Research On Thrombolytic Effects Of Fermentation Broth Of Lyophyllum Ulamarium And Its Mechamism

Objective:Objective:To investigate thrombolytic effects of fermentation broth of Lyophyllum ulamarium and its mechamism.Method:(1) cellular experiments:MTT assay was used to determine the effects of fermentation broth of L.u on viability in HUVEC; H2O2was used to induce apoptosis in HUVEC. The effects of fermentation broth of L.u and drug extract serum on viability were measured by MTT assay in HUVEC; The effect of fermentation broth of L.u on apoptosis was detected by DNA ladder; Flow cytometry was performed to exam apoptosis rate; Western Blot was used to exam the protein levels of Bcl-2、Bax、 Caspase-9、Caspase-3; the LDH levels in medium were determined by colorimetry.(2) animal experiments:The capability to hydrolyze fibrin and blood clot lysis time were measured in vitro; The changes of bleeding time and coagulation time were observed after adding fermentation broth of L.u Experimental hyperlipidemia mice were estabilished, following the effects of fermentation broth of L.u. on the levels of TG and TC in serum and SOD、GSH、MDA in liver homogenate were measured; Carotid artery thrombosis were formed by stimulating with20%Fecl3in rats, following the effects of fermentation broth of L.u on thrombolytic effects were measured. Elisa assay was used to detect t-PA、PAI-1、ET-1level in plasma. NO levels were detected by the method of nitric acid reductase.Result:(1) cellular experiments:MTT assay results showed that fermentation broth of L.u protect HUVEC from apoptosis at certainly concentrations. DNA ladder results demonstrated that clear apoptosis ladders were observed in model group, while the bands in high-dose drug groups were hardly detected. Apoptosis rates were significantly decreased in drug groups got from flow cytometry results, compared to model group. Western Blot results have showed that Bcl-2protein expression were increased while the protein levels of Bax, Caspase-9and Caspase-3were decreased in drug groups. The LDH levels in the medium detected by colorimetry showed that fermentation broth of L.u suppress the levels of LDH in a concentration-dependent manner.(2) Animal experimental results indicated that fermentation broth of L.u has the ability to hydrolyzed fibrin. The bleeding time was prolonged and thrombolytic rate was increased after fermentation broth of L.u treatment. Fermentation broth of L.u also significantly lowered the blood lipid levels and decrease the concentration of MDA, while enhance the activity of GSH and SOD. Rat Carotid artery thrombosis experimental results showed that fermentation broth of L.u significantly reduced thrombus Weight. Compared to model group, the levels of NO and ET-1were increased to normal after fermentation broth of L.u treatment, while PAI-1and t-PA were also increased and their ratio was decreased.Conclusion:(1) Fermentation broth of L.u performed anticoagulatory function and fibrinolysis.(2) Fermentation broth of L.u showed hypoglycemic antioxidant effects.(3) Fermentation broth of L.u has the ability to regulate vasoactive substance.(4) the protective effects of Fermentation broth of L.u on HUVEC may be related with its inhibition of cell apoptosis.