Mume Fructus (Prunus mume Sieb. et Zucc.) extract accelerates colonic mucosal healing of mice with colitis induced by dextran sulfate sodium through potentiation of cPLA2-mediated lysophosphatidylcholine synthesis.

BACKGROUND: Mume Fructus (MF) is the fruit of Prunus mume Sieb. et Zucc, a plant of Rosaceae family. Previous studies demonstrated that MF was capable of ameliorating ulcerative colitis (UC) in mice, its action mechanism needs to be clarified. PURPOSE: This study deciphered whether and how MF extract accelerates colonic mucosal healing, the therapeutic endpoint of UC. METHODS: Biochemical, histopathological and qRT-PCR analyses were utilized to define the therapeutic efficacy of MF on dextran sulfate sodium (DSS)-induced colitis in mice. UHPLC-QTOF-MS/MS-based metabolomics technique was adopted to explore the changes of endogenous metabolites associated with UC and responses to MF intervention. qRT-PCR analysis was performed to confirm the molecular pathway in vivo. The effects of MF and lysophosphatidylcholine (LPC) on cell viability, wound healing, proliferation, and migration were examined through a series of in vitro experiments. Moreover, the effects of different subtypes of phospholipase A2 (PLA2) inhibitors on MF-treated colonic epithelial cells were detected by wound healing test and transwell assay. RESULTS: Orally administered MF could alleviate colitis in mice mainly by accelerating the healing of colonic mucosa. Guided by an unbiased metabolomics screen, we identified LPC synthesis as a major modifying pathway in colitis mice after MF treatment. Notably, MF facilitated the synthesis of LPC by enhancing the expression of PLA2 in colitis mice. Mechanistically, MF and LPC accelerated wound closure by promoting cell migration. Moreover, the promotion of MF on wound healing and migration of colonic epithelial cells was blunted by a cytosolic phospholipase A2 (cPLA2) inhibitor. CONCLUSION: MF can facilitate colonic mucosal healing of mice with colitis through cPLA2-mediated intestinal LPC synthesis, which may become a novel therapeutic agent of UC.

3, 3'-diindolylmethane enhances macrophage efferocytosis and subsequently relieves visceral pain via the AhR/Nrf2/Arg-1-mediated arginine metabolism pathway.

BACKGROUND: 3, 3'-diindolylmethane (DIM), a classical aryl hydrocarbon receptor (AhR) agonist, has been shown to relieve neuropathic pain, but few studies have reported the efficacy of DIM in visceral pain under colitis condition. PURPOSE: This study aimed to investigate the effect and mechanism of DIM on visceral pain under colitis condition. METHODS: Cytotoxicity was performed using the MTT assay. RT-qPCR and ELISA assays were applied to determine the expression and release of algogenic substance P (SP), nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF). Flow cytometry was used to examine the apoptosis and efferocytosis. The expression of Arg-1-arginine metabolism-related enzymes was detected using western blotting assays. ChIP assays were used to examine the binding of Nrf2 to Arg-1. Mouse models of dextran sulfate sodium (DSS) were established to illustrate the effect of DIM and validate the mechanism in vivo. RESULTS: DIM did not directly affect expressions and release of algogenic SP, NGF and BDNF in enteric glial cells (EGCs). However, when co-cultured with DIM-pre-treated RAW264.7 cells, the release of SP and NGF was decreased in lipopolysaccharides-stimulated EGCs. Furthermore, DIM increased the number of PKH67+ F4/80+ cells in the co-culture system of EGCs and RAW264.7 cells in vitro and alleviated visceral pain under colitis condition by regulating levels of SP and NGF as well as values of electromyogram (EMG), abdominal withdrawal reflex (AWR) and tail-flick latency (TFL) in vivo, which was significantly inhibited by efferocytosis inhibitor. Subsequently, DIM was found to down-regulate levels of intracellular arginine, up-regulate levels of ornithine, putrescine and Arg-1 but not extracellular arginine or other metabolic enzymes, and polyamine scavengers reversed the effect of DIM on efferocytosis and release of SP and NGF. Moving forward, Nrf2 transcription and the binding of Nrf2 to Arg-1-0.7 kb was enhanced by DIM, AhR antagonist CH223191 abolished the promotion of DIM on Arg-1 and efferocytosis. Finally, nor-NOHA validated the importance of Arg-1-dependent arginine metabolism in DIM-alleviated visceral pain. CONCLUSION: DIM enhances macrophage efferocytosis in an arginine metabolism-dependent manner via "AhR-Nrf2/Arg-1" signals and inhibits the release of SP and NGF to relieve visceral pain under colitis condition. These findings provide a potential therapeutic strategy for the treatment of visceral pain in patients with colitis.

Galangin Targets HSP90ß to Alleviate Ulcerative Colitis by Controlling Fatty Acid Synthesis and Subsequent NLRP3 Inflammasome Activation.

SCOPE: The purpose of this research is to investigate the specific role of HSP90 paralogs in ulcerative colitis (UC), and to explore the mechanisms behind the inhibitory effects of galangin (Gal) on UC by inhibiting HSP90ß in vivo. METHODS AND RESULTS: In order to achieve this, publicly available gene expression data and molecular biology techniques are used. The results show that the expression of HSP90ß is significantly increased in the mucosal biopsies of UC patients and in the colons of colitis mice, and that there is a significant correlation between HSP90ß levels and disease severity. Then, Gal is found to bind directly to HSP90ß and downregulates the level of p-AKT, as well as the stability and oligomerization of HSP90ß, indicating Gal as an HSP90ß inhibitor. Moreover, the findings reveal that HSP90ß plays a critical role in controlling UC, and that Gal can alleviate colitis by inhibiting HSP90ß and perturbing fatty acid synthesis-mediated NLRP3 inflammasome activation. CONCLUSION: These results not only provide insight into the potential therapeutic use of Gal in the treatment of UC, but also offer new perspectives on the role of HSP90ß in this disease.

Sinomenine Ameliorates Colitis-Associated Cancer by Modulating Lipid Metabolism via Enhancing CPT1A Expression.

Colitis-associated cancer (CAC), arising from long-lasting intestinal inflammation, is a common type of colorectal cancer. Sinomenine (SIN), the major active compound of Sinomenium acutum, displays excellent antitumor activity. In modern pharmacological research, SIN has been proved to arrest proliferation of human colon cancer cells in vitro, but its functional role and specific mechanism in CAC were still elusive. This study explored the molecular mechanism of SIN on CAC. The results showed that orally administered SIN could decrease the occurrence and development of CAC. Metabolomics results revealed SIN could reprogram metabolism in CAC mice by reversing 34 endogenous metabolites. Importantly, the most prominent metabolic alteration was lipid metabolism. Mechanistically, SIN improved lipid metabolism by enhancing the expression of CPT1A in CAC mice. Moreover, the inhibitory effect of SIN on the proliferation of human colon cancer cells was blunted via CPT1A inhibitor. The results of this study added further evidence of the molecular mechanisms that allow SIN to exert anti-CAC effect by facilitating lipid metabolism and reaffirmed its potential and distinctive role as a chemopreventive agent in CAC.

Tripterygium glycoside tablet attenuates renal function impairment in diabetic nephropathy mice by regulating triglyceride metabolism.

Tripterygium glycoside tablet (TGT) has been used clinically to alleviate diabetic nephropathy (DN) for decades. However, the mechanism of its anti-DN has not been fully clarified. The aim of this study was to elucidate molecular mechanism of TGT in repairing renal function injury. The results of biochemical parameters and renal histopathology implied that TGT intervention could attenuate creatinine, albumin excretion rate and histological injury of kidney in DN mouse model. Moreover, UHPLC-QTOF-MS/MS-based untargeted metabolomic analysis indicated that 11 metabolites in kidney of mice with DN were restored after TGT treatment, and the most prominent metabolic alteration was triglyceride (TG) metabolism. Mechanistically, TGT effectively improved the function of impaired kidney by promoting TG catabolism via modulation of adipose triglyceride lipase in DN mice. Our findings identified the link between circulating metabolites and DN, suggesting that it might be a possibility to intervene in DN by targeting metabolism.

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.

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.

Kinetic study of Reactive Black 5 degradation by Fe2+/S2O82- process via interactive model-based response surface methodology.

This study aimed to kinetically discover optimal conditions on characteristics of Reactive Black 5 decolorization/degradation via ferrous (Fe2+)-activated potassium persulfate (PS). Monod-like kinetics and interactive model-based response surface methodology (RSM) were applied to fitting and predict optimize treatment. Biodegradability of the intermediates was also tested by shaking culture with two species (Proteus hauseri ZMd44 and Shewanella sp. WLP72). Results showed that the optimal degradation efficiency was predicted (through RSM) as pH 3.72, (PS) = 0.39 mM, and (Fe2+) = 0.29 mM. The transformation products (dl-4-hydroxymandelic acid, benzoic acid, benzene, formic acid, oxalic acid and acetic acid) were less toxic than the original dye solution. According to those results, clean-up of dye pollutants by the Fe2+/S2O82- process is feasible as a pre-processing for the biodegradation, and the predicted optimal conditions are meaningful for further industry utilization.

The absorption enhancement of norisoboldine in the duodenum of adjuvant-induced arthritis rats involves the impairment of P-glycoprotein.

Lindera aggregata (Sims) Kosterm root has been used in traditional Chinese medicine for the treatment of rheumatism palsy, dyspepsia and frequent urination for a long time. Norisoboldine, the main active constituent of this herb drug, possesses outstanding anti-arthritis activity. However, the in vivo disposition of norisoboldine is known to a limited extent, especially under the pathological condition of rheumatoid arthritis (RA). The aim of this study is to investigate whether and how the absorption of norisoboldine is altered in adjuvant-induced arthritis (AIA) rats. Comparative studies of the intestinal absorption of norisoboldine in normal and AIA rats at different pathological stages of the arthritis were performed using in situ single-pass intestinal perfusion, and the effects of an inhibitor of efflux proteins were also investigated. Norisoboldine was shown to be a substrate of P-glycoprotein (P-gp), as P-gp inhibitor verapamil markedly increased the permeability coefficient (Peff ) of norisoboldine by 88% in the intestine of normal rats. Compared with normal rats, AIA rats displayed increased Peff values of norisoboldine by 84% and 86% on day 5 and day 10 after the appearance of the secondary response of arthritis, respectively. Verapamil could eliminate the difference of intestinal absorption of norisoboldine between normal and AIA rats. Further studies showed that impaired expression and activity of P-gp in AIA rats play a decisive role in the absorption enhancement of norisoboldine. Notably, the impairment of P-gp function positively correlated with the severity of arthritis. Copyright © 2016 John Wiley & Sons, Ltd.

Mechanism and toxicity research of benzalkonium chloride oxidation in aqueous solution by H2O2/Fe(2+) process.

As widely used disinfectants, the pollution caused by benzalkonium chloride (BAC) has attracted a lot of attention in recent years. Since it is not suitable for biodegradation, BAC was degraded firstly by Fenton advanced oxidation technologies (AOTs) in this research to enhance the biodegradability of the pollutions. The result revealed that the optimal molar ratio of H2O2/Fe(2+) for BAC degradation was 10:1, and the COD removal rate was 32 %. To clarify the pathway of degradation, the technique of GC-MS was implemented herein to identify intermediates and the toxicity of those BAC intermediates were also novelty tested through microbial fuel cells (MFC). The findings indicated that ten transformation products including benzyl dimethyl amine and dodecane were formed during the H2O2/Fe(2+) processes, which means the degradation pathway of BAC was initiated both on the hydrophobic (alkyl chain) and hydrophilic (benzyl and ammonium moiety) region of the surfactant. The toxicity of BAC before and after treated by Fenton process was monitored through MFC system. The electricity generation was improved 337 % after BAC was treated by H2O2/Fe(2+) oxidation processes which indicated that the toxicity of those intermediates were much lower than BAC. The mechanism and toxicity research in this paper could provide the in-depth understanding to the pathway of BAC degradation and proved the possibility of AOTs for the pretreatment of a biodegradation process.

Paeoniflorin suppresses TGF-ß mediated epithelial-mesenchymal transition in pulmonary fibrosis through a Smad-dependent pathway.

AIM: Paeoniflorin has shown to attenuate bleomycin-induced pulmonary fibrosis (PF) in mice. Because the epithelial-mesenchymal transition (EMT) in type 2 lung endothelial cells contributes to excessive fibroblasts and myofibroblasts during multiple fibrosis of tissues, we investigated the effects of paeoniflorin on TGF-ß mediated pulmonary EMT in bleomycin-induced PF mice. METHODS: PF was induced in mice by intratracheal instillation of bleomycin (5 mg/kg). The mice were orally treated with paeoniflorin or prednisone for 21 d. After the mice were sacrificed, lung tissues were collected for analysis. An in vitro EMT model was established in alveolar epithelial cells (A549 cells) incubated with TGF-ß1 (2 ng/mL). EMT identification and the expression of related proteins were performed using immunohistochemistry, transwell assay, ELISA, Western blot and RT-qPCR. RESULTS: In PF mice, paeoniflorin (50, 100 mg·kg(-1)·d(-1)) or prednisone (6 mg·kg(-1)·d(-1)) significantly decreased the expression of FSP-1 and α-SMA, and increased the expression of E-cadherin in lung tissues. In A549 cells, TGF-ß1 stimulation induced EMT, as shown by the changes in cell morphology, the increased cell migration, and the increased vimentin and α-SMA expression as well as type I and type III collagen levels, and by the decreased E-cadherin expression. In contrast, effects of paeoniflorin on EMT disappeared when the A549 cells were pretreated with TGF-ß1 for 24 h. TGF-ß1 stimulation markedly increased the expression of Snail and activated Smad2/3, Akt, ERK, JNK and p38 MAPK in A549 cells. Co-incubation with paeoniflorin (1-30 µmol/L) dose-dependently attenuated TGF-ß1-induced expression of Snail and activation of Smad2/3, but slightly affected TGF-ß1-induced activation of Akt, ERK, JNK and p38 MAPK. Moreover, paeoniflorin markedly increased Smad7 level, and decreased ALK5 level in A549 cells. CONCLUSION: Paeoniflorin suppresses the early stages of TGF-ß mediated EMT in alveolar epithelial cells, likely by decreasing the expression of the transcription factors Snail via a Smad-dependent pathway involving the up-regulation of Smad7.

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.

Comparative pharmacokinetics of bergenin, a main active constituent of Saxifraga stolonifera Curt., in normal and hepatic injury rats after oral administration.

Bergenin, isolated from the herb of Saxifrage stolonifera Curt. (Hu-Er-Cao) has hepatoprotective, anti-inflammatory, antitussive, and neuroprotective activities. The aim of the present study was to establish a simple, rapid, and sensitive RP-HPLC method for determination of bergenin in rat plasma and compare its oral pharmacokinetic behaviors in normal and CCl4-induced hepatic injury rats. With norisoboldine as an internal standard, chromatographic separation was performed on a C18 analytical column with acetonitrile and water (11 : 89, V/V) containing 0.1% formic acid as the mobile phase. A good linearity was obtained over the range of 100-10 000 ng·mL-1. The lower limit of quantification was 50 ng·mL-1. The developed method was successfully applied to a study of the pharmacokinetic difference of bergenin (100 mg·kg-1) between normal and hepatic injury rats after oral administration. Marked alterations of pharmacokinetic parameters in hepatic injury rats were observed. Compared to normal rats, the AUC(0-∞) of bergenin in hepatic injury rats was elevated to 2.11-fold and Cmax was increased by 130%, whereas CL value was only 55% of the normal rats, suggesting that the systemic exposure of bergenin was significantly increased under hepatic injury status.

Tetrandrine inhibits migration and invasion of rheumatoid arthritis fibroblast-like synoviocytes through down-regulating the expressions of Rac1, Cdc42, and RhoA GTPases and activation of the PI3K/Akt and JNK signaling pathways.

Tetrandrine (Tet), the main active constituent of Stephania tetrandra root, has been demonstrated to alleviate adjuvant-induced arthritis in rats. The present study was designed to investigate the effects of Tet on the migration and invasion of rheumatoid arthritis fibroblast-like synoviocytes (RA-FLS) and explore the underlying mechanisms. By using cultures of primary FLS isolated from synoviums of RA patients and cell line MH7A, Tet (0.3, 1 µmol·L(-1)) was proven to significantly impede migration and invasion of RA-FLS, but not cell proliferation. Tet also greatly reduced the activation and expressions of matrix degrading enzymes MMP-2/9, the expression of F-actin and the activation of FAK, which controlled the morphologic changes in migration process of FLS. To identify the key signaling pathways by which Tet exerts anti-migration effect, the specific inhibitors of multiple signaling pathways LY294002, Triciribine, SP600125, U0126, SB203580, and PDTC (against PI3K, Akt, JNK, ERK, p38 MAPK and NF-κB-p65, respectively) were used. Among them, LY294002, Triciribine, and SP600125 were shown to obviously inhibit the migration of MH7A cells. Consistently, Tet was able to down-regulate the activation of Akt and JNK as demonstrated by Western blotting assay. Moreover, Tet could reduce the expressions of migration-related proteins Rho GTPases Rac1, Cdc42, and RhoA in MH7A cells. In conclusion, Tet can impede the migration and invasion of RA-FLS, which provides a plausible explanation for its protective effect on RA. The underlying mechanisms involve the reduction of the expressions of Rac1, Cdc42, and RhoA, inhibition of the activation of Akt and JNK, and subsequent down-regulation of activation and/or expressions of MMP-2/9, F-actin, and FAK.

Norisoboldine ameliorates DSS-induced ulcerative colitis in mice through induction of regulatory T cells in colons.

Norisoboldine (NOR), the main active constituent of Radix Linderae, was previously demonstrated to ameliorate collagen-induced arthritis in rats through regulating the imbalance of T cells in intestines, which implied its therapeutic potential in inflammatory bowel disease. Here, we investigated the effect of NOR on ulcerative colitis (UC) induced by dextran sulfate sodium (DSS) in mice. Results showed that NOR (20, 40mg/kg) markedly reduced the symptoms of colitis, the levels of IL-1ß and TNF-α, and the activation of ERK, p38 MAPK and NF-κB-p65. NOR only slightly decreased the levels of IFN-γ and IL-17A in mouse colons, but it dramatically increased the level of IL-10 at both protein and mRNA grades. Consistently, NOR increased the number of CD4(+)CD25(+)Foxp3(+) Treg cells more obviously than it decreased that of CD4(+)IL-17(+) Th17 cells in mesenteric lymph nodes (MLNs) and colonic lamina proprias (LPs) of colitis mice, and promoted the expression of Foxp3 mRNA in colon tissues. It could facilitate the in vitro differentiation of Treg cells from naive T cells and promote the phosphorylations of Smad2/3 in colon tissues of colitis mice. On the other hand, NOR did not affect the expressions of homing receptors CCR9 and α4ß7 in SPs, and homing ligands CCL25 and Madcam-1 in MLNs and colonic LPs, suggesting that the increase of Treg cells in colons by NOR was not due to gut homing. In conclusion, NOR can ameliorate DSS-induced UC in mice, and the mechanisms involve reduction of pro-inflammatory cytokines and selective induction of Treg cells in colons.

[Effect of Madecassoside on Intestinal Mucosal Immunity in Collagen-Induced Arthritis Rats].

OBJECTIVE: To explore the changes of intestinal mucosal immunity in collagen-induced arthritis rats and the impact of madecassoside on these changes. METHODS: Collagen-induced arthritis was established in female Wistar rats. Treatment group was orally administrated madecassoside once daily for consecutive 21 days, while blank control and model groups were orally administered saline at the same volume. The concentrations of sIgA in small intestine content and IFN-γ in small intestinal tissue homogenate were determined by ELISA. The proportions of CD4+ T and CD8+ T in the epithelium and laminar propria of small intestine were detected by flow cytometry, and the ratios of CD4+/CD8+ were calculated. The relative expressions of CD80, CD86, IL-6, IL-12 and Foxp3 mRNA in the small intestine were determined by real-time PCR. RESULTS: Compared with blank control rats, the concentrations of sIgA in small intestine content and IFN-γ in small intestinal tissue homogenate from model rats were increased, the ratios of CD4+/CD8+ in the epithelium and laminar propria of small intestine were higher and the relative expressions of CD80, CD86, IL-6 and IL-12 mRNA in the small intestine were increased. Madecassoside treatment decreased the concentrations of sIgA in small intestine content and IFN-γ in small intestinal tissue, downregulated the ratios of CD4+/CD8+ in the epithelium and laminar propria and decreased the relative expressions of CD80, CD86, IL-6 and IL-12 mRNA, while upregulated the relative expression of Foxp3 mRNA in the small intestine. CONCLUSION: The intestinal mucosal immune response is enhanced in collagen-induced arthritis rats, the antigen presenting cells are activated abnormally and the immune tolerance is disturbed. Madecassoside treatment can downregulate the intestinal mucosal immune response and benefit for the induction and maintenance of intestinal immune tolerance.

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.

Comparative pharmacokinetics of arctigenin in normal and type 2 diabetic rats after oral and intravenous administration.

Arctigenin is the main active ingredient of Fructus Arctii for the treatment of type 2 diabetes. In this study, the pharmacokinetics of arctigenin in normal and type 2 diabetic rats following oral and intravenous administration was investigated. As compared to normal rats, Cmax and AUC(0-10h) values of oral arctigenin in diabetic rats increased by 356.8% and 223.4%, respectively. In contrast, after intravenous injection, the Cmax and AUC(0-10h) values of arctigenin showed no significant difference between diabetic and normal rats. In order to explore how the bioavailability of oral arctigenin increased under diabetic condition, the absorption behavior of arctigenin was evaluated by in situ single-pass intestinal perfusion (SPIP). The results indicated that arctigenin was a substrate of P-glycoprotein (P-gp). The absorption difference of arctigenin in the normal and diabetic rats could be eliminated by the pretreatment of classic P-gp inhibitor verapamil, suggesting that P-gp might be the key factor causing the absorption enhancement of arctigenin in diabetic rats. Further studies revealed that the uptake of rhodamine 123 (Rho123) in diabetic rats was significantly higher, indicating that diabetes mellitus might impair P-gp function. Consistently, a lower mRNA level of P-gp in the intestine of diabetic rats was found. In conclusion, the absorption of arctigenin after oral administration was promoted in diabetic rats, which might be partially attribute to the decreased expression and impaired function of P-gp in intestines.

SC-III3, a novel scopoletin derivative, induces cytotoxicity in hepatocellular cancer cells through oxidative DNA damage and ataxia telangiectasia-mutated nuclear protein kinase activation.

BACKGROUND: Natural products from plants have been proven to be important resources of antitumor agents. In this study, we exploited the antitumor activity of (E)-3-(4-chlorophenyl)-N-(7-hydroxy-6-methoxy-2-oxo-2H-chromen-3-yl) acrylamide (SC-III3), a newly synthesized derivative of scopoletin, by in vitro and in vivo experiments. METHODS: Human hepatocellular carcinoma cell line HepG2 cells and xenograft of HepG2 cells in BALB/c nude mice were used to investigate the effects of SC-III3 on hepatocellular cancers. Cell cycle arrest and apoptosis were analyzed by flow cytometry. Cell cycle arrest, apoptosis and ATM-Chk pathway-related proteins were characterized by western blot. RESULTS: SC-III3 selectively inhibited the viability of HepG2 cells without significant cytotoxicity against human normal liver cells LO2. In mouse xenograft model of HepG2 cells, SC-III3 showed a marked inhibition of tumor growth in a dose-dependent manner. Cell cycle analysis revealed that SC-III3 induced cells to accumulate in S phase, which was accompanied by a marked decrease of the expressions of cyclin A, cyclin B, cyclin E and Cdk2 proteins, the crucial regulators of S phase cell cycle. SC-III3 treatment resulted in DNA breaks in HepG2 cells, which might contribute to its S phase arrest. The S arrest and the activation of ATM-Chk1/Chk2-Cdc25A-Cdk2 pathways induced by SC-III3 in HepG2 cells could be efficiently abrogated by pretreatments of either Ku55933 (an inhibitor of ATM) or UCN-01 (an inhibitor of Chk1/Chk2). The activation of p53-p21 pathway by SC-III3 was also reversed by Ku55933 treatment. SC-III3 led to significant accumulation of intracellular reactive oxygen species (ROS), a breaker of DNA strand, in HepG2 cells but not LO2 cells. Pretreatment with N-acetyl-l-cysteine (NAC), a ROS scavenger, could reverse SC-III3-caused ROS accumulation, DNA damage, activation of signal pathways relevant to DNA damage, S phase arrest and cell viability decrease in HepG2 cells. CONCLUSION: SC-III3 is able to efficiently inhibit the growth of hepatocellular carcinoma through inducing the generation of intracellular ROS, DNA damage and consequent S phase arrest, but lack of significant cytotoxicity against normal liver cells. This compound deserves further studies as a candidate of anticancer drugs.

A comparative study on improved Arrhenius-type and artificial neural network models to predict high-temperature flow behaviors in 20MnNiMo alloy.

The stress-strain data of 20MnNiMo alloy were collected from a series of hot compressions on Gleeble-1500 thermal-mechanical simulator in the temperature range of 1173 ∼ 1473 K and strain rate range of 0.01 ∼ 10 s(-1). Based on the experimental data, the improved Arrhenius-type constitutive model and the artificial neural network (ANN) model were established to predict the high temperature flow stress of as-cast 20MnNiMo alloy. The accuracy and reliability of the improved Arrhenius-type model and the trained ANN model were further evaluated in terms of the correlation coefficient (R), the average absolute relative error (AARE), and the relative error (η). For the former, R and AARE were found to be 0.9954 and 5.26%, respectively, while, for the latter, 0.9997 and 1.02%, respectively. The relative errors (η) of the improved Arrhenius-type model and the ANN model were, respectively, in the range of -39.99% ∼ 35.05% and -3.77% ∼ 16.74%. As for the former, only 16.3% of the test data set possesses η-values within ± 1%, while, as for the latter, more than 79% possesses. The results indicate that the ANN model presents a higher predictable ability than the improved Arrhenius-type constitutive model.