脂质体包裹Clodronate对单核/巨噬细胞选择性清除作用的研究

为探讨脂质体色裹clodronate清除单核 /巨噬细胞的作用 ,将clodronate用脂质体包裹 ,导入具有吞噬作用的单核 /巨噬细胞内 ,使其在单核/巨噬细胞内浓集 ,并形成具有毒性作用的ATP类似物。结果显示 ,所制备的脂质体直径 2 0 0nm ,表面呈负电荷 (-40mV) ,clodronate包裹率高 (17 6 %～ 19 0 %) ,透射电镜及激光扫描共聚焦显微镜检查均见其大小均匀 ;体外实验证实clodronate溶液、未包裹clodronate脂质体对培养巨噬细胞、血管内皮细胞及血管平滑肌细胞增殖无明显影响 (P >0 0 5 ) ,clodronate脂质体包裹后对内皮细胞、平滑肌细胞增殖仍无影响 ,但明显抑制巨噬细胞增殖 (P <0 0 1) ,激光扫描共聚焦显微镜观察发现 ,罗丹明标记的脂质体与单核细胞及上述细胞孵育 4h后 ,平滑肌细胞内无脂质体 ,而脂质体可快速进入单核细胞 /巨噬细胞内 ,且包裹clodronate的脂质体对巨噬细胞有明显损伤作用。上述结果表明 ,clodronate经脂质体包裹后可选择性清除单核 /巨噬细胞 ,而对非吞噬细胞无明显影响。

Protective effect of clodronate-containing liposomes on intestinal mucosal injury in rats with severe acute pancreatitis

BACKGROUND: Severe acute pancreatitis (SAP) can result in intestinal mucosal injury. This study aimed to demonstrate the protective effect of clodronate-containing liposomes on intestinal mucosal injury in rats with SAP. METHODS: Liposomes containing clodronate or phosphate buffered saline (PBS) were prepared by the thin-film method SAP models were prepared by a uniform injection of sodium taurocholate (2 mL/kg body weight) into the subcapsular space of the pancreas. Sprague-Dawley rats were randomly divided into a control group (C group), a SAP plus PBS-containing liposomes group (P group) and a SAP plus clodronate-containing liposomes group (T group). At 2 and 6 hours after the establishment of SAP models, 2 mL blood samples were taken from the superior mesenteric vein to measure the contents of serum TNF-α and IL-12. Pathological changes in the intestine and pancreas were observed using hematoxylin and eosin staining, while apoptosis was detected using TUNEL staining. In addition, the macrophage markers cluster of differentiation 68 (CD68) in the intestinal tissue was assessed with immunohistochemistry. RESULTS: At the two time points, the levels of TNF-α and IL-12 in the P group were higher than those in the C group (P<0.05) Compared with the P group, the levels of TNF-α and IL-12 decreased in the T group (P<0.05). The pathological scores of the intestinal mucosa and pancreas in the T group were lower than those of the P group. In the T group, large numbers of TUNEL-positive cells were observed, but none or few in the C and P groups. The number of CD68-positive macrophages decreased in the T group.CONCLUSIONS: Clodronate-containing liposomes have prote- ctive effects against intestinal mucosal injury in rats with SAP. The blockade of macrophages may provide a novel therapeutic strategy in SAP.

MRI shows clodronate-liposomes attenuating liver injury in rats with severe acute pancreatitis

BACKGROUND: Studies have revealed that macrophages play an important role in the development of severe acute pancreatitis (SAP). Activated macrophages can lead to a systemic inflammatory response, induce lipid peroxidation, impair membrane structure, result in injury to the liver and the other extrahepatic organs, and eventually result in multiple organ dysfunction syndrome by promoting excessive secretion of cytokines. Liver injury can further aggravate the systemic inflammatory response and increase mortality by affecting the metabolism of toxins and the release of excessive inflammatory mediators. Clodronate is a synthetic bisphosphonate, which is often used for treating bone changes caused by osteoporosis and other factors. In the current study, we created liposomes containing superparamagnetic iron oxide particles (SPIOs) for macrophage labeling and magnetic resonance imaging, using a novel method that can bind the clodronate to induce apoptosis and deplete macrophages. METHODS: Superparamagnetic Fe(3)O(4) nanoparticles were prepared by chemical coprecipitation. SPIO-containing liposomes and SPIO-clodronate-containing liposomes were prepared by the thin film method. SAP models were prepared by injection of sodium taurocholate (2 ml/kg body weight) into the subcapsular space of the pancreas. Sprague-Dawley rats were randomly divided into a control group, a SAP plus SPIO-liposome group, and a SAP plus SPIO-clodronate-containing group. Two and six hours after SAP models were available, T2-weighted MRI scans (in the same plane) of the livers of rats in each group were performed. At the end of the scans, 2 ml of blood was taken from the superior mesenteric vein to measure the levels of serum amylase, ALT, AST, TNF-alpha, and IL-6. Pathological changes in the liver and pancreas were assessed. RESULTS: Transmission electron microscopy showed that the liposomes had a uniform size. No pathological changes in the pancreata of rats in the control group were noted. The pathological changes in the pancreata and livers of rats in the SAP plus SPIO-clodronate-containing liposome group were milder than those in the SAP plus SPIO-liposome group. The MRI signal intensity of the livers in the SAP plus SPIO-liposome and SAP plus SPIO-clodronate-containing groups was significantly lower than that in the control group. There were significant changes in the two experimental groups (P<0.01). In addition, the levels of serum amylase, ALT, AST, TNF-alpha, and IL-6 in rats in the SAP plus SPIO-liposome group were higher than those in the control group (P<0.01), while the corresponding levels in the SPIO-clodronate-containing liposome group were significantly lower than those in the SAP plus SPIO-liposome group (P<0.01). CONCLUSION: Clodronate-containing liposomes protect against liver injury in SAP rats, and SPIO can be used as a tracer for MRI examination following liver injury in SAP rats. (Hepatobiliary Pancreat Dis Int 2010; 9: 192-200)

Clodronate脂质体的制备及诱导重症急性胰腺炎大鼠腹腔巨噬细胞凋亡的实验研究

目的 探讨clodronate脂质体的制备及其对重症急性胰腺炎（severe acute pancreatitis,SAP）大鼠腹腔巨噬细胞（peritoneal Macrophages,PM）凋亡的诱导作用.方法 利用薄膜法制备clodronate脂质体;用5%牛磺胆酸钠逆行胰胆管注射制备SAP模型,分离培养SAP大鼠PM.PM经不同剂量的clodronate脂质体（50、100、150μl）作用后,用MTT法检测其对PM增殖的影响.用Annexin V/PI双标记法和DNA琼脂糖凝胶电泳技术检测PM凋亡情况.结果 制备的脂质体直径200 nm,clodronate包裹率为5.8%.透射电镜检查见其大小均匀.在3种不同剂量clodronate脂质体干预下,MTT结果显示巨噬细胞的生长受到明显抑制（P＜0.01;流式细胞术检测显示,在3种不同剂量clodronate脂质体干预组,PM凋亡百分率分别为（10.32±0.34）%、（18.16±0.49）%和（29.87±0.35）%,差异均有显著性（P＜0.01）.1.2%琼脂糖凝胶电泳显示,随着clodronate脂质体剂量的增高出现渐清晰的特征性梯状条带.结论 clodronate脂质体可诱导SAP大鼠PM的凋亡.

Clodronate-containing liposomes attenuate lung injury in rats with severe acute pancreatitis

Objectives:Severe acute pancreatitis(SAP) can lead to acute lung injury(ALI) .The purpose of this paper is to investigate the protective effect of clodronate-containing liposomes on ALI in rats with SAP.Methods:The thin film method was used to prepare liposomes.Sprague-Dawley rats were randomly divided into three groups.After the SAP model was established by injecting 5%(w/v) sodium taurocholate(2 ml/kg body weight) into the subcapsular space of the pancreata,normal saline was administered to the control(C) group,phosphate buffer solution(PBS) -containing liposome to the Pgroup,and clodronate-containing liposome to the Tgroup through tail veins.Blood samples were obtained from the superior mesenteric vein at 2 and 6 h to measure the levels of amylase,interleukin-6(IL-6) ,and tumor necrosis factor-α(TNF-α) .Morphological changes in the pancreata and lung were observed using hematoxylin and eosin(H&E) staining,while cell apoptosis was detected using terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling(TUNEL) .In addition,the macrophage marker cluster of differentiation 68(CD68) in lung tissue was detected with immunohistochemistry.Results:Blood levels of amylase,IL-6,and TNF-αwere significantly increased in the Pgroup compared to those in the Tgroup(P<0.05) .In the Tgroup,large numbers of TUNEL-positive cells were observed,but no or few in the C and Pgroups.Gross inspection and H&E staining of pancreata and lung showed dramatic tissue damage,including inflammation and necrosis in the Pgroup.Less remarkable changes were noted in the Tgroup,and the C group exhibited normal histology.The histological scores according to Kaiser's criteria were consistent with H&E findings.The number of CD68-positive macrophages decreased in the Tgroup.Conclusions:Clodronate-containing liposomes have a protective effect against ALI in rats with SAP.Blockade of macrophages may represent a novel therapeutic strategy in SAP.

Pre-treatment twice with liposomal clodronate protects against acetaminophen hepatotoxicity through a pre-conditioning effect

Background and aim:Acetaminophen(APAP)overdose is a major cause of acute liver injury,but the role of macrophages in the propagation of the hepatotoxicity is controversial.Early research revealed that macrophage inhibitors protect against APAP injury.However,later work demonstrated that macrophage ablation by acute pre-treatment with liposomal clodronate(LC)exacerbates the toxicity.To our surprise,during other studies,we observed that pre-treatment twice with LC seemed to protect against APAP hepatotoxicity,in contrast to acute pre-treatment.The aim of this study was to confirm that observation and to explore the mechanisms.Methods:We treated mice with empty liposomes(LE)or LC twice per week for 1 week before APAP overdose and collected blood and liver tissue at 0,2,and 6 h post-APAP.We then measured liver injury(serum alanine aminotransferase activity,histology),APAP bioactivation(total glutathione,APAP-protein adducts),oxidative stress(oxidized glutathione(GSSG)),glutamate-cysteine ligase subunit c(Gclc)mRNA,and nuclear factor erythroid 2-related factor(Nrf2)immunofluorescence.We also confirmed the ablation of macrophages by F4/80 immunohistochemistry.Results:Pre-treatment twice with LC dramatically reduced F4/80 staining,protected against liver injury,and reduced oxidative stress at 6 h post-APAP,without affecting APAP bioactivation.Importantly,Gclc mRNA was higher in the LC group at 0 h and total glutathione was higher at 2 h,indicating accelerated glutathione re-synthesis after APAP overdose due to greater basal glutamate-cysteine ligase.Oxidative stress was lower in the LC groups at both time points.Finally,total Nrf2 immunofluorescence was higher in the LC group.Conclusions:We conclude that multiple pre-treatments with LC protect against APAP by accelerating glutathione re-synthesis through glutamate-cysteine ligase.Investigators using twice or possibly more LC pre-treatments to deplete macrophages,including peritoneal macrophages,should be aware of this possible confounder.

辅剂clodronate治疗对减少乳腺癌新转移的作用

据《The New England Journal ofMedicine》1998年339卷第6期报道 二磷酸酯是焦磷酸盐的同功异构体,因其抑制破骨细胞活力,故能有效治疗某些疾病引起的骨吸收增加。在乳腺癌和乳腺癌骨转移患者,二磷酸酯clodronate能减少骨骼并发症的发生。

Macrophage depletion and Schwann cell transplantation reduce cyst size after rat contusive spinal cord injury

Schwann cell transplantation is a promising therapy for the treatment of spinal cord injury（SCI） and is currently in clinical trials.In our continuing efforts to improve Schwann cell transplantation strategies,we sought to determine the combined effects of Schwann cell transplantation with macrophage depletion.Since macrophages are major inflammatory contributors to the acute spinal cord injury,and are the major phagocytic cells,we hypothesized that transplanting Schwann cells after macrophage depletion will improve cell survival and integration with host tissue after SCI.To test this hypothesis,rat models of contusive SCI at thoracic level 8 were randomly subjected to macrophage depletion or not.In rat subjected to macrophage depletion,liposomes filled with clodronate were intraperitoneally injected at 1,3,6,11,and 18 days post injury.Rats not subjected to macrophage depletion were intraperitoneally injected with liposomes filled with phosphate buffered saline.Schwann cells were transplanted 1 week post injury in all rats.Biotinylated dextran amine（BDA） was injected at thoracic level 5 to evalute axon regeneration.The Basso,Beattie,and Bresnahan locomotor test,Gridwalk test,and sensory test using von Frey filaments were performed to assess functional recovery.Immunohistochemistry was used to detect glial fibrillary acidic protein,neurofilament,and green fluorescent protein（GFP）,and also to visulize BDA-labelled axons.The GFP labeled Schwann cell and cyst and lesion volumes were quantified using stained slides.The numbers of BDA-positive axons were also quantified.At 8 weeks after Schwann cell transplantation,there was a significant reduction in cyst and lesion volumes in the combined treatment group compared to Schwann cell transplantation alone.These changes were not associated,however,with improved Schwann cell survival,axon growth,or locomotor recovery.Although combining Schwann cell transplantation with macrophage depletion does improve histopathology of the injury site,the effect on axon growth and behavioral recovery appears no better than what can be achieved with Schwann cell transplants alone.

Are gastric mucosal macrophages responsible for gastric injury in acute pancreatitis?

AIM:To investigate the protective effect of clodronatecontaining liposomes against severe acute pancreatitis(SAP)-triggered acute gastric mucosal injury(AGMI) in rats.METHODS:Clodronate- and phosphate-buffered saline(PBS)-containing liposomes were prepared by reverse-phase evaporation.The SAP rat model was established by injecting sodium taurocholate into the pancreatic subcapsular space.Sprague-Dawley rats were randomly divided into three groups:control(C),SAP plus PBS-containing liposome(P) and SAP plus clodronate-containing liposome(T).Serum tumor necrosis factor(TNF)-α levels were estimated by ELISA.Pathological changes in the gastric mucosa and pancreas were observed by hematoxylin and eosin(HE) staining.Apoptotic cells were detected by terminal deoxynucleotidyl transferase d UTP nick end labeling staining.The numbers of macrophages in the gastric mucosa were analyzed by CD68 immunohistochemical staining.RESULTS:The liposomes had a mean diameter of 150 ± 30 nm.The TNF-α levels were significantly higher in the P group than that in the C group(2 h,145.13 ± 11.50 vs 23.2 ± 2.03; 6 h,245.06 ± 12.11 vs 30.28 ± 6.07,P < 0.05),and they were significantly lower in the T group than that in the P group(2 h,93.24 ± 23.11 vs 145.13 ± 11.50; 6 h,135.18 ± 13.10 vs 245.06 ± 12.11,P < 0.05).The pathological scores of the pancreas were lower in the T group than in the P group(2 h,1.88 ± 0.83 vs 4.13 ± 0.83; 6 h,2.87 ± 0.64 vs 6.25 ± 0.88,P < 0.01).The pathological scores of the gastric mucosa were also lower in the T group than in the P group(2 h,1.12 ± 0.64 vs 2 ± 0.75; 6 h,1.58 ± 0.53 vs 3 ± 1.31,P < 0.05).In addition,increased CD68 levels were observed in the gastric mucosa of the P group compared with the C group.Clodronate-containing liposomes decreased the CD68 levels in the mucosa of the T group.The apoptotic indexes of the gastric mucosa were higher in the T group than in the P group(2 h,15.7 ± 0.92 vs 11.5 ± 1.64; 6 h,21.12 ± 1.06 vs 12.6 ± 2.44,P < 0.01).CONCLUSION:Gastric macrophages contribute to the pathogenesis of gastric injury in SAP.Clodronatecontaining liposomes have protective effects against AGMI in rats with SAP.

Don’t know what you got till it’s gone:microglial depletion and neurodegeneration

In the central nervous system,immunologic surveillance and response are carried out,in large part,by microglia.These resident macrophages derive from myeloid precursors in the embryonic yolk sac,migrating to the brain and eventually populating local tissue prior to blood-brain barrier formation.Preserved for the duration of lifespan,microglia serve the host as more than just a central arm of innate immunity,also contributing significantly to the development and maintenance of neurons and neural networks,as well as neuroregeneration.The critical nature of these varied functions makes the characterization of key roles played by microglia in neurodegenerative disorders,especially Alzheimer’s disease,of paramount importance.While genetic models and rudimentary pharmacologic approaches for microglial manipulation have greatly improved our understanding of central nervous system health and disease,significant advances in the selective and near complete in vitro and in vivo depletion of microglia for neuroscience application continue to push the boundaries of research.Here we discuss the research efficacy and utility of various microglial depletion strategies,including the highly effective CSF1R inhibitor models,noteworthy insights into the relationship between microglia and neurodegeneration,and the potential for therapeutic repurposing of microglial depletion and repopulation.

STUDYING THE ROLE OF MACROPHAGES IN CIRCULATING PROSTATE CANCER CELLS BY IN VIVO FLOW CYTOMETRY

Metastasis is a very complicated multi-step process and accounts for the low survival rate of the cancerous patients.To metastasize,t he malignant cells must detach from the primary tumor and migrate to secondary sites in the body through either blood or lymph circulation.Macrophages appear to be directly involved in tumor progression and metastasis.However,the role of macrophages in affecting cancer metast asis has not been fully elucidated.Here,we have utilized an emerging technique,namely in vivo flow cytometry(IVFC)to study the depletion kinetics of circulating prostate cancer cells in mice and determine how depletion of macrophages by the liposome encapsulated clodronate affects the depletion kinetics.Our results show diferent depletion kinetics of PC-3 cells between the macrophagedeficient group and the control group.The number of circulating tumor cells(CTCs)in the macrophage-deficient group decreases in a slower manner compared to the control mice group.The differences in depletion kinetics indicate that the absence of macrophages facilitates the stay of prostate cancer cells in circulation.In addition,our imaging data suggest that macrophages might be able to arrest,phagocytose and digest PC-3 cells.Therefore,phagocy tosis may mainly contribute to the de-pletion kinetic diferences.The developed methods elaborated here would be useful to study the relationship between macr ophages and tumor metastasis in small animal cancer models.

Monocyte-derived fibrocytes elimination had little contribution on liver fibrosis

Background:Monocyte-derived fibrocytes play an important role in the progression of fibrosis in the skin,lungs,heart and kidney.However,the contribution of fibrocytes to liver fibrosis is unclear.The aim of this study was to investigate whether fibrocytes contributed to fibrosis progression in the livers of carbon tetrachloride(CCl 4)-treated mice.Methods:C57BL/6J mice were divided into 4 groups:normal control group,CCl 4-treated group,CCl 4+control liposome-treated group,and CCl 4+clodronate liposome-treated group.For the elimination of systemic monocyte and monocyte-derived fibrocyte,one group was treated with clodronate liposome,and another group with control liposome as a control.After 4 weeks of treatment,hepatic mononuclear cells were subjected to immunofluorescent(IF)staining and fluorescence-activated cell sorter(FACS)analysis to detect fibrocytes.Measurement of collagen-positive Sirius red stained area and collagen-I mRNA expression in the liver were performed to evaluate the degree of liver fibrosis quantitatively.Results:In the liver of the CCl 4-treated and CCl 4+control liposome-treated groups,the number of fibrocytes,the area positive for Sirius red staining and collagen-I mRNA expression significantly increased compared with those in the normal control group.In the liver of the CCl 4+clodronate liposome-treated group,few fibrocytes was observed as in the normal control group,but Sirius red staining positive area and collagen-I mRNA expression were increased and equivalent to the CCl 4-treated and CCl 4+control liposome-treated groups.Conclusion:Monocyte-derived fibrocytes play a minimal role in CCl 4-induced liver fibrosis.Cells other than fibrocytes such as hepatic stellate cells play a central role in liver fibrosis.