Corticotropin-releasing factor receptor agonists decrease interstitial cells of Cajal in murine colon.

BACKGROUND: Peripheral corticotropin-releasing factor (CRF) has been reported to affect gastrointestinal motility through corticotropin-releasing factor receptor located in enteric nervous system (ENS), but less is known about of the relationship between peripheral CRF and interstitial cells of Cajal (ICC). METHODS: Mice were intraperitoneally injected with CRF receptor agonists to determine their effects on colonic ICC. Chronic heterotypic stress (CHeS) was applied to mice to determine endogenous CRF-CRF receptor signaling on colonic ICC. RESULTS: We found that stressin1, a selective CRF receptor 1 (CRF1 ) agonist, significantly increased the expression of CRF1 but had no effect on the expression of CRF2 in the smooth muscles of murine colon. The protein expression of c-Kit, Anoctamin-1 (ANO1), and stem cell factor (SCF) in the colonic smooth muscles was significantly decreased in stressin1-treated mice. Accordingly, 2-(4-Chloro-2-methylphenoxy)-N'-(2-methoxybenzylidene) acetohydrazide (Ani 9), a selective ANO1 blocker, had a less significant inhibitory effect on CMMC in stressin1-treated mice compared to the saline-treated ones. Similarly, we also found that ICC and ANO1 were reduced in the colonic smooth muscles of mice by treatment with sauvagine (ip), a CRF2 agonist. However, different with stressin1, sauvagine decreased the expression of CRF2 besides increasing CRF1 expression in the colonic smooth muscles. Similar results of CRF1 and c-Kit expressions were also obtained from the colon of CHeS-treated mice. CONCLUSION: All these results suggest that CRF may be involved in the abnormality of colonic motility through peripheral CRF1 to decrease the number and function of ICC, which provides a potential target for treating stress-induced gastrointestinal motility disorder.

Involvement of PAR2 in platelet-derived growth factor receptor-α-positive cell proliferation in the colon of diabetic mice.

Our previous study indicated that streptozotocin (STZ)-induced diabetes leads to colonic platelet-derived growth factor receptor-α-positive (PDGFRα+ ) cell proliferation accompanied by slow colonic transit in mice; however, the mechanism of this effect is unclear. The present study used western blotting, immunohistochemistry, and quantitative PCR to investigate whether proteinase-activated receptor 2 (PAR2) mediates PDGFRα+ cell proliferation. Our results showed that PDGFRα, PAR2, and Ki-67 coexpression was increased in the diabetic colonic muscle layer. PDGFRα and PAR2 mRNA and protein expression levels were also markedly enhanced in the diabetic colonic muscle layer. Mice treated with 2-furoyl-LIGRLO-amide (2-F-L-a), a PAR2 agonist, exhibited significant colon elongation and increased smooth muscle weight. In the 2-F-L-a-treated mice, PDGFRα, PAR2, and Ki-67 coexpression was increased and PDGFRα and PAR2 mRNA and protein expression was significantly enhanced in the colonic smooth muscle layer. 2-F-L-a also increased proliferation and PDGFRα expression in NIH/3T3 cells cultured in high glucose, while LY294002, a PI3K antagonist, decreased cell proliferation and PDGFRα expression. PI3K and Akt protein and mRNA expression and p-Akt protein expression in diabetic and 2-F-L-a-treated mice were markedly reduced in colonic smooth muscle. 2-F-L-a also reduced PI3K, Akt, and p-Akt protein expression in NIH/3T3 cells, while the PI3K antagonist LY294002 increased this expression. The results indicate that PAR2 is involved in the proliferation of PDGFRα+ cells through the PI3K/Akt signaling pathway in the colon of STZ-induced diabetic mice, which may contribute to the slow transit and constipation that are associated with diabetes.

Cynandione A Alleviates Neuropathic Pain Through α7-nAChR-Dependent IL-10/ß-Endorphin Signaling Complexes.

Cynandione A, an acetophenone isolated from Cynanchum Wilfordii Radix, exhibits antineuropathic pain effect. This study further explored the target molecule and signaling mechanisms underlying cynandione-A-induced antineuropathic pain. Intrathecal injection of cynandione A significantly attenuated mechanical allodynia in neuropathic rats and substantially increased spinal expression of IL-10 and ß-endorphin but not dynorphin A. Cynandione A treatment also enhanced expression of IL-10 and ß-endorphin but not α7 nicotinic acetylcholine receptors (nAChRs) in cultured microglia. The IL-10 antibody attenuated cynandione-A-induced spinal or microglial gene expression of ß-endorphin and mechanical allodynia, whereas the ß-endorphin antiserum blocked cynandione-A-induced mechanical antiallodynia but not spinal or microglial IL-10 gene expression. The α7 nAChR antagonist methyllycaconitine significantly reduced cynandione-A-induced mechanical antiallodynia and spinal or microglial expression of IL-10 and ß-endorphin. Furthermore, cynandione A stimulated microglial phosphorylation of PKA, p38, and CREB in an α7-nAChR-dependent manner, and treatment with their inhibitors attenuated cynandione-A-induced mechanical antiallodynia and spinal or microglial expression of IL-10 and ß-endorphin. In addition, cynandione A stimulated spinal phosphorylation of the transcription factor STAT3, which was inhibited by methyllycaconitine, the PKA activation inhibitor or IL-10 antibody. The STAT3 inhibitor NSC74859 also abolished cynandione-A-induced mechanical antiallodynia and spinal expression of ß-endorphin. These findings suggest that cynandione A suppresses neuropathic pain through α7-nAChR-dependent IL-10/ß-endorphin signaling pathway in spinal microglia.

Lappaconitine, a C18-diterpenoid alkaloid, exhibits antihypersensitivity in chronic pain through stimulation of spinal dynorphin A expression.

Lappaconitine is a representative C18-diterpenoid alkaloid extracted from Aconitum sinomontanum Nakai and has been prescribed as a pain relief medicine in China for more than 30 years. This study evaluated its antihypersensitivity activity in the rat models of neuropathic and cancer pains and explored its underlying mechanisms. Subcutaneous injection of cumulative doses of lappaconitine produced dose-dependent mechanical antiallodynia and thermal antihyperalgesia in spinal nerve ligation-induced neuropathic rats. The cumulative dose-response analysis exhibited their Emax values of 53.3 and 58.3% MPE, and ED50 values of 1.1 and 1.6 mg/kg. Single intrathecal lappaconitine dose in neuropathy also dose- and time-dependently blocked mechanical allodynia, with an Emax of 66.1% MPE and an ED50 of 0.8 µg. Its multiple twice-daily intrathecal administration over 7 days did not induce mechanical antiallodynic tolerance. Subcutaneous cumulative doses of lappaconitine also produced dose-dependent blockade of mechanical allodynia in the rat bone cancer pain model induced by tibia implantation of cancer cells, with the Emax of 57.9% MPE and ED50 of 2.0 mg/kg. Furthermore, lappaconitine treatment stimulated spinal dynorphin A expression in neuropathic rats, and in primary cultures of microglia but not neurons or astrocytes. Intrathecal pretreatment with the specific microglia depletor liposome-encapsulated clodronate, dynorphin A antibody, and κ-opioid receptor antagonist GNTI totally suppressed intrathecal and subcutaneous lappaconitine-induced mechanical antiallodynia. This study suggests that lappaconitine exhibits antinociception through directly stimulating spinal microglial dynorphin A expression. Graphical Abstract ᅟ.

Silencing of MICAL-L2 suppresses malignancy of ovarian cancer by inducing mesenchymal-epithelial transition.

Ovarian cancer remains the disease with the highest associated mortality rate of gynecologic malignancy due to cancer metastasis. Rearrangement of actin cytoskeleton by cytoskeleton protein plays a critical role in tumor cell metastasis. MICAL-L2, a member of MICAL family, can interact with actin-binding proteins, regulate actin cross-linking and coordinate the assembly of adherens junctions and tight junctions. However, the roles of MICAL-L2 in tumors and diseases have not been explored. In this study, we found that MICAL-L2 protein is significantly up-regulated in ovarian cancer tissues along with FIGO stage and associated with histologic subgroups of ovarian cancer. Silencing of MICAL-L2 suppressed ovarian cancer cell proliferation, migration and invasion ability. Moreover, silencing of MICAL-L2 prevented nuclear translocation of ß-catenin, inhibited canonical wnt/ß-catenin signaling and induced the mesenchymal-epithelial transition (MET). Taken together, our data indicated that MICAL-L2 may be an important regulator of epithelial-mesenchymal transition (EMT) in ovarian cancer cells and a new therapeutic target for interventions against ovarian cancer invasion and metastasis.

Silencing of WISP3 suppresses gastric cancer cell proliferation and metastasis and inhibits Wnt/ß-catenin signaling.

CCN6/Wnt1-inducible signaling protein-3 (CCN6/WISP3) is a cysteine-rich protein that belongs to the CCN (Cyr61, CTGF, Nov) family of matricellular proteins, which are often dysregulated in cancers. However, the functional role and clinical significance of WISP3 in gastric cancer remain unclear. In this study, we found that silencing of WISP3 suppressed gastric cancer cell proliferation, migration and invasion. Cell adhesion to collagens (collagen I and IV), but not to fibronectin, were significantly inhibited by silencing of WISP3. Furthermore, silencing of WISP3 prevented ß-catenin transferring from cell cytoplasm to nuclear, and suppressed canonical Wnt/ß-catenin signaling and its downstream target genes, cyclin D1 and TCF-4. By immunohistochemical analysis of 379 patients, we found that the expression of WISP3 is closely associated with gastric cancer size and tumor invasion, and indicates a poor prognosis in both test cohort (253 patients) and validation cohort (126 patients). Moreover, the expression of WISP3 was positively correlated with the expression of cyclin D1 and TCF-4 in gastric cancer tissues. Taken together, our data suggests that WISP3 might be a promising prognostic factor and WISP3-Wnt/ß-catenin axis may be a new therapeutic target for the intervention of gastric cancer growth and metastasis.

DNA methylation-mediated silencing of matricellular protein dermatopontin promotes hepatocellular carcinoma metastasis by α3ß1 integrin-Rho GTPase signaling.

Dermatopontin (DPT), a tyrosine-rich, acidic matricellular protein, has been implicated in several human cancers. However, its biological functions and molecular mechanisms in cancer progression, particular hepatocellular carcinoma (HCC), remain unknown. We demonstrated that DPT was significantly down-regulated in 202 HCC clinical samples and that its expression level was closely correlated with cancer metastasis and patient prognosis. The overexpression of DPT dramatically suppressed HCC cell migration in vitro and intrahepatic metastasis in vivo. We further revealed that the down-regulation of DPT in HCC was due to epigenetic silencing by promoter DNA methylation. And the inhibitory effects of DPT on HCC cell motility were associated with dysregulated focal adhesion assembly, decreased RhoA activity and reduced focal adhesion kinase (FAK) and c-Src tyrosine kinase (Src) phosphorylation, and all of these alterations required the involvement of integrin signaling. Furthermore, we determined that the inhibitory effects of DPT on HCC cell motility were primarily mediated through α3ß1 integrin. Our study provides new evidence for epigenetic control of tumor microenvironment, and suggests matricellular protein DPT may serve as a novel prognostic marker and act as a HCC metastasis suppressor.

Dysregulated cell mechanical properties of endometrial stromal cells from endometriosis patients.

Endometriosis, diagnosed with ectopically implanted endometrial stromal cells (ESC) and epithelial cells to a location outside the uterine cavity, seriously threaten the quality of life and reproductive ability of women, yet the mechanisms and the pathophysiology of the disease remain unclear. Specially, the functional changes of ESC during endometriosis progression need in-depth investigation. In this study, we characterized mechanical properties of normal ESC (NESC) from healthy women and eutopic ESC (EuESC) and ectopic ESC (EcESC) from endometriosis patients. We found the collagen lattice contractile ability of EuESC was significantly stronger than that of NESC, and the cell mobility of EuESC and EcESC was significantly greater than that of NESC. Furthermore, the expression of F-actin and vinculin in NESC, EuESC and EcESC cells progressively increased, and the Rho GTPase activity, of which RhoA exhibited the highest activity, in the three cells gradually increased. Collectively, these results suggest that the mechanical characteristics of NESC, EuESC and EcESC cells exhibited progressive abnormalities. Therefore, the biomechanics of endometrial stromal cells may be a potent target for intervention in patients with endometriosis.

Decreased hepatic peroxisome proliferator-activated receptor-γ contributes to increased sensitivity to endotoxin in obstructive jaundice.

AIM: To investigate the role of hepatic peroxisome proliferator-activated receptor-γ (PPAR-γ) in increased susceptibility to endotoxin-induced toxicity in rats with bile duct ligation during endotoxemia. METHODS: Male Sprague-Dawley rats were subjected to bile duct ligation (BDL). Sham-operated animals served as controls. DNA binding were determined by polymerase chain reaction, Western blotting analysis, and electrophoretic mobility shift assay, respectively. BDL and sham-operated rats received a non-lethal dose of intraperitoneal lipopolysaccharide (LPS) injection (3 mg/kg, i.p.). Additionally, the potential beneficial effects of the PPAR-γ agonist rosiglitazone were determined in BDL and sham-operated rats treated with a non-lethal dose of LPS. Survival was assessed in BDL rats treated with a non-lethal dose of LPS and in sham-operated rats treated at a lethal dose of LPS (6 mg/kg, i.p.). RESULTS: PPAR-γ activity in rats undergoing BDL was significantly lower than in the sham-controls. Hepatic PPAR-γ gene expression was downregulated at both the mRNA and protein levels. In a parallel group, serum levels of pro-inflammatory cytokines were nearly undetectable in the sham-operated rats. When challenged with a non-lethal dose of LPS (3 mg/kg), the BDL rats had approximately a 2.4-fold increase in serum IL-6, a 2.7 fold increase in serum TNF-α, 2.2-fold increase in serum IL-1 and 4.2-fold increase in serum ALT. The survival rate was significantly lower as compared with that in sham-operated group. Additionally, rosiglitazone significantly reduced the concentration of TNF-α, IL-1ß, IL-6 and ALT in sham-operated rats, but not in BDL rats, in response to LPS (3 mg/kg). Also, the survival was improved by rosiglitazone in sham-operated rats challenged with a lethal dose of LPS, but not in BDL rats, even with a non-lethal dose of LPS (3 mg/kg). CONCLUSION: Obstructive jaundice downregulates hepatic PPAR-γ expression, which in turn may contribute to hypersensitivity towards endotoxin.

[A study of the bacteriophage-based assay for the detection of pyrazinamide resistance in Mycobacterium tuberculosis].

OBJECTIVE: To set up and evaluate the method of phage amplified biologically assay (PhaB) in rapid detection of pyrazinamide (PZA) resistance. METHODS: The PhaB assay was developed and applied in detecting PZA resistance in 108 clinical isolates of Mycobacterium tuberculosis and the results were compared with those of the absolute concentration method. The minimum inhibitory concentration (MIC) was detected for all discrepancy isolates. RESULTS: The results showed that the optimal detecting condition was pH 5.5, PZA 200 microg/ml and 48 h. Of the 108 strains of Mycobacterium tuberculosis, 28 strains were PZA-susceptible and 80 strains were PZA-resistant detected by PhaB; while 32 strains were PZA-susceptible and 76 strains were PZA-resistant by absolute concentration method. Twenty-eight of the 108 strains were PZA-susceptible and 71 were PZA-resistant by the two methods. The concordant isolates of determination of PZA resistance were 99 by the two methods and the concordance rates was 91.7%. There were 9 strains in discordant isolates, of which 7 were the same with MIC method and gene chip in drug susceptibility. If the results of absolute concentration method was the gold standard, the sensitivity, specificity, positive and negative predictive values, as well as accuracy of PhaB assay was 94.7%, 84.8%, 93.4%, 87.5% and 91.7% respectively. CONCLUSION: The PhaB assay can be used as a rapid screening method for detection of drug susceptibility of PZA in clinical isolates of Mycobacterium tuberculosis.