Downregulation of the CXCR4/CXCL12 axis blocks the activation of the Wnt/ß-catenin pathway in human colon cancer cells.

Chemokine CXCL12 is an extracellular chemokine, which binds to its cell surface receptor CXCR4. High expressions of CXCR4 and CXCL12 are associated with biological malignant potential in colon cancers. We aimed to investigate the roles of the CXCR4/CXCL12 axis in activation of the Wnt/ß-catenin pathway in the development of colon cancers. Using colon cancer cell line, we performed the RNA interference assay to downregulate the expression of CXCR4. Cells were exposed to CXCL12 and their growth and metastatic activity were examined. Matrix metalloproteinases (MMPs) activity were analyzed by the gelatin zymography assay. Cell migration ability was estimated by assays of scratch wound and transwell chamber. The expression of CXCR4 and molecules relevant to the Wnt/ß-catenin pathway were analyzed by the western blotting and real-time PCR assays. Human colon cancer HT-29 cells identified high expression of CXCR4. HT-29 cells highly responded to CXCL12 stimulation, showing the increase of cell proliferation, invasion and migration through the Matrigeal. The secretion and activity of MMP-2 and MMP-9 were also stimulated in HT-29 cells exposure to CXCL12. However, the CXCR4 knockdown HT-29 cells did not response to CXCL12 stimulation. We suggested that the activation of the CXCR4/CXCL12 axis be blocked in the CXCR4 knockdown cells. This study indicated that one key to the role of the CXCR4/CXCL12 axis is activation of the Wnt/ß-catenin pathway. Downregulation of the CXCR4/CXCL12 axis thus reduces cancer growth and metastasis. Targeted therapy utilizing the CXCR4/CXCL12 axis could be an effective strategy for treatment of colon cancers.

Synthesis and evaluation of trehalose-based compounds as novel inhibitors of cancer cell migration and invasion.

As a continuous research for the discovery of trehalose-based anti-invasive agents, we developed a convenient synthetic approach for the preparation of 6,6'-dideoxy-6,6'-bis(acylamino)-α,α-D-trehaloses. A series of trehalose-based amides were prepared through the trityl protection of the two primary hydroxyls of α,α-D-trehalose, benzoylation, the removal of the trityl protective group, mesylation, azidation, catalytic hydrogenation in the presence of hydrochloride, coupling reaction with a variety of acids, and subsequent debenzoylation and deacetylation in some cases. Compound 8b, 6,6'-dideoxy-6,6'-bis(2-hydroxybenzamide)-α,α-D-trehalose, was just as potent as the natural brartemicin against the invasion of murine colon 26-L5 cells. It exhibited no cytotoxicity on human breast adenocarcinoma MDA-MB-231 and murine colon 26-L5 cells. It can significantly inhibit the migration and invasion of the MDA-MB-231 cells. The anti-invasive effect of 8b was possibly related to its inhibitory activity on MMP-9, its suppression on the expression of MMP-9 and VEGF, and its deactivation of Akt.

Synthesis and structure-activity relationships studies of brartemicin analogs as anti-invasive agents.

Brartemicin is a trehalose-based inhibitor of tumor cell invasion produced by the actinomycete of the genus Nonomuraea. In order to find more potent anti-invasive agents and study the structure-activity relationships, a series of 19 brartemicin analogs were prepared via two synthetic routes from α,α-D-trehalose and evaluated for their anti-invasive activities. Compound 4f, 6,6'-bis(2,3-dimethoxybenzoyl)-α,α-D-trehalose, was more potent than the natural brartemicin. It inhibited the invasion of murine colon 26-L5, colon carcinoma SW620, melanoma B16-BL6 and breast MDA-MB-231 cells with IC50 values of 0.15, 2.35, 4.12 and 2.61 µM, respectively. Analog 4p, 6,6'-bis(3,4-dimethoxycinnamoyl)-α,α-D-trehalose, was as potent as brartemicin against invasion of murine colon 26-L5 carcinoma cells in vitro. The structure-activity relationships of these novel trehalose-based compounds were summarized.

[Effects of budesonide on HIF-1α and VEGF expression and airway remodeling in an asthmatic mouse model].

OBJECTIVE: To study the effects of budesonide on hypoxia inducible factor 1α(HIF-1α) and vascular endothelial growth factor (VEGF) expression, angiogenesis and airway remodeling in the chronic asthmatic mouse model. METHODS: Thirty female BALB/c mice were randomly divided into normal control, asthma model and treatment groups (10 in each group).The asthmatic mouse model was established via OVA challenge test. Mice in the treatment group were administered with aerosol budesonide (100 µg/kg) an hour before the OVA challenge test from the 28th day. Mice in the control group were treated with PBS instead of OVA. Hematoxylin and eosin staining was performed to observe thickness of the airway wall. Masson staining was used for examing collagen deposition of lung tissues. Angiogenesis and HIF-1α and VEGF expression were measured using immunohistochemistry and Western blot. The relationship of airway wall thickness and vessel area to HIF-1α and VEGF expression was investigated. RESULTS: Vessel area, collagen deposition of lung tissues and airway wall thickness increased in the asthma model group. Levels of HIF-1α and VEGF were also elevated. Administration of budesonide significantly reduced angiogenesis, collagen deposition of lung tissues and airway wall thickening, as well as expression of HIF-1α and VEGF. The vessel area and airway wall thickness were positively correlated with expression of HIF-1α and VEGF. A positive correlation was also found between the expression of HIF-1α and VEGF. CONCLUSIONS: Budesonide can decease angiogenesis and airway remodeling by inhibiting HIF-1α and VEGF expression in asthmatic mice.

Flavonoids from Dracocephalum tanguticum and their cardioprotective effects against doxorubicin-induced toxicity in H9c2 cells.

Two new flavonoids, ladanetin-6-O-ß-(6″-O-acetyl)glucoside (1) and pedalitin-3'-O-ß-glucoside (2), together with 15 known compounds (3-17), were isolated from the whole plants of Dracocephalum tanguticum. Their structures were established on the basis of extensive spectroscopic (IR, MS, 2D NMR) data analysis and by the comparison with spectroscopic data reported in the literature. Antioxidant capacities of the isolated substances were determined using the 1,1-diphenyl-2-picrylhydrazyl (DPPH), ferrous ions, and ABTS(·)(+) radical in vitro assay, and their cytoprotective activities were also tested on doxorubicin (DOX)-induced toxicity in H9c2 cardiomyocytes. Among all the tested compounds, luteolin-7-O-ß-D-glucopyranoside (7) exhibited both strong antioxidative effect and high protective activity against DOX-induced toxicity. Further investigation found 7 could decrease DOX-induced death of H9c2 cell, reduce LDH and CK level, and inhibit the elevated intracellular concentration of ROS and [Ca(2+)](i). The preliminary structure-activity relationships (SAR) of these compounds revealed the Δ(2,3) double bond on C-ring and 3',4'-di-OHs on B-ring with a flavone skeleton such as luteolin and its derivatives, were necessary for their cardioprotective effects.

[Mechanism of conditioned immune response in curing bronchial asthma in mice].

OBJECTIVE: To understand the mechanism of effect of conditioned immune response in curing bronchial asthma. METHODS: An experimental asthma modal was produced on healthy BALB/C mice (female, 4 - 6 weeks old) by sensitization and stimulation with ovalbumin (OV A). Totally 105 mice were divided into 7 groups randomly with 15 in each and treated differently: in group CIR(1), noise was used as conditioned stimulus (CS) and budesonide and salbutamol as unconditioned stimulus (UCS) respectively, a conditioned immune response model of mice with asthma was established by the combination of CS and UCS 7 times (7 days), then the mice were given CS only, and the combination were given once a week for 20 weeks. In group CIR(2) saccharin (SAC) was taken as CS, and the other treatments were the same as the group CIR(1). In the group of conventional therapy, the mice were given inhalation of nebulized budesonide and salbutamol only for 20 weeks. In the group of lower dose conventional therapy, the mice were given nebulized inhalation of budesonide and salbutamol for the first 7 days, then once a week for 20 weeks. In the noise group the mice were given noise only everyday for 20 weeks. In SAC group the mice were treated with SAC only everyday for 20 weeks. In the blank control group the mice were treated with placebo for 20 weeks. The mice in all the groups were stimulated with OVA once a day. The mice in the healthy control group were given PBS inhalation for 20 weeks. After 20 weeks therapy, the bronchoalveolar lavage fluid (BALF) was taken for eosinophils (EOS) counting. The spleens were taken to obtain CD4(+)T lymphocytes and the expression of neuronal acetylcholine receptor alpha 7 (nAChRalpha7), IL-4, IFN-gamma and IL-17 were detected by flow cytometry. RESULTS: (1) The percent of EOS of groups CIR(1), CIR(2), conventional therapy and healthy control was much lower than that of blank control (P < 0.01), and there was no significant difference among groups CIR(1), CIR(2) and conventional therapy (P > 0.05). (2) The expression of nAChRalpha7, IL-4 and IL-17 of groups CIR(1), CIR(2), conventional therapy and healthy control was much lower than that in blank control group, IFN-gamma was much higher (P < 0.01), and no significant difference was found among groups CIR(1), CIR(2) and conventional therapy (P > 0.05). There was a positive correlation between nAChRalpha7 and IL-4 (r = 0.76, P < 0.01), nAChRalpha7 and IL-17 (r = 0.46, P < 0.01). There was a negative correlation between nAChRalpha7 and IFN-gamma (r = 0.69, P < 0.01). (3) In the groups treated with lower dose of conventional therapy, noise, SAC and blank control, the epithelial tissue of airway were much thicker, the lumens were much narrower, and inflammatory cells and collagen fibers were much more than in the healthy control group, and after therapy, the inflammation in groups CIR(1), CIR(2) and conventional therapy was significantly improved. CONCLUSION: The conditioned immune response models established by both noise and SAC as CS and budesonide and salbutamol as UCS can downregulate nAChRalpha7 on CD4(+)T lymphocytes, regulate the function of CD4(+)T lymphocytes, and achieve the same therapeutic efficacy in treatment of asthma.

[Therapeutic mechanisms of interferon-beta and intravenous immunoglobulin for experimental peripheral neuropathy].

OBJECTIVE: To explore the therapeutic mechanisms of interferon-beta (IFN-beta) and intravenous immunoglobulin (IVIG) for experimental peripheral neuropathy induced by Campilobacter jejuni (Cj) lipopolysaccharide (LPS). METHOD: Forty healthy Wistar rats weighing 205 - 230 g were divided into IFN-beta, IVIG, IFN-beta plus IVIG and control groups. After the immune neuropathy was induced in the rats by Cj LPS, IFN-beta (1.3 microg/kg) was given by subcutaneous injection to the rats every other day for 6 weeks; IVIG [400 mg/(kg x d)] was given to the rats for five days, every other week for two times and IFN-beta [1.3 microg/(kg x d)] and IVIG [400 mg/(kg x d)] were given to the rats on the same days. Meanwhile, the control group was given PBS. The sera were collected in the 2nd, 4th and 6th week after therapy, the titers of anti-GM(1) IgG, MMP-9 and TNF-alpha in sera of immunized rats were measured by ELISA; histological study of sciatic nerve was performed and IgG on sciatic nerve was detected by immunohistochemistry in the 6th week. RESULTS: (1) There were no significant differences in titers of anti-GM(1) IgG, MMP-9 and TNF-alpha among the 3 therapeutic groups and control group after therapy for 2 weeks (P > 0.05). (2) The titers of anti- GM(1) IgG, MMP-9 or TNF-alpha in the control group were much higher than those of the IFN-beta group, the IVIG group or the IFN-beta and IVIG group after therapy for 4 weeks (P > 0.01) and there were no significant differences in titers of antibody among the 3 therapeutic groups (P > 0.05); the titers of MMP-9 or TNF-alpha in the IFN-beta and IVIG group were lower than those of the IFN-beta group or the IVIG group (P < 0.05). (3) The titers of anti-GM(1) IgG, MMP-9 or TNF-alpha in the control group were much higher than those of the IFN-beta group, the IVIG group or the IFN-beta with IVIG group after therapy for 6 weeks (P > 0.01); the IFN-beta with IVIG group had much lower levels of all indexes than the IFN-beta group or the IVIG group (P < 0.01). CONCLUSION: IFN-beta and IVIG showed therapeutic effects on immune peripheral neuropathy through inhibiting the humoral and cellular immunity simultaneously in the peripheral neuropathy induced by CJ LPS, treatment with combined IFN-beta and IVIG was more effective.

Oligomers of resveratrol and ferulic acid prepared by peroxidase-catalyzed oxidation and their protective effects on cardiac injury.

Peroxidase extracted from Momordica charantia was used for the oligomerization of trans-resveratrol and ferulic acid on a preparative scale. One new heterocoupling oligomer, trans-3 E-3-[(4-hydroxy-3-methoxyphenyl)methylene]-4-(3,5-dihydroxyphenyl)-5-(4-hydroxyphenyl)tetrahydro-2-franone (6), and six resveratrol dimers, leachianol G (1), restrytisol B (2), parthenostilbenins A (3) and B (5), 7- O-acetylated leachianol G (4), and resveratrol trans-dehydrodimer (8), and one known ferulic acid dehydrodimer, (3alpha,3aalpha,6alpha,6aalpha)tetrahydro-3,6-bis(4-hydroxy-3-methoxyphenyl)-1 H,4 H-furo[3,4-c]furan-1,4-dione (7) were obtained. Bioactive experiments showed that compounds 6- 8 have strong free radical scavenging effects and also have protective effects on doxorubicin-induced cardiac cell injury when tested in vitro.

[The mechanisms responsible for the therapeutic effects of anti-Fas ligand antibody on viral myocarditis in mice].

OBJECTIVE: Viral myocarditis (VM) is one of the most common acquired myocardial diseases in children. However, its pathogenesis is not clear. Recent studies indicate that the cytotoxicity mediated by cytotoxic T lymphocyte (CTL) plays an important role in the development of myocardial injury involved in VM. Apoptosis mediated by Fas/FasL pathway is an essential mechanism of target cells damage by CTL. In this study, the authors investigated the regulatory effects of neutralizing anti-Fas ligand (anti-FasL) antibody on apoptosis and caspase-3 expression in experimental coxsackievirus B3 myocarditis and the role of the CTL mediated apoptosis in myocardium through Fas/FasL pathway in the development of VM. METHODS: A total of 80 BALB/c mice were used in the experiments. They were divided randomly into the following groups: normal control group (Gr1), CVB3 control group (Gr2), IgG control group (Gr3) and anti-FasL antibody therapy group (Gr4). The mice in Gr2, Gr3 and Gr4 were inoculated with 0.15 ml of TCID(50) 10(9)/ml coxsackie virus B3 (CVB3) and the mice in Gr1 with 0.15 ml of Eagle reagent. The mice in Gr3 and Gr4 were inoculated with IgG (0.1 mg/kg) and FasL antibody (0.1 mg/kg) on days 0 and days 3 after inoculation (p.i.), respectively. Eight mice in each group were sacrificed on day 10 p.i. Histopathological studies and terminal transferase-mediated dUTP-biotin nick end labeling (TUNEL) assays were used to quantify inflammation, necrosis and apoptosis in myocardium. The expression of active caspase-3 in myocardium was determined by immunohistochemistry. Caspase-3 mRNA and CVB3 mRNA were analyzed by reverse-transcription polymerase chain reaction (RT-PCR). RESULTS: (1) Caspase-3 activation and apoptosis were seen in the myocardium of mice with myocarditis. They had a significantly positive correlation with the changes of myocardial histopathologic scores (r = 0.81, P < 0.05; r = 0.73, P < 0.05). (2) The pathologic scores, average percentages of apoptotic cardiomyocytes, expression of active caspase-3 (protein and mRNA) and expression of CVB3 mRNA in myocardium of mice in Gr4, were significantly reduced compared to those in the myocardium of mice in Gr2 (P < 0.01, P < 0.01, P < 0.01, P < 0.01, and P < 0.05, respectively) and Gr3 (P < 0.01, P < 0.01, P < 0.05, P < 0.01, and P < 0.05, respectively). CONCLUSION: Myocytic apoptosis is a key mechanism responsible for myocardial damage in viral myocarditis. Anti-FasL antibody can effectively reduce expression of active caspase-3 protein and mRNA, viral replication, cardiomyocytic apoptosis and myocardial injury in the experimental CVB3 myocarditis.

Dynamic changes in myocardial matrix metalloproteinase activity in mice with viral myocarditis.

BACKGROUND: Matrix metalloproteinases (MMPs) are the major regulators of collagen degradation involved in the pathogenesis of several diseases of the heart. The purpose of this study was to investigate the dynamic changes in myocardial MMP activity in mice with viral myocarditis (VM), the relationship between MMP activity and both cardiac function and the quantity of myocardial collagen, and the role MMPs playing in the pathological lesions of VM. METHODS: Sixty-five six-week-old male DBA/2 mice were divided into two groups. Mice in the infected group (n = 50) were inoculated intraperitoneally with 0.14 ml of Coxsackievirus B3 (CVB3, Nancy strain). Control mice (n = 15) were inoculated intraperitoneally with 0.14 ml of Eagle's medium. Eight infected mice and three control mice were sacrificed on each of days 3, 7, 10, 21 and 30 after inoculation. MMP activity was measured on an SDS-PAGE substrate gel embedded with type I gelatin (zymography). Echocardiographic studies were performed under anesthesia with 3% chloralhydrate administered intraperitoneally (0.01 ml/g - 0.015 ml/g). Cardiac systolic function indices, such as peak velocity of the aorta (Vp), flow velocity integral of the aorta (Vi), ejection fraction (EF), and fractional shortening (FS) were determined by echocardiography. Histological cross sections of the hearts were stained with hematoxylin-eosin and myocardial histopathological scores were determined under an optical microscope. The amount of myocardial collagen was measured by means of hydroxyproline quantification. RESULTS: In virus-infected mice, both MMP-2 and MMP-9 activities were significantly higher than in control mice, reaching a peak on day 10 (P < 0.01). On day 10, cardiac systolic function indices (EF, FS, Vp, and Vi) were all significantly lower compared both to other stages following viral inoculation and to the control group (P < 0.05). In the acute stage, the amount of myocardial collagen in mice with VM was not significantly different from normal control mice (P > 0.05). However, the amount of myocardial collagen in infected mice at the recovery stage (on days 21 and 30) was significantly greater than those of the control mice. MMP-2 and MMP-9 activities positively correlated with myocardial histopathological scores (r = 0.801, 0.821, P < 0.01) and negatively correlated with Vp (r = -0.649, -0.683, P < 0.01) and Vi (r = -0.711, -0.755, P < 0.01). However, Vp negatively correlated with myocardial histopathological scores (r = -0.756, P < 0.01). CONCLUSIONS: In mice with VM, the activities of myocardial MMP-2 and MMP-9 increase significantly during the acute stage, and the total quantity of myocardial collagen increases by the time of recovery. These changes are associated with myocardial interstition remodeling and cardiac dysfunction. MMP activity is an important reference marker for myocardial pathological lesions and can be used to evaluate the severity of myocardial interstitial damage and cardiac dysfunction.

[Myocardial matrix metalloproteinases activities in mice with viral myocarditis and their relationship with cardiac function and myocardial collagen amount].

OBJECTIVE: To investigate the dynamic changes of myocardial matrix metalloproteinases (MMPs) activities in mice with viral myocarditis (VM) and their relationships with cardiac function and myocardial collagen amount and to explore the role of MMPs in the pathologic lesion of VM. METHODS: Sixty-five six-week-old male DBA/2 mice were obtained from the Chinese Academy of Medical Sciences. They were divided into two groups randomly. Mice in infected group (n=50) were inoculated intraperitoneally with 0.14 ml of coxsackievirus B3 (CVB3, Nancy strain). Control mice (n=15) were inoculated intraperitoneally with 0.14 ml of Eagle's solution. Eight infected mice were sacrificed on day 3, 7, 10, 21 and 30, respectively and fifteen control mice were killed on day 30 after inoculation. Total protein concentration was determined according to the method of Bradford, while MMPs activities were measured with SDS-PAGE type substrate gels embedded with type I gelatin (zymography). Echocardiographic studies were performed under anesthesia with 3% chloralhydrate intraperitoneally (0.01-0.015 ml/g). Cardiac systolic function indexes, such as peak velocity of aorta (Vp) and flow velocity integral of aorta (Vi) were determined by echocardiography. Histological cross sections of hearts were stained with hematoxylin-eosin and myocardial histopathologic scores were counted under optical microscope. Myocardial collagen amount was measured by determination of hydroxyproline quantification. RESULTS: In virus-infected mice, both MMP-2 and MMP-9 activities were increased significantly compared with those in controls and reached the peak on day 10 (P < 0.01). On day 10, cardiac systolic function indexes (Vp and Vi) were all significantly lower than those at other stages after virus inoculation and in control group (P < 0.05). There was no obvious elevation in myocardial collagen amount in mice with VM at acute stage (P > 0.05). While the myocardial collagen amount in infected group at recovery stage (on day 21 and 30) increased significantly compared with controls. MMP-2 and MMP-9 activities positively correlated with myocardial histopathological scores, respectively (r =0.801, 0.821 P < 0.01), while they negatively correlated with Vp (r = -0.649, -0.683, P < 0.01) and Vi, respectively (r = -0.711, -0.755, P < 0.01). However, Vp and Vi negatively correlated with myocardial histopathological scores (r = -0.756, -0.584, P < 0.01). CONCLUSIONS: In mice with VM, the activities of myocardial MMP-2 and MMP-9 at acute stage increased significantly, then myocardial collagen amount elevated in recovery stage. These changes were associated with myocardial remodeling and cardiac dysfunction. Myocardial MMP activities are important markers of myocardial pathologic lesion. They are of value in the evaluation of the severity of myocardial damage and cardiac dysfunction in mice with VM.