日本医蛭摄食前后不同组织中水蛭素基因(hirudin)的时空表达模式

【目的】探究日本医蛭在不同摄食阶段、各个组织(唾液腺、肠、嗉囊、皮肤及精/卵巢)中水蛭素基因(hirudin)的时空表达模式,为水蛭药材的科学合理利用提供理论依据。【方法】基于水蛭唾液腺转录组数据筛选结果及基因克隆获得的水蛭素基因序列信息,利用实时荧光定量PCR方法,对水蛭关键活性成分的编码基因hirudin在不同摄食阶段、不同组织中的表达量进行检测分析,研究该基因的时空表达模式。【结果】水蛭素基因(hirudin)在日本医蛭的唾液腺、肠、嗉囊、皮肤及精/卵巢组织中均有表达,且在唾液腺中的表达量显著高于其他组织;Hirudin基因在不同摄食阶段的唾液腺组织中表达结果显示,摄食后第2天表达量显著升高,之后随着摄食时间的推延而呈下降趋势;Hirudin基因在嗉囊、肠中的表达量呈先升高后下降趋势,分别于摄食后第1、第2天达到最高,之后会随着停食时间延长呈下降趋势,尤其是在摄食后第7、第8天的表达量呈显著性降低;Hirudin基因在皮肤组织中也有表达,并随着停食时间的延长呈先升后降趋势,最高峰出现在摄食后第2天;在精/卵巢组织中,摄食期间hirudin基因的表达量最高,与其他摄食阶段的表达呈显著性差异。【结论】水蛭素基因(hirudin)在日本医蛭的唾液腺、肠、嗉囊、皮肤和精/卵巢组织中均有不同程度的表达,在唾液腺中的表达量显著高于其他组织。摄食行为及食物刺激可能影响水蛭素基因(hirudin)的表达,在不同摄食阶段的表达量存在显著差异。研究结果可为水蛭药材的科学采收加工、提取部位的科学选取、合理入药提供理论依据和技术支撑,对未来水蛭药材资源的可持续利用具有重要的参考价值。

Interventional effect of hirudin on the expression of microtubule-associated protein 2 in peripheral tissue of hematom of model rats with acute intracerebral hemorrhage

BACKGROUND： It is suspected that dissociation, destruction or synthetic disorder of microtubule-associated protein 2 （MAP-2） may participate in secondary injury of intracerebral hemorrhage （ICH）, and the reason may be related to thrombin in high concentration after ICH; therefore, the mechanism should be studied further. OBJECTIVE： To explore the effect of hirudin on expression of MAP-2 in peripheral tissue of hematom after ICH and changes of water content in brain tissue and analyze pathogenesis of thrombin in secondary injury after ICH. DESIGN ： Completely randomized grouping design and controlled animal study SEn-ING ： Department of Neurology, the First Affiliated Hospital of Jilin University MATERIALS ： The experiment was carried out in the Neurological Laboratory of the First Affiliated Hospital of Jilin University from April 2003 to April 2004. A number of 80 healthy Wistar rats, of both genders, aged 3-4 months, weighing 250-350 g, were randomly divided into 8 groups： normal control group, 6-hour ICH group, 1-day ICH group, 2-day ICH group, 3-day ICH group, 7-day ICH group, 3-day hirudin group and 7-day hirudin group with 10 in each group. Five rats from each group were selected to measure their water content, and the others were undertaken immunohistochemical stain. Hirudin was produced by Sigma Company, USA, and MAP-2 rabbit-rat polyclonal antibody was provided by Fuzhou Maixin Biotechnology Company Limited. METHODS： ① Model establishing and grouping intervention： Rats in simple ICH group were collected their blood from tails and then inserted with 50 μL non-anticoagulant auto-arterial blood into the cauda of the putamen in right brain within 5 minutes. Rats in hirudin groups were inserted with 10 U hirudin （which was diluted with saline to 20 μL） into local hematom regions within 5 minutes, and the needle was pulled out after 10 minutes. Rats in normal control group were untouched. ② Water content in peripheral tissue of hematom： Based on the ratio between dry weight and wet weight, brain tissue at bleeding side and in right frontal lobe was selected to measure dry and wet weights so as to calculate the water content [（wet weight - dry weight） /wet weight] × 100%.③ Positive expression of MAP-2： Based on immunohistochemical stain, positive MAP-2 cells were regarded as neurons and they were buffy morphological. Positive rate of MAP-2 was calculated, i.e., percentage of positive cells in each sight to total cells in all sights. ④ Statistical analysis： Data among groups were compared with one-way analysis of variance, averages were compared with SNK-q test by each other, and relation between water content and MAP-2 was analyzed with linear regression technique. MAIN OUTCOME MEASURES： Changes of water content and MAP-2 expression in peripheral tissue of hematorn at various time points after ICH and intervention of hirudin. RESULTS： All 80 rats were involved in the final analysis. ①Water content： Water content was increased at day 1, reached peak at day 3 and decreased at day 7. It was （72.31±0.32）%, （77.42±0.53）%, （78.44±0.28）%, （74.10±0.13）%, （74.85±0.51）% and （70.07±0.36）%, respectively in 1-day, 2-day, 3-day and 7-day ICH groups and 3-day and 7-day hirudin groups, which was higher than that in normal control group （63.85±0.41, q=-4.684 3 to -7.262 0, P〈 0.05）; that in 2-day and 3-day ICH groups was higher than that in 7-day ICH group （q=-3.053 4, -3.727 0, P 〈 0.05）; and that in 3-day and 7-day ICH groups was higher than that in hirudin groups at the same time points （q=-2.965 6, -2.726 4, P 〈 0.05）. ②Positive expression of MAP-2： Positive expression of MAP-2 was decreased at 6 hours after ICH, reached the lowest value at day 3 and increased at day 7. Positive rate was （78.60±0.42）%, （60.56±0.74）%, （44.60±0.26）%, （25.45±0.85）%, （32.55±0.64）%, （37.69＋0.76）%, （41.75±0.68）%, respectively in 6-hour, 1-day, 2-day, 3-day and 7-day ICH groups and 3-day and 7-day hirudin groups, which was lower than that in normal control group [（96.50±0.33）%, q= -3.074 5 to -8.128 5, P 〈 0.05]. In addition, positive cells of MAP-2 disappeared plentifully at 3-7 days after ICH, stain of positive cells were light, and only stain of plasma was positive. That in 3-day and 7-day hirudin groups was higher than that in ICH groups at the same time points （q= -3.391 8, -2.967 9, P 〈 0.05）. Moreover, positive cells of MAP-2 was formed slightly but deeply stained. ③ Results of linear regression： Water content was negatively related to MAP-2 changes at 7 days after ICH （r= -0.894 9, P〈 0.01）, i.e., water content was increased with decrease of MAP-2 expression. CONCLUSION ： The deterioration of MAP-2 may be involved in the pathogenesis of thrombin within the first week after ICH, and the local administration of hirudin can protect neurons.

Surface Characterization and in Vitro Blood Compatibility of Poly (Ethylene Terephthalate) Immobilized with Hirudin

Poly （ethylene terephthalate）（dacron, PET） films were exposed under argon plasma glow discharge with different glows and induced polymerization of acrylic acid（AA） in order to in- troduce carboxylic acid group onto PET （PET-AA） assisted by ultraviolet radiation（UV）. Hirudin- immobilized PET （PET-HRD） films were prepared by the grafting of PET-AA, followed by chem- ical reaction with hirudin. The surface structure of the treated PET was determined by X-ray photoelectron spectroscopy （XPS）. The wettability, surface free energy, and interface free energy of the films were investigated by contact angle measurement. The blood compatibility of the films was assessed by platelet-adhesion test and fibrinogen conformational change measurements to eval- uate the viability of the materials in biomedical engineering. Measurement by scanning electron microscopy （SEM） revealed that the amounts of adhered, aggregated and morphologically changed platelets were reduced on the hirudin-immobilized PET films. Enzyme-linked-immunoassay mea- surements that disclosed fibrinogen conformational changes showed results consistent with the platelets＇ behavior.

In vitro Blood Compatibility of Polyethylene Terephthalate with Covalently Bounded Hirudin on Surface

Polyethylene terephthalate （PET,Dacron） was modified by surface immobilization of hirudin with glutaraldehyde（GA） as coupling reagent to improve the blood compatibility.Hirudin-immobilized PETs were characterized by X-ray photoelectron spectroscopy （XPS） and contact angle measurements.The blood compatibility of the PETs was evaluated by platelet adhesion evaluation and fibrinogen conformational change measurements in vitro.The results showed the decrease of platelet adhesion and activation on hirudin-immobilized PET with increasing of glutaraldehyde concentration.Fibrinogen experiment showed that fibrinogen adherence and conformational changes of PET-HRD were less than those of untreated PET,which made the materials difficult to form thrombus.The proper reason of blood compatibility improvement was low interface tension between hirudin-immobilized PETs and blood,as well as blood proteins,and low ratio of dispersive/polar component of the surface energy（γsd/γsp） and high hydrophilicity.

Distribution and excretion of N-Ile1 Thr2-63-desulfatohirudin in rats

Objective To investigate distribution and excretion of N-Ile1Thr2-63-desulfatohirudin(rH)a recombinant hirudin newly developed in China,in rats for its development as a novel anticoagulant agent.Methods ELISA was used to determine the rH concentration in related tissues and body fluids.Tissues were collected at 15,60 and 180min respectively,after iv administration of rH 1.0 mg·kg-1 to 3 groups of 5 rats,and homogenized.Urine,bile and feces were collected at pre-selected intervals of time after iv dosing 1.0 mg·kg-1 to 3 groups of 5 rats and assayed.Results rH following iv dosing was distributed rapidly,the rH levels in all tissues being found to be the highest at 15 min post-injection,afterwards gradually reduced.The highest concentration of rH was found in blood,the next in lung and heart,the lowest in brain.With 15 min post dose as an example,the rH contents in tissues were ranked in order of plasma>lung>heart >adipose>skeletal muscles>kidney>liver>spleen>brain.The 12 h-cumulative excretion amount of rH in urine and feces accounted for 0.03%and 0.001% of administered dose,respectively;the 6 h-cumulative excretion amount in bile was 0.02%of the dose.Conclusions The rH is distributed mainly in blood circulation system with very low content in other tissues.The drug is excreted from urine,feces and bile of rats in extremely minute amount(only 0.051% dose),suggesting that rH undergoes extensive metabolic elimination in rat body.

Research Progress on Pharmacology and adverse reactions of hirudin

Hirudin is an active ingredient extracted from leeches(Hirudo).At present,there are many hirudin preparations on the market,which are roughly divided into three categories,the first is natural hirudin,the second is hirudin derivatives such as lepirudin,desirudin and bivalirudin,and the third is new hirudin preparations such as hirudin-bovine serum albumin(BSA)nanoparticles,polydopamine fitted titanium dioxide nanoparticles systems and recombinant hirudins-2(rhv2)-loaded picmice.The pharmacological effects and adverse reactions of hirudin were reviewed to evaluate its safety,efficacy and quality control.Hirudin has obvious pharmacological effects on cardio-cerebrovascular diseases(coronary atherosclerotic heart disease,myocardial infarction,hyperlipidemia,cerebral infarction,arteriosclerosis obliterans of lower extremities),angiogenesis(fracture,skinflaptransplantation),tissue fibrosis,tumor,ophthalmopathy,hyperuricemia and female infertility.However,attention should be paid to clinical adverse reactions(bleeding,allergic reaction,infection,cutaneous pseudolymphoma).

Effect of Hirudin on farnesol X receptor pathway during acute intrahepatic cholestasis

Objective:The purpose of this study is to explore the effect of Hirudin on the farnesoid X receptor(FXR)pathway during acute intrahepatic cholestasis in vivo and in vitro.Method:In vivo,sixty male Sprague-Dawley rats were randomly divided into six groups:regular group,model group,ursodeoxycholic acid(UDCA)group(60 mg/kg),hirudin treatment group(84 u/kg),hirudin treatment group(63 u/kg)and hirudin treatment group(42 u/kg).The male Sprague-Dawley rats of UDCA group were intragastrically administered with a corresponding concentration of 0.005 mL/g body weight for seven days,once a day;and the hirudin treatment group was injected subcutaneously with different concentrations of Hirudin for seven days,once a day;Except for the normal group,other groups of rats were given 100 mg/kg ANIT by gavage on the 5th day.The model was administered by gavage once a day for three days.In vitro,(Z)-Guggulsterone was used to stimulate the L02 cells(0.05μmol/ml),with or without different concentrations of Hirudin(2,4 and 8 u/ml)for 24 h.The liver tissue was examined by HE microscope and the pathological state of the rat liver was observed;FXR,Small heterodimeric chaperone receptor(SHP),uridine diphosphate glucuronide transfer 2B4(UGT2B4),bile salt output pump(BSEP)mRNA and protein expressions were tested by real-time fluorescent quantitative PCR and Western blot test.And immunohistochemistry(IHC)was used to analyze the expression of FXR.Results:Compared with the model group,the hirudin group can improve liver tissue damage,and promote FXR,SHP,BSEP and UGT2B4 proteins and mRNA expression in vivo and in vitro.Conclusion:Hirudin can alleviate intrahepatic cholestasis,reduce liver tissue damage.Hirudin can up-regulate the expression of FXR gene,promote the up-regulation of SHP,BSEP and UGT2B4 genes,and inhibit the cholestasis pathway to protect liver cells.The study may provide an effective drug for clinical treatment of intrahepatic cholestasis.

PEGylation of Hirudin and Analysis of Its Antithrombin Activity in vitro

Hirudin 是在自然发现的大多数抗凝剂药，但是它的短浆液半衰期显著地禁止它的临床的申请。hirudin 的 PEGylation，最有希望的抗凝剂药，在这篇论文被执行。为 PEGylated hirudin 的最佳的反应条件被调查。PEGylation 反应什么时候在 10h 以后在 4 ° C 下面被进行，在在最适 PH 的硼酸盐缓冲区 8.5 与到 hirudin 的木钉的摩尔比率 250:1，更高的修正程度被完成。最后， PEGylated hirudin 的 bioactrvity 在 vitro 被测量。与未修改的 hirudin 相比， 26% 反凝血酵素活动被保留。

Design, Preparation and in vitro Bioactivity of Mono-PEGylated Recombinant Hirudin

Hirudin，凝血酵素的大多数有势力禁止者在自然发现了，在浆液有短半衰期，它显著地作为抗凝剂限制它的临床的申请。最近， PEGylation 通常被用作一个有效方法在浆液延长它的半衰期。与在随机指向离氨酸残余的基本条件下面的非特定的 PEGylation 相对照，在略微酸的条件下面的 PEGylation 地点最好指向 histidine 残余，并且仅仅在 r-hirudin 在 51 点有一 histidine 残余；因此， succinimidyl 羰基 methoxy 聚乙烯乙二醇(SC-mPEG， 20000 ) 在略微酸的 pH 被纳入 r-hirudin 赞成 mono-PEGylated r-hirudin 的形成。有高 mono-PEGylated 比率的反应混合被一步舞离子交换容易分开色析法(IEC ) 过程。约 79.71% mono-PEGylated r-hirudin 是在 His51 的 PEGylated，它证明酸的 PEGylation 操作原则上阻止了 r-hirudin 的活跃中心(Lys47 ) 的 PEGylation。有比 95% 高的纯净的 Mono-PEGylated 产品作为占优势的产品被获得，并且 34% 抗凝剂活动在 vitro 被保留。为钠 dodecyl 硫酸盐 polyacrylamide 胶化电气泳动(SDS 页) 的染色的方法被改进在不到 5min 获得完美的 electrophoretic 模式。更精确的分子的重量作为分子的重量标准由于木钉的使用被推出。

Pharmacokinetic study with N-Ile^1-Thr^2-63-desulfato-r-hirudin in rabbits by means of bioassay

Aim: To study the pharmacokinetic (PK) properties in rabbits treated with N-Ile1-Thr2-63-desulfato-r-hirudin (rH) newly developed in China by means of bioassay in order to provide preclinical experiment basis for its development as a novel anticoagulant agent. Methods: rH plasma concentration was determined using bioassay based on ex vivo antithrombin activity of rH. Normal rabbits received iv rH 4.0, 2.0 and 1.0 mg/kg or sc rH 2.0 mg/kg, respectively. The rabbits with acute severe renal failure were given iv rH 2.0 mg/kg. Results: The bioassay described in this paper met requirements for study of PK in rabbits. The major PK parameters after iv dosing were as follows: t1/2β 58.4~59 min. Vd 0.09~0.12 L/kg, CL 0.0035~0.0040 L/(kgmin); AUC were proportional to the doses, t1/2 and CL did not change significantly with the doses. The sc bioavailability reached 94%. The rabbits suffering from acute severe renal failure presented 11-fold longer t1/2β and 13-fold greater AUC than normal healthy rabbits. Conclusion: rH exhibited rapid elimination, distribution was only limited to extracellular space and good absorption from sc site. The excretion of rH by kidneys played a very important role in the elimination of rH. The PK of rH could be described by the two- and one-compartment model after iv and sc dosing, respectively, and followed linear kinetics.

Effects of fused hirudin on activity of thrombin and function of platelets

Objective: To investigate whether fused hirudin peptide has both antithrombin and antiplatelet functions. Methods: The core region of fused hirudin was the C-terminal tail of hirudin(hirudin_ 53-64),which could bind to the anion binding exosite (ABE) of thrombin.Arg-Pro-Pro-Gly-Phe(RPPGF) amino acid sequence,a metabolite of bradykinin,was added to the N-terminus of hirudin_ 53-64.It bound to the active site of thrombin.Additionally,Arg-Gly-Asp(RGD)amino acid sequence,an inibitor of glycoprotein Ⅱb/Ⅲa( GP Ⅱb/Ⅲa) receptor,was linked to C-terminus of hirudin_ 53-64.This 26-animo acid-fused hirudin peptide was artificially synthesized,purified and analysed. Results: Fused hirudin peptide significantly lengthened the activated partial thromboplastin time(APTT),thrombin time(TT)and prothrombin time(PT) and inhibited the amidolytic activity of thrombin.The ADP-induced platelet aggregation was markedly inhibited by fused hirudin peptide. Conclusion: Fused hirudin peptide has activity of antithrombin as well as antiplatelet.Therefore bifunctional anticoagulation peptide has capacity to target various components of haemostatic process and may become more powerful antithrombosis agent.

PCL-based and Hirudin-containing Composite Nanofibers for Prolonged Anticoagulation Effect

More and more concerns about health bring the increasing demand for blood contact tissue engineering alternatives.In this paper,nanoparticles of poly(lactic-co-glycolic acid)/polyethyleneimine mixed with recombinant hirudin(rHNPs)were prepared by a double emulsion solvent volatilization method,which were then loaded onto the polycaprolactone(PCL)with polydopamine(PDA)coating to form the composite nanofibers of PCL/PDA/rHNPs.The hydrophilicity and mechanical properties of the composite nanofibers were improved significantly compared with pure PCL.The morphology kept almost unchanged after 30 d of degradation in phosphate buffer saline(PBS).The anticoagulant molecule of hirudin could be gradually released from the composite scaffolds through the degradation of rHNPs in vitro.When the concentration of rHNPs suspension was 5.0 mg/mL,the composite nanofibers could better promote the growth and proliferation of human umbilical vein endothelial cells(HUVECs).The anticoagulant ability of the composite nanofibers was also significantly improved in comparison with that of pure PCL.The design of controlled release anticoagulant materials would alleviate the sudden release of simple fixed hirudin,which could also provide a new idea for the development of novel blood contact materials.

Chemical protein synthesis elucidates key modulation mechanism of the tyrosine-O-sulfation in inducing strengthened inhibitory activity of hirudin

Tyrosine sulfation is an important post-translational modification that enhances the inhibitory activity of hirudin.Herein,we developed a facile synthetic strategy to afford the sulfated hirudins with up to three modifications and in multi-milligram scales,after a single HPLC purification step.Through these synthetic proteins,a novel type of modulation mechanism exhibited by tyrosine sulfation was proposed,which would help to delineate the structure-function relationships in other sulfated proteins and more importantly,to serve as a basis for the development of related antithrombotic agents.

疏血通抑制Bim依赖的小脑颗粒神经元凋亡

【目的】探究疏血通及其主要成分水蛭素对Sprague-Dawley(SD)大鼠小脑颗粒神经元(CGNs)凋亡的影响及机制。【方法】体外成熟7 d的CGNs分为存活对照组(用含K+浓度为25 mmol/L的培养基,即25 K组)、凋亡组(用含K+浓度为5 mmol/L的培养基,即5 K组)、以1/50、1/40、1/30、1/20、1/10浓度(稀释50、40、30、20、10倍)疏血通注射液处理组(25 K以及5 K合并疏血通处理组)以及对应不同浓度(2 U/mL、2.5 U/mL、3.34 U/mL、5 U/mL、10 U/mL)水蛭素处理组(25 K以及5 K合并水蛭素处理组),用Hoechst染色法观察并统计凋亡率。在蛋白印迹实验中,在25 K和5 K条件下用1/50、1/10浓度疏血通注射液以及对应2 U/mL、10 U/mL浓度水蛭素处理细胞,用Western blot法检测Cleaved Caspase-3、Bim、VEGF的表达水平。【结果】核染色结果显示,与25 K存活对照组比较,5 K凋亡组凋亡率增加;与25 K存活对照组比较,不同浓度疏血通注射液与水蛭素处理细胞后凋亡率无明显变化;与5 K凋亡组比较,不同浓度疏血通注射液与水蛭素处理细胞后凋亡率下降,且随着浓度的升高,凋亡率下降越明显。Western blot结果显示,与5 K凋亡组比较,不同浓度疏血通与水蛭素处理细胞后Cleaved Caspase-3、Bim蛋白表达水平均下降,VEGF蛋白表达水平升高。【结论】疏血通及其主要成分水蛭素通过抑制Bim表达,进而抑制线粒体依赖的小脑颗粒神经元凋亡。

水蛭素的HPLC色谱分析条件优化研究

为研究水蛭素的HPLC最佳色谱分析条件,通过对比重组水蛭素的HPLC分析方法的洗脱条件、检测波长、色谱柱的分离效果,拟合水蛭素浓度与峰面积的线性方程,优选最佳水蛭素色谱分析条件。结果显示,ODSC18柱分析最佳条件为:采用梯度洗脱,流动相A和B洗脱程序:0~60 min,B:0%~100%,进样量20μL,柱温35℃,流速为1 mL/min,波长205 nm;TSKgelG2000SW柱最佳分析条件为:流动相磷酸缓冲液(0.02 mol/L,pH 7.0),流速0.5 mL/min,检测波长205 nm,进样量20μL。研究表明,水蛭素浓度与峰面积具有良好的直线线性相关关系,采用TSKgelG2000SW色谱柱的分析效果优于ODSC18柱,可为动物活性多肽的分析提供依据。

基于转录组学探讨水蛭素对HK-2细胞的影响机制

目的采用转录组学技术探讨水蛭素对人肾皮质近曲小管上皮细胞(HK-2细胞)的影响及机制。方法利用CCK-8法检测水蛭素处理之后的HK-2细胞活力,通过转录组学的技术对溶媒对照组和水蛭素组进行转录组测序;通过RSEM的方法计算不同样品中的基因表达水平,差异基因的筛选采用DEseq2方法,筛选标准为|log2FC值|≥1,P<0.05。随后根据差异表达的基因进行富集分析。结果水蛭素5 mg/mL下提高HK-2细胞活力效果最佳,转录组学研究结果表明,与正常HK-2细胞相比,水蛭素处理后的HK-2细胞有1723个基因发生差异表达。GO和KEGG分析结果表明,炎症通路的调控是水蛭素治疗高尿酸血症的主要作用机制。结论水蛭素作用于多条炎症信号通路,进而促进尿酸排泄,促进集体代谢和尿酸排出体外,起到降尿酸的作用,表明水蛭素在治疗高尿酸血症方面效果显著。

Effects of Hirudin on High Glucose-Induced Oxidative Stress and Inflammatory Pathway in Rat Dorsal Root Ganglion Neurons

Objective:To investigate protective effects of hirudin on oxidative stress and apoptosis of spinal dorsal root ganglion cells in high-glucose rats at the cellular and molecular level.Methods:Dorsal root ganglion neurons(DRGn)were harvested from embryonic day in 15 SD rats,purified and identificated after primary culture.They were divided into the normal control group,high-glucose(HG)group,positive control(alpha-lipoic acid,ALA)group,low-dose hirudin group(H1),medium-dose hirudin group(H2)and high-dose hirudin group(H3).The control group was cultured by neuron specific culture medium,while the HG group was cultured by neuron specific culture medium and 20 mmol/L glucose(HG medium).The hirudin groups were cultured by HG medium+0.25 IU/mL hirudin(H1),HG medium+0.5 IU/mL hirudin(H2)and HG medium+1 IU/mL hirudin(H3).The ALA group was cultured by HG medium +100μmol/L ALA.3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylt etrazolium bromide(MTT)assay was used to explore the optimum concentration and intervention time.Flow cytometry assay was used to detect the level of reactive oxygen series(ROS).Western blot and quantificational realtime polymerase chain reaction(qRT-PCR)were used to detect the expression of protein and mRNA of nuclear factor erythroid 2-related factor 2(Nrf-2),hemeoxygence-1(HO-1),nuclear factor-κB(NF-κB)and Caspase-3.TUNEL assay was used to test the apoptosis rate of different groups.Results:After 24 h of culture,the cell activity of hirudin and ALA groups were higher than that of HG group,and there was a statistical difference between the H1 group and HG group(P<0.05).In hirudin groups,the apoptosis rate of cells,the expression of activated Caspase-3 protein and Caspase-3 mRNA were lower than those of HG group(P<0.01),higher than those of ALA group(P<0.01 or P<0.05).The ROS level of hirudin groups was higher than that of ALA group(P<0.01),lower than that of HG group(P<0.01 or P<0.05).The expression of NF-κB(P65)protein in H3 group were lower than those of HG group(P<0.05).The expression of Nrf-2 protein in hirudin groups was higher than that of HG group(P<0.01),lower than that of ALA group(P<0.01 or P<0.05).The expression of HO-1 protein in hirudin groups was lower than that of ALA group(P<0.01 or P<0.05),higher than that of HG group(P<0.01 or P<0.05).Conclusions:The activity of DRGn cells can be promoted by hirudin under HG conditions.The effects of hirudin on the inhibition of HG on DRGn cells damage mainly include scavenging ROS,up-regulating Nrf-2/HO-1 pathway,inhibiting activation of NF-κB pathway,down-regulating the expression of and Caspase-3 and reducing DRGn cell apoptosis.

重组水蛭素抗ApoE^(-/-) 小鼠动脉粥样硬化的研究

目的探讨重组水蛭素对ApoE^(-/-)小鼠动脉粥样硬化血脂水平和粥样斑块的影响。方法高脂饲料喂养雄性ApoE^(-/-)小鼠随机分为模型组和重组水蛭素低、中、高剂量给药组,正常饮食的C57BL/6J小鼠作为正常对照组。连续给药8周后,检测血脂水平;HE染色以及Masson染色观察主动脉血管病理学改变;CD68、α-SMA免疫组织化学染色分别观察重组水蛭素对粥样斑块中巨噬细胞浸润和平滑肌肌动蛋白的影响。结果与模型组比较,重组水蛭素各剂量给药组的血清总胆固醇、三酰甘油和低密度脂蛋白胆固醇水平均有不同程度的降低(P<0.05);各给药组与模型组的高密度脂蛋白胆固醇的差异无统计学意义(P>0.05);Masson染色显示模型组主动脉血管胶原异常增生,而各给药组胶原含量均有不同程度减少;CD68、α-SMA免疫组织化学染色显示,模型组主动脉粥样斑块处有大量CD68表达和少量α-SMA表达,而重组水蛭素各给药组粥样斑块处CD68表达减少(表示巨噬细胞浸润减少)、α-SMA表达增加(P<0.05)。结论重组水蛭素能降低ApoE^(-/-)小鼠血清血脂水平,延缓平滑肌细胞凋亡,抑制血管胶原增生,从而抑制动脉粥样硬化的发展。

水蛭素对脓毒症大鼠血小板功能的影响

目的研究水蛭素对脓毒症大鼠血小板功能的影响。方法将45只大鼠分为假手术组、模型组和水蛭素组。术后6 h和24 h对三组大鼠进行采血并检测血小板计数、血小板聚集率和P-选择素表达率水平。结果水蛭素组术后6 h血小板计数较模型组下降(P<0.05)。模型组术后24 h血小板计数较水蛭素组和假手术组降低(P<0.05),但假手术组术后24 h血小板计数较水蛭素组明显上升(P<0.05)。模型组术后6 h血小板聚集率较假手术组和水蛭素组升高(P<0.05),而水蛭素组术后6 h血小板聚集率较假手术组明显更高(P<0.05)。假手术组术后24 h血小板聚集率较模型组及水蛭素组明显更高(P<0.05)。水蛭素组术后24 h血小板聚集率较模型组更高(P<0.05)。术后6 h及24 h,模型组P-选择素表达率较假手术组及水蛭素组明显增加(P<0.05)。水蛭素组术后6 h及24 h的P-选择素表达率较假手术组更高(P<0.05)。结论水蛭素能提高脓毒症大鼠的血小板数量,增强聚集功能并下调P-选择素表达水平以发挥抗炎作用。

EXPRESSION OF SYNTHESIZED HIRUDIN GENE IN YEAST

Hirudin is a sort of polypeptides secreted from the salivary gland of medicinal leech. It is of potential importance in medicine. We designed and synthesized the hirudin gene based on the amino acid sequence of hirudin HV2, and expressed it using the yeast alpha factor expression system. The yeast strain stably carrying the hirudin expression plasmid was deduced by mutagenesis. After its cultivation in rich nutritious medium for 36—48 h, the hirudin expression product secreted into the culture fluid was 10—20 ATU/ml. The HPLCpure hirudin product could be obtained through a simpler purification procedure, its N-terminal amino acid sequence was identical with the natural product, and it showed potent anticoagulant and antithrombin activity. About 3000 ATU of pure hirudin witha specific activity of 6600 ATU/mg could be obtained from 500 ml of culture fluid.