Hesheng Luo

Neurogenic and myogenic properties of pan-colonic motor patterns and their spatiotemporal organization in rats.

Background and Aims Better understanding of intrinsic control mechanisms of colonic motility will lead to better treatment options for colonic dysmotility. The aim was to investigate neurogenic and myogenic control mechanisms underlying pan-colonic motor patterns. Methods Analysis of in vitro video recordings of whole rat colon motility was used to explore motor patterns and their spatiotemporal organizations and to identify mechanisms of neurogenic and myogenic control using pharmacological tools. Results Study of the pan-colonic spatiotemporal organization of motor patterns revealed: fluid-induced or spontaneous rhythmic propulsive long distance contractions (LDCs, 0.4–1.5/min, involving the whole colon), rhythmic propulsive motor complexes (RPMCs) (0.8–2.5/min, dominant in distal colon), ripples (10–14/min, dominant in proximal colon), segmentation and retrograde contractions (0.1–0.8/min, prominent in distal and mid colon). Spontaneous rhythmic LDCs were the dominant pattern, blocked by tetrodotoxin, lidocaine or blockers of cholinergic, nitrergic or serotonergic pathways. Change from propulsion to segmentation and distal retrograde contractions was most prominent after blocking 5-HT3 receptors. In the presence of all neural blockers, bethanechol consistently evoked rhythmic LDC-like propulsive contractions in the same frequency range as the LDCs, indicating the existence of myogenic mechanisms of initiation and propulsion. Conclusions Neurogenic and myogenic control systems orchestrate distinct and variable motor patterns at different regions of the pan-colon. Cholinergic, nitrergic and serotonergic pathways are essential for rhythmic LDCs to develop. Rhythmic motor patterns in presence of neural blockade indicate the involvement of myogenic control systems and suggest a role for the networks of interstitial cells of Cajal as pacemakers.

Down-Regulation of miR-126 Is Associated with Colorectal Cancer Cells Proliferation, Migration and Invasion by Targeting IRS-1 via the AKT and ERK1/2 Signaling Pathways

Background Colorectal carcinoma (CRC) is one of the leading causes of cancer-related mortality worldwide. MicroRNAs (miRNAs, miRs) play important roles in carcinogenesis. MiR-126 has been shown to be down-regulated in CRC. In this study, we identified the potential effects of miR-126 on some important biological properties of CRC cells and clarified the regulation of insulin receptor substrate 1 (IRS-1) and its possible signaling pathway by miR-126. Methods The effect of miR-126 on IRS-1, AKT, and ERK1/2 expression was assessed in the CRC cell lines HT-29 and HCT-116 with a miR-126 mimic or inhibitor to increase or decrease miR-126 expression. Furthermore, the roles of miR-126 in regulation of the biological properties of CRC cells were analyzed with miR-126 mimic or inhibitor-transfected cells. The 3′-untranslated region (3′-UTR) of IRS-1 regulated by miR-126 was analyzed by using a dual-luciferase reporter assay. Results We found that IRS-1 is the functional downstream target of miR-126 by directly targeting the 3′-UTR of IRS-1. Endogenous miR-126 and exogenous miR-126 mimic inhibited IRS-1 expression. Furthermore, gain-of-function or loss-of-function studies showed that over-expression of miR-126 down-regulated IRS-1, suppressed AKT and ERK1/2 activation, CRC cells proliferation, migration, invasion, and caused cell cycle arrest, but had no effect on cell apoptosis. Knockdown of miR-126 promoted these processes in HCT-116 cells and promoted AKT and ERK1/2 activation by up-regulating the expression of the IRS-1 protein. Conclusions MiR-126 may play roles in regulation of the biological behavior of CRC cells, at least in part, by targeting IRS-1 via AKT and ERK1/2 signaling pathways.

MicroRNA-126 functions as a tumor suppressor in colorectal cancer cells by targeting CXCR4 via the AKT and ERK1/2 signaling pathways

Recent evidence shows that altered microRNA-126 (miR-126) expression is implicated in the progression of colorectal cancer (CRC). However, the precise roles and mechanisms of miR-126 in CRC remain unclear. The aim of this study was to investigate the roles of miR-126 in CRC cells and to elucidate miR-126-mediated mechanisms in CRC cells. First, miR-126 expression was analyzed using qRT-PCR in 4 human CRC cell lines (SW480, SW620, HT-29 and HCT-116). Furthermore, the biological properties of miR-126 in CRC cells in vitro were examined by applying Cell Counting Kit 8, cell cycle, cell apoptosis and transwell assays. The mechanisms and pathways of miR-126-mediated in CRC cells were detected by using qRT-PCR, western blotting and luciferase reporter assay. We found that miR-126 overexpression inhibited cell proliferation, migration and invasion, and induced cell arrest in the G0/G1 phase of CRC cells, suggesting that miR-126 functions as a tumor suppressor in CRC cells. Furthermore, we identified the CXC chemokine receptor 4 (CXCR4) as a target of miR-126, and showed that it was negatively regulated by miR-126. We demonstrated that miR-126-mediated tumor suppression might be partly dependent on AKT and ERK1/2 signaling pathways. In conclusion, our data revealed that miR-126 functions as a tumor suppressor in CRC cells by regulating CXCR4 expression via the AKT and ERK1/2 signaling pathways and might be a novel target for therapeutic strategies in CRC.

Low expression of MicroRNA‐126 is associated with poor prognosis in colorectal cancer

MicroRNA‐126 (miR‐126) has been reported to be a tumor suppressor that targets CXCR4 in colorectal cancer (CRC) cells. This study investigated whether miR‐126 has any prognostic impact in patients with CRC. MiR‐126 and CXCR4 mRNA expression in 92 pairs of CRC and adjacent nontumorous tissues was examined using quantitative real‐time PCR, and CXCR4 protein expression was assessed by immunohistochemistry (IHC) and Western blotting. The correlation between miR‐126 and CXCR4 protein expression and clinicopathological features and overall survival rate was determined. MiR‐126 was downregulated in CRC tissues that expressed high levels of CXCR4 mRNA. IHC and Western blotting detected high expression of CXCR4 protein in CRC tissues. An inverse correlation was observed between miR‐126 and CXCR4 protein expression in CRC tissues. Moreover, low miR‐126 and high CXCR4 protein expression was associated with distant metastasis, clinical TNM stage, and poor survival. Multivariate analysis indicated that miR‐126 was an independent prognostic factor for overall survival, suggesting its clinical significance as a prognostic predictor in CRC patients.

Differential diagnosis between Crohn's disease and intestinal tuberculosis in China.

To evaluate the values of clinical findings in differential diagnosis between Crohns disease (CD) and intestinal tuberculosis (ITb), so that better diagnosis strategies could be found, we analysed the records of 30 patients with ITb and 30 patients with CD retrospectively. We reviewed the cardinal symptomatic and physical, radiological, colonoscopic, pathological findings in the patients. Some cardinal symptomatic and physical, radiographic and colonscopic features of CD and ITb were positive correlation. Its Pearson correlation coefficients were 0.976, 0.953 and 0.961, respectively (p = 0.000, 0.003 and 0.000). Some histological features of CD and ITb had no correlation. Its Pearson correlation coefficient was 0.140 (p = 0.765). It is difficult to differentiate CD from ITb in clinical practice. The differential diagnosis depends largely upon colonoscopy and histopathology.

Osteopontin promotes the progression of gastric cancer through the NF-κB pathway regulated by the MAPK and PI3K

To elucidate the biological functions of osteopontin (OPN) in gastric tumors and to better understand the molecular events of OPN responsible for the malignancy, the present studies were performed. Growth curve, apoptosis assay, invasion assay and migration assay revealed that OPN status significantly affected proliferation, apoptosis, invasion and migration in gastric cancer cell lines. In mouse xenograft models of human gastric cancer, OPN silencing significantly inhibited tumor growth and the incidence of metastasis compared with non-silenced control. Mechanistic investigations revealed that OPN silencing inhibited the mitogen-activated protein kinase (MAPK), phosphoinositide 3-kinase (PI3K) and phosphoinositide 3-kinase (NF-κB) pathways, and OPN-mediated NF-κB activity was reduced in the presence of MAPK or PI3K inhibitor. Our findings also indicated that OPN through the NF-κB pathway promotes the expression of matrix metalloproteinase 2 (MMP-2), MMP-9 and urokinase-type plasminogen activator (uPA), the activation of MMP-2 and MMP-9, and the inhibition of caspase-3. Taken together, our results reveal that OPN promotes the progression of gastric cancer through the NF-κB pathway, which is regulated by the MAPK and PI3K pathways and leads to MMP-2, MMP-9 and uPA activation and caspase-3 inhibition. These findings identify OPN as a novel oncogene in gastric cancer and suggest that OPN is an attractive therapeutic target for this aggressive malignancy.

The DNA methyltransferase inhibitor zebularine induces mitochondria-mediated apoptosis in gastric cancer cells in vitro and in vivo.

DNA methyltransferase (DNMT) inhibitor zebularine has been reported to potentiate the anti-tumor effect by reactivating the expression of tumor suppressor genes and apoptosis-related genes in various malignant cells. However, the apoptotic signaling pathway in gastric cancer cells induced by zebularine is not well understood. In the study, the effects of zebularine on the growth and apoptosis of gastric cancer cells were investigated by MTT assay, Hoechst assay, Western blot analysis, flow cytometric analysis of annexin V-FITC/PI staining, and TUNEL assay. Zebularine was an effective inhibitor of human gastric cancer cells proliferation in vitro and in vivo. The effects were dose dependent. A zebularine concentration of 50 μM accounted for the inhibition of cell proliferation of 67% at 48 h. The treatment with zebularine upregulated Bax, and decreased Bcl-2 protein. Caspase-3 was activated, suggesting that the apoptosis is mediated by mitochondrial pathways. Moreover, zebularine injection successfully inhibited the tumor growth via apoptosis induction which was demonstrated by TUNEL assay in xenograft tumor mouse model. These results demonstrated that zebularine induced apoptosis in gastric cancer cells via mitochondrial pathways, and zebularine might become a therapeutic approach for the treatment of gastric cancer.

Plasma Hormones Facilitated the Hypermotility of the Colon in a Chronic Stress Rat Model

Objective To study the relationship between brain-gut peptides, gastrointestinal hormones and altered motility in a rat model of repetitive water avoidance stress (WAS), which mimics the irritable bowel syndrome (IBS). Methods Male Wistar rats were submitted daily to 1-h of water avoidance stress (WAS) or sham WAS (SWAS) for 10 consecutive days. Plasma hormones were determined using Enzyme Immunoassay Kits. Proximal colonic smooth muscle (PCSM) contractions were studied in an organ bath system. PCSM cells were isolated by enzymatic digestion and IKv and IBKca were recorded by the patch-clamp technique. Results The number of fecal pellets during 1 h of acute restraint stress and the plasma hormones levels of substance P (SP), thyrotropin-releasing hormone (TRH), motilin (MTL), and cholecystokinin (CCK) in WAS rats were significantly increased compared with SWAS rats, whereas vasoactive intestinal peptide (VIP), calcitonin gene-related peptide (CGRP) and corticotropin releasing hormone (CRH) in WAS rats were not significantly changed and peptide YY (PYY) in WAS rats was significantly decreased. Likewise, the amplitudes of spontaneous contractions of PCSM in WAS rats were significantly increased comparing with SWAS rats. The plasma of WAS rats (100 µl) decreased the amplitude of spontaneous contractions of controls. The IKv and IBKCa of PCSMs were significantly decreased in WAS rats compared with SWAS rats and the plasma of WAS rats (100 µl) increased the amplitude of IKv and IBKCa in normal rats. Conclusion These results suggest that WAS leads to changes of plasma hormones levels and to disordered myogenic colonic motility in the short term, but that the colon rapidly establishes a new equilibrium to maintain the normal baseline functioning.

Actions of hydrogen sulfide and ATP-sensitive potassium channels on colonic hypermotility in a rat model of chronic stress.

Objective To investigate the potential role of hydrogen sulphide (H2S) and ATP-sensitive potassium (KATP) channels in chronic stress-induced colonic hypermotility. Methods Male Wistar rats were submitted daily to 1 h of water avoidance stress (WAS) or sham WAS (SWAS) for 10 consecutive days. Organ bath recordings, H2S production, immunohistochemistry and western blotting were performed on rat colonic samples to investigate the role of endogenous H2S in repeated WAS-induced hypermotility. Organ bath recordings and western blotting were used to detect the role of KATP channels in repeated WAS. Results Repeated WAS increased the number of fecal pellets per hour and the area under the curve of the spontaneous contractions of colonic strips, and decreased the endogenous production of H2S and the expression of H2S-producing enzymes in the colon devoid of mucosa and submucosa. Inhibitors of H2S-producing enzymes increased the contractile activity of colonic strips in the SWAS rats. NaHS concentration-dependently inhibited the spontaneous contractions of the strips and the NaHS IC50 for the WAS rats was significantly lower than that for the SWAS rats. The inhibitory effect of NaHS was significantly reduced by glybenclamide. Repeated WAS treatment resulted in up-regulation of Kir6.1 and SUR2B of KATP channels in the colon devoid of mucosa and submucosa. Conclusion The colonic hypermotility induced by repeated WAS may be associated with the decreased production of endogenous H2S. The increased expression of the subunits of KATP channels in colonic smooth muscle cells may be a defensive response to repeated WAS. H2S donor may have potential clinical utility in treating chronic stress- induced colonic hypermotility.

Hydrogen Sulfide Regulates the Colonic Motility by Inhibiting Both L-Type Calcium Channels and BKCa Channels in Smooth Muscle Cells of Rat Colon

Objective To examine the hypothesis that hydrogen sulfide (H2S) regulates the colonic motility by modulating both L-type voltage-dependent calcium channels and large conductance Ca2+-activated K+ (BKCa) channels. Methods Immunohistochemistry was performed on rat colonic samples to investigate the localization of the H2S-producing enzymes cystathionine-β-synthase (CBS) and cystathionine-γ-lyase (CSE). The contractions of proximal colonic smooth muscle were studied in an organ bath system. The whole-cell patch-clamp technique was used to record both L-type calcium currents (I Ca,L) and BKCa currents in colonic smooth muscle cells (SMCs) isolated from male Wistar rats. Results Immunohistochemistry revealed the presence of CBS and CSE in mucosa, smooth muscle cells and myenteric neurons. The H2S donor NaHS inhibited spontaneous contractions of the longitudinal muscle and circular muscle strips in a dose-dependent manner, and the inhibitory effects were not blocked by tetrodotoxin. NaHS inhibited the peak I Ca,L in colonic SMCs at a membrane potential of 0 mV. The current-voltage (I-V) relationship of L-type calcium channels was modified by NaHS, and the peak of the I-V curve was shifted to the right. NaHS (200μΜ) evoked a significant rightward shift of the steady-state activation curve and inhibited the inactivation of L-type calcium channels. Furthermore, NaHS reversibly decreased the peak I Ca,L in a dose-dependent manner. Likewise, BKCa channels were significantly inhibited by NaHS, and the addition of NaHS caused a time- and dose-dependent reduction in the BKCa current. Conclusion The relaxant effect of H2S on colonic muscle strips may be associated with the direct inhibition of H2S on L-type calcium channels. H2S may be involved in the regulation of calcium homeostasis in colonic SMCs of rat colon.