| Hepatocellular carcinoma(Hepatocellmular carcinoma,HCC)(hereinafter referred to as cancer of the liver)incidence is second only to lung cancer was ranked the second of malignant tumors,the incidence of liver cancer is different due to regional differences,the incidence of Asian countries than the United States and Western European countries,the incidence in China has more than 25/100000,of which high incidence of liver cancer standardized incidence of 49.17/100000 population.According to statistics,the world's annual increase of liver cancer patients more than 60 million people,of which nearly half of distribution in China.In addition,China's annual death of liver cancer patients is about 300000 people,5-year survival rate of only 2.7% of patients.Liver cancer onset occult,rapid progress,poor quality of life,short survival time.Surgery is the preferred method of early liver cancer treatment,radiation therapy,chemotherapy is advanced liver cancer and metastatic liver cancer commonly used treatment.Over the years,the research and development of natural herbal medicine has received extensive attention in cancer treatment.In the previous research work,I have carried out a systematic study on the anti-hepatocarcinogenesis effect of Sapporo extract in August,and found that it can significantly reduce the weight of tumor in H22tumor-bearing mice and has significant effect on the growth and proliferation of HCC cells Inhibitory effect.At the same time,the water extract in August can also significantly improve the immune level of H22tumor-bearing mice,improve the body's antioxidant stress,scavenging free radicals and maintaining the dynamic balance of reactive oxygen species within the environment.It is speculated that the dietary extract of August may play an anti-tumor effect by inducing the apoptosis of hepatocarcinoma cells and up-regulating the immune level.In this study,Hederagenin(HD),a main ingredient of August,was used as a research object,and the anti-hepatocarcinogenesis effect of HD was studied systematically by H22 tumor-bearing mice in vivo and HepG2 cells in vitro.The MTT,RT-PCR,immunofluorescence,Western Blot,AO/EB apoptosis staining,Hoechst33342 apoptosis staining,enzyme-linked immunosorbent assay,NK cell activity assay and flow cytometry were used to detect the anti-in the regulation of tumor cells,induction of apoptosis and immune regulation and other aspects of the study,trying to explore its anti-tumor effect and related mechanisms,and get the following results.1.In the study of anti-hepatocarcinoma effect of H22 tumor bearing mice,H22 tumor bearing mice model was established and randomly divided into 5 groups(12 rats in each group),control group,cyclophosphamide(CTX),positive control group,HD low dose group(100 mg/kg),HD middle dosegroup(200mg/kg)and HD high dose group(400 mg/kg).After 14 days of H22 tumor-bearing mice,the growth and proliferation of H22 tumor-bearing mice were significantly inhibited,and the survival status and vital signs of mice were significantly improved compared with the control group The(100 mg/kg,200 mg / kg and 400 mg / kg)could significantly inhibit the tumor growth of H22 tumor-bearing mice(P <0.01).The tumor inhibition rates were 29.67%,42.75% and 40.26%.The histological structure of liver and kidney of H22 tumor-bearing mice was observed by HE staining,and the liver and renal function related indexes were detected.It was found that HD had no obvious side effects on mice.The expression of mutant P53,P21 and Bcl-2 proteins in tumor tissues of H22 tumor-bearing mice were detected by immunofluorescence.The results showed that the mutant P53 and Bcl-2 proteins in the control group.And P21 protein showed low expression.The expression of P53 and Bcl-2 protein was significantly decreased in mice treated with HD and CTX(P <0.01),while the expression of P21 protein was significantly increased(P <0.01).These results suggest that HD may play an anti-tumor effect by inducing tumor cell apoptosis.2.The expression of Bcl-2,Bax,Fas,Fasl,Caspase-3 and Caspase-8 in tumor tissue were detected by immunofluorescence,RT-PCR and Western blot in HD-induced tumor cell apoptosis in H22 tumor bearing mice.The changes in gene transcription and translation levels have been studied.Compared with the control group,the expression of Bcl-2 in the tumor tissue of H22 tumor-bearing mice was significantly lower than that of the control group(P<0.01)at the level of transcription and translation,while the expression of Bax,Fas,FasL,Caspase-3 and Caspase-8 was significantly higher(P<0.01)at the level of transcription and translation.These results suggest that HD may induce tumor cell apoptosis by activating the mitochondrial pathway and death receptor pathway of apoptosis,and thus play its anti-tumor effect.3.In the study of the effect of HD on the immune function of H22 tumor-bearing mice,it was found that HD test group could significantly improve the thymus of H22 tumor-bearing mice by analyzing the changes of immune organ tissue structure and function before and after HD treatment Index and spleen index(P <0.05).The preliminary determination of HD could promote the repair of two immune organs of thymus and spleen in H22 tumor bearing mice(P <0.05).HD could significantly increase the number of CD4+ T cells and enhance the proliferation of CD4+ T cells in the immune organs of mice(P <0.01),and significantly increased the proliferation of spleen lymphocytes(P <0.01)CD8+ ratio(P <0.05),indicating that HD can improve the cellular immune response by adjusting the number and proportion of T cell subsets.In addition,HD can also regulate the expression of IL-2 and TNF-?in peripheral blood of tumor-bearing mice,especially HD 200 mg /kg and 400 mg /kg group can significantly increase the expression of IL-2 and TNF-?Expression level(P <0.05).These results suggest that HD may play an anti-tumor effect by repairing immune organs,regulating cellular immune responses and cytokine levels.4.HepG2 cells were treated with different concentrations of HD(concentrations 0,30,60,90,120 and180 ?g/m L)for 24 h and 48 h after HD in vitro inhibition of HepG2 cells.The inhibitory effect of proliferation is increasing and in a dose-dependent manner.HD inhibited the growth of HepG2 cellssignificantly(P <0.01),and the inhibition rates were 5.26%,8.73%,11.00%,16.44% and 27.72%respectively at 24 h,respectively,and the inhibition rates were 1.80%,11.01%,19.38%,27.76% and42.01% respectively.HE staining and laser confocal microscopy showed that HepG2 cells were treated with HD for 48 h,and the membrane vesicles and apoptotic bodies were found.The distribution of nuclear chromatin was uneven and the nuclear membrane was damaged.Morphological changes.In addition,by Apoptosis of HepG2 cells treated with Annexin V-FITC double staining,it was further proved that HD could induce apoptosis of HepG2 cells in HepG2 cells treated with HD for 48 h.When the concentrations were 120 and 180?g/mL,the percentages of HD on Hep G2 cells were 12.10% and 17.90%,respectively,which were significantly higher than those in the control group(P <0.05).These results suggest that HD may play an anti-hepatocarcinogenic role by inducing HepG2 cell apoptosis.5.Expression of mutant P53,Bcl-2,Bax,Fas,Caspase-3 and Caspase-8 in HepG2 cells by immunofluorescence and Western blot in the study of the mechanism of apoptosis induced by HD in vitro Change the situation to study.Compared with the control group,the expression of mutant P53 and Bcl-2protein in HepG2 cells treated with HD was significantly decreased(P <0.01),while the expression of Bax,Fas,Fasl,Caspase-3 and Caspase-8 protein(P <0.01),suggesting that HD anti-HepG2 may be associated with the mitochondrial pathway and death receptor pathway that activate apoptosis.In this study,the anti-tumor effect and related mechanism of HD on H22 tumor-bearing mice and HepG2 cells were investigated by in vivo test and in vitro culture of cell culture.Through the pharmacology,pathology,biochemistry and molecular biology and other related technologies and methods,and tumor development and development are closely related to cytokines,mRNA,protein and apoptosis(apoptosis-related regulatory pathway)and other indicators were The detection and analysis of the system demonstrated that HD has a significant inhibitory effect on the growth and proliferation of tumor cells in vivo and in vitro,which may be closely related to the mitochondrial pathway and death receptor pathway that can activate apoptosis.Through this study,Clinical use of HD to help treat liver cancer to provide theoretical and experimental basis... |
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