胆碱能抗炎通路的机制及其在脓毒症的应用

胆碱能抗炎通路是近年来发现的对全身性炎症反应有调节作用的通路,可通过迷走神经抑制促炎细胞因子的合成从而抑制机体炎症反应.核因子-κB和Janus激酶/信号转导与转录激活子是该通路细胞内信号转导中最重要的两条信号通路.脓毒症以全身炎症反应为特点,是危重症患者的常见死因.根据胆碱能抗炎通路作用迅速有效的特点,推测其可应用于...     

Vagal function indexed by respiratory sinus arrhythmia and cholinergic anti-inflammatory pathway

The autonomic nervous system, in particular vagal function, plays an important role in a wide range of somatic and mental disorders. Cardiac vagal function can be indexed by the respiratory sinus arrhythmia (RSA) – oscillations in heart rate linked to respiration mediated predominantly by fluctuations of vagus nerve efferent traffic originating in the nucleus ambiguus. Moreover, the neurocardiac vagal modulation has been shown to be related to physiological adaptability/flexibity and emotional regulation. Thus, greater vagal withdrawal during stressors and subsequent recovery should be indicative of a more flexible physiological response system.     

MicroRNA-132通过调控胆碱能通路减轻肺泡巨噬细胞炎症反应

 目的: 探讨microRNA-132(miR-132)通过调控胆碱能通路减轻肺泡巨噬细胞炎症反应的作用机制。方法: 向大鼠肺泡巨噬细胞NR8383中转染miR-132 mimic、mimic阴性对照(NC)、miR-132 inhibitor或inhibitor NC,转染后用脂多糖(LPS)和(或)乙酰胆碱(ACh)处理细胞;采用real-time PCR检测乙酰胆碱酯酶(AChE)的mRNA表达;采用Western blot检测细胞中AChE、信号转导及转录激活因子3(STAT3)和磷酸化STAT3(p-STAT3),以及细胞浆和细胞核中核因子κB(NF-κB)蛋白的改变;采用AChE活性测试盒检测细胞上清液中AChE活性的改变;采用免疫荧光检测NF-κB的核移位变化。结果: 上调或下调miR-132不影响AChE的mRNA相对表达水平;上调miR-132可使AChE蛋白及活性的水平均显著降低(P < 0.05),下调miR-132可使AChE蛋白及活性的水平均显著升高(P < 0.05)。当给予LPS+ACh作用时,miR-132 mimic组对NF-κB p65核移位的抑制作用较mimic NC组更强(P < 0.05),miR-132 inhibitor组较inhibitor NC组更弱(P < 0.05);miR-132 mimic组对STAT3及p-STAT3蛋白的抑制作用较mimic NC组更强(P < 0.05)。结论: miR-132通过调控胆碱能通路减轻肺泡巨噬细胞炎症反应的作用机制可能是miR-132靶向作用AChE,抑制AChE对ACh的水解作用,从而增强ACh的抗炎作用,抑制NF-κB及STAT3的活化。     

Central cholinergic activation of a vagus nerve-to-spleen circuit alleviates experimental colitis

The cholinergic anti-inflammatory pathway is an efferent vagus nerve–based mechanism that regulates immune responses and cytokine production through α7 nicotinic acetylcholine receptor (α7nAChR) signaling. Decreased efferent vagus nerve activity is observed in inflammatory bowel disease. We determined whether central activation of this pathway alters inflammation in mice with colitis and the mediating role of a vagus nerve-to-spleen circuit and α7nAChR signaling. Two experimental models of colitis were used in C57BL/6 mice. Central cholinergic activation induced by the acetylcholinesterase inhibitor galantamine or a muscarinic acetylcholine receptor agonist treatments resulted in reduced mucosal inflammation associated with decreased major histocompatibility complex II level and pro-inflammatory cytokine secretion by splenic CD11c⁺ cells mediated by α7nAChR signaling. The cholinergic anti-inflammatory efficacy was abolished in mice with vagotomy, splenic neurectomy, or splenectomy. In conclusion, central cholinergic activation of a vagus nerve-to-spleen circuit controls intestinal inflammation and this regulation can be explored to develop novel therapeutic strategies.     

可乐定通过激活胆碱能抗炎通路抑制肺损伤小鼠炎性反应

目的:探讨可乐定对肺损伤小鼠炎性反应的影响及其作用机制。方法:以脂多糖(lipopolysac charide,LPS)诱导的肺损伤小鼠为模型,静脉注射可乐定,取左肺上叶组织,检测肺湿干重比(W/D)和总肺含水量(TLW),ELISA试剂盒检测炎性因子IL-1β、IL-6和TNF-α的含量,RT-PCR和Western blot检测α7烟碱型乙酰胆碱受体(α7nAChR)、高迁移率族蛋白B1(HMGB1)表达的变化;鼻内吸入法将α7nAChR基因RNA干扰慢病毒载体导入肺损伤模型小鼠肺内,或者静脉注射HMGB1外源性蛋白,检测炎性因子和HMGB1的变化。结果:可乐定可减轻LPS诱导的小鼠肺损伤,并通过降低细胞因子IL-6、IL-10和TNF-α水平从而抑制LPS诱导的炎性反应;可乐定促进α7nAChR表达从而激活胆碱能抗炎通路,并抑制HMGB1的表达。结论:可乐定对肺损伤小鼠具有抗炎作用,这可能与激活胆碱能抗炎通路,抑制HMGB1的表达有关。     

Suppressive effect of berberine on experimental dextran sulfate sodium-induced colitis

The anti-inflammatory effect of berberine was evaluated in murine model of acute experimental colitis induced by dextran sulfate sodium (DSS). Berberine, given orally at 40, 20, 10?mg/kg for 10 days, ameliorated all the supposed inflammatory symptoms of the induced colitis, such as body weightloss, blood hemoglobin reduction, high myeloperoxidase levels, and malondialdehyde content-inflamed mucosa. Furthermore, the cytokine production of splenic lymphocytes was analyzed. The results showed the IFN-γ and IL-12 were increased, but IL-4 and IL-10 were decreased in DSS-induced colitis,when those were compared with the normal control. But the administration of berberine to DSS-induced colitis mice showed lower production of IFN-γ and IL-12 and higher production of IL-4 and IL-10 than the DSS-induced colitis mice. The results suggest that the protective effects of berberine against the DSS-induced colitis may be associated with the regulation of cytokine production.     

Suppressive effect of berberine on experimental dextran sulfate sodium-induced colitis

The anti-inflammatory effect of berberine was evaluated in murine model of acute experimental colitis induced by dextran sulfate sodium (DSS). Berberine, given orally at 40, 20, 10?mg/kg for 10 days, ameliorated all the supposed inflammatory symptoms of the induced colitis, such as body weightloss, blood hemoglobin reduction, high myeloperoxidase levels, and malondialdehyde content-inflamed mucosa. Furthermore, the cytokine production of splenic lymphocytes was analyzed. The results showed the IFN-γ and IL-12 were increased, but IL-4 and IL-10 were decreased in DSS-induced colitis,when those were compared with the normal control. But the administration of berberine to DSS-induced colitis mice showed lower production of IFN-γ and IL-12 and higher production of IL-4 and IL-10 than the DSS-induced colitis mice. The results suggest that the protective effects of berberine against the DSS-induced colitis may be associated with the regulation of cytokine production.     

The cholinergic pathway to cerebral blood vessels

The application of cobalt chloride to the peripheral cut end of the greater superficial petrosal nerve (g.s.p.n.) in rats revealed that only a few fibers in the plexus of nerves on the adventitial surface of the internal carotid artery were in axonal continuity with the g.s.p.n. A similarly small contribution of cholinergic fibers to cerebral blood vessels from this nerve was suggested by the observation that section of the g.s.p.n. resulted in an insignificant reduction in the density of the AChE-staining plexus in the internal carotid and cerebral arteries and in the incidence of at most 2% degenerate terminals of those observed on the middle cerebral artery. Alternative explanations of the results are discussed: that the AChE-staining fibers are postganglionic, that the time course for degeneration is unusually slow and that non-cholinergic fibers stain non-specifically for AChE. It is concluded that a cholinergic dilator pathway is most probably carried by the g.s.p.n. but that it is not unique.     

The cholinergic pathway to cerebral blood vessels

Summary The effect of stimulating the greater superficial petrosal nerve (g.s.p.n.) upon retroglenoid venous blood flow has been tested in anaesthetized, paralysed and artificially ventilated rats. In 11 out of 15 tests, blood flow increased by an average of 25% with a time to peak response of 28 s. This response was abolished with the injection of atropine 0.1 mg kg^–1 injected intra-arterially. With both petrosal nerves intact, the administration of 6–7% CO₂ in air or 15% O₂ in N₂ caused average increases in blood flow of 105% and 45% respectively. These responses were not affected by bilateral section of the g.s.p.n. Similar experiments were carried out in 5 anaesthetized, spontaneously breathing rabbits in which, in addition toPaCO₂ andPaO₂,PO₂,PCO₂ and blood flow in the caudate nucleus were measured continuously using chronically implanted mass spectrometer catheters and heated thermistors. Caudate nucleus blood flow increased in response to hypoxia and hypercapnia and this response was not significantly affected by section of one or both g.s.p.n., sinus or vagus nerves. With section of sinus and vagus nerves, blood flow changed passively with arterial pressure.     

胆碱能抗炎通路在电针预处理调控脑缺血再灌注大鼠小胶质细胞活化状态中的作用

目的:观察电针预处理对大鼠脑缺血再灌注后小胶质细胞活化状态的影响并探讨其机制。方法:成年雄性SD大鼠随机分为7组,分别为假手术组(Sham),脑缺血再灌注组(MCAO),电针(EA)+MCAO组,烟碱型乙酰胆碱受体α7亚单位(α7nAChR)PHA-543,613+MCAO组,溶剂(Vehicle)+MCAO组,α7nAChR抑制剂α-BGT+EA+MCAO组,溶剂+EA+MCAO组。脑缺血再灌注后3 d取材,运用Western Blot技术以及免疫荧光技术检测脑缺血半暗带小胶质细胞经典激活状态(M1型)及选择性激活状态(M2型)特异性标志分子的表达水平。结果:(1)与MCAO组比较,电针预处理组(EA+MCAO)M1型小胶质细胞标志分子iNOS表达下调(P0.05),M2型小胶质细胞标志分子Arginase表达上调(P0.05);(2)α7nAChR激动剂PHA-543,613可模拟电针预处理的作用,使小胶质细胞由M1型向M2型转化;α7nAChR抑制剂α-BGT逆转了电针促进小胶质细胞由M1型向M2型转化的作用。结论:电针预处理可以使脑缺血再灌注后小胶质细胞由M1型向M2型转化,α7nAChR参与了电针预处理对小胶质细胞活化状态改变的过程。     

The effect of atorvastatin and its role on systemic cytokine network in treatment of acute experimental colitis

Inflammatory bowel diseases are characterized by disabilities in gastrointestinal system and defects in mucosal immune system. Statins are 3-hydroxy-3-methyl glutaryl coenzyme A reductase inhibitor and are used to treat hypercholesterolemia in patients with coronary artery and atherosclerotic diseases. Recent studies have demonstrated that statins have immunomodulatory role by effecting different pathways in immune system. In this study, we investigated the effect of atorvastatin and its mechanism on systemic immune response in treatment of trinitrobenzene sulfonic acid (TNBS)-induced colitis mice. We observed that atorvastatin significantly suppressed the severity of TNBS-induced colitis in BALB/c mice. This was manifested in reduced rectal bleeding, decrease in colon length, reduction of histological damage, and improved survival. Concurrently, we investigated the immunomodulatory role of atorvastatin on systemic immune system. We investigated the proinflammatory (IL-1α, IL-6, TNF-α), Th1 (IFN-γ, IL-2), Th2 (IL-4, IL-5, IL-10), and Th17 (IL-17, IL-23) cytokine levels in serum samples of colitis and atorvastatin-administered mice. We discovered that administration of atorvastatin significantly down-regulates systemic TNF-α level and Th17 cytokine levels. Furthermore, atorvastatin treatment switches Th1 type T-cell response toward/to Th2 (IL-4, IL-10) type response.     

The effect of atorvastatin and its role on systemic cytokine network in treatment of acute experimental colitis

Inflammatory bowel diseases are characterized by disabilities in gastrointestinal system and defects in mucosal immune system. Statins are 3-hydroxy-3-methyl glutaryl coenzyme A reductase inhibitor and are used to treat hypercholesterolemia in patients with coronary artery and atherosclerotic diseases. Recent studies have demonstrated that statins have immunomodulatory role by effecting different pathways in immune system. In this study, we investigated the effect of atorvastatin and its mechanism on systemic immune response in treatment of trinitrobenzene sulfonic acid (TNBS)-induced colitis mice. We observed that atorvastatin significantly suppressed the severity of TNBS-induced colitis in BALB/c mice. This was manifested in reduced rectal bleeding, decrease in colon length, reduction of histological damage, and improved survival. Concurrently, we investigated the immunomodulatory role of atorvastatin on systemic immune system. We investigated the proinflammatory (IL-1α, IL-6, TNF-α), Th1 (IFN-γ, IL-2), Th2 (IL-4, IL-5, IL-10), and Th17 (IL-17, IL-23) cytokine levels in serum samples of colitis and atorvastatin-administered mice. We discovered that administration of atorvastatin significantly down-regulates systemic TNF-α level and Th17 cytokine levels. Furthermore, atorvastatin treatment switches Th1 type T-cell response toward/to Th2 (IL-4, IL-10) type response.     

金纳多对小鼠急性实验性结肠炎的保护作用及可能机制

目的观察金纳多对小鼠急性实验性结肠炎的保护机制并对比分析其与柳氮磺胺吡啶(SASP)的疗效。方法用3%DSS建立小鼠急性实验性结肠炎动物模型。小鼠随机分为4组:正常组、造模组、金纳多治疗组、柳氮磺胺吡啶(SASP)治疗组。记录各组小鼠一般情况(饮食、便血、体重)、DAI评分、肠管长度和肠组织学损伤评分;通过免疫组化、Real-time PCR和Western blot检测各组小鼠肠组织中炎症因子(gp130,STAT3, p-STAT3, ROR-γt,IL-6, IL-17, IL-23)的表达。结果金纳多可改善小鼠一般情况(饮食、便血、体重),与造模组比较,可有效降低小鼠DAI评分、组织学损伤评分、炎症因子(gp130, STAT3, p-STAT3, ROR-γt,IL-6, IL-17, IL-23)的表达水平(P0.01)。与SASP治疗组比较,金纳多治疗组小鼠DAI评分、组织学损伤评分、炎症因子(gp130, STAT3, p-STAT3, ROR-γt,IL-6, IL-17, IL-23)的表达均高于SASP治疗组(P0.05)。结论金纳多对小鼠急性实验性结肠炎的保护作用与抑制IL-6/STAT3通路和IL-23/IL-17轴相关,但疗效弱于柳氮磺胺吡啶。     

Suppressive effects of a new anti-inflammatory agent, IS-741, on dextran sulfate sodium-induced experimental colitis in rats

A novel anti-inflammatory drug, IS-741, blocked the adhesion of inflammatory cells to microvascular endothelial cells in vivo and in vitro. We examined the efficacy of IS-741 in a dextran sulfate sodium (DSS)-induced colitis model. DSS colitis was induced by the oral administration of 3% DSS for 10 days in rats. The rats were then divided in two groups: a 1% DSS plus IS-741 group and a 1% DSS plus water group. IS-741 was dissolved in water and administered orally (10 mg/kg) once per day for 14 days. The rats treated with DSS plus IS-741 remained healthy, and their body weight increased. The wet weight of the colon was significantly lower and the total colon length was significantly longer in the IS-741-treated group. Histological examinations revealed a marked infiltration of inflammatory cells into both the mucosa and submucosa in the DSS plus water group, but these changes were attenuated in the IS-741-treated group. The mucosal damage score was significantly reduced by treatment with IS-741. IS-741 also significantly reduced the mucosal myeloperoxidase activity. FACS analysis revealed that IS-741 significantly reduced Mac-1 expression on blood neutrophils. In conclusion, IS-741 suppressed DSS-induced experimental colitis in rats. Some of the action of IS-741 may be associated with its inhibitory effects on the Mac-1 expression of neutrophils in association with the blockade of their adhesion to endothelial cells. The findings in this study suggest that IS-741 may be a useful new therapeutic agent for inflammatory bowel disease.     

Pharmacological manipulations of the substantia innominata-cortical cholinergic pathway

The present study examined the effects of various pharmacological agents on the activity of cholinergic neurons within the nucleus basalis that are located in the substantia innominata (SI) and send afferent projections to the neocortex. Male Sprague-Dawley rats received microinfusions of selected compounds into the SI via a chronic indwelling cannula. In vitro sodium-dependent high affinity choline uptake (SDHACU) was determined in the frontal cortex 50 min later as an indicator of the changes in in vivo activity of cholinergic cells in the SI. GABAergic and opiate agonists, muscimol and enkephalin respectively, decreased neocortical SDHACU. Glutamate increased neocortical SDHACU. Noradrenergic, dopaminergic and serotonergic manipulations did not produce significant changes. These results suggest a regulatory role for enkephalinergic, GABAergic and glutamatergic afferents onto the basal forebrain cholinergic system within the SI.     

绿原酸对溃疡性结肠炎的治疗作用及对IL-6/STAT3信号通路的影响

目的 探讨绿原酸对溃疡性结肠炎（ulcerative colitis, UC）小鼠的治疗作用及对IL-6/STAT3信号通路的影响。方法 SPF级C57BL/6小鼠随机分为对照组（control）、模型组（model）、绿原酸低及高剂量组（CGA-L、CGA-H）,每组8只。检测小鼠疾病活动指数（DAI）评分;HE染色观察小鼠结肠病理损伤;ELISA检测结肠组织中白介素-17A(IL-17A)、肿瘤坏死因子-α（TNF-α）、IL-4及IL-10水平;Western blot检测小鼠结肠组织中孤独核受体γt(RORγt)、叉头状转录因子（Foxp3）、IL-6、信号传导与转录激活因子3(STAT3)及磷酸化STAT3(p-STAT3)蛋白的表达。结果 CGA-L与CGA-H能降低小鼠DAI评分,明显减轻结肠组织病理损伤;降低IL-17A与TNF-α水平,上调IL-4与IL-10水平（P<0.05）;CGA-L与CGA-H能降低RORγt蛋白表达,上调FOXP3蛋白表达（P<0.05）;降低小鼠结肠组织中IL-6与及p-STAT3蛋白表达（P<0.05）。结论 绿原酸可有效治疗DSS诱导的小鼠UC,调控Th17/Treg平衡,其作用机制可能与抑制IL-6/STAT3信号通路有关。     

The effect of cholinergic stimulation on rat pup ultrasonic vocalizations

As cholinergic stimulation increases vocalizations in adult rats, the present study investigated the effects of systemic oxotremorine, a cholinergic agonist, on the production of separation calls in rat pups of different ages and whether these effects are in response to central versus peripheral stimulation. The first experiment examined the dose-response effects of oxotremorine on the number of vocalizations and acoustic parameters of 10-, 15-, and 17-day-old rat pups. In contrast to other studies on adult rats, pup vocalizations were decreased while marginally changing acoustic parameters. The second experiment, using muscarinic antagonists, showed that pretreatment with atropine sulfate, which can cross the blood-brain barrier (BBB), reversed the call-reducing effect of oxotremorine whereas pretreatment with atropine methyl nitrate, which does not cross BBB, did not. Suppression of vocalizations by oxotremorine may be explained by central activation and not the peripheral effects of the drug. Dissimilar effects of cholinergic stimulation of infant and adult rat brains may be attributed to a differential role of the cholinergic system during development and maturity.     

Peripheral bee venom's anti-inflammatory effect involves activation of the coeruleospinal pathway and sympathetic preganglionic neurons

There are several reports indicating that the locus coeruleus (LC) is capable of altering immune responses. Moreover, it is well established that the LC is the major source of descending noradrenergic system. Recently we have demonstrated that subcutaneous bee venom (BV) injection dramatically suppressed peripheral inflammation through activation of sympathetic preganglionic neurons (SPNs) leading to release of adreno-medullary catecholamines. Importantly, this 'BV-induced anti-inflammatory effect' (BVAI) is also associated with an increase of the activity of LC. Based on these data, present study examined whether BV-induced LC activation increased the activity of SPNs and this pathway played a role in BVAI using a zymosan-induced inflammatory air pouch model in mice. Unilateral BV injection into left hind limb produced anti-inflammation and specifically increased Fos expression in SPNs of the T7-T11 (which mainly project to adrenal medulla), but not those of the T1-T6 or T12-L2 spinal cord. 6-Hydroxydopamine-induced unilateral lesion of the contralateral, but not ipsilateral (to the BV injection site) LC significantly blocked BVAI and BV-induced Fos expression in SPNs. Additionally, intrathecal administration of idazoxan (alpha2-adrenoceptor antagonist), blocked BVAI. These results indicate that BV-induced activation of the contralateral LC-descending noradrenergic pathway increased the activity of SPNs that project to the adrenal medulla and this pathway is necessary for BVAI.     

Kallistatin抗炎作用的研究进展

Kallistatin(KS)是一种激肽释放酶结合蛋白(kallikrein-binding protein),广泛分布于多种组织及体液中。KS不但具有抑制组织型激肽释放酶的作用,还具有抗炎、抗血管生成、抗肿瘤等多种生物学作用。KS生物学功能与其分子结构中不同的结构域有关。而KS抗炎作用被认为主要与肝素结合结构域有关。KS通过其肝素结合结构域,能竞争性抑制肿瘤坏死因子(tumor necrosis factor α,TNF-α)与它的受体的结合,从而起到影响TNF-α-NF-κB信号通路,发挥抗炎作用。KS也能与Kruppel样因子4(Kruppel-like Factor 4,KLF4)结合,激活内皮型一氧化氮合酶(endothelial nitric oxide synthase,eNOS),抑制TNF-α活化核因子-κB(nuclear factor-κB,NF-κB),发挥抗炎作用。但这与肝素结合结构域的机制不同。不同的机制可能为抗炎治疗提供新的思路,本文将就Kallistatin抗炎作用的相关研究进展进行阐述。