Mechanism Of Scutellaria Baicalensis And Its Main Components Inhibiting Angiogenesis By Improving The Inflammatory Microenvironment Of Pancreatic Cancer

Objective:In this study,the macrophage-conditioned medium was used to simulate the inflammatory microenvironment of pancreatic cancer,to explore the effect of baicalein and wogonin on pancreatic cancer inflammatory microenvironment on pancreatic tumor angiogenesis and its mechanism.And compares different effects of pancreatic cancer inflammatory microenvironment on angiogenesis under the action of Scutellaria baicalensis,Radix Bupleuri Scutellariae,baicalein and wogonin.Methods:1.Explore the effect of pancreatic cancer inflammatory microenvironment on pancreatic tumor angiogenesis under the action of baicalein and wogoninBy extracting macrophage-conditioned medium to simulate the inflammatory microenvironment of pancreatic cancer,in vitro cell experiments were designed to explore the effect of the inflammatory microenvironment of pancreatic cancer under the action of baicalein and wogonin on pancreatic tumor angiogenesis.The experimental design is as follows:(1)Collection of conditioned medium: RAW264.7 was induced with lipopolysaccharide(LPS)and given different concentrations of baicalein and wogonin to interfere with macrophage activity.The collected RAW264.7 conditioned medium was applied to pancreatic ductal adenocarcinoma Bx PC-3 cells,the conditioned medium of Bx PC-3 was collected to simulate the inflammatory microenvironment of pancreatic cancer after the action of baicalein and wogonin.(2)To explore the effect of Bx PC-3 conditioned medium on human umbilical vein endothelial cells(HUVECs)under different inflammatory environments in vitro: Bx PC-3 conditioned medium was applied to HUVECs,and the MTT,Scratch test,and Matrigel experiments were used to explore baicalein and wogonin on vascular endothelial cells in the inflammatory microenvironment of pancreatic cancer.(3)To explore the effect of Bx PC-3 conditioned medium on chorioallantoic membrane(CAM)angiogenesis under different inflammatory environments in vivo: Bx PC-3 conditioned medium was applied to the CAM model to explore the effect of pancreatic cancer inflammatory microenvironment on pancreatic tumor angiogenesis under the action of baicalein and wogonin in vivo.2.Predict the mechanism of pancreatic cancer inflammatory microenvironment on angiogenesis under the action of baicalein and wogoninThrough TCMSP,Gene Cards,Genclip and OMIM database,the target collection of baicalein and wogonin were obtained as drug targets,and the protein function and KEGG pathway of the drug targets were analyzed.Then the intersection of pancreatic cancer,inflammation and angiogenesis-related targets was obtained as disease targets,and the intersection of drugs and disease targets is taken as the relevant target of the inflammatory microenvironment of pancreatic cancer under the action of baicalein and wogonin on angiogenesis.It was imported into “Wei Sheng Xin” for visual analysis,the David database conducts bioinformatics analysis on its GO and KEGG pathways,and uses the STRING database to build a drug-disease-target PPI network.Finally,the top 30 targets among the six algorithms are calculated through the analysis of local characteristic parameters of the network.The intersection serves as the final target.3.In vitro experiments to verify the mechanism of pancreatic cancer inflammatory microenvironment under the action of baicalein and wogonin on pancreatic tumor angiogenesis.In view of the prediction results of network pharmacology,we designed vitro-cell experiments to test the mechanism of pancreatic cancer inflammatory microenvironment on angiogenesis under the action of baicalein and wogonin.The experimental process is as follows:(1)Western blot analysis was performed to analyze the protein expression of HIF-1α,MMP-9,VEGFA,TNF-α,IL-6 and PI3K/AKT/NF-κB,STAT3 pathways in Bx PC-3 cells treated with RAW264.7 conditioned medium.(2)The function of VEGFA in Bx PC-3conditioned medium was reduced by neutralizing antibody,and the effect of Bx PC-3conditioned medium after neutralizing antibody on HUVEC migration was explored by scratch experiment.4.Compares effects of pancreatic cancer inflammatory microenvironment on angiogenesis under the action of Scutellaria baicalensis,Radix Bupleuri Scutellariae,baicalein,wogoninThe clinical drug application of traditional Chinese medicine is mostly its water extract,and the drug pair is the smallest compatibility in the clinical use of traditional Chinese medicine.In order to make the experiment closer to the clinical practical application of traditional Chinese medicine,in the later experiments,by using the macrophage conditioned medium to simulate the inflammatory microenvironment of pancreatic cancer,in vitro cell experiments were designed to compare the different effects of Scutellaria baicalensis,Radix Bupleuri Scutellariae,baicalein and wogonin on angiogenesis of pancreatic cancer in an inflammatory microenvironment.The experimental design is as follows:(1)NO detection by Griess method: According to the content of baicalein and wogonin in the water extract of Scutellaria baicalensis and Radix Bupleuri Scutellariae detected by UPLC technology,the concentration of baicalein and wogonin were prepared to be 60 μM in Scutellaria baicalensis and Radix Bupleuri Scutellariae,and Griess method were used to detect the effects of these drugs on NO secretion of macrophages.(2)Collection of conditioned medium: RAW264.7was induced with LPS and given Scutellaria baicalensis,Radix Bupleuri Scutellariae,baicalein and wogonin to interfere with macrophage activity,and the collected RAW264.7conditioned medium was applied to pancreatic ductal adenocarcinoma Bx PC-3 cells,Bx PC-3conditioned medium was collected to simulate the inflammatory microenvironment of pancreatic cancer after different drugs.(3)To explore the effect of Bx PC-3 conditioned medium on HUVEC under different inflammatory environments in vitro: Bx PC-3 conditioned medium was applied to HUVECs,and MTT,scratch test,and Matrigel experiments were used to explore the effects of different drugs on vascular endothelial cells in the inflammatory microenvironment of pancreatic cancer.(4)To explore the effect of Bx PC-3 conditioned medium on the angiogenesis of rat abdominal aortic rings under different inflammatory environments in vivo: Bx PC-3 conditioned medium was applied to SD rat abdominal aortic rings to explore different drugs efects of the inflammatory microenvironment of pancreatic cancer after action on angiogenesis in vivo.(5)Western blot was used to detect the protein expression of HIF-1α,MMP-9,VEGFA,TNF-α,IL-6 and PI3K/AKT/NF-κB,STAT3 pathways in Bx PC-3 cells under different inflammatory conditions.Result:1.Compared with the model group,60 μM baicalein and wogonin-treated Bx PC-3conditioned medium can significantly inhibit the proliferation,migration,number of branches and total branch length formed by tubules of HUVECs(P<0.05)and angiogenesis in the CAM model in vivo(P<0.01).2.The target of baicalein and wogonin on angiogenesis in pancreatic cancer inflammatory microenvironment was predicted by network pharmacology in total of 24 as follows: IL6,MAPK14,MAPK1,MYC,CCND1,ESR1,JUN,AKT1,MAPK8,TNF,PTGS2,RELA,VEGFA,CXCL8,MMP-9,HIF1 A,AR,MCL1,CASP9,NFKB1,TGFB1,XIAP,NOS2,CATA.3.Compared with the model group,the inflammatory microenvironment under the action of baicalein and wogonin at 60 μM can significantly inhibit the protein expression of p-IκB/IκB,p-p65/p65,p-AKT/AKT and PI3 K,STAT3,HIF-1α,MMP-9,VEGFA,TNF-α,IL-6(P<0.05);compared with the model group,the ability of Bx PC-3 conditioned medium to promote HUVEC scratch healing after neutralization of VEGFA function was significantly inhibited(P<0.05).4.Compared with the model group,when the concentration of baicalein is 60 μM,Scutellaria baicalensis and Radix Bupleuri Scutellariae can significantly inhibit the secretion of NO from RAW264.7(P<0.05).The Bx PC-3 conditioned medium under the action of Scutellaria baicalensis and Radix Bupleuri Scutellariae had no significant effect on the proliferation,migration,number of branches and total branch length formed by tubules of HUVEC and angiogenesis of the abdominal aortic ring of SD rats(P>0.05);compared with the model group,Scutellaria baicalensis with a concentration of 60 μM baicalein can significantly inhibit the protein expression of p-AKT/AKT,PI3 K and MMP-9 in Bx PC-3cells(P<0.05),Radix Bupleuri Scutellariae with a concentration of 60 μM baicalein can significantly inhibit the protein expression of p-AKT/AKT,PI3 K,IL-6(P<0.05)and MMP-9(P<0.01).Conclusion:1.In this study,macrophage conditioned medium and pancreatic tumor cell conditioned culture was extracted to simulate the inflammatory microenvironment of pancreatic cancer,and it was found that 60 μM baicalein and wogonin could inhibit angiogenesis in the inflammatory microenvironment of pancreatic cancer,through the prediction of network pharmacology and the verification of Western blotting experiments,it is suggested that the effect of baicalein and wogonin on angiogenesis may be related to HIF-1α,MMP-9,VEGFA,TNF-α,IL-6 and PI3K/AKT/NF-κB,STAT3 pathways,and through neutralizing antibody experiments and scratch experiments,it was suggested that VEGFA may be a key target for pancreatic tumor cells to regulate endothelial cell migration.2.Scutellaria baicalensis and Radix Bupleuri Scutellariae with baicalein content of 60μM can inhibit the inflammatory microenvironment of macrophages and the protein expression of PI3K/AKT pathway and MMP-9 in Bx PC-3 cells under the action of inflammatory microenvironment,however,there was no significant inhibitory effect on the angiogenesis of pancreatic cancer in the inflammatory microenvironment under the experimental period and conditions.