| Background: Hepatocellular carcinoma is a common malignant tumor with a high incidence and mortality.Although the choice of surgical resection is the preferred treatment for patients with liver cancer,many patients miss the best treatment time due to insidious onset.In addition to local treatments such as TACE,PEI,etc.,the overall prognosis is still poor with more side effects,the effect is not satisfactory.However,at present,traditional Chinese medicine has a good effect in improving the quality of life of patients and reducing the pain of patients,and has great potential value in prolonging the survival period.Asiatic acid(AA)is found in many edible and medicinal plants.A large number of clinical studies have shown that asiatic acid has anticancer potential.Basic research shows that asiatic acid can reduce inflammation,inhibit tumor cell proliferation,induce mitochondrial apoptosis and other effects.It has been reported that asiatic acid can inhibit the proliferation of hepatoma cells and promote apoptosis,but the specific mechanism of inhibition of liver cancer cell metastasis,proliferation and apoptosis has not been reported in detail.Our laboratory has long been engaged in the research of traditional Chinese medicine for the treatment of liver cancer and has a good working basis.The main content of this study is to investigate the effects of asiatic acid on proliferation,apoptosis and invasion of hepatoma cells and its molecular mechanism.It provides a theoretical basis for the clinical application of asiatic acid in liver cancer.Part ?: Effect of asiatic acid on proliferation and apoptosis of hepatoma cellsObjective: To investigate the effects of asiatic acid on proliferation and apoptosis of hepatoma cells and its related mechanismsMethods: 1.The effects of different concentrations of asiatic acid on the proliferation of human hepatoma cell line Huh7 were detected by CCK8 and EdU kits.4.Flow cytometry was used to detect the effect of asiatic acid on cell cycle and apoptosis of human hepatoma cell line Huh7.3.Western blot analysis was used to detect the effects of different concentrations of asiatic acid on human hepatoma cell line Huh7 on major protein molecules and related apoptotic proteins on MAPK and AKT signaling pathways,including P-Akt,P-ERK1/2,P-P38,Akt,ERK,P38;related apoptosis protein molecules include cleaved-caspase 3,cleaved-caspase 9,bax,bcl-2,pro-caspase 3,pro-caspase 9.4.in vivo Experiment: Human hepatoma cell Huh7 was implanted into the skin of mice,and intraperitoneal injection of asiatic acid was observed after tumor formation.And then verified the inhibitory effect of asiatic acid on subcutaneous tumors in nude mice and the safety of liver and kidney biochemical function.Results: 1.The results of CCK8 showed that with the increase of time and drug concentration,asiatic acid could significantly inhibit the activity of human hepatoma cell line Huh7,and the effect was stronger at 50 ?M and 100 ?M.Normal human liver cells had no significant effect,only when the concentration of asiatic acid was 100 ?M,and it had a slight inhibitory effect after 24 h;EdU showed that with the increase of the concentration of asiatic acid,the human hepatoma cell line Huh7 was significantly inhibited.At a concentration of 100 ?M,approximately 80.37 ± 2.34% of cell proliferation was inhibited.2.Cell cycle results by flow cytometry showed that asiatic acid induced the accumulation of human hepatoma cells Huh7 in G1 phase and the decrease of G2/M phase cells in a dose-dependent manner,indicating that asiatic acid inhibited cells from G1 to G2/M phase transformation;detection of apoptosis showed that the apoptotic rate of human hepatoma cell line Huh7 increased with the increase of asiatic acid concentration.3.Western blot results showed that the protein levels of cleaved-caspase 3 and cleaved-caspase 9 in human hepatoma cell line Huh7 increased with the dose-dependent manner,and the protein levels of pro-caspase 3 and pro-caspase 9 were almost unchanged.The expression level of bax increased in a dose-dependent manner,while bcl-2 expression decreased.The level of P-Akt was significantly decreased at 100 ?M,the level of P-ERK1/2 was significantly decreased in a dose-dependent manner,and the expression of P-P38 was significantly increased at a concentration of 10 ?M.The levels of Akt,ERK1/2 and P38 remain essentially unchanged.4.the results of in vivo experiments showed that the asiatic acid group can inhibit the growth of subcutaneous tumors well.The results of immunohistochemistry showed that with the increase of the concentration of asiatic acid,the level of proliferation marker ki-67 was inhibited.The level of the apoptosis marker bcl-2 was inhibited and the level of bax was elevated,indicating that asiatic acid can effectively inhibit the proliferation of subcutaneous tumor cells in nude mice and promote apoptosis.Conclusion: Asiatic acid can inhibit the proliferation of human hepatoma cells Huh7 and promote their apoptosis.Molecular mechanisms are involved in the regulation of cleaved-caspase 3,cleaved-caspase 9,bax,bcl-2 expression,and regulation of MAPK and AKT signaling pathways.And asiatic acid can inhibit the growth of subcutaneous tumors in nude mice,and it is safe.Part ?: Effect of asiatic acid on migration and invasion of liver cancer cellsObjective: To investigate the effects of asiatic acid on migration and invasion of hepatocarcinoma cells and its related mechanismsMethods: 1.In order to improve the effect of asiatica acid on hepatocellular carcinoma cells,the effects of asiatica acid on proliferation and apoptosis of MHCC-97 H cells were also examined.The effect of different concentrations of asiatic acid on the proliferation of human hepatoma cell line MHCC-97 H was detected by CCK8.2.Flow cytometry was used to detect the effect of asiatic acid on apoptosis of human hepatoma cell line MHCC-97 H.3.Transwell chamber migration assay was used to detect the effects of different concentrations of asiatic acid on the migration of highly metastatic human hepatoma cell line MHCC-97H(choose the concentration that had less effect on proliferation and apoptosis),and the transwell chamber invasion assay of pre-made Matrige gel to detect asiatic acid for human hepatoma cell line MHCC-97 H cell invasion.4.Using western blot to detect the effects of different concentrations of asiatic acid on human hepatoma cell line MHCC-97 H,the main protein molecules in MAPK signaling pathway and related invasion and migration proteins,the key molecular proteins in MAPK signaling pathway include P-ERK1/2,P-P38,P-JNK,ERK1/2,P38,JNK;protein molecules involved in invasion and migration include MMP-2 and MMP-9.Results: 1.The results of CCK8 showed that with the increase of time and drug concentration,asiatic acid inhibited the activity of human hepatoma cell line MHCC-97 H cells.2.Flow cytometry showed that the apoptosis rate of MHCC-97 H cells increased with the concentration of asiatic acid,which was concentration-dependent.3.Transwell chamber experiments showed that when the concentration was greater than 10 ?M,it could effectively inhibit the migration and invasion of human hepatoma cell line MHCC-97 H.;4,Western blot results showed that the protein level of MMP-2 and MMP-9 decreased with the dose of asiatic acid after 24 h of human hepatoma cell line MHCC-97 H,MMP-9 The level changes little after10 ?M.P-ERK1/2 levels were reduced in a dose-dependent manner and P-P38 expression was significantly increased at 25 ?M concentration.The expression of P-JNK was significantly inhibited at a concentration of 25 ?M,and the change at other concentrations was not significant.The levels of ERK1/2,P38 and JNK remained essentially unchanged.Conclusion: Asiatic acid can inhibit the invasion and migration of human hepatoma cell line MHCC-97 H.The molecular mechanism is related to the regulation of MMP-2 and MMP-9 expression and regulation of MAPK signaling pathway. |
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