A Study Of Akebiae Fructus Seeds Ethanol Extract Inhibits Proliferation Of Hepatoma Cells And Induces Cytoplasmic Vacuoles

Objective:A study of Akebiae Fructus which is a representative traditional Chinese medicine having the effects of soothing the liver,regulating qi and activating blood.To study the mechanism of the ethanol extract of Akebiae fructus seeds?AFSEE?inhibiting the proliferation of hepatoma cells,and to study the effect of the combination of AFSEE with cytotoxic drugs,targeted drugs and anti-cancer monomers extracted from traditional Chinese medicine,and to explore the mechanism of cytoplasmic vacuoles induced by AFSEE.Methods:?1?Analyze the relevant knowledge of traditional Chinese medicine Akebiae fructus through ancient herbal literature and modern clinical and experimental research reports.?2?MTT assay,cell morphology observation,flow cytometry,gene array,PCR and other methods were used to observe the effects of AFSEE on proliferation,cell cycle,apoptosis,necrosis and cell senescence of SMMC7721 hepatoma cells.?3?Using MTT method and cell morphology observation to evaluate the effects of AFSEE combined with common chemotherapy drugs,targeted drugs and anti-cancer monomers extracted from traditional Chinese medicine.?4?Using cell morphology observation,PCR and other methods to assess the phenomenon and mechanism of cytoplasmic vacuoles induced by AFSEE.To detect whether the AFSEE acts through endoplasmic reticulum stress or paraptosis by detecting the expression of genes involved in the signaling pathway involved in the above phenomenon.Results:?1?Clarified the academic origin of the clinical application of the AFSEE and sorted out the historical understanding and modern understanding of its property,flavor,efficacy and treatment.?2?After 72 hours'intervention of the AFSEE,the proliferation of SMMC7721liver cancer cells was significantly inhibited,and the dose of IC₅₀ is 917.4?g/ml.After 72 hours'intervention of the AFSEE at 375?g/ml and 750?g/ml,the proportion of cells in G0/G1 phase decreased,the proportion of cells in S phase decreased,and the proportion of cells in G2/M phase increased;the proportion of apoptotic and necrotic cells increased slightly from 6.6%to 12.6%;and?-galactosidase dyeing become positive.?3?Confirmed the combined drug effects of AFSEE with epirubicin,oxaliplatin,5-FU,vincristine,norvinblastine,Gleevec,mitomycin,paclitaxel,celastrol,curcumin and sorafenib.?4?AFSEE induces typical intracellular cytoplasmic vacuolation;the concentration of vacuolation inducing in SMMC7721 cells is much lower than that of L-02 normal hepatocytes;and the induced vacuolization can recover spontaneously after withdrawal,but it takes long time.The phenomenon of cytoplasmic vacuolation is related to the structure of saponin E.The vacuoles are non-lipid and their contents are different from the cytoplasm and nucleus.AFSEE caused up-regulation of GDF15?PPP1R15A?JUN?IGF1 and other gene expression in 72h;caused the down-regulation of ATF6?HSP90B1,WARS?PPIA?PPIB?HSPA4?PDCD5?PDCD6IP?TP53?WNT1and other genes.Conclusion:?1?YUZHIZI in the ancient literature before 1936 was not the name of Akebiae fructus.In the modern literature after 1936,YUZHIZI is the name of Akebiae fructus under most circumstances.There are some errors in some ways such as basic source,property,flavor,efficacy and treatment.?2?AFSEE can effectively inhibit the malignant proliferation of SMMC7721 cells,affect the cell cycle,slightly promote apoptosis and necrosis,and induce cell senescence.?3?In vitro cytology observation,AFSEE combined with oxaliplatin,5-FU,vincristine,norvinblastine,mitomycin is harmful.The combination of AFSEE with sorafenib is beneficial.There are certain prospects for combination of AFSEE with epirubicin,Gleevec,paclitaxel,celastrol or curcumin.?4?AFSEE-induced cytoplasmic vacuolation may be related to genes and proteins such as ATF6?GDF15?PPP1R15A and ATF4 associated with endoplasmic reticulum stress;JUN?PDCD6IP?WNT1?PPIA?PPIB associated with paraptosis.