Inhibitory Effect And Mechanism Of Ophiopogonin B On Human Lung Adenocarcinoma A549 Xenog Rafted In Nude Mice

Background:Lung cancer is the abbreviation of primary bronchial lung cancer and is currently one of the most common malignant tumors in clinical practice.Non-small cell lung cancer(NSCLC)accounts for more than 85%of newly diagnosed lung cancer cases each year.Modern medicine mainly uses surgery,chemotherapy,and radiotherapy.For treatment with technical methods,due to the side effects of chemotherapy drugs such as cytotoxicity and drug resistance,the final prognosis of NSCLC patients is poor,and the 5-year survival rate of patients is only 15%.Therefore,the prevention and treatment of NSCLC has become a research hotspot in the medical field.The malignant degree of tumor depends on the efficiency of tumor cell growth and proliferation,and sufficient energy supply is the prerequisite and necessary condition for tumor cell malignant proliferation.The "Warburg",effect is aerobic glycolysis,which is one of the important pathways of energy metabolism in tumor cells.The enhanced glycolysis of tumor cells is related to the enhanced expression or activity of related enzymes.Studies have shown that inhibiting tumor cell glycolysis can effectively inhibit tumor cell proliferation and effectively kill tumor cells.In recent years,domestic and foreign studies have shown that traditional Chinese medicine and its biologically active ingredients can inhibit tumors at multiple levels and multiple targets,providing new ideas and directions for tumor prevention and treatment.Ophiopogon saponin B(OP-B)is a key indicator for the quality control of Ophiopogon japonicus and an important active ingredient for its medicinal effect.It can significantly inhibit the growth and differentiation of a variety of tumor cell lines and has potential anti-tumor effects.Objectives:To study the inhibitory effect of OP-B on the proliferation of human lung adenocarcinoma A549,explore its effect on the Warburg effect and its regulatory mechanism,provide an experimental basis for the clinical application of OP-B.Methods:SPF male BALB/c-nu nude mice,human lung adenocarcinoma A549 cells were inoculated into the right side of the skin,and A549 nude mice xenograft model was prepared.After screening,they were randomly divided into 4 groups:blank group(group A),OP-B low dose group(group B),OP-B high dose group(group C),and cisplatin group(group D).B and C groups were intragastrically administered with the corresponding concentration of OP-B,once a day for 21 days.Cisplatin group was injected intraperitoneally with cisplatin(2mg/kg)in D group,once every 3 days.The blank group was intragastrically administered with an equal volume of physiological saline.The tumor growth curve and tumor inhibition rate were observed.HE staining was used to detect tumor pathological changes.Colorimetric method was used to detect lactic acid,Na+-K+-ATPenzymeand Ca2+-Mg2+-ATPenzymelevels in tumor tissues.TUNEL method was used to detect apoptosis.Immunohistochemistry method for expressions of apoptosis-related proteins BCL-2,BAX and CASPASE-3 were detected.Western blot was used to detect the expression of glucose transporter-1(GLUT1),hexokinase-2(hK2),pyruvate kinase-2(pkm2),lactate dehydrogenase A(LDH-A)and hypoxia inducible factor-1α(HIF-1α)in tumor tissues.Results:1.Compared with the control group,the tumor volume and weight of each administration group decreased to various degrees(P<0.01),and the tumor volume and weight of each administration group decreased concomitantly with the increase of OP-B dose,and the tumor inhibition rate increased significantly.HE staining results showed that compared with the control group,the number of tumor cells in each administration group was significantly reduced,the nucleus pyknosis,cytoplasm swelling or fusion,loose connective tissue was increased and surrounded the compartmentalized tumor cells,capillaries were significantly reduced.OP-B high dose group showed the most obvious pathological changes.2.Compared with the control group,the number of tumor cells undergoing apoptosis,the protein expression of BAX and CASPASE-3 in each administration group were significantly increase(P<0.05),the protein expression of BCL-2 was significantly decreased(P<0.01),and the changes of the above indicators were more obvious with the increase of OP-B dosage.3.Compared with the control group,the levels of lactic acid secretion and the activities of Na+-K+-ATPase and Ca2+-Mg2+-ATPase in the tumor tissues of each administration group were significantly(P<0.01)lower,and their above indexes decreased more significantly with increasing doses of OP-B.Compared with the control group,the protein expressions of GLUT1,HK2,PKM2,LDH-A and HEF-1αin tumor tissues of each administration group were significantly decreased(P<0.01),and the differences in the changes of the above indexes in tumor tissues were more obvious with the increase of OP-B dosage.Conclusions:1.OP-B exhibited obvious inhibitory effects on human lung cancer A549 cell growth.2.OP-B can promote the occurrence and development of apoptosis in human lung cancer A549 cells by regulating the expression of apoptosis related proteins BCL-2,BAX and CASPASE-3.3.OP-B can reduce lactate secretion and ATPase activity in human lung cancer A549 cells,thereby exerting an inhibitory effect on the energy metabolism of tumor cells.OP-B can block the glycolytic process in human lung cancer A549 cells by inhibiting the expression of GLUT1,HK2,PKM2,LDH-A and HIF-1α proteins.