Mechanistic studies on stress, brain inflammation and neuroprotection

我们的第一个具体目标是阐明化合物有益作用的机制，直到最近，这些化合物才被认为对治疗脑部疾病感兴趣。第二个具体目标是进一步确定此类化合物对脑部疾病的治疗益处的程度。 我们研究了一组统称为沙坦类药物或血管紧张素 II AT1 受体阻滞剂 (ARB) 的化合物。沙坦类药物是联苯衍生物，具有极好的安全性，广泛用于治疗心血管和代谢紊乱，因为它们可以拮抗血管紧张素 II 诱导的血管收缩和病理性细胞生长和纤维化，因为它们可以减少外周炎症，并且可以提高胰岛素敏感性。我们最初发现沙坦类药物可以减少高血压引起的脑血管炎症，后来我们发现沙坦类药物治疗可以减少啮齿类动物模型中的脑缺血、压力和焦虑，并延长寿命。最近，我们确定沙坦类药物的有益作用包括显着改善大脑周围炎症的负面影响。我们的结论是，多种机制可能导致 ARB 治疗的主要神经保护作用，我们继续研究以进一步阐明这些机制。 在本财年，我们进一步阐明了沙坦类药物在大脑中的抗炎作用。我们假设至少部分中枢抗炎和神经保护作用是 ARB 对脑细胞直接作用的结果。此外，我们还讨论了大脑炎症的减少可能与行为相关的可能性，并使用经过验证的动物模型考虑了沙坦类药物对焦虑和抑郁的影响。 沙坦类药物的抗炎和神经保护作用（减少炎症诱导的活化 B 细胞核因子 kappa-轻链增强子 (NFkappaBalpha) 和激活蛋白 1 (AP-1) 的激活，诱导型一氧化氮合酶、环氧合酶-2 和烟酰胺腺嘌呤二核苷酸磷酸 (NADPH) 氧化酶的表达，减少过量一氧化氮、前列腺素的产生E2（以及导致脑部炎症和神经元损伤的活性氧）广泛存在于脑实质中。这表明沙坦类药物可能影响多种脑细胞类型。使用小胶质细胞、初级皮质神经元、初级小脑颗粒细胞和脑微血管内皮细胞培养物，我们发现 ARB 可以改善所有研究细胞类型的炎症。 ARB 对细菌内毒素脂多糖 (LPS)、过量谷氨酸和促炎细胞因子 IL-1β 具有神经保护作用。涉及的机制包括几种蛋白激酶的激活减少和转录因子 NFkappaBalpha 的激活减少。 我们假设沙坦类药物的主要抗炎和神经保护作用可能不是 AT1 受体抑制的唯一结果。在人循环单核细胞（表达很少 AT1 受体的细胞）中，沙坦类药物的抗炎作用部分依赖于过氧化物酶体增殖物激活受体 γ (PPARgamma) 的激活。我们发现一些沙坦类药物可能具有双重作用机制：与抑制 AT1 受体相关的抗高血压、抗生长和抗炎作用，以及代谢和抗炎作用，部分是直接 PPARgamma 激活的结果。我们现在证实，PPARgamma 激活作为 ARB 的主要组成部分，参与了 THP-1 细胞（一种人类急性单核细胞白血病细胞系）和缺乏显着 AT1 受体表达的大鼠皮质小胶质细胞的原代培养物中的作用， 我们早期的研究结果表明，ARB 可能会限制激素和交感神经对压力的过度反应，这开辟了第二个研究方向。我们初步确定，ARB 可以限制应激引起的皮质 γ-氨基丁酸 (GABAA) 功能的改变，这一点是通过皮质苯二氮卓结合来确定的。最近我们发现，ARB 在炎症应激期间可以保护 GABAA 功能。这一发现解释了 ARB 在我们的炎症应激模型中的抗焦虑和抗抑郁作用（预防 LPS 引起的疾病行为、厌食和体重减轻，以及减少 LPS 引起的焦虑）。 我们最近在合作研究中发现，在具有抑郁和焦虑遗传易感性的啮齿动物模型中，ARB 可以减轻与慢性轻度可变应激相关的抑郁和焦虑。 我们不断阐明终身沙坦给药产生的神经保护和延长寿命的机制。我们发现，长期服用沙坦可以减少与年龄相关的脑部炎症、血管重塑和焦虑，并且 ARB 的神经保护作用会持续一生。我们的研究结果可能至少部分解释了其他研究小组报告的沙坦疗法对阿尔茨海默病的主要有益作用。 另一项新发现是，在啮齿动物模型中施用 ARB 可显着保护大脑免受创伤性脑损伤。 在该模型中，ARB 可以减小病变大小，减少神经元损伤并保护神经功能。 这是首次证明 ARB 在创伤性脑损伤中具有神经保护作用。 我们的工作继续进行机制和转化研究，以进一步阐明 ARB 诱导的神经保护和抗抑郁作用的机制。 我们的目标是检验我们的假设，即 ARB 在治疗脑部疾病（包括重度抑郁症、阿尔茨海默病和创伤性脑损伤）方面具有主要治疗优势，并为进一步开发更有效、具有转化价值的 ARB 衍生神经保护和抗抑郁化合物奠定更坚实的基础。

Our first specific aim is to clarify the mechanisms involved in the beneficial effects of compounds which have not, until very recently, been considered of interest to the therapy of brain disorders. The second specific aim is to further establish the extent of therapeutic benefits of such compounds in diseases of the brain. We study a group of compounds collectively named sartans, or Angiotensin II AT1 receptor blockers (ARBs). Sartans are biphenyl derivatives with an excellent margin of safety, extensively used to treat cardiovascular and metabolic disorders because they antagonize Angiotensin II-induced vasoconstriction and pathological cellular growth and fibrosis, because they reduce peripheral inflammation and because they improve insulin sensitivity. Following our initial finding that sartans decrease hypertension-induced cerebrovascular inflammation, we later discovered that sartan treatment reduces brain ischemia, stress, and anxiety, and increases lifespan in rodent models. More recently, we established that the beneficial effects of sartans include a major amelioration of the negative effects of peripheral inflammation in the brain. Our conclusion was that several mechanisms may be responsible for the major neuroprotective effects of ARB treatment, and we continued studies to further clarify such mechanisms. During the current fiscal year, we advanced on the clarification of the anti-inflammatory effects of sartans in the brain. We hypothesized that at least part of the central anti-inflammatory and neuroprotective effects were the consequence of direct actions of ARBs on brain cells. In addition we addressed the possibility that reduction of brain inflammation may have behavioral correlates, and, using validated animal models, considered the effects of sartans on anxiety and depression. The anti-inflammatory and neuroprotective effects of sartans (decline in inflammation-induced activation of the transcription factors nuclear factor kappa-light-chain-enhancer of activated B cells (NFkappaBalpha) and activator protein-1 (AP-1), expression of inducible nitric oxide synthase, cyclooxygenase-2 and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, reduction in the production of excess nitric oxide, prostaglandin E2, and reactive oxygen species leading to brain inflammation and neuronal injury) are widespread in the brain parenchyma. This suggested that sartans may influence multiple brain cell types. Using microglia, primary cortical neuron, primary cerebellar granule cell, and cerebral microvascular endothelial cell cultures, we discovered that ARBs ameliorate inflammation in all cell types studied. ARB neuroprotective effects were demonstrated against the bacterial endotoxin lipopolysaccharide (LPS), against excess glutamate and against the pro-inflammatory cytokine IL-1beta. Mechanisms involved include decreased activation of several protein kinases and reduced activation of the transcription factor NFkappaBalpha. We hypothesized that the major anti-inflammatory and neuroprotective effects of sartans may not be the exclusive result of AT1 receptor inhibition. In human circulating monocytes, cells expressing very few AT1 receptors, the anti-inflammatory effects of sartans were partially dependent on peroxisome proliferator-activated receptor gamma (PPARgamma) activation. We have found that some sartans may have dual mechanisms of action: anti-hypertensive, anti-growth and anti-inflammatory effects related to their inhibition of AT1 receptors, and metabolic and anti-inflammatory effects, partially the consequence of direct PPARgamma activation. We now confirm that participation of PPARgamma activation as a major component of ARB effects in THP-1 cells, a human acute monocytic leukemia cell line, and in primary cultures of rat cortical microglia devoid of significant AT1 receptor expression, Our earlier findings that ARBs may limit the exaggerated hormonal and sympathetic response to stress opened a second direction of study. We have initially established that ARBs restrict the stress-induced alterations in cortical gamma-aminobutyric acid (GABAA) function, as determined by cortical benzodiazepine binding. We found, more recently, that ARBs protect GABAA function during inflammatory stress. This finding explains the anti-anxiety and anti-depressant effects of ARBs in our model of inflammatory stress (prevention of LPS-induced sickness behavior, anorexia and weight loss, and reduction of LPS-induced anxiety). We have more recently discovered, in collaborative studies, that ARBs reduce depression and anxiety associated with chronic mild variable stress in a rodent model of genetic vulnerability to depression and anxiety. We have continued to clarify the mechanisms of neuroprotection and prolongation of lifespan produced by life-long sartan administration. We found that long-term sartan administration reduces age-associated brain inflammation, vascular remodeling and anxiety, and that the neuroprotective effects of ARBs persist throughout life. Our findings may, at least in part, explain the major beneficial effects of sartan therapy in Alzheimers disease reported by other groups. An additional novel finding is that ARB administration in a rodent model significantly protects the brain from traumatic brain injury. ARBs decrease lesion size, reduce neuronal injury and protect neurological function in this model. This is the first demonstration of the neuroprotective effect of ARBs in traumatic brain injury. Our work continues with mechanistic and translational studies to further clarify the mechanisms of ARB-induced neuroprotection and anti-depressant effects. Our goals are to test our hypothesis of major therapeutic advantages of ARB use in brain disorders,including major depression, Alzheimer's disease and traumatic brain injury, and to establish a more solid base for further development of more effective, ARB-derived neuroprotective and anti-depressant compounds of translational value.