Inhibitory Effect of Hirudin on Hepatocellular Carcinoma Cells

[Objectives]This study was conducted to explore the proliferation inhibition of hirudin on hepatocellular carcinoma HepG 2 cells and Huh-7 cells.[Methods]Hirudin solutions of different concentrations(2.0,2.5,3.0,3.5,4.0 mg/ml)were used to treat HepG 2 cells and Huh-7 cells.The effects of different hirudin concentrations on the proliferative activity of HepG2 and Huh-7 cells were detected by CCK-8 assay,and the IC 50 values were calculated.A living/dead cell double staining experiment was conducted to observe the fluorescence of cells under a fluorescent microscope,so as to assess the inhibitory effect of different concentrations of hirudin on the proliferation of HepG2 and Huh-7 cells.A cell scratch assay was carried out,and an inverted microscope was employed to observe the healing of the scratched areas,so as to assess the impact of hirudin on the migratory and invasive capabilities of hepatocellular carcinoma HepG2 and Huh-7 cells.[Results](i)The results of CCK-8 assay indicated that compared with the blank control group,the proliferation inhibition rates of both hepatocellular carcinoma HepG2 and Huh-7 cells increased with the concentration of hirudin increasing,demonstrating that hirudin had an inhibitory effect on the proliferative activity of these cells.Specifically,the IC 50 values for HepG2 and Huh-7 were found to be 3.5 and 4.0 mg/ml.(ii)The living/dead cell double staining experiment revealed that the number of living cells in the hirudin-treated group decreased significantly compared with the control group,while the number of dead cells increased markedly,indicating an inhibitory effect of hirudin on the proliferation of HepG2 and Huh-7 cells.(iii)The results of cell scratch assay showed that the healing degree of the scratched areas in the hirudin-treated groups was significantly lower than that of the control group,indicating a reduction in cell growth and migration capabilities.[Conclusions]Hirudin exhibited a significant inhibitory effect on the proliferation of hepatocellular carcinoma HepG2 and Huh-7 cells.

Separation and Purification of Recombinant Hirudin Variant 3 from Bacillus subtilis

[ Objective] The research aimed to get the optimized separation and purification conditions of the hirudin produced from Bacillus subtilis DB403 （pUBH5）. [Method] Through the systemic pretreatment, preliminary chromatography and fine chromatography. [Result]The optimized separation and purification conditions were that： Supernatant was treated by trichloroacetic acid, then by ultrafiltration desalt and anion exchange chromatography. Strong anion Q F. F. was better than weak anion DEAE F.F. The proper balanced solution was Tris-HCI （ pH 8.0）. The proper conductivity was 6 ms/cm. The maximum applied sample was 240 ATU/ml to matrix of strong anion Q F. F. This optimized procedure was magnified in strong anion exchange HiPrep 16/10Q with the 90% recovery and 70.2% purity. The purification of gel filtration of Sephacryl S-100 to hirudin was not relative to flow rate within certain scope. The application size of sample was 10 ml. The purity checked by HPLC was 95.1%, and the recovery was 93%, and the band of SDS-PAGE was single. [ Conclusion] The research provided the reference of the further industrialization separation and purification of hiruin.

Optimization for Purification and Characterization of Recombinant Hirudin Ⅲ from E. coli

Aim To optimize purification conditions of recombinant hirudin 3 in thefermentation broth and characterize the product. Methods Reambinant hirudin 3 was isolated andpurified from the fermentation broth by three column chromatography steps with macroporous resin,DEAE cellulose DES2 and preparative RP-HPLC, respectively, and the optimal conditions were obtained.Purity of the product was determined by SDS-PAGE and analytical RP-HPLC. The molecular weight wasdetermined by mass spec-trometry. The structure of the product was analyzed by peptide map.ResultsThe product with purity of 95.4786% was obtained after three purification steps in the optimumconditions with a total yield of 39%. The molecular weight of the product was 6 913.32 ± 6.55 Da,coincident to the theoretical molecular weight of r-hirudin 3. The structure of the product wascoincident to r-hirudin 3 either. Conclusion The optimized purification steps can be successfullyemployed for purification of r-hirudin 3 from E. coli using batch-type approaches. The productobtained with high purity was confirmed to be r-hirudin 3.

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。

PEGylation of Hirudin and Analysis of Its Antithrombin Activity in vitro

Hirudin is the most anticoagulant drug found in nature, but its short serum half-life significantly inhibits its clinical anpplication. The PEGvlation of hirudin, the most promising anticoagulant drug, was performed in this paper. The optimal reaction conditions for PEG ylated hirudin were investigated, wh.en the PEGylation react, on.wasconducted under 4℃ after 10h, in the borate buffer at pH 8.5 .with the molar ratio 230 ： 1 of PEG to hirudin, a higher modification extent was achieved. Finally, the bioactivity of PEGylated hirudin was measured in vitro.Compared with unmodified hirudin, 26% of anti-thrombin activity was retained.

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.

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.

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.

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.

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.

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.

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.

Exploring the mechanism of hirudin in the treatment of diabetic kidney disease using network pharmacology combined with molecular docking verification

OBJECTIVE:To explore the mechanism of hirudin in the treatment of diabetic kidney disease(DKD).METHOD:Cytoscape software was used to analyze the network between hirudin targets and active components in the treatment of DKD.The biological function and mechanism of effective targets of hirudin for DKD treatment were analyzed by the Database for Annotation,Visualization and Integrated Discovery(DAVID)database.Molecular docking technology was used to simulate the docking of key targets,and the DKD rat model was used to verify the first 4 key targets with high"Hydrogen number"among the top 10 targets verified by molecular docking.RESULTS:Total of 12334 DKD targets were screened in Gene Cards,OMIM and other databases,Hirudin and DKD had 247 common target genes,and the protein interaction network got 2115 edges.The DAVID database was used for the Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analysis,confirming that hirudin in treatment of DKD involves multiple signaling pathways such as the forkhead box O signaling pathway,the phosphatidylinositol 3-kinase-protein kinase B signaling pathway,the vascular endothelial-derived growth factor signaling pathway and other signaling pathways.The top ten key targets of hirudin in treatment of DKD were verified by molecular docking.Animal experiments showed that hirudin could decrease the expression of caspase-3 in renal tissue of DKD rats,and increase the expression of RAC-alpha serine/threonineprotein kinase,Catalase,and Heat shock protein HSP 90-alpha in renal tissue of DKD rats.CONCLUSION:This study preliminarily reveals that hirudin treats DKD through multiple targets and pathways,and molecular docking and animal experiments indicates the feasibility of this study.Hirudin may be directly or indirectly involved in the regulation of cell metabolism,oxidative stress and other mechanisms in the treatment of DKD,which will lay the foundation for future molecular biological experiments of hirudin in the treatment of DKD.

SA-hirudin-RGD融合蛋白的原核表达及其抗凝血酶与抗血小板聚集功能的研究

目的:构建SA-hirudin-RGD重组载体,表达和纯化融合蛋白,并对其抗凝血酶、抗血小板聚集功能进行初步验证。方法:利用基因重组技术将链霉亲和素(SA)核心区与hirudin-RGD序列连接,并克隆到原核表达载体PET-44b中,Westernblotting鉴定经IPTG诱导后纯化的融合蛋白。抗凝血酶和抗血小板聚集作用分析证明该融合蛋白既有抗凝血酶又有抗血小板聚集的功能。结果:重组载体p ET44b-SA-hirudin-RGD经限制性酶切鉴定和基因测序证实构建成功;经IPTG诱导后SA-hirudin-RGD融合蛋白在大肠杆菌中高效表达;纯化得到该目的蛋白,相对分子质量经Westernblotting鉴定约为70000。抗凝血酶和抗血小板聚集的实验证明,融合蛋白SA-hirudin-RGD既有抗凝血酶又有抗血小板聚集的功能。结论:具有抗凝血酶和抗血小板聚集双重功能的SA-hirudin-RGD融合蛋白被成功表达和纯化,为下一步SA-hirudin-RGD的功能研究及临床应用确立了基础。

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。

水蛭素对缺氧诱导心脏微血管内皮细胞间质转分化的作用及机制研究

目的探讨通络药物水蛭素对缺氧诱导的人心脏微血管内皮细胞(HCMECs)间质转分化(EndMT)的作用及可能机制。方法取常规培养的HCMECs细胞,随机分为对照组、缺氧组、水蛭素组(包括0、20、40、80、100μg/ml 5个浓度)。对照组常规培养不做任何处理,缺氧组置入低氧培养箱72 h,水蛭素组预加入Hirudin工作液,4 h后置入低氧培养箱72 h。MTS比色法检测HCMECs增殖能力;倒置显微镜观察HCMECs形态;免疫荧光鉴定HCMECs间质转分化情况,Western-blot检测内皮间质转分化相关蛋白:包括内皮细胞标记血小板—内皮细胞黏附分子(PECAM-1/CD31)、血管内皮钙黏蛋白(VE-cadherin),间质细胞标记α-平滑肌肌动蛋白(α-SMA)、成纤维细胞特异性蛋白-1(FSP-1),以及低氧诱导因子1α(HIF-1α)、转化生长因子β1(TGF-β1)、Smad同源物2/3(Smad2/3)、锌指转录因子(snail)等相关信号通路的蛋白表达。结果MTS法检测显示,缺氧显著抑制细胞活性(P<0.01),水蛭素在20~100μg/ml浓度范围内可提高细胞活性,且呈现浓度依赖性,当浓度为100μg/ml时细胞活性最强(P<0.01)。各组细胞培养72 h后,倒置显微镜下观察发现:对照组细胞呈铺路石样或鹅卵石状结构,缺氧组细胞由鹅卵石状结构变为分散的长梭形,接近成纤维细胞形态,水蛭素组长梭形细胞形态明显改善,细胞恢复鹅卵石样。Western-blot与免疫荧光结果显示:与对照组比较,缺氧组CD31、VE-cadherin蛋白水平降低(P<0.01),且vWF表达减少,α-SMA、FSP-1蛋白水平升高(P<0.01),且vimentin表达增强;与缺氧组比较,水蛭素明显增加CD31、VE-cadherin蛋白表达(P<0.01),并增强vWF表达,下调α-SMA、FSP-1蛋白表达(P<0.01),并减弱vimentin表达;与对照组相比,缺氧组信号通路HIF-1α、TGF-β1、p-smad2/3、snail蛋白表达均升高(P<0.01),与缺氧组比较,水蛭素下调HIF-1α、TGF-β1、p-smad2/3、snail蛋白表达(P<0.01)。结论水蛭素可以改善缺氧诱导的HCMECs细胞发生EndMT,其机制可能与调控HIF-α/TGF-β1/smad/snail通路有关。