| Objective: The Research will investigate the anti-cancer effect of Haizao Yuhu Decoction and fucoxanthin on the model of human hepatocellular cancer xenograft in nude mice and the effect on human liver cancer cells,and investigate the expression level of key proteins and explore the anti-hepatoma effect pathway of fucoxanthin.Methods: 1.The xenograft model of nude mice was established and divided into model group(intragastric administration of 0.9% sodium chloride solution once daily),positive control group(intraperitoneal injection of doxorubicin hydrochloride 1 mg/kg once daily),high,medium and low dose groups(intragastric administration of 660,330 and 165 mg/kg once daily)and high,medium and low dose groups(intragastric administration of 50,25 and 12.5 mg/kg once daily)of fucoxanthin(FX).After 21 days of treatment,tumor tissues were collected for tumor volume measurement and routine HE staining;Cyclin D1 and Bax in tumor tissues were detected by immunohistochemistry;serum alanine aminotransferase(ALT)and aspartate aminotransferase(AST)were detected.2.Identification of Fucoxanthin in Haizao Yuhu Decoction by Ultra Performance Liquid Chromatography-Mass Spectrometry.3.The cell growth curve was drawn;the effect of FX on cell viability was detected by CCK-8 assay;the effect of FX on cell colony formation was detected by colony formation assay;the cell cycle was analyzed by flow cytometry;and apoptosis was detected by flow cytometry Annexin V-FITC/PI double staining.4.Proteomic analysis: Human hepatoma Hep3B cells were treated with FX(10 ?M)for 24 and 48 h,respectively,and total cellular proteins were extracted,analyzed and identified the differentially expressed proteins using iTRAQ technology combined with quantitative proteomics method of high performance liquid chromatography-tandem mass spectrometry,and bioinformatics analysis was performed;Western blot was used to verify the key differential protein expression.Results: 1.Effects of Haizao Yuhu Decoction and Fucoxanthin on the Growth of Human Hepatoma Hep3B Cells in Mice.Compared with the model group,the volume of transplanted tumor in the high,medium and low dose groups of Haizao Yuhu Decoction was smaller,and the difference was statistically significant(P < 0.01).The tumor inhibition rates in the high,medium and low dose groups of Haizao Yuhu Decoction were 51.74%,46.93% and 25.17%,respectively.The tumor volume in the low dose group was higher than that in the model group and high dose group(P < 0.05).Compared with model group,the volume of transplanted tumor in FX high,middle and low dose groups was smaller,with statistical significance(P < 0.05).The tumor inhibition rate of high and medium dose fucoxanthin was 51.34% and 43.50%.Compared with the model group,the ALT in the high dose group was significantly lower(P < 0.05).HE staining was used to observe the histopathological changes of tumors: In the treatment group,tumor cells showed focal necrosis and pyknotic hyperchromatic nuclei in varying degrees.Immunohistochemistry showed that the number of Cyclin D1 protein positive cells in each group of Haizao Yuhu Decoction and fucoxanthin high and medium dose groups decreased,and Bax protein positive expression increased.2.The results of liquid chromatography-mass spectrometry(LC-MS)showed that the retention time of fucoxanthin was 10.94 min.In the detection results of Haizao Yuhu Decoction,the chromatographic peak can be extracted with the same m/z value and mass deviation at the same retention time;the secondary spectrum corresponding to this peak has a good match with the secondary spectrum of fucoxanthin reference.3.Effects of fucoxanthin(FX)on cell viability,colony formation,cell cycle and apoptosis of Hep3B and MHCC97 h cells.(1)FX inhibited the proliferation of Hep3B and MHCC97 h cells in a time and concentration dependent manner.Compared with the control group,treatment with FX 25 ?M for 24 h and FX 5 ?M for 48 h reduced cell viability(P<0.05),and the IC35 and IC50 values of Hep3B cells treated with FX for 72 h were 10 and 14 ?M,respectively;compared with the control group,treatment with FX 25 ?M for 24 h reduced cell viability(P<0.05),and the IC35 and IC50 values of MHCC97 cells treated with FX for 72 h were 16.9 and 20.5 ?M,respectively.(2)After treatment of Hep3B cells with FX 10 and 14 ?M,the colony formation rate was reduced to 40.00% and 13.47%;after treatment of MHCC97 H cells with FX 10 and 14 ?M,the colony formation rate was reduced to 52.60% and 35.13%,and the differences were statistically significant compared with the control group(P < 0.01).(3)Hep3B,MHCC97 h cells were treated with FX to block the cell cycle in G1 phase.After Hep3B cells were treated with FX for 24 h,compared with the control group,the content of cells in G0/G1 phase increased in the FX 14 ?M group(P < 0.01).After treatment for 48 h,compared with the control group,the content of cells in G0/G1 phase in FX 10 ?M group and FX 14 ?M group increased(P < 0.01).After MHCC97 h cells were treated with FX for 24 h,compared with the control group,the content of cells in G0/G1 phase of FX 10 ?M group and FX 14 ?M group increased(P<0.01).After treatment for 48 h,compared with the control group,the content of cells in G0/G1 phase in FX 10 ?M group and FX 14 ?M group increased(P<0.01).(4)Compared with the control group,the apoptotic rate of Hep3B cells treated with FX for 24 h increased(P<0.01),while the apoptotic rate of Hep3B cells treated with FX for 48 h increased(P<0.01).Compared with the FX 10 ?M group,the apoptotic rate of Hep3B cells treated with FX 14 ?M increased(P<0.01).After treated with FX for 24 h,the apoptotic rate of MHCC97 h cells was increased in FX 14 ?M group compared with control group(P<0.01),and the apoptotic rate was increased in FX 14 ?M group compared with FX 10 ?M group(P<0.01);after treated for 48 h,the apoptotic rate was increased in FX 10 ?M group and FX 14 ?M group compared with control group(P<0.01).4.Proteomic analysis(1)A total of 77,064 peptides matched to the map were identified in the experiment,with 27,883 unique peptides and a total number of 5,092 high-confidence proteins.Setting more than 2-fold difference as significant difference,97 differential proteins were up-regulated and 87 were down-regulated after the FX treatment with 24 h;30 differential proteins were up-regulated and 43 were down-regulated after the FX treatment with 48 h.(2)Gene ontology analysis of differential proteins after FX treatment.Biological process analysis revealed that the differential proteins were mainly concentrated in the metabolic process,biological regulation and so on.The analysis of cellular components revealed that the differential proteins were mainly distributed in organelles and membranes.In addition,after exposure to FX for 24 h,the differential protein cellular components were also distributed on the cell junctions.Molecular function analysis suggested that the differential protein molecules mainly have catalytic activity,binding and other functions.Signaling pathway analysis revealed that the differential protein enrichment signaling pathways were mainly pathways in cancer.Protein interaction analysis of the differential proteins revealed that proteins such as RPS6KA1,NOP2,MIF,CDK12,SQSTM1,STAT3,MDH2,and ATP5 I play a key role,and the protein levels of RPS6KA1,NOP2,MIF,CDK12,STAT3,MDH2,and ATP5 I were downregulated and the protein level of SQSTM1 was upregulated after FX treatment,as verified by Western blot.Conclusion: 1.Haizao Yuhu Decoction and its active ingredient fucoxanthin significantly inhibited Hep3B xenograft growth in nude mice in a dose-dependent manner.2.Fucoxanthin significantly inhibited the viability of human hepatoma Hep3B and MHCC97 h cells in vitro,and decreased the number of cell colonies,which may play an anticancer role by inducing G1 cell cycle block and promoting apoptosis.3.Fucoxanthin can regulate a variety of proteins: organelles,membranes,containing protein complexes and so on,most of these proteins have binding,catalytic activity,transport activity and other functions,involved in metabolic process,biological regulation,tissue cell composition or biosynthesis,and other biological processes;related pathways mainly include pathways in cancer.Protein interaction analysis combined with experimental validation showed that the key protein of protein interaction network has the effects of regulating cell cycle progression,inhibiting macrophage movement,mediating signal transduction and transcriptional activation,and promoting autophagy;FX maybe play a role in inducing cell cycle block and apoptosis by regulating this protein,thereby inhibiting the proliferation,viability,and colony formation of Hep3B cells. |
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