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Stroke is one of the most common cerebrovascular diseases， including hemorrhagic stroke and ischemic stroke. From a modern medical perspective， stroke is caused by cerebrovascular damage or embolism leading to impaired blood circulation. From the traditional Chinese medicine （TCM） perspective， the pathogenesis of this disease is mainly due to the disorder of Qi and blood， which ascend to the brain， causing either blood extravasation or blockage of brain collaterals. Stasis is a pathological factor that runs throughout the entire course of stroke， and the method of promoting blood circulation and resolving stasis has been a core treatment for stroke for a long time. Hirudo， as a traditional insect drug， has shown good effects in promoting blood circulation and resolving stasis. Modern pharmacological research has confirmed that Hirudo contains anticoagulant components， which provide significant advantages in dissolving thrombi in ischemic stroke and facilitating hematoma absorption in hemorrhagic stroke. Hirudo and its related preparations have been proven to exert an anti-stroke effect through anticoagulation， anti-thrombosis， and protection of vascular endothelium. As a result， they have been widely used in the treatment of stroke. This article explored the theoretical basis and research status of using Hirudo for treating stroke based on its main active components and hemostatic properties and summarized the current research status of commonly used Hirudo-based formulations and preparations， aiming to provide references for the involvement of Hirudo in stroke treatment.
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Aim To observe the effect of hirudin on pulmonary inflammation and fibrosis in rats with bleo-myc in-induced pulmonary fibrosis and the underlying mechanism.Methods Sixty male SD rats were ran¬domly divided into control group, model group, hirudin treatment group ( low,medium and high concentration) and prednisone group.The control group received en-dotracheal injection of saline, while the remaining five groups carried out endotracheal one-time injection of blemycin to establish rat pulmonary fibrosis model.From the second day after modeling, hirudin treatment groups were respectively administered different concen¬trations of hirudin subcutaneous injection, while control group was given saline, and prednisone was gavaged with 5 mg • kg~1 prednisone acetate, then all rats were sacrificed on day 28.Lung lesions were observed by HE and Masson staining.The relative expression levels of COL 1 and ot-SMA mRNA were detected by real¬time fluorescence quantitative PCR.The content of hydroxy proline ( HYP) in lung tissues was determined by kit.The expression levels of p38MAPK/NF-KB sig¬naling pathway related proteins in lung tissues were de¬tected by Western blot, and IL-6 and TNF-cx levels were detected by ELISA.Results Compared with model group, the inflammatory response and interstitial fibrosis of lung tissues were improved, the content of hvdroxvproline decreased, the expression of p-p38 MAPK,NF-kB p-p65and p~IkB protein decreased, and the concentration of TNF-cx and IL-6 decreased after hirudin intervention.Conclusions Hirudin can effec¬tively inhibit alveolar inflammation and reduce the de¬velopment of pulmonary fibrosis, which may be related to regulating p38 MAPK/NF-kB signaling pathway,re¬ducing the inflammatory response of lung tissues and reducing the deposition of extracellular matrix.
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Objective: To investigate the effect of natural hirudin on revascularization of ischemic skin flap in rats using Micro-CT and three-dimensional (3D) reconstruction. Methods: Thirty-two Sprague Dawley rats were prepared a ischemic skin flap (8.0 cm×1.8 cm) model on the back and randomly divided into hirudin group and control group (16 rats in each group). At immediate and within 3 days after operation, the rats were treated with hypodermic injection of natural hirudin 0.3 mL (including natural hirudin 6 ATU) every day in hirudin group and the equal amount of normal saline in control group. At 6 days after operation, the survival rate of skin flap was evaluated, histological changes were observed by HE staining, and the volemia, length of blood vessels, and number of blood vessels were analyzed with Micro-CT 3D reconstruction. Results: Both groups of rats survived to the end of the experiment without infection. Different degrees of necrosis occurred in the distal part of the skin flaps in both groups at 6 days after operation, but the flap survival rate of the hirudin group (72.11%±8.97%) was significantly higher than that of control group (58.94%±4.02%) ( t=3.280, P=0.008). Histological observation showed that the histological hierarchy of the hirudin group was clearer than that of the control group, with more microangiogenesis and less inflammatory response and inflammatory cell infiltration. Micro-CT 3D reconstruction showed that the flap vessels in the hirudin group were more and denser, and the volemia, length of blood vessels, and number of blood vessels were significantly higher than those in the control group ( P<0.05). Conclusion: Natural hirudin can reduce the inflammation of tissue, promote the regeneration and recanalization of blood vessels in ischemic skin flap, so as to improve the survival rate of the flap.
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Objective: To explore the effect of hirudin on hyperuricemia rats and its mechanism. Methods: Male Wistar rats were randomly divided into control group, model group, allopurinol (30 mg/kg) group, hirudin low-, middle- and high-dose (0.2, 0.4, 0.8 g/kg) group. Rats were ig potassium oxonate (0.75 g/kg) to induce hyperglycemia model, once a day for five weeks. And all administration groups were respectively ig corresponding doses of drugs. The level of uric acid in serum and urine of rats were measured by biochemical method; The level of organic anion transporter 1 (OAT1) in kidney was measured by immunohistochemistry; The protein expressions of glucose transporter 9 (GLUT9), OAT1 and urate transporter 1 (URAT1) in kidney were measured by western blotting; The expression levels of GLUT9, OAT1 and URAT1 mRNA in kidney were detected by qRT-PCR. Results: Compared with control group, the level of uric acid in serum and urine of rats in model group was significantly increased (P < 0.01), the expressions of GLUT9, URAT1 mRNA and protein were significantly increased (P < 0.01), the expressions of OAT1 mRNA and protein were significantly decreased (P < 0.01). Compared with model group, the level of uric acid in serum and urine of rats in hirudin group were significantly decreased (P < 0.01), the expressions of GLUT9, URAT1 mRNA and protein were significantly reduced (P < 0.01), the expressions of OAT1 mRNA and protein were significantly increased (P < 0.01). Conclusion Hirudin can reduce the uric acid by regulating the expressions of renal urate transporters OAT1, URAT1 and GLUT9 in hyperuricemia rats.
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The Siddha system of medicine is an unique traditional system of medicine practiced in Tamil speaking countries particularly India. In Siddha system of medicine the diseases were treated through internal and external medicine. In external medicine, the leech therapy is one which is used to treat various non-surgical and surgical cases. The leeches have been used for therapeutic purposes since their beginning of civilisation. Ancient Indian, Egyptian, Greek and Arab physicians were used the leeches for wide range of diseases such as inflammatory condition, skin diseases, respiratory disorders, eye diseases, cardiac diseases, urinary and reproductive diseases and dental diseases. The leeches are also used as one of the bloodletting methods in Unani, Ayurveda, and Modern science for various clinical conditions. Recently, researches on leech saliva revealed the presence nearly 100 biologically active compounds such as Hirudin, vasodilators, hyaluronidase, anaesthetics, fibrinases, antibacterial, collagenase etc. The above mentioned pharmacological compounds are injected into human body while sucking of the blood and are responsible for the anaesthetic, anti-inflammatory, anticoagulant and analgesic effect of leech application. In current scenario, the leech therapy is used for various diseases including life threatening diseases such as cardiovascular diseases, cancer, etc. in different system of medicine including modern medicine. Leech therapy is used in plastic and microsurgery as a protective tool against vascular congestion in modern medicine. The Scientific studies on leech saliva were proven and support the leech application in Siddha system of medicine.
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Objective: To investigate the effect of natural hirudin combined with hyperbaric oxygen therapy on the survival of transplanted random-pattern skin flap in rats.
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Objective: To explore the effect of natural hirudin on proliferation of human microvascular endothelial cells (HMVECs) and its preliminary mechanism of promoting angiogenesis. Methods: Three-dimensional culture models of HMVECs were established in vitro and observed by inverted phase contrast microscopy after 24 hours of culturing. Then, the three-dimensional culture models of HMVECs were treated with different concentrations (1, 4, and 7 ATU/mL) of the natural hirudin, respectively, and Dulbecco's modified Eagle's medium containing 10% fetal bovine serum as control. The cell proliferations of 4 groups were detected by cell counting kit 8 (CCK-8) method at 24, 48, and 72 hours; the angiogenesis of 4 groups were observed by tube formation assay at 24 hours; the expressions of vascular endothelial growth factor (VEGF) and Notch1 of HMVECs in 4 groups were observed by immunofluorescence staining at 24 hours. Results: The observation of cells in three-dimensional culture models showed that HMVECs attached to Matrigel well, and the cells formed tube structure completely after 24 hours. The results of CCK-8 test showed that the absorbance ( A) value of 1 and 4 ATU/mL groups were higher than that of control group at each time point ( P0.05). The results of immunofluorescence staining showed that compared with control group, the Notch1 expression was higher in 1 and 4 ATU/mL groups and lower in 7 ATU/mL group; and there was significant difference between 4 and 7 ATU/mL groups and control group ( P<0.05). The VEGF expression was higher in 1, 4, and 7 ATU/mL groups than in control group, in 4 ATU/mL group than in 1 and 7 ATU/mL groups, showing significant differences ( P<0.05). Conclusion: Natural hirudin can promote angiogenesis at low and medium concentrations, but suppress angiogenesis at high concentrations. Its mechanism may be related to the VEGF-Notch signal pathway.
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Objective: To investigate the anti-gout effect of hirudin and its mechanism. Methods: Hypoxanthine was used to replicate mouse model of hyperuricemia. Sodium uric induced acute gouty inflammation in rats to observe the effect of hirudin on the level of uric acid and inflammation induced by acute hyperuricemia. The serum uric acid, serum urea nitrogen, serum xanthine oxidase activity, and liver xanthine oxidase activity were observed in chronic hyperuricemia mouse model induced by potassium oxonate. The changes of renal pathology, the level of C-reactive protein (CRP) and the expression of glucose transporter 9 (GLUT9) were also be tested. Results: Hirudin could significantly reduce the serum uric acid level in hyperuricemia mice induced by hypoxanthine and significantly inhibit acute toe swelling induced by sodium uric acid in rats. It could significantly reduce the level of serum hyperuricemia, decrease the level of blood urea nitrogen (BUN), significantly inhibit the expression of GLUT9 and alleviate the pathological changes of kidney in chronic hyperuricemia mice induced by potassium oxazinate. Conclusion: Hirudin has significant anti-hyperuricemia and anti-gout effects, and its mechanism may be related to the protection of kidney and the regulation of GLUT9 expression.
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OBJECTIVE@#To investigate the antithrombotic effects of recombinant hirudin and its mechanism.@*METHODS@#Sixty male Kunming mice were randomly divided into 6 group (=10):control group, model group, aspirin (25 mg/kg) group, recombinant hirudinlow, middle and high dose (0.05, 0.1, 0.2 mg/kg) groups.Except mice in control group, 2.5 mg/kg carrageenan was injected intraperitoneallyto mice in the other groups to produce thrombosis on the mice tail. The mice in aspirin group were administrated intraperitoneally 25 mg/kg aspirin, the mice in recombinant hirudinlow, middle and high dose groups were administrated intraperitoneally 0.05, 0.1, 0.2 mg/kg combinanthirudin, the mice in control group and model group were administrated intraperitoneallynormal saline at the same volume respectively at 24 h, 0.5 h before injecting carrageenan and 24 h after injecting carrageenan. The black tail length of mice and the incidence of black tail were observed at 48h after injection of carrageenan; prothrombin time (PT), activated partial thromboplastin time (APTT), tissue plasminogen activator (t-PA), type-1 plasminogen activator inhibitor (PAI-1), 6-keto-PGF1α, and thromboxane B2 (TXB2) level in mice plasma were determined.@*RESULTS@#As compared with control group, the mice in model group presented tail thrombosis; PT level in plasma was significantly shortened (<0.01), PAI-1 and TXB2levels in plasma were significantly increased (<0.01), while the t-PA and 6-keto-PGF1α levels in plasma in model group were significantly decreased (<0.01). As compared with model group, the thrombus length in the tail was significantly shortened (<0.05, <0.01), PT level was obviously prolonged (<0.01), and the plasma levels of PAI-1 and TXB2 were significantly decreased (<0.01), while the plasma levels of t-PA and 6-keto-PGF1α were significantly increased (<0.01)in the mice of recombinant hirudin low dose, middle dose, high dose groups and aspirin group. As compared with aspirin group, the thrombus length in the tail was significantly increased (<0.05), PT level was obviously shortened (<0.01), and the plasma levels of PAI-1 and TXB2 were significantly increased (<0.01)in the mice of recombinant hirudin low dose group; the plasma level of 6-keto-PGF1α was significantly decreased (<0.01, <0.05) in the mice of recombinant hirudin low dose and middle dose groups; the plasma levels of PAI-1 and TXB2 were significantly increased (<0.01, <0.05)in the mice of recombinant hirudin middle dose group.@*CONCLUSIONS@#The recombinant hirudin can fight against thrombosis, its antithrombotic mechanisms may be related to its influence on the exogenous coagulation system and the promotion of fibrinolysis function.
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Animals , Male , Mice , Blood Coagulation , Fibrinolytic Agents , Hirudins , Pharmacology , Recombinant Proteins , Thromboxane B2 , Tissue Plasminogen ActivatorABSTRACT
Objective: To study the anti-gout effect of active ingredients in Poecilobdella manillensis. Methods: Hypoxanthine was used to replicate mouse model of hyperuricemia, and xylene was used to induce mouse auricle swelling model. The hot plate method and writhing method were used to screen the active site of Poecilobdella manillensis, and then the active ingredients were screened. The material basis of anti-gout effect of Poecilobdella manillensis was observed. Results: The water-soluble fraction of Poecilobdella manillensis was the active site against gout, which could reduce the level of serum uric acid in hypoxanthine-induced hyperuricemic mice and inhibit xylene-induced auricular swelling in mice, deduce acetic acid-induced writhing reaction in mice and increase the hot plate pain threshold in mice; Hirudin was the main active ingredient in water-soluble parts. Poecilobdella manillensis active ingredient 0.8 g/kg and 0.4 g/kg and Poecilobdella manillensis residue 2.0 g/kg could significantly reduce serum uric acid levels. The serum uric acid levels decreased from232.73 ± 50.93 umol/L in model group to 140.70 ± 25.97 umol/L, 149.07 ± 39.28 umol/L, 176.45 ± 44.33 umol/L, respectively (P ＜ 0.01) . Poecilobdella manillensis active ingredients 0.8 g/kg, 0.4 g/kg and 0.2 g/kg and Poecilobdella manillensis residue 2.0 g/kg could significantly inhibit xylene-induced ear auricle swelling in mice. The swelling degree was inhibited from 22.80 ± 2.86 mg to 20.10 ± 2.18 mg, 19.80 ± 2.57 mg, 20.10 ± 1.66 mg and 20.85 ± 1.60 mg respectively (P ＜ 0.05) . Poecilobdella manillensis 0.8 g/kg active ingredient could significantly reduce the number of writhing mice caused by acetic acid. The number of times was reduced from 22.80 ± 2.86 times to 20.10 ± 2.18 times (P ＜0.05) . Conclusion: Poecilobdella manillensis anti-gout activity is in water-soluble parts, and Hirudin is the main active ingredient.
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OBJECTIVE:To study the pharmacokinetic characteristics of Recombinant hirudin enteric-coated capsule by single and multiple administration in Beagle dogs. METHODS:12 Beagle dogs were divided into single ig group and single iv group by random control method,6 in each group. Recombinant hirudin 0.2 mg/kg was intragastrically administrated or intravenously inject-ed,blood sample was collected;after 2 weeks of cleaning,12 dogs were intragastrically administrated recombinant hirudin 0.2 mg/kg,for 7 d. Sample blood was collected,referred to multiple ig group. Recombinant hirudin concentration in plasma was deter-mined by enzyme-linked immunosorbent assay,and pharmacokinetic parameters were calculated by DAS 2.0 software. RESULTS:The results showed that pharmacokinetics by ig and iv recombinant hirudin in Beagle dogs fitted to two-compartment model,abso-lute bioavailability of ig Recombinant hirudin enteric-coated capsule was(14.908±1.868)%;the pharmacokinetic parameters in sin-gle ig group and multiple ig group were tpeak of(2.105±0.243),(3.000±0.000)h,t1/2β of(8.660±2.965),(14.870±2.710)h, cmax of(10.700±0.872),(12.05±1.587)ng/mL,AUC0-1440 min of(55.250±4.386),(58.978±6.002)ng·h/mL,without statistical significances in two groups(P>0.05). CONCLUSIONS:The ig Recombinant hirudin enteric-coated capsule can be absorbed into the blood to a certain extent. There is no accumulation for ig Recombinant hirudin enteric-coated capsule for several days,and it dose not change the pharmacokinetic characteristics.
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Hirudin as a novel anticoagulant and antithromotic agent, is a kind of protein that is obtained from the salivary gland secretion of one kind blood-sucking leech.It increases the difficulty extraction and purification of hirudin that composition of no anticoagulant activity of peseudo-hirudin in the body of leech and a large number of complex material from the salivary gland secretion of one kind blood-sucking leech.This article focuses on reviewing the structure of hirudin, genetic makeup, extraction and purification of hirudin according to the related research.Analysis of the pros and cons of various extraction and purification methods.Looking forward to its developing tendency, so as to provide reference for extraction and purification of Hirudin.
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Objective To investigate the effect of hirudin on janus kinase 2 / signal transducer and activator of transcription 3 (JAK2 / STAT3)signaling pathway in rats with intracerebral hemorrhage. Methods A total of 216 healthy male Wistar rats were divided into sham operation group,model group, and hirudin group (n =72 in each group)using random digit table. The model of cerebral hemorrhage was induced by injecting autologous arterial blood into the caudate nucleus,and the equal volume isotonic saline was used instead of autologous arterial blood in the sham operation group. Hirudin were injected at 6,24, 72,and 120 h,respectively in the hirudin group,while the sham operation group and the model group were injected with isotonic saline during same period. Neurobehavioral scores,brain water content and brain coefficients of the 3 groups at the different time points were compared. The changes of brain cell apoptosis were detected with annexin V-fluorescein isothiocyanate/ propidium iodide double staining flow cytometry. The expression levels of phosphorylated JAK2 (p-JAK2)and phosphorylated STAT3 (p-STAT3)in brain tissue were detected with Western blot. Results (1)Compared with the sham operation group at same time period,neurobehavioral score,brain water content,brain coefficients,brain cell apoptosis rate,and the expression levels of p-JAK2 and p-STAT3 were increased significantly in the rats of the model group. The differences between the two groups were statistically significant (all P < 0. 05). Compared with the model group at same time period,neurobehavioral score,brain water content,brain coefficients,and brain cell apoptosis rate were decreased significantly in the rats of the hirudin group. The differences between the two groups were statistically significant (all P <0. 05). (2)At 6,24,72,and 120 h after modeling,the expression levels of p-JAK2 were 0. 632 ±0. 036,0. 783 ± 0. 045,1. 250 ± 0. 071,and 1. 006 ± 0. 052,respectively,and those of p-STAT3 were 0. 155 ±0. 005,0. 193 ±0. 006,0. 379 ±0. 012,and 0. 317 ± 0. 010,respectively in model group. The expression levels of p-JAK2 were 0. 267 ± 0. 014,0. 248 ± 0. 013,0. 329 ± 0. 018,and 0. 283 ±0. 016,respectively,and those of p-STAT3 were 0. 139 ± 0. 004,0. 081 ± 0. 001,0. 283 ± 0. 009, and 0. 174 ± 0. 005,respectively in the hirudin group. The expression levels of p-JAK2 and p-STAT3 in hirudin group were lower than those in the model group. There were significant differences between the two groups (all P < 0. 05). Conclusion Hirudin can reduce apoptosis of brain cells after intracerebral hemorrhage in rats,its protective mechanism may be associated with the inhibition of JAK2 / STAT3 signaling pathway.
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Objective To analyse the clinical effect and mechanism of hirudin in the treatment of patients with lower limb venous thrombosis after fracture.Methods 56 patients who were diagnosed with lower limb venous thrombosis after fracture in our hospital were collected.All patients were randomly divided into experimental group (n:28) and control group (n:28).12h after fracture operation, patients in control group were treated with subcutaneous injection of low molecular weight heparin calcium, patients in experimental group were treated with Maixuekang capsule ( hirudin) orally on the basis of control group.Before and after the treatment, the plasma levels of thrombin antithrombin complex ( TAT) , thrombin activated fiber soluble inhibitor (TAFI), D-Dimer (D-D), fibrinogen (FIB), thrombin time (TT), prothrombin time (PT), activated partial thromboplastin time (APTT) and clinical effect were detected in all patients.Results After treatment, the total effective rate of experimental group ( 92.86%) was significantly higher than that of the control group ( 71.43%) ( P<0.05 );compared with control group, the plasma levels of TAT, TAFI were lower in the experimental group (P<0.05); the plasma levels of D-D, FIB were lower in the experimental group (P<0.05); the plasma levels of TT,PT,APTT were higher in the experimental group ( P<0.05 ) .Conclusion The hirudin could inhibit thrombin activation and coagulation function in fracture patients with lower limb venous thrombosis, decrease the plasma D-D and FIB levels for improving blood hypercoagulable state and preventing thrombosis,thus improve the clinical efficacy.
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Objective To investigate the applying comparative values of anticoagulant and thrombolytic Sak-hirudin fusion protein used in cardiopulmonary bypass.Methods From february 2013 to July 2014,selected nitinol sheet into uncoated group (group A),base coated group (chitosan,group B),chitosan/ Sak group (group C),chitosan/hirudin group (group D),chitosan/Sak-hirudin fusion protein group (group E),there were included in the fresh round of blood hemolysis,the blood cell hemolysis rates were calculated.Meanwhile used the healthy newborns umbilical vein endothelial cells were added into various categories nitinol sheet for cell compatibility testing.Results The samples hemolysis rates in the 5 groups were around 1.5% and the RBC,WBC and PLT counts compared in the 5 groups were showed no differences (P 0.05).Randomized double-blind cell count result shows that the group C,D and E compared to the group A,B were more significant differences (P <0.05), at the same time,the group E compared to the group C,D differences were statistically significant (P <0.05).Conclusion Compared to Staphylokinase and hirudin alone,Sak-hirudin fusion protein used in cardiopulmonary bypass can play stronger anticoagulant and antithrombotic effects,its safety and ensures the effectiveness that interventional treatment of cardiovascu-lar disease.
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Objective To investigate the protective effect and mechanism of recombinant hirudin on avulsed flap and to explore the application of recombinant hirudin in clinical treating avulsed flap.Methods 108 Sprague-Dawley rats were randomly divided into three groups:local injecting recombinant hirudin (group A),local injecting low molecular heparin (group B),and local injecting physiological saline (group C).The far ends were sutured after forming dorsal avulsed flap with local freeing.The levels of P-selectin and VEGF were detected,and the inflammatory cell aggregation and formation of microthrombus were observed in pathological section.On 14th postsurgery day the survival rate of flap was calculated.Results The content of P-selectin in group A was lower than that of groups B and C,with a statistical difference (P＜0.05).The content of VEFG in group A was higher than that of groups B and C,with a statistical difference (P＜0.05).On 14th postsurgery day,group A was higher than that in groups B (P＜0.05)and C (P＜0.01) in the survival rate of flap.Conclusions Locally injected recombinant hirudin can actively suppress the production of P-selectin,reduce accumulation of inflammatory cells,prevent information of microthrombus,improve ischemia and anoxia of avulsed flaps,decrease injuries of endotheliocyte,enhance the expression of VEGF,and stimulate hyperplasia of neogenesis capillaries that elevate survival rate of avulsed flaps.
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OBJECTIVE: To evaluate the anti-thrombus activitity of EPR-hirudin (EH), a derivative of hirudin obtained by adding three amino acids at the nitrogen terminus. METHODS: The anti-thrombus activitity of EH was investigated by chromophore substrate method in vitro as well as by rat models of carotid arterial thrombosis and inferior vena cava thrombosis in vivo. RESULTS: The result of anti-thrombin experiment in vitro showed that EH did not produced anti-thrombin activity unless it was cleaved by blood coagulation factors. Then the anti-thrombus feature of EH was confirmed in vivo separately in the rat models of carotid arterial thrombosis and inferior vena cava thrombosis. The formation of arterial clot was inhibited and the weight of venous clot was decreased after administration of EH in a dose-independent manner. However, the blooding time, activated partial thromboplastin time (APTT) and thrombin time (TT) did not significantly increase in EH groups compared with HV and low molecular heparin. CONCLUSION: EPR-hirudin can inhibit the formation of arterial and venous clot and has low risk of blooding.
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OBJECTIVE: To establish an extraction method of hirudin by double aqueous phase combined with gel chromatography. METHODS: Anti-thrombin activity units (ATU) was adopted as the index. On the basis of optimizing the double aqueous phase extraction conditions, a new extraction technology of hirudin was established by employing double aqueous phase system combined with gel chromatography. The double aqueous phase was composed of polyethylene glycol and ammonium sulfate and took the digestive juice of Poecilobdella manillensis from Guangxi Province as the raw material. RESULTS: In the studied experimental range, the optimum technological conditions of double aqueous phase extraction of hirudin reference substantce were as follows: the contents of PEG 2000, (NH4)2SO4 and NaCl were 22%, 20% and 0.04%, respectively. The extraction temperature was 35°C, and pH was 6.0. The obtained extracts were further purified by gel chromatography. The recoveries of ATU reached 81.92%, 80.34% and 80.36% for hirudin reference substance, hirudin crude extracts and scale-up experiment, respectively. The specific activity of obtained products reached 3210.27 ATU · mg-1. The results of discontinuous polyacrylate gel electrophoresis and spectral scan for the extract were the same with for the reference substance. CONCLUSION: The double aqueous phase extraction combined with gel chromatography is good for hirudin. Copyright 2012 by the Chinese Pharmaceutical Association.
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Background Recombinant hirudin variant Ⅲ(rHV3) can effectively prevent galactose-induced human lens epithelial cells LECs injury,but little is known about the molecular mechanism of its action.Objective The present study was to investigate the effects of rHV3 on the expression of apoptosis-related genes in damaged LECs induced by galactose.Methods The rHV3 was extracted by our research group,and the biological activity of rHV3 was identified by titration of thrombase according to Markwardt's method.Human LECs (SRA01/04) were cultured using 125×10-3 mol/L D-galactose+10% FBS+D/F12 medium to establish the damaged human LECs model.rHV3 was added into the medium of the damaged human LECs model.Human LECs were cultured in D/F12 medium containing 10% FBS as normal control.The expression of apoptosis-related genes,such as aldose reductase (AR),bax,bcl2 and p53,in LECs at the mRNA level was detected using RT-PCR.The abundance ratio of target genes was presented with the absorbance (A) of gene mRNA/GAPDH mRNA.Results Compared to the normal control group,the A values of AR mRNA/GAPDH mRNA,bax mRNA/GAPDH mRNA and p53 mRNA/GAPDH mRNA were significantly elevated in model group (t=3.90E-06,t=8.44E-04,t=5.15E-08,P＜0.01).However,in the rHV3-treated group,the A values of AR mRNA/GAPDH mRNA,bax mRNA/GAPDH mRNA and p53 mRNA/GAPDH mRNA were lower than those of model group (t=5.90E-06,t=1.51E-04,t=3.42E-06,P＜0.01).The bcl2 mRNA/GAPDH mRNA was markedly downregulated in the model group when compared with the normal control group (t=1.86E-05,P＜0.01);while after rHV3 addition,bcl2 mRNA/GAPDH mRNA increased in comparison with the model group (t=8.56E-05,P＜0.01).Conclusion 125×10-3mol/L D-galactose induces the damage and apoptosis of human LECs.rHV3 likely plays a protective function on D-galactose-induced damage of human LECs by inhibiting the polyol pathway and mitochondria-mediated pathway.
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Objective To study the biological effects of hirudin on the cell circle of fibroblasts in hypertrophic scar.Methods Fibroblasts were taken from normal skin and hypertrophic scar,and cultured in vitro with various concentrations of hirudin.Then,flow cytometry was used for observing cellcycles,and Western blot for some proteins (p27 and cyclin E) related with cell circle.Results With the increasing of the concentrations of hirudin,fibroblasts on G1 phase increased and on S phase decreased,and the expression of protein p27 increased,but that of protein cyclin E decreased.Conclusion Hirudin can influence the cell circle of fibroblasts derived from normal skin and hypertrophic scar by means of the expression of some proteins related with celI circle.Hirudin causes G1 phase arrest and inhibits the production of fibroblasts.Therefore,it seems that hypertrophic scar could be prevented and treated with hirudin.