| Background: Currently,liver cancer is the second leading cause of cancer-related death in the world,seriously threatening human life and health.The incidence and mortality of liver cancer in Asia are the highest in the world.Liver cancer is an important public health problem in my country.Hepatocellular carcinoma(HCC)accounts for about90% of liver cancer,and is most closely related to liver cirrhosis,hepatitis B virus(HBV)and hepatitis C virus(HCV)infection.Traditional Chinese medicine has a long history and rich experience in the treatment of liver cancer.,phlegm coagulation,toxin aggregation;deficiency is related to righteousness deficiency,liver and kidney yin deficiency,etc.,traditional Chinese medicine has unique advantages in the treatment of liver cancer,and research on its anticancer mechanism is increasing.Sanlengjiashen Decoction(SLS)is composed of the classic anti-cancer prescription Sanleng Pills and Blind Ginseng.It is an independent and innovative treatment prescription that combines the characteristics of liver cancer itself and the concept of treating cancer with both TCM attack and supplement,and both qi and blood.The patients have definite curative effect,and modern Chinese medicine pharmacological studies have also confirmed that the four traditional Chinese medicines in the SLS have anti-tumor activity.On the basis of the exact anticancer effect of SLS in clinical practice,this topic systematically studied the intervention effect of SLS in hepatocellular carcinoma from the level of cells and animals.The current status of the disease caused by toxic hepatitis,and the possible mechanism of SLS anti-hepatocellular carcinoma was preliminarily explored from the JAK2/STAT3 signaling pathway mediated by the inflammatory factor IL-6.Chapter 1: Prediction of the possible mechanism of action of SLS participating in hepatocellular carcinoma based on network pharmacologyObjective: To explore the possible mechanism of SLS Participant’s intervention in hepatocellular carcinoma,and to provide scientific basis for subsequent experiments and clinical application.Methods: Using the Traditional Chinese Medicine System Pharmacology Database and Analysis Platform(TCMSP),the core components and action targets of SLS were screened;the network diagram of "component-target-disease" of SLS prescription for the treatment of hepatocellular carcinoma was constructed;Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analyses were performed.Results: Through the TCMSP database screening,103 potential targets of Sanlengjia Participant were screened,and 6363 HCC disease targets were screened through the Genecards database,and 80 targets were co-acted by the two;KGEE analysis showed that the inflammation-related pathways included hepatitis B.Viral infection(HBV),hepatitis C virus infection(HCV),interleukin 17(IL-17),herpes virus infection,etc.;lipid and atherosclerosis,diabetic complications and AGE-RAGE signaling pathway are related to metabolism,non-alcoholic Fatty liver,etc.;tumor-related pathways include chemical carcinogenesis-receptor activation signaling pathway,small cell lung cancer,tumor necrosis factor signaling pathway(TNF),Apoptosis,prostate cancer,P53 signaling pathway,etc.,among which HBV signaling pathway The most significant difference was in the pathway,which included the classical signaling pathway most closely related to the occurrence and development of liver cancer,namely the JAK2/STST3 signaling pathway mediated by IL-6.Conclusion: SLS has anti-hepatocellular carcinoma effect mainly by regulating HBV signaling pathway and inhibiting inflammatory response.Chapter 2: Research on the effect and mechanism of SLS against hepatocellular carcinomaObjective:1.CCK-8 method was used to observe the effect of SLS on the proliferation of human hepatoma cell lines Hep G2 and HCCLM3 cells,as well as the cytotoxicity of human hepatocyte L02;plate colony formation assay,cell counting method to determine cell proliferation;flow cytometry detection Cell cycle and apoptosis;Western blot was used to detect the expression of cell proliferation,apoptosis,migration and other related proteins.2.Western blot was used to detect the expression levels of JAK2/STAT3 pathway key proteins JAK2,P-JAK2,STAT3,P-STAT3,etc.,the expression level of the upstream inflammatory factor IL-6 and the expression levels of its downstream key proteins MMP2,MMP9,and the detection of PI3 K,P-PI3 K,AKT,P-AKT and other protein expression levels.3.A mouse model of subcutaneously transplanted tumor was established.After SLS intervention,the tumor volume was measured.Immunohistochemical staining was used to detect the tumor tissue cell morphology and the expression of proliferation and apoptosis markers.Western blot was used to detect apoptosis-related proteins and proteins in the JAK2/STAT3 pathway.express the situation.Results: 1.CCK-8 assay showed that SLS inhibited the proliferation of human hepatoma cells in a time-and dose-dependent manner(P<0.05),but had no significant effect on the cell viability of human hepatocyte L02;Plate colony formation experiments and cell counting experiments showed that SLS could inhibit the proliferation of hepatoma cells.2.Flow cytometry showed that SLS could induce the cycle arrest of human hepatoma cell line Hep G2,arrest the cell cycle in the G1 phase,and induce apoptosis of human hepatoma cell lines Hep G2 and HCCLM3,and with the subsequent development of the cell cycle.With the increase of dose,the number of late apoptosis increased(P<0.05).3.Western blot detection showed that SLS could inhibit the proliferation of human hepatoma cells,and had an inhibitory effect on the expression level of the proliferation-related protein Ki67.The expression levels of Caspase3,Cleaved Caspase3,and BAX increased,which induced apoptosis of human hepatoma cells.Detection of cell cycle-related protein levels found that the expression levels of cycle-related proteins Cyclin D1,Cyclin B1,and C-myc were down-regulated.4.The results of Western blot experiments showed that in addition to promoting cell apoptosis,inhibiting proliferation and inducing cell cycle arrest,SLS down-regulated the expression levels of inflammatory factors such as IL-6,AKT,P-AKT,and NF-κB.At the same time,the expression levels of related proteins in the JAK2/STAT3 signaling pathway and downstream key proteins MMP2,MMP9 were down-regulated 5.Animal experiments showed that the tumor volume and tumor weight of the SLS treatment group were reduced compared with the control group,and the results were significantly;At the same time,HE staining showed no abnormality in mouse organs.and immunohistochemical staining showed that the expression level of proliferation-related protein Ki67 was significantly decreased after SLS intervention,the expression levels of apoptosis proteins BAX and Cleaved Caspase3 were significantly increased,and the expression level of BCL2 was decreased;Western blot was used to detect the expression of inflammatory factors in tumor tissue The expression levels of IL-6,NF-κ B,and AKT were all down-regulated,and the expression levels of JAK2,STAT3,and their downstream MMP2,MMP9 were inhibited.Conclusion:1.SLS can inhibit the proliferation of hepatoma cells and induce cell cycle arrest and apoptosis.2.Its mechanism may play an anti-tumor effect by inhibiting the inflammatory response and down-regulating the IL-6-mediated JAK2/STAT signaling pathway.Chapter 3: Interventional effect of ginsenoside Rg3,one of the active components of SLS on hepatocellular carcinomaObjective: To investigate the antitumor effect and possible mechanism of ginsenoside Rg3,an active component of SLS,on hepatocellular carcinoma.Methods: 1.The effect of ginsenoside Rg3 on the proliferation ability of human hepatoma cells Hep G2 and HCCLM3 was detected by CCK-8 method and plate cloning assay;2.Cell cycle and apoptosis were detected by flow cytometry,and proliferation,cycle and apoptosis were detected by Western blot;3.To establish a mouse subcutaneously transplanted tumor model,observe the changes in body weight and tumor volume after 27 days of ginsenoside Rg3 intervention,and detect the expression of proliferation and apoptosis-related proteins by Western blot and immunohistochemistry.Result:1.CCK-8 assay showed that ginsenoside Rg3 could inhibit the proliferation of human hepatoma cells in a time-and dose-dependent manner.Compared with the control group,the difference was statistically significant(P<0.05).The cell viability of L02 had no significant effect;the results of colony formation and cell counting experiments showed that ginsenoside Rg3 could inhibit the proliferation of Hep G2 and HCCLM3 cells(P<0.01).2.Flow cytometry showed that ginsenoside Rg3 could induce the cycle arrest of human Hep G2 cells and induce the apoptosis of human hepatoma cells at the same time(P<0.05).3.Western blot detection showed that in Hep G2 and HCCLM3 cells,the expression of proliferation-related protein Ki67 was inhibited;the level of apoptosis-related protein BCL2 decreased(P<0.01),and the expression levels of Cleaved Caspase3 and BAX proteins increased(P<0.01);The expression levels of cell cycle-related proteins Cyclin D1,Cyclin B1 and C-myc were down-regulated.4.Western blot detection further found that the expression levels of IL-6,AKT,P-AKT,NF-κB were all down-regulated(P<0.01).At the same time,the expression of related proteins in the JAK2/STAT3 signaling pathway,which is closely related to the occurrence of HCC The levels were down-regulated,and the expression levels of the downstream key proteins MMP2 and MMP9 were down-regulated(P<0.01).5.Animal experiments showed that the tumor volume and tumor weight in the ginsenoside Rg3 group were smaller than those in the negative control group,and there was a significant difference in the high-dose group(P<0.05);immunohistochemical staining showed that the expression level of the proliferation-related protein Ki67 was significantly reduced,the expression levels of apoptosis proteins BAX and Cleaved Caspase3 were significantly increased,and the expression level of BCL2 was decreased;Western blot results showed that the expression levels of IL-6,NF-κB and AKT were down-regulated in tumor tissue,JAK2,STAT3,and their downstream MMP2,MMP9 and other expression levels were inhibited.Conclusion: Ginsenoside Rg3 has good anticancer effects in vitro and in vivo,can inhibit the proliferation of human hepatoma cells,induce cycle arrest and apoptosis in hepatoma cells,and regulate the JAK2/STAT3 signaling pathway.Chapter 4: The effect and mechanism of ginsenoside Rg3 and Emodin of SLS in combination on hepatocellular carcinomaObjective: To study the effect of combined use of the active ingredients Rg3 and Emodin of SLS on hepatocellular carcinoma.Methods: 1.CCK-8 method and cell counting method were used to detect the effect of ginsenoside and emodin on the proliferation of human hepatoma cells;flow cytometry was used to detect the effect of combined use on cell cycle and apoptosis;Changes in the levels of related proteins such as cell proliferation and apoptosis after administration.2.To establish a mouse model of human hepatocellular carcinoma xenograft,observe the tumor volume after 25 days of combined drug intervention,and detect the expression of proliferation and apoptosis-related proteins by immunohistochemistry.Result:1.The detection of CCK-8 found that emodin had a relatively obvious inhibitory effect on the proliferation of human liver cancer cell lines Hep G2 and HCCLM3 in a time-and dose-dependent manner.All cell lines showed strong tumor suppressor effect,and the cell viability of Hep G2 and HCCLM3 was significantly decreased(P<0.001).2.The results of the cell counting method showed that the cell viability of the two cell lines was significantly inhibited after 24,48,72 and 96 hours of combined treatment,and the differences were statistically significant compared with the Control control group and the single drug control group(P <0.001).3.Western blot showed that after 48 hours of intervention,the expression level of cell proliferation protein Ki67 in the combination group was significantly decreased(P<0.001);the expression levels of BAX,Cleaved Caspase3 and Caspase3 were significantly up-regulated(P<0.001);compared with the Rg3 group and Emodin group,the The expression levels of Cleaved Caspase3 and Caspase3 in the combination group were significantly up-regulated,and the difference was statistically significant(P<0.01).The expression levels were significantly down-regulated,and the difference was statistically significant(P<0.01).4.The results of flow cytometry showed that compared with the single addition group,the number of apoptotic cells increased significantly after the combined treatment,and the difference was statistically significant(P<0.01).Animal experiments showed that the tumor body shrank significantly(P<0.01)after the combination treatment.Immunohistochemical staining showed that the expression level of Ki67 was significantly decreased,the expression levels of apoptosis proteins BAX and Cleaved Caspase3 were significantly increased,and the expression level of BCL2 was decreased.The anti-tumor effect was significantly enhanced after treatment.Conclusion:1.The combination of ginsenoside Rg3 and Emodin can significantly inhibit the proliferation of liver cancer cells,induce their apoptosis,and inhibit the expression levels of inflammatory factors such as IL-6 and NF-κB.2.Compared with the single-drug intervention group,the combined anti-cancer effect of ginsenoside Rg3 and emodin was significantly enhanced. |
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