Objective To observe the relationship between endothelial constitutive nitric oxide synthase (ecNOS) genetic polymorphism and diabetic retinopathy（DR）of non insulindependent diabetes mellitus (NIDDM) patients of the Han nationality.Methods A total of 166 patients who clinical diagnosed with NIDDM as case group, 85 cases of patients (cataract or fracture) and healthy subjects without diabetes, hypertension and kidney disease,over 40 years old of age and without consanguinity between each other were selected as normal control group. Case group were divided into non-DR (NDR) group, nonproliferative-DR (BDR) group and proliferativeDR (PDR) group according to the result of fundus fluorescein angiography. Case group and normal control group subjects all were Han nationality. DNA was extracted from peripheral venous blood; the fourth 27 base pairs (bp) repeat polymorphism of ecNOS gene by was measured by polymerase chain reaction (PCR). Results The 27 bp repeat sequences within the ecNOS gene present in the Han nationality,allele b repeat 5 times, alleles a repeat 4 times. PCR results showed that there are 2 alleles and 3 genotypes in normal control, NDR, BDR and PDR group. The frequency of genotype bb、ab、aa were 80%, 16.5%, 3.5% in normal subjects; 77.2％, 13.9%, 8.9％ in NDR group; 80.5％, 17.1%,2.4％ in BDR group;78.3％, 13%, 8.7％ in PDR group,respectively. The allele frequency (chi;2 =1.841) and gene frequency (chi;2=3.847) were not statistically significant (P＞0.5) in normal control,NDR,BDR and PDR group. Logistic regression analysis showed that there is no relation between DR and ecNOS duplicated gene polymorphism. Conclusions There is 27 bp repeated polymorphism in 4th intron of ecNOS gene, which may not be associated with the DR of NIDDM in the Han nationality.
Objective
To study the relationship between the expression ratio of induced nitric oxide synthase (iNOS) over glial fibrillary acidic protein (GFAP) and the time of injury after brain concussion in rat, in order to acquire a new visual angle for determining injury time of cerebral concussion.
Methods
Eighty-five healthy Sprague-Dawley rats were divided into three groups randomly: model group (n=25), experimental group (n=55), and control group (n=5). The rats in the model group were used to confirm the attack hight to make the model of brain concussion; according to the time of execution, rats in the experimental group were then subdivided into 11 groups with 5 rats in each subgroup, and their execution time was respectively hour 0.5, 1, 3, 6, 12, 24, 48, 96, 168, 240, and 336; the rats in the control group were executed after fed for 24 hours. After the model of cerebral concussion was established through freefalling dart method, hematoxylin-eosin staining and immunohistochemistry staining of iNOS and GFAP were conducted for the brain of the rats. All related experimental results were studied by using microscope with image analytical system and homologous statistics.
Results
The ratio of positive expression of iNOS over that of GFAP increased gradually during hour 0.5- 3 after injury in brain (from 5.03 to 10.47). At the same time, the positive expression of iNOS increased significantly (from 14.61% to 37.45%). However, the increase of the positive expression of GFAP was not obvious. Between hour 3 and 12, the ratio began to decline to 4.98, which was still at a high level, and during the same time period, the positive expressions of iNOS and GFAP also experienced the same change pattern. Later, the ratio began to decline between hour 12 and 336 after injury (from 4.98 to 0.95). All ratios at this time were lower than those between hour 0.5 and 12. The positive expression of iNOS and GFAP both increased to a climax before declining.
Conclusions
The ratio of positive expression of iNOS over GFAP and the respective change pattern of iNOS and GFAP can be used as the evidence of estimating the injury time of cerebral concussion. We can use the ratio of two or more markers to provide a new visual angle for concluding the concussion injury time.
Objective To study the effect of the competitive inhibitor of nitric oxide synthase NG-nitro-L-arginine methyl ester (LNAME) on thedenervated muscle atrophy. Methods A model of the denervated gastrocnemius atthe right lower limb was established in 36 SD adult rats. The rats were randomly divided into two groups: the L-NAMEgroup (Group A) and the control group(Group B). L-NAME 10 mg/ kg daily was injected into the denervated gastrocnemius inGroup A, and normal saline was injected into the denervated gastrocnemius in Group B. At 2, 4 and 8 weeks after operation, the rate of the muscle wet weight preservation, the cross section area of the myocyte, the protein amount, and the percentage of the apoptotic muscle cells were measured respectively and the ultramicrostructure of the myocyte was observed. Results At 2 and 4 weeks after operation, the rate of the muscle wet weight preservation, the cross section area of themyocyte, and the protein amount were significantly greater in Group A than in Group B; however, the percentage of the apoptotic muscle cells was significantly smaller in Group A than in Group B. The observation of the ultramicrostructure of themyocyte showed that an injection of L-NAME could protect the ultramicrostructure of themyocyte. At 8 weeks after operation, there was no significant difference between the two groups in the abovementioned parameters. Conclusion The nitric oxide synthase inhibition can delay the denervated muscle atrophy.
Objective
To observe the expression of matrix metalloproteinase-9 (MMP-9), its tissue inhibitor of matrix metalloproteinase (TIMP-1), inducible nitric oxide synthase (iNOS) and contents of nitric oxide (NO) in the ocular tissues of Sprague-Dawley (SD) rats with endotoxin induced uveitis（EIU）.
Methods
Ninety SD rats were randomly divided into experimental (81 rats) and control group (9 rats). The model of EIU was induced in rats in experimental group by injecting with lipoplysaccharide (LPS) 200 μl into the hind feet pads, while the rats in the control group were not injected. Nine rats were executed 0, 6, 12, 18, 24, 48, 72, 96 hours and 7 days, respectively, after injecting with LPS; the NO content and concentration of protein in the aqueous humor in blood plasma, aqueous humor, and uveal tissues were detected. The expressions of MMP-9, TIMP-1 and iNOS in the ocular tissues were detected by immunohistochemistry, and the average absorbance (A) value was evaluated by computer medical image analysis system.
Results
iNOS, MMP-9 and TIMP-1 expressed in the epithelial cells of iris and ciliary body and exudated inflammatory cells of rats. The concentration of protein in the aqueous humor, the contents of NO in blood plasma, aqueous humor, and uveal tissues, and A value of MMP-9 had obvious relativity with the inflammatory extent, while no positive correlation was found between the inflammatory extent and the A value of iNOS and TIMP-1. Expression of iNOS was found 6 hours after injection, reached the peak after 12 hours, and then dropped gradually. The expression of TIMP-1 could be seen 24 hours after injection, and reached its peak after 72 hours.
Conclusion
The content of NO and expressions of iNOS, MMP-9 and TIMP-1 changes from the beginning and during the development of EIU, which suggests that NO, iNOS, MMP-9 and TIMP-1 are involved in the pathologic process of EIU.
(Chin J Ocul Fundus Dis, 2005, 21: 371-374)
【Abstract】ObjectiveTo investigate the protective effect of melatonin on renal injury induced by bile duct ligation in rats. MethodsSixtyfour rats were randomly divided into four experimental groups (n=16 rats per group): the control group (CN), sham operative group (SO), bile duct ligation group (BDL) and bile duct ligation melatonin treatment group (BDL+Mel). Obstructive jaundice was induced by common bile duct ligation. Plasma level of nitric oxide (NO), total bilirubin (TB), direct bilirubin (DB), alanine aminotransferase (ALT), aspartate aminotransferase (AST), urea nitrogen (BUN) and creatinine (Cr) were measured 4 d and 8 d after operation. NO and inducible nitric oxide synthase (iNOS) in renal tissue were detected at the same time point, too. Histopathological changes of kidneys were examined by HE staining. ResultsIn BDL group, the plasma levels of NO, TB, DB, ALT, AST, BUN and Cr were higher than those of SO group (P＜0.01）, and the level of NO and activities of iNOS in renal tissue were significantly increased (P＜0.01）. However, in BDL+Mel group, the plasma levels of NO, ALT, AST, BUN and Cr were lower than those of the BDL group (P＜0.01）, and the level of NO and activities of iNOS in renal tissue were significantly suppressed (P＜0.01）; histopathological changes of kidneys were improved.ConclusionAugmentation of iNOS activities and increasing of NO production in local tissue contributed to renal injury induced by bile duct ligation, and the mode of melatonin’s protective actions, at least in part, relates to interference with no pathways.
Objective
To analyse the changes of nitric oxide and nitric oxide synthase in rat retina under acute high ocular pressure and study the effect of nitric oxide in rat retinal damage under hypertension.
Methods
Sixty Wistar rats were divided randomly into five groups:Ocular hypertension 30 min,60 min,90 min and 12 h,24 h after reperfusion．Elevation of the ocular pressure in the anterior chamber of the rat eye ca used retina ischemic damage．The changes of retinal nitric oxide content were ob served indirectly by measuring NO2－/NO3－ content in retina.The distribution and changes of neuronal constitutive nitric oxide synthase (ncNOS)were studied by immunocytochemical localization of ncNOS.
Results
ncNOS positive neurons were distributed in the inner nuclear layer (INL),ganglion cell layer (GCL) and the inner plexiform layer of the normal and ischemic rat retina.During acute high IOP 30 min,60 min and 90 min,NO content decreased gradually and ncNOS immune activity weakens.During reperfusion,NO content increased remarkably (Plt;0.05) as compared with the groups of hypertension 90 min and decreased remarkably as compared with the normal rat retina.But ncNOS positive neurons continue to decrease compared with the groups of hypertension 90 min.
Conclusion
NO participates the rat retinal injury by acute elevated intraocular pressure, and nitric oxide synthetized by ncNOS may play an important role in protecting the retina from ischemic and post-ischemic injury.
ObjectiveTo detect the induction of inducible nitric oxide synthase (iNOS) and nitric oxide (NO) production in immunostimulated retinal pigment epithelial (RPE) cells to seek for the supplying of the arginine, a substrate for NOS; as well as the effects of produced NO on the tight junction of RPE-J cells.
MethodsRat′s RPE-J cells were treated with interferon-γ(INF-γ), tumor necrosis factor-α(TNF-α) and lipopolysaccharide (LPS), and Northern and Western blotting were applied to analyze the expression of the citrulline-NO cycle enzymes and related enzymes and the effect of dexamethasone and cyclic adenosine monophosphate (camp) on the expression of iNOS. Immunocytochemistry reaction and Western blotting were used to evaluate the effect of produced NO on the tight junctions of RPE-J cells.ResultsiNOS and argininosuccinate synthetase (AS) were highly induced at both mRNA and protection levels in immunostimulated RPE cells while arginiosuccinate lyase (AL) was not induced. NO was produced by cells after stimulation with TNFα, IFNγ and LPS. The induction of iNOS mRNA and the production of NO by these immunostimulated cells was further enhanced by cAMP. NO was produced from citrulline as well as from arginine. And the produced NO impaired the tight junction of RPE-J cells, decreased the production of tight junction related protein ZO-1.ConclusionIn activated RPE-J cells, citrullinearginine recycling is important for NO production, and the produced NO weakened the function of tight junction of RPE-J cells.(Chin J Ocul Fundus Dis, 2005,21:32-36)
