| Lung cancer is a type of tumor with low degree of differentiation and high degree of malignancy.Its morbidity and mortality ranks among the forefront of all malignant tumors worldwide.Although the current clinical first-line chemotherapy drugs can inhibit the occurrence and development of lung cancer,but there are serious side effects.Traditional Chinese medicine can synergize with chemotherapy drugs,reduce the toxic and side effects of chemotherapy,and improve the quality of life of patients.Thus it has great application prospects in the treatment of tumors.Based on the therapeutic principles of promoting blood circulation and removing blood stasis,invigorating qi and strengthening the body,the reserach group conducted deconstraction and antitumor activity detection of Huoxue Yiqi decoction in "Synopsis of the Golden Chamber" in the early stage,and found that the formula for promoting blood circulation and nourishing qi(ginseng,salvia,licorice)has anti-tumor effect,and this effect may be related to the inhibition of PD-L1 expression.In this subject,the chemical constituents of Huoxue Yiqi recipe will be analyzed by ultra-high performance liquid chromatography-quadrupole-time-of-flight mass spectrometry(UPLC-Q/TOFMS)method.In vitro experiments were carried out to verify the anti-tumor effect and explore its mechanism of action of Glycyrrhetic acid,the main component in plasma.Objective:In this study,UPLC-Q/TOF-MS was used to analyze the chemical constituents of Huoxue Yiqi recipe in vitro and in vivo.This study confirmed the anti-tumor effect of glycyrrhetinic acid,the main component of Huoxue Yiqi recipe,and explored that its anti-tumor effect may down-regulate the expression of PD-L1 protein by inhibiting the activity of IL-1β/NF-κB.Methods:1.Based on UPLC-Q/TOF-MS technology research on the chemical constituents of Huoxue Yiqi prescription in vitro and in vivo,the data of Huoxue Yiqi recipe was collected,and the database of TCMSP,Pubchem,Chemspider and other databases were used to search,and the chemical composition database of ginseng,salvia and licorice was built by ourselves.Combine the information of primary MS and fragment information of secondary MS,compare its structure with secondary fragments,and use the information of characteristic fragments and neutral ion loss to determine whether the broken group is a functional group in the structure,and analyze the Huoxue Yiqi formula.Chemical constituents in decoctions.2.The MetabolitePilotTM(v2.2)software was used to determine the original components and possible metabolites of Huoxue Yiqi recipe into the blood.3.MTT method and the effect of Glycyrrhetic acid on the proliferation of lung cancer cells and normal lung cells was detected by clone formation assay.4.The cell scratch test was used to observe the effect of Glycyrrhetic acid on the migration ability of lung cancer H1975 cells,and the Transwell test was used to detect the effect of Glycyrrhetic acid on the migration and invasion ability of lung cancer H1975 cells.5.qRT-PCR detection of Glycyrrhetic acid on the gene expression of IL-1β,TNF-α,VEGF,IL-8,NF-κB,PD-L1 in lung cancer cells H1975.6.The effect of Glycyrrhetic acid on the expression of PD-L1 protein in lung cancer H1975 cells was detected by Western Blot,and the effect of Glycyrrhetic acid on the nuclear entry of NFκB p65 in lung cancer H1975 cells was detected by cellular immunofluorescence.ELISA detected the effect of Glycyrrhetinic acid on the secretion of IL-1β in lung cancer H1975 cells.Results:1.UPLC-Q/TOF-MS data with the self-built database and literature,85 compounds were preliminarily identified from Huoxue Yiqi recipe,2 7 were derived from licorice,40 were derived from Salvia miltiorrhiza,and 18 were derived from ginseng.These include licorice flavonoids,insenosides,salvianolic acids,Diterpene quinones,etc.2.MetabolitePilotTM(v2.2)was determined in mouse plasma to obtain 6 prototype components and 11 metabolites in Huoxue Yiqi recipe.3.HPLC and UPLC-Q/TOF-MS experiments found that glycyrrhizic acid,the main active component of licorice,exists in Huoxue Yiqi recipe,and the metabolite glycyrrhetinic acid of glycyrrhizic acid was found in plasma.4.The results of MTT assay showed that Glycyrrhetinic acid showed an increase in the inhibition rate of lung cancer cells A549,H1299,H1975,H460,and 95D with the increase of concentration and time.Clone formation experiments showed that Glycyrrhetinic acid significantly decreased the proliferation of lung cancer cells A549,H1299,H1975,H460 and 95D(p<0.05 or p<0.01).5.Scratch test and Transwell results showed that Glycyrrhetic acid could significantly inhibit the migration ability and invasion ability of lung cancer cells H1975(p<0.05 orp<0.01)6.The results of qRT-PCR experiment showed that Glycyrrhetinic acid could significantly reduce the gene expression levels of cytokines IL-1β,TNF-α,VEGF and IL-8 in H1975 lung cancer cells(p<0.01 or p<0.001).7.The results of qRT-PCR and Western Blot experiments showed that Glycyrrhetic acid could significantly reduce the gene and protein expression levels of H1975 PD-L1 in lung cancer cells(p<0.01 or p<0.001).8.The results of immunofluorescence detection showed that Glycyrrhetic acid could significantly reduce the gene and protein expression levels of H1975 PD-L1 in lung cancer cells(p<0.05 or p<0.01).Conclusion:Huoxue Yiqi recipe contains a variety of active ingredients,and 85 ingredients derived from Ginseng,Salvia miltiorrhiza,and Licorice were preliminarily identified,mainly representative components of corresponding traditional Chinese medicine such as flavonoids,saponins,and phenolic acids.The main active component of licorice,Glycyrrhizic acid,was found in the decoction of Huoxue Yiqi Fang,and the main metabolite of Glycyrrhizic acid,Glycyrrhetinic acid,was found in plasma samples after Huoxue Yiqi Decoction.Glycyrrhetinic acid,a representative component in the plasma of mice treated with Huoxue Yiqi Decoction,has a significant inhibitory effect on the proliferation,migration and invasion of various lung cancer cells.Glycyrrhetinic acid may exert its ability to inhibit the proliferation,migration and invasion of lung cancer cells by inhibiting the expression of inflammatory cytokines in lung cancer H1975 cells,thereby inhibiting the nuclear translocation of NF-κB p65,and finally inhibiting the expression of PD-L1. |
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