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Objective To explore the effects of sumatriptan on the modulation of calcitonin generelated peptide(CGRP) expression and its involving intracellular signaling transduction mechanisms in rat trigeminal ganglion(TG) after organ culture.Methods Using organ culture in vitro model,54 isolated TGs of SD rats were randomly divided into fresh group ( n =6 ),control group ( n =6 ) and experimental group (n =42,6 TGs for each subgroup).Experimental group included seven subgroups,which were respectively pretreated with four different concentrations of sumatriptan,specific inhibitors of extracellular signalregulated kinases 1/2 (ERK1/2) pathway (U0126 and PD98059 ),and the inhibitor of c-Jun N-terminal kinase (JNK) (SP600125).After co-cultured with above intervention agents for 24 h,CGRP-immunoreactivity (CGRP-ir) positive neurons and CGRP-mRNA expression levels were quantified by immunohistoehemistry stain and real-time polymerase chain reaction,respectively.Phosphorylated ERK1/2 (pERK1/2) and JNK (pJNK) proteins levels were determined by Western-blotting method.Results The CGRP-ir ( + ) neurons expression levels were significantly increased after 24 h organ culture.However,0.10 and 0.50 mg/ml concentrations of sumatriptan remarkably decreased the CGRP-ir ( + ) neurons expression levels.The positive cell percentage,positive optic area,integrated optical density,mean optical density and CGRP-mRNA expression level in TG were significantly reduced than control groups (tPCP =8.652,26.382; tarea =6.220,13.917; tIA =5.606,15.904; tM14 =2.661,21.748; tmRNA =8.032,15.675.P ＜ 0.05 ).The CGRP-mRNA expressions were significantly down-regulated after co-incubation with concentration of 0.50 mg/ml sumatriptan for 24 h in TG of SD rat ( P ＜0.05 ).The levels of pERK1/2 and pJNK protein kinase detected by Western-blotting were significantly reduced by 0.50 mg/ml concentration of sumatriptan,the degrees of which were closed to the ERK1/2 and JNK pathway specific blockers.Conclusion It suggests that the optimal concentration of sumatriptan significantly down-regulates CGRP over-expression via intracellular ERK1/2 and JNK signaling transduction pathways in TG after organ culture.
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The Chinese herbal medicine Tianma (Gastrodia elata) has been used for treating and preventing primary headache over thousands of years, but the exact pharmacological mechanism of the main bioactive ingredient gastrodin remains unclear. In present study, the effects of gastrodin on calcitonin gene-related peptide (CGRP) and phosphorylated extracellular signal-regulated kinase1/2 (pERK1/2) expression were observed in rat trigeminal ganglion (TG) after in vitro organ culture to explore the underlying intracellular mechanism of gastrodin on primary vascular-associated headache. CGRP-immunoreactivity (CGRP-ir) positive neurons count, positive area, mean optical density and integrated optical density by means of immunohistochemistry stain were compared at different concentrations of gastrodin, which was separately co-incubated with DMEM in SD rat TG for 24 hours. Only at 5 or 10 mmol L(-1) concentration, gastrodin demonstrated significantly concentration-dependent reduction of CGRP-ir (+) expression and its action closed to 1.2 mmol L(-1) sumatriptan succinate. While at 2.5, 20, and 40 mmol L(-1) concentration, gastrodin did not show remarkable effects on CGRP-ir (+) expression. The optimal concentration of gastrodin (5 and 10 mmol L(-1)) similarly inhibited CGRP-mRNA expression level separately compared with 1.2 mmol L(-1) sumatriptan succinate and 10 micromol L(-1) flunarizine hydrochloride, which was quantitatively analyzed by real-time PCR (RT-PCR). pERK1/2 level was examined by Western blotting after co-cultured with optimal concentration of gastrodin and effective specific ERK1/2 pathway inhibitors PD98059, U0126. The result indicated that gastrodin significantly reduced pERK1/2 protein actions similarly to ERK1/2 pathway specific blockade. It suggests ERK1/2 signaling transduction pathway may be involved in gastrodin intracellular mechanism. This study indicates gastrodin (5 and 10 mmol L(-1)) can remarkably reduce CGRP-ir (+) neuron, CGRP-mRNA and pERK1/2 expression level in cultured rat TG, with its actions similar to the effective concentration of sumatriptan succinate, flunarizine hydrochloride and specific ERK1/2 pathway blocker. The intracellular signaling transduction ERK1/2 pathway may be involved in the gastrodin reducing CGRP up-regulation in rat TG after organ culture.
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Objective To investigate the serum coxsackie virus B(CVB) infection and nitric oxide (NO)level of the patients suffer from latent or chronic Keshan disease and their characteristics in the etiopathology of Keshan disease. Methods Sera were isolated from 30 patients with latent or chronic Keshan disease in Huangling county.Shaanxi Province, and the CVB-specific IgM antibody and NO were tested. Control groups were health subjects in Huangling county or Xi'an city, Shaanxi Province. Results The percentage of CVB-specific IgM positive in patients in Huangling county was significantly higher than that of both control groups in Huangling county and Xi'an city (P＜0. 05). The serum level of NO in patients was significantly higher than that of the control group in Huangling county (P＜0.05) ,however,compared with control group in Xi'an city, there was no difference (P＞0.05). In CVB-specific IgM positive patients,the serum level of NO was significantly higher than that of CVB-specific IgM negative group(P＜0.05).Conclusion CVB infection and serum NO level might be related to the etiopathology and the development of Keshan disease.
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Objective To definite the interactions between the human gastric carcinoma cell and the human vascular endothelial cell during the establishment and maintenance of the tumor vascular system and the tumor hematogenous metastasis.Methods We prepared the conditioned mediums of each cell so as to study the effect of the conditioned medium on itself or others by MTT colorimetry. The comprehensive effect of interactions between two cells was determined by stratified transfilter co-culture or direct contact co-culture.Results The conditioned medium of human gastric carcinoma cell can stimulate the proliferation of the human vascular endothelial cell, but the CM of HVEC can inhibit the growth of HGCC. Both kinds of cells can inhibit the growth of itself. The ultimate comprehensive effect of the interactions between two kinds of cells was increase of total cell numbers.Conclusion There exist the complicated interactions between the human gastric carcinoma cell and the human vascular endothelial cell during the tumor angiogenesis and the tumor hematogenous metastasis. The ultimate comprehensive effect of the interactions is increase of total cells numbers and tumor volume.
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Objective To study the releases of endothelin-1 and prostacyclin by endothelial cells in culture and to elucidate how these releases were influenced by smoke-treated low density lipoprotein. Methods We exposed endothelial cell cultures to native or oxidized Iow density lipoproteins,low density lipoproteins treated by dimethylsulfoxide-soluble particles from cigarette smoke or dimethylsulfoxide alones. The release of endothelin-1 was assayed by bioassay and the release of prostacyclin was assayed by radioimmunoassay. Results Low density lipoproteins treated by smoke significantly increased the release of endothelin-l (P＜0. 025) and decreased the release of prostacyclin (P＜0. 02) by endothelial cells in culture, contrast to native or dimethylsulfoxide-treated lipoproteins. Conclusion The main part of vasoconstrictor activity in conditioned medium from bovine aortic EC is endothelin-1.
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Objective To ascertain whether the growth inhibitor in conditioned medium from cultured rabbit arte rial cells is distinct from TGF-β. Methods Rabbit aortic smooth muscle ceils were grown from explained segments of the aorta. Conditioned medium from cultured rabbit aortic smooth muscle ceils and anti-TGF-β were employed in this study. Smooth muscle cell proliferation was measured by XTT detection (Boehringer Mannheim). Results Acidified conditioned medium from smooth muscle ceils had significantly stronger effects of growth inhibition than controls, and anti-TGF-β did not affect the growth inhibitory effect of conditioned medium from cultured rabbit arterial smooth muscle cells. Conclusion The growth inhibiting substance in conditioned medium from cultured rabbit aortic smooth muscle cells is distinct from TGF-β.
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AIM: To investigate the injuring effect of DMSO-soluble particles from cigarette smoke(DSP) on human umbilical vein endothelial cells. METHODS: Human umbilical vein endothelial cell line EA. hy 926 was used as target cells in the study. The growth and viability of the cells treated with various dosages (1, 2, 4 or 4 mL/L) of DSP and low dose (2 mL/L) of DSP at different time points were evaluated by MTT colorimetric assay and celllular protein assay in 96-well plates. Transmission electron microscopy study was carried out to observe the ultrastructure of human umbilical vein endothelial cells under DSP treatment.RESULTS: DSP inhibited the proliferation of human umbilical vein endothelial cell line EA. hy 926. Under DSP treatment, the reducing cellular protein and increasing cell death(mainly necrosis) were observed in time-dependent and dosage-dependent manners.CONCLUSIONS: These results indicated that the toxic effect of DSP caused functional disturbance and structural damage of human endothelial cells.
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Whether InfusaSleeve(IS) catheter can deliver antisense oligodeoxynucleotide (ODNs) following arterial denuation is unknown. We evaluate the feasibility of local endoluminal delivery of C-myc ODNs to the site of arterial denudation by using IS catheter and to determine the biological importance of these effects. IS catheter was introduced into right side of iliac artery of 21 rabbits after angioplasty of iliac artery. Animals were randomized to the control group (n＝6) receiving saline injection and the treated group receiving c-myc antisense (n＝15, 1 mg ODNs per vessel). In two weeks and 40 days following the operation, angiography was performed. Morphometric analyses were carried out in balloon-denuded iliac arteries. The expression of c-myc protein was detected by using a mouse monoclonal antibody to c-myc. Morphometric analyses carried out at 40 days after transcatheter c-myc antisense oligomer administration. The results showed that maximal neointimal area was reduced from 7.66±3.7(×105 μm2) in the control group (n＝6) to 4.04±1.02(×105 μm2) in the antisense treated group (n＝6, P＜0.05). These changes in vascular remodeling following denuding injury resulted in an increase in residual luman from 20～50% in the control group to 70～90% in the antisense-treated group. C-myc protein expression was virtually undetectable at baseline in locally ODNs-delivered arteries and detectable in control denuded arteries. The results show that: ①Single IS transcatheter administration allowed endoluminal delivery of ODNs to the site of arterial injury; ② c-myc antisense oligomer reduced the formation of neointime in denuded arteries, implying a therapeutic potential of this approach.