| Primary liver cancer(PLC)is one of the most fatal malignancies,including Hepatocellular Carcinoma(HCC),Intrahepatic cholangiocarcinoma(ICC)and other types.Among all malignant tumors,the incidence of PLC ranks sixth,and tumorrelated mortality rate ranks third.PLC is the fourth most common malignant tumor and the second leading cause of tumor death in China,due to lots of liver disease patients in China.At present,hepatectomy or liver transplantation is the main method for radical treatment of HCC,but the postoperative recurrence rate is high.HCC is a typical vascular rich tumor,its growth and metabolism require continuous angiogenesis,moreover neovascularization vessels will aggravate tumor growth,invasion and metastasis.Therefore,anti-angiogenic targeted drugs are the standard drugs for systemic therapy in patients with advanced stage of HCC.However,due to the poor general condition of the patients,tumor heterogeneity and the toxic and side effects of targeted drugs,it is easy to lead to drug resistance and treatment limitation.The high cost of targeted drugs has also deterred many patients.Exploring the mechanism of HCC angiogenesis and finding new therapeutic targets have become important directions in the field of HCC research.Traditional Chinese Medicine has rich clinical experience in the treatment of tumors.Traditional Chinese Medicine holds that the bases of tumor formation are deficiency of vital qi.The vital qi of five zang-organs is deficient,and then dysfunction of organs and meridians,all kinds of pathologic products are produced gradually,including phlegm turbidity,wet poison,blood stasis,etc.All kinds of pathological products are produced gradually,the imbalance of Qi with pathological products for a long time,resulting in tumor.Method of hardness-softing and masses-resolving is one of the tumor treatment methods,many clinical trails and basic research have proved that herbal medicine which have hardness-softing and masses-resolving effect has obvious anti-tumor and anti-angiogenesis efficacy,but its potential mechanism and molecular targets are still unclear,which need to be confirmed by further scientific research.Turtle shell is a representative drug of hardness-softng and masses-resolving drugs.Existing studies have shown that turtle shell has good anti-tumor and anti-tumor angiogenesis effects.Therefore,studying the mechanism of anti-tumor and antiangiogenesis of turtle shell is of great significance in clinical tumor treatment.In this study,the effect of turtle shell water extract on zebrafish embryos was analyzed through high-throughput sequencing and bioinformatics analysis.Then observe the coculture model of human hepatoma cell HepG2 and human umbilical vein endothelial cell HUVEC treated by water extract of turtle shell,observe the changes of HepG2 cells,study the specific molecular mechanism,and provide experimental research basis for turtle shell treatment of tumor.Objectives:Clinical studies show that turtle shell has good antitumor and angiogenesis effects,but the mechanism of action is still unclear.In this study,the model animal zebrafish was used to study the effect of turtle shell on the angiogenesis of zebrafish embryos,and the possible mechanism was analyzed by biological methods.Finally,the co-culture model of human hepatocellular carcinoma cell Line HepG2 and human umbilical vein endothelial cell line HUVEC was constructed to observe the effect of turtle shell water extract on the co-culture model cells,so as to discover the potential mechanism of turtle shell water extract in the treatment of tumor and angiogenesis.Methods:(Ⅰ)、Effect of water extract of turtle shell on angiogenesis of zebrafish embryo.1、The decoction method of traditional Chinese medicine was adopted,and then the water extract powder of turtle shell was prepared by means of concentration,sieving and drying.The fingerprint of water extract of Turtle shell was determined by ULTRA performance liquid chromatography.2、Tg(flila: EGFP transgenic zebrafish(EGFP)were used as models.Turtle shell extract(working concentration 200μg/m L)was added into the fish water of the experimental group,and PTK787(working concentration 5μg/m L)was added into the fish water of the control group.The zebrafish embryos were respectively fertilized for22 h for 26 h.To evaluate the effect of water extract of turtle shell on neovascularization of transgenic zebrafish embryos.3、High-throughput sequencing of RNA in zebrafish embryo tissues after turtle shell water extract intervention was conducted to construct a full transcription library,and comprehensive determination of gene changes in zebrafish after turtle shell intervention was conducted,followed by GO analysis and KEGG analysis.4、Zebrafish embryo proteins were extracted for protein quantitative detection parallel response monitoring(PRM)to monitor the changes of differentially expressed proteins related to the glutamine pathway.(Ⅱ)、Effect and mechanism of water extract of turtle shell on HepG2 cells in coculture of HepG2 cells and HUVEC cells.1、CCK-8 method was used to detect the activity of water extract of Turtle shell on human umbilical vein endothelial cells.The cells were divided into 4 groups.The water extract group was divided into three concentration groups(low(10μg/m L),medium(20μg/m L)and high(40μg/m L)and blank control group.2、HepG2 and HUVEC co-culture models were constructed,and cells were divided into three groups: HepG2 group,coculture +20μg/m L turtle shell and co-culture group.HepG2 cells separately cultured at the same time and HepG2 cells in the coculture model were detected by scratched healing assay,Transwell chamber chemotaxis assay and FDA fluorescence adhesion staining.Real-time PCR was used to detect glutamine metabolisation-related genes ASNS,GPT2 and ADSSL.The kit detects the contents of glutamine,glutamic acid,α-ketoglutaric acid,ATP and glutathione in cells.Western Blot was used to detect the contents of p-AMPK,AMPK,p-4EBP1,4EBP1,p-M TOR and m TOR.AMPK and m TOR were detected by immunofluorescence staining.3、Cells were divided into four groups: In HepG2 group,HepG2+AMPK inhibitor Dorsomorphin,co-culture +20μg/m L turtle shell,co-culture +20μg/m L turtle shell +AMPK inhibitor Dorsomorphin,real-time PCR detection of glutamine metabolization-related genes ASNS,GPT2,ADSSL;The kit was used to detect the contents of α-ketoglutaric acid,ATP and glutathione in cells.The contents of P-AMPK,AMPK,P-4EBP1,4EBP1,P-M TOR and m TOR were detected by Western Blot.AMPK and m TOR were detected by immunofluorescence staining.4、Cells were divided into four groups: HepG2 group,HepG2+m TOR activator3 BDO,co-culture +20μg/m L turtle shell,co-culture +20μg/m L turtle shell +m TOR activator 3BDO,real-time PCR detection of glutamine metabolization-related genes ASNS,GPT2,ADSSL;The kit was used to detect the contents of α-ketoglutaric acid,ATP and glutathione in cells.The contents of p-AMPK,AMPK,p-4EBP1,4EBP1,pm TOR and m TOR were detected by Western Blot.AMPK and m TOR were detected by immunofluorescence staining.Results:1、In this experiment,the water extract powder of turtle shell was 20.6828 g,the extract yield was 10.3414%,and the weight of crude drug per gram of the extract was9.66987 g.The water extract powder prepared by this experiment was light gray,fine,no clumps,dry,moisture content ≤5%.Ultra-performance liquid chromatography(UHPLC)was used to determine the chromatographic peak with the same retention time as the corresponding amino acid reference and the similarity was greater than 0.9.2、Turtle shell water extract could inhibit angiogenesis of transgenic zebrafish embryos.Compared with blank control group,after the intervention of turtle shell water extract,ISVs fluorescence expression was partially lost,most OF DLAV was lost,and only a small amount of DLAV could be observed.There was no teratogenic effect on zebrafish embryos at the concentration of 200μg/m L water extract and 26 h intervention time.The positive control drug vatalanib hydrochloride(PTK787)can inhibit the angiogenesis of transgenic zebrafish embryos.PTK787 at 5μg/m L had no teratogenic effect on zebrafish embryos.3、High-throughput sequencing and KEGG Pathway analysis showed that 32 pathways were enriched with down-regulated differentially expressed genes,among which 3 genes involved in glutamine metabolism were down-regulated: ASNS,GPT2 and ADSSL;PRM assay verified the down-regulation of glutamine metabolisationrelated genes: ANSN,GPT2 and ADSSL protein expression levels.4、CCK-8 assay showed that the water extract of turtle shell had inhibitory effects on HUVEC activity and invasion and migration of HUVEC.The 24-HOUR IC50 value of CCK-8 assay was 39.96μg/m L.5、Co-culture of human hepatoma cell line HepG2 and human umbilical vein endothelial cells(HUVEC)can enhance the viability of HepG2 cells.Scratch healing assay,Transwell chemotaxis assay and FDA adhesion staining showed that the water extract of tripod beetle had certain inhibitory effects on the viability,migration and invasion of HepG2 cells in co-culture of HepG2 and HUVEC.6、In the co-culture state of HepG2 and HUVEC cells,glutamine metabolism test kit showed that the co-culture state could enhance the glutamine metabolism level of cells.RT-PCR showed that the expression of glutamine metabolity-related genes ASNS,GPT2 and ADSSL increased.The expression of glutamine related genes ASNS,GPT2 and ADSSL in HepG2 cells under co-culture condition were significantly inhibited by water extract of turtle shell.Western Blot and immunofluorescence staining showed that AMPK protein expression level was increased and m TOR protein expression level was decreased.7、RT-PCR showed that AMPK inhibitor Dorsomorphin or m TOR activator3 BDO enhanced the m RNA expression levels of glutamine metabolity-related genes ASNS,GPT2 and ADSSL in HepG2 cells under coculture.Western Blot and immunofluorescence staining showed that Dorsomorphin inhibited AMPK protein expression and enhanced m TOR expression,while 3BDO enhanced m TOR expression and inhibited AMPK expression.Conclusion:1、The water extract of turtle shell can inhibit the angiogenesis of Tg(Flila: EGFP)transgenic zebrafish embryos,and the mechanism may be related to the inhibition of the expression of ASNS,GPT2 and ADSSL genes related to glutamine metabolism.2、The water extract of turtle shell can inhibit the activity,migration and invasion of HepG2 cells which are co-cultured with human umbilical vein endothelial cells HUVEC.And its potential mechanism is to inhibit the metabolism and utilization of glutamine by regulating AMPK/m TOR signaling pathway. |
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