Study On The Substance Bases Of Anti-thrombi Activity And The Mechanism Of Anti-apoptosis Of Human Umbilical Vein Endothelial Cells(HUVECs) Of Total Flavonoids From Folium Apocyni Venet (TFF)

Objective:To establish a standard HPLC fingerprinting of total flavonoids of Folium Apocyni veneti(TFF). To study anti-thrombi pharmacological action and intracorporal process(substance bases)of TFF, and investigate the protective effects and mechanism of TFF and hyperin on H₂O₂-induced human umbilical vein endothelial cells(HUVECs)insults through cultured in vitro.Methods:(1)The standard fingerprint of TFF was established by HPLC-DAD,which can supply samples of controllable quality for further experiments with animals and research mechanism of action. (2) Rat model of venous thrombosis was made to measure the thrombus weight. Mouse model of thrombosis by injecting collagen-adrenaline to the tail vein was applied to observe numbers of dead mice in 5 minutes and recovery mice from hemiplegia in 15 minutes. The clotting time(CT) of mouse, and the prothrombin time (PT) and partial thromboplastin time(PTT) of rabbits were tested by Clotting Method.. The contents of TXB₂,6-keto-PGF1αin rat plasma were detected by radioactive immunization. (3)The gastrointestinal absorption and biotransformation, correlation ingredient of rat plasma and serums and the initiative pharmacokinetics of TFF were investigated by HPLC,LC/MS/MS and UPLC-MS/MS techniques. (4) The cultured HUVECs were used as the experimental model. H₂O₂ was used as the inducer of oxidative stress. Cell viability was measured by MTT assay. Cell apoptosis, cell circles and the level of ROS were detected by flow cytometry(FCM). The cell morphology change was assessed by AO/EB fluorescent staining. The contents of MDA and LDH, the activities of SOD and NO were determined by spectrophotometry. The immunohistochemistry was used to observe the expression of NF-κB and VEGF. The concentration of TF and TFPI were determined by ELISA.(5)Intracellular Ca²⁺ and the mitochondria transmembrane potential variance (ΔΨm) were measured with Flou3-AM and Rh123 fluorescent staining respectively by LSCM. The gene expression of Bcl-2, Bax and caspase 3 were analysized by RT-PCR. The protein expression of cytochrome C, caspase-9 and caspase-3 were analysized by western blotting to further investigatation of the mechanism of TFF and hyperin in inhibiting HUVECs apoptosis induced by H₂O₂.Results:(1)The chromatographic fingerprinting of TFF was established which showed 17 charavcteristic peaks from 7 patches of total flavonoids products. The similarity from different lots was 0.95¹.00 by the software of"Computer-aided Similarity Evaluation"and showed high similitude in peak numbers and the retention time. Moreover, comparison of the HPLC profiles of total flavonoids and the Folium Apocyni veneti indicated that they were closely related to each other. The chromatographic fingerprinting with high specificity can be used to control its quality and assure to supply samples of controllable quality for further experiments with animals and research mechanism of action. (2)The study indicated that TFF had obvious restriction to venous thrombosis in the test of phlebothrombosis. Compared with the control group , the high and middle dose group of TFF had notable difference in dry weight and wet weight of thrombosis (p<0.001) and the restrain rate of thrombus wet weigh were 73.0% and 83.4% respectively. The hemiplegy and death rate was decreased in mouse by injecting collagen-adrenaline to the tail vein with different dosage TFF compared with control group, but has no notable difference. The high and middle dose group can obviously prolong the clotting time(CT) of mouse (p<0.05), can prolong the prothrombin time (PT) and partial thromboplastin time(PTT) of rabbits, which show TFF has anticoagulant action through the restrain to intrinsic and extrinsic coagulation pathway. The high and middle dose group can decline the content of TXB2 and increase the 6-keto-PGF1α(p<0.05 or p<0.01 vs control group) in the detection of TXB2,6-keto-PGF1αin rat plasma by radioactive immunization. (3)The absorptivity of hyperin and isoquercitrin in gastrointestinal was counted through detecting the content of hyperin and isoquercitrin in former and behind of perfusion TFF by HPLC. The results showed that the average absorptivity in stomach of hyperin and isoquercitrin were 36.92% and 36.49%, while in intetines were 47.7% and 48.25% respectively. All these indicated that hyperin and isoquercitrin have good bsorptivity in intetines. The detective results of correlation ingredient of rat plasma indicated that hyperin, isoquercitrin, glucuronic acid complex compound of quercetin and sulphating complex compoundof quercetin (the phaseâ…¡metabolites of the quercetin) can be detected in vivo after ig and iv TFF. Hyperin, isoquercitrin and measly phaseâ…¡metabolites of the quercetin can be detected after iv TFF 5 and 15 min. However, only substantive phaseâ…¡metabolites of the quercetin can be detected after ig TFF 4 h and 15 min. The detective results of correlation ingredient of rat serums indicated that the prototype ingredient(hyperin and isoquercitrin) can not be detected after ig TFF 30 min, while can be detected after iv TFF 30 min. The results indicated biotransformation have happened after ig TFF. The potential inclusive substance included quercetin, quercetin+glucuronic acid , astragalin, tamarisktwig, tamarisktwig-7-O-glucuronic acid and so on through initiative speculate based mass spectrum by UPLC-MS analysis. The biotransformation pathways in vivo perhaps comprised of methylation reaction, sulphating reaction and metabolic product association reaction with glucuronic acid etc. Preliminary pharmacokinetics study showed that quercetin concentration were low in no hydrolyzing blood sample,while were high in hydrolyzing blood sample, which confered that quercetin was obtained by absorption of hyperin and isoquercitrin after hydrolysis.The oral bioavailability of flavonoid was 59.8% through area under concentration (AUC). (4) The apoptosis model of human umbilicus vascular endothelial cell (HUVECs) with cell volume reduction, cytoplasm shrinkage, condensation and fragmentation of chromatin can be induced by 200μmol/l H₂O₂. It offered a good instrument for further study the protective effect and mechanism of TFF and hyperin on H₂O₂-induced HUVECs injury. Compared with the normal group, H₂O₂ can obviously increase the release of ROS, decrease cell viability, induce cell apoptosis, increase contents of LDH and MDA, decrease the activities of SOD and NO of the cultured medium, reduce the percentage of cells of S phase and G2/M phase, down-regulate the expression VEGF, up-regulated the expression of NF-κB, (P<0.01). Compared with the H₂O₂ model group, different concentrations of TFF and hyperin were shown to alike distinctly attenuate these responses elicited by H₂O₂(P<0.05或P<0.01). The mechanism may be related to its exact antioxidant effects and regulate the expression of NF-κB and VEGF. At the same time, the concentration determination of TF and TFPI indicated that different concentrations of TFF and hyperin can remarkably inhibit the increase of TF content, while the change of TFPI content was reversed with TF. The concentration change of TF and TFPI was may be a mechanism of TFF and hyperin inhibit the H₂O₂-induced apoptosis of HUVECs.(5) Compared with the normal group, the intracellular Ca²⁺ influx content was severely overloaded , while mitochondria membrane potential△Ψm was obviously decreased (P<0.01), Bcl-2 down regulation and Bax up-regulation, the protein expression of caspase-3, caspase-9 and Cyt C remarkably increased (P<0.05 or P<0.01). The study indicated that HUVECs apoptosis by H₂O₂ induced was carried out mainly by activating the mitochondrial pathway, according to the literature reporting. Compared with the H₂O₂ model group, different concentrations of TFF and hyperin were shown to distinctly inhibit the increase of intracellular Ca²⁺content , Bcl-2 up-regulation and Bax down-regulation, depress the mitochondrion permeability, increase the mitochondria membrane potential△Ψm (P<0.05 or P<0.01). At the same time, the protein expression of caspase-3, caspase-9 and Cyt C remarkably decreased (P<0.05 or P<0.01). The investigation indicated that the mechanism of TFF and hyperin in inhibiting the HUVECs apoptosis by H₂O₂ induced may be related to inhibit the increase of intracellular Ca²⁺content, Bcl-2 up-regulation and Bax down-regulation, depress the mitochondrion permeability, increase the mitochondria membrane potential△Ψm, prevent mitochondria to release cytochrome C, prevent the activation of Caspase-9 and Caspase-3 and inhibite the HUVECs apoptosis by H₂O₂ induced by preventing the mitochondria caspases signal transduction pathway.Conclusions:(1) The standard fingerprint of TFF was established which can ensure to supply samples of controllable and uniform quality for further experiments with animals and research mechanism of action of TFF. (2) In results, TFF exerted remarkable effects against thrombosis by the major pharmacology research of resist thrombosis. (3)The absorption, biotransformation and its chief metabolites were initiatively illuminated by HPLC /MS/MS and UPLC/MS/MS analysis. The substance bases of pharmacological action were discussed.(4) The study first indicated that TFF and hyperin had a potent protective effect on damage in cultured HUVECs, and obviously inhibit the H₂O₂-induced apoptosis of HUVECs.The mechanism may be related to its exact antioxidant effects and the regulative expression of NF-κB and VEGF, or may be related to the concentration changes of TF and TFPI.(5) The study indicated that HUVECs apoptosis by H₂O₂ induced was carried out mainly by activating the mitochondrial pathway. So, the research focal point was the mitochondria caspases signal transduction pathway. The dissociative Ca²⁺ was one of prerequisite to activate caspase-3, while homeostatic destroying of Ca²⁺ could directly cause mitochondria to release cytochrome C and some apoptosis factor. Cyt-C released from mitochondria can combine with apoptosis enzyme promoter and then activate Apaf-1 by deoxidation triphosadenine. Eventually, the activated Apaf-1 binding to procaspase-9 would start a series of caspases cascade reaction, thus activates typical mitochondria apoptosis pathway. The investigation indicated that different doses of TFF and hyperin were shown to distinctly inhibit the increase of intracellular Ca²⁺content , Bcl-2 up-regulation and Bax down-regulation, depress the mitochondrion permeability, increase the mitochondria membrane potential△Ψm, prevent mitochondria to release cytochrome C, prevent the activation of Caspase-9 and Caspase-3 and inhibit the H₂O₂ induced HUVECs apoptosis by preventing the mitochondria caspases signal transduction pathway.