Study On Anti-hepatocarcinoma Effect Of Isoalantolactone Through AMPK Signaling Pathway

Liver cancer is the most common cancer in the world.It is also the second leading cause of death in cancer.The incidence of liver cancer in our country is relatively high at the world level,and it has created a serious economic burden on China's medical and social security,which is still under development.Liver cancer has the characteristics of difficulty in discovery and rapid progression.At present,there are no markers that can accurately detect early stage liver cancer.Usually,patients reach the advanced stage when they are diagnosed,and miss the best opportunity for surgical treatment.At present,the specificity of targeted drugs for treating liver cancer is not high,and targeted drugs that target a single target are likely to produce drug resistance after continuous drug use.It is still necessary to find drugs targeting more targets to increase the therapeutic effects of liver cancer and extend the patients The survival period improves the quality of life.AMPK is a protein associated with energy metabolism.When the ATP in a cell is below a threshold,it activates AMPK and inhibits ATP consumption,including the synthesis of lipids,RNA,and proteins.Tumor cells are highly proliferating cells that consume a large amount of ATP for the synthesis of various substances.The expression of AMPK in tumor tissues is significantly lower than that around tumors,suggesting that AMPK is a potential antitumor target.Traditional Chinese medicine is the product of traditional wisdom in our country,which has proved its effectiveness for nearly several thousand years.Modern medical research has found that the active ingredient of traditional Chinese medicine is an important new drug source.The latest discovery is that isoalantolactone has antibacterial and insecticidal effects.In addition,there are anti-tumor effects.Therefore,the purpose of this study is to explore whether anti-hepatocarcinoma effects of isoalantolactone in vivo and in vitro,whether through the AMPK signaling pathway,and to explore the relevant mechanisms.This research content is divided into three parts.Part?:Anti-hepatocarcinoma effect of isoalantolactone in vitro.MTT assay was used to study the anti-hepatocarcinoma effect of isoalantolactone in vitro,and the dose-dependence and time-dependence of its antitumor activity was clarified,and its IC₅₀ was calculated.The IC₅₀values of isoclaran lactones against Bel-7402 at 24 hours,48 hours,and 72hours were 37.4,25.46,and 22.36?M,respectively.HepG2 is 34.82,26.32,20.22?M.Part?:Anti-hepatocarcinoma Effect of isoalantolactone in vivoA tumor-bearing BALB/c nude mouse model of hepatocarcinoma Bel-7402 was established.The liposome was prepared with isoalantolactone,administered orally once a day for 14 consecutive days.The tumor volume and the weight of the nude mice were measured every other day.The animals were sacrificed on the 15th day and the tumor tissues were weighed.The volume inhibition rate of 90 mg·kg^-1isoalantolactone was 67.32%,the weight inhibition rate was 70.15%,and its anti-tumor effect in vivo has a certain degree of safety.Part?:the anti-tumor mechanism of isoalantolactoneHoechst 33258 was used to observe the morphological changes of hepatocarcinoma cells by isoalantolactone.Annexin V-FITC,PI,DCFH-DA staining and flow cytometry were used to detect apoptosis,cycle inhibition,and ROS production.The results showed that 40?M isoalantolactone can induce apoptosis of hepatocarcinoma,arrest the proliferation of hepatocarcinoma cells in G2/M phase,and significantly increase ROS level.Western blot and immunohistochemistry were used to detect key signaling molecules.The results showed that 40?M isoalantolactone can induce the expression of p-AMPK,p53,Bax,Cleaved-Caspase3 and Cleaved-Caspase9,and inhibit the expression of CyclinB1,CDK1 and Bcl-2 protein.The results of immunohistochemistry showed that the expression of p-AMPK,p53 and Caspase3 proteins in tumor tissues were also up-regulated.In summary,isoalantolactone has an anti-hepatocarcinoma effect both in vivo and in vitro.The mechanism is to induce apoptosis of hepatocarcinoma cells by generating ROS,activating the AMPK pathway,upregulating p53 regulatory factors,and arresting the growth cycle of hepatocarcinoma cells in the G2/M phase,thereby inhibiting hepatocarcinoma proliferation through multiple targets.This study will lay the theoretical foundation for the development and clinical application of isocitrate as a new anti-hepatocarcinoma drug.