Mechanism of GLP-1 Receptor Agonists-Mediated Attenuation of Palmitic Acid-Induced Lipotoxicity in L6 Myoblasts.

Methods: Cells were divided into 5 groups-control, high-fat, 10 nmol/L LR + 0.6 mmol/L palmitic acid (PA) (10LR), 100 nmol/L LR + 0.6 mmol/L PA (100LR), and 1000 nmol/L LR + 0.6 mmol/L PA (1000LR). CCK-8 method to detect cell viability, GPO-PAP enzymatic method to detect intracellular triglyceride content, and reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) and western blotting methods to detect fatty acid translocase CD36 (FAT/CD36) and fatty acid binding protein 4 (FABP4) in L6 cells, glucose-regulated protein 78 (GRP78), glucose transporter 4 (GLUT4) expression at the mRNA and protein levels, respectively, were performed. Results: We found that after PA intervention for 24 h, the cell viability decreased significantly; the cell viability of the LR group was higher than that of the high-fat group (P < 0.01). After PA intervention, compared with those in the high-fat group, GRP-78, FAT/CD36, FABP4 mRNA ((4.36 ± 0.32 vs. 8.15 ± 0.35); (1.00 ± 0.04 vs. 2.46 ± 0.08); (2.88 ± 0.55 vs. 8.29 ± 0.52), P < 0.01) and protein ((3338.13 ± 333.15 vs. 4963.98 ± 277.29); (1978.85 ± 124.24 vs. 2676.07 ± 100.64); (3372.00 ± 219.84 vs. 6083.20 ± 284.70), both P < 0.01) expression decreased in the LR group. The expression levels of GLUT4 mRNA ((0.75 ± 0.04 vs. 0.34 ± 0.03), P < 0.01) and protein ((3443.71 ± 191.89 vs. 2137.79 ± 118.75), P < 0.01) increased. Conclusion: Therefore, we conclude that LR can reverse PA-induced cell inactivation and lipid deposition, which may be related to the change in GRP-78, FAT/CD36, FABP4, GLUT4, and other factors.

BRAFV600E mutant colorectal cancer cells mediate local immunosuppressive microenvironment through exosomal long noncoding RNAs.

BACKGROUND: BRAFV600E mutated colorectal cancer (CRC) is prone to peritoneal and distant lymph node metastasis and this correlates with a poor prognosis. The BRAFV600E mutation is closely related to the formation of an immunosuppressive microenvironment. However, the correlation between BRAFV600E mutation and changes in local immune microenvironment of CRC is not clear. AIM: To explore the effect and mechanism of BRAFV600E mutant on the immune microenvironment of CRC. METHODS: Thirty patients with CRC were included in this study: 20 in a control group and 10 in a treatment group. The density of microvessels and microlymphatic vessels, and M2 subtype macrophages in tumor tissues were detected by immunohistochemistry. Screening and functional analysis of exosomal long noncoding RNAs (lncRNAs) were performed by transcriptomics. The proliferation and migration of human umbilical vein endothelial cells (HUVECs) and human lymphatic endothelial cells (HLECs) were detected by CCK-8 assay and scratch test, respectively. The tube-forming ability of endothelial cells was detected by tube formation assay. The macrophage subtypes were obtained by flow cytometry. The expression of vascular endothelial growth factor (VEGF)-A, basic fibroblast growth factor (bFGF), transforming growth factor (TGF)-ß1, VEGF-C, claudin-5, occludin, zonula occludens (ZO)-1, fibroblast activation protein, and α-smooth muscle actin was assessed by western blot analysis. The levels of cytokines interleukin (IL)-6, TGF-ß1, and VEGF were assessed by enzyme-linked immunosorbent assay. RESULTS: BRAFV600E mutation was positively correlated with the increase of preoperative serum carbohydrate antigen 19-9 (P < 0.05), and with poor tumor tissue differentiation in CRC (P < 0.01). Microvascular density and microlymphatic vessel density in BRAFV600E mutant CRC tissues were higher than those in BRAF wild-type CRC (P < 0.05). The number of CD163+ M2 macrophages in BRAFV600E mutant CRC tumor tissue was markedly increased (P < 0.05). Compared with exosomes from CRC cells with BRAF gene silencing, the expression of 13 lncRNAs and 192 mRNAs in the exosomes from BRAFV600E mutant CRC cells was upregulated, and the expression of 22 lncRNAs and 236 mRNAs was downregulated (P < 0.05). The biological functions and signaling pathways predicted by differential lncRNA target genes and differential mRNAs were closely related to angiogenesis, tumor cell proliferation, differentiation, metabolism, and changes in the microenvironment. The proliferation, migration, and tube formation ability of HUVECs and HLECs induced by exosomes in the 1627 cell group (HT29 cells with BRAF gene silencing) was greatly reduced compared with the HT29 cell group (P < 0.05). Compared with the HT29 cell group, the expression levels of VEGF-A, bFGF, TGF-ß1, and VEGF-C in the exosomes derived from 1627 cells were reduced. The expression of ZO-1 in HUVECs, and claudin-5, occludin, and ZO-1 in HLECs of the 1627 cell group was higher. Compared with the 1627 cell group, the exosomes of the HT29 cell group promoted the expression of CD163 in macrophages (P < 0.05). IL-6 secretion by macrophages in the HT29 cell group was markedly elevated (P < 0.05), whereas TGF-ß1 was decreased (P < 0.05). The levels of IL-6, TGF-ß1, and VEGF secreted by fibroblasts in the 1627 cell group decreased, compared with the HT29 cell group (P < 0.05). CONCLUSION: BRAFV600E mutant CRC cells can reach the tumor microenvironment by releasing exosomal lncRNAs, and induce the formation of an immunosuppressive microenvironment.

DL-3-n-butylphthalide protects the blood-brain barrier against ischemia/hypoxia injury via upregulation of tight junction proteins.

BACKGROUND: The increased permeability of the blood-brain barrier (BBB) induced by ischemia/hypoxia is generally correlated with alteration of tight junctions (TJs). DL-3-n-butylphthalide (NBP) has been shown to exert neuroprotective effects after ischemic injury. However, few studies have assessed the correlation between NBP and TJs. This study aimed to investigate the potential effect of NBP on the TJ proteins claudin-5, zonula occludens-1 (ZO-1), and occludin during brain ischemia. METHODS: A chronic cerebral hypoperfusion (CCH) Sprague-Dawley rat model was established, and NBP (20, 40, or 80 mg/kg, gavage, once a day) treatment was performed for 14 days. NBP (0.1 or 1.0 µmol/L) pre-treatment was applied to an in vitro hypoxia microvascular endothelial cell model (1% O2, 24 h). BBB permeability was assessed by performing the Evans blue assay. The expressions and localization of claudin-5, ZO-1, occludin, phosphorylated/total protein kinase B (p-Akt/Akt), phosphorylated/total glycogen synthase kinase 3ß (GSK-3ß)/GSK-3ß, and ß-catenin/ß-actin were evaluated by Western blotting or immunofluorescence. Reactive oxygen species (ROS) generation was measured by flow cytometry analysis. TJ ultrastructure was observed by transmission electron microscopy. RESULTS: In CCH rats, treatment with 40 and 80 mg/kg NBP decreased the Evans blue content in brain tissue (9.0 ±â€Š0.9 µg/g vs. 12.3 ±â€Š1.9 µg/g, P = 0.005; 6.7 ±â€Š0.6 µg/g vs. 12.3 ±â€Š1.9 µg/g, P < 0.01), increased the expression of claudin-5 (0.79 ±â€Š0.08 vs. 0.41 ±â€Š0.06, P < 0.01; 0.97 ±â€Š0.07 vs. 0.41 ±â€Š0.06, P < 0.01), and elevated the ZO-1 protein level (P < 0.05) in brain microvascular segments in a dose-dependent manner in comparison with the corresponding values in the model group. There was no significant difference in occludin expression (P > 0.05). In the hypoxia cell model, NBP pre-treatment improved TJ ultrastructure, decreased intracellular ROS level, and increased the expression of claudin-5 (P < 0.01) and ZO-1 (P < 0.01) in comparison with the corresponding values in the hypoxia group. NBP treatment also elevated the relative expression levels of p-Akt/Akt, p-GSK-3ß/GSK-3ß, and ß-catenin/ß-actin in comparison with the corresponding values in the hypoxia group (all P < 0.05). CONCLUSION: NBP improves the barrier function of BBB against ischemic injury by upregulating the expression of TJ proteins, possibly by reducing oxidative stress and activating the Akt/GSK-3ß/ß-catenin signaling pathway.

Role of X-Box Binding Protein-1 in Fructose-Induced De Novo Lipogenesis in HepG2 Cells.

BACKGROUND: A high consumption of fructose leads to hepatic steatosis. About 20-30% of triglycerides are synthesized via de novo lipogenesis. Some studies showed that endoplasmic reticulum stress (ERS) is involved in this process, while others showed that a lipotoxic environment directly influences ER homeostasis. Here, our aim was to investigate the causal relationship between ERS and fatty acid synthesis and the effect of X-box binding protein-1 (XBP-1), one marker of ERS, on hepatic lipid accumulation stimulated by high fructose. METHODS: HepG2 cells were incubated with different concentrations of fructose. Upstream regulators of de novo lipogenesis (i.e., carbohydrate response element-binding protein [ChREBP] and sterol regulatory element-binding protein 1c [SREBP-1c]) were measured by polymerase chain reaction and key lipogenic enzymes (acetyl-CoA carboxylase [ACC], fatty acid synthase [FAS], and stearoyl-CoA desaturase-1 [SCD-1]) by Western blotting. The same lipogenesis-associated factors were then evaluated after exposure of HepG2 cells to high fructose followed by the ERS inhibitor tauroursodeoxycholic acid (TUDCA) or the ERS inducer thapsigargin. Finally, the same lipogenesis-associated factors were evaluated in HepG2 cells after XBP-1 upregulation or downregulation through cell transfection. RESULTS: Exposure to high fructose increased triglyceride levels in a dose- and time-dependent manner and significantly increased mRNA levels of SREBP-1c and ChREBP and protein levels of FAS, ACC, and SCD-1, concomitant with XBP-1 conversion to an active spliced form. Lipogenesis-associated factors induced by high fructose were inhibited by TUDCA and induced by thapsigargin. Triglyceride level in XBP-1-deficient group decreased significantly compared with high-fructose group (4.41 ± 0.54 µmol/g vs. 6.52 ± 0.38 µmol/g, P < 0.001), as mRNA expressions of SREBP-1c (2.92 ± 0.46 vs. 5.08 ± 0.41, P < 0.01) and protein levels of FAS (0.53 ± 0.06 vs. 0.85 ± 0.05, P = 0.01), SCD-1 (0.65 ± 0.06 vs. 0.90 ± 0.04, P = 0.04), and ACC (0.38 ± 0.03 vs. 0.95 ± 0.06, P < 0.01) decreased. Conversely, levels of triglyceride (4.22 ± 0.54 µmol/g vs. 2.41 ± 0.35 µmol/g, P < 0.001), mRNA expression of SREBP-1c (2.70 ± 0.33 vs. 1.00 ± 0.00, P < 0.01), and protein expression of SCD-1 (0.93 ± 0.06 vs. 0.26 ± 0.05, P < 0.01), ACC (0.98 ± 0.09 vs. 0.43 ± 0.03, P < 0.01), and FAS (0.90 ± 0.33 vs. 0.71 ± 0.02, P = 0.04) in XBP-1s-upregulated group increased compared with the untransfected group. CONCLUSIONS: ERS is associated with de novo lipogenesis, and XBP-1 partially mediates high-fructose-induced lipid accumulation in HepG2 cells through augmentation of de novo lipogenesis.

[Oxymatrine alleviates oxidative stress in fat-induced insulin resistance mice by suppressing p38MAPK pathway].

This paper was aimed to investigate the effect of oxymatrine on fat-induced insulin resistance mice（IR）, and to explore the effects of oxymatrine on oxidative stress and on p38 mitogen activated protein kinase (p38MAPK) pathway. ApoE-/- mice with high fat diet for 16 weeks were selected as IR animal model and randomly divided into the model group, oxymatrine 25, 50, 100 mg•kg⁻¹ group. C57BL/6J mice were selected as the normal control group. Mice were gavage for 8 weeks. Fasting blood glucose (FBG), cholesterol (TC), triglyceride (TG), fatty acid (FFA) and serum insulin (FINS) in the plasma were detected. Activity of superoxide dismutase (SOD), glutathione peroxidase, glutathione peroxidase (GSH-Px) and content of malondialdehyde (MDA) in liver were detected. Reactive oxygen species (ROS) content in liver cells were detected by Flow cytometry. The expression of heme oxygenase-1(HO-1), γ-glutamyl cysteine synthetase (γ-GCS) of liver was examined by Real time PCR and Western blot. The protein expression of p38MAPK, p-p38MAPK was examined by Western blot. In the study, the authors found that oxymatrine reduced the levels of FBG, TC, TG and FFA, increased SOD and GSH-Px contents, decreased MDA and ROS content. Compared with model group, HO-1, γ-GCS mRNA and protein expression significantly increased in 50, 100 mg•kg⁻¹ oxymatrine group. The expression of p-p38MAPK decreased in oxymatrine group. The results showed that oxymatrine alleviate oxidative stress in hepatic by inhibiting the phosphorylation of p38MAPK, to ameliorate fat-induced insulin resistance mice.

[Effects of Tongxinluo capsule on sciatic nerve apoptosis in spontaneous type II diabetic KK/Upj-Ay mice and mechanism research].

To investigate the effects of Tongxinluo capsule on sciatic nerve apoptosis in spontaneous type II diabetic KK/Upj-Ay mice, in order to explore its mechanism for improving diabetic peripheral neuropathy (DPN). KK/Upj-Ay mice were selected as the DPN animal model and randomly divided into the model, Tongxinluo low, middle and high group (1, 2, 4 g x kg(-1)). C57BL/6 mice were selected as the control group. Mice were given intragastrically for 12 weeks. Paw withdrawal latency, motor nerve conduction velocity (MNCV) and sensory nerve conduction velocity (SNCV) were detected. Apoptotic rate were detected by FCM. Bcl-2, Bax, Caspase-3 mRNA and protein expression in sciatic nerve were examined by Real-time PCR and Western blot. p38MAPK, p-p38MAPK expression were examined by Western blot. In this study,the authors found that Tongxinluo capsule could increase paw withdrawal latency, MNCV and SNCV. Apoptotic rate of sciatic, the expression of Bax and caspase-3 were lower, while Bcl-2 expression was higher in Tongxinluo group than those in model mice. The expression of p-p38MAPK significantly decreased in Tongxinluo group. The results showed that Tongxinluo capsule has protective effects on diabetic peripheral neuropathy of mice via inhibiting cell apoptosis and suppressing the expression of p-p38MAPK.

[Heart-type fatty acid-binding protein alteration in rat's cardiocyte after acute myocardial infarction].

AIM: To explore the changes of mRNA and protein expressions of heart-type fatty acid binding protein (H-FABP) in rat ischemic myocardium at different intervals ischemia. METHODS: 60 SD male rats weighing 250-350 g, were randomly divided into one sham-operated group and five study groups (group A1, A2, A3, A4, A5, the left coronary artery of rats has been ligated for 1 h, 2 h, 4 h, 6 h, 12 h respectively). Myocardil samples from infarct zone, ischemic and non-ischemic zone, were obtained for histology examination, and the mRNA for H-FABP in ischemic myocardial tissue were determined by RT-PCR. Serum free fatty acid(FFA) was determined by colorimetric method. RESULTS: Compared to sham hearts, H-FABP mRNA expression were significantly decreased in ischemia zone of AMI rat hearts (P < 0.05), especially in rats underwent 4 h ischemia and 6 h ischemia (P < 0.01). Serum FFA were significantly increased in AMI rats relative to sham rats (P < 0.05). CONCLUSION: Significant down-regulated heart-type fatty acid binding protein after myocardial ischemia might play an important role in myocardial injury and energy metabolism disorder.

Hemin, a heme oxygenase-1 inducer, improves aortic endothelial dysfunction in insulin resistant rats.

BACKGROUND: Under an insulin resistance (IR) state, overproduction of reactive oxygen species (ROS) may be playing a major role in the pathogenesis of endothelial dysfunction, hypertension and atherosclerosis. Recently, increasing attention has been drawn to the beneficial effects of heme oxygenase-1 (HO-1) in the cardiovascular system. This study aimed to investigate the effects of HO-1 on vascular function of thoracic aorta in IR rats and demonstrate the probable mechanisms of HO-1 against endothelial dysfunction in IR states. METHODS: Sprague-Dawley (SD) rats fed with high-fat diet for 6 weeks and the IR models were validated with hyperinsulinemic-euglycemic clamp test. Then the IR rat models (n = 44) were further randomized into 3 subgroups, namely, the IR control group (n = 26, in which 12 were sacrificed immediately and evaluated for all study measures), a hemin treated IR group (n = 10) and a zinc protoporphyrin-IX (ZnPP-IX) treated IR group (n = 8) that were fed with a high-fat diet. Rats with standardized chow diet were used as the normal control group (n = 12). The rats in IR control group, hemin treated IR group and ZnPP-IX treated IR group were subsequently treated every other day with an intraperitoneal injection of normal saline, hemin (inducer of HO-1, 30 micromol/kg) or ZnPP-IX (inhibitor of HO-1, 10 micromol/kg) for 4 weeks. Rats in the normal control group remained on a standardized chow diet and were treated with intraperitoneal injections of normal saline every other day for 4 weeks. Systolic arterial blood pressure (SABP) was measured by tail-cuffed microphotoelectric plethysmography. The blood carbon monoxide (CO) was measured by blood gas analysis. The levels of nitric oxide (NO), inducible nitric oxide synthase (iNOS), endothelial nitric oxide synthase (eNOS), blood glucose (BG), insulin, total cholesterol (TC) and triglyceride (TG) in serum, and the levels of total antioxidant capacity (TAOC), malondialdehyde (MDA) and superoxide dismutase (SOD) in the aorta were measured. The expression of HO-1 mRNA and HO-1 protein in aortal tissue were detected by semi-quantitative RT-PCR and Western blot. The vasoreactive tensometry was performed with thoracic aortic rings (TARs). RESULTS: Compared with the normal control group, the levels of SABP, BG, insulin, TC, TG, NO, iNOS and MDA were higher, while the levels of CO, TAOC, SOD and eNOS were lower in IR control rats. After treatment of IR rats for 4 weeks a more intensive expression of HO-1 mRNA and HO-1 protein were observed in hemin treated IR group compared with the normal control group. And compared with 4-week IR control rats, the levels of CO, TAOC, SOD and eNOS were increased, while the levels of SABP and iNOS activity were lower in the hemin treated IR group. Administration of hemin in IR rats appeared to improve the disordered vasorelaxation of TARs to acetylcholine (ACh). Alternatively, the reverse results of SABP, CO, TAOC, SOD, iNOS and vasorelaxation responses to ACh were observed in IR rats with administration of ZnPP-IX. CONCLUSIONS: The endothelial dysfunction in the aorta is present in the IR state. The protective effects of HO-1 against aortic endothelial dysfunction may be due to its antioxidation and regulative effect of vasoactive substances. It is proposed that hemin, inducer of HO-1, could be a potential therapeutic option for vascular dysfunction in IR states.

Role of CXCL12 in metastasis of human ovarian cancer.

BACKGROUND: In a previous study, we have verified that CXCR4 expression is correlated with tumor aggressive progression and poor prognosis in patients with epithelial ovarian cancer. The aim of this study was to explore the effect of CXCL12-CXCR4 axis on the metastasis of human ovarian cancer. METHODS: The expressions of CXCR4 and CXCL12 mRNA and protein in human ovarian cancer cell line CAOV-3 was detected by RT-PCR and immunocytochemistry. Methythiazolyltetrazolium (MTT) was used to analyze the effect of different concentrations of CXCL12 on the proliferation of CAOV-3 cells. Transwell invasion chamber and matrigel were used to evaluate the effect of various concentrations of CXCL12 and ascites on the migration and invasion of CAOV-3 cells. The expressions of integrin beta(1) and vascular endothelial growth factor-C (VEGF-C) mRNA were detected by RT-PCR. Data were analyzed using ANOVA by SAS 6.12. RESULTS: Under serum-free suboptimal culture conditions, CXCL12 (100 ng/ml) significantly enhanced the proliferation of CAOV-3 cells compared with the control and 10 ng/ml CXCL12 groups (0.428 +/- 0.051 vs. 0.325 +/- 0.045 and 0.328 +/- 0.039, P < 0.05). This enhancing effect of CXCL12 was significantly inhibited by 10 microg/ml neutralizing CXCR4 antibody or 1 microg/ml CXCR4 antagonist AMD3100. However, 10 microg/ml neutralizing CXCR4 antibody could not inhibit cell proliferation without CXCL12. The levels of migration and invasion of the CAOV-3 cells treated with 100 ng/ml CXCL12 were significantly higher than those in the control (migration: 523.3 +/- 25.2 vs 108.0 +/- 7.2; invasion: 39.3 +/- 4.0 vs. 4.0 +/- 1.0). The enhancing effect of CXCL12 on cell migration and invasion increased with the concentration of CXCL12 (100 ng/ml vs10 ng/ml: migration, 523.3 +/- 25.2 vs 211.7 +/- 24.7; invasion, 39.3 +/- 4.0 vs 15.7 +/- 3.1, P < 0.05), and was strongly inhibited by 10 microg/ml neutralizing CXCR4 antibody or 1 microg/ml AMD3100. The number of migrated and invading cells in the CAOV-3 added with ascites was significantly higher than those in the 100 ng/ml CXCL12 group (migration: 706.6 +/- 30.6 vs 523.3 +/- 25.2, invasion: 61.7 +/- 7.6 vs 39.3 +/- 4.0, P < 0.05). The level of integrin beta(1) mRNA was greatly increased at 3 hours after being treated with CXCL12 (0.53 +/- 0.10 vs. 1.53 +/- 0.16, P < 0.05), and VEGF-C mRNA displayed significant augment at 24 hours after being treated with CXCL12 (0.52 +/- 0.09 vs 1.11 +/- 0.15, P < 0.05). CONCLUSIONS: CXCL12 and its receptor CXCR4 can promote the proliferation, migration, invasion of ovarian cancer cell line CAOV-3 and enhance its secretion of integrin beta(1) and VEGF-C. These effects can be inhibited by neutralizing CXCR4 antibody or AMD3100. CXCL12-CXCR4 axis plays an important role in ovarian cancer growth and metastasis.

[Effect of chemokine CXCL12 and its receptor CXCR4 on proliferation, migration and invasion of epithelial ovarian cancer cells].

OBJECTIVE: To explore the effect of chemokine CXCL12 and its receptor CXCR4 on proliferation, migration and invasion of epithelial ovarian cancer cells. METHODS: CXCR4 and CXCL12 mRNA and protein expression of human ovarian cancer cell line CAOV3 was detected by RT-PCR and immunocytochemistry. Integrin beta1 and vascular endothelial growth factor-C (VEGF-C) mRNA expression were detected in CAOV3 cells stimulated by CXCL12. The CAOV3 cells were divided into 6 groups: control group (un-stimulated), experimental group 1 (stimulated by 100 ng/ml CXCL12), experimental group 2 (stimulated by 10 ng/ml CXCL12), experimental group 3 (100 ng/ml CXCL12 and 10 microg/ml neutralizing CXCR4 antibody), experimental group 4 (100 ng/ml CXCL12 and 1 microg/ml CXCR4 antagonist AMD3100), experimental group 5 (10 microg/ml neutralizing CXCR4 antibody or ascites). Methyl thiazolyl tetrazolium (MTT) was used to analyze the effects of different concentrations of CXCL12 on CAOV3 cell proliferation. Transwell invasion chamber and reconstructed basement membrane (Matrigel) were used to evaluate effect of various concentrations of CXCL12 and ascites on CAOV3 cell migration and invasion. RESULTS: CAOV3 cells expressed CXCR4 mRNA (0.70 +/- 0.10) and protein, but did not express CXCL12 mRNA or protein. Immunostaining of CXCR4 was mainly located in cytoplasm. CXCR4 mRNA was up-regulated after 100 ng/ml CXCL12 stimulation (1.24 +/- 0.14; t = -7.1088, P = 0.0021). Integrin beta1 mRNA was greatly increased at 3 hours by stimulation of 100 ng/ml CXCL12 (before and after stimulation 0.53 +/- 0.10, 1.53 +/- 0.16; P < 0.01), and VEGF-C mRNA showed significant increase at 24 hours by treatment with CXCL12 (before and after stimulation 0.52 +/- 0.09, 1.11 +/- 0.15; P < 0.05). Under serum-free sub-optimal culture conditions, experimental group 1 greatly enhanced cell proliferation in CAOV3 cells compared with control group and experimental group 2 (respectively 0.428 +/- 0.051, 0.325 +/- 0.045, 0.328 +/- 0.039; P < 0.05). Experimental group 1 was strongly inhibited compared with experimental groups 3 and 4 (the latter two groups respectively 0.356 +/- 0.031, 0.373 +/- 0.029; P < 0.05). There was no significant difference between experimental group 5 (0.349 +/- 0.038) and control group (P > 0.05). Experimental group 1 stimulated the migration and invasion of CAOV3 cells in chemotaxis assay compared with control group and experimental group 2 (number of cell migration respectively 523.3 +/- 25.2, 108.0 +/- 7.2, 211.7 +/- 24.7, number of cell invasion respectively 39.3 +/- 4.0, 4.0 +/- 1.0, 15.7 +/- 3.1; P < 0.01). This enhancing effect of experimental group 1 was strongly inhibited compared with experimental groups 4 and 5 (P < 0.05). The number of migrating and invading cells in experimental group 5 (migration: 706.6 +/- 30.6, invasion: 61.7 +/- 7.6) was significantly higher than that of experimental group 1 (P < 0.05). CONCLUSIONS: This in vitro study shows CXCL12 promote proliferation, migration, invasion of ovarian cancer cell line CAOV3, and up-regulate integrin beta1 and VEGF-C expression, and these effects are strongly inhibited by neutralizing CXCR4 antibody. It suggests CXCL12 and its receptor CXCR4 may play important roles in ovarian cancer growth and metastasis.

[Roles of endogenous carbon monoxide in vasorelaxation disorder of femoral artery in diabetic rats].

OBJECTIVE: To explore the effects of CO on femoral vasorelaxation in diabetic rats. METHODS: Using isolated vascular ring tension detecting technique, cumulative relaxation responses of femoral arteries to 10(-8) - 10(-4) mol/L acetylcholine (ACh) were measured. The content of COHb in blood was detected. RESULTS: Diabetic animals expressed lower weight [(249.38+/-7.58) g] than control rats [(345.83+/-12.14) g , P<0.01]. The blood sugar levels in diabetic rats [(20.28+/-0.35) mmol/L] were significantly higher than that in control rats [(5.56+/-0.19) mmol/L]. In addition,diabetic animals demonstrated elevated blood pressure [(118.75+/-8.33) mm Hg] after 4 weeks, (132.43+/-10.98) mm Hg after 8 weeks, (139.0+/-10.41) mm Hg after 12 weeks compared with control (108.43+/-4.18) mm Hg, P<0.01, 1 mm Hg=0.133 kPa] in a time-dependent manner. The COHb content in the blood was decreased in 4 weeks [(1.50%+/-0.21%) vs (2.50%+/-0.61%)], and restored to normal in 8 weeks and 12 weeks in diabetic rats. The dose cumulative vasorelaxation-response to ACh in diabetic rats was diminished (63.46%+/-2.48% after 4 weeks; 69.76%+/-7.61% after 8 weeks; 49.37%+/-4.74% after 12 weeks compared with control 96.81%+/-3.15%). Treatment with hemin did not affect weight, blood sugar and blood pressure in diabetic rats, markedly increased the COHb content in the blood (3.20%+/-0.73%, P<0.01) and improved the vasorelaxation disturbance of femoral arteries (69.76%+/-7.60%, P<0.01) in diabetic rats. While administration with ZnPP-IX could inhibit the production of COHb (0.93%+/-0.35%), and worse the hypertension [(130.84+/- 8.56) mm Hg] and aggravate the vasorelaxation disturbance (37.70%+/-5.65%) vs diabetes (63.46%+/-2.48%) in diabetic rats. CONCLUSION: The results suggested that the decrease of blood CO in early stage of diabetic rats were related to the vasorelaxation disorder of femoral arteries, and that lead to hypertension in diabetic rats,which may be one of the important mechanisms of diabetes company hypertension.

Cholecystokinin octapeptide improves cardiac function by activating cholecystokinin octapeptide receptor in endotoxic shock rats.

AIM: To explore the effect of sulfated cholecystokinin octapeptide (sCCK-8) on cardiac functions and its receptor mechanism in endotoxic shock (ES) rats. METHODS: The changes of the mean arterial pressure (MAP), heart rate (HR), the left ventricular pressure (LVP) and the maximal/minimum rate of LVP (+/-LVdp/dt(max))) were measured by using physiological record instrument in eight groups of rats. The expression of cholecystokinin-A receptor (CCK-AR) and cholecystokinin-B receptor (CCK-BR) mRNA of myocardium in ES rats was examined by reverse transcription polymerase chain reaction (RT-PCR). RESULTS: (1) Low doses of sCCK-8 (0.4 microg/kg) caused tachycardia (441+/-27, normal control 391+/-22 s/min) and slight increase in MAP, LVP and +/-LVdp/dt(max) (16.96+/-1.79, 18.21+/-1.69 and +768.85+/-31.28/-565.04+/-27.71 kPa, respectively, all P<0.01), while medium doses (4.0 microg/kg) and high doses of sCCK-8 (40 microg/kg) elicited bradycardia and marked increase in MAP, LVP and +/-LVdp/dt(max) (17.29+/-1.63, 19.46+/-2.57 and +831.46+/-22.57/-606.08 +/-31.32; 17.46+/-1.08, 19.83+/-2.91 and +914.52+/-35.95/-639.15+/-30.23 kPa, respectively, all P<0.01). Proglumide (1.0 mg/kg), a nonselective antagonist of CCK-receptor (CCK-R), significantly inhibited the pressor effects of sCCK-8 (15.96+/-1.38, 17.36+/-0.66 and +748.18+/-19.29/-512.12+/-14.39 kPa, respectively, all P<0.01), whilst reversing the bradycardiac responses. (2) High doses of LPS (8 mg/kg) elicited marked decrease in MAP, LVP and +/-LVdp/dt(max). (7.16+/-0.59, 7.6+/-0.68 and +298.01+/-25.52/-166.96+/-19.25 kPa, respectively, all P<0.01). Pretreatment with sCCK-8 (40 microg/kg) could reverse the decline of cardiac functions (10.71+/-0.45, 11.7+/-1.26 and +446.04+/-67.18/-347.90+/-36.98 kPa, respectively, all P<0.01), while proglumide could cause further decline of cardiac function in ES rats (4.71+/-0.67, 5.58+/-1.25 and +226.48+/-15.84/-142.83+/-20.23 kPa, respectively, all P<0.01). (3) CCK-A/BR mRNAs were expressed in myocardium of control rats. Gene expression of CCK-AR and CCK-BR significantly increased in myocardium of ES rats. The increase of CCK-AR mRNA induced by LPS began at 0.5 h, peaked at 2 h, kept a high level at 6 h and declined at 12 h, respectively. Similar to CCK-AR mRNA, the expression of CCK-BR mRNA peaked at 2 h and kept a high level at 6 h, but it did not change at the first 0.5 h and was stable at a high level at 12 h. CONCLUSION: The above results indicate that endogenous and exogenous sCCK-8 may significantly improve cardiac function and intractable hypotension of ES rats, which was likely related to high expression of CCK-A/BR in myocardium induced by LPS.

[Melatonin improves vascular reactivity of endotoxemia rats].

The purpose of the present study was to investigate the effect of melatonin (MT) on the abnormal reactivity of thoracic aorta and pulmonary artery induced by lipopolysaccharide (LPS) in rats. Sprague-Dawley rats were divided into four groups randomly: (1) Vehicle group; (2) LPS group: LPS (4 mg/kg, i.p.); (3) LPS+MT group: MT (5 mg/ml, i.p.) was given 30 min before LPS and 60 min after LPS (4 mg/kg ,i.p); (4) MT group: received two doses of MT, 90 min after the first injection of MT another dose of MT was given. Six hours after LPS injection,the rats were killed and both thoracic aortic rings (TARs) and pulmonary artery rings (PARs)were prepared. The reactivity of TARs and PARs in the four subgroups was tested separately. The contraction response to phenylephrine (PE) and the endothelium-dependent relaxation response (EDRR) to ACh were observed with the isolated artery ring technique. Concentration-response curves were generated with ACh or PE (1 x 10(-8) - 1 x 10(-5) mol/L). Superoxide dismutes (SOD) activity and the content of malondialhyde (MDA) in artery tissues were detected. For TARs, LPS significantly reduced the contraction response to PE compared with the vehicle group (P<0.01) and the curve of cumulative dose responses to PE in the LPS group shifted downward. Although EDRR to ACh in the LPS group had the tendency to decrease but still showed no significant difference compared with the vehicle group (P>0.05). For PARs, EDRR to ACh was depressed significantly in the LPS group (P<0.01), while no effect on contraction response to PE in the LPS group was observed, compared with the vehicle group (P> 0.05). Compared with the LPS group, TARs in the LPS+MT group exhibited an increased contraction response to PE, but were still lower than that in the vehicle group. Similarly, EDRR to ACh of PARs in the LPS+MT group was improved significantly and there was no difference between the LPS+MT group and the vehicle group. The vascular reactivity was unaffected in MT group compared with the vehicle group in both TARs and PARs. SOD activity in the LPS +MT group increased significantly and the content of MDA decreased markedly compared with the LPS group. These results suggest that MT may improve the vascular reactivity in endotoxemia rats due to its antioxidant properties.