The individual and combined effects of deoxynivalenol and aflatoxin B1on primary hepatocytes of Cyprinus carpio.

Aflatoxin B(1) (AFB(1)) and deoxynivalenol (DON) are important food-borne mycotoxins that have been implicated in animal and human health. In this study, individual and combinative effects of AFB(1) and DON were tested in primary hepatocytes of Cyprinus carpio. The results indicated that the combinative effects of AFB(1) and DON (0.01 µg/mL AFB(1) and 0.25 µg/mL DON; 0.02 µg/mL AFB(1) and 0.25 µg/mL DON; 0.02 µg/mL AFB(1) and 0.5 µg/mL DON) were higher than that of individual mycotoxin (P < 0.05). The activity of AST, ALT and LDH in cell supernatant was higher than that of control group (P < 0.05) when the mycotoxins were exposed to primary hepatocytes for 4 h. The decreased cell number was observed in tested group by inverted light microscopy. The mitochondrial swelling, endoplasmic reticulum dilation and a lot of lipid droplets were observed in primary hepatocytes by transmission electron microscope. Therefore, this combination was classified as an additive response of the two mycotoxins.

Atorvastatin reduces calcification in rat arteries and vascular smooth muscle cells.

To examine the in vivo effects of atorvastatin (AT) on arterial calcification in rats, arterial calcification was established by subcutaneous injection of vitamin D3 and Warfarin. Intragastric administration of AT began 4 days before establishment of arterial calcification in the AT group (n=6). Blood samples were taken and abdominal aortas were collected and stained. After induction of calcification, plasma Ca(2+) levels in the CA and AT groups were significantly higher than those before treatment and in the untreated controls. Plasma Ca(2+) levels in the AT group were significantly lower than in the CA group. The relative calcification area in aortic specimens from the AT group was significantly smaller than in the CA group. Rat aortic vascular smooth muscle cells (VMSC) were isolated from abdominal aortic segments and pre-treated with AT (1, 5, or 10 µM) for 24 hr. Cells in the calcification (CA) group and the AT group were cultured with ß-glycerophosphate, insulin and vitamin C for 14 days to induce cell calcification. Calcium deposition and alkaline phosphatase activity were significantly increased in the CA group compared to untreated controls (p<0.01). This effect was ameliorated by AT (all p<0.01). In vivo administration of AT reduced arterial calcification and plasma Ca(2+) concentration. In vitro, AT reduced calcification markers in rat aortic vascular smooth muscle cells.

Repeated electroacupuncture preconditioning attenuates matrix metalloproteinase-9 expression and activity after focal cerebral ischemia in rats.

OBJECTIVE: This study investigates the effects of electroacupuncture (EA) preconditioning on blood-brain barrier (BBB) integrity and matrix metalloproteinase-9 (MMP-9) expression in subsequent ischemic hemisphere. METHODS: Focal cerebral ischemia was induced by middle cerebral artery occlusion (MCAO) in rats. Animals were randomly divided into four groups: normal, sham-operated, MCAO and EA groups. In EA group, rats received electroacupuncture stimuli at the Baihui acupoint (GV 20) 30 minutes/day for 5 days. Twenty-four hours after last treatment, the MCAO was performed. The brain water content and BBB permeability were measured 24 hours after MCAO. MMP-9 expression and activity were measured at 6, 12 and 24 hours after MCAO. RESULTS: The results showed that the brain water content of ischemic hemisphere was lower in EA group (81.45 +/- 1.09%) compared with MCAO group (83.98 +/- 1.30%; p<0.05). Similarly, the Evans blue content in EA group (4.90 +/- 1.77 microg/g) was lower compared with MCAO group (9.41 +/- 2.87 microg/g; p<0.05). The protein expression and enzyme activity of MMP-9 increased and reached maximum at 24 hours after reperfusion. However, the protein expression was lower in EA group at 12 and 24 hours after reperfusion (p<0.01, versus MCAO group), and enzyme activity was lower in EA group only at 24 hours (p<0.01, versus MCAO group). DISCUSSION: EA preconditioning could attenuate brain edema and BBB disruption caused by subsequent cerebral ischemia. EA preconditioning could decrease MMP-9 expression and activity, which may be an important mechanism of cerebral ischemic tolerance.

Arginine vasopressin increases iNOS-NO system activity in cardiac fibroblasts through NF-kappaB activation and its relation with myocardial fibrosis.

Previous studies have shown that arginine vasopressin (AVP) promotes myocardial fibrosis (MF), whereas nitric oxide (NO) inhibits MF. Cardiac fibroblasts (CFs) are the main target cells of MF. However, the modulatory effect of AVP on NO production in CFs and the role of this effect in MF are still unknown. In the present study, CFs obtained from Sprague-Dawley rats were stimulated with or without AVP and pyrrolidine dithiocarbamate (PDTC), a specific inhibitor of nuclear factor kappa-B (NF-kappaB). NO production and NOS activity were detected with absorption spectrometry, inducible nitric oxide synthase (iNOS) protein with Western blot analysis, iNOS mRNA with real-time PCR, CF collagen synthesis with [(3)H]proline incorporation, and NF-kappaB activation with immunofluorescence staining and Western blot analysis. The results showed that AVP increased NO production in a dose- and time-dependent manner, with maximal effects at 10(-7) mol/l after 24-h stimulation. AVP also increased NOS activity, protein and mRNA levels of iNOS in a coincident manner. Furthermore, AVP also increased CF collagen synthesis in a dose- and time-dependent manner. In addition, it was found that NF-kappaB was activated by AVP, and that PDTC could inhibit NO production, NOS activity, protein and mRNA levels of iNOS stimulated by AVP in a dose-dependent manner. The inhibitory effects of PDTC on NF-kappaB translocation were coincident with the effects of PDTC on iNOS-NO system activity. It is suggested that AVP increases NO production via the regulation of iNOS gene expression, and the upregulation of iNOS gene expression stimulated by AVP is mediated through NF-kappaB activation. NO production induced by AVP may counteract the profibrotic effects of AVP, thus the development of MF perhaps depends on the balance between profibrotic AVP and antifibrotic NO effects on MF.

The opposite-direction modulation of CD4+CD25+ Tregs and T helper 1 cells in acute coronary syndromes.

Different subsets of T lymphocytes have different functions in atherosclerosis advancement. T helper 1 cells and T regulatory 1 cells have been demonstrated to play opposite roles in rupture of atherosclerotic lesion. However, the role of novel subset of T regulatory cells, known as CD4+CD25+Foxp3+ T cells, remains largely unknown in coronary artery disease (CAD). In this study, we investigated the peripheral CD4+CD25+Foxp3+ T cells of patients with CAD and controls. The patients submitted were divided into three groups: stable angina pectoris (SA) group, unstable angina pectoris (UA) group and acute myocardial infarction (AMI) group. We analyzed the frequencies of peripheral CD4+CD25+Foxp3+ T cells and T helper 1/T helper 2 cells, expression of Foxp3 in CD4+CD25+ T subsets and cytokines pattern in patients and controls. We found that the reduction of CD4+CD25+Foxp3+ T lymphocytes was consistent with the expansion of Th1 cells in patients with unstable CAD. The reversed development between CD4+CD25+ Tregs and Th1 cells might contribute to plaque destabilization.

[Effects of IL-1beta on inducible nitric oxide synthase-nitric oxide system activity in arginine vasopressin-induced rat cardiac fibroblasts].

AIM: To explore the effects of interleukin-1beta (IL-1beta) on inducible nitric oxide synthase (iNOS)-nitric oxide (NO) system activity in arginine vasopressin (AVP)-induced rat cardiac fibroblasts (CFs). METHODS: CFs were isolated by trypsin digestion method. Nitric acid reductase method, spectrophotometry and reverse transcription-polymerase chain reaction (RT-PCR) were used to detect NO contents, NOS activity and iNOS mRNA expression. RESULTS: AVP significantly increased iNOS mRNA expressions, NOS activity and NO contents (P < 0.05) in CFs. IL-1beta enhanced the effects of AVP on iNOS-NO system activity in a concentration-dependent manner, moreover the iNOS mRNA expressions, NOS activity and NO contents of AVP + 3 ng/ml, AVP + 5 ng/ml IL-1beta group were both significantly higher than those of AVP group (P < 0.05). But when IL-1beta concentration increased to 5 ng/ml, the iNOS mRNA expressions, NOS activity and NO contents did not increase accordingly, slightly decreased instead. CONCLUSION: Within certain range of concentrations IL-1beta cooperates with AVP to increase iNOS-NO system activity in CFs.

[Arginine vasopressin-induced nitric oxide content changes in cultured cardiac fibroblasts and its relation to nuclear factor-kappaB].

To investigate the changes in the nitric oxide (NO) contents, nitric oxide synthase (NOS) activity and inducible nitric oxide (iNOS) mRNA expression in arginine vasopressin (AVP)-induced cardiac fibroblasts (CFs) in vitro and its relation to nuclear factor-kappaB (NF-kappaB), CFs were isolated by trypsin digestion method. Nitric acid reductase method, spectrophotometry, reverse transcription-polymerase chain reaction (RT-PCR), immunofluorescence-interactive laser cytometer techniques and Western blotting were used respectively to detect NO contents, NOS activity, iNOS mRNA expression and the activation of NF-kappaB in CFs. AVP increased NO contents, NOS activity and iNOS mRNA expressions in a concentration-dependent manner; NF-kappaB was activated and mobilized from cytoplasm to nucleus in AVP-induced CFs; PDTC, one of the inhibitors of NF-kappaB, could inhibit aforementioned increments. It is suggested that the increases in NO contents, elevation of NOS activity and increment of iNOS mRNA expression may be mediated through NF-kappaB activation pathway in cultured CFs induced by AVP, and that NF-kappaB is involved in the occurrence and development of myocardial fibrosis.

[Effects of chelerythrine on cell proliferation and p27 expression of cardiac fibroblasts modulated by arginine vasopressin].

OBJECTIVE: To study the effects of chelerythrine, a protein kinase C (PKC) inhibitor, on the cell proliferation and p27 expression of cardiac fibroblasts (CFs) modulated by arginine vasopressin (AVP) and to investigate the intracellular signal transduction mechanisms of AVP in CFs. METHODS: The cultured CFs of neonatal Sprague-Dawley rats were divided into 3 groups: AVP group (10(-7) mol/L AVP was added into the culture), chelerythrine group (10(-6) mol/L chelerythrine and 10(-7) mol/L AVP were added into the culture), and control group. MTT assay was used to evaluate the cell proliferation. The cultured cells were collected and propidium iodide was used to label the DNA so as to identify the cell cycle. Specific mouse-versus-rat p27 protein monoclonal antibody and fluorescein isothiocyanate-labeled secondary antibody were added into the cell suspension to label the p27 protein in the cells. Flow cytometry was used to determine the distribution of cell cycles and p27 expression. RESULTS: (1). The A value of CFs measured by MTT assay in AVP + chelerythrine group was 0.32 +/- 0.01, significantly lower than that of the AVP group (0.39 +/- 0.01, P < 0.01). (2). The percentage of CFs in S stage was 4.4% +/- 1.7% in the AVP + chelerythrine group, lower than those of the AVP group (15.5% +/- 1.4%, P < 0.01) and control group (7.5% +/- 1.0%). The PI of CFs was 20.9% +/- 1.2% in the AVP + chelerythrine group, significantly lower than that of the AVP group (31.4% +/- 1.5%, P < 0.01). The PI of the AVP group was significantly lower than that of the control group (26.0% +/- 1.0%, P < 0.01). The percentage of CFs in G(0)/G(1) stage was 79.1% +/- 1.2% in the AVP + 1 chelerythrine group, significantly higher than that in the AVP group (68.6% +/- 1.5%, P < 0.01). The percentage of CFs in G(0)/G(1) stage in the AVP group was significantly lower than that in the control group (74.0% +/- 1.0%, P < 0.01) too. (3). The expression rate of p27 protein was 91.7% +/- 2.2% in the AVP + chelerythrine group, significantly higher than that in the AVP group (63.3% +/- 1.9%, P < 0.01). CONCLUSION: PKC inhibitor remarkably reverses the CFs proliferation and p27 downregulation induced by AVP. It may be involved in the intracellular signal transduction pathway of AVP in CFs.

[Effects of atorvastatin on the proliferation and collagen synthesis of rat cardiac fibroblasts].

OBJECTIVE: To investigate the effects of atorvastatin on the proliferation and collagen synthesis of rat cardiac fibroblasts (CFs). METHODS: Isolated and cultured CFs of neonatal Sprague-Dawley (SD) rats were isolated and cultured. The DNA synthesis of CFs was measured by (3)H-TdR uptake test. CFs proliferation was measured by thiazolyl blue (MTT) assay. Cell cycle distribution was determined with flow cytometer (FCM). Collagen synthesis was measured by 3H-proline uptake test. RESULTS: (1) The (3)H-TdR uptake in the 10(-7) mol/L, 10(-6) mol/L, 10(-5) mol/L, and 10(-4) mol/L atorvastatin groups was (cpm/5,000 cells) 314 +/- 68, 253 +/- 52, 201 +/- 53, and 170 +/- 48 respectively, all significantly lower than that of the control group (378 +/- 65, all P < 0.001). (2) MTT colorimetry showed that the A(490) values were 0.288 +/- 0.008, 0.252 +/- 0.007, 0.225 +/- 0.008, and 0.216 +/- 0.013 respectively in the 10(-7) mol/L, 10(-6) mol/L, 10(-5) mol/L and 10(-4) mol/L atorvastatin groups, all significantly lower than that in the control group (0.311 +/- 0.005, all P < 0.01). (3) The percentage of cells in G(0)/G(1) phase was 54.8% +/- 2.5%, 61.4% +/- 2.7%, 70.4% +/- 3.2%, and 82.0% +/- 4.0% in the 10(-7) - 10(-4) mol/L atorvastatin groups, all significantly higher than that in the control group (46.5 +/- 2.9, all P < 0.01). The percentage of cells in S phase was 18.8% +/- 2.3%, 15.8% +/- 2.1%, 12.5% +/- 1.8%, and 7.3% +/- 2.0% in the 10(-7) - 10(-4) mol/L atorvastatin groups, all significantly lower than that in the control group (23.1% +/- 2.0%, all P < 0.01). The percentage of cells in G(2)/M phase was 26.5% +/- 0.8%, 22.8% +/- 1.2%, 17.2% +/- 1.4%, and 10.7% +/- 2.0% in the 10(-7) - 10(-4) mol/L atorvastatin groups respectively, all significantly lower than that in the control group (30.5% +/- 1.4%, all P < 0.01). The proliferation index (PI) was 45.3% +/- 2.5%, 38.6% +/- 2.7%, 29.6% +/- 3.2%, and 18.0% +/- 4.0% in the 10(-7) - 10(-4) mol/L atorvastatin groups respectively, all significantly lower than that in the control group (53.5% +/- 2.9%, all P < 0.01). (4) The (3)H-proline uptake was (cpm/5 000 cells) 422 +/- 59, 332 +/- 67, 252 +/- 53, and 184 +/- 48 in the 10(-7) mol/L, 10(-6) mol/L, 10(-5) mol/L, and 10(-4) mol/L atorvastatin groups respectively, all significantly lower than that in the control group (566 +/- 62, all P < 0.01). CONCLUSION: Atorvastatin inhibits effectively the proliferation of cultured rat CFs and the collagen synthesis therein, which may play a role in the regression of heart remodeling.

Effect of simvastatin on the proliferation of rat cardiac fibroblasts.

OBJECTIVE: To investigate the effects of simvastatin on the proliferation of rat cardiac fibroblasts (CFs) induced by arginine vasopressin (AVP). METHODS: CFs of neonatal Sprague-Dawley (SD) rats were isolated by trypsin digestion method and growth-arrested CFs were stimulated with 1x10-7 mol/L AVP in the presence of simvastatin (Sim) with varied concentrations. MTT assay was employed to measure CFs proliferation and determine the cell number, and the cell cycle distribution was determined with flow cytometer (FCM). RESULTS: With the increase of Sim concentration, D490 of CFs as shown by MTT assay gradually decreased, and for the cells treated with 1x10-6 mol/L Sim or 1x10-5 mol/L Sim, D490 (0.215+/-0.041and 0.163+/-0.018, respectively) was significantly lower than that of the control (0.939+/-0.048, P<0.01). In a dose-dependent manner, Sim decreased the cell percentage at S stage and the proliferation index (PI) as its concentration increased, but acted to the contrary effect with the percentage of cells at G0/G1 stage, and in CFs treated with 1x10-5 mol/L or 1x10-6 mol/l Sim, the 3 parameters were significantly different from those measured in the CFs with 1x10-7 mol/L AVP treatment (P<0.01). CONCLUSION: The results indicate that Sim can inhibit the proliferation of CFs induced by AVP, possibly through the mechanism of regulating the cell cycle distribution.