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.

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.

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.

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.