| ObjectiveTo observe the effect of Banxia Xiexin Decoction medicated intestinal absorption solution on the proliferation and migration of PMN-MDSCs in gastric cancer microenvironment,and to explore the mechanism of action.To reveal the action rule of Banxia Xiexin Decoction intervening the migration and invasion of PMN-MDSCs in the gastric cancer microenvironment through FAK signaling pathway,so as to explore the action mechanism of Banxia Xiexin Decoction preventing and treating gastric cancer from the perspective of intervention of PMNMDSCs,and provide ideas for the diagnosis and treatment of gastric cancer in clinical Chinese medicine.MethodsBanxia Xiexin Decoction medicated intestinal absorption solution containing 0.63 g/m L of raw drug was prepared,and non-contact co-culture of gastric cancer cells and PMN-MDSCs was conducted in Transwell chamber to establish the gastric cancer microenvironment model.Cell proliferation and activity detection(CCK-8)method was used to screen the optimal intervention concentration and time of PMN-MDSCs in Banxia Xiexin Decoction medicated intestinal absorption solution for follow-up experiments.The experiments were divided into blank group,model group,FAK inhibitor group and Banxia Xiexin Decoction medicated intestinal absorption solution group with 26%,18% and 10% concentration.The proliferation of PMN-MDSCs was detected by CCK-8 method.Apoptosis of PMN-MDSCs was detected by flow cytometry.The migration and invasion ability of PMN-MDSCs were detected by cell scratch assay and Transwell assay.The expressions of vascular endothelial cell growth factor(VEGF)and matrix metalloproteinase-9(MMP-9)cytokines in tumor microenvironment were detected by enzyme-linked immunosorbent assay(ELISA).The expression levels of local adhesion spot kinase(FAK),phosphorylated(p)-FAK,protein tyrosine kinase(Src)and phosphorylated(p)-Src of PMN-MDSCs pathway related proteins were detected by Western blot.Results1.CCK-8 method: Compared with 24 h,after 48 h treatment,the inhibition rates of PMNMDSCs were increased by 5%,50%,75% and 100% Banxia Xiexin Decoction medicated intestinal absorption solution(P<0.05).Compared with 48 h,after 72 h treatment,the inhibition rate of PMN-MDSCs in 50% Banxia Xiexin Decoction medicated intestinal absorption solution was lower than that in 48 h(P<0.01),and that in 5% and 100% Banxia Xiexin Decoction medicated intestinal absorption solution was slightly higher than that in 48 h(P<0.05).There was no significant difference in the inhibition rate of other concentrations.And 48 h half inhibitory concentration(IC50)was 18.09%,indicating that 18% concentration of Banxia Xiexin Decoction containing intestinal absorption solution was the best intervention concentration and time at 48 h.In vitro gastric cancer cells were co-cultured with PMN-MDSCs without contact,and treated with Banxia Xiexin Decoction medicated intestinal absorption solution for 48 h.Compared with blank group,the survival rate of PMN-MDSCs in model group was significantly increased,with statistical significance(P<0.05).Compared with model group,the survival rate of PMN-MDSCs in FAK inhibitor group,Banxia Xiexin Decoction26%,18% and 10% medicated intestinal absorption solution groups decreased significantly,with statistical significance(P<0.05).All concentration groups of Banxia Xiexin decoction could inhibit PMN-MDSCs in gastric cancer microenvironment.Among them,26% medicated intestinal absorption solution group was the most significant(P<0.05).2.Flow cytometry: In vitro gastric cancer cells were co-cultured with PMN-MDSCs without contact,and treated with Banxia Xiexin Decoction medicated intestinal absorption solution for 48 h.Compared with blank group,apoptosis rate of PMN-MDSCs in model group was significantly decreased(P<0.05).Compared with model group,the apoptosis rate of PMNMDSCs in FAK inhibitor group,Banxia Xiexin Decoction 26%,18% and 10% medicated intestinal absorption solution groups was significantly increased(P<0.05).In this study,all concentration groups of Banxia Xiexin Decoction could promote the apoptosis of PMNMDSCs in gastric cancer microenvironment,among which 26% medicated intestinal absorption solution group was the most significant(P<0.05).3.Cell scratch test: After 48 h of external intervention with Banxia Xiexin Decoction containing intestinal absorption liquid,migration distance of PMN-MDSCs in model group was significantly increased compared with blank group(P<0.01).Compared with model group,the migration distance of PMN-MDSCs in FAK inhibitor group,Banxia Xiexin Decoction 26%,18% and 10% medicated intestinal absorption solution groups decreased significantly(P<0.01).All concentration groups of Banxia Xiexin decoction could inhibit the migration of PMNMDSCs in gastric cancer microenvironment.26% intestinal absorption solution group was the most significant(P<0.01).4.Transwell experiment: After 48 h of external intervention with Banxia Xiexin Decoction containing intestinal absorption liquid,migration and invasion of PMN-MDSCs increased in model group(P<0.01)compared with blank group(P<0.01).Compared with model group,migration number of PMN-MDSCs in FAK inhibitor group,Banxia Xiexin Decoction 26%,18%and 10% medicated intestinal absorption solution groups decreased(P<0.01)and invasion number decreased(P<0.01).In the present study,all concentration groups of Banxia Xiexin Decoction could inhibit the migration and invasion of PMN-MDSCs in the gastric cancer microenvironment,and 26% medicated intestinal absorption solution group was the most significant(P<0.01).5.Enzyme-linked immunosorbent assay(ELISA): After 48 h intervention with Banxia Xiexin Decoction and its different concentration groups,compared with blank group,the expressions of VEGF and MMP-9 factors of PMN-MDSCs in model group were significantly increased(P<0.01).Compared with model group,the expressions of PMN-MDSCs VEGF and MMP-9 factors were decreased in FAK inhibitor group,Banxia Xiexin Decoction 26%,18%and 10% medicated intestinal absorption solution groups(P<0.01),and the most significant was in 26% medicated intestinal absorption solution group(P<0.01).6.Western blot: After 48 h intervention with Banxia Xiexin Decoction and its different concentration groups,the expressions of PMN-MDSCs FAK,P-fak,Src and P-src in model group were significantly increased compared with blank group(P<0.01).Compared with model group,FAK,p-fak,Src and protein expressions of PMN-MDSCs were decreased in FAK inhibitor group and Banxia Xiexin Decoction 26%,18% and 10% medicated intestinal absorption solution groups(P<0.01),and P-src protein expressions were decreased in FAK inhibitor group and Banxia Xiexin decoction 18% medicated intestinal absorption solution group(P<0.01).ConclusionIn this study,it was found that different concentrations of Banxia Xiexin Decoction medicated intestinal absorption solution could inhibit the proliferation of PMN-MDSCs and induce their apoptosis,and reduce the ability of cell migration and invasion.The mechanism of action was related to the down-regulation of the expression of cytokines VEGF and MMP-9and the inhibition of FAK signaling pathway in the gastric cancer microenvironment.The biological mechanism of PMN-MDSCs proliferation and migration in gastric cancer microenvironment was preliminarily clarified. |
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