Expression of c-Fos protein and nitricoxide synthase in neurons of cerebral cortex from fetal rats in hypoxia and protective role of Angelica sinensis

BACKGROUND： Both c-Fos protein and nitricoxide synthase （NOS） have been used as general indexes in relative research about neurons, but it is lack of reports that c-Fos protein and NOS are applied synchronously to study the neurons of hypoxic fetal rats in uterus. OBJECTIVE： To study the effect of hypoxia in uterus on the expression of c-Fos protein and NOS in neurons of cerebral cortex from fetal rats and whether Angelica sinensis has the protective effect on these neurons in hypoxia. DESIGN： Randomized control experiment.SETTING ： Department of Histology and Embryology, Luzhou Medical College.MATERIALS ： Twelve adult female Wistar rats in oestrum and 1 male Wistar rat with bodymass from 220 to 250 g were chosen. Parenteral solution of Angelica sinensis mainly contained angelica sinensis, 10 mL/ampoule, was provided by Department of Agent of the Second Hospital Affiliated to Hubei Medical University （batch number： 01062310）. METHODS ： This experiment was completed in the Department of Histology and Embryology of Luzhou Medical College from September 2003 to June 2004. ①Twelve adult female Wistar rats in oestrum and 1 male Wistar rat were housed in one rearing cage. Vaginal embolus was performed on conceive female rat at 8： 00 am next day. On the 15^th conceiving day, all conceiving rats were divided randomly into three groups： control group, hypoxia group and Angelica group with 4 in each group. Rats in hypoxia group and Angelica group were modeled with hypotonic hypoxia in uterus. Angelica group： Rats were injected with 8 mL/kg Angelica sinensis injection through caudal veins before hypoxia. Hypoxia group： Rats were injected with the same volume of saline. Control group： Rats were not modeled and fed with normal way. ② Twenty embryos of rats were chosen randomly from each group and then routinely embedded in paraffin. Paraffin sections were cut from the brain of embryos to anterior fontanelle. Double-label staining was used to detect the expression of nNOS and c-Fos in neurons of cerebral cortex from embryos of rats. OLYMPUS Bx-50 microscope was used to observe sections and DP12 digit camera was also used under 400 times to detect types of cells. Under microscope, the number of c-Fos, NOS, c-Fos/NOS positive neurons in cerebral cortex from embryos of rats were counted in 2 fields with magnification of 400 in one section per animal. ③ The data in experiments were analyzed by one-way analysis of variance （ANOVA） followed by q test. MAIN OUTCOME MEASURES： ① Results of immunohistochemical double-label staining of c-Fos/NOS from cerebral cortex; ② Comparison of amount immunohistochemical double-label staining of c-Fos/NOS positive cells from cerebral cortex. RESULTS：① The positive NOS cells and c-Fos/NOS cells in the three groups were mainly distributed in cerebral cortex, but positive c-Fos neurons were not observed. ② Positive NOS cells and c-Fos/NOS cells in hypoxia group were more than those in control group （76.55±12.02, 50.45±10.39; 33.35±7.42, 26.35±6.67, P 〈 0.05）, but those in Angelica group were less than those in hypoxia group （51.70±9.82, 35.65±8.37, P 〈 0.05）. CONCLUSION： Hypoxia can stimulate the increase of expression of c-Fos protein and NOS in neurons of cerebral cortex. However, Angelica sinensis can decrease this expression so as to play a protective role in cerebral neurons of hypoxic fetal rats.

一氧化氮合酶、氧化应激与糖尿病心肌病

心血管疾病是糖尿病患者最普遍的发病率和致死率的病因。高血糖不仅引起血管损害,同时也直接损害心肌细胞。已经证明糖尿病心肌病导致缺血性事件的死亡率增加。

桃核承气改良方对兔血管内皮剥脱术后一氧化氮和血管内皮生长因子的影响

[目的]通过与西药作用对比,观察中药桃核承气改良方对兔血管损伤后内皮细胞生长及功能的影响。[方法]40只兔随机数字表法分为四组,模型组(n=11)、中药组(n=12)、西药组(n=11)和空白组(n=6)。造模后模型组、中药组、西药组正常饲料喂养,中药组予桃核承气改良方,西药组予舒降之、阿司匹林混合液,模型组、空白组予生理盐水各灌胃4周。[结果]32只白兔进入结果分析,模型组(n=8)、中药组(n=10)、西药组(n=8)和空白组(n=6)。①与空白组相比,其他三组造模后1d血清一氧化氮含量下降(P<0.05)。造模后28d兔中药组、西药组较模型组NO明显升高(P<0.05)。②模型组VEGF表达明显高于其他3组(P<0.05)。[结论]桃核承气改良方能够促进血管内皮细胞的修复,防治再狭窄。

Liver plays a central role in asymmetric dimethylargininemediated organ injury

Asymmetric-dimethylarginine(ADMA) competes with L-arginine for each of the three isoforms of nitric oxide synthase:endothelial;neuronal;inducible.ADMA is synthesized by protein methyltransferases followed by proteolytic degradation.ADMA is metabolized to citrulline and dimethylamine,by dimethylarginine dimethylaminohydrolase(DDAH) and enters cells through cationic amino-acid transporters extensively expressed in the liver.The liver plays a crucial role in ADMA metabolism by DDAH-1 and,as has been recently demonstrated,it is also responsible for ADMA biliary excretion.A correlation has been demonstrated between plasma ADMA levels and the degree of hepatic dysfunction in patients suffering from liver diseases with varying aetiologies:plasma ADMA levels are increased in patients with liver cirrhosis,alcoholic hepatitis and acute liver failure.The mechanism by which liver dysfunction results in raised ADMA concentrations is probably due to impaired activity of DDAH due to severe inflammation,oxidative stress,and direct damage to DDAH.High plasma ADMA levels are also relevant as they are associated with the onset of multiorgan failure(MOF).Increased plasma concentration of ADMA was identified as an independent risk factor for MOF in critically-ill patients causing enhanced Intensive Care Unit mortality:a significant reduction in nitric oxide synthesis,leading to malperfusion in various organs,eventually culminating in multi organs dysfunction.

Protective effect of inducible nitric oxide synthase inhibitor on pancreas transplantation in rats

AIM: To investigate the effect of inducible nitric oxide synthase inhibitor, aminoguanidine, on pancreas transplantation in rats. METHODS: A model of pancreas transplantation was established in rats. Streptozotocin-induced diabetic male Wistar rats were randomly assigned to sham-operation control group (n= 6), transplant control group (n= 6), and aminoguanidine (AG) treatment group (n=18). In the AG group, aminoguanidine was added to intravascular infusion as the onset of reperfusion at the dose of 60 mg/kg, 80 mg/kg, 100 mg/kg body weight, respectively. Serum nitric oxide (NO) level, blood sugar and amylase activity were detected. Nitric oxide synthase (NOS) test kit was used to detect the pancreas cNOS and inducible NOS (iNOS) activity. Pancreas sections stained with HE and immunohistochemistry were evaluated under a light microscope. RESULTS: As compared with the transplant control group, the serum NO level and amylase activity decreased obviously and the evidence for pancreas injury was much less in the AG group. The AG (80 mg/kg body weight) group showed the most signifi cant difference in NO and amylase (NO: 66.0 ± 16.6 vs 192.3 ± 60.0, P < 0.01 and amylase: 1426 ± 177 vs 4477 ± 630, P < 0.01). The expression and activity of tissue iNOS, and blood sugar in the AG (80 mg/kg body weight) group were much lower than those in the transplant control group (iNOS: 2.01 ± 0.23 vs 26.59 ± 5.78, P < 0.01 and blood sugar: 14.2 ± 0.9 vs 16.8 ± 1.1, P < 0.01). CONCLUSION: Selective iNOS inhibitor, aminoguanidine as a free radical, has a protective effect on pancreas transplantation in rats by inhibiting NO and reducing its toxicity.

Topiramate as a neuroprotective agent in a rat model of spinal cord injury

Topiramate（TPM） is a widely used antiepileptic and antimigraine agent which has been shown to exert neuroprotective effects in various experimental traumatic brain injury and stroke models. However, its utility in spinal cord injury has not been studied extensively. Thus, we evaluated effects of TPM on secondary cellular injury mechanisms in an experimental rat model of traumatic spinal cord injury（SCI）. After rat models of thoracic contusive SCI were established by free weight-drop method, TPM（40 mg/kg） was given at 12-hour intervals for four times orally. Post TPM treatment, malondialdehyde and protein carbonyl levels were significantly reduced and reduced glutathione levels were increased, while immunoreactivity for endothelial nitric oxide synthase, inducible nitric oxide synthase, and apoptotic peptidase activating factor 1 was diminished in SCI rats. In addition, TPM treatment improved the functional recovery of SCI rats. This study suggests that administration of TPM exerts neuroprotective effects on SCI.

Temporal dynamic changes of connexin 43 expression in C6 cells following lipopolysaccharide stimulation

Connexin 43, a gap junction protein, is expressed mainly in glia in the central nervous system. Neuroinflammation plays an important role in central nervous system injury. Changes to glial connexin 43 levels and neuroinflammation may trigger brain injury and neurodegenerative diseases To illustrate the relationship between connexin 43 and neuroinflammation, this study investigated how connexin 43 expression levels change in lipopolysaccharide-stimulated rat C6 glioma cells. C6 cells were treated with 0.05, 0.25, 0.5, 1,2.5 and 5 IJg/mL lipopolysaccharide for 24 hours. The nitrite estimation-detected nitric oxide release level was elevated substantially after lipopolysaccharide stimulation. To test the transcriptional level changes of inducible nitric oxide synthase, tumor necrosis factor-a and connexin 43 mRNA, C6 cells were treated with 5 pg/mL lipopolysaccharide for 3 48 hours. Reverse transcription-PCR showed that the expression of inducible nitric oxide synthase and tumor necrosis factor-a mRNA increased over time, but connexin 43 mRNA levels increased in lipopolysaccharide-stimulated C6 cells at 3 and 6 hours, and then decreased from 12 to 48 hours. Connexin 43 protein expression was detected by immunofluorescence staining, and the protein levels matched the mRNA expression levels. These results suggest that connexin 43 expression is biphasic in lipopo^ysacchadde-induced neuroinflammation in C6 cells, which may be correlated with the connexin 43 compensatory mechanism.

Nitrogen monoxide vector of ultrasonic atomizing inhalation improves vertebro-basilar artery insufficiency Hemodynamic changes are detected by transcranial Doppler test

BACKGROUND： Latest researches at home and abroad indicate that glycerol trinitrate plays its function because it can metabolize into nitrogen monoxide （NO） in vivo. OBJECTIVE： To study the therapeutic effects of NO vector of ultrasonic atomizing inhalation on vertebro-basilar artery insufficiency （VBI） through transcranial Doppler （TCD） detection and serum NO content and indirect effect of TCD on cerebral blood flow changes. DESIGN： Randomized grouping and controlled clinical study. SETTING： Department of Neurology, the Fourth People＇s Hospital of Jinan. PARTICIPANTS： A total of 130 patients who were diagnosed as VBI were selected from Department of Neurology, the Fourth People＇s Hospital of Jinan from December 2001 to December 2005. The involved inpatients were checked by CT and MRI, and met the VBI diagnostic standard enacted by the Fourth National Academic Meeting of Cerebrovascular Disease in 1995. All patients and their relatives provided the confumed consent. They were randomly divided into low-dose treatment group （n =60）, high-lose treatment group （n =30） and control group （n =40）. METHODS： Patients in the low-dose and high-dose treatment groups were given ultrasonic atomizing inhalation of 3 mg and 5 mg glycerol trinitrate, respectively, for 20 minutes, once a day. In addition, ligustrazine and energy mixture were used once a day for three days in a course. Cases in the control group were only given ligustrazine and energy mixture. All selected cases accepted TCD, blood NO content was checked at the time of beginning, after the first time and after a period of treatment. According to the TCD test, VBI patients were divided into two groups （high-low flow velocity）. The vertebral artery （VA） and basal artery （BA） of left or right sides were detected by 2 Hz detector via occipital window. MAIN OUTCOME MEASURES： ①Blood flow velocity of systolic phase, blood flow velocity of diastole phase and vascular resistance in left and right VA and BA detected by using TCD before treatment, after treatment for one course; ②content of serum NO indirectly measured by using nitric acid disoxidation technique. RESULTS： All 130 VBI patients were involved in the final analysis. ①Changes of hemodynamic indexes： Systolic phase of VA and diastole phase of BA were higher in low-dose treatment group than that in the control group after first treatment, and there was significant difference （P 〈 0.05）; meanwhile, systolic phase and diastole phase of VA and systolic phase of BA were also higher in treatment group than that in the control group after one course （P 〈 0.05）. However, both systolic phase and diastole phase of VA and BA were lower in high-dose treatment group than that in the control group after first treatment and one course, and there was significant difference （P 〈 0.05）. ②Content of serum NO： After first treatment, there was no significant difference between low-dose treatment group and high-dose treatment group （P 〉 0.05）; but both groups were higher than control group, and there was significant difference （P 〈 0.05, 0.01）. CONCLUSION： NO vector of ultrasonic atomizing inhalation can improve VBI so as to improve cerebral blood-supply state.

Nitric oxide and hydrogen sulfide share regulatory functions in higher plant events

Nitric oxide(NO)and hydrogen sulfide(HS)are two molecules that share signaling properties in plant and animal cells NO and H2S originate two farmilies of de rived mol ecules designated reactive nitrogen and sulfur species(RNS and RSS,respectively).These molecules are responsible for certain protein regulatory processes through posttranslational modifications(PTMs),being the most remarkable S nitrosation and persufidation,which afect the thiol group of cysteine residues.NO and H2S can also exert regulatory functions due to their interaction through the iron present in proteins that contain heme groups or iron-sulfur dlusters,as reported mainly in animal cells.Howewer,the available information in plant cells is still very limited thus far.In higher plants,NO and H2S are involved in a myriad of physiological events from seed germination to fruit ripening,but also the mec hanism of response to biotic and abiotic stress conditions.This vie wpoint manuscript highlights the functional regulatory parllelism of these two molecules which also interact with the metabolism of reactive oxygen species(ROS)in plant cells.

Interaction between enteric epithelial cells and Peyer's patch lymphocytes in response to Shigella lipopolysaccharide: Effect on nitric oxide and IL-6 release

AIM： TO investigate the effect of interaction between enteric epithelial cells and lymphocytes of Peyer＇s patch on the release of nitric oxide （NO） and IL-6 in response to Shigella lipopolysaccharide （LPS）. METHODS： Human colonic epithelial cells （Caco-2） were mixed cocultured with lymphocytes of Peyer＇s patch from wild-type （C57 mice） and inducible NO synthase knockout mice, and challenged with Shigella F2a-12 LPS. Release of NO and raiL-6 was measured by Griess colorimetric assay and enzyme-linked immunosorbent assay （ELISA）, respectively. RESULTS： In the absence of LPS challenge, NO was detected in the culture medium of Caco-2 epithelial cells but not in lymphocytes of Peyer＇s patch, and the NO release was further up-regulated in both cocultures with lymphocytes from either the wild-type or iNOS knockout mice, with a significantly higher level observed in the coculture with iNOS knockout lymphocytes. After Shigella F2a-12 LPS challenge for 24-h, NO production was significantly increased in both Caco-2 alone and the coculture with lymphocytes of Peyer＇s patch from the wild-type mice but not from iNOS knockout mice. LPS was found to stimulate the release of mIL-6 from lymphocytes, which was suppressed by coculture with Caco-2 epithelial cells. The LPS-induced mIL-6 production in lymphocytes from iNOS knockout mice was significantly greater than that from the wild-type mice. CONCLUSION： Lymphocytes of Peyer＇s patch maintain a constitutive basal level of NO production from the enteric epithelial cell Caco-2. LPS-induced mIL-6 release from lymphocytes of Peyer＇s patch is suppressed by the cocultured epithelial cells. While no changes are detectable in NO production in lymphocytes from both wild-type and iNOS knockout mice before and after LPS challenge, NO from lymphocytes appears to play an inhibitory role in epithelial NO release and their own mIL-6 release in response to LPS.