Study Of Correlation Between Expressions Of NNOS And CGMP And Pathological Grades Of Glioma

IntroductionBlood - brain barrier ( BBB ) , made up of capillary endothelial cell, intercellular tight junction, base membrane, and astroglial cell, not only exists in normal brain tissue, but also in tumor tissue forming blood - tumor barrier (BTB). Though it is not well developed, BTB limits adequate delivery of antitumor agents to tumor tissue and reduces the pharmacological effect.Normal brain capillaries appear to prevent vasoactive compounds from increasing the permeability of blood vessels, while brain tumor capillaries or injured brain capillaries lose the ability to resist such increasing. Recently, many scientists studied the biochemical opening of the BTB with vasoactive compounds with rat glioma model. Many studies have demonstrated that these compounds do not increase drug delivery in normal brain tissue, but selectively increase the capillary permeability of the brain tumor, which decreases the cytotoxic effects to normal brain tissue. Currently, the most effective compound that selectively opens the BTB is bridykinin ( BK) or its analog, receptor - mediated permeavil-izer-7 (RMP-7).Studies indicate that the selective increase of BTB permeability by BK is mediated by nitric oxide ( NO) , which is correlated with the high expressing level of nitric oxide synthase ( NOS) in tumor. NO has the ability to activate soluble guanylate cycalse ( sGC) which increases the production of cyclic guanosine 3', 5'- monophosphate (cGMP) , and cGMP can regulate the opening of the BTB. The American and European researchers have carried out a series of clinical experiments and found that there were significant differences in the changes of BTB permeability by BK or RMP - 7 in brain tumors with thesame diagnoses. Such difference limits the wide clinical use of BK or RMP -7. We suggest the differences are relevant with the expressing level of nNOS and cGMP in giloma, and the causes of such differences are urgent task to study.This study compared the expressing level of nNOS and cGMP in human glioma with the different pathological grades and analyzed the correlation between them, in order to make clear the theoretical causes that lead to the notable differences of opening of BTB by BK or RMP -7. For applying to the clinical therapy of glioma, this study may help to select out the glioma patients who are sensitive to BK or RMP - 7 and indeed increase the curative effects and living qualities.Materials1. Experimental specimens: fresh glioma tissues were obtained from 36 patients who underwent surgery in the Department of Neurosurgery, the Affiliated Hospital of China Medical University, Shenyang, China. After resection, the samples were immediately frozen in liquid nitrogen, then kept at -70℃2. Experimental reagents: rabbit anti - human nNOS antibody, goat anti -human β-actin antibody, enhanced chemiluminescence (ECL) kit, (Zhongs-han Biotechnology CO. , LTD. , Beijing) , nitrocellulose membrane (Sino - A-merican Biotechnology CO. , Shanghai) , immunohistochemistry kit (Boster Biological Technology LTD. , Wuhan) , radioimmunoassay kit of cGMP (Shanghai University of T. C. M).3. Experimental instruments; stir apparatus, low temperature centrifugal machine, electrophoresis apparatus, transmark apparatus, freeze microtome, gamma RIA counter, luzex - F image analysis system.Methods1. The clinicopathologic findings were diagnosed by reviewing all tissue sections stained by hematoxylin and eosin ( H&E).2. According to the results of H&E, the specimens were devided into 3groups. Every grade was as one group.3. The expression of nNOS in glioma with different pathological grades was determined by western blot.4. The expression of nNOS in glioma with different pathological grades was determined by immunohistochemistry.5. The expression of cGMP of the same tissues was detected by radioimmunoassay.6. Statistical analysisIntegrated density value (IDV) of the nNOS and β - actin protein band were abtained from scanning immunoblot by chemi imger 5500 V2.03 software, and the data were calculated by fluor chen 2.0 software. The distribution of cells which immunopositive to anti - nNOS antibody was analysed semiuantitatively by luzex - F real - time image analysis system.Data from immunohistochemical and western blot analysis were expressed as mean standard deviation ( SD). The differences among groups were determined by using one - way analysis of variance ( One - way ANOVA). When the P value was less than 0.05, the result was considered significant. The correlation and linear regression were used to compare the difference of expressing level of nNOS and cGMP in glioma with different pathological grades by SPSS 11.0 statistic software.Results1. The result of H&E stainingIn 36 cases of glioma, there were 11 cases of grade 1, 14 cases of grade Ⅱ, 11 cases of grade　Ⅲ, 0 cases of grade Ⅳ.2. The result of western blotThere was significant difference between the ratio of IDV of nNOS to IDV of β - actin and pathological grades of glioma, which was measured by western blot, grade Ⅰ vs. grade Ⅱ (1.0900 ±0.0347 vs. 1. 2242 ±0.0292, p <0.01); grade Ⅰ vs. grade Ⅲ (1. 0900 ± 0. 0347 vs. 1. 5398 ± 0. 0433 , P < 0. 01 ) ; grade Ⅱ vs. grade Ⅲ ( 1. 2242 ± 0. 0292 vs. 1. 5398 ± 0. 0433 , P < 0. 01).Comparison of the result showed different expressions in the three groups of glioma, and the order was of grade Ⅰ < grade Ⅱ < grade Ⅲ. There was a significant positive correlation between expression of nNOS and pathological grades of glioma(y=0.830+0.225x, r=0.953, P<0.01).3. The result of immunohistochemistryImmunohistochemical studies clearly showed positive staining in every grade of glioma cells by the polyclonal anti - nNOS antibody. There was significant difference of nNOS expressing level among glioma with different pathological grades, grade Ⅰ vs. grade Ⅱ (0. 1717 ±0. 0136 vs. 0. 2744 ±0. 0150, P < 0.01); grade Ⅰ vs. grade Ⅲ (0. 1717 ±0. 0136 vs. 0. 3684 ±0. 0150, P < 0.01) ; grade Ⅱ vs. grade Ⅲ (0. 2744 ±0. 0150 vs. 0. 3684 ±0. 0150, P < 0.01 ). Comparison of the result showed different expressions in the three groups of glioma, and the order was grade Ⅰ < grade Ⅱ < grade Ⅲ. There was a significant positive correlation between expression of nNOS and pathological grades of glioma (y =0.075+0.098x, r=0.984, P<0.01).4. The result of radioimmunoassayThere was remarkable difference of cGMP expression in three groups which was determined by radioimmunoassay, grade Ⅰ vs. grade Ⅱ (0. 0034 ±0. 0009 vs. 0.0063 ±0.0013, p<0.01); grade Ⅰ vs. grade Ⅱ(0.0034 ±0.0009 vs. 0.0132 ±0. 0049, p < 0. 01); grade Ⅱ vs. grade Ⅲ (0. 0063 ± 0. 0013 vs. 0. 0132 ±0.0049, p <0.05 ). Comparison of the result showed different expression of the three groups of glioma, the order was grade Ⅰ < grade Ⅱ < grade Ⅲ. There was a significant positive correlation between expression of cGMP and pathological grades of glioma (y=0.0049x - 0.0022, r = 0.796, P <0.05).DiscussionFor the first time using human specimens, this study demonstrated that there was obviously different expressing level of nNOS and cGMP in glioma with different pathological grades, and the consequence was grade Ⅰ < grade Ⅱ < grade Ⅲ. There was a significant positive - correlation between the expressing level of nNOS and cGMP and the pathological grades of glioma.The precise mechanisms that BK or its analog selectively opens the BTB are not completely understood. The previous study has reported that activation of bradykinin B2 receptors upon binding BK elicits a cascade of signal transduction reaction involving transient increase in intracellular Ca2+ concentration. The transient increase in the cytosolic Ca2 + mainly comes from intracellular pools through the activation of phosphoinositide specific phospholipase C and influxes of Ca2+ from the extracellular spase. Elevated cytosolic Ca2+ stimulates Ca2+/ calmodulin -dependent activation of constitutive nitric oxide synthase (cNOS). NO is generated. The production of cGMP is upon activation of sGG by NO. The increase of intracellular Ca2+ and cGMP leads to contraction of capillary endothelial cells with a resultant deformation of tight junctions and an increase in vascular permeability. The permeability of BTB is increased consequently.Utepbergenov had proved that until the concentration of NO reached a certain level it couldnt increase the permeability of BBB by using the cell culture model of BBB ( coculture of rat brain endothelial cells with rat astrocytes). This study found that there were differences in expressing level of nNOS in glioma concerning for different pathological grades, the order was grade Ⅰ < grade Ⅱ < grade Ⅲ. In vivo, the synthesis of NO is based on the L - arginine as donor and catalyzed by NOS. This process suggests that with different pathological glioma grades, the concentration of NO is different, which may contribute to the remarkable difference in opening BTB by BK or its analog RMP-7.Intracellular cGMP is modulated by two kinds of enzymes: synthesized by sGC while broken down by phosphodiesterase (PDE). It has been reported that the biological effect of NO is to activate the sGC to increase the intracellular level of cGMP, therefore increase the permeability of BTB. And professor Liu and Matsukado k found respectively that there were two kinds of inhibitors of PDE, which increase the permeability of BTB significantly by inhibiting the decomposing of cGMP. All of above shows that the increasing level of cGMP is likely the one of important factors leading to the selective opening of BTB. This study indicated that the expression of cGMP was different in glioma with different pathological grades, grade Ⅰ < grade Ⅱ < grade Ⅲ, which may also contribute to the remarkable difference in opening BTB by BK or its analog RMP -7.