The Interaction Between Platelets And Lung Adenocarcinoma Cells On The Formation Of Endothelial Procoagulant Phenotype And Intervention Of Taohong Siwu Tang

Lung Cancer is the most common malignant tumor in China,with its incidence and mortality increasing year by year.Non-Small Cell Lung Cancer(NSCLC)accounts for about 85%of lung cancer,and lung adenocarcinoma,as a common NSCLC,accounts for about 60%of this group.In recent years,surgery and chemotherapy have been used to treat lung adenocarcinoma in order to improve the survival of patients.However,recurrence and metastasis of lung adenocarcinoma remain significant barriers to survival.Hypercoagulation is common in patients with malignant tumors,mainly manifested as abnormal coagulation indexes,slow blood flow and other symptoms.Vascular endothelial cells,as an important physiological barrier of blood vessels and blood,can maintain the balance of coagulation and fibrinolysis system.The interaction between tumor cells and platelets can promote the formation of the procoagulant phenotype of vascular endothelial cells,thus causing blood hypercoagulable state.The hypercoagulable state of blood can affect the adhesion of tumor cells and vascular endothelial cells,the generation of tumor blood vessels,accelerate microthrombus and microcirculation disorders,and promote the progress of tumor.Taohong Siwu Tang(TST)is derived from the Golden Seal of Medical Emperor written by Qing Wu Qian.It has the effects of promoting blood circulation,removing blood stasis,nourishing blood,and improving blood stasis syndrome associated with the tumor.Although the hypercoagulable state in the blood of tumor patients is not completely equivalent to the blood stasis syndrome,both of them refer to the state of thick,sticky,coagulated,and converged blood.Modern pharmacological studies have confirmed that most of the drugs in traditional Chinese medicine to promote blood circulation and remove blood stasis,such as TST and Salvia miltiorrhiza,have the effect of reducing the viscosity of whole blood and inhibiting platelet aggregation.They are widely used in cardiovascular and cerebrovascular diseases,demonstrating the unique advantages of traditional Chinese medicine.However,no studies have focused on the effect of the interaction between lung adenocarcinoma cells and platelets on the formation of the pro-coagulant phenotype of vascular endothelial cells and the mechanism of TST.Therefore,this project aims to explore the role of lung adenocarcinoma cell H1299 co-incubation with platelets in promoting coagulation phenotype formation of Human Umbilical Vein Endothelial Cells(HUVEC).And whether TST interferes with the formation of coagulation-promoting phenotype of vascular endothelial cells by inhibiting platelet activation and regulating the interaction between tumor cells and platelets,so as to inhibit tumor progression,providing theoretical basis for exploring new tumor treatment strategies.Purpose:To explore the role of lung adenocarcinoma cell H1299 co-incubation with platelets in promoting coagulation phenotype formation of HUVEC and the effect of TST in promoting coagulation phenotype formation of HUVEC.Methods:In this study,the effect of H1299 and platelet co-incubation on the formation of HUVEC coagulant phenotype and the intervention of Taohong Siwu Tang were investigated in three parts.Part Ⅰ:The co-culture system of H1299 cells and platelets was established,and the co-culture supernatant was collected and co-incubated with HUVEC.CCK-8 assay was used to detect cell viability,ghost pen cyclic peptide staining was used to detect cytoskeleton,and ELISA was used to detect Phosphatidylserine(Phosphatidylserine,PS)level.The expressions of intercellular adhesion molecule(Intercellular adhesion molecule 1,ICAM-1),P-selectin receptor(P-selectin glycoprotein ligand 1,PSGL-1)and vascular cell adhesion molecule(Vascular cell adhesion protein 1,VCAM-1)were detected by Western blot.Part Ⅱ:On the basis of H1299 and platelet co-culture system of HUVEC,plus Peach red Siwu Tang,CCK-8 experiment was used to determine the optimal drug concentration of peach red Siwu Tang;EDU proliferation assay was used to observe cell proliferation.The expression levels of ICAM-1,VCAM-1 and P-selectin were detected by ELISA.Cytoskeletal changes were detected by ghost pen cyclic peptide staining.The expressions of ICAM-1,VCAM-1,VEGFR2,Ve-Cadherin and PSGL-1 were detected by Western blot.Part Ⅲ:As for tumor growth measurement of lung adenocarcinoma transplant tumor model,it was observed that the tumor inoculated with H1299 and platelets co-incubated in vitro was tested through in vivo experiment to detect the effects of TST combined with cisplatin on blood hypercoagulable gold index and tumor metastasis.Healthy male nude mice were randomly divided into 6 groups after adaptive feeding for one week:H1299 group,H1299+PLT group,H1299+PLT+CLO group,H1299+DDP group,H1299+TST(0.8g/l)+DDP group,H1299+LMWHD+DDP group.Serum gold index of hypercoagulable state,such as D-dimer(D-D)and Fibrinogen(FIB),was detected by ELISA.The growth of lung adenocarcinoma grafts was recorded.The expressions of HUVEC adhesion protein and EMT protein were detected by Western blot.Results:1.The effect of H1299 co-incubation with platelets on the formation of coagulationpromoting phenotypes in HUVEC(1)The results of CCK-8 showed that HUVEC activity was significantly enhanced after 24h treatment with H1299 and platelet co-culture supernatant(P<0.05).Nnnj(2)The results of ghost pen cyclic peptide staining showed that the cytoskeleton was not clearly arranged in the HUVEC+CM_HP group,with partial absence of microfilaments and filamentous pseudopodia.The HUVEC+PLT group showed smooth edges,reduced microfilaments and changes in cell morphology.(3)ELISA results showed that H1299 and platelets promoted the expression of PS in HUVEC supernatant(P<0.01).(4)Western Blot showed that the expression levels of ICAM-1,PSGL-1 and VCAM-1 in HUVEC+CM_HP group were increased(P<0.001).2.Intervention effect of Taohong Siwu Tang on endothelial procoagulant phenotype formation caused by interaction between tumor cells and platelets(1)The results of CCK-8 showed that the proliferative power of HUVEC treated with H1299 and platelet co-culture supernatant decreased in a concentration-dependent manner when treated with0.1g/l、0.2g/l、0.4g/l、0.8g/l、2.0g/l TST for 24h.The difference was statistically significant(P<0.01).After statistical analysis,0.2g/l and 0.8g/l were selected as the low and high doses of the optimal concentration of TST,and the following experiments were conducted.(2)The results of EDU proliferation experiment showed that H1299 and platelet coculture supernatant promoted the proliferation of HUVEC.On this basis,TST concentrations of 0.2g/l and 0.8g/l inhibited the proliferation of HUVEC(P<0.05).(3)Ghost-pen cyclic peptide staining showed that H1299 and platelet co-culture promoted the formation of filamentous pseudopods in HUVEC.On this basis,0.2g/l and 0.8g/l TST improved the number of pseudopods and the morphology of HUVEC cells.(4)ELISA results showed that the interaction between H1299 and platelets promoted the expression of ICAM-1,VCAM-1 and P-selectin in endothelial cell supernatants.The expression levels of PS decreased after 0.2g/l and 0.8g/l TST treatment,in a dosedependent manner(P<0.05).(5)Western Blot showed that the expressions of ICAM-1,VCAM-1,VEGFR2,VeCadherin and PSGL-1 in HUVEC+CM_HP group were increased.After 0.2g/l and 0.8g/l TST intervention,The expression of ICAM-1,VCAM-1,VEGFR2,Ve-Cadherin and PSGL-1 decreased significantly(P<0.05).3.Effects of TST combined with cisplatin on blood hypercoagulability and metastasisrelated indexes of H1299 nude mouse transplanted tumor model(1)ELISA results showed that peach red Siwuke combined with cisplatin decreased the expression levels of D-D and FIB in the blood of nude mouse lung adenocarcinoma transplantation model(P<0.05).(2)As for the growth measurement of lung adenocarcinoma grafts in nude mice,it was observed that the growth of lung adenocarcinoma grafts inoculated with H1299 and platelets co-incubated in vitro grew faster,and TST combined with cisplatin interfered with the growth of lung adenocarcinoma grafts(P<0.05).(3)Western Blot results showed that TST(0.8g/l)combined with cisplatin decreased the expression of ICAM-1,VCAM-1,PSGL-1,N-cadherin and Vimentin,and increased the expression of E-cadherin(P<0.05).Conclusions:The co-incubation of lung adenocarcinoma cells and platelets may promote the formation of thrombogenic phenotypes of vascular endothelial cells.Peach red Siwu Tang may regulate the interaction between tumor cells and platelets by inhibiting platelet activation,thus affecting the formation of HUVEC pro-coagulant phenotype,improving the hypercoagulable state of tumors,and thus inhibiting tumor progression.To provide theoretical basis for exploring new strategies of tumor treatment.