Morin, the PPARγ agonist, inhibits Th17 differentiation by limiting fatty acid synthesis in collagen-induced arthritis.

T helper (Th) 17 cells highly contribute to the immunopathology of rheumatoid arthritis. Morin, a natural flavonoid, owns well anti-arthritic action but unclear effect on Th17 differentiation. This study tried to solve this issue and explore the mechanisms in view of cellular metabolism. Naïve CD4+ T cells were treated with anti-CD3/CD28 along with Th17-inducing cytokines. Morin was shown to block Th17 differentiation without affecting cell viability even when Foxp3 was dampened. The mechanisms were ascribed to the limited fatty acid synthesis by restricting FASN transcription, as indicated by metabolomics analysis, nile red staining, detection of triglycerides, FASN overexpression, and addition of palmitic acid. Moreover, morin had slight effect on cell apoptosis and protein palmitoylation during Th17 differentiation, but blocked the binding of RORγt to promoter and CNS2 region of Il17a gene. Oleic acid rescued the inhibition of morin on RORγt function, and Th17-inducing cytokines could not induce RORγt function in SCD1-defficient cells, suggesting that oleic acid but not palmitic acid was the direct effector in the action of morin. Then, PPARγ was identified as the target of morin, and GW9662 or PPARγ CRISPR/Cas9 KO plasmid weakened its above-mentioned effects. The transrepression of FASN by morin was owing to physical interaction between PPARγ and Sp1, and the importance of Sp1 in Th17 differentiation was confirmed by siSp1. Finally, the effects and mechanisms for morin-dampened Th17 responses were confirmed in collagen-induced arthritis (CIA) mice. Collectively, morin inhibited Th17 differentiation and alleviated CIA by limiting fatty acid synthesis subsequent to PPARγ activation.

Fenpropathrin induces neurotoxic effects in common carp (Cyprinus carpio L.).

Fenpropathrin (FEN) is a synthetic pyrethroid that has been frequently detected in aquatic environments, yet the neurotoxic impacts and underlying mechanisms on nontarget organisms are lacking. In this experiment, common carp were exposed to 0.45 and 1.35 µg/L FEN for 14 d and exhibited abnormal locomotor behaviour. Biochemical and molecular analysis results indicated that FEN altered the contents of tight junction proteins (claudin-1, occludin, and ZO-1), disturbed Na+-K+-ATPase and AChE activities, caused abnormal expression of neurotransmitters (ACh, DA, GABA, 5-HT, and glutamate) and caused histological damage in the brain, suggesting that FEN may damage the blood-brain barrier and induce neurotoxicity in carp. Furthermore, FEN also promoted an increase in ROS, changed SOD and CAT activities, and generally upregulated the contents of MDA, 8-OHdG, and protein carbonyl in the brain, indicating that FEN can induce oxidative stress and cause damage to lipids, DNA, and proteins. Moreover, inflammation-related indicators (TNF-α, IL-1ß, IL-6, and IL-10), mitophagy-related genes (PINK1, parkin, ulk1, beclin1, LC3, p62, tfeb, and atg5), and apoptosis-related parameters (p53, bax, bcl-2, caspase-3, caspase-8, and caspase-9) were also significantly changed, suggesting that inflammation, mitophagy, and apoptosis may participate in FEN-induced neurotoxicity in carp. This study refines the understanding of the toxicity mechanism of FEN and thus provides data support for the risk assessment of FEN.

The activation of PPARγ enhances Treg responses through up-regulating CD36/CPT1-mediated fatty acid oxidation and subsequent N-glycan branching of TßRII/IL-2Rα.

BACKGROUND: Peroxisome proliferator-activated receptor gamma (PPARγ) is an enhancer of Treg responses, but the mechanisms remain elusive. This study aimed to solve this problem in view of cellular metabolism. METHODS: Three recognized PPARγ agonists (synthetic agonist: rosiglitazone; endogenous ligand: 15d-PGJ2; natural product: morin) were used as the tools to activate PPARγ. The fatty acid oxidation (FAO) was evaluated through the detection of fatty acid uptake, oxygen consumption rate, mitochondrial mass, mitochondrial membrane potential and acetyl-CoA level. The involvement of UDP-GlcNAc/N-linked glycosylation axis and the exact role of PPARγ in the action of PPARγ agonists were determined by flow cytometry, Q-PCR, western blotting, a commercial kit for enzyme activity and CRISPR/Cas9-mediated knockout. RESULTS: Rosiglitazone, 15d-PGJ2 and morin all increased the frequency of CD4+Foxp3+ Treg cells generated from naïve CD4+ T cells, boosted the transcription of Foxp3, IL-10, CTLA4 and TIGIT, and facilitated the function of Treg cells. They significantly promoted FAO in differentiating Treg cells by up-regulating the levels of CD36 and CPT1 but not other enzymes involved in FAO such as ACADL, ACADM, HADHA or HADHB, and siCD36 or siCPT1 dampened PPARγ agonists-promoted Treg responses. Moreover, PPARγ agonists enhanced UDP-GlcNAc biosynthesis and subsequent N-linked glycosylation, but did not affect the expressions of N-glycan branching enzymes Mgat1, 2, 4 and 5. Notably, the enzyme activity of phosphofructokinase (PFK) was inhibited by PPARγ agonists and the effect was limited by siCD36 or siCPT1, implying PFK to be a link between PPARγ agonists-promoted FAO and UDP-GlcNAc biosynthesis aside from acetyl-CoA. Furthermore, PPARγ agonists facilitated the cell surface abundance of TßRII and IL-2Rα via N-linked glycosylation, thereby activating TGF-ß/Smads and IL-2/STAT5 signaling, and the connection between N-linked glycosylation and Treg responses was revealed by tunicamycin. However, the increased surface abundance of CD36 was demonstrated to be mainly owing to PPARγ agonists-up-regulated overall expression. Finally, PPARγ antagonist GW9662 or CRISPR/Cas9-mediated knockout of PPARγ constrained the effects of rosiglitazone, 15d-PGJ2 and morin, confirming the exact role of PPARγ. CONCLUSIONS: The activation of PPARγ enhances Treg responses through up-regulating CD36/CPT1-mediated fatty acid oxidation and subsequent N-glycan branching of TßRII/IL-2Rα, which is beneficial for inflammatory and autoimmune diseases. Video Abstract.

Bergenin, a PPARγ agonist, inhibits Th17 differentiation and subsequent neutrophilic asthma by preventing GLS1-dependent glutaminolysis.

Bergenin is a natural PPARγ agonist that can prevent neutrophil aggregation, and often be used in clinics for treating respiratory diseases. Recent data show that Th17 cells are important for neutrophil aggregation and asthma through secreting IL-17A. In this study, we investigated the effects of bergenin on Th17 differentiation in vitro and subsequent neutrophilic asthma in mice. Naïve T cells isolated from mouse mesenteric lymph nodes were treated with IL-23, TGF-ß, and IL-6 to induce Th17 differentiation. We showed that in naïve T cells under Th17-polarizing condition, the addition of bergenin (3, 10, 30 µM) concentration-dependently decreased the percentage of CD4+ IL-17A+ T cells and mRNA expression of specific transcription factor RORγt, and function-related factors IL-17A/F, IL-21, and IL-22, but did not affect the cell vitality and apoptosis. Furthermore, bergenin treatment prevented GLS1-dependent glutaminolysis in the progress of Th17 differentiation, slightly affected the levels of SLC1A5, SLC38A1, GLUD1, GOT1, and GPT2. Glutamine deprivation, the addition of glutamate (1 mM), α-ketoglutarate (1 mM), or GLS1 plasmid all significantly attenuated the above-mentioned actions of bergenin. Besides, we demonstrated that bergenin (3, 10, and 30 µM) concentration-dependently activated PPARγ in naïve T cells, whereas PPARγ antagonist GW9662 and siPPARγ abolished bergenin-caused inhibition on glutaminolysis and Th17 differentiation. Furthermore, we revealed that bergenin inhibited glutaminolysis by regulating the level of CDK1, phosphorylation and degradation of Cdh1, and APC/C-Cdh1-mediated ubiquitin-proteasomal degradation of GLS1 after activating PPARγ. We demonstrated a correlation existing among bergenin-affected GLS1-dependent glutaminolysis, PPARγ, "CDK1-APC/C-Cdh1" signaling, and Th17 differentiation. Finally, the therapeutic effect and mechanisms for bergenin-inhibited Th17 responses and neutrophilic asthma were confirmed in a mouse model of neutrophilic asthma by administration of GW9662 or GLS1 overexpression plasmid in vivo. In conclusion, bergenin repressed Th17 differentiation and then alleviated neutrophilic asthma in mice by inhibiting GLS1-dependent glutaminolysis via regulating the "CDK1-APC/C-Cdh1" signaling after activating PPARγ.

Oral administration of curcumin ameliorates pulmonary fibrosis in mice through 15d-PGJ2-mediated induction of hepatocyte growth factor in the colon.

Oral administration of curcumin has been shown to inhibit pulmonary fibrosis (PF) despite its extremely low bioavailability. In this study, we investigated the mechanisms underlying the anti-PF effect of curcumin in focus on intestinal endocrine. In bleomycin- and SiO2-treated mice, curcumin (75, 150 mg· kg-1 per day) exerted dose-dependent anti-PF effect when administered orally or rectally but not intravenously, implying an intestinal route was involved in the action of curcumin. We speculated that curcumin might promote the generation of gut-derived factors and the latter acted as a mediator subsequently entering the lungs to ameliorate fibrosis. We showed that oral administration of curcumin indeed significantly increased the expression of gut-derived hepatocyte growth factor (HGF) in colon tissues. Furthermore, in bleomycin-treated mice, the upregulated protein level of HGF in lungs by oral curcumin was highly correlated with its anti-PF effect, which was further confirmed by coadministration of c-Met inhibitor SU11274. Curcumin (5-40 µM) dose-dependently increased HGF expression in primary mouse fibroblasts, macrophages, CCD-18Co cells (fibroblast cell line), and RAW264.7 cells (monocyte-macrophage cell line), but not in primary colonic epithelial cells. In CCD-18Co cells and RAW264.7 cells, curcumin dose-dependently activated PPARγ and CREB, whereas PPARγ antagonist GW9662 (1 µM) or cAMP response element (CREB) inhibitor KG-501 (10 µM) significantly decreased the boosting effect of curcumin on HGF expression. Finally, we revealed that curcumin dose-dependently increased the production of 15-deoxy-Δ12, 14-prostaglandin J2 (15d-PGJ2) in CCD-18Co cells and RAW264.7 cells, which was a common upstream of the two transcription factors. Moreover, both the in vitro and in vivo effects of curcumin were diminished by coadministration of HPGDS-inhibitor-1, an inhibitor of 15d-PGJ2 generation. Together, curcumin promotes the expression of HGF in colonic fibroblasts and macrophages by activating PPARγ and CREB via an induction of 15d-PGJ2, and the HGF enters the lungs giving rise to an anti-PF effect.

Alpinetin improves intestinal barrier homeostasis via regulating AhR/suv39h1/TSC2/mTORC1/autophagy pathway.

The injury of intestinal epithelial barrier is considered as the key pathophysiological process in response to gastrointestinal infection and inflammation, and plays an important role in the initiation and development of colitis. Alpinetin has been shown to improve intestinal barrier homeostasis under colitis condition, but the mechanism is still unclear. Here, we showed that alpinetin significantly improved transepithelial electrical resistance (TEER) in TNF-α-stimulated Caco-2 cells, which was mainly mediated by inhibiting the apoptosis. Mechanistic studies demonstrated that alpinetin markedly increased the production of autophagosomes, along with obvious regulation of LC3B-II, beclin-1, p62, Atg7 and Atg5 expressions. In addition, it also markedly repressed the activation of mTORC1 signaling pathway, which was ascribed to TSC2 rather than p-AKT, p-ERK, p-AMPKα or PTEN expressions in Caco-2 and NCM460 cells. Furthermore, the enrichment of H3K9me3 at TSC2 promoter region was decreased and ubiquitin proteasome degradation of suv39h1 was increased. Additionally, alpinetin activated aryl hydrocarbon receptor (AhR) and promoted co-localization of AhR with suv39h1 in the cytoplasm. The relationship between alpinetin-regulated AhR/suv39h1/TSC2/mTORC1 signals, autophagy and apoptosis of Caco-2 and NCM460 cells was confirmed by using CH223191, siAhR, siTSC2 and chloroquine. Finally, CH223191 and leucine abolished alpinetin-mediated inhibition of intestinal epithelial cells apoptosis, improvement of intestinal epithelial barrier and amelioration of colitis.

Tetrandrine attenuates the bone erosion in collagen-induced arthritis rats by inhibiting osteoclastogenesis via spleen tyrosine kinase.

Tetrandrine, a bisbenzylisoquinoline alkaloid, was previously demonstrated to attenuate inflammation and cartilage destruction in the ankles of mice with collagen-induced arthritis (CIA). Here, we explored the underlying mechanism by which tetrandrine prevented arthritis-induced bone erosion by focusing on the differentiation and function of osteoclasts. We found that daily administration of tetrandrine (30 mg/kg) markedly reduced the bone damage and decreased the number of osteoclasts in CIA rats. In vitro, tetrandrine inhibited receptor activator of NF-κB ligand (RANKL)-induced osteoclastogenesis at the early stage and reduced the expressions of osteoclast-related marker genes. In bone marrow-derived macrophages and RAW264.7 cells, tetrandrine inhibited RANKL-induced translocation of NF-κB-p65 and nuclear factor of activated T cell 1 (NFATc1) through suppressing spleen tyrosine kinase (Syk)-Bruton's tyrosine kinase-PLCγ2-Ca2+ signaling. Of interest, tetrandrine did not affect the phosphorylation of immunoreceptor tyrosine-based activation motifs, the conventional upstream of Syk, but it inhibited the activity of Syk by enhancing its ubiquitination and degradation. The anti-osteoclastogenesis effect of tetrandrine nearly disappeared when it was used in combination with the Syk inhibitor piceatannol or in constitutively activated Syk-overexpressing cells. Taken together, tetrandrine attenuated CIA-induced bone destruction by inhibiting osteoclastogenesis through hindering the translocation of NF-κB-p65 and NFATc1 via reducing the activation of Syk.-Jia, Y., Miao, Y., Yue, M., Shu, M., Wei, Z., Dai, Y. Tetrandrine attenuates the bone erosion in collagen-induced arthritis rats by inhibiting osteoclastogenesis via spleen tyrosine kinase.

Nicotinamide adenine dinucleotide metabolism: driving or counterbalancing inflammatory bowel disease?

Nicotinamide adenine dinucleotide (NAD) is an important electron and hydrogen carrier for oxidative metabolism and involved in energy production, antioxidant responses, and signal transduction within cells. NAD-consuming enzymes can mediate post-translational modifications, such as deacylation and ADP-ribosylation, and the production of Ca2+ -mobilizing second messengers, including OAADPR, ADPR, cADPR, and NAADP, to regulate metabolic homeostasis, DNA damage and gene expression. Inflammatory bowel disease (IBD) is a condition characterized by impaired intestinal barrier, disturbed intestinal mucosal immunity, and abnormal intestinal repair, that is caused by genetic susceptibility and environmental factors. Although various components involved in NAD biosynthesis and metabolism are upregulated in IBD, it remains disputed whether increased NAD turnover drives or counterbalances IBD progression. This review discusses the significance of increased NAD turnover in the intestinal barrier integrity and the inflammation resolution in IBD conditions. We propose that a better understanding of the reasons for the altered NAD metabolism in IBD may help identify novel treatment approaches.

3, 3'-diindolylmethane, a natural aryl hydrocarbon receptor agonist, alleviates ulcerative colitis by enhancing "glycolysis-lactate-STAT3″ and TIP60 signals-mediated Treg differentiation.

BACKGROUND: Aryl hydrocarbon receptor (AhR) plays an important role in the occurrence and development of ulcerative colitis (UC). In this study, the effect and mechanism of 3, 3'-diindolylmethane (DIM), the classical AhR agonist, on UC was investigated from the angle of recovering the balance of Th17/Treg. METHODS: The in vivo colitis model was established in mice by using dextran sulfate sodium, and CD4+ T cells were used to simulate the in vitro differentiation of Treg and Th17 cells. The proportions and related factors of Th17 and Treg cells were measured using flow cytometry, Q-PCR and western blotting. The glycolysis was evaluated by examining the glucose uptake, glucose consumption and lactate production using kits or immunofluorescence. The activation of AhR was detected by western blotting and the XRE-luciferase reporter gene. The co-immunoprecipitation, transfection or other methods were selected to investigate and identify the signaling molecular pathway. RESULTS: DIM significantly attenuated symptoms of colitis mice by rebuilding the balance of Th17/Treg in anoxic colons. In hypoxia, a more potent promotion of Treg differentiation was showed by DIM relative to normoxia, and siFoxp3 prevented DIM-suppressed Th17 differentiation. DIM repressed the excessive glycolysis in hypoxia evidenced by down-regulated glucose uptake, lactate production, Glut1 and HK2 levels. Interestingly, IL-10, the function-related factor of Treg cells, showed the feedback effect of DIM-suppressed glycolysis. Besides, 2-deoxy-D-glucose, HK2 plasmid and IL-10 antibody prevented increase of DIM on the expression of Foxp3 at the transcriptional level and subsequent Treg differentiation through the lactate-STAT3 pathway, and reasons for the direct improvement of DIM on Foxp3 protein was attributed to promoting the formation of HIF-1α/TIP60 complexes as well as subsequent acetylation and protein stability. Finally, AhR dependence and mechanisms for DIM-improved Treg differentiation in vitro and in vivo were well confirmed by using plasmids or inhibitors. CONCLUSIONS: DIM enhances activation of AhR and subsequent "glycolysis-lactate-STAT3″ and TIP60 signals-mediated Treg differentiation.

Morin inhibits the transformation of fibroblasts towards myofibroblasts through regulating "PPAR-γ-glutaminolysis-DEPTOR" pathway in pulmonary fibrosis.

Morin, a natural flavonoid exists in many foods and dietary plants, owns good bioactivities. Herein, we investigated its effect on pulmonary fibrosis (PF), and further explored the mechanisms. Results showed that morin remarkably improved the pathologic alterations, and inhibited the transformation of fibroblasts towards myofibroblasts in lungs of mice with bleomycin-induced PF as well as TGF-ß1 or hypoxia-stimulated NIH-3T3 cells. Mechanistic studies revealed that morin activated peroxisome proliferator activated receptor-gamma (PPAR-γ), and GW9662 or siPPAR-γ significantly weakened the inhibition of morin on the transformation of NIH-3T3 cells. Furthermore, morin restricted glutaminolysis by down-regulating the level of glutaminase 1 (GLS1), which was confirmed by glutamine deprivation, and GLS1 overexpression. Replenishment of metabolite α-ketoglutarate (α-KG) and 2-hydroxyglutarate (2-HG) inhibited morin-prevented transformation of fibroblasts, but neither TGF-ß1 nor hypoxia could induce the transformation of IDH2-knockdown fibroblasts, suggesting 2-HG was directly involved in the action of morin. Then, ubiquitination of DEPTOR was demonstrated to be prevented by morin, which was attributed to KDM4A, an enzyme inactivated by 2-HG, and leucine as well as KDM4A inhibitor obstructed the effect of morin. Finally, the mechanisms of morin were further confirmed in vivo. Collectively, morin inhibited PF through intervening in "PPAR-γ-glutaminolysis-DEPTOR" signals, and subsequent restriction on the transformation of fibroblasts towards myofibroblasts.

Morin Acts as a USP7 Inhibitor to Hold Back the Migration of Rheumatoid Arthritis Fibroblast-Like Synoviocytes in a "Prickle1-mTORC2" Dependent Manner.

INTRODUCTION: The aim of this study is to investigate the effect and detailed mechanisms of morin, an anti-arthritis compound widely distributed in foods of plant origin, on the pathological migration of fibroblast-like synoviocytes (FLS). METHODS AND RESULTS: The migration of FLS collected from arthritis rats and MH7A cells is induced by platelet-derived growth factor, and an arthritis model in rats is established by Freund's complete adjuvant. The results show that morin remarkably restrains FLS migration but slightly affects FLS apoptosis and proliferation. Moreover, in the progression of FLS migration, focal adhesion (FA) turnover is inhibited by morin via lowering the activation of Paxillin and focal adhesion kinase (FAK) and internalization of integrin ß1. Morin disrupts the formation of mTOR complex 2 (mTORC2) and the activation of AKT (S473) and PKCα (S657), and MHY1485 reverses morin-limited FLS migration. Of note, the protein stability of Prickle1, a binding factor of Rictor, is reduced by morin, and MG132 but not Baf A1 shows a repressive effect. Finally, the target protein is identified as ubiquitin-specific protease 7 (USP7) but not USP9X. USP7 overexpressing plasmid weakens morin-affected protein and ubiquitination of Prickle1, and mechanisms are confirmed in vivo by using an overexpressing plasmid and inhibitor. CONCLUSION: Morin restricts FLS migration and arthritis by intervening in "USP7-Prickle1-mTORC2" signaling and FA turnover.

The role of GLS1-mediated glutaminolysis/2-HG/H3K4me3 and GSH/ROS signals in Th17 responses counteracted by PPARγ agonists.

Background: Peroxisome proliferator-activated receptor gamma (PPARγ) has the ability to counter Th17 responses, but the full mechanisms remain elusive. Herein, we aimed to elucidate this process in view of cellular metabolism, especially glutaminolysis. Methods: MTT, CCK-8, Annexin V-FITC/PI staining or trypan blue exclusion assays were used to analyze cytotoxicity. Flow cytometry and Q-PCR assays were applied to determine Th17 responses. The detection of metabolite levels using commercial kits and rate-limiting enzyme expression using western blotting assays was performed to illustrate the metabolic activity. ChIP assays were used to examine H3K4me3 modifications. Mouse models of dextran sulfate sodium (DSS)-induced colitis and house dust mite (HDM)/lipopolysaccharide (LPS)-induced asthma were established to confirm the mechanisms studied in vitro. Results: The PPARγ agonists rosiglitazone and pioglitazone blocked glutaminolysis but not glycolysis under Th17-skewing conditions, as indicated by the detection of intracellular lactate and α-KG and the fluorescence ratios of BCECF-AM. The PPARγ agonists prevented the utilization of glutamine and thus directly limited Th17 responses even when Foxp3 was deficient. The mechanisms were ascribed to restricted conversion of glutamine to glutamate by reducing the expression of the rate-limiting enzyme GLS1, which was confirmed by GLS1 overexpression. Replenishment of α-KG and 2-HG but not succinate weakened the effects of PPARγ agonists, and α-KG-promoted Th17 responses were dampened by siIDH1/2. Inhibition of KDM5 but not KDM4/6 restrained the inhibitory effect of PPARγ agonists on IL-17A expression, and the H3K4me3 level in the promoter and CNS2 region of the il-17 gene locus down-regulated by PPARγ agonists was rescued by 2-HG and GLS1 overexpression. However, the limitation of PPARγ agonists on the mRNA expression of RORγt was unable to be stopped by 2-HG but was attributed to GSH/ROS signals subsequent to GLS1. The exact role of PPARγ was proved by GW9662 or PPARγ knockout, and the mechanisms for PPARγ-inhibited Th17 responses were further confirmed by GLS1 overexpression in vivo. Conclusion: PPARγ agonists repressed Th17 responses by counteracting GLS1-mediated glutaminolysis/2-HG/H3K4me3 and GSH/ROS signals, which is beneficial for Th17 cell-related immune dysregulation.

Arctigenin disrupts NLRP3 inflammasome assembly in colonic macrophages via downregulating fatty acid oxidation to prevent colitis-associated cancer.

Arctigenin, the major active constituent of Fructus Arctii, has been reported to inhibit the growth of various tumors and alleviate colitis. This study aimed to prove the protective effect of arctigenin on colitis-associated cancer (CAC) and explore its mechanisms. Orally administered arctigenin prevented the progression of colitis and protected against colon carcinogenesis in azoxymethane (AOM)/dextran sulfate sodium (DSS)-induced CAC mice. Arctigenin downregulated NLRP3 inflammasome activation and fatty acid oxidation (FAO) metabolism in macrophages, as determined by untargeted metabolomics. Arctigenin also inhibited the expression of carnitine palmitoyltransferase 1 (CPT1), reduced the acetylation of α-tubulin, and disrupted NLRP3 complex formation, which in turn inactivated the NLRP3 inflammasome. Downregulation of the CPT1-FAO-acetyl-coenzyme A (acetyl-CoA)-acetylated α-tubulin pathway was observed to inhibit the effect of arctigenin on NLRP3 inflammasome assembly, as confirmed by CPT1 overexpression. Lastly, arctigenin was shown to inhibit NLRP3 inflammasome activation and improve CAC in mice, and the effect was significantly diminished by the overexpression of adeno-associated virus (AAV)9-CPT1. Taken together, these results show that the inhibition of NLRP3 inflammasome assembly in macrophages due to FAO downregulation contributes to the preventative effect of arctigenin against CAC. Our findings highlight the potential value of arctigenin to reduce the risk of CAC in patients with colitis.

Bergenin impedes the generation of extracellular matrix in glomerular mesangial cells and ameliorates diabetic nephropathy in mice by inhibiting oxidative stress via the mTOR/ß-TrcP/Nrf2 pathway.

Bergenin, a plant polyphenol, has been reported to lower the blood glucose level and ameliorate kidney function in streptozotocin (STZ)-induced diabetic rats. Herein, its protective effect on diabetic nephropathy (DN) was explored in view of extracellular matrix (ECM) generation in glomerular mesangial cells. Glomerular mesangial cells were treated with high glucose, and Q-PCR as well as western blot were used to determine the expression of ECM. To establish the participation and role of mammalian target of rapamycin (mTOR) and nuclear factor erythroid-derived 2-related factor 2 (Nrf2) in ECM generation, a combination of l-leucine (activator of mTOR) and Nrf2 shRNA transfection were performed, respectively. Moreover, a DN model was established in mice using high-glucose/high-fat diet and STZ. Bergenin impeded the generation of TGF-ß1 and ECM, decreased the levels of intracellular superoxide anion and hydrogen peroxide, and increased the activity of antioxidant enzymes in the glomerular mesangial cells (HBZY-1 and HRMC cells) treated with high glucose. The inhibition of ECM generation by bergenin was dependent on the down-regulation of oxidative stress as confirmed via a superoxide overexpression system. The activation of Nrf2 was required for bergenin to inhibit the oxidative stress and ECM generation. Moreover, bergenin was found to inhibit the phosphorylation of mTOR, which is located at the upstream of Nrf2. Bergenin did not interfere with the expression of Nrf2 mRNA and Keap1 (the classic degradation control factor of Nrf2), but markedly inhibited the protein expression of the ß-TrcP, an effect which could be abolished by l-leucine. In DN model mice, l-leucine diminished the ability of bergenin to reduce the levels of superoxide anion, hydrogen peroxide and ECM, which contributed to the eradication of the ameliorative effect of bergenin on nephropathy. Bergenin can inhibit glucose-induced ECM production in glomerular mesangial cells through the down-regulation of oxidative stress via the mTOR/ß-TrcP/Nrf2 pathway, and it might be a candidate drug for the prevention and treatment of DN.

Cardamonin, a natural flavone, alleviates inflammatory bowel disease by the inhibition of NLRP3 inflammasome activation via an AhR/Nrf2/NQO1 pathway.

The present study aimed to evaluate the anti-colitis effect and underlying mechanisms of cardamonin, a natural flavone isolated from Alpinia katsumadai Hayata. The results showed that oral cardamonin significantly inhibited dextran sulfate sodium (DSS)- and 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis in mice, evidenced by improvement of disease activity index scores, myeloperoxidase activity, length shortening and histopathological changes of colons. A rectal administration of cardamonin also exhibited marked anti-colitis effect, suggesting that oral cardamonin might function in a prototype form. Cardamonin down-regulated levels of IL-1ß, TNF-α, IL-6, NLRP3, cleaved caspase-1, ASC, cleaved IL-1ß in colons of colitis mice. In vitro, cardamonin inhibited NLRP3 inflammasome activation in THP-1 and bone marrow-derived macrophages. It acted as an AhR activator, enhanced dissociation of AhR/HSP90 complexes, association of AhR/ARNT complexes, AhR nuclear translocation, XRE reporter gene activity, and AhR/ARNT/XRE DNA binding activity in THP-1 cells. The AhR antagonist CH223191 obviously abolished NLRP3 inflammasome activation inhibited by cardamonin. Furthermore, cardamonin elevated levels of Nrf2 and its target genes NQO1, Trx1, SOD2, HO-1, and the effect on NQO1 was the most obvious. The relationship of cardamonin-adjusted AhR activation, expressions of Nrf2 and NQO1, and NLRP3 inflammasome activation was confirmed by using CH223191, siAhR, ML385 and siNQO1, respectively. Finally, CH223191 was shown to abolish amelioration of cardamonin on DSS- and TNBS-induced colitis, inhibition of NLRP3 inflammasome activation and up-regulation of Nrf2 and NQO1 levels in colons. Taken together, cardamonin ameliorated colitis in mice through the activation of AhR/Nrf2/NQO1 pathway and consequent inhibition of NLRP3 inflammasome activation.