血脂康对新生大鼠心肌成纤维细胞增殖及胶原合成的影响

目的观察血脂康对血管紧张素Ⅱ(AngⅡ)诱导的新生大鼠心肌成纤维细胞(CFs)增殖及胶原合成的影响,并探讨可能的分子机制。方法采用胰酶消化法分离培养Sprague-Dawley大鼠心肌成纤维细胞,建立AngⅡ诱导CFs增殖的模型,以MTT比色法和流式细胞仪检测细胞周期分析法观察血脂康对CFs数目和细胞周期的影响。AngⅡ及不同浓度血脂康作用48 h后,用天狼星红染色法检测培养上清中胶原的含量;ELISA法检测细胞培养上清液中TGF-β1蛋白表达;RT-PCR法检测胶原和TGF-β1 mRNA表达。结果AngⅡ对CFs增殖有明显促进作用,血脂康可明显抑制AngⅡ诱导的CFs增殖,且呈剂量依赖性;血脂康可增加G0/G1期细胞百分率,降低S、G2/M期细胞百分率,降低胶原含量、TGF-β1蛋白及胶原和TGF-β1 mRNA的表达。结论血脂康能抑制AngⅡ诱导的CFs增殖和胶原的产生,其作用可能是通过抑制TGF-β1表达实现的。     

积雪草提取物对成纤维细胞胶原合成的影响

应用不同剂量的积雪草苷和羟基积雪草作用于体外培养成纤维细胞（Human Skin Fibroblast,HSFb）后，通过放射免疫的方法测定实验组和空白对照组细胞上清中Ⅰ型前胶原氨基端须原（PⅠNP）和Ⅲ型前胺原氨基端肽原（PⅢNP）含量。结果显示：①与空白对照组相比，积雪草苷和羟基积雪草苷可有效刺激体外培养HSFb PⅠNP和PⅢNP的分泌；②积雪草苷和羟基积雪草苷刺激靶细胞PⅠNP和PⅢNP分泌的生物学作用呈现明显的量效关系；③在10μg/mL～150μg/mL剂量内，Ad对HSFb刺激PⅠNP的分泌显示出比Md更强的生物活性（ρ＜0.01）。     

经皮植入体对大鼠皮肤成纤维细胞的作用研究

对自行研制的新型经皮通路植入材料致密增强羟基磷灰石基陶瓷、医用硅橡胶/羟基磷灰石复合材料进行体外细胞毒性评价.原代培养大鼠皮肤成纤维细胞,并与材料的浸提液相互作用,用MTT法测定新型经皮通路材料的细胞毒性.结果表明,致密增强羟基磷灰石基陶瓷、医用硅橡胶/羟基磷灰石复合材料均无细胞毒性.     

水溶性富勒烯对人皮肤成纤维细胞活性的影响

为探讨水溶性富勒烯对人皮肤成纤维细胞的毒性,将人皮肤成纤维细胞与不同浓度水溶性富勒烯共孵育不同时间后,采用MTS法对人皮肤成纤维细胞的活性进行检测。结果表明,富勒烯浓度为7.5μg·mL~(-1)且共孵育时间在48 h以内时,富勒烯对人皮肤成纤维细胞产生微小的毒性;若浓度大于7.5μg·mL~(-1)或共孵育时间超过48 h,富勒烯对人皮肤成纤维细胞有较明显的毒性。因此,开发基于水溶性富勒烯产品时,应考虑一定条件下富勒烯材料可能存在的细胞毒性问题,当富勒烯材料与皮肤接触时,建议控制富勒烯材料的浓度在7.5μg·mL~(-1)以下,同时与人体皮肤的接触时间控制在48 h内为宜。     

小鼠肾脏发育中iNOS及iNOSmRNA的表达

目的本研究通过观察胚胎及生后小鼠肾脏iNOSmRNA及iNOS的表达,探讨iNOS在小鼠肾脏发生发育中意义。方法取昆明小鼠,按胚龄12,14,16,18d和新生1,3,5,7,14,21,42d分组。取肾脏,Western-blot检测iNOS,原位杂交检测iNOSmRNA,结果用CIAS—1000细胞图像分析仪分析。结果原位杂交结果显示:iNOSmRNA产生于胚胎14d,分布在输尿管芽;胚胎16d至成年iNOSmRNA主要分布在近曲小管,而远曲小管表达较少。各组iNOSmRNA呈持续表达,无明显差异。Western-blot结果显示:iNOS的光密度百分数在胚胎14d组最高,随肾脏发育iNOS含量逐渐减少,各组差异有显著性。结论肾脏发生发育中iNOSmRNA是持续稳定的,iNOS含量随发生发育过程逐渐减少,因此iNOS表达调节可能发生转录和转录后水平。     

两种脂质体制备方法对碱性成纤维细胞生长因子活性的影响

目的考察两种脂质体制备方法对碱性成纤维细胞生长因子(bFGF)活性的影响。方法采用薄膜分散法和冻干重建法制备bFGF脂质体,MTT法检测两种方法制备的脂质体中bFGF的活性。结果薄膜分散法和冻干重建法制备的bFGF脂质体活性分别为1·99×107和1·94×107IU/ml。与对照bFGF水溶液相比,两种脂质体制备方法均未降低bFGF活性,且包封率与粒径差异无显著意义。结论采用薄膜分散法和冻干重建法制备bFGF脂质体,对其活性均无影响。     

积雪草苷对成纤维细胞胶原相关基因的作用

应用积雪草苷（Asiaticoside,Ad）作用于体外培养成纤维细胞（Human Skin Fibroblast,HSFb）后，通过基因芯片杂交技术，研究Ad对HSFb基因表达谱的影响；进一步，通过Northem杂交和放射免疫技术研究Ad对胶原代谢相关可疑靶基因在mRNA水平和蛋白水平的影响，以探讨Ad在皮肤损伤修复中的作用机制。结果显示：①细胞外基质（Extracellular Matrix,ECM）合成相关基因在不同时相点基因表达谱明显示不同；②在蛋白水平和mRNA水平，Ad具有刺激HSFbⅠ型和Ⅲ型胶原合成的作用。提示，Ad对胶原合成相关基因具有明显的调节作用。     

一氧化氮与代谢综合症相关性的研究进展

代谢综合症(MS)是代谢系统疾病中一组慢性进行性疾病,高血脂症、高血压及肥胖是其主要的病因。一氧化氮(NO)作为重要的细胞信使和效应分子,它介导并调节多种机体代谢功能,同时一氧化氮合酶(NOS)是NO合成过程中的关键酶,现就NO的功能特性及其参与MS相关疾病诊治的研究进展进行概述。     

内皮素-1对人子宫肌层成纤维细胞表型分化的影响

目的探讨内皮素-1(endothelin-1,ET-1)对人子宫肌层成纤维细胞向成肌纤维细胞转化的影响。方法原代培养人子宫肌层成纤维细胞,经免疫细胞化学法鉴定后,将细胞分为实验组和对照组,实验组分别以0.1、1、10、100 nmol/L ET-1作用24 h,另以10 nmol/L ET-1分别作用6、12、24、48 h,设未处理细胞作为对照,采用实时定量PCR(real-time quantitative polymerase chain reaction,RT-qPCR)和Western blot法检测各组细胞α-SMA mRNA及蛋白的表达水平。结果人子宫肌层成纤维细胞的α-SMA蛋白染色呈阴性,波形蛋白染色呈阳性;不同浓度ET-1作用细胞24 h后,均可检测到α-SMA mRNA及蛋白的表达,与对照组相比,差异有统计学意义(P0.05),其中10 nmol/L ET-1实验组表达量最高,与0.1和1 nmol/L ET-1实验组相比,差异有统计学意义(P0.05);经10 nmol/L ET-1作用不同时间的实验组,α-SMA mRNA及蛋白均有表达,与对照组相比,差异均有统计学意义(P0.05),其中以作用48 h实验组表达最高,与作用6、12、24 h实验组相比,差异均有统计学意义(P0.05)。结论 ET-1可促进子宫肌层成纤维细胞向成肌纤维细胞的转化。     

丹红注射液对心肌缺血再灌注大鼠血清一氧化氮合酶、内皮素-1的影响

目的观察丹红注射液对心肌缺血再灌注损伤大鼠血清一氧化氮合酶、内皮素-1含量的变化的作用。方法建立大鼠心肌缺血再灌注损伤动物模型,研究丹红注射液对心肌缺血再灌注损伤大鼠血清一氧化氮合酶、内皮素-1水平的影响。结果与正常对照组相比较,心肌缺血再灌注损伤组内皮素-1明显升高(P<0.05);丹红注射液可抑制内皮素-1的升高(P<0.05)。与正常对照组相比较,各组的一氧化氮合酶均有升高(P<0.05);丹红注射液组与心肌缺血再灌注损伤组比较未见显著性差异。与正常对照组相比较,心肌缺血再灌注损伤组ET-1/NOS明显升高(P<0.05);丹红注射液可抑制ET-1/NOS的升高(P<0.05)。结论丹红注射液能调整ET-1/NOS比值,对心肌缺血再灌注损伤有一定的保护作用。     

Pu-erh Tea Reduces Nitric Oxide Levels in Rats by Inhibiting Inducible Nitric Oxide Synthase Expression through Toll-Like Receptor 4

Pu-erh tea undergoes a unique fermentation process and contains theabrownins, polysaccharides and caffeine; although it is unclear about which component is associated with the down regulation of nitric oxide levels or how this process is mediated. To address this question we examined the effects of pu-erh tea on nitric oxide synthase (NOS) genes. Cohorts of rats were separately given four-week treatments of water as control, pu-erh tea, or the tea components: theabrownins, caffeine or polysaccharides. Five experimental groups were injected with lipopolysaccharides (LPS) to induce nitric oxide (NO) production, while the corresponding five control groups were injected with saline as a negative control. The serum and liver NO concentrations were examined and the NOS expression of both mRNA and protein was measured in liver. The results showed that the rats which were fed pu-erh tea or polysaccharides had lower levels of NO which corresponded with the down-regulation of inducible nitric oxide synthase (iNOS) expression. We further demonstrate that this effect is mediated through reduction of Toll-like receptor 4 (TLR4) signaling. Thus we find that the polysaccharide components in pu-erh tea reduce NO levels in an animal model by inhibiting the iNOS expression via signaling through TLR4.     

Nitric Oxide,Nitric Oxide Formers and Their Physiological Impacts in Bacteria

Nitric oxide (NO) is an active and critical nitrogen oxide in the microbe-driven nitrogen biogeochemical cycle, and is of great interest to medicine and the biological sciences. As a gas molecule prior to oxygen, NO respiration represents an early form of energy generation via various reactions in prokaryotes. Major enzymes for endogenous NO formation known to date include two types of nitrite reductases in denitrification, hydroxylamine oxidoreductase in ammonia oxidation, and NO synthases (NOSs). While the former two play critical roles in shaping electron transport pathways in bacteria, NOSs are intracellular enzymes catalyzing metabolism of certain amino acids and have been extensively studied in mammals. NO interacts with numerous cellular targets, most of which are redox-active proteins. Doing so, NO plays harmful and beneficial roles by affecting diverse biological processes within bacterial physiology. Here, we discuss recent advances in the field, including NO-forming enzymes, the molecular mechanisms by which these enzymes function, physiological roles of bacterial NOSs, and regulation of NO homeostasis in bacteria.     

Circulating Microvesicle-Associated Inducible Nitric Oxide Synthase Is a Novel Therapeutic Target to Treat Sepsis: Current Status and Future Considerations

To determine whether mitigating the harmful effects of circulating microvesicle-associated inducible nitric oxide (MV-A iNOS) in vivo increases the survival of challenged mice in three different mouse models of sepsis, the ability of anti-MV-A iNOS monoclonal antibodies (mAbs) to rescue challenged mice was assessed using three different mouse models of sepsis. The vivarium of a research laboratory Balb/c mice were challenged with an LD₈₀ dose of either lipopolysaccharide (LPS/endotoxin), TNFα, or MV-A iNOS and then treated at various times after the challenge with saline as control or with an anti-MV-A iNOS mAb as a potential immunotherapeutic to treat sepsis. Each group of mice was checked daily for survivors, and Kaplan–Meier survival curves were constructed. Five different murine anti-MV-A iNOS mAbs from our panel of 24 murine anti-MV-A iNOS mAbs were found to rescue some of the challenged mice. All five murine mAbs were used to genetically engineer humanized anti-MV-A iNOS mAbs by inserting the murine complementarity-determining regions (CDRs) into a human IgG_1,kappa scaffold and expressing the humanized mAbs in CHO cells. Three humanized anti-MV-A iNOS mAbs were effective at rescuing mice from sepsis in three different animal models of sepsis. The effectiveness of the treatment was both time- and dose-dependent. Humanized anti-MV-A iNOS rHJ mAb could rescue up to 80% of the challenged animals if administered early and at a high dose. Our conclusions are that MV-A iNOS is a novel therapeutic target to treat sepsis; anti-MV-A iNOS mAbs can mitigate the harmful effects of MV-A iNOS; the neutralizing mAb’s efficacy is both time- and dose-dependent; and a specifically targeted immunotherapeutic for MV-A iNOS could potentially save tens of thousands of lives annually and could result in improved antibiotic stewardship.     

Carbon Monoxide and Nitric Oxide as Examples of the Youngest Class of Transmitters

The year 2021 is the 100th anniversary of the confirmation of the neurotransmission phenomenon by Otto Loewi. Over the course of the hundred years, about 100 neurotransmitters belonging to many chemical groups have been discovered. In order to celebrate the 100th anniversary of the confirmation of neurotransmitters, we present an overview of the first two endogenous gaseous transmitters i.e., nitric oxide, and carbon monoxide, which are often termed as gasotransmitters.     

Chrysin Suppressed Inflammatory Responses and the Inducible Nitric Oxide Synthase Pathway after Spinal Cord Injury in Rats

Chrysin (CH), a natural plant flavonoid, has shown a variety of beneficial effects. Our present study was conducted to evaluate the therapeutic potential of CH three days after spinal cord injury (SCI) in rats and to probe the underlying neuroprotective mechanisms. SCI was induced using the modified weight-drop method in Wistar rats. Then, they were treated with saline or CH by doses of 30 and 100 mg/kg for 26 days. Neuronal function was assessed with the Basso Beattle Bresnahan locomotor rating scale (BBB). The water content of spinal cord was determined after traumatic SCI. The NF-κB p65 unit, TNF-α, IL-1β and IL-6 in serums, as well as the apoptotic marker, caspase-3, of spinal cord tissues were measured using commercial kits. The protein level and activity of inducible nitric oxide synthase (iNOS) were detected by western blot and a commercial kit, respectively. NO (nitric oxide) production was evaluated by the determination of nitrite concentration. The rats with SCI showed marked reductions in BBB scores, coupled with increases in the water content of spinal cord, the NF-κB p65 unit, TNF-α, IL-1β, IL-6, iNOS, NO production and caspase-3. However, a CH supplement dramatically promoted the recovery of neuronal function and suppressed the inflammatory factors, as well as the iNOS pathway in rats with SCI. Our findings disclose that CH improved neural function after SCI in rats, which might be linked with suppressing inflammation and the iNOS pathway.     

激活素A对L929细胞活性的调节作用

目的探讨激活素A对小鼠纤维母细胞L929活性的调节作用。方法用不同剂量的激活素A刺激L929细胞,应用还原酶法分析细胞分泌一氧化氮(NO)的水平,RT-PCR法检测细胞诱导型一氧化氮合成酶(iNOS)、纤维连接蛋白(FN)和金属蛋白酶组织抑制物(TIMP)mRNA的表达,应用中性红检测细胞的吞饮活性,MTT法检测细胞的增殖活性。结果L929细胞经激活素A刺激后,与对照组细胞比较,分泌NO的水平明显升高,iNOS mRNA及FN mRNA的表达水平也升高,而TIMP mRNA的表达水平无明显改变。激活素A可以促进L929细胞的吞饮活性,但对L929细胞的增殖活性无影响。结论激活素A具有促进L929细胞分泌炎性介质和形成细胞外基质的作用,这可能是其促进炎症发展、诱导组织纤维化的原因。     

N-Acetylcysteine Administration Attenuates Sensorimotor Impairments Following Neonatal Hypoxic-Ischemic Brain Injury in Rats

Hypoxic ischemic (HI) brain injury that occurs during neonatal period has been correlated with severe neuronal damage, behavioral deficits and infant mortality. Previous evidence indicates that N-acetylcysteine (NAC), a compound with antioxidant action, exerts a potential neuroprotective effect in various neurological disorders including injury induced by brain ischemia. The aim of the present study was to investigate the role of NAC as a potential therapeutic agent in a rat model of neonatal HI brain injury and explore its long-term behavioral effects. To this end, NAC (50 mg/kg/dose, i.p.) was administered prior to and instantly after HI, in order to evaluate hippocampal and cerebral cortex damage as well as long-term functional outcome. Immunohistochemistry was used to detect inducible nitric oxide synthase (iNOS) expression. The results revealed that NAC significantly alleviated sensorimotor deficits and this effect was maintained up to adulthood. These improvements in functional outcome were associated with a significant decrease in the severity of brain damage. Moreover, NAC decreased the short-term expression of iNOS, a finding implying that iNOS activity may be suppressed and that through this action NAC may exert its therapeutic action against neonatal HI brain injury.     

The Inhibition of the Inducible Nitric Oxide Synthase Enhances the DPSC Mineralization under LPS-Induced Inflammation

Nitric oxide (NO) is a key messenger in physiological and pathological processes in mammals. An excessive NO production is associated with pathological conditions underlying the inflammation response as a trigger. Among others, dental pulp inflammation results from the invasion of dentin by pathogenic bacteria. Vital functions of pulp mesenchymal stem cells (DPSCs, dental pulp stem cells), such as mineralization, might be affected by the inducible NOS (iNOS) upregulation. In this context, the iNOS selective inhibition can be considered an innovative therapeutic strategy to counteract inflammation and to promote the regeneration of the dentin-pulp complex. The present work aims at evaluating two acetamidines structurally related to the selective iNOS inhibitor 1400W, namely CM544 and FAB1020, in a model of LPS-stimulated primary DPSCs. Our data reveal that CM544 and even more FAB1020 are promising anti-inflammatory compounds, decreasing IL-6 secretion by enhancing CD73 expression-levels, a protein involved in innate immunity processes and thus confirming an immunomodulatory role of DPSCs. In parallel, cell mineralization potential is retained in the presence of compounds as well as VEGF secretion, and thus their angiogenetic potential. Data presented lay the ground for further investigation on the anti-inflammatory potential of acetamidines selectively targeting iNOS in a clinical context.     

The Roles of Nitric Oxide Synthase/Nitric Oxide Pathway in the Pathology of Vascular Dementia and Related Therapeutic Approaches

Vascular dementia (VaD) is the second most common form of dementia worldwide. It is caused by cerebrovascular disease, and patients often show severe impairments of advanced cognitive abilities. Nitric oxide synthase (NOS) and nitric oxide (NO) play vital roles in the pathogenesis of VaD. The functions of NO are determined by its concentration and bioavailability, which are regulated by NOS activity. The activities of different NOS subtypes in the brain are partitioned. Pathologically, endothelial NOS is inactivated, which causes insufficient NO production and aggravates oxidative stress before inducing cerebrovascular endothelial dysfunction, while neuronal NOS is overactive and can produce excessive NO to cause neurotoxicity. Meanwhile, inflammation stimulates the massive expression of inducible NOS, which also produces excessive NO and then induces neuroinflammation. The vicious circle of these kinds of damage having impacts on each other finally leads to VaD. This review summarizes the roles of the NOS/NO pathway in the pathology of VaD and also proposes some potential therapeutic methods that target this pathway in the hope of inspiring novel ideas for VaD therapeutic approaches.