Global MicroRNA Expression Profiling of Mouse Livers following Ischemia-Reperfusion Injury at Different Stages

Hepatic ischemia-reperfusion injury is a dynamic process consisting of two stages: ischemia and reperfusion, and triggers a cascade of physiological and biochemical events. Given the important role of microRNAs in regulating gene expression, we analyzed gene expression changes in mouse livers at sham control, ischemia stage, and reperfusion stage. We generated global expression profiles of microRNA and mRNA genes in mouse livers subjected to ischemia-reperfusion injury at the three stages, respectively. Comparison analysis showed that reperfusion injury had a distinct expression profile whereas the ischemia sample and the sham control were clustered together. Consistently, there are 69 differentially expressed microRNAs between the reperfusion sample and the sham control whereas 28 differentially expressed microRNAs between the ischemia sample and the sham control. We further identified two modes of microRNA expression changes in ischemia-reperfusion injury. Functional analysis of both the differentially expressed microRNAs in the two modes and their target mRNAs revealed that ischemia injury impaired mitochondrial function, nutrient consumption, and metabolism process. In contrast, reperfusion injury led to severe tissue inflammation that is predominantly an innate-immune response in the ischemia-reperfusion process. Our staged analysis of gene expression profiles provides new insights into regulatory mechanisms of microRNAs in mouse hepatic IR injury.     

Neuroprotection of exercise: P2X4R and P2X7R regulate BDNF actions

The neurotrophin brain-derived neurotrophic factor (BDNF), which acts as a transducer, is responsible for improving cerebral stroke, neuropathic pain, and depression. Exercise can alter extracellular nucleotide levels and purinergic receptors in central nervous system (CNS) structures. This inevitably activates or inhibits the expression of BDNF via purinergic receptors, particularly the P2X receptor (P2XR), to alleviate pathological progression. In addition, the significant involvement of sensitive P2X4R in mediating increased BDNF and p38-MAPK for intracerebral hemorrhage and pain hypersensitivity has been reported. Moreover, archetypal P2X7R blockade induces mouse antidepressant-like behavior and analgesia by BDNF release. This review summarizes BDNF-mediated neural effects via purinergic receptors, speculates that P2X4R and P2X7R could be priming molecules in exercise-mediated changes in BDNF, and provides strategies for the protective mechanism of exercise in neurogenic disease.     

高压氧预处理对急性减压所致的大鼠肺组织细胞凋亡及Bcl-2/Bax表达的影响

目的:探讨高压氧预处理对减压病大鼠肺组织细胞凋亡的影响相关蛋白表达的影响。方法:雄性SD大鼠24只,随机分为3组,正常对照组(NC group)、HBO预处理组(HBOP group)、减压组(DCS group),每组8只。连续进行HBO预处理5天后进行减压病模型制备,取左侧肺组织进行湿干重比值测定,右侧肺组织用于病理实验;HE染色观察肺组织病理学改变,免疫组织化学法标记Bcl-2、Bax、Caspase-3与MMP-9阳性细胞表达,并对bcl-2/bax值进行分析。结果:减压组肺组织Bax、Caspase-3与MMP-9阳性细胞数明显增加(P0.05),而Bcl-2阳性细胞表达减少(P0.05);高压氧预处理组与减压组相比,Bax、Caspase-3与MMP-9阳性细胞数明显减少(P0.05),而Bcl-2阳性细胞表达增加(P0.05);大鼠肺组织减压组与高压氧预处理组Bcl-2/Bax值较对照组明显降低(P0.05);与减压组相比,高压氧预处理组明显升高(P0.05)。结论:HBO预处理可以减轻减压对肺组织的病理损伤,减轻肺泡和支气管上皮细胞的变性坏死,抑制细胞凋亡,从而起到对减压病的保护作用。     

Cysteine substitution mutagenesis and the effects of methanethiosulfonate reagents at P2X2 and P2X4 receptors support a core common mode of ATP action at P2X receptors

The agonist binding site of ATP-gated P2X receptors is distinct from other ATP-binding proteins. Mutagenesis on P2X(1) receptors of conserved residues in mammalian P2X receptors has established the paradigm that three lysine residues, as well as FT and NFR motifs, play an important role in mediating ATP action. In this study we have determined whether cysteine substitution mutations of equivalent residues in P2X(2) and P2X(4) receptors have similar effects and if these mutant receptors can be regulated by charged methanethiosulfonate (MTS) compounds. All the mutants (except the P2X(2) K69C and K71C that were expressed, but non-functional) showed a significant decrease in ATP potency, with >300-fold decreases for mutants of the conserved asparagine, arginine, and lysine residues close to the end of the extracellular loop. MTS reagents had no effect at the phenylalanine of the FT motif, in contrast, cysteine mutation of the threonine was sensitive to MTS reagents and suggested a role of this residue in ATP action. The lysine-substituted receptors were sensitive to the charge of the MTS reagent consistent with the importance of positive charge at this position for coordination of the negatively charged phosphate of ATP. At the NFR motif the asparagine and arginine residues were sensitive to MTS reagents, whereas the phenylalanine was either unaffected or showed only a small decrease. These results support a common site of ATP action at P2X receptors and suggest that non-conserved residues also play a regulatory role in agonist action.     

Combinations of TLR and NOD2 ligands stimulate rat microglial P2X4R expression

As ATP-gated ion channels, P2X4 receptors (P2X4R) of microglial cells play a crucial role in central nervous system (CNS) inflammation. In this study, we used rat microglial cell cultures to examine P2X4R expression in response to stimulation by combination of toll-like receptors (TLRs) and nucleotide-binding oligomerization domain 2 (NOD2) receptors. Various TLR1-9 ligands and NOD2 agonist muramyldipeptide (MDP) were investigated. Our results showed that certain combination of ligands had additive effects on upregulating microglial P2X4R at both mRNA and protein levels, and induced nitric oxide increase and tumor necrosis factor-α production. Thus TLRs and NOD2 combinations are contributors to the signaling cascades resulting in purinergic microglial activation.     

慢性溴氰菊酯摄取对大鼠胃黏膜损伤及P物质的表达的影响

目的:探索溴氰菊酯慢性中毒对大鼠胃黏膜的病理损伤及损伤后P物质表达的影响.方法:实验组大鼠分别给予不同荆量溴氰菊酯灌胃,对照组仅给予橄榄油.取胃体部组织,制成石蜡切片,HE及免疫组织化学染色,镜下观察胃黏膜损伤及胃黏膜组织P物质的表达情况,并对损伤程度进行累计积分、壁细胞计数.结果:高剂量组大鼠胃粘膜慢性损害病理积分与各组相比差异均有显著性(P＜0.05);壁细胞计数与对照组相比较其差异有P＜0.05).免疫组化发现SP在实验组大鼠胃黏膜组织中呈强阳性表达,在对照组则呈弱阳性表达;平均光密度(OD)值显示高、中剂量组之间及与其余各组之间差异均有显著(P＜0.05l.结论:溴氰菊酯对大鼠胃黏膜有较明显的慢性损伤,SP可能参与胃黏膜局部病变.     

超早期高压氧对脊髓完全横断损伤大鼠血液生化及后肢运动功能的影响

目的探讨超早期高压氧（HBO）治疗对脊髓完全横断损伤模型血液生化及后肢运动功能的影响。方法 55只SD大鼠随机分为A组（假手术组,15只）、B组（模型组,20只）及C组（高压氧组,20只）,A组仅行椎板切除术,其余2组均行T10椎板水平脊髓完全横向切断术。B、C组均予常规护理,C组于术后3 h置于动物舱内开始高压氧治疗,10 d一疗程,共3疗程。分别于建立模型后第1～6周末,用BBB运动功能评分法评价并比较两组大鼠后肢运动功能恢复程度,术后第6周过量麻醉处死大鼠,以40 g/L多聚甲醛行心室-主动脉灌注,取脊髓损伤区标本,光镜观察损伤脊髓的组织病理学改变。检测血钙（Ca）、血磷（P）、血清碱性磷酸酶（ALP）改变情况。结果 B、C两组大鼠术后第1～6周BBB运动功能评分逐渐增高,C组在3～6周末的BBB运动功能评分均明显高于B组。B组、C组血钙、血磷在术后1、3周高于A组,血清碱性磷酸酶（ALP）术后1、3周低于A组;C组血钙、血磷在术后5、6周低于B组。病理组织切片观察C组较B组组织水肿减轻,炎性细胞浸润减轻。结论超早期高压氧治疗能促进脊髓完全横断损伤大鼠后肢运动功能的部分恢复,降低血钙、血磷含量,对脊髓完全横断损伤大鼠具有保护和治疗作用。     

不同时期喷施NaHS对盐碱胁迫下裸燕麦H2S产生和活性氧代谢的影响

为了解气体信号硫化氢（H₂S）对盐碱胁迫下裸燕麦（Avena nuda）活性氧（ROS）代谢的调节效应,筛选和确定H₂S最佳的施用时期和适宜浓度。采用盆栽土培试验,研究了在裸燕麦不同时期（幼苗期、拔节期、抽穗期、开花期和灌浆期）喷施0、25、50、100、200、400 μmol·L^-1 H₂S供体硫氢化钠（NaHS）对3.0 g·kg^-1盐碱混合 （NaCl∶Na₂SO₄∶NaHCO₃∶Na₂CO₃摩尔比为12∶8∶9∶1）胁迫下裸燕麦叶片H₂S含量、H₂S生成关键酶L-半胱氨酸脱巯基酶（LCD）活性和ROS代谢相关物质含量和酶活性的影响。结果表明：喷施时期和NaHS浓度及其交互作用对盐碱胁迫下裸燕麦叶片中H₂S、超氧阴离子（）、过氧化氢（H₂O₂）、丙二醛（MDA）、抗坏血酸（AsA）和谷胱甘肽（GSH）含量及LCD、超氧化物歧化酶（SOD）、过氧化氢酶（CAT）、过氧化物酶（POD）、抗坏血酸过氧化物酶（APX）和谷胱甘肽还原酶（GR）活性存在显著影响。与喷施0 μmol?L^-1 NaHS相比,喷施一定浓度NaHS能够提高H₂S、AsA、GSH含量和LCD、SOD、CAT、POD、APX和GR活性,减少、H₂O₂和MDA积累,但以上各指标最佳的喷施时期和NaHS浓度存在差异。隶属函数综合分析显示,在幼苗期和拔节期喷施25~200 μmol?L^-1 NaHS的综合评价值（D）最高,表明在幼苗—拔节期喷施25~200 μmol?L^-1 NaHS能更好提高ROS清除能力,从而缓解盐碱胁迫诱导ROS对裸燕麦的氧化伤害。     

不同年龄白发性高血压大鼠肾脏AT1R和AT2R的表达

目的本研究观察不同月龄自发性高血压大鼠（SHR）肾脏ALR和ALR表达,初步探讨AT1R和AT2R在高血压发生、发展过程中的可能作用。方法1月龄组（S1）、2月龄组（S2）、3月龄组（S3）、6月龄组（S6）和9月龄组（墨）雄性SHR共5组,每组各6只,各组均有相应月龄匹配的Wistar-Kyoto大鼠（WKY）作对照。采用RBP-I型大鼠血压心率测定仪测量大鼠尾动脉收缩压（SBP）;放免法测定血浆血管紧张素Ⅱ（AngⅡ）;免疫组化染色结合计算机图像分析方法测定肾脏AT1R和ALR表达水平。结果（1）SHR SBP随着月龄的增加而上升,S6后趋于稳定。（2）1个月后SHR血浆AngⅡ浓度均高于S1（P〈0.05）,而S2、S3、S6和S9之间无明显差别（P〉0.05）;1个月后SHR血浆AngⅡ浓度均高于相应配对的WKY组（P〈0.05）;而WKY各月龄组均无明显差别（P〉0.05）。（3）SHR肾脏AT1R随着月份的增加而增加（P〈0.05）,且高于相应配对的WKY组（P〈0.05）。SHR肾脏ABR随着月份的增加而降低,S6明显降低（P〈0.05）,S6和S9比较无明显差别（P〉0.05）;且均低于相应配对的WKY组（P〈0.05）。WKY各月龄组AT1R和AT2R无明显差别（P〉0.05）。结论SHR肾脏AT1R表达水平比WKY高,并随着年龄的增加而递增;AT2R表达水平比WKY低,并随着年龄的增加而降低。     

Expression, purification, electron microscopy, N-glycosylation mutagenesis and molecular modeling of human P2X4 and Dictyostelium discoideum P2XA

The recent publication of the apo-, closed-state 3D crystal structure of zebrafish (zf) P2X4.1 has not only revolutionized the P2X research field, but also highlighted the need for further crystal structures, of receptors in different activation states, so that we can gain a complete molecular understanding of ion channel function. zfP2X4.1 was selected as a 3D-crystallization candidate because of its ability to form stable trimers in detergent solution, and purified from over-expression in baculovirus-infected Spodoptera frugiperda (Sf9) insect cells. In this work, we have used a similar approach to express both human P2X4 (hP2X4) and Dictyostelium discoideum P2XA (DdP2XA) in Sf9 cells. Although hP2X4 did not form stable trimers in detergent solution, both receptors bound to ATP-coupled resins, indicating that their extracellular domains were folded correctly. DdP2XA formed strong trimers in detergent solution, and we were able to selectively purify trimers using preparative electrophoresis, and build a 21?-resolution 3D structure using transmission electron microscopy and single particle analysis. Although the structure of DdP2XA possessed similar dimensions to those of the previously determined low-resolution hP2X4 structure and the zfP2X4.1 crystal structure, N-glycosylation mutagenesis and molecular modeling indicated differences between N-glycan usage and predicted accessibility in models of DdP2XA based on the zfP2X4.1 crystal structure. Our data demonstrate that DdP2XA expressed in insect cells retains ATP-binding capacity after detergent solubilization, is an ideal candidate for structural study, and possesses a significantly different 3D structure to that of both hP2X4 and zfP2X4.1.     

Expression, regulation, and function of P2X4 purinergic receptor in human cervical epithelial cells

Micromolarconcentrations of ATP stimulate biphasic change in transepithelialconductance across CaSki cultures on filters, an acute transientincrease (phase I response; triggered by P2Y₂ receptor and mediated by calcium mobilization-dependent cell volume decrease) followed by a slower decrease in permeability (phase II response). Phase II response is mediated byaugmented calcium influx and protein kinase C-dependent increase intight junctional resistance. The objective of the study was todetermine the role of P2X₄ receptor as a mediator ofphase II response. Human cervical epithelial cells expressP2X₄ receptor mRNA (1.4-, 2.2-, and 4.4-kb isoforms byNorthern blot analysis) and P2X₄ protein. Depletion ofvitamin A reversibly downregulated P2X₄ receptor mRNA andprotein and ATP-induced calcium influx. Depletion of vitamin Aabrogated phase II response, and the effect could bepartially reversed only with retinoic acid receptor (RAR)-selectiveretinoids but not retinoid X receptor (RXR) agonists. Depletion ofvitamin A also abrogated protein kinase C increase in tight junctionalresistance, and the effect could not be reversed with retinoids.Depletion of vitamin A also abrogated phase I increase inpermeability and reversibly downregulated P2Y₂ receptormRNA and ATP-induced calcium mobilization. However, in contrast tophase II response, both RAR and RXR agonists could fullyreverse those effects. These results suggest that phase IIresponse is mediated by a P2X₄ receptor mechanism.

     

不同剂量法舒地尔对压力负荷心力衰竭大鼠心肌细胞凋亡和凋亡相关基因表达的影响

目的从细胞凋亡角度探讨不同剂量法舒地尔(fasudil)对升主动脉缩窄压力超负荷心力衰竭大鼠的影响及作用机制。方法采用升主动脉缩窄术建立大鼠心力衰竭模型。观察不同剂量fasudil治疗心力衰竭时对心肌细胞凋亡指数(AI)、bcl-2、c-myc蛋白表达水平的影响。结果fasudil干预心功能不全可以使心肌细胞凋亡指数降低,使bcl-2蛋白表达水平上调,c-myc蛋白表达水平降低,且剂量大时效果更明显。结论fasudil能有效减少心力衰竭大鼠心肌细胞凋亡指数,使bcl-2蛋白表达水平上调,c-myc蛋白表达水平降低,防治心力衰竭,这是其治疗心力衰竭的重要机制之一,且剂量大时更明显。     

P2X3 Receptors Mediate Visceral Hypersensitivity during Acute Chemically-Induced Colitis and in the Post-Inflammatory Phase via Different Mechanisms of Sensitization

Objectives

Experiments using P2X₃ knock-out mice or more general P2X receptor antagonists suggest that P2X₃ receptors contribute to visceral hypersensitivity. We aimed to investigate the effect of the selective P2X₃ antagonist A-317491 on visceral sensitivity under physiological conditions, during acute colitis and in the post-inflammatory phase of colitis.

Methods

Trinitrobenzene sulphonic-acid colitis was monitored by colonoscopy: on day 3 to confirm the presence of colitis and then every 4 days, starting from day 10, to monitor convalescence and determine the exact timepoint of endoscopic healing in each rat. Visceral sensitivity was assessed by quantifying visceromotor responses to colorectal distension in controls, rats with acute colitis and post-colitis rats. A-317491 was administered 30 min prior to visceral sensitivity testing. Expression of P2X₃ receptors (RT-PCR and immunohistochemistry) and the intracellular signalling molecules cdk5, csk and CASK (RT-PCR) were quantified in colonic tissue and dorsal root ganglia. ATP release in response to colorectal distension was measured by luminiscence.

Results

Rats with acute TNBS-colitis displayed significant visceral hypersensitivity that was dose-dependently, but not fully, reversed by A-317491. Hypersenstivity was accompanied by an increased colonic release of ATP. Post-colitis rats also displayed visceral hypersensitivity that was dose-dependently reduced and fully normalized by A-317491 without increased release of ATP. A-317491 did not modify visceral sensitivity in controls. P2X₃ mRNA and protein expression in the colon and dorsal root ganglia were similar in control, acute colitis and post-colitis groups, while colonic mRNA expression of cdk5, csk and CASK was increased in the post-colitis group only.

Conclusions

These findings indicate that P2X₃ receptors are not involved in sensory signaling under physiological conditions whereas they modulate visceral hypersensitivity during acute TNBS-colitis and even more so in the post-inflammatory phase, albeit via different mechanisms of sensitization, validating P2X₃ receptors as potential new targets in the treatment of abdominal pain syndromes.     

CXCR4 Chemokine Receptor Signaling Induces Apoptosis in Acute Myeloid Leukemia Cells via Regulation of the Bcl-2 Family Members Bcl-XL,Noxa, and Bak

The CXCR4 chemokine receptor promotes survival of many different cell types. Here, we describe a previously unsuspected role for CXCR4 as a potent inducer of apoptosis in acute myeloid leukemia (AML) cell lines and a subset of clinical AML samples. We show that SDF-1, the sole ligand for CXCR4, induces the expected migration and ERK activation in the KG1a AML cell line transiently overexpressing CXCR4, but ERK activation did not lead to survival. Instead, SDF-1 treatment led via a CXCR4-dependent mechanism to apoptosis, as evidenced by increased annexin V staining, condensation of chromatin, and cleavage of both procaspase-3 and PARP. This SDF-1-induced death pathway was partially inhibited by hypoxia, which is often found in the bone marrow of AML patients. SDF-1-induced apoptosis was inhibited by dominant negative procaspase-9 but not by inhibition of caspase-8 activation, implicating the intrinsic apoptotic pathway. Further analysis showed that this pathway was activated by multiple mechanisms, including up-regulation of Bak at the level of mRNA and protein, stabilization of the Bak activator Noxa, and down-regulation of antiapoptotic Bcl-X_L. Furthermore, adjusting expression levels of Bak, Bcl-X_L, or Noxa individually altered the level of apoptosis in AML cells, suggesting that the combined modulation of these family members by SDF-1 coordinates their interplay to produce apoptosis. Thus, rather than mediating survival, SDF-1 may be a means to induce apoptosis of CXCR4-expressing AML cells directly in the SDF-1-rich bone marrow microenvironment if the survival cues of the bone marrow are disrupted.     

Effects of Acupuncture at GV20 and ST36 on the Expression of Matrix Metalloproteinase 2, Aquaporin 4, and Aquaporin 9 in Rats Subjected to Cerebral Ischemia/Reperfusion Injury

Background/Purpose

Ischemic stroke is characterized by high morbidity and mortality worldwide. Matrix metalloproteinase 2 (MMP2), aquaporin (AQP) 4, and AQP9 are linked to permeabilization of the blood-brain barrier (BBB) in cerebral ischemia/reperfusion injury (CIRI). BBB disruption, tissue inflammation, and MMP/AQP upregulation jointly provoke brain edema/swelling after CIRI, while acupuncture and electroacupuncture can alleviate CIRI symptoms. This study evaluated the hypothesis that acupuncture and electroacupuncture can similarly exert neuroprotective actions in a rat model of middle cerebral artery occlusion (MCAO) by modulating MMP2/AQP4/APQ9 expression and inflammatory cell infiltration.

Methods

Eighty 8-week-old Sprague-Dawley rats were randomly divided into sham group S, MCAO model group M, acupuncture group A, electroacupuncture group EA, and edaravone group ED. The MCAO model was established by placement of a suture to block the middle carotid artery, and reperfusion was triggered by suture removal in all groups except group S. Acupuncture and electroacupuncture were administered at acupoints GV20 (governing vessel-20) and ST36 (stomach-36). Rats in groups A, EA, and ED received acupuncture, electroacupuncture, or edaravone, respectively, immediately after MCAO. Neurological function (assessed using the Modified Neurological Severity Score), infarct volume, MMP2/AQP4/AQP9 mRNA and protein expression, and inflammatory cell infiltration were all evaluated at 24 h post-reperfusion.

Results

Acupuncture and electroacupuncture significantly decreased infarct size and improved neurological function. Furthermore, target mRNA and protein levels and inflammatory cell infiltration were significantly reduced in groups A, EA, and ED vs. group M. However, MMP2/AQP levels and inflammatory cell infiltration were generally higher in groups A and EA than in group ED except MMP2 mRNA levels.

Conclusions

Acupuncture and electroacupuncture at GV20 and ST36 both exercised neuroprotective actions in a rat model of MCAO, with no clear differences between groups A and EA. Therefore, acupuncture and electroacupuncture might find utility as adjunctive and complementary treatments to supplement conventional therapy for ischemic stroke.