脂多糖诱导肝细胞HepG2释放HMGB-1的研究

观察小剂量LPS(lipopolysaccharide)刺激下非坏死HepG2细胞是否存在HMGB1(high mobility group box-1 protein)移位及释放.以终浓度为100μg/L的LPS作用HepG2和RAW264.7细胞0、4、8、12、16、20、24h.LPS作用16~24h,HepG2和RAW264.7细胞培养上清中均可以检测到明显的HMGB1,与对照组差别有显著性(P<0.05).而MTT法和上清中LDH(lactate dehydrogenase)含量测定显示,LPS处理24h的HepG2存活率仍然非常高.免疫荧光观察到HMGB1的释放伴随着其从细胞核向胞浆的移位.由此可见,经LPS诱导,非坏死状态的HepG2细胞可发生HMGB1的移位及释放.     

砷暴露诱导人肝细胞氧化应激过程中NADPH氧化酶作用机制

目的：探讨砷暴露诱导细胞氧化应激的分子机制。方法：采用人正常肝细胞进行亚砷酸钠和砷酸钠的暴露处理,并设相应对照组,采用SOD模拟物MnTMPyP和还原型谷胱甘肽（reducedglutathione,GSH）预处理,检测细胞超氧阴离子（02。）和细胞整体ROS的水平。WestemBlot方法检测细胞氧化／抗氧化重要酶微粒体谷胱甘肽硫转移酶（microsomalglutathioneS-transferase-l,Mgst．1）、半胱氨酸双加氧酶l（cysteinedioxygenasel,Cd01）和NADPH氧化酶的催化亚基NOX4的表达。针对NADPH氧化酶,采用特异性抑制剂（diphenyleneiodoniumchloride,DPI）进行预处理,观察对砷暴露引起的细胞ROS水平及细胞凋亡的影响。结果：砷暴露能够显著诱导细胞超氧阴离子的产生,提高细胞整体ROS水平,其中三价砷（亚砷酸钠,A矿）诱导氧化应激作用显著强于五价砷（砷酸钠,As5＋）。亚砷酸钠能够显著提高NOX4的表达。针对NADPH氧化酶的抑制剂DPI能够显著抑制砷暴露引起的细胞ROS水平升高以及细胞凋亡的增加。结论：NADPH氧化酶是砷暴露诱导人肝细胞的作用靶点,砷能够通过NADPH氧化酶产生大量超氧阴离子,提高ROS水平,造成氧化应激,诱导人正常肝细胞凋亡。     

蛋白酶体抑制剂乳胞素(LAC)对LPS诱导的人关节软骨细胞炎症反应的抑制作用

目的:探讨蛋白酶体抑制剂乳胞素(LAC)对LPS诱导的人关节软骨细胞炎症反应的抑制作用.方法:体外培养原代人关节软骨细胞,分为四组,即对照组、LPS组(100 ng/mL LPS处理6小时)、LAC组(5μM LAC处理1小时)和LAC+LPS组(5μMLAC预处理1小时后,给予LPS处理6小时),分别用ELISA和western blot检测和比较各组细胞内20s蛋白酶体,NF-κB,TNF-α和iNOS的含量.结果:LPS处理后的人关节软骨细胞内20s蛋白酶体,NF-κB,TNF-α和iNOS的表达均较对照组显著升高(P＜0.05),LAC+LPS组以上指标较LPS组均显著降低(P＜0.05),而LAC组和LAC+LPS组以上指标与对照组比较均无显著性差异(P＞0.05).结论:LAC可显著抑制LPS诱导的人关节软骨细胞炎症反应.     

白桦脂酸通过抗氧化应激作用抑制脂多糖诱导的血管收缩功能损伤

白桦脂酸（betulinic acid,BA）有良好的抗心血管氧化应激损伤作用。然而,BA对脂多糖lipopolysaccharide,LPS）诱导血管收缩功能损伤是否具有保护作用,该保护作用是否与抗氧化应激有关,尚不清楚。本研究给予雄性SD大鼠白桦脂酸灌胃（25 mg/kg/d,3 d）预处理,于第4 d腹腔注射LPS（10 mg/kg）,4 h后麻醉处死,分离血浆及胸主动脉,测定大鼠胸主动脉环收缩性,测定炎症因子白细胞介素6（interleukin-6,IL-6）及氧化应激指标。结果显示,白桦脂酸明显抑制LPS诱导的血浆及胸主动脉IL-6水平（P<0.01）,降低LPS对苯肾上腺素、KCl及Ca²⁺血管收缩反应的抑制作用（84.8%±9.09% vs 42.80%±9.00%,P<0.01;127.48%±12.02% vs 99.78%±6.02%,P<0.01;52.07%±13.48% vs 20.83%±5.04%,P<0.01）,减少LPS诱导的胸主动脉丙二醛水平（P<0.01）及诱导型一氧化氮合酶（inducible nitric oxide synthase,iNOS）活性（P<0.01）,缓解LPS对超氧化物歧化酶（superoxide dismutase,SOD）的抑制作用（P<0.01）。上述结果提示,白桦脂酸抑制LPS诱导血管收缩功能障碍的机制可能与增加机体SOD活性,抑制氧化应激及iNOS活性有关。     

加兰他敏对间歇性低氧引起的认知损害的预防作用

目的:探究加兰他敏对间歇性低氧引起的认知损伤是否有保护作用,从而说明其对睡眠呼吸暂停综合症引起的认知损害是否有预防作用。方法:建立间歇低氧大鼠模型,行水迷宫试验检测行为功能变化,免疫组化检测海马神经元及胶质细胞数目的变化。结果:加兰他敏与间歇低氧模型组相比,行水迷宫的平均逃避潜伏期缩短,游泳总距离减少;免疫组化的结果海马神经元的数目有所增加,胶质细胞的数目减少。结论:加兰他敏对间歇性低氧引起的认知损伤有明显的改善作用,可能与减少神经元的丢失及减少胶质细胞的再生有关。所以对于诊断了睡眠呼吸暂停综合征(ASA)的患者,如果同时合并其他痴呆的易感因素,可预防性应用加兰他敏。     

加兰他敏对间歇性低氧引起的认知损害的预防作用

目的：探究加兰他敏对间歇性低氧引起的认知损伤是否有保护作用,从而说明其对睡眠呼吸暂停综合症引起的认知损害是否有预防作用。方法：建立间歇低氧大鼠模型,行水迷宫试验检测行为功能变化,免疫组化检测海马神经元及胶质细胞数目的变化。结果：加兰他敏与间歇低氧模型纽相比,行水迷宫的平均逃避潜伏期缩短,游泳总距离减少;免疫组化的结果海马神经元的数目有所增加,胶质细胞的数目减少。结论：加兰他敏对间歇性低氧引起的认知损伤有明显的改善作用,可能与减少神经元的丢失及减少胶质细胞的再生有关。所以对于诊断了睡眠呼吸暂停综合征（ASA）的患者,如果同时合并其他痴呆的易感因素,可预防性应用加兰他敏.     

KLF4在内毒素血症小鼠中的表达模式及其意义

目的探讨Kruppel样因子4（Kruppel-like factor 4,KLF4）在内毒素血症小鼠中的表达模式及意义。方法运用实时荧光PCR技术和Western blot技术,分别从mRNA水平和蛋白水平探讨内毒素血症小鼠肝脏和肺脏中KLF4的表达;运用生物信息学技术,对启动子区含有KLF4的结合位点的炎症介质基因进行了预测;运用RT-PCR技术,从mRNA水平探讨内毒素血症小鼠肝脏和肺脏中IL1β的表达模式。结果内毒素血症小鼠肝脏和肺脏中KLF4 mRNA的表达下凋,KLF4蛋白的表达先下凋后升高;IL-18、IL-15、IL-12、IL-18、IL-10等炎症介质基因的启动子区均含有KLF4的结合元件,这些炎症基因的表达可能直接受到KLF4的调控;内毒素血症小鼠肝脏和肺脏中IL-IB的表达模式与KLF4的表达模式呈相反趋势。结论内毒素血症小鼠肝脏和肺脏中KLF4表达下调,KLF4在炎症介质基因表达调控中可能具有重要作用。     

N-Acetylcysteine Prevents Spatial Memory Impairment Induced by Chronic Early Postnatal Glutaric Acid and Lipopolysaccharide in Rat Pups

Background and Aims

Glutaric aciduria type I (GA-I) is characterized by accumulation of glutaric acid (GA) and neurological symptoms, such as cognitive impairment. Although this disease is related to oxidative stress and inflammation, it is not known whether these processes facilitate the memory impairment. Our objective was to investigate the performance of rat pups chronically injected with GA and lipopolysaccharide (LPS) in spatial memory test, antioxidant defenses, cytokines levels, Na+, K+-ATPase activity, and hippocampal volume. We also evaluated the effect of N-acetylcysteine (NAC) on theses markers.

Methods

Rat pups were injected with GA (5umol g of body weight-1, subcutaneously; twice per day; from 5th to 28th day of life), and were supplemented with NAC (150mg/kg/day; intragastric gavage; for the same period). LPS (2mg/kg; E.coli 055 B5) or vehicle (saline 0.9%) was injected intraperitoneally, once per day, from 25th to 28th day of life. Oxidative stress and inflammatory biomarkers as well as hippocampal volume were assessed.

Results

GA caused spatial learning deficit in the Barnes maze and LPS potentiated this effect. GA and LPS increased TNF-α and IL-1β levels. The co-administration of these compounds potentiated the increase of IL-1β levels but not TNF-α levels in the hippocampus. GA and LPS increased TBARS (thiobarbituric acid-reactive substance) content, reduced antioxidant defenses and inhibited Na+, K+-ATPase activity. GA and LPS co-administration did not have additive effect on oxidative stress markers and Na+, K+ pump. The hippocampal volume did not change after GA or LPS administration. NAC protected against impairment of spatial learning and increase of cytokines levels. NAC Also protected against inhibition of Na+,K+-ATPase activity and oxidative markers.

Conclusions

These results suggest that inflammatory and oxidative markers may underlie at least in part of the neuropathology of GA-I in this model. Thus, NAC could represent a possible adjuvant therapy in treatment of children with GA-I.     

Impairment of Central Chemoreception in Neonatal Rats Induced by Maternal Cigarette Smoke Exposure during Pregnancy

It has been postulated that prenatal cigarette smoke exposure (CSE) increases the risk for sudden infant death syndrome. The victims of infant death syndrome suffer from respiratory abnormalities, such as central apnea, diminished chemoreflex and alteration in respiratory pattern during sleep. However, no experimental evidence on CSE model exists to confirm whether prenatal CSE gives rise to reduction of neonatal central chemoreception in in vitro preparations in absence of peripheral sensory feedback. The aim of the present study was to test the hypothesis that maternal CSE during pregnancy depresses central chemoreception of the neonatal rats. The pregnant rats were divided into two groups, control (n = 8) and CSE (n = 8). Experiments were performed on neonatal (0–3days) rat pups. Fictive respiratory activity was monitored by recording the rhythmic discharge from the hypoglossal rootlets of the medullary slices obtained from the neonatal rats. The burst frequency (BF) and integrated amplitude (IA) of the discharge were analyzed. Their responses to acidified artificial cerebrospinal fluid (aCSF) were tested to indicate the change of the central chemosensitivity. Under condition of perfusing with standard aCSF (pH 7.4), no significant difference was detected between the two groups in either BF or IA (P>0.05). Under condition of perfusing with acidified aCSF (pH 7.0), BF was increased and IA was decreased in both groups (P<0.01). However, their change rates in the CSE group were obviously smaller than that in the control group, 66.98 ± 10.11% vs. 143.75 ± 15.41% for BF and −22.38 ± 2.51% vs. −44.90 ± 3.92% for IA (P<0.01). In conclusion, these observations, in a prenatal CSE model, provide important evidence that maternal smoking during pregnancy exerts adverse effects on central chemoreception of neonates.     

Exercise Prevents Memory Impairment Induced by Arsenic Exposure in Mice: Implication of Hippocampal BDNF and CREB

High concentrations of arsenic, which can be occasionally found in drinking water, have been recognized as a global health problem. Exposure to arsenic can disrupt spatial memory; however, the underlying mechanism remains unclear. In the present study, we tested whether exercise could interfere with the effect of arsenic exposure on the long-term memory (LTM) of object recognition in mice. Arsenic (0, 1, 3, and 10 mg/ kg, i.g.) was administered daily for 12 weeks. We found that arsenic at dosages of 1, 3, and 10 mg/kg decreased body weight and increased the arsenic content in the brain. The object recognition LTM (tested 24 h after training) was disrupted by 3 mg/ kg and 10 mg/ kg, but not 1 mg/ kg arsenic exposure. Swimming exercise also prevented LTM impairment induced by 3 mg/ kg, but not with 10 mg/ kg, of arsenic exposure. The expression of brain-derived neurotrophic factor (BDNF) and phosphorylated cAMP-response element binding protein (pCREB) in the CA1 and dentate gyrus areas (DG) of the dorsal hippocampus were decreased by 3 mg/ kg and 10 mg/ kg, but not by 1 mg/ kg, of arsenic exposure. The decrease in BDNF and pCREB in the CA1 and DG induced by 3 mg/ kg, but not 10 mg/ kg, of arsenic exposure were prevented by swimming exercise. Arsenic exposure did not affect the total CREB expression in the CA1 or DG. Taken together, these results indicated that swimming exercise prevented the impairment of object recognition LTM induced by arsenic exposure, which may be mediated by BDNF and CREB in the dorsal hippocampus.     

Anatomical Substrates of Behavioral Impairment Induced by Developmental Lead Exposure in Monkeys: Inferences from Brain Lesions

There is a substantial literature exploring the behavioral consequencesof developmental lead exposure in the monkey; deficits havebeen observed on a number of tasks assessing learning and memoryincluding spatial delayed alternation, discrimination reversal,matching to sample, and concurrent discrimination. Differencesin performance between control and lead-exposed monkeys havealso been observed on intermittent schedules of reinforcement.Comparison of the effects of lead with the extensive literatureon the consequences of lesions in discrete areas of brain onthe same tasks may provide insight into the possible sites ofbrain damage responsible for lead-induced behavioral impairment.Available data strongly suggest that prefrontal cortical areasare damaged by lead, based on the pattern of performance deficitsacross specific tasks. In addition, a constellation of globaldeficits including perseveration, increased distractibility,inability to change response strategy, and inability to inhibitinappropriate responding are hallmarks of both prefrontal damageand developmental lead exposure. Evidence also implicates basalforebrain structures in behavior impairment produced by leadbased on the pattern of deficits across numerous tasks, althoughthe evidence is much weaker than for prefrontal cortex. In contrast,the pattern of behavioral impairment produced by limbic systemlesions is different in many respects from that produced bylead; in addition, the scant neuropathological data availablesuggest that limbic structures are not a target of lead evenat high blood lead levels in the monkey. Comparison of the patternof damage following lead exposure with the effects of lesions,presented here, provides direction for further morphologicalor neurochemical exploration of lead-induced brain damage inthe monkey.     

Grape Seed Procyanidin B2 Inhibits Human Aortic Smooth Muscle Cell Proliferation and Migration Induced by Advanced Glycation End Products

Advanced glycation end product (AGE)-induced vascular smooth muscle cell (VSMC) proliferation is vital to the progression of diabetic vasculopathy. A grape seed procyanidin extract has been reported to possess anti-oxidative and anti-inflammatory properties and to display a significant cardiovascular protective effect, but little is know about the underlying mechanism. The objective of this present study was to determine whether GSPB2 (grape seed procyanidin B2), which is a dimeric procyanidin and more biologically active, could inhibit AGE-induced VSMC proliferation by affecting the production of ubiquitin COOH-terminal hydrolase 1 (UCH-L1), the degradation of IκB-α and nuclear translocation of NF-κB in human aortic smooth muscle cells (HASMCs). Our data show that GSPB2 preincubation markedly inhibited AGE-induced proliferation and migration of HASMCs in a dose-dependent manner and upregulated the protein level of UCH-L1. Further studies revealed that the GSPB2 pretreatment markedly attenuated the degradation of IκB-α and nuclear translocation of NF-κB by modulating ubiquitination of IκB-α in AGE-exposed HASMCs. These results collectively suggest that AGE-induced HASMC proliferation and migration was suppressed by GSPB2 through regulating UCH-L1 and ubiquitination of IκB-α. GSPB2 may therefore have therapeutic potential in preventing and treating vascular complications of diabetes mellitus.     

Role of Oxygen-Derived Free Radicals in Gastric Mucosal Injury Induced by Ischemia or Ischemia-Reperfusion in Rats

Oxygen-derived free radicals have been implicated as possible mediators in the development of tissue injury induced by ischemia and reperfusion. Clamping of the celiac artery in rats reduced the gastric mucosal blood flow to 10% of that measured before the clamping. The area of gastric erosions and thiobarbituric acid (TBA) reactants in gastric mucosa were significantly increased 60 and 90 min after clamping. These changes were inhibited by treatment with SOD and catalase. Thirty and 60 min after reoxyganation, produced by removal of the clamps following 30 min of ischemia, gastric mucosal injury and the increase in TBA reactants were markedly aggravated compared with those induced by ischemia alone. SOD and catalase significantly inhibited these changes. The serum a-tocopherol/cholesterol ratio, an index of in vivo lipid peroxidation, was significantly decreased after long periods of ischemia (60 and 90 min), or after 30 and 60 min of reperfusion following 30 min of ischemia. These results indicated that active oxygen species and lipid peroxidation may play a role in the pathogenesis of gastric mucosal injury induced by both ischemia alone and ischemia-reperfusion. Although, allopurinol inhibited the formation of gastric mucosal injury and the increase in TBA reactants in gastric mucosa, the depletion of polymorphonuclear leukocytes (PMN) counts induced by an injection of anti-rat PMN antibody did not inhibit these changes. As compared with the hypoxanthine-xanthine oxidase system, PMN seem to play a relatively small part in the formation of gastric mucosal injury induced by ischemia-reperfusion.     

Role of CD14 in a Mouse Model of Acute Lung Inflammation Induced by Different Lipopolysaccharide Chemotypes

Background

Recognition of lipopolysaccharide (LPS) is required for effective defense against invading gram-negative bacteria. Recently, in vitro studies revealed that CD14 is required for activation of the myeloid differentiation factor (MyD)88-dependent Toll-like receptor (TLR)4 signaling pathway by smooth (S)-LPS, but not by rough (R)-LPS. The present study investigated the role of CD14 in induction of lung inflammation in mice by these different LPS chemotypes.

Methodology/Results

Neutrophil accumulation and tumor necrosis factor (TNF) release in bronchoalveolar lavage fluid were determined 6 hours after intranasal treatment of wild type (WT) and CD14 knock-out (KO) mice with different doses S-LPS or R-LPS. The contribution of CD14 to lung inflammation induced by S-LPS or R-LPS depended on the LPS dose. At low doses, S-LPS and R-LPS induced neutrophil influx in a CD14-dependent manner. Low dose S-LPS-induced cytokine release also depended on CD14. Strikingly, neutrophil influx and TNF release induced by high dose S-LPS or R-LPS was diminished in the presence of CD14. Intranasal administration of sCD14 to CD14 KO mice treated with S-LPS partially reversed the inflammatory response to the response observed in WT mice.

Conclusions

In conclusion, CD14 modulates effects of both S-LPS and R-LPS within the lung in a similar way. Except for R-LPS-induced TNF release, S-LPS and R-LPS at low dose induced acute lung inflammation in a CD14-dependent manner, while the inflammatory response triggered by high dose S-LPS or R-LPS was diminished by CD14.     

Antiproliferative Effect and Cell Cycle Alterations Induced by Salvia officinalis Essential Oil and Its Three Main Components in Human Colon Cancer Cell Lines

Colon cancer is one of the most common human malignancies, and chemotherapy cannot yet prevent recurrence in all patients. Essential oils are phytocomplexes with antiproliferative properties. In this study, we elucidated the antiproliferative properties and the effect on cell cycle progression of Sicilian Salvia officinalis essential oil and its three main compounds, α‐thujone, 1,8‐cineole (eucalyptol) and camphor, on three human colon cancer cell lines. The essential oil was obtained by hydrodistillation and analyzed by gas chromatography. Cell proliferation was evaluated by MTT assay, and the cell cycle distribution was determined by flow cytometry. Thirty‐four compounds were identified in the tested essential oil. Growth inhibition was observed after 72 h, with an impact on cell cycle progression and no effect on the viability of normal colonic epithelial cells. The study shows that S. officinalis essential oil and its three main components have an in vitro antiproliferative effect on colon cancer cells.     

rMCP-2, the Major Rat Mucosal Mast Cell Protease,an Analysis of Its Extended Cleavage Specificity and Its Potential Role in Regulating Intestinal Permeability by the Cleavage of Cell Adhesion and Junction Proteins

Mast cells of the rat intestinal mucosa express three chymotryptic enzymes named rMCP-2, -3 and 4. rMCP-2, the most abundant of these enzymes, has been shown to increase the permeability of the intestinal epithelium, most likely by cleavage of cell adhesion and junction proteins and thereby play a role in intestinal parasite clearance. However, no target for this effect has yet been identified. To address this question we here present its extended cleavage specificity. Phage display analysis showed that it is a chymase with a specificity similar to the corresponding enzyme in mice, mMCP-1, with a preference for Phe or Tyr in the P1 position, and a general preference for aliphatic amino acids both upstream and downstream of the cleavage site. The consensus sequence obtained from the phage display analysis was used to screen the rat proteome for potential targets. A few of the most interesting candidate substrates were cell adhesion and cell junction molecules. To see if these proteins were also susceptible to cleavage in their native conformation we cleaved 5 different recombinant cell adhesion and cell junction proteins. Three potential targets were identified: the loop 1 of occludin, protocadherin alpha 4 and cadherin 17, which indicated that these proteins were at least partly responsible for the previously observed prominent role of rMCP-2 in mucosal permeability and in parasite clearance.     

Defining the Role of Vascular Smooth Muscle Cell Apoptosis in Atherosclerosis

Vascular smooth muscle cell (VSMC) apoptosis occurs in many arterial diseases, including aneurysm formation, angioplasty restenosis and atherosclerosis. Although VSMC apoptosis promotes vessel remodelling, coagulation and inflammation, its precise contribution to these diseases is unknown, given that apoptosis frequently accompanies vessel injury or alterations to flow. Using transgenic mice with selective induction of VSMC apoptosis, a recent study has precisely determined the direct consequences of VSMC apoptosis in both normal vessels and atherosclerotic plaques. Surprisingly, normal arteries can withstand huge cell losses with little change in active or passive properties. Normal vessels demonstrate highly efficient clearance of apoptotic bodies, even in the absence of professional phagocytes. In contrast, VSMC apoptosis alone is sufficient to induce multiple features of vulnerability to rupture in plaques, identifying VSMC apoptosis as a critical process determining plaque stability.