Steam explosion (SE) is a potential method to modify pectin structure, which might be connected to its emulsifying characteristics and the bioavailability of encapsulated polymethoxyflavone like nobiletin. However, the relationship between SE-modified pectin and the bioavailability of encapsulated nobiletin is still unclear. In this study, nobiletin-loaded emulsion was fabricated using citrus pectin modified with SE (0.15-0.9 MPa, 3 min) as emulsifier for in vitro digestion study, and the transport and absorption of nobiletin in Caco-2 cells to investigate the bioavailability-promoting effect. The results showed that SE treatment lowered the droplet size of emulsion from 21.38 ± 2.30 μm to 2.14 ± 0.12 μm, enhanced the nobiletin encapsulation efficiency from 23.73 ± 0.78 % to 86.27 ± 3.81 %, improved the nobiletin bioaccessibility in vitro from 2.48 ± 0.10 % to 25.42 ± 0.10 % and increased the intracellular accumulation of nobiletin by over 10 times, even higher than that of Tween 80. In conclusion, pectin from SE-treated citrus peel exhibited good emulsion properties and bioavailability-promoting effect in vitro of nobiletin.

BACKGROUND: Macrophage dysregulation is a common pathogenic feature of viruses that provides extensive targets for antiviral therapy. Nobiletin, a polymethoxylated flavonoid found in citrus fruits, has a multitude of effects.
METHODS: We investigated the effect of nobiletin on polyinosinic-polycytidylic acid (poly(I:C))-induced inflammation in RAW264.7 cells. Nobiletin inhibited the production of poly(I:C)-induced inflammatory factors, including tumor necrosis factor (TNF)-α, interleukin (IL)-6, and CXCL10. High-throughput sequencing revealed that nobiletin inhibited the expression of TNF-α, IL-6, and CXCL10 and promoted the expression of CD206, Chil3, and Vcam1. In the Kyoto Encyclopedia of Genes and Genomes enrichment analyses, the upregulated differential genes were significantly enriched in the peroxisome proliferator-activated receptor (PPAR) signaling pathway.
RESULTS: The PPAR-γ inhibitor T0070907 significantly reversed the inhibitory effects of nobiletin on IL-6 and CXCL10 but had no significant effect on TNF-α secretion.
CONCLUSIONS: Thus, nobiletin regulated poly(I:C)-induced inflammatory responses in RAW264.7 cells partially via the PPAR-γ signaling pathway.

Nobiletin is an active compound extracted from citrus fruits. Research has indicated that nobiletin has a potential inhibitory effect on ovarian cancer (OV). However, the mechanism of action remains unclear. The OV A2780 cells were treated using nobiletin, cell viability was examined using a cell counting kit-8 experiment, and cell migration was examined with a wound healing experiment. Nobiletin targets were retrieved from target databases. Differentially expressed genes (DEG) and weighted gene co-expression network analysis (WGCNA) were conducted on GSE26712 (OV). The intersection of the critical genes for nobiletin\'s action on OV and gene enrichment and immune infiltration analyses were performed. The Cancer Genome Atlas-OV data and molecular docking helped validate the findings. After adding nobiletin, cell viability and migration significantly decreased (P < 0.01). A total of 88 nobiletin targets and 1288 DEG were identified. The intersection genes were enriched inflammatory response and response to hypoxia. The most related module obtained from WGCNA contained 414 genes (correlation coefficient = 0.77, P < 0.01). DPP4 and TXNIP were recognized as the hub genes. The abundance of macrophages M2 and mast cells activated significantly enhanced with increased DPP4 expression (P < 0.05). The binding energy between DPP4/TXNIP and nobiletin was - 7.012/ - 7.184 kcal/mol, forming 5/2 hydrogen bonds. Nobiletin effectively suppresses the viability and migration of OV A2780 cells. In this process, DPP4 and TXNIP are the key target, immune regulation, and oxidative stress playing significant roles.

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, β-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, β-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 has been reported to protect against obesity-related metabolic disorders by enhancing the circadian rhythm; however its effects on lipid metabolism in adipose tissue are unclear. In this study, mice were fed with high-fat diet (HFD) for four weeks firstly and gavaged with 50 or 200 mg/kg bodyweight/day nobiletin at Zeitgeber time (ZT) 4 for another four weeks while still receiving HFD. At the end of the 8-week experimental period, the mice were sacrificed at ZT4 or ZT8 on the same day. Mature 3T3-L1 adipocytes were treated with nobiletin in the presence or absence of siBmal1, siRora, siRorc, SR8278 or SR9009. Nobiletin reduced the weight of white adipose tissue (WAT) and the size of adipocytes in WAT. At ZT4, nobiletin decreased the TG, TC and LDL-c levels and increased serum FFA level and glucose tolerance. Nobiletin triggered the lipolysis of mesenteric and epididymal WAT at both ZT4 and ZT16. Nobiletin increased the level of RORγ at ZT16, that of BMAL1 and PPARγ at ZT4, and that of ATGL at both ZT4 and ZT16. Nobiletin increased lipolysis and ATGL levels in 3T3-L1 adipocytes in Bmal1- or Rora/c- dependent manner. Dual luciferase assay indicated that nobiletin enhanced the transcriptional activation of RORα/γ on Atgl promoter and decreased the repression of RORα/γ on PPARγ-binding PPRE. Promoter deletion analysis indicated that nobiletin inhibited the suppression of PPARγ-mediated Atgl transcription by RORα/γ. Taken together, nobiletin elevated lipolysis in WAT by increasing ATGL levels through activating the transcriptional activity of RORα/γ and decreasing the repression of RORα/γ on PPARγ-binding PPRE.

Possible protective and therapeutic effects of nobiletin on kidney in a cisplatin-induced nephrotoxicity rat model were investigated. Forty male albino rats were divided into four groups: control, cisplatin (CIS), cisplatin+nobiletin (CIS+NOB), and nobiletin+cisplatin (NOB+CIS). At the end of the study, the rats were subjected to biochemical, histological and immunohistochemical analyzes. Compared to the control group, tGSH (p < 0.05) levels, and G6PD (p < 0.05) and GPx (p < 0.001) activities, were increased in the CIS group; while significant (p < 0.05) decreases occurred in the MDA and TOC levels. Histopathologically, the kidneys of the groups administered nobiletin (CIS+NOB, NOB+CIS) were significantly different from the CIS group, being closer to control group in terms of degeneration and hyaline cylinder formation in the tubules (p < 0.05). While dilatation in the tubules, protein-rich fluid and hyaline cylinder formation in the lumen were most common in the CIS group, a significant decrease (p < 0.05) of these parameters was seen in the nobiletin groups (CIS+NOB, NOB+CIS). This study suggests that nobiletin can be effective in preventing and ameliorating toxic effects of cisplatin on the kidney.

Nobiletin (NOB), a polymethoxylated flavone isolated from citrus peels, is a promising dietary treatment for lung diseases, such as pulmonary fiborsis. In this work, the underlying mechanisms of NOB\'s preventative effect on pulmonary fibrosis were explored using bleomycin-exposed mice and IL-4-induced M2 polarization of the macrophages. Results showed that NOB treatment could significantly ameliorate lung fibrosis by suppressing pathological damages, collagen deposition, and fibroblat activation. Moreover, NOB obviously reduced the M2 macrophage-related proteins, including CD206, Arg1, and pro-fibrotic mediators such as TGF-β and CTGF, which might contribute to the antifibrosis effect of NOB. Network analysis of the differentially expressed genes in NOB-treated M2 macrophages showed that autophagy, mTOR signaling pathway, and AMPK signaling pathway might be involved in the effects of NOB. Further exploration illustrated that autophagy was enhanced in NOB-treated lung and M2 macrophages.The addition of 3MA, an autophagy inhibitor, could significantly weaken the effect of NOB on lung fibrosis and macrophage M2 polarization. Additionally, NOB also markedly decreased the expression of p-AMPK, p-mTOR, and p-P70S6K in the M2 macrophages and lung tissues of BLM-exposed mice. Compound C, an AMPK agonist, significantly suppressed NOB-induced activation of AMPK and mTOR signals, as well as its inhibitory effect on autophagy, M2 macrophages and lung fibrosis both in vitro and in vivo, supporting the requirement of AMPK-mTOR-mediated autophagy for the NOB\'s antifibrosis activity. Taken together, this study suggests that NOB ameliorates pulmonary fibrosis likely involving the inhibition of M2 macrophage via activating AMPK-mTOR-mediated autophagy.

This study aimed to investigate a synergistic anti-inflammatory effect of a citrus flavonoid nobiletin and docosahexaenoic acid (DHA), one of n-3 long-chain polyunsaturated fatty acids, in combination. Simultaneous treatment with nobiletin and DHA synergistically inhibited nitric oxide production (combination index < 0.9) by mouse macrophage-like RAW 264.7 cells stimulated with lipopolysaccharide (LPS) without cytotoxicity. On the other hand, the inhibitory effect of nobiletin and DHA in combination on proinflammatory cytokine production was not synergistic. Neither nobiletin nor DHA affected the phagocytotic activity of RAW 264.7 cells stimulated with LPS. Immunoblot analysis revealed that the inhibition potency of DHA on the phosphorylation of ERK and p38 and nuclear translocation of NF-κB is markedly enhanced by simultaneously treating with nobiletin, which may lead to the synergistic anti-inflammatory effect. Overall, our findings show the potential of the synergistic anti-inflammatory effect of nobiletin and DHA in combination.

The reproductive performance of lactating dairy cows is gradually declining, and one of the causes of this problem is the presence of long-term infertility repeat breeder cows (RBCs). The causes of RBCs are largely thought to be maternal factors, including the uterine environment. This study aimed to accurately investigate the uterine environment of RBCs using uterine tissue and fluid. Next, we investigated the effect of nobiletin in bovine endometrial epithelial cells to explore the possibility of improving the uterine environment of RBCs. Uterine fluid was collected by flushing the uterus and endometrial tissues were collected by biopsy on day 7 of the estrous cycle from both normal fertile cows and RBCs (n = 5 in each group). A comprehensive analysis of the uterus revealed that gene expression and altered pathways differed between normal fertile cows and RBCs. Especially, pathways of natural killer cell-mediated cytotoxicity, cell cycle, and calcium signaling pathway were picked up in the uterine tissues of RBCs. In the uterine fluid, the levels of lipopolysaccharide were higher in the RBC than in normal group (P = 0.08). In in vitro experiment, treatment with the uterine fluid from RBCs upregulated inflammation-related pathways and molecules such as interleukin-8 (IL-8) in bovine endometrial epithelial cells. The treatment with nobiletin suppressed IL-8 induced by the treatment with uterine fluid. In conclusion, the uterine environment of RBCs was found to be in inflammatory condition, causing the lower reproductive performance. It is necessary to develop methods to improve to the anti-inflammatory state in the uterine environment of RBCs.

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-β 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.