Phytoestrogen arctigenin preserves the mucus barrier in inflammatory bowel diseases by inhibiting goblet cell apoptosis via the ERß/TRIM21/PHB1 pathway.

Intestinal mucus barrier dysfunction is closely involved in the pathogenesis of inflammatory bowel diseases (IBD). To investigate the protective effect and underlying mechanism of arctigenin, a phytoestrogen isolated from the fruits of Arctium lappa L., on the intestinal mucus barrier under colitis condition. The role of arctigenin on the intestinal mucus barrier and the apoptosis of goblet cells were examined by using both in vitro and in vivo assays. Arctigenin was demonstrated to promote the mucus secretion and maintain the integrity of mucus barrier, which might be achieved by an increase in the number of goblet cells via inhibiting apoptosis. Arctigenin selectively inhibited the mitochondrial pathway-mediated apoptosis. Moreover, arctigenin elevated the protein level of prohibitin 1 (PHB1) through blocking the ubiquitination via activation of estrogen receptor ß (ERß) to competitively interact with PHB1 and disrupt the binding of tripartite motif 21 (TRIM21) with PHB1. ERß knock down in the colons of mice with DSS-induced colitis resulted in significant reduction of the protection of arctigenin and DPN against the mucosal barrier. Arctigenin can maintain the integrity of the mucus barrier by inhibiting the apoptosis of goblet cells through the ERß/TRIM21/PHB1 pathway.

Regulation of gut microbiota substantially contributes to the induction of intestinal Treg cells and consequent anti-arthritis effect of madecassoside.

Previously, we reported that oral administration of madecassoside, the main active triterpene in Centella asiatica L., exerted anti-arthritis effect by inducing the generation of regulatory T (Treg) cells in small intestine. This study investigates the action site and mechanism of madecassoside to induce Treg cells. In collagen-induced arthritis (CIA) of rats, oral administration of madecassoside significantly alleviated arthritis symptoms, but its main metabolite madecassic acid did not, suggesting that madecassoside functions in the parent form. Madecassoside was shown to increase the number of Treg cells and promote the expression of Foxp3 and IL-10 in rat ileum rather than duodenum and jejunum, as detected using the immunohistochemistry assay and quantitative PCR assay, respectively. Unexpectedly, madecassoside was absent of significant effect on in vitro Treg cell differentiation and the expression of Foxp3 and IL-10. A combined use of broad-spectrum antibiotics resulted in significant reduction of the anti-arthritis effect of madecassoside in CIA rats. The 16S rRNA gene sequence showed that madecassoside could reverse the changes of gut microbiota under arthritis condition, and enrich several bacteria such as Lachnospiraceae, Butyricicoccus, Faecalibacterium, Butyricicoccus pullicaecorum and so on. GC-MS assay showed that madecassoside elevated the levels of acetic acid and butyric acid, but not other short chain fatty acids (SCFAs) in the cecum contents of CIA rats. Butyric acid rather than acetic acid could induce the in vitro differentiation of Treg cells and the expression of Foxp3 and IL-10. Accordingly, when madecassoside was co-administered with heptanoyl CoA, the competitive inhibitor of butyrate synthase, its effect on butyric acid production, Treg cell proportion and arthritis nearly disappeared. These findings indicate that oral madecassoside induces the generation of Treg cells and therefore displays anti-arthritis effect in the parent form but not metabolites, and the ileum is the main action site. The mechanism of madecassoside can be summarized as: expansion of the richness of butyrate-producing bacteria-up-regulation of intestinal butyrate level-induction of Treg cell differentiation and IL-10 expression.

Pharmacological activation of ERß by arctigenin maintains the integrity of intestinal epithelial barrier in inflammatory bowel diseases.

Intestinal epithelial barrier dysfunction is deeply involved in the pathogenesis of inflammatory bowel diseases (IBD). Arctigenin, the main active constituent in Fructus Arctii (a traditional Chinese medicine), has previously been found to attenuate colitis induced by dextran sulfate sodium (DSS) in mice. The present study investigated whether and how arctigenin protects against the disruption of the intestinal epithelial barrier in IBD. Arctigenin maintained the intestinal epithelial barrier function of mice with DSS- and TNBS-induced colitis. In Caco-2 and HT-29 cells, arctigenin lowered the monolayer permeability, increased TEER, reversed the abnormal expression of tight junction proteins, and restored the altered localization of F-actin induced by TNF-α and IL-1ß. The specific antagonist PHTPP or shRNA of ERß largely weakened the protective effect of arctigenin on the epithelial barrier function of Caco-2 and HT-29 cells. Molecular docking demonstrated that arctigenin had high affinity for ERß mainly through hydrogen bonds as well as hydrophobic effects, and the protective effect of arctigenin on the intestinal barrier function was largely diminished in ERß-mutated (ARG346 and/or GLU305) Caco-2 cells. Moreover, arctigenin-blocked TNF-α induced increase of the monolayer permeability in Caco-2 and HT-29 cells and the activation of myosin light chain kinase (MLCK)/myosin light chain (MLC) pathway in an ERß-dependent manner. ERß deletion in colons of mice with DSS-induced colitis resulted in a significant attenuation of the protective effect of arctigenin on the barrier integrity and colon inflammation. Arctigenin maintained the integrity of the intestinal epithelial barrier under IBD by upregulating the expression of tight junction proteins through the ERß-MLCK/MLC pathway.

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.

The gut microbiota modulator berberine ameliorates collagen-induced arthritis in rats by facilitating the generation of butyrate and adjusting the intestinal hypoxia and nitrate supply.

The commensal microbiota is one of the environmental triggers of rheumatoid arthritis (RA). Recent studies have identified the characteristics of the gut microbiota in patients with RA. However, it is still unclear how the microbiota can be modulated to slow down disease progression. In the present study, berberine, a modulator of gut microbiota with substantial anti-RA effect, was chosen to explore the mechanisms by which the microbiota modulators ameliorate RA. The results showed that oral administration of berberine alleviated collagen-induced arthritis (CIA) in rats in a gut microbiota-dependent manner. Berberine down-regulated the diversity and richness of the gut bacteria, reduced the abundance of Prevotella, and elevated the abundance of butyrate-producing bacteria in CIA rats as determined by the 16S rRNA gene sequence, which might function through limiting the generation of nitrate and stabilizing the physiologic hypoxia in the intestine. Moreover, berberine treatment significantly increased the intestinal butyrate level and promoted the expression and activity of butyryl-CoA:acetate-CoA transferase (BUT). The coadministration of a BUT inhibitor largely diminished the adjustment of intestinal environment and the antiarthritic effect of berberine. In conclusion, modulators of the gut microbiota might serve as therapeutic agents for RA by inducing the butyrate generation through promoting the expression and activity of BUT.-Yue, M., Tao, Y., Fang, Y., Lian, X., Zhang, Q., Xia, Y., Wei, Z., Dai, Y. The gut microbiota modulator berberine ameliorates collagen-induced arthritis in rats by facilitating the generation of butyrate and adjusting the intestinal hypoxia and nitrate supply.

Morin Exerts Anti-Arthritic Effects by Attenuating Synovial Angiogenesis via Activation of Peroxisome Proliferator Activated Receptor-γ.

SCOPE: Morin, a flavonoid occurring in many dietary plants, can reduce the number of synovial blood vessels and ameliorate collagen-induced arthritis (CIA) in rats. Herein, its underlying mechanisms in view of the peroxisome proliferator activated receptor-γ (PPARγ) pathway are addressed. METHODS AND RESULTS: In vitro, wound-healing and transwell assays are conducted to explore the effect of morin on HUVECs migration. Morin inhibits HUVECs migration and tube formation induced by VEGF, which is reversed by PPARγ antagonist GW9662 or siPPARγ. Molecular docking and competitive binding assays show that morin could bind to PPARγ. Morin increases the expression of PDK4 and CD36 in a PPARγ-dependent manner and increases the luciferase activity in cells transfected with PPARγ plasmid, which indicates that morin could activate PPARγ after binding. In addition, morin increases the expression of PTEN, a target gene of PPARγ that suppresses angiogenesis and inhibits PI3K/Akt signaling. The effects of morin on the PTEN-PI3K/Akt pathway are diminished by GW9662 and siPPARγ. In vivo studies show that morin ameliorates rat CIA, reduces synovial angiogenesis, and upregulates the expression of PTEN in the synovium, which is almost completely abolished by GW9662. CONCLUSIONS: Morin is a potential agonist of PPARγ, which attenuates synovial angiogenesis and arthritis via the PPARγ-PTEN-PI3K/Akt pathway.

Gut-Sourced Vasoactive Intestinal Polypeptide Induced by the Activation of α7 Nicotinic Acetylcholine Receptor Substantially Contributes to the Anti-inflammatory Effect of Sinomenine in Collagen-Induced Arthritis.

Sinomenine has long been used for the treatment of rheumatoid arthritis in China. However, its anti-inflammatory mechanism is still debatable because the in vitro minimal effective concentration (≥250 µM) is hardly reached in either synovium or serum after oral administration at a therapeutic dose. Recent findings suggest that the α7 nicotinic acetylcholine receptor (α7nAChR) might mediate the inhibitory effect of sinomenine on macrophage activation, which attracts us to explore the anti-arthritis mechanism of sinomenine by taking neuroendocrine-inflammation axis into consideration. Here, we showed that orally administered sinomenine ameliorated the systemic inflammation of collagen-induced arthritis (CIA) rats, which was significantly diminished by either vagotomy or the antagonists of nicotinic acetylcholine receptors (especially the antagonist of α7nAChR), but not by the antagonists of muscarinic receptor. Sinomenine might bind to α7nAChR through interacting with the residues Tyr184 and Tyr191 in the pocket. In addition, the generation of vasoactive intestinal polypeptide (VIP) from the gut of CIA rats and cultured neuron-like cells was selectively enhanced by sinomenine through the activation of α7nAChR-PI3K/Akt/mTOR pathway. The elevated levels of VIP in the serum and small intestine of rats were negatively correlated with the scores of joint destruction. The crucial role of VIP in the anti-arthritic effect of sinomenine was confirmed by using VIP hybrid, a non-specific antagonist of VIP receptor. Taken together, intestine-sourced VIP mediates the anti-arthritic effect of sinomenine, which is generated by the activation of α7nAChR.

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.

Berberine ameliorates collagen-induced arthritis in rats by suppressing Th17 cell responses via inducing cortistatin in the gut.

Berberine, an isoquinoline alkaloid, has been reported to ameliorate various autoimmune diseases including rheumatoid arthritis by oral administration. However, its mechanism remains mysterious due to an extremely low bioavailability. The fact that berberine readily accumulates in the gut, the largest endocrine organ in the body, attracted us to explore its anti-arthritic mechanism in view of the induction of intestinal immunosuppressive neuropeptides. In this study, berberine (200 mg·kg-1 , i.g.) was shown to ameliorate collagen-induced arthritis in rats, which was manifested by the reduction of clinical signs and joint destruction, as well as marked down-regulation of Th17 cell frequency and interleukin-17 level in blood. In contrast, an intravenous injection of berberine failed to affect arthritis in rats, implying that its anti-arthritic effect was gut-dependent. Further studies revealed that oral berberine selectively elevated the levels of cortistatin, of five gut-derived neuropeptides tested, in the intestines and sera of arthrititic rats. Antagonists of ghrelin/growth hormone secretagogue receptor 1 (a subtype of cortistatin receptor) almost completely abolished the ameliorative effect of berberine on arthritis and Th17 cell responses in rats. In vitro, berberine showed a moderate ability to promote the expression of cortistatin in nerve cells, which was strengthened when the nerve cells were cocultured with enteroendocrine cells to induce an autocrine/paracrine environment. In summary, oral berberine exerted anti-arthritic effect through inhibiting the Th17 cell response, which was closely associated with the induction of cortistatin generation from gut through augmenting autocrine/paracrine action between enteric nerve cells and endocrine cells.

Arctigenin functions as a selective agonist of estrogen receptor ß to restrict mTORC1 activation and consequent Th17 differentiation.

Arctigenin was previously proven to inhibit Th17 cell differentiation and thereby attenuate colitis in mice by down-regulating the activation of mechanistic target of rapamycin complex 1 (mTORC1). The present study was performed to address its underlying mechanism in view of estrogen receptor (ER). The specific antagonist PHTPP or siRNA of ERß largely diminished the inhibitory effect of arctigenin on the mTORC1 activation in T cell lines and primary CD4+ T cells under Th17-polarization condition, suggesting that arctigenin functioned in an ERß-dependent manner. Moreover, arctigenin was recognized to be an agonist of ERß, which could bind to ERß with a moderate affinity, promote dissociation of ERß/HSP90 complex and nuclear translocation and phosphorylation of ERß, and increase the transcription activity. Following activation of ERß, arctigenin inhibited the activity of mTORC1 by disruption of ERß-raptor-mTOR complex assembly. Deficiency of ERß markedly abolished arctigenin-mediated inhibition of Th17 cell differentiation. In colitis mice, the activation of ERß, inhibition of mTORC1 activation and Th17 response by arctigenin were abolished by PHTPP treatment. In conclusion, ERß might be the target protein of arctigenin responsible for inhibition of mTORC1 activation and resultant prevention of Th17 cell differentiation and colitis development.

Madecassoside ameliorates bleomycin-induced pulmonary fibrosis in mice through promoting the generation of hepatocyte growth factor via PPAR-γ in colon.

BACKGROUND AND PURPOSE: Madecassoside has potent anti-pulmonary fibrosis (PF) effects when administered p.o., despite having extremely low oral bioavailability. Herein, we explored the mechanism of this anti-PF effect with regard to gut hormones. EXPERIMENTAL APPROACH: A PF model was established in mice by intratracheal instillation of bleomycin. Haematoxylin and eosin stain and Masson's trichrome stain were used to assess histological changes in the lung. Quantitative-PCR and Western blot detected mRNA and protein levels, respectively, and cytokines were measured by ELISA. Small interfering RNA was used for gene-silencing. EMSA was applied to detect DNA-binding activity. KEY RESULTS: Administration of madecassoside, p.o., but not its main metabolite madecassic acid, exhibited a direct anti-PF effect in mice. However, i.p. madecassoside had no anti-PF effect. Madecassoside increased the expression of hepatocyte growth factor (HGF) in colon tissues, and HGF receptor antagonists attenuated its anti-PF effect. Madecassoside facilitated the secretion of HGF from colonic epithelial cells by activating the PPAR-γ pathway, as shown by an up-regulation of PPAR-γ mRNA expression, nuclear translocation and DNA-binding activity both in vitro and in vivo. Also GW9662, a selective PPAR-γ antagonist, almost completely prevented the madecassoside-induced increased expression of HGF and amelioration of PF. CONCLUSIONS AND IMPLICATIONS: The potent anti-PF effects induced by p.o. madecassoside in mice are not mediated by its metabolites or itself after absorption into blood. Instead, madecassoside increases the activity of PPAR-γ, which subsequently increases HGF expression in colonic epithelial cells. HGF then enters into the circulation and lung tissue to exert an anti-PF effect.

Norisoboldine, an Anti-Arthritis Alkaloid Isolated from Radix Linderae, Attenuates Osteoclast Differentiation and Inflammatory Bone Erosion in an Aryl Hydrocarbon Receptor-Dependent Manner.

Norisoboldine (NOR), the primary isoquinoline alkaloid constituent of the root of Lindera aggregata, has previously been demonstrated to attenuate osteoclast (OC) differentiation. Accumulative evidence has shown that aryl hydrocarbon receptor (AhR) plays an important role in regulating the differentiation of various cells, and multiple isoquinoline alkaloids can modulate AhR. In the present study, we explored the role of NOR in the AhR signaling pathway. These data showed that the combination of AhR antagonist resveratrol (Res) or α-naphthoflavone (α-NF) nearly reversed the inhibition of OC differentiation through NOR. NOR could stably bind to AhR, up-regulate the nuclear translocation of AhR, and enhance the accumulation of the AhR-ARNT complex, AhR-mediated reporter gene activity and CYP1A1 expression in RAW 264.7 cells, suggesting that NOR might be an agonist of AhR. Moreover, NOR inhibited the nuclear translocation of NF-κB-p65, resulting in the evident accumulation of the AhR-NF-κB-p65 complex, which could be markedly inhibited through either Res or α-NF. Although NOR only slightly affected the expression of HIF-1α, NOR markedly reduced VEGF mRNA expression and ARNT-HIF-1α complex accumulation. In vivo studies indicated that NOR decreased the number of OCs and ameliorated the bone erosion in the joints of rats with collagen-induced arthritis, accompanied by the up-regulation of CYP1A1 and the down-regulation of VEGF mRNA expression in the synovium of rats. A combination of α-NF nearly completely reversed the effects of NOR. In conclusion, NOR attenuated OC differentiation and bone erosion through the activation of AhR and the subsequent inhibition of both NF-κB and HIF pathways.