Treadmill exercise suppresses nigrostriatal dopaminergic neuronal loss in 6-hydroxydopamine-induced Parkinson's rats

In Parkinson's disease, the progressive loss of dopaminergic neurons in the pars compacta of the substantia nigra leads to debilitating motor dysfunction. In the present study, we investigated the effects of treadmill exercise on the dopaminergic neuronal cell death in the substantia nigra and on the dopaminergic fiber loss in the striatum of Parkinson's rats. Parkinson's rats were made by injecting 6-hydroxydopamine into the striatum with using a stereotaxic instrument. The rats in the exercise groups were put on the treadmill to run for 30 min once a day for 14 consecutive days after 6-hydroxydopamine administration into the striatum. Two weeks after the intrastriatal injection of 6-hydroxydopamine, the rats without treadmill exercise displayed rotational asymmetry following injection of apomorphine (0.5 mg/kg, s.c.). In contrast, the rats undergoing treadmill exercise showed a significant reduction of rotational asymmetry. Analysis via immunohistochemistry for the tyrosine hydroxylase expression revealed a substantial loss of cell bodies in the substantia nigra and their projected fibers in the striatum ipsilateral to the lesion following 6-hydroxydapamine injection into the striatum. However, treadmill running enhanced the survival of dopaminergic neurons in the substantia nigra and also their fibers projecting into the striatum. The results of the present study show that treadmill exercise may provide therapeutic value for the treatment of Parkinson's disease patients.     

α-MSH对脂多糖诱导小鼠腹腔巨噬细胞CD14和TLR4 mRNA 表达的影响

目的：观察α-黑色素细胞刺激素(α-MSH)对脂多糖（LPS）诱导小鼠腹腔巨噬细胞CD14和TLR4 mRNA表达的影响，探讨α-MSH拮抗LPS的作用机制。方法：用半定量逆转录多聚酶链反应（RT-PCR）的方法检测LPS诱导体外培养的小鼠腹腔巨噬细胞CD14和TLR4 mRNA表达水平和给予α-MSH后对CD14和TLR4 mRNA表达的影响。结果：正常静息小鼠腹腔巨噬细胞只表达少量的CD14和TLR4 mRNA,给予LPS刺激后6 h，两者表达明显强于正常对照（P<0.01），并且其表达量随着LPS刺激时间的增加维持在高水平，24 h达到峰值，在48 h CD14 mRNA的表达降到正常水平，而TLR4 mRNA的表达仍然维持在高水平。在LPS刺激的同时给予α-MSH，CD14和TLR4 mRNA的表达则明显低于LPS组（P<0.05），而且α-MSH这种效应与其使用浓度有关，0.1 nmol/L α-MSH不影响LPS诱导的CD14和TLR4 mRNA的表达，而当α-MSH的浓度达到1、10、100 nmol/L则能显著影响CD14和TLR4 mRNA的表达（P<0.05），但各个浓度组之间的作用没有明显差别（P>0.05）。结论：α-MSH抗LPS的效应可能与其下调LPS信号转导通路关键受体CD14和TLR4 mRNA的表达有关，从而干扰LPS跨膜信号转导，阻碍巨噬细胞活化。     

Effects of endurance exercise on ventral tegmental area neurons in the chronic 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine and probenecid-treated mice

Loss of dopaminergic neurons in the substantia nigra (A9 cells) and ventral tegmental area (VTA) (A10 cells) has been reported in Parkinson's disease with reference to causing motor and non-motor deficits, although clinical and laboratory animal studies on the degeneration of VTA neurons are less emphasized comparative to the degeneration of substantia nigra neurons. In the present study, we examined the VTA dopaminergic neurons in a chronic mouse model of Parkinson's disease induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine and probenecid at a level showing moderate neurodegeneration and studied the impact of endurance exercise on VTA neurons in this model. In comparison to the normal control animals, the chronic mouse model of Parkinson's disease with moderate neurodegeneration demonstrated a significant reduction of VTA neurons (52% loss), when these animals were kept sedentary throughout the study. Morphologically, the VTA dopaminergic neurons in this model displayed a decrease in cell volume and showed irregular or disparaging axonal and dendritic projections. When the chronic Parkinsonian mice were exercised on a motorized rodent treadmill up to 15 m/min, 40 min/day, 5 days/week for 10 and 18 weeks, the total number of VTA dopaminergic neurons were significantly higher than the sedentary Parkinsonian animals. Especially noted with the 18-week exercised Parkinsonian mice, the number of VTA neurons returned to normal range and the cells were densely populated and displayed distinctive axons and dendritic arborization. These results demonstrate that prolonged exercise training is neuroprotective to the dopaminergic neurons in the VTA of the chronic mouse model of Parkinson's disease with moderate neurodegeneration.     

人正常肠上皮细胞内毒素信号转导相关受体mRNA的表达

目的 研究人正常肠上皮细胞内毒素信号转导相关受体TLR4、TLR2、CDl4和MD2 mRNA的表达情况，以及内毒素刺激对其表达的影响。方法 提取有、无内毒素刺激的培养人肠上皮细胞和阳性对照THP1细胞的总RNA，用RT—PCR方法检测TLR4、TLR2、CDl4和MD2 mRNA的表达。结果 和THP1细胞相比，人肠上皮细胞TLR4、TLR2、CDl4和MD2 mRNA的表达均较弱。内毒素刺激后，TLR4、CDl4和MD2 mRNA表达明显减弱，而TLR2的表达无显著变化。结论 人正常肠上皮细胞组成性低表达TLR4、CDl4和MD2 mRNA，且在受脂多糖(LPS)刺激后反应性下调其表达，这可能是肠上皮细胞(IEC)耐受LPS的关键机制之一，而TLR2在IEC耐受LPS的机制中可能不起主要作用。     

Peptidoglycan of Staphylococcus aureus upregulates monocyte expression of CD14, Toll-like receptor 2 (TLR2), and TLR4 in human blood: possible implications for priming of lipopolysaccharide signaling

Previous studies have indicated that peptidoglycan (PepG) from gram-positive bacteria can exert a priming effect on the innate immune response to lipopolysaccharide (LPS) from gram-negative bacteria. Here, we hypothesized that this priming effect may be preceded by enhanced expression of monocyte CD14, Toll-like receptor 2 (TLR2), and TLR4. In an ex vivo whole human blood model, we observed a substantial synergy between LPS and PepG in the release of tumor necrosis factor alpha and interleukin-1beta (IL-1beta) over the 24-h experimental period, whereas the effect on IL-8 and IL-10 release was more time dependent. The priming effect of PepG on cytokine release was preceded by a rapid upregulation of CD14, TLR2, and TLR4 expression on monocytes: at 3 hours there was a twofold increase in CD14 expression (P < 0.03), a fivefold increase in TLR2 expression (P < 0.03), and a twofold increase in TLR4 expression (P < 0.03). CD14 and TLR2 remained upregulated throughout the experimental period following exposure to PepG (P < 0.05). Only a transient upregulation of these monocyte receptors was observed following treatment with LPS or LPS plus PepG. In conclusion, the synergistic effect of LPS and PepG on cytokine release is preceded by a reciprocal upregulation of TLR2 and TLR4 by both bacterial cell wall components.     

甘露糖受体在正常和炎症小鼠脑内小胶质细胞的表达

目的 确定脑内小胶质细胞是否表达甘露糖受体,以及在不同脑区甘露糖受体的表达是否存在差异,以进一步明确小胶质细胞的功能.方法 C57小鼠26只,分为侧脑室炎症模型组(10只)、全身炎症模型组(6只)和正常对照组(10只).通过注射细菌脂多糖(LPS)建立全脑急性炎症模型,用免疫荧光双标技术对小鼠脑组织冷冻切片进行染色,激...     

GM-CSF increases LPS-induced production of proinflammatory mediators via upregulation of TLR4 and CD14 in murine microglia

Background

Microglia are resident macrophage-like cells in the central nervous system (CNS) and cause innate immune responses via the LPS receptors, Toll-like receptor (TLR) 4 and CD14, in a variety of neuroinflammatory disorders including bacterial infection, Alzheimer’s disease, and amyotrophic lateral sclerosis. Granulocyte macrophage-colony stimulating factor (GM-CSF) activates microglia and induces inflammatory responses via binding to GM-CSF receptor complex composed of two different subunit GM-CSF receptor α (GM-CSFRα) and common β chain (βc). GM-CSF has been shown to be associated with neuroinflammatory responses in multiple sclerosis and Alzheimer’s disease. However, the mechanisms how GM-CSF promotes neuroinflammation still remain unclear.

Methods

Microglia were stimulated with 20 ng/ml GM-CSF and the levels of TLR4 and CD14 expression were evaluated by RT-PCR and flowcytometry. LPS binding was analyzed by flowcytometry. GM-CSF receptor complex was analyzed by immunocytechemistry. The levels of IL-1β, IL-6 and TNF-α in culture supernatant of GM-CSF-stimulated microglia and NF-κB nuclear translocation were determined by ELISA. Production of nitric oxide (NO) was measured by the Griess method. The levels of p-ERK1/2, ERK1/2, p-p38 and p38 were assessed by Western blotting. Statistically significant differences between experimental groups were determined by one-way ANOVA followed by Tukey test for multiple comparisons.

Results

GM-CSF receptor complex was expressed in microglia. GM-CSF enhanced TLR4 and CD14 expressions in microglia and subsequent LPS-binding to the cell surface. In addition, GM-CSF priming increased LPS-induced NF-κB nuclear translocation and production of IL-1β, IL-6, TNF-α and NO by microglia. GM-CSF upregulated the levels of p-ERK1/2 and p-p38, suggesting that induction of TLR4 and CD14 expression by GM-CSF was mediated through ERK1/2 and p38, respectively.

Conclusions

These results suggest that GM-CSF upregulates TLR4 and CD14 expression in microglia through ERK1/2 and p38, respectively, and thus promotes the LPS receptor-mediated inflammation in the CNS.

     

MPTP treatment increases expression of pre-pro-nociceptin/orphanin FQ mRNA in a subset of substantia nigra reticulata neurons

Antagonists selectively inhibiting activation of the nociceptin/orphanin FQ (N/OFQ) receptor reduce motor symptoms in experimental models of Parkinson's disease, and genetic deletion of the ppN/OFQ gene offers partial protection of mid-brain dopamine neurons against the neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). MPTP increased ppN/OFQ mRNA expression in the substantia nigra (SN). We have evaluated the temporal relationship of dopamine cell loss to increased ppN/OFQ mRNA expression in the substantia nigra after MPTP treatment, and characterized the cellular locations in which increased ppN/OFQ mRNA expression was observed after MPTP treatment. MPTP increased by about 5-fold the number of neurons expressing ppN/OFQ mRNA in the pars reticulata of SN (SNr) by 24 h after treatment and the elevation remained significant for at least 7 days. This period coincided with the timing of the loss of dopamine neurons from the pars compacta of substantia nigra (SNc) after MPTP. The increased expression of ppN/OFQ mRNA co-localized with a neuronal marker in the SNr. MPTP treatment resulted in a small increase in the numbers of neurons expressing ppN/OFQ in the SNc in mice from one mouse colony but the increase did not reach statistical significance in mice from another colony. No changes in ppN/OFQ-mRNA expression were observed in the ventral tegmental area (VTA), the caudate-putamen, the subthalamic nucleus, or in two other brains areas. These results demonstrate that increased N/OFQ expression in the SNr is closely associated with the MPTP-induced loss of dopamine neurons in the SNc in a widely used animal model of Parkinson's disease.     

Regulation of Toll-like receptor (TLR)2 and TLR4 on CD14dimCD16+ monocytes in response to sepsis-related antigens

Rapid overproduction of proinflammatory cytokines are characteristic of sepsis. CD14(dim)CD16(+) monocytes are thought to be major producers of cytokine and have been shown to be elevated in septic patients. Toll-like receptors (TLR) are pattern recognition receptors important in mediating the innate immune response and their activation can lead to production of cytokines. Using whole blood culture and flow cytometry we have investigated TLR2 and TLR4 regulation after stimulation with sepsis-relevant antigens [lipopolysaccharide (LPS), Staphylococcal enterotoxin B (SEB) and peptidoglycan (PGN)]. The percentage of CD14(dim)CD16(+) monocyte population expanded at 20 h post-stimulation, after a rise in tumour necrosis factor (TNF)-alpha and interleukin (IL)-6 at 2 h. A strong positive correlation between the percentage of CD14(dim)CD16(+) monocytes and secreted TNF-alpha was demonstrated (r = 0.72). Furthermore, we were able to induce expansion of the CD14(dim)CD16(+) population to approximately 35% of all monocytes with the addition of recombinant TNF-alpha to the whole blood culture. TLR4 was found to be expressed 2.5 times higher on CD14(dim)CD16(+) compared to CD14(+) CD16(-) monocytes, while TLR2 expression was similar in both subpopulations. The CD14(dim)CD16(+) and CD14(+) CD16(-) monocyte populations were different in their response to various antigens. LPS down-regulated TLR4 by 4.9 times in CD16(+) monocytes compared to only 2.3 times in CD16(-) monocytes at 2 h. LPS was able to up-regulate TLR2 by 6.2 times after 2 h, with no difference between the subpopulations. LPS further up-regulated TLR2 by 18.4 times after 20 h only in the CD14(+) CD16(-) population. PGN and SEB induced no significant changes in TLR2 or TLR4 expression. We hypothesize that following exposure to bacterial antigens, subsequent TNF-alpha drives a differentiation of monocytes into a CD14(dim)CD16(+) subpopulation.     

Aging-related dysregulation of dopamine and angiotensin receptor interaction

It is not known whether the aging-related decrease in dopaminergic function leads to the aging-related higher vulnerability of dopaminergic neurons and risk for Parkinson's disease. The renin-angiotensin system (RAS) plays a major role in the inflammatory response, neuronal oxidative stress, and dopaminergic vulnerability via type 1 (AT1) receptors. In the present study, we observed a counterregulatory interaction between dopamine and angiotensin receptors. We observed overexpression of AT1 receptors in the striatum and substantia nigra of young adult dopamine D1 and D2 receptor-deficient mice and young dopamine-depleted rats, together with compensatory overexpression of AT2 receptors or compensatory downregulation of angiotensinogen and/or angiotensin. In aged rats, we observed downregulation of dopamine and dopamine receptors and overexpression of AT1 receptors in aged rats, without compensatory changes observed in young animals. L-Dopa therapy inhibited RAS overactivity in young dopamine-depleted rats, but was ineffective in aged rats. The results suggest that dopamine may play an important role in modulating oxidative stress and inflammation in the substantia nigra and striatum via the RAS, which is impaired by aging.     

Reduced expression of TLR4 is associated with the metastatic status of human colorectal cancer

Signaling mediating colorectal cancer (CRC) progression is incompletely understood. Previously, we identified lipopolysaccharide (LPS), an endotoxin of ubiquitously existing colonic bacteria, as a pivotal stimulus increasing the metastatic potential of human CRC. Since the ubiquitous colonic bacteria release large amounts of LPS this observation could be of enormous relevance for the progression of CRC. In this study we present data contributing to the elucidation of its mode of action. Since both receptors CD14 and TLR4 act as LPS mediators, we determined their expression in various CRC cell lines and in 115 non-metastatic, lymphogenous-metastatic and haematogenous-metastatic CRC specimens. Here we showed that CD14 was not expressed in normal colon epithelium, in non-metastatic and metastatic CRC. Furthermore, we showed that diverse CRC cell lines did not express CD14 under normal conditions and after LPS stimulation. Thus, CD14 can be ruled out as a mediator of LPS-induced signaling related to CRC progression. In contrast, we found that normal colon epithelium and CRC cell lines were positive for TLR4. Furthermore, both lymphogenous and haematogenous metastatic cases showed either loss of expression or strong downregulation of TLR4 as compared to normal tissue and to non-metastatic tumors. We found that LPS stimulation resulted in significant TLR4 upregulation in cells expressing lower constitutive TLR4 levels such as CaCo2, whereas no significant response to LPS was observed in cells characterized by relatively high amounts of constitutive TLR4. Our data suggest that TLR4 expression may be associated with mechanisms preventing CRC progression.     

Molecular hydrogen is protective against 6-hydroxydopamine-induced nigrostriatal degeneration in a rat model of Parkinson's disease

Molecular hydrogen serves as an antioxidant that reduces hydroxyl radicals, but not the other reactive oxygen and nitrogen species. In the past year, molecular hydrogen has been reported to prevent or ameliorate eight diseases in rodents and one in human associated with oxidative stress. In Parkinson's disease, mitochondrial dysfunction and the associated oxidative stress are major causes of dopaminergic cell loss in the substantia nigra. We examined effects of ∼50%-saturated molecular hydrogen in drinking water before or after the stereotactic surgery on 6-hydroxydopamine-induced nigrostrital degeneration in a rat model of Parkinson's disease. Methamphetamine-induced behavioral analysis showed that molecular hydrogen prevented both the development and progression of the nigrostrital degeneration. Tyrosine hydroxylase staining of the substantia nigra and striatum also demonstrated that pre- and post-treatment with hydrogen prevented the dopaminergic cell loss. Our studies suggest that hydrogen water is likely able to retard the development and progression of Parkinson's disease.     

Distinct roles of pattern recognition receptors CD14 and Toll-like receptor 4 in acute lung injury

Acute lung injury (ALI) induced by lipopolysaccharide (LPS) is a major cause of mortality among humans. ALI is characterized by microvascular protein leakage, neutrophil influx, and expression of proinflammatory mediators, followed by severe lung damage. LPS binding to its receptors is the crucial step in the causation of these multistep events. LPS binding and signaling involves CD14 and Toll-like receptor 4 (TLR4). However, the relative contributions of CD14 and TLR4 in the induction of ALI and their therapeutic potentials are not clear in vivo. Therefore, the aim of the present study was to compare the roles of CD14 and TLR4 in LPS-induced ALI to determine which of these molecules is the more critical target for attenuating ALI in a mouse model. Our results show that CD14 and TLR4 are necessary for low-dose (300-microg/ml) LPS-induced microvascular leakage, NF-kappaB activation, neutrophil influx, cytokine and chemokine (KC, macrophage inflammatory protein 2, tumor necrosis factor alpha, interleukin-6) expression, and subsequent lung damage. On the other hand, when a 10-fold-higher dose of LPS (3 mg/ml) was used, these responses were only partially dependent on CD14 and they were totally dependent on TLR4. The CD14-independent LPS response was dependent on CD11b. A TLR4 blocking antibody abolished microvascular leakage, neutrophil accumulation, cytokine responses, and lung pathology with a low dose of LPS but only attenuated the responses with a high dose of LPS. These data are the first to demonstrate that LPS-induced CD14-dependent and -independent (CD11b-dependent) signaling pathways in the lung are entirely dependent on TLR4 and that blocking TLR4 might be beneficial in lung diseases caused by LPS from gram-negative pathogens.     

Ageing and Parkinson's disease: Why is advancing age the biggest risk factor?

As the second most common age related neurodegenerative disease after Alzheimer's disease, the health, social and economic impact resulting from Parkinson's disease will continue to increase alongside the longevity of the population. Ageing remains the biggest risk factor for developing idiopathic Parkinson's disease. Although research into the mechanisms leading to cell death in Parkinson's disease has shed light on many aspects of the pathogenesis of this disorder, we still cannot answer the fundamental question, what specific age related factors predispose some individuals to develop this common neurodegenerative disease. In this review we focus specifically on the neuronal population associated with the motor symptoms of Parkinson's disease, the dopaminergic neurons of the substantia nigra, and try to understand how ageing puts these neurons at risk to the extent that a slight change in protein metabolism or mitochondrial function can push the cells over the edge leading to catastrophic cell death and many of the symptoms seen in Parkinson's disease. We review the evidence that ageing is important for the development of Parkinson's disease and how age related decline leads to the loss of neurons within this disease, before describing exactly how advancing age may lead to substantia nigra neuronal loss and Parkinson's disease in some individuals.     

脂多糖对人正常肝细胞株L02损伤的实验研究

目的探讨脂多糖(lipopolysaccharide,LPS)对人正常肝细胞株L02的损伤作用及其机制。方法采用流式细胞术分别检测LPS诱导L02细胞凋亡和线粒体膜电位变化的作用,测定L02细胞膜上CD14、Toll样受体4(TLR4)、Toll样受体2(TLR2)的表达水平;采用酶联免疫吸附法(ELISA)测定细胞培养上清液中肿瘤坏死因子α(TNF-α)含量;生化法测定细胞培养上清液中丙氨酸转氨酶(ALT)、门冬氨酸转氨酶(AST)及乳酸脱氢酶(LDH)含量。结果10、20、40、80mg/L剂量的LPS作用于L02细胞后0、6、12、24和36h,细胞的凋亡率和线粒体膜电位无显著性差异(P>0.05),各组上清液中ALT、AST、LDH和TNF-α含量亦无明显变化(P>0.05),L02细胞膜上LPS受体CD14、TLR4、TLR2表达分别为(2.28±0.60)%,(1.04±0.80)%,(2.07±0.50)%。结论L02细胞膜上LPS受体CD14、TLR4、TLR2表达水平低,致使LPS不能直接引起L02细胞损伤。     

Alterations of Hrd1 expression in various encephalic regional neurons in 6-OHDA model of Parkinson's disease

The ubiquitin-proteasome system plays a central role in regulated degradation of cellular proteins under different physiological conditions. Accumulation of misfolded proteins is involved in the pathogenesis of many neurodegenerative diseases, such as Parkinson's disease (PD), Alzheimer's disease (AD) and Huntington's disease (HD). Hrd1 is a newly identified ubiquitin ligase involved in degradation of misfolded proteins from the endoplasmic reticulum (ER), thereby protecting cells against ER stress. Increasing evidence has linked ER stress to PD pathogenesis. However, the expression of Hrd1 in PD brain remains elusive. In the present study, the expression of Hrd1 in different encephalic regions was studied in 6-OHDA model of Parkinson's disease by immunohistochemistry. The results showed that Hrd1 was up-regulated in 6-OHDA-treated mice in various encephalic regional neurons, especially those in hippocampus, substantia nigra (SN), subthalamic nucleus (STN), striatum and frontal lobe. It suggested that Hrd1 up-regulation may represent a protective response against neurodegeneration in PD.     

DJ-1 mRNA anatomical localization and cell type identification in the mouse brain

Mutations in DJ-1 cause familial Parkinson's disease (PD). The expression pattern of DJ-1 in the brain remains controversial. In the present study, we used DJ-1 deficient mice as negative controls and examined DJ-1 mRNA expression in mouse brains. In sequential double labeling on the same sections, in situ hybridization of DJ-1 mRNA was followed by immunofluorescence detection of cell type markers. We found that DJ-1 mRNA was expressed in the majority of neurons in all brain areas examined. In particular, all dopamine neurons in the ventral midbrain expressed DJ-1 mRNA. In contrast, the choroid plexus and ependymal cells lining the ventricles were the only non-neuronal regions strongly expressing DJ-1 mRNA. DJ-1 mRNA was not detected in astrocytes. The fact that DJ-1 mRNA is expressed in all nigra dopamine neurons but not in astrocytes suggests that its potential neuroprotective role could be cell-autonomous. Moreover, that DJ-1 expression is not restricted to substantia nigra dopamine neurons suggests that PD-linked mutant DJ-1 may interact with other predisposing factors to cause the relatively selective dopamine neuron degeneration in Parkinson's disease.