| Isodon amethystoides(Benth)Cy Wu et Hsuan is a traditional medicine and food homologous,whole grass medicine plants.The Pharmacopoeia records that it can eliminate inflammation,antibacterial,phlegm,heat,and blood stasis,and is commonly used to treat hemorrhoids,snake bites,tuberculosis and acute infectious hepatitis.Recent studies have shown that the internal natural active ingredients are complex and rich in content,and contain a large number of anti-tumor steroids.In the early stage,the research team extracted,isolated and purified two kinds of natural active substances,inflexuside A and glaucocalyxin A.In addition,found that it has certain anticancer activity.Therefore,in this study,A549(Lung cancer)and HepG2(Liver cancer)cells were used as the research object,and the two active substances were studied in vitro,and the cell viability,reactive oxygen species(ROS),and dehydrogenation of lactate were studied.The detection of enzyme(lacte dehydrogenase,LDH),observation of JC-1,AV/PI staining,Western Blot assay and other experiments,explore the role of inhibition of tumor activity and its mechanism of action,for the Chinese medicine can it provides theoretical basis and technical support as a new potential anti-tumor drug in clinical practice.(1)MTT assay showed thatinflexuside A and glaucocalyxin A could inhibit the proliferation of A549 and HepG2 cells,and inhibit the growth of tumor cells in different cell lines.Among them,inflexuside A inhibited the growth of A549 cells,while glaucocalyxin A inhibited HepG2 cells.The anti-proliferative effect is stronger,and the viability of both tumor cells gradually decreases with the increase of the concentration of the drug in vitro and the increase of the action time of the drug.The study found that afterinflexuside A and glaucocalyxin A,the cell morphology and structure of the two tumor cells were destroyed to varying degrees.(2)After the action of inflexuside A and glaucocalyxin A,the ROS level in tumor cells increased significantly,causing oxidative stress damage of tumor cells.(3)By detecting the release rate of LDH in the supernatant of HepG2 and A549 cells before and after drug treatment,it was found that the release rate of LDH was significantly increased after drug treatment.(4)Through AV/PI staining and flow cytometry analysis,it was found that the active concentration of the low concentration of Wangzaozi on the tumor cells showed that the number of early apoptotic cells increased;the effect of high concentration of drugs,early apoptosis gradually turned into late stage.Apoptosis,this study shows that the inhibition of the activity of the two drugs on tumor cells is caused by the initiation of apoptosis.(5)It can be seen from the changes of mitochondrial membrane potential of inflexuside A and glaucocalyxin A after treatment of two kinds of tumor cells for 12 h,the drug action causes the mitochondrial membrane potential to decrease.The effects of two active substances on the content of Cytochromsome C(Cyto-C)in A549 and HepG2 cytoplasm were investigated.The results showed that the cytoplasm of the control group contained almost no Cyto-C,and the cytoplasm was added after the drug was added.The content of Cyto-C is increased.(6)After treated with different concentrations of inflexuside A for 12 h,the expression of Bax was significantly increased and the anti-apoptotic protein Bcl-2 was decreased.The effect of glaucocalyxin A on both cells is almost identical to that of ISAA.This experiment preliminarily proved that the inflexuside A and glaucocalyxin A extracted from Wangzaozi can inhibit the mitochondrial membrane potential,enhance the permeability,and release Cyto-C into the cells,triggering the apoptotic signal pathway.High Bax expression,attenuates Bcl-2 expression and ultimately leads to apoptosis. |
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