Effects of Danhong Injection (丹红注射液) and its main components on anticoagulation and fibrinolysis in cultured vein endothelial cells.

OBJECTIVE: To observe the effects of Danhong Injection (丹红注射液) and its main components, including daiclzein and hydroxysafflor yellow A (HSYA), on the anticoagulation, fibrinolysis, anti-apoptosis in hypoxia model of vein endothelial cells (VECs). METHODS: VECs were prepared and were put in a hypoxia environment, which consisted of mixed gas of 95% N and 5% CO mixed gas, when reached confluent culture. Five groups used different treatments, including normal control group, hypoxia group, daiclzein group, HSYA group and Danhong Injection group. The VECs were identified by fluorescence double labeling methods. The morphology was observed by a phase contrast microscopy. The effects of Danhong Injection, daiclzein and HSYA on 6 keto prostaglandin F1α (6-keto-PGF1α) level was measured by the method of radioimmunoassay (RIA). Superoxide dismutase (SOD) activity was tested by water soluble tetrazolium salt. The content of malondialdehyde (MDA) was measured by thiobarbituric acid. The activities of tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor (PAI) were measured by the method of chromogenic substrate. The contents of endothelin (ET) and nitric oxide (NO) were detected by non-equilibrium RIA and enzymelinked immunosorbent assay. Cells apoptosis rate was determined by flow cytometry. RESULTS: Compared with the normal control group, the floating cells number, PAI activity, ET and MDA contents, and cells apoptosis rate in the culture solution of hypoxia group were all significantly increased, whereas the 6-keto-PGF1α and NO contents, and t-PA and SOD activities were decreased significantly (P<0.01). Compared with the hypoxia group, Danhong Injection markedly increased the 6-keto-PGF1α content and SOD activity, regulated PAI and t-PA activities, ET and NO contents, and decreased MDA content and cells apoptosis rate (P<0.05 or P<0.01). CONCLUSIONS: Danhong Injection and its main components played an important role in protecting primary VECs from hypoxic damage by regulating the secretion and vasomotor function of VECs. The function of Danhong Injection was most remarkable.

Prevention of experimental postoperative peritoneal adhesions through the intraperitoneal administration of tanshinone IIA.

Postoperative adhesions develop after nearly every abdominal surgery. The formation of adhesions is associated with the inflammatory response, fibrinolytic system, and extracellular matrix deposition in response to injury. Tanshinone IIA is one of the major extracts obtained from Salvia miltiorrhiza, which has anti-inflammatory effects on many diseases. Postoperative adhesions were induced by injuring the parietal peritoneum and cecum in Wistar rats, followed by the administration of various dosages of tanshinone IIA. The adhesion scores for each group were collected seven days after the initial laparotomy. The activity of the tissue-type plasminogen activator in the peritoneal lavage fluid was measured. The messenger ribonucleic acid expression levels of the tissue-type plasminogen activator, plasminogen activator inhibitor-1, and cyclooxygenase-2 in the ischaemic tissues were measured by quantitative real-time polymerase chain reaction. The intraperitoneal administration of tanshinone IIA is effective for the prevention of the formation of postoperative adhesions in rats. Tanshinone IIA increased fibrinolytic activity in the peritoneal lavage fluid and tissue-type plasminogen activator messenger ribonucleic acid expression in ischaemic peritoneal tissues but decreased the plasminogen activator inhibitor and cyclooxygenase-2 messenger ribonucleic acid expression significantly. These results revealed that tanshinone IIA was a potent postoperative adhesion preventer by enhancing fibrinolytic activity and decreasing cyclooxygenase-2 activity.

Inhibitory effects of Physalis angulata on tumor metastasis and angiogenesis.

UNLABELLED: ETHNOPHARMACOLOGICAL RELAVENCE: Physalis angulata is well-known in traditional Chinese medicine as a ingredient for various herbal formulation; also, it has been shown to exhibit anti-cancer and anti-inflammatory effects. In this study, the ability of P. angulata to inhibit tumor metastasis and angiogenesis was investigated. MATERIALS AND METHODS: Anti-proliferative activity of ethyl acetate extracts of P. angulata (PA extracts), was determined against human oral squamous carcinoma (HSC-3) and human umbilical vein endothelial cells (HUVECs) by trypan blue exclusion method. Wound-healing migration, trans-well invasion, Western blotting and chick chorioallantoic membrane assay were carried out to determine the anti-metastatic and anti-angiogenic effects of PA extracts in vitro and in vivo. RESULTS: We demonstrated that at sub-cytotoxic concentrations of PA extracts (5-15 µg/mL) markedly inhibited the migration and invasion of highly metastatic HSC-3 cells as shown by wound-healing repair assay and trans-well assay. Gelatin zymography assay showed that PA extracts suppressed the activity of matrix metalloproteinase (MMP)-9 and -2, and urokinase plasminogen activator (u-PA) in HSC-3 cells. In addition, Western blot analysis confirmed that PA extracts significantly decreased MMP-2 and u-PA protein expression in HSC-3 cells. Notably, PA extracts significantly augmented the expression of their endogenous inhibitors, including tissue inhibitors of MMP (TIMP-1 and -2), and plasminogen activator inhibitors (PAI-1 and -2). Further investigations revealed that non-cytotoxic concentration of PA extracts (5-15 µg/mL) inhibited vascular endothelial growth factor (VEGF)-induced proliferation, and migration/invasion of HUVECs in vitro. PA extracts also suppressed the activity of MMP-9, but not MMP-2, in HUVECs. Further, we observed, PA extracts strongly suppressed neovessel formation in the chorioallantoic membrane of chick embryos in vivo. CONCLUSIONS: These results strongly support an anti-metastatic and anti-angiogenic activity of P. angulata that may contribute to the development of better chemopreventive agent for cancer and inflammation.

Salvianolic acid B modulates hemostasis properties of human umbilical vein endothelial cells.

Salviae miltiorrhizae (SM), a clinical, commonly used herb, can activate blood circulation and resolve stasis. We have investigated the effects of salvianolic acid B (Sal B), a pure compound extracted from the dried SM roots, on fibrinolytic (tissue-type plasminogen activator and plasminogen activator inhibitor, t-PA and PAI) and anticoagulant (thrombomodulin,TM) properties of cultured human umbilical vein endothelial cells (HUVECs). When HUVECs were treated with Sal B, a dose- (0.0125-0.5 mg/ml) and a time-dependent decrease in PAI activity were observed. PAI type 1 (PAI-1) antigen and PAI-1 mRNA expression significantly decreased compared to control values in the conditioned media of HUVECs pretreated with Sal B for 12 h. Moreover, TM activity reached a maximum stimulation of 1.25-fold over control levels in the pretreatment of Sal B for 12 h and t-PA and TM specific mRNA expression also increased (1.7- and 1.8-fold, respectively). In conclusion, Sal B increased the fibrinolytic and anticoagulant potential of cultured HUVECs by up-regulating the expression of t-PA and TM and by down-regulating the expression of PAI-1. These data suggest that Sal B is clinically effective because of its ability to change the gene expression profile of endothelial cells thereby preventing vascular events.

[Curative machanism of Shenle capsule on 5/6 nephrectomy rats].

OBJECTIVE: To explore curative machanism of Shenle capsule on the 5/6 nephrectomy rats. METHOD: Fibrin plate method was applied to examine activity of urinary plasminogen activator(PA). Semi-quantitative analysis was used to observe stained intensity and area of tissue-type plasminogen activator, urokinas-type plasminogen activator/ plasminogen activator inhibitor(tPA, uPA/PAI-1)in remnant renal tissue. Northern blot was employed to analyze the expression of transforming growth factor (TGF-beta) mRNA. RESULT: In model control group, the urinary PA activity and protein expression of tPA, uPA were down-regulated, but protein expression of PAI-1, TGF-beta mRNA was up-regulated in remnant renal tissue. In each treated group, the urinary PA activity and protein expression of tPA/uPA were enhanced,but the protein expression of PAI-1, TGF-beta mRNA decreased simultaneously. CONCLUSION: Shenle capsule can delay glomerulosclersis and tubulointerstitial fibrotic lesions of remnant kidney by improving the activity of urinary PA and modulating the expression of tPA, uPA/PAI-1 and TGF-beta mRNA.

Applying a highly specific and reproducible cDNA RDA method to clone garlic up-regulated genes in human gastric cancer cells.

AIM: To develop and optimize cDNA representational difference analysis (cDNA RDA) method and to identify and clone garlic up-regulated genes in human gastric cancer (HGC) cells. METHODS: We performed cDNA RDA method by using abundant double-stranded cDNA messages provided by two self-constructed cDNA libraries (Allitridi-treated and paternal HGC cell line BGC823 cells cDNA libraries respectively). Bam H I and Xho I restriction sites harbored in the library vector were used to select representations. Northern and Slot blots analyses were employed to identify the obtained difference products. RESULTS: Fragments released from the cDNA library vector after restriction endonuclease digestion acted as good marker indicating the appropriate digestion degree for library DNA. Two novel expressed sequence tags (ESTs) and a recombinant gene were obtained. Slot blots result showed a 8-fold increase of glia-derived nexin/protease nexin 1 (GDN/PN1) gene expression level and 4-fold increase of hepatitis B virus x-interacting protein (XIP) mRNA level in BGC823 cells after Allitridi treatment for 72h. CONCLUSION: Elevated levels of GDN/PN1 and XIP mRNAs induced by Allitridi provide valuable molecular evidence for elucidating the garlic's efficacies against neurodegenerative and inflammatory diseases. Isolation of a recombinant gene and two novel ESTs further show cDNA RDA based on cDNA libraries to be a powerful method with high specificity and reproducibility in cloning differentially expressed genes.

Effect of alpha-tocopherol on plasma testosterone and plasminogen activator activity or inhibition in ram spermatozoa.

The objective of this study was to evaluate the effect of alpha-tocopherol on blood testosterone and specific proteolytic enzymes in spermatozoa and seminal plasma, with final aim the increase of sperm fertilizing ability with a nutritional supplement. The effect of alpha-tocopherol supplementation on testosterone parameters (mean value, basal level, peak number, mean concentration value during peaks, peak amplitude, peak duration) and plasminogen activator activity (PAA), plasminogen activator inhibition (PAI) and plasmin inhibition (PI) of spermatozoa and seminal plasma was studied in the ram during autumn (estrous period for the sheep in Greece) and spring (anestrous period). Treated animals showed a marked increase in serum alpha-tocopherol. Testosterone parameters were not affected by the alpha-tocopherol in either autumn or spring, however, the spermatozoal PAA and PAI (anti-tPA) were increased in the spring but not in autumn. These enzymes and their inhibitors are normally increased in autumn (the breeding season) and low in spring. If PAA can improve the fertilizing ability of spermatozoa in the spring, our finding may mean that a nutritional supplement, such as alpha-tocopherol, could provide rams for an accelerated onset of the breeding season in the ewe.

[Exploration on parameters of TCM syndrome in acute cerebral infarction through investigating active factors of vascular endothelium cells].

OBJECTIVE: To explore the multiple parameters of TCM Syndrome-types and the acute cerebral infarction (ACI) with blood stasis type. METHODS: Sixty-six acute cerebral infarction patients with blood stasis Syndrome, various vascular active factors such as tissue plasminogen activator (t-PA), the activity of plasminogen activator inhibitor (PAI), the concentration of prostaglandin F1 alpha (6-keto-PGF1 alpha) etc. were determined. RESULTS: (1) In Incidental Syndrome, those "Phlegm" and "stasis" predominant, mainly manifested as Wind-Phlegm-Blood stasis (WPBS), Qi deficiency-blood stasis (QDBS) and Phlegm-Heat-bowel excess (PHBE) Syndrome all showed t-PA activity lowered, among them, QDBS Syndrome lowered more obviously (P < 0.01); and in fundamental deficiency predominant Syndrome such as Yin-deficiency and Wind-move (YDWM) Syndrome, the active t-PA content increased (P < 0.05); in Liver Yang ascending (LYA) Syndrome and YDWM Syndrome, the 6-keto-PGF1 alpha lowered very significantly. (2) Through regression analysis, although influencing the severity of acute blood stasis was related with 3 factors (t-PA activity, nervous system defect score and age growth), but single factor linear relationship analysis indicated that did not existed positive-negative relationship. (3) Through statistical analysis, the factor influencing nervous system defect scores was positively related with blood stasis score (r = 0.70, P < 0.01). CONCLUSION: (1) The basis of WPBS, QDBS and PHBE Syndrome mainly was fibrinolytic system activity lowering, and YDWM and LYA Syndrome prostaglandin system activity lowering. Comprehensive analysis of multiple parameters would be helpful to differentiate the ACI blood stasis stage. (2) Single parameter would not help to differentiate the types of ACI blood stasis stage, its change merely denoted the existence of blood stasis, its type should be differentiated after comprehensive analysis. (3) Those influencing nervous system scoring, mainly was blood stasis score, which suggested that the importance of activating blood circulation to remove stasis in preventing and treating ACI. (4) Put forward ACI blood stasis, and the quantification for new standard of Syndrome for discussion.

[Effect of puerarin on fibrinolytic activity and lipid peroxide in patients with coronary heart disease].

OBJECTIVE: To observe the effect of puerarin on superoxide dismutase (SOD), lipid peroxidation (LPO), tissue plasminogen (TPA) and plasminogen activator inhibitor (PAI-1) of coronary heart disease (CHD) patients. METHODS: Forty-eight patients were divided into two groups (treated group and control group). Treated group was given intravenously 600 mg puerarin, once daily for one week, also the same treatment as that of control was given. Nitrite colorimetric method, radioimmunoassay (RIA), TBA fluorescent development process and fibrinolysin specific colorbase substrate decomposition development process were used to determine SOD activity, SOD, LPO, TPA and PAI-1. RESULTS: After the treatment with puerarin, SOD activity was increased, LPO reduced, while without any changes in the control group. There was insignificant changes of SOD between the treated and the control groups. TPA and PAI-1 were increased significantly in the treated group, but was insignificantly changed in the control group. CONCLUSION: Puerarin can increase the SOD activity, decrease LPO level and enhance the activity of fibrinolysis.

[Effect of qigong on plasma coagulation fibrinolysis indices of hypertensive patients with blood stasis].

The levels of plasma tissue-type plasminogen activator (tPA), plasminogen activator inhibitor (PAI), VIII factor related antigen (VIIIR:Ag) and anti-thrombin (AT-III) were determined in 40 hypertensive patients with Blood Stasis. The results indicated that the function of coagulation-fibrinolytic system was disturbed. After one year of practising Qigong, plasma PAI and VIII R:Ag levels were decreased, while plasma tPA and AT-III levels increased. It suggested that Qigong could improve the function of coagulation-fibrinolytic system.

Effects of a dried garlic preparation on fibrinolysis and platelet aggregation in healthy subjects.

The acute and chronic effects of a preparation of dried garlic powder (Sapec) in a total daily dose of 900 mg on fibrinolysis and platelet aggregation have been evaluated in a randomized, double-blind, placebo controlled cross-over study of 12 healthy subjects. Total euglobulin fibrinolytic activity and t-PA (tissue plasminogen activator) activity were significantly higher 4 and 6 h after garlic and placebo ingestion, and no differences were recorded between treatments. After 14 days of treatment, t-PA activity was significantly higher after garlic, with inter-treatment significance. No significant changes in PAI (Plasminogen Activator Inhibitor) activity and fibrinogen levels were recorded. Platelet aggregation induced by adenosine diphosphate and collagen, and especially beta-thromboglobulin (beta-TG) release after collagen stimulation were significantly inhibited 2 and 4 h after garlic ingestion; platelet aggregation values were also significantly lower after 7 and 14 days of garlic treatment. No significant changes were found in adenosine triphosphate release and serum TXB2 levels after acute garlic administration.

Saturation mutagenesis of the plasminogen activator inhibitor-1 reactive center.

Plasminogen activator inhibitor-1 (PAI-1) is a specific inhibitor of the serine proteases tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA). To systematically investigate the roles of the reactive center P1 and P1' residues in PAI-1 function, saturation mutagenesis was utilized to construct a library of PAI-1 variants. Examination of 177 unique recombinant proteins indicated that a basic residue was required at P1 for significant inhibitory activity toward uPA, whereas all substitutions except proline were tolerated at P1'. P1Lys variants exhibited lower inhibition rate constants and greater sensitivity to P1' substitutions than P1Arg variants. Alterations at either P1 or P1' generally had a larger effect on the inhibition of tPA. A number of variants that were relatively specific for either uPA or tPA were identified. P1Lys-P1'Ala reacted 40-fold more rapidly with uPA than tPA, whereas P1Lys-P1'Trp showed a 6.5-fold preference for tPA. P1-P1' variants containing additional mutations near the reactive center demonstrated only minor changes in activity, suggesting that specific amino acids in this region do not contribute significantly to PAI-1 function. These findings have important implications for the role of reactive center residues in determining serine protease inhibitor (serpin) function and target specificity.

Plasmatic factors of haemostasis remain essentially unchanged except for PAI activity during n-3 fatty acid intake in type I diabetes mellitus.

Diabetic patients are prone to develop vascular complications. Increased procoagulatory factors and a reduced fibrinolytic potential are considered as thrombogenic risk factors, although controversy remains. In epidemiological and dietary intervention studies fish or fish oil, rich in the two n-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), have demonstrated a potential to reduce cardiovascular disease. We compared the plasmatic coagulatory and fibrinolytic profile of 13 near normoglycaemic type I diabetics almost free of cardiovascular disease with healthy volunteers, matched for age and sex. Except for fibrinogen levels and tPA activity being elevated and soluble fibrin and fibrinopeptide A being reduced, no differences could be discerned between type I diabetics and controls in all investigated plasmatic parameters. In a dietary intervention study we investigated the effects of 5.4 g EPA and 2.7 g DHA per day during and after a 4-week dietary supplementation in the diabetic patients. The factors, inhibitors and activation products of coagulation and fibrinolysis measured were at best transiently affected by the diet. Only plasminogen activator inhibitory activity in plasma significantly increased during the dietary supplementation and returned to prediet values after cessation of n-3 fatty acids. Changes in PAI activity were negatively correlated to changes in serum triglycerides. We conclude that well adjusted type I diabetics show an almost unchanged haemostatic profile compared to matched healthy controls. A dietary intervention with n-3 fatty acids in these patients does not affect the plasmatic haemostatic pattern except for an increase in PAI activity.

Regulation of plasminogen activator activity in arthritic joints.

The plasminogen activator (PA)/plasmin system has been implicated in the inflammation and connective tissue remodelling occurring in arthritic joints. PA activity is detected in cultures of human monocytes, synoviocytes and chondrocytes and can be regulated by a variety of cytokines found in diseased joints; PA inhibitors (PAI-1 and/or PAI-2) are also produced by these cells. We have shown that human monocytes can synthesize both urokinase-type PA (u-PA) and tissue-type PA (t-PA). One cytokine present in rheumatoid synovial fluids, granulocyte macrophage colony stimulating factor (GM-CSF), stimulates monocyte u-PA production; since this cytokine can also be produced by activated monocytes and other cell types in joints, than a "CSF network" can be produced leading to u-PA production. Another monocyte cytokine, interleukin 1, causes human synoviocytes to increase their u-PA expression, a response which can be dependent on the presence of endogenous cyclooxygenase products; this cytokine also causes human chondrocytes and cartilage tissue to produce increased u-PA and t-PA activity, i.e., under conditions during which cartilage is resorbed.