation. In conclusion, nobiletin regulates lipid metabolism and inflammation in obese mice. Trem2 is a potential target of nobiletin for ENS protection in obese mice.

Nobiletin activates thermogenesis of brown and white adipose tissue in high-fat diet-fed C57BL/6 mice by shaping the gut microbiota.

Increasing energy expenditure by activating thermogenesis in brown and beige adipocytes is a critical approach to protect against obesity. Here, we investigated the action and mechanism of a natural polymethoxyflavone on adaptive thermogenesis in high-fat diet-induced obesity mouse model. Nobiletin treatment significantly ameliorated obesity, alleviated the whitening of brown adipose tissue, and promoted browning of white adipose tissue in mice fed a high-fat diet. Gut microbiota analysis and metabolomic profiling revealed that nobiletin treatment resulted in a composition shift in the gut microbiota thereby altering fermentation products acetate levels in the host feces and serum. Further, transplantation of the microbiota from nobiletin-treated mice to microbiota-depleted mice activated brown adipose tissue activity, promoted beige adipocytes formation, and improved high-fat diet-induced obesity. Our results indicate that nobiletin could be used as a dietary therapy to prevent HFD-induced obesity, and provide a potential target-specific gut microbial species-driven mechanism for activating thermogenesis in brown and beige adipocytes.

Nobiletin protects against diabetes-induced testicular injury via hypophysis-gonadal axis upregulation and amelioration of oxidative stress.

BACKGROUND: Testicular injury is one of the most serious problems associated with diabetes mellitus. The present study aimed to compare the effects of two different doses of nobiletin and analyze its mechanisms of action against diabetes-induced testicular impairment in rats. METHODS AND RESULTS: Streptozotocin injection was used to induce diabetes. Diabetic rats received nobiletin orally at 10 or 25 mg/kg daily for 30 days. Diabetic rats displayed significant elevations in glucose, glycosylated hemoglobin (HbA1c), Homeostatic Model of Insulin Resistance (HOMA-IR), and pro-inflammatory cytokines, while the serum levels of insulin, testosterone, luteinizing hormone (LH), and follicle-stimulating hormone (FSH) were significantly reduced. Histological changes to positivity for caspase-3 and decreased androgen receptors (AR) immunoexpression were observed in diabetic rats. Both doses of nobiletin improved hyperglycemia, reduced pro-inflammatory cytokines, and augmented insulin, testosterone, LH, and FSH levels. LH and FSH receptors and cytochrome P450 17 α-hydroxylase (CYP17A1) were markedly downregulated in terms of both gene and protein expression in testicular tissues of the diabetic group, effects that were markedly ameliorated with both doses of nobiletin. In addition, both doses significantly reduced lipid peroxidation and caspase-3 immunoexpression and improved the activity of the antioxidant enzymes and AR in testicular tissues of the diabetic group. CONCLUSION: Both nobiletin doses showed protective effects against diabetes-induced testicular injury by reducing oxidative stress, hyperglycemia, inflammation, and caspase-3 and upregulating the hypophysis-gonadal axis and AR. The high dose of nobiletin was more effective than the lower one.

Novel treatment for refractory rheumatoid arthritis with total glucosides of paeony and nobiletin codelivered in a self-nanoemulsifying drug delivery system.

Patients with refractory rheumatoid arthritis (RA) remain a substantial clinical problem, while the overexpression of P-glycoprotein (P-gp) on their lymphocytes may contribute to resistance to anti-rheumatic drugs. This study aims to develop a novel treatment for refractory RA consisting of the combination of total glucosides of paeony (TGPs) and the P-gp inhibitor nobiletin (N), which are codelivered in a self-nanoemulsifying drug delivery system (SNEDDS). Based on the solubility, compatibility, and pseudoternary phase diagram tests, a nano-SNEDDS formulation composed of capryol 90-cremophor EL35-tcranscutol HP (CET) to codeliver TGP and N was developed, and this formulation increased the bioavailability of TGP by 435.04% (indicated with paeoniflorin). A modified adjuvant-induced arthritis (AIA) rat model was verified for the overexpression of P-gp in lymphocytes and resistance to methotrexate (MTX) treatment at the reported anti-inflammatory dosage. CET formulation not only increased the solubility and permeability of TGP but also inhibited the function and expression of P-gp, leading to enhanced bioavailability and intracellular concentration in the lymphocytes of AIA rats and consequently boosting the anti-arthritic effects of TGP. Moreover, TGP and N coloaded CET reduced the expression of P-gp in AIA rats partly by inhibiting the phosphorylated AKT and HIF-1α pathways. In summary, TGP-N coloaded SNEDDS is a novel and effective treatment for refractory RA.