| Lung cancer is the malignant tumor with the highest mortality rate in china.Studies have shown that lung cancer patients are often accompanied by abnormal blood coagulation function and inflammatory level.This abnormal state is not only the inducement of complications such as venous embolism,but also can promote tumor growth and metastasis through various ways.Blood stasis syndrome is an important syndrome of lung cancer patients,and its essence refers to the slow running,block and coagulation of blood in the vessels.The abnormal coagulation function of lung cancer patients is mainly manifested as hypercoagulable state of blood,which is similar to the pathological manifestations of blood stasis syndrome,Exudation,metamorphism and proliferation caused by inflammatory injury also play an important role in the process of blood stasis syndrome.The formation of this abnormal state of coagulation and inflammation is affected by the tumor itself on the one hand,and is related to the joint action of body immunity and tumor microenvironment on the other hand,Specifically,it can be manifested as abnormal blood coagulation and inflammationrelated indicators in patients with lung cancer.In this study,patients with non-small cell lung carcinoma(NSCLC)were taken as the research objects,the correlation between specific coagulation and inflammatory indicators and tumor metastasis and traditional Chinese medicine(TCM)syndrome was analyzed,the microscopic manifestations of lung cancer syndromes including blood stasis syndrome were discussed.Tumor angiogenesis is one of the important steps in the metastasis of malignant tumors.In recent years,cancer stem cells(CSCs)have been found to play an important role in the process of angiogenesis,and the hypoxic environment is one of the factors that promote angiogenesis.The theory of TCM believes that the accumulation of pathological products such as blood stasis causes "hyperactivity of the collaterals",which is similar to the pathological process of tumor angiogenesis.Previous studies have found that the bloodactivating drugs Danshen and Chuanxiong can inhibit the formation of Vasculogenic mimicry(VM)and the expression of VEGF in lung cancer stem-like cells(CSI Cs).Tanshinone ⅡA and tetramethylpyrazine also have inhibitory effects on angiogenesis-related factors VEGF,HIF-1α,MMP-2 and MMP-9.Therefore,we speculate that tanshinone ⅡA and tetramethylpyrazine,monomers of activating blood drugs,can inhibit CSCs-related angiogenesis,and may have different mechanisms under different oxygen environments.We chose to explore the possible targets of active components of blood activating drugs on lung cancer metastasis from the perspectives of VM and endothelial-related angiogenesis.Objectives:1 To explore the correlation of blood coagulation and inflammation-related indexes with lymph node metastasis,distant metastasis and distribution of TCM syndromes in NSCLC patients2 To explore the effects and mechanisms of tanshinone ⅡA and tetramethylpyrazine,the active ingredients of danshen and chuanxiong,on the angiogenesis of CSLCs in different oxygen environments.Methods:1 Clinical research:268 patients with NSCLC were retrospectively collected,and the general and clinical data of the patients were collected,including tumor stage,pathological type,lymph node and distant metastasis,etc.;collected laboratory indicators including inflammation-related indicators:NLR,PLR,LMR,NEUT,EO,BASO,MONO,LYMPH,coagulation indexes:PLT,PDW,MPV,P-LCR,PCT,FIB,FDP,D-dimer,INR,APTT,ATⅡI,a total of 19 items;and collected TCM four diagnostic Information and TCM syndrome composition,analyzed the distribution of coagulation and inflammation-related indexes in NSCLC patients and the relationship between each index and NSCLC lymph node metastasis,distant metastasis and TCM syndromes;2 Experimental research:(1)A549 CSLCs were isolated and enriched by serum-free spheroidization method,and the enriched A549 CSLCs were identified by soft agar cloning assay,Transwell migration and invasion assay,qPCR and Western Blot detection of CSLCs stemness markers.(2)A co-culture model of A549/A549 CSLCs and human umbilical vein endothelial cells(HUVEC)was established,and the pro-angiogenic effect of A549 CSLCs was verified by CCK8 assay,scratch assay,Transwell migration assay,and tubule formation assay.(3)The hypoxia model was established and the level of HIF-la was verified by Western Blot method,and the effect of hypoxic environment on A549 CSLCs VEGF was verified by ELISA method;(4)The cytotoxicity of tanshinone ⅡA and tetramethylpyrazine on A549 CSLCs was detected by CCK8 method,and the tubule Formation experiment was used to detect the effect of tanshinone ⅡA and tetramethylpyrazine on A549 CSLCs VM,and Western Blot method was used to detect the effect of the two drugs on the expression of A549 CSLCs VM-related proteins;(5)Scratch and tubule formation experiments were used to test the effect of tanshinone ⅡA and tetramethylpyrazine on A549 CSLCs in promoting endothelial-related angiogenesis,and qPCR method to detect the effect of tanshinone ⅡA and tetramethylpyrazine on A549 CSLCs impacting the co-culture system HUVEC’s VEGF,FLT1,KDR mRNA.Results:1 Clinical research:(1)Among the 268 patients,34.7%showed increased PLT,6.7%showed decreased LYMPH,and 19.2%showed increased NEUT.10.1%of patients had decreased PDW,8.6%of patients had decreased MPV,3.0%of patients had increased PCT,9.3%of patients had prolonged APTT,59%of patients had increased FIB levels,and 34.3%of patients had increased FDP,65.3%of the patients had increased D-dimer value,and 21.3%of the patients had decreased AT-Ⅲ activity;(2)The differences in the distribution of PLT,MPV and PCT among the N staging groups were statistically significant(P<0.05);The differences in the distributions of FDP and D-dimer among the M staging groups were statistically significant(P<0.01);(3)Compared with non-phlegm syndrome,MONO,NEUT,NLR and LMR were increased in NSCLC patients with phlegm syndrome(P<0.05);Compared with non-blood stasis syndrome,FDP increased in blood stasis syndrome patients(P<0.05);compared with non-heat syndrome patients,NEUT increased in heat syndrome patients(P<0.05);compared with non-fluid syndrome patients,MONO decreased and FDP、D-dimer increased in fluid syndrome patients(P<0.05).2 Experimental study:(1)The A549 sphere cell cultured by serum-free spheroidization were more three-dimensional and regular after three passages.The results of soft agar cloning experiments showed that the isolated and enriched A549 CSLCs had stronger cloning ability than A549 parental cells;The results of migration and invasion experiments showed that A549 CSLCs had stronger migration and invasion abilities than A549 parental cells;Western Blot results showed that compared with A549,A549 CSLCs OCT-4,SOX-2,Nanog protein expression levels were significantly up-regulated(P<0.05);qPCR results showed that compared with A549,A549 CSLCs OCT-4,SOX-2,Nanog,CD133 mRNA levels were significantly up-regulated(P<0.05);(2)The results of CCK8 experiment showed that compared with A549,HUVEC co-cultured with A549 CSLCs had a faster proliferation rate(P<0.0001);scratch and migration experiments showed that compared with A549,HUVEC co-cultured with A549 CSLCs had stronger migration ability;the results of tubule formation experiment showed that compared with A549,HUVEC co-cultured with A549 CSLCs had stronger vascular network formation ability;(3)A549 CSLCs hypoxia model was effectively established by 150 μM CoCl2 intervention for 24 h;ELISA results showed that the level of VEGF secreted by A549 CLSCs in the hypoxia group was significantly higher than that in the normoxia group after being treated with 150 μM CoCl2 for 24 h and 48 h(P<0.05);(4)Under different oxygen environments,both tanshinone ⅡA and tetramethylpyrazine could inhibit the formation of VM in A549 CSLCs to different degrees(P<0.05).In normoxic environment,tanshinone ⅡA may play an anti-VM effect by down-regulating the expression of MMP-9 and VEGFA proteins.Under hypoxic environment,tanshinone ⅡA may exert its anti-VM effect by down-regulating the protein expressions of VE-Cadherin,Integrin β1,HIF-lα,MMP-2,MMP-9,EphA2 and VEGFA.Tetramethylpyrazine may exert its anti-VM effect by downregulating the expression of EphA2 protein;(5)Under normoxia condition,tanshinone ⅡA and tetramethylpyrazine groups could significantly reduce the migration rate of HUVEC in the coculture system(P<0.05);under hypoxia condition,tanshinone ⅡA could significantly reduce the migration rate of HUVEC in the co-culture system(P<0.01).Under normoxia condition,both tanshinone ⅡA and tetramethylpyrazine could inhibit the formation of HUVEC tubules in the co-culture system(P<0.05);under hypoxia condition,tanshinone ⅡA could inhibit the formation of HUVEC tubules(P<0.01).Under normoxic environment,tanshinone ⅡA acts on A549 CSLCs,which may play an anti-angiogenic effect by down-regulating HUVEC VEGFA mRNA levels in the co-culture system,while tetramethylpyrazine may play its antiangiogenic effect by down-regulating VEGFA,FLT1,and KDR mRNA levels.TanshinoneⅡA may exert its anti-angiogenic effect by down-regulating the mRNA levels of VEGFA,FLT1 and KDR under hypoxia condition.Conclusions:1 Clinical study:(1)Abnormal coagulation function and inflammatory state are commonly found in patients with advanced NSCLC;(2)PLT and PCT elevations,and MPV decreases indicate higher lymph node metastasis stages in NSCLC patients;increased FDP and D-dimer indicate higher levels of high distant metastasis stage;(3)MONO,NEUT,NLR are elevated,LMR reduction is closely related to phlegm syndrome,NEUT reduction is related to qi deficiency syndrome,NEUT elevation is related to heat syndrome,FDP elevation is related to blood stasis syndrome,The decrease of MONO and the increase of FDP and Ddimer were related to fluid syndrome.2 Experimental study:(1)A549 CSLCs obtained by serum-free spheroid culture have stemness characteristics;(2)A549 CSLCs have stronger angiogenesis ability than A549 cells;(3)CoCl2 can simulate hypoxic environment and promote A549 CSLCs VEGF secretion(4)Tanshinone ⅡA and tetramethylpyrazine can inhibit the formation of A549 CSLCs VM in different oxygen environments,and the two drugs may achieve this process through different mechanisms;(5)Tanshinone ⅡA can inhibit angiogenesis of A549 CSLCs in both normoxic and hypoxic environments.Tetramethylpyrazine can inhibit the angiogenesis of A549 CSLCs in normoxic environment.The anti-angiogenesis mechanism of the two drugs is not completely same. |
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