A protease-activated receptor 1 antagonist protects against global cerebral ischemia/reperfusion injury after asphyxial cardiac arrest in rabbits

Cerebral ischemia/reperfusion injury is partially mediated by thrombin, which causes brain damage through protease-activated receptor 1（PAR1）. However, the role and mechanisms underlying the effects of PAR1 activation require further elucidation. Therefore, the present study investigated the effects of the PAR1 antagonist SCH79797 in a rabbit model of global cerebral ischemia induced by cardiac arrest. SCH79797 was intravenously administered 10 minutes after the model was established. Forty-eight hours later, compared with those administered saline, rabbits receiving SCH79797 showed markedly decreased neuronal damage as assessed by serum neuron specific enolase levels and less neurological dysfunction as determined using cerebral performance category scores. Additionally, in the hippocampus, cell apoptosis, polymorphonuclear cell infiltration, and c-Jun levels were decreased, whereas extracellular signal-regulated kinase phosphorylation levels were increased. All of these changes were inhibited by the intravenous administration of the phosphoinositide 3-kinase/Akt pathway inhibitor LY29004（3 mg/kg） 10 minutes before the SCH79797 intervention. These findings suggest that SCH79797 mitigates brain injury via anti-inflammatory and anti-apoptotic effects, possibly by modulating the extracellular signal-regulated kinase, c-Jun N-terminal kinase/c-Jun and phosphoinositide 3-kinase/Akt pathways.

Protease-activated receptors in neuropathic pain:an important mediator between neuron and glia

Chronic neuropathic pain is a refractory symptom in clinical practice due to nervous injury or inflammation, and affects millions of people all over the world. Although the neuronal functioning of pain pathways has been studied for many years, the induction and maintenance of this non-adaptive, pathological pain is still poorly understood. Recent evidence indicates that protease-activated receptors (PARs) participate in the initiation and maintenance of neuropathic pain and play a key role in mediating the interactions of nerve cells. Firstly, following nerve injury, alterations in neuron and neuron function induce an abnormal increase of some neurotransmitters and neuromodulators, such as substance P (SP), calcitonin gene-related peptide (CGRP), prostaglandins, kinins, and so on. Such abnormal factors can act on neuron reversely and then induce pain sensation directly, or activate glial cells (astrocytes and microglia) mediated by PARs, which trigger and accelerate the progress of neuropathic pain. Secondly, when the noxious factors invade, glial cells are activated as the first barrier of nervous system and secret many neuroinflammatory factors. These inflammatory factors have effects on PARs (especially PAR1 and PAR2) in the neurons around, and then aggravate the status of pain. Thirdly, in the progress of neuroinflammatory pain, microglia is activated first and initiates the status of pain, and then inflammatory factors and complements from microglia activate astrocytes and maintain or make the pain worse. All of these actions is protective to neurons first, but then turns to a kind of nociception and forms the feeling of pain under the continuous noxious stimuli. Conclusively, PARs may play an important role in the formation and maintenance of chronic pain through mediating the interactions among nerve cells, which may be a novel target in the study and control of neuropathic pain. This article focuses on recent developments of PARs in the progress of neuropathic pain, and provides a framework for addressing the major questions for the future.

Antagonism of Protease-Activated Receptor 4 Protects Against Traumatic Brain Injury by Suppressing Neuroinflammation via Inhibition of Tab2/NF-κB Signaling

Traumatic brain injury(TBI)triggers the activation of the endogenous coagulation mechanism,and a large amount of thrombin is released to curb uncontrollable bleeding through thrombin receptors,also known as protease-activated receptors(PARs).However,thrombin is one of the most critical factors in secondary brain injury.Thus,the PARs may be effective targets against hemorrhagic brain injury.Since the PAR1 antagonist has an increased bleeding risk in clinical practice,PAR4 blockade has been suggested as a more promising treatment.Here,we explored the expression pattern of PAR4 in the brain of mice after TBI,and explored the effect and possible mechanism of BMS-986120(BMS),a novel selective and reversible PAR4 antagonist on secondary brain injury.Treatment with BMS protected against TBI in mice.mRNA-seq analysis,Western blot,and qRT-PCR verification in vitro showed that BMS significantly inhibited thrombin-induced inflammation in astrocytes,and suggested that the Tab2/ERK/NF-κB signaling pathway plays a key role in this process.Our findings provide reliable evidence that blocking PAR4 is a safe and effective intervention for TBI,and suggest that BMS has a potential clinical application in the management of TBI.

Baicalin Attenuates Focal Cerebral Ischemic Reperfusion Injury by Inhibition of Protease-Activated Receptor-1 and Apoptosis

Objective： To investigate the neuro-protective effects of baicaiin in Wistar rats with focal cerebral ischemic reperfusion injury. Methods： Ninety adult male Wistar rats weighing 320-350 g were randomly divided into the following groups （n=5）： （a） sham control group; （b） vehicle group, subjected to middle cerebral artery occlusion and received vehicle intraperitoneally; （c-e） baicalin groups, which were subjected to the middle cerebral artery occlusion and treated with baicalin 25, 50 and 100 mg/kg, respectively. The neurological scores were determined at postoperative 1, 3 and 7 d after the treatment. The expression of protease-activated receptor-1 （PAR-1）, PAR-1 mRNA and Caspase-3 were determined using Western blot, reverse transcription polymerase chain reaction （RT- PCR） analysis and immunohistochemistry, respectively. Results： Significant decrease was noted in the neurological score in the baicalin group compared with that of the vehicle group （P〈0.01）. Additionally, down-regulation of PAR-1 mRNA, PAR-1 and Caspase-3 was observed in the baicalin groups compared with those obtained from the vehicle group （P〈0.01）. Compared with the low-dose baicalin group （25 mg/kg）, remarkable decrease was noted in neurological score, and the expression of PAR-1 mRNA, PAR-1 as well as Caspase-3 in the high-dose group （P〈0.05）. Conclusion： Baicalin showed neuro-protective effects in focal cerebral ischemic reperfusion injury through inhibiting the expression of PAR-1 and apoptosis.

Expression of protease-activated receptors on platelets in healthy individuals

This study aimed to investigate the expression of protease-activated receptors(PARs)on platelets in healthy individuals and preliminarily elucidate physiolo-gical functions of PARs.Thirty healthy volunteers,who did not take any anticoagulants and antiplatelet agents within 10 days before the examination,were recruited.Fasting venous blood(5 mL)was taken from the medial cubital vein in each individual and platelet-rich plasma(PRP)was prepared.The expression of PAR1 mRNA and PAR4 mRNA in PRP was determined by RT-PCR analysis.The results showed that the average levels of PAR1 mRNA and PAR4 mRNA on platelets in healthy individuals were 0.1601�0.0269 and 0.1073�0.0194 respectively.In combination with literature analysis,it was concluded that the thrombin signaling pathway plays a vital role in the development of hemostasis and thrombosis,and the selective PARs antagonist has the potential for extensive application in clinical practice.

GW4064, a farnesoid X receptor agonist, upregulates adipokine expression in preadipocytes and HepG2 cells

AIM: To investigate the effect of GW4064 on the expression of adipokines and their receptors during differentiation of 3T3-L1 preadipocytes and in HepG2 cells.

Argatroban promotes recovery of spinal cord injury by inhibiting the PAR1/JAK2/STAT3 signaling pathway

Argatroban is a synthetic thrombin inhibitor approved by U.S.Food and Drug Administration for the treatment of thrombosis.However,whether it plays a role in the repair of spinal cord injury is unknown.In this study,we established a rat model of T10 moderate spinal cord injury using an NYU Impactor ModerⅢand performed intraperitoneal injection of argatroban for 3 consecutive days.Our results showed that argatroban effectively promoted neurological function recovery after spinal cord injury and decreased thrombin expression and activity in the local injured spinal cord.RNA sequencing transcriptomic analysis revealed that the differentially expressed genes in the argatroban-treated group were enriched in the JAK2/STAT3 pathway,which is involved in astrogliosis and glial scar formation.Western blotting and immunofluorescence results showed that argatroban downregulated the expression of the thrombin receptor PAR1 in the injured spinal cord and the JAK2/STAT3 signal pathway.Argatroban also inhibited the activation and proliferation of astrocytes and reduced glial scar formation in the spinal cord.Taken together,these findings suggest that argatroban may inhibit astrogliosis by inhibiting the thrombin-mediated PAR1/JAK2/STAT3 signal pathway,thereby promoting the recovery of neurological function after spinal cord injury.

电针对去势大鼠血清E_2及外周血淋巴细胞雌激素受体的影响

目的 :从生殖内分泌 -免疫网络角度探讨电针治疗围绝经期综合征的作用机制。方法 :以去势大鼠为实验对象 ,取双侧“三阴交”、“太溪”、“后三里”穴 ,采用放射免疫法和流式细胞分析技术 ,观察电针治疗对去势大鼠血清E2 及外周血淋巴细胞内ER水平的影响。结果 :电针可使去势大鼠血清E2 明显升高 ,外周血淋巴细胞内ER水平明显升高 ,与造模对照组相比 ,有显著性差异 ,而且大鼠血清E2 水平与外周血淋巴细胞内ER表达密切相关。结论 :电针可明显改善去势大鼠的生殖内分泌功能 ,并能调节免疫细胞的雌激素受体表达。

GnRHa超排卵对小鼠子宫内膜形态学和雌、孕激素受体表达的影响

探讨 Gn RHa长周期辅助超排卵对小鼠分泌期子宫内膜组织形态和性激素受体表达的影响。方法 :将 40只小鼠随机分成 4组 ,其中 2组给 Gn RHa+h MG+h CG为用药组 ,另 2组为对照组给等体积生理盐水 ,在促排卵和排卵后 2 4、48h利用多功能图象分析仪定量测定子宫内膜厚度、腺体和腺腔面积、周长、最大直径、腺上皮细胞总面积和平均高度 ;SP免疫组化法定量测定腺上皮细胞雌、孕激素受体表达。结果 :( 1 )用药组在注 h CG后 2 4、48h子宫内膜厚度与对照组相比差异无显著性 ( P>0 .0 5 )。 ( 2 )用药组在注 h CG后此两时段腺体和腺腔面积、周长、最大直径以及腺上皮细胞总面积和平均直径均显著小于对照组 ( P均 <0 .0 5 )。同时用药组部分内膜间质和腺体发育不同步。( 3 )用药组在注 h CG后两时段内膜腺上皮细胞 ER、PR免疫组化阳性率和染色强度均显著低于对照组同期值 ( P均 <0 .0 5 )。结论 :Gn RHa辅助超排卵对小鼠子宫内膜组织结构和 ER、PR表达有不利影响 ,提示该方案并不能完全促进生理状态的子宫内膜形成 。

腹腔注射5-HT(2A)受体拮抗剂MDL11939对小鼠急性及慢性疼痛的影响

目的探讨5-羟色胺(5-hydroxytryptamine,5-HT)2A受体拮抗剂MDL11939在急性乙酸内脏痛、急性切口痛和坐骨神经慢性缩窄性损伤(chronic constrictive injury,CCI)模型中发挥的作用及意义。方法选用♂昆明小鼠,建立急性乙酸内脏痛、急性切口痛和CCI神经病理性痛模型,通过腹腔注射给药,观察乙酸致小鼠扭体反应次数变化、切口痛和CCI痛患侧热缩足反射潜伏期(thermal withdrawal latency,TWL)变化。结果在小鼠急性乙酸内脏痛模型中,与溶剂对照组相比,MDL11939(0.25、0.5、1.0 mg·kg^(-1),i.p.)呈剂量依赖性地减轻乙酸内脏痛,扭体反应次数明显减少。在急性切口痛和CCI模型中,与模型组和自身给药前相比,MDL11939(0.5 mg·kg^(-1),i.p.)可以明显提高小鼠患侧的热缩足阈值,减轻疼痛。结论 MDL11939通过腹腔系统性给药可以有效地减轻急性乙酸内脏痛、急性切口痛和CCI慢性神经病理性痛。

蛋白酶激活受体-1拮抗剂对兔心脏骤停后综合征中肾损伤的保护作用及机制

目的:探讨蛋白酶激活受体-1(PAR-1)拮抗剂对心脏骤停后综合征(PCAS)中肾损伤的影响及机制。方法:将日本大耳白兔随机分为假手术组(n=5)、PCAS组(n=10)、PAR-1拮抗剂组(n=10)。采用窒息性心脏骤停法制备兔PCAS模型。PAR-1拮抗剂组于心肺复苏后10 min给予静脉滴注PAR-1拮抗剂SCH79797(25μg/kg),其余各组给予等量生理盐水静脉滴注。72 h后取股静脉血检测血清肌酐和胱抑素C水平;取肾组织制备石蜡切片后行HE染色;采用TUNEL法测定肾脏细胞凋亡情况;应用Western blot测定肾组织半胱天冬酶(casepase)-3、细胞外调节蛋白激酶(ERK)活化情况。结果:与假手术组相比,PCAS组血清肌酐和胱抑素C水平明显上升,肾组织有大量炎性细胞浸润,凋亡细胞数显著增多,有催化活性的caspase-3裂解片段(cleaved caspase-3)表达增高,磷酸化ERK(p-ERK)表达减少(P<0.05)。与PCAS组相比,PAR-1拮抗剂组肾组织损伤减轻,血清肌酐和胱抑素C水平明显下降,凋亡细胞数减少,cleaved caspase-3表达降低,而p-ERK表达显著增高(P<0.05)。结论:PAR-1拮抗剂SCH79797能抑制PCAS兔肾组织的炎症反应和细胞凋亡,可能与ERK信号通路激活有关。

口服型VEGFR-2 DNA疫苗的研制及其对小鼠的免疫效应

目的:血管内皮生长因子受体-2(vascular endothelial factor recepto-2,VEGFR-2)在肿瘤生长和转移过程中起着重要作用,制备口服型VEGFR-2DNA疫苗并研究其生物免疫效应。方法:采用基因重组技术构建VEGFR-2胞外区基因表达载体pcDNA3.1-VR-2,DNA序列分析证实后,脂质体法转染COS-7细胞,Western blotting检测其VEGFR蛋白表达;以氯化钙法将pcDNA3.1-VR-2转化减毒沙门菌SL3261,制备口服型VEGFR-2DNA疫苗。口服型疫苗免疫C57BL/6小鼠,ELISA法检测免疫后小鼠外周血VEGFR-2抗体水平,MTT法检测小鼠脾淋巴细胞对小鼠血管内皮细胞的体外杀伤活性。结果:成功制备口服型VEGFR-2DNA疫苗。ELISA法检测显示,疫苗组小鼠抗体滴度随着免疫时间和免疫次数的增加而增高,在免疫6周后产生了高水平抗VEGFR-2IgG类抗体(1.07±0.018),明显高于生理盐水组(0.14±0.033)和质粒对照组(0.14±0.038),差异均有统计学意义(F=853.17,P=0.000)。MTT法检测显示,疫苗组小鼠脾淋巴细胞对靶细胞MS1的杀伤率随着效靶比的增加而增加,在效靶比8∶1时CTL活性(89.38±1.51)明显高于生理盐水组(15.17±2.54)和空质粒对照组(12.99±4.31),差异有统计学意义(F=315.42,P=0.000)。结论:成功制备了口服型VEGFR-2DNA疫苗,该疫苗可激活小鼠特异性的细胞免疫和体液免疫,为进一步抗肿瘤研究奠定了基础。

外用维甲酸对表皮生长因子受体表达作用的研究

目的：了解维甲酸外用前后表皮生长因子受体的分布，以便有助于进一步探讨维甲酸治疗银屑病的机理。方法：应用免疫组化技术，观察分析正常人正常皮肤、0．1％维甲酸乳膏外用4天后表皮生长因子受体的分布，并与银屑病皮损及未受累皮肤作对照。结果：正常皮肤中表皮生长因子受体间断性“串珠样”分布于基底层及基底上层，外用维甲酸后阳性细胞数显著增加，失去“串珠样”特征。银屑病皮损中表皮生长因子受体遍布表皮全层，在基底层呈连续性分布，而在未受累皮肤，阳性细胞数明显减少。结论：外用维甲酸可增强表皮生长因子受体的表达，这种增强是否是对银屑病表皮生长因子过度表达的负反馈抑制需进一步研究。

快速筛选雌激素类环境激素的方法探讨

目的 开发快速筛选环境激素的检测方法。方法 将构建的表达人α雌激素受体的载体pcDNA 3 1-ER和含雌激素受体反应元件的荧光素酶报告基因载体pGL3 -tk -ERE共转染到HeLa细胞中 ,以雌二醇或被检样品双酚A诱导雌激素受体表达后检测荧光素酶活性。结果 报告基因载体中的荧光素酶的表达受雌激素受体反应元件的调控 ,且在一定浓度范围内荧光素酶的活性与雌二醇的加入量成线性关系 ;疑为有雌激素样作用的双酚A也显著诱导荧光素酶基因的表达。结论 本方法经济、简便、快速 。

Liver myofibroblasts activate protein C and respond to activated protein C

AIM:To study the protein C activation system in human liver myofibroblasts,and the effects of activated protein C(APC)on these cells.METHODS:Human liver myofibroblasts were obtained by outgrowth.Expression of protease activated receptor 1(PAR-1),endothelial protein C receptor(EPCR) and thrombomodulin(TM)was analyzed by flow cytometry.Extracellular signal-regulated kinase(ERK)1/2 activation was assessed by Western blotting using anti-phospho-ERK antibodies.Collagen synthesis was studied with real-time reverse transcription-polymerase chain reaction(RT-PCR).Activation of protein C was studied by incubating liver myofibroblasts with zymogen protein C in the presence of thrombin and detecting the generation of APC with a colorimetric assay using a peptide substrate. RESULTS:Primary cultures of human liver myofibroblasts expressed EPCR on their surface,together with PAR-1 and TM.This receptor system was functional since exposure of myofibroblasts to APC inducedERK1/2 phosphorylation in a dose-and time-dependent manner.Furthermore,APC significantly upregulated the expression of collagen mRNA,as shown by real-time RT-PCR.Collagen upregulation was controlled through the ERK pathway as it was inhibited when using the mitogen-activated protein/extracellular signal-regulated kinase kinase inhibitor PD98059.Finally,using a cell-based colorimetric assay,we showed that intact myofibroblasts converted protein C into APC in the presence of thrombin.CONCLUSION:These data suggest that APC is a new modulator of liver myofibroblast activity and contributes to the pathophysiology of chronic liver diseases.

福辛普利对自发性高血压大鼠主动脉脂联素受体1表达的影响

目的:探讨福辛普利对自发性高血压大鼠(SHR)主动脉脂联素受体1表达的影响。方法:24只14周龄SHR大鼠随机分成高血压组、福辛普利10 mg组和福辛普利20 mg组;8只同系魏凯式(WKY)大鼠做对照组。从14周龄起,WKY组和SHR组给予蒸馏水灌胃,福辛普利10 mg组和福辛普利20 mg组分别给予福辛普利10 mg/(kg.d)和20 mg/(kg.d)灌胃。8周后采血测定脂联素浓度,取血管组织HE染色后测定中膜厚度(MT)与胸主动脉内径(LD)比值,通过免疫组化法检测脂联素受体1蛋白的表达。结果:8周后,与高血压组相比,福辛普利低剂量组和高剂量组血压水平明显降低,高血压血管重构改善,血清脂联素水平以及主动脉脂联素受体1蛋白的表达明显升高,差异均有统计学意义(P<0.05)。结论:SHR大鼠主动脉脂联素受体1表达下调,福辛普利可通过升高SHR大鼠胸主动脉脂联素受体1的表达而改善血管重构。

六甲氧苄嗪对离体豚鼠乳头状肌生理特性的影响

六甲氧苄嗪(hexametazidine)具有浓度依赖的负性变力作用(IC 50=81.8±16.9μmol/L).细胞外Ca^(2+)从1.8mmol/L提高到5.4mmol/L,可完全对抗六甲氧苄嗪(30.8μmol/L)的负性变力作用,六甲氧苄嗪(30.8μmol/L)可使肾上腺素诱导自律性的阈浓度从3.95±1.80提高到6.67±4.84μmol/L(P<0.05);61.6μmol/L使心肌的功能不应期从218±14延长至254±20ms(P<0.05),六甲氧苄嗪6.2μmol/L可竞争性地拮抗异丙肾上腺素的正性变力效应(PA_2=5.45);15.41μmol/L非竞争性地拮抗组胺的正性变力效应(pD_2′=4.13)。

Effect of Thrombin on the Apoptosis of Hippocampal Neurons in vitro

Hippocampal neurons were treated by thrombin and thrombin receptor activatingpeptides (TRAP). Cell survival rate was decreased in a dose-dependent manner by MTT assay. Thenumbers of apoptotic cell and apoptotic rate of hippocampal neurons treated bydifferentconcentrations of thrombin were increased in a dose-dependent manner by terminal deoxynucleotidyltransferase (TdT) mediated dUTP-biotin nick end-labeling (TUNED method and Flow Cytometry. When theconcentration of thrombin is 40 U/mL, TUNEL positive cells and apoptotic rate of hippocampal neuronsreached peak value, were 27. 3 +- 4. 0 and (29. 333 +- 4. 633 ) % , respectively.Immunocytochemistry assay show that Bcl-2 protein expression was down- regulated and Bax proteinexpression was up-regulated with the concentration of thrombin increased. TRAP can mimic the effectof thrombin to induce apoptosis on hippocampal neurons. These data demonstrated that thrombininduced hippocampal neuron apoptosis in a dose-dependent manner through activatingprotease-acti-vated protein-1 (PAR-1). The change in expression of Bcl-2 and Bax was related withthe effect of high concentration thrombin induced apoptosis on hippocampal neurons.

2型糖尿病患者ABCA1、PPARγ、SREBP、ADPN、LXRα表达水平及临床价值研究

目的探讨2型糖尿病患者中三磷酸腺苷(ATP)结合盒转运体A1(ABCA1)、过氧化物酶体增殖物激活受体(PPARγ)、固醇调节元件结合蛋白(SREBP)、脂联素(ADPN)、小鼠肝X受体α(LXRα)表达水平及其临床价值。方法选自该院于2015年6月至2017年6月期间收治的2型糖尿病患者71例作为观察组;另选自该院于2015年6月至2017年6月期间健康体检者60例作为对照组。清晨空腹采集受试者外周静脉血,分离血清,采用放射免疫法测定血清人ADPN含量,采用酶联免疫吸附双抗体夹心法测定血清ABCA1、PPARγ、SREBP及LXRα含量。结果观察组血清ABCA1和ADPN含量低于对照组,而血清PPARγ、SREBP和LXRα含量高于对照组,差异均有统计学意义(P<0.05);ABCA1+PPARγ+SREBP+ADPN+LXRα五项联合检测灵敏度和特异度高于ABCA1、PPARγ、SREBP、ADPN和LXRα单独检测。结论 2型糖尿病患者ABCA1和ADPN表达下降,而PPARγ、SREBP和LXRα表达上升,ABCA1+PPARγ+SREBP+ADPN+LXRα五项联合检测具有较高的灵敏度和特异度,具有重要的临床研究价值。

血浆置换在控制肾移植排斥反应中的应用

目的观察血浆置换（ＰＥ）控制肾移植术后排斥反应的疗效，并初步探讨其可能的作用机制。方法对肾移植前淋巴细胞毒试验阳性患者９例与术后出现排斥反应患者１０例１１例次行ＰＥ治疗，并于ＰＥ前后监测ｓＩＬ－２Ｒ、ＴＮＦ水平。结果９例移植前淋巴细胞毒试验阳性者ＰＥ后全部转阴雨顺利行肾移植手术，术后排斥反应低，只有１例次急性排斥反应。术后１１例次排斥反应ＰＥ后１０例次得到控制。ＰＥ后血清中ｓＩＬ－２Ｒ、ＴＮＦ水平较前下降。结论 ＰＥ配合常规药物疗法在预防和治疗肾移植术后排斥反应是有效的，其机制与ＰＥ下调ＴＮＦ、ｓＩＬ－２Ｒ水平有关。