Jianqun Xie

Curcumin induces apoptosis in human gastric carcinoma AGS cells and colon carcinoma HT-29 cells through mitochondrial dysfunction and endoplasmic reticulum stress.

In the present study, we investigate the effect of curcumin, a major active component isolated from rhizomes of Curcuma longa, on the cytotoxicity of three human carcinoma cell lines (AGS, HT-29 and MGC803) in gastrointestinal tract and a normal gastric epithelial cell line GES-1, and the mechanism of curcumin-induced apoptosis. The results indicated that curcumin inhibited the gastrointestinal carcinoma cell growth in a dose-dependent manner and cytotoxicity was more towards the gastric carcinoma cell AGS and colon carcinoma cell HT-29 compared to normal gastric cell GES-1, and increased externalization of phosphatidylserine residue was observed by Annexin V/PI staining in the two cell lines. Treatment of AGS and HT-29 cells with curcumin enhanced the cleavage of procaspase-3, -7, -8 and -9. Meanwhile, curcumin induced endoplasmic reticulum (ER) stress and mitochondrial dysfunction as evidenced by up-regulation of CCAAT/enhancer binding protein homologous protein (CHOP), phosphorylation of JNK and down-regulation of SERCA2ATPase, release of cytochrome c, decrease of Bcl-2 and reduction of mitochondrial membrane potential in both AGS and HT-29 cells. Overexpression of bax, total JNK, phospho-FADD and total FADD were also observed in curcumin-treated HT-29 cells. Moreover, curcumin decreased cytosolic and ER Ca2+, but increased mitochondrial Ca2+ in the two cell lines. 2-Aminoethoxydiphenyl borate, an antagonist of inositol 1, 4, 5-triphosphate receptor, partly blocked curcumin-induced cytosolic Ca2+ decrease in AGS and HT-29 cells. Additionally, carbonyl cyanide m-chlorophenylhydrazone, an inhibitor of mitochondrial Ca2+ uptake, reversed curcumin-triggered AGS and HT-29 cells growth inhibition. siRNA to CHOP markedly reduced curcumin-induced apoptosis. These results suggest that curcumin can impact on ER stress and mitochondria functional pathways in AGS and HT-29 cells, death receptor pathway was also involved in curcumin-treated HT-29 cells, thus identifying specific well-defined molecular mechanisms that may be targeted by therapeutic strategies.

Endotoxemia unrequired in the pathogenesis of pediatric nonalcoholic steatohepatitis

Nonalcoholic steatohepatitis (NASH), the severe form of nonalcoholic fatty liver disease, is a serious liver complication associated with obesity. Several studies suggest that endotoxemia is associated with nonalcoholic fatty liver disease and NASH. We aimed to study the correlation of gut microbiome composition and the incidence of endotoxemia in obese patients and NASH patients in comparison with normal controls.

Tangeretin, a citrus polymethoxyflavonoid, induces apoptosis of human gastric cancer AGS cells through extrinsic and intrinsic signaling pathways

Tangeretin, a natural polymethoxyflavone present in citrus peel oil, is known to have anticancer activities in breast cancer, colorectal carcinoma and lung carcinoma, yet, the underlying mechanisms of tangeretin in human gastric cancer AGS cells have not been investigated to date. In the present study, the apoptotic mechanisms of tangeretin in AGS cells were explored. It was observed that tangeretin increased the apoptotic rates of AGS cells following treatment with tangeretin for 48 h in a dose-dependent manner by Annexin V-FITC and PI double staining. In addition, characteristic apoptotic morphology such as nuclear shrinkage and apoptotic bodies was observed after Hoechst 33258 staining. Flow cytometric assay showed that treatment of AGS cells with tangeretin decreased the mitochondrial membrane potential (MMP) in a dose-dependent manner, which indicated that mitochondrial dysfunction was involved in the tangeretin-induced apoptosis. Caspase-3, -8 and -9 activities were increased by tangeretin in a dose-dependent manner. Western blotting showed that the protein levels of pro-apoptotic proteins including cleaved caspase-3, cleaved caspase-8, cleaved caspase-9, Bax, Bid, tBid, p53, p21/cip1, Fas and FasL were significantly upregulated by tangeretin. In addition, PFT-α (a p53 inhibitor) reduced the apoptotic rates and the expression of p53, p21, caspase-3 and caspase-9 induced by tangeretin, indicating that tangeretin-induced apoptosis was p53-dependent. In conclusion, these results suggest that tangeretin induces the apoptosis of AGS cells mainly through p53-dependent mitochondrial dysfunction and the Fas/FasL-mediated extrinsic pathway.

Berberine hydrochloride IL-8 dependently inhibits invasion and IL-8-independently promotes cell apoptosis in MDA-MB-231 cells.

Breast cancer, the leading cause of cancer-related mortality worldwide in females, has high metastastic and recurrence rates. The aim of the present study was to evaluate the anti-metastatic and anticancer in situ effect of berberine hydrochloride (BER) in MDA-MB-231 cells. BER dose-dependently inhibited proliferation and the IL-8 secretion of MDA-MB-231 cells. Additional experiments revealed that the inactivation of PI3K, JAK2, NF-κB and AP-1 by BER contributed to the decreased IL-8 secretion. BER abrogated cell invasion induced by IL-8 accompanied with the downregulation of the gene expression of MMP-2, EGF, E-cadherin, bFGF and fibronectin. In addition, BER reduced cell motility but induced G2/M arrest and cell apoptosis in an IL-8‑independent manner. BER modulated multiple signaling pathway molecules involved in the regulation of cell apoptosis, including activation of p38 MAPK and JNK and deactivation of JAK2, p85 PI3K, Akt and NF-κB. The enhanced cell apoptosis induced by BER was eliminated by inhibitors of p38 MAPK and JNK but was strengthened by activator of p38 MAPK. Thus, BER inhibited cell metastasis partly through the IL-8 mediated pathway while it induced G2/M arrest and promoted cell apoptosis through the IL-8 independent pathway. Apoptosis induced by BER was mediated by crosstalks of various pathways including activation of p38 MAPK and JNK pathways and inactivation of Jak2/PI3K/NF-κB/AP-1 pathways. The results suggested that BER may be an efficient and safe drug candidate for treating highly metastatic breast cancer.

The effects of jatrorrhizine on contractile responses of rat ileum.

This study was designed to evaluate the effect of jatrorrhizine on smooth muscle contractions isolated from rat ileum longitudinal muscles. Jatrorrhizine increased the amplitude of spontaneous contractions of ileum longitudinal muscles in concentration-dependent manner with an EC(50) of 30.0±8.4μM. Preincubation of ileum strips with atropine (1μM), 4-diphenyllacetoxy-N (2-chloriethyl)-piperidine (4-DAMP, 1μM) or darifenacin (1μM) significantly inhibited acetylcholine (0.1μM)- and jatrorrhizine (100μM)-induced ileum longitudinal muscle contractions, whereas they were not affected by AF-DX116 (1μM) or hexamethonium (100μM). Pretreatment with SB204070 (1μM) rather than 3-tropanyl-indole-3-carboxyleat (tropisetron, 1μM) significantly inhibited 5-HT (10μM)-induced ileum longitudinal muscle contractions. In contrast, jatrorrhizine-induced ileum longitudinal muscle contractions were not inhibited by tropisetron or SB204070. Furthermore, jatrorrhizine-induced ileum longitudinal muscle contractions were strongly inhibited by nifedipine (1μM), and also attenuated by removal of extracellular Ca(2+), U73122 (1μM), ruthenium red (50μM) or 2-aminoethoxydiphenylborate (2-APB, 10μM). Taken together, jatrorrhizine-elicited spontaneous contractions in rat ileum longitudinal muscles are mediated by activation of acetylcholine receptors, mostly the M(3) receptor. Ca(2+) influx through L-type Ca(2+) channel is significantly contributed to jatrorrhizine-elicited spontaneous contractions, and Ca(2+) release via IP(3) and ryanodine pathways are also involved.

Alterations in serotonin, transient receptor potential channels and protease-activated receptors in rats with irritable bowel syndrome attenuated by Shugan decoction

AIM To determine the molecular mechanisms of Shugan decoction (SGD) in the regulation of colonic motility and visceral hyperalgesia (VHL) in irritable bowel syndrome (IBS). METHODS The chemical compounds contained in SGD were measured by high-performance liquid chromatography. A rat model of IBS was induced by chronic water avoidance stress (WAS). The number of fecal pellets was counted after WAS and the pain pressure threshold was measured by colorectal distension. Morphological changes in colonic mucosa were detected by hematoxylin-eosin staining. The contents of tumor necrosis factor (TNF)-α in colonic tissue and calcitonin-gene-related peptide (CGRP) in serum were measured by ELISA. The protein expression of serotonin [5-hydroxytryptamide (5-HT)], serotonin transporter (SERT), chromogranin A (CgA) and CGRP in colon tissue was measured by immunohistochemistry. RESULTS SGD inhibited colonic motility dysfunction and VHL in rats with IBS. Blockers of transient receptor potential (TRP) vanilloid 1 (TRPV1) (Ruthenium Red) and TRP ankyrin-1 (TRPA1) (HC-030031) and activator of protease-activated receptor (PAR)4 increased the pain pressure threshold, whereas activators of PAR2 and TRPV4 decreased the pain pressure threshold in rats with IBS. The effect of SGD on pain pressure threshold in these rats was abolished by activators of TRPV1 (capsaicin), TRPV4 (RN1747), TRPA1 (Polygodial) and PAR2 (AC55541). In addition, CGRP levels in serum and colonic tissue were both increased in these rats. TNF-α level in colonic tissue was also significantly upregulated. However, the levels of 5-HT, SERT and CgA in colonic tissue were decreased. All these pathological changes in rats with IBS were attenuated by SGD. CONCLUSION SGD alleviated VHL and attenuated colon motility in IBS, partly by regulating TRPV1, TRPV4, TRPA1, PAR2, 5-HT, CgA and SERT, and reducing CGRP and TNF-α level.

Potassium channel KCNJ15 is required for histamine-stimulated gastric acid secretion

Gastric acid secretion is mediated by the K(+)-dependent proton pump (H(+),K(+)-ATPase), which requires a continuous supply of K(+) at the luminal side of the apical membrane. Several K(+) channels are implicated in gastric acid secretion. However, the identity of the K(+) channel(s) responsible for apical K(+) supply is still elusive. Our previous studies have shown the translocation of KCNJ15 from cytoplasmic vesicles to the apical membrane on stimulation, indicating its involvement in gastric acid secretion. In this study, the stimulation associated trafficking of KCNJ15 was observed in a more native context with a live cell imaging system. KCNJ15 molecules in resting live cells were scattered in cytoplasm but exhibited apical localization after stimulation. Furthermore, knocking down KCNJ15 expression with a short hairpin RNA adenoviral construct abolished histamine-stimulated acid secretion in rabbit primary parietal cells. Moreover, KCNJ15, like H(+),K(+)-ATPase, was detected in all of the parietal cells by immunofluorescence staining, whereas only about half of the parietal cells were positive for KCNQ1 under the same condition. Consistently, the endogenous protein levels of KCNJ15, analyzed by Western blotting, were higher than those of KCNQ1 in the gastric mucosa of human, mouse, and rabbit. These results provide evidence for a major role of KCNJ15 in apical K(+) supply during stimulated acid secretion.

The ethanol extract isolated from Weiqi Decoction (胃祺饮) induces G2/M arrest and apoptosis in AGS cells

ObjectiveTo evaluate the effects of the ethanol extract isolated from Weiqi Decoction (胃祺饮, WQD-EE) on AGS cell proliferation and apoptosis.MethodsBy using high-performance liquid chromatography with ultraviolet detectors (HPLC-UV) assay and MTT method, the main compounds in WQD-EE and cell viability were detected. And cell cycle distributions were determined by flow cytometry with propidium iodine (PI) staining while apoptosis was detected by flow cytometry with annexin V/PI double staining. Finally, caspase-3 activities were measured by colorimetric method and protein expression was determined by Western blotting.ResultsHPLC analysis showed that naringin (35.92 μg/mg), nobiletin (21.98 μg/mg), neohesperidin (17.98 μg/mg) and tangeretin (0.756 μg/mg) may be the main compounds in WQD-EE. WQD-EE not only inhibited AGS and MCF 7 cell proliferation in a dose-dependent manner, but also blocked cell cycle progression at G2/M stage as well as inducing cell apoptosis at concentrations triggering significant inhibition of proliferation and cell cycle arrest in AGS cells. While at 0.5 mg/mL, WQD-EE significantly increased caspase-3 activity by 2.75 and 7.47 times at 24 h and 48 h, respectively. Moreover, WQD-EE in one hand reduced protein expressions of p53 and cyclin B1, and in other hand enhanced protein expressions of cytochrome c and Bax. Protein levels of Bcl-2, Fas L and Fas were not significantly affected by WQD-EE.ConclusionsWQD-EE inhibits AGS cell proliferation through G2/M arrest due to down-regulation of cyclin B1 protein expression, and promotes apoptosis by caspase-3 and mitochondria-dependent pathways, but not by p53-dependent pathway.

Tu1011 Serum Endotoxin Is Not Associated With Pediatric Non-Alcoholic Steatohepatitis

Introduction: Esophageal varices (EV) bleeding is a severe complication of portal hypertension and can be fatal. The standard diagnostic screening tool for EV is endoscopy, which could be used as treatment of large esophageal varices, but it is still considered an invasive procedure in pediatric patients. Aim: to evaluate clinical and laboratory parameters in predicting large EV in children with intra-hepatic portal hypertension. Methods: We studied retrospectively eighty eight children (mean age: 10.1 ±7.7) with intra-hepatic portal hypertension All patients had no bleeding history and they underwent upper GI endoscopy for EV screening. We recorded variceal size (F1, F2 and F3), according to The Japan Society for Portal Hypertension classification. Patients were classified into two groups: small (F1) and no varices and large varices (F2 and F3). We evaluate seven noninvasive markers in predicting large EV: (1) platelet count; (2) spleen z score, expressed as a standard deviation score relative to normal values for age; (3) platelets count: spleen z score ratio; (4) platelets count: spleen size (cm) ratio; (5) the Clinical Prediction Rule (CPR); (6)APRI test and (7) risk score. Results: Thirty one children had large EV in the first endoscopy. In univariate analyses, platelet count, CPR, risk score, platelet count: spleen z score ratio and Child-Pugh Classification were statistically significant. The best noninvasive predictors of large varices were: platelets (AUROC 0.67; IC 95%, 0.57-0.78), CPR (AUROC 0.65; IC 95%, 0.54-0.76) and risk score (AUROC 0.66; IC 95%: 0.56-0.76). A logistic regression model was applied with large esophageal varices as the dependent variable and corrected by albumin, Child-PughClassification, bilirubin and spleen size z score. Children with CPR under 114 were 8.59-fold more likely to have large esophageal varices compared to children with CPR.114. Risk score .1.2 increased the likelihood of large varices (odds ratio 6.09; 95% CI,1.43-25.90; p=0.014). Using Child-Pugh Classification (CPC), a negative predictive value (NPV) of 92.3% for risk score over -1.2 and NPV of 88.5% for CPR under 114 in children with CPC-A for large varices. Conclusions: Children with intra-hepatic portal hypertension with CPR below 114 and risk score greater than-1.2 have more chance to present large EV. According to these results, patients with CPC-A and risk score under -1.2 and CPR over 114 have low risk of large EV. Therefore those two tests could be helpful to select cirrhotic children to endoscopic treatment.