Anticoagulant Effect And Mechanism Of Polysaccharides From Curcuma Kwangsiensis

Objective:To study the anticoagulant effect and mechanism of different doses of polysaccharides from Curcuma kwangsiensis in vitro and in vivo experiments,provide scientific basis for clinical study of Curcuma kwangsiensis.Methods:(1)The protective effect of polysaccharides from Curcuma kwangsiensis on H2O2-induced injury of human umbilical vein endothelial cells(HUVECs)and its mechanism:HUVECs were cultured in vitro,HUVECs were injured with different concentrations of H2O2,and the best concentration of H2O2 was 500?mol/L by CCK-8 method.Then,different concentration of polysaccharides from Curcuma kwangsiensis were used to protect the damaged HUVECs.The CCK-8 method was used to determine 62.5?g/mL,125?g/mL and 250?g/mL as the low,medium and high dose groups.Determination of NO,LDH,SOD,GSH-Px,MDA by kit.The levels of 6-keto-PGF1?,ET-1,PGI2,eNOS,t-PA,PAI-1,TNF-? and IL-6 in cells were detected by enzyme-linked immunosorbent assay(ELISA).Hoechst 33258 fluorescence staining was used to observe the cell apoptosis.The expression of Bax,Bcl-2 and Caspase-3 genes were detected by qRT-PCR(2)The anticoagulant effect and mechanism of polysaccharides from Curcuma kwangsiensis on rats:1.SPF rats were randomly divided into normal group,aspirin(20mg/kg),polysaccharides from Curcua kwangsiensis 30mg/kg,150mg/kg,300mg/kg five groups,the determination of plasma PT,TT,APTT.2.The levels of TXB2,PGI2,t-PA,PAI-1,TNF-?and IL-6 in rats were detected by enzyme linked immunosorbent assay(ELISA)Results:(1)The result of CCK-8 assay showed that administration of 24h,polysaccharides from Curcuma kwangsiensis in 15.625?g/mL-500?g/mL concentration range,can promote cell proliferation,while the concentration increased,polysaccharides from Curcuma kwangsiensis has certain inhibitory effect on cells.The experiment found that different concentrations of polysaccharides from Curcuma kwangsiensis against H2O2 caused by the damage of HUVECs cells.Compared with the model group,polysaccharides from Curcuma kwangsiensis can make the content of LDH,MDA,ET-1,PAI-1,IL-6,TNF-?,the contents of SOD,GSH-Px,NO,eNOS,6-keto-PGF1?,PGI2 and t-PA increased.Hoechst 33258 fluorescence staining results showed that compared with the normal group,the nucleus of HUVECs showed bright blue fluorescence in the model group,dense and dense granular lumps of fluorescence were observed in the cytoplasm,and the cell density was decreased.Compared with the model group,Blue fluorescence intensity decreased,the typical morphological apoptosis decreased.The qRT-PCR results showed that compared with the normal group,the model group decreased the expression of Bcl-2 and increased the expression of Bax and Caspase-3.Compared with the model group,reduce Bax,Caspase-3 expression.(2)The results of the experiment showed that polysaccharides from Curcuma kwangsiensis could prolong rat plasma TT,PT,APTT time.The results of enzyme-linked immunosorbent assay(ELISA)showed that the polysaccharides from Curcuma kwangsiensis could increase the content of serum PGI2,t-PA,reduce the content of TXB2,IL-6,TNF-?,PAI-1Conclusions:(1)The protective effect of Polysaccharides from Curcuma kwangsiensis on oxidative injury of HUVECs might be related to the inhibition of the production of thrombotic substances LDH,PAI-1,and vasoconstrictor ET-1;the increase of vasodilators PGI2,NO,eNOS and anticoagulant t-PA generation,reduce the inflammatory cytokines IL-6,TNF-? and so on.(2)Polysaccharides from Curcuma kwangsiensis could protect H2O2-induced HUVECs from oxidative damage,which might be related to regulating the expression of Bcl-2,Bax and Caspase-3(3)Polysaccharides from Curcuma kwangsiensis with anti-clotting effect,the mechanism might be related to increased anticoagulant substances t-PA and vasodilators PGI2;reduce thrombotic substances TXB2,PAI-1 formation At the same time Polysaccharides from Curcuma kwangsiensis could reduce IL-6,TNF-? production of inflammatory cytokines(4)In vivo and in vitro studies showed that Polysaccharides from Curcuma kwangsiensis can improve the cell activity,platelet and endothelial function,fibrinolytic system,oxidative stress,inflammation,apoptosis,inhibit thrombosis,and thus play an anti clotting effect.