| Objective:At present,liver cancer is one of the most common and malignant tumors in the world.The effect of existing treatment methods such as surgery and chemotherapy is not obvious,so it is urgent to develop new treatment methods and drugs for liver cancer.6-Shogaol is one of the main active components of ginger,and its content in dried ginger is relatively high.It has a variety of pharmacological effects,such as anti-tumor,anti-inflammation,anti-oxidation and so on,especially in anti-tumor,such as colon cancer,gastric cancer,non-small cell lung cancer and breast cancer.However,because the content of6-Shogaol in ginger is very low,and many compounds with similar molecular structure will interfere with the process of extraction,purification and separation,the preparation is difficult and the efficiency is not high.At present,there are few reports on the anti-liver cancer of 6-Shogaol,and the research on the anti-liver cancer effect of combination with chemotherapeutic drugs has not been reported.The research on the mechanism of anti-liver cancer is not deep enough,and because of its insolubility in water,it is not conducive to preparation and clinical application.In order to solve the above problems,this paper first combines flash extraction with high-speed countercurrent chromatography to construct an efficient method for preparing 6-Shogaol,then makes an in-depth study on the anti-hepatoma effect of 6-Shogaol and its synergism with chemotherapeutic drugs,and explains its mechanism from the cellular and molecular levels.finally,nanotechnology is used to prepare nanoemulsion for carrying 6-Shogaol to solve the problem of its low solubility.And to study the anti-liver cancer effect of 6-Shogaol nanoemulsion,in order to promote the study of this component and its anti-liver cancer effect in ginger traditional Chinese medicine,and to provide a necessary basis for the research and development and clinical application of new drugs for the treatment of liver cancer and adjuvant chemotherapy.Method:1.Establishment of a method for determination and analysis of 6-Shogaol in vitro;flash extraction and heating reflux extraction were used to extract6-Shogaol from dried ginger from different areas,and the advantages and disadvantages of the two extraction methods and the content of 6-Shogaol in dried ginger from different areas were compared.the effects of ethanol concentration,liquid-to-material ratio,extraction times and extraction time on6-Shogaol extraction were investigated by single factor method.The extraction conditions of 6-Shogaol were optimized by orthogonal experimental design,the crude extract of 6-Shogaol was purified by ethyl acetate extraction,the solvent system was screened by thin layer chromatography（TLC）,and the purified 6-Shogaol extract was separated by high speed countercurrent chromatograph.The molecular weight and structure of the separated target compound were determined by LC-MS/MS and NMR,and compared with the standard,the purity of the separated target compound was determined by HPLC,which provided the raw material for the follow-up preparation research.2.The effects of 6-Shogaol on the viability of Bel-7402 and HepG2hepatoma cells were detected by MTT method,the effects of 6-Shogaol on proliferation and apoptosis of hepatoma cells were investigated by Brd U staining and Annexin V-FITC/PI double staining,and the effects of different concentrations of 6-Shogaol on the expression of TLR4,FOXO3a,Wnt1 andβ-catenin genes or proteins in hepatocellular carcinoma cells were investigated by q RT-PCR and Westernblotting methods.Through lentivirus infection and cell transfection experiments,a hepatoma cell model with overexpression of TLR4 and FOXO3a genes was constructed.The effects of 6-Shogaol on the proliferation,apoptosis and expression of TLR4,FOXO3a,Wnt1 andβ-catenin genes or proteins in hepatoma cells overexpressing TLR4 and overexpressing TLR4 and FOXO3a were investigated by MTT,Brd U staining,q RT-PCR,Westernblotting and Annexin V-FITC/PI double staining.The effects of 6-Shogaol and SKL2001（activator of Wnt/β-catenin pathway）on proliferation,apoptosis and gene or protein expression of FOXO3a,Wnt1 andβ-catenin in hepatocellular carcinoma cells were investigated by MTT method,Brd U staining,q RT-PCR method,Westernblotting method and Annexin V-FITC/PI double staining method.The tumor model of BALB/c nude mice was established by inoculating hepatoma cells（Bel-7402）or hepatoma cells stably expressing TLR4（Bel-7402）.The effects of different administration groups on tumor size after 31 days were investigated,and the effects of different treatment groups on the expression of Ki-67,TLR4,Wnt1,β-catenin and FOXO3a proteins in nude mice were investigated by immunohistochemical（IHC）and Westernblotting methods.3.The effects of 6-Shogaol combined with 5-FU on proliferation,cell cycle and apoptosis of HepG2 and LI-7 hepatoma cells were investigated by MTT method and flow cytometry.The expression of Cyclin A2,Cyclin D1 and Cyclin E1 proteins was detected by Westernblotting method,and the effect of6-Shogaol combined with 5-FU on cyclin expression of hepatoma cells was investigated.Set up different concentrations of 6-Shogaol and 5-FU,calculate the survival rate of hepatoma cells treated alone and jointly by MTT method,and calculate the joint index（CI）to investigate whether there is a synergistic effect between 6-Shogaol and 5-FU.The effects of 6-Shogaol combined with5-FU on proliferation,cycle and apoptosis of hepatocellular carcinoma cells through AKT/mTOR pathway were investigated by MTT,flow cytometry and Westernblotting methods.According to the methods of lentivirus infection and transfection,the hepatoma cell model with overexpression of MRP1 was established,and different administration groups were set up.MTT,flow cytometry,q RT-PCR and Westernblotting were used to investigate the effects of 6-Shogaol combined with 5-FU on proliferation,cycle and apoptosis of hepatocellular carcinoma cells through MRP1 pathway,and the effects of6-Shogaol combined with 5-FU on proliferation,cycle and apoptosis of hepatocellular carcinoma cells by regulating AKT/mTOR/MRP1 axis.The tumor model of BALB/c nude mice was established by inoculating hepatoma cells（HepG2）.The effects of different administration groups on tumor size after 35 days were investigated,and the effects of different treatment groups on the expression of MRP1,AKT,mTOR and MRP1 proteins in nude mice tumor were investigated by immunohistochemical method and Westernblotting method.4.According to the pre-experimental results,the effects of oil phase dosage,mixed surfactant dosage,ultrasonic time and ultrasonic power on the preparation of blank nanoemulsion were investigated by single factor experiment and taking particle size as the evaluation index.According to the results of single factor prescription screening of blank nanoemulsion,the prescription and process parameters which have great influence on it were selected as the investigation factors,the particle size and drug loading after milk formation were selected as the response value,the blank nanoemulsion prescription was optimized by star design-effect surface method,and the drug loading was determined according to the experimental results,and the appearance of 6-Shogaol nanoemulsion was evaluated by observing the color and fluidity of the prescription.The particle size and Zeta potential of6-Shogaol nanoemulsion were measured by Malvern laser particle size analyzer.The morphology was observed under transmission electron microscope,the type of emulsion was identified by staining,the entrapment efficiency of 6-Shogaol nanoemulsion was determined by centrifugation,and the drug release in vitro was studied by dialysis.Result:1.A method for the determination of HPLC content of 6-Shogaol in vitro was established.in the methodological investigation,the specificity,linearity,precision,repeatability,stability and sample recovery experiments all met the requirements of determination,and the method can detect the content of6-Shogaol quickly and accurately.According to the chromatographic conditions,the content of 6-Shogaol in the extracts of different producing areas was determined.The results showed that there was no significant difference in the amount of 6-Shogaol extracted by two different methods of dried ginger from the same area,but the extraction time of flash extraction was shorter.Under the same extraction method,dried ginger from Yishui County,Linyi City,Shandong Province had the largest amount of 6-Shogaol extraction,and the single factor results of flash extraction showed that ethanol concentration,liquid-material ratio,extraction times and extraction time would affect the extraction amount of 6-Shogaol in a certain range.The optimum extraction conditions obtained by orthogonal design were as follows:80%ethanol as extraction solvent,the ratio of material to liquid at 1:30,extraction twice,1.5min extraction each time.The verification results of the optimal process showed that the average extraction amount of 6 batches of samples was 0.685±0.007 and the RSD was 0.96%.The results of separation and purification of 6-Shogaol by high-speed countercurrent chromatography showed that after 6-Shogaol was extracted with ethyl acetate,6-Shogaol with high purity could be separated by high-speed countercurrent chromatograph using petroleum ether-ethyl acetate-methanol-water as solvent system.The target compound was identified as 6-Shogaol after molecular weight estimation and structure analysis by LC-MS and NMR.2.The effects of 6-Shogaol on the proliferation and apoptosis of Bel-7402 and HepG2 hepatoma cells were studied.the results showed that6-Shogaol decreased the viability and inhibited the proliferation of hepatoma cells in a dose-dependent manner,and the effect was the most significant at the concentration of 40μM.The apoptosis rate of hepatoma cells was significantly increased by different concentrations of 6-Shogaol,and the effect of 6-Shogaol on the gene or protein expression of TLR4,FOXO3a,Wnt1 andβ-catenin in hepatoma cells showed that compared with normal hepatocytes,the expression of TLR4 gene in hepatoma cells was significantly up-regulated,while the expression of FOXO3a gene was down-regulated.The expression of TLR4,Wnt1 andβ-catenin protein was significantly up-regulated,while the expression of FOXO3a protein was down-regulated in HCC cells.6-Shogaol significantly down-regulated the expression of TLR4,Wnt1 andβ-catenin genes or proteins in hepatocellular carcinoma cells in a dose-dependent manner.In contrast,FOXO3a gene or protein in hepatoma cells was activated by 6-Shogaol,and the effects of 6-Shogaol on proliferation,apoptosis and expression of TLR4,FOXO3a,Wnt1 andβ-catenin genes or proteins in hepatoma cells overexpressing TLR4 showed that the effect of 6-Shogaol on TLR4 and FOXO3a gene expression was reversed by TLR4 upregulation.In6-Shogaol-treated hepatoma cells,the overexpression of TLR4 significantly increased the expression of TLR4,Wnt1 andβ-catenin protein,while inhibited the expression of FOXO3a protein.In addition,the overexpression of TLR4can reverse the inhibitory effect of 6-Shogaol on the proliferation of hepatocellular carcinoma cells.Overexpression of TLR4 inhibits the apoptotic effect of 6-Shogaol on hepatocellular carcinoma cells.The simultaneous effects of 6-Shogaol and SKL2001（activator of Wnt/β-catenin pathway）on proliferation,apoptosis and gene or protein expression of FOXO3a,Wnt1 andβ-catenin in hepatocellular carcinoma cells showed that SKL2001 activated Wnt/β-catenin pathway and significantly reversed the activation of FOXO3a induced by 6-Shogaol.The existence of SKL2001 restored the proliferation and decrease of hepatoma cells induced by 6-Shogaol,while the apoptosis-promoting effect of 6-Shogaol on hepatocellular carcinoma cells was inhibited by SKL2001.The effects of 6-Shogaol on the proliferation,apoptosis and the expression of TLR4,FOXO3a,Wnt1 andβ-catenin genes or proteins in overexpressed hepatoma cells showed that when TLR4overexpressed cells were treated with 6-Shogaol,the expression of FOXO3a gene in hepatocytes was significantly down-regulated,while the expression of TLR4,Wnt1 andβ-catenin protein was activated.After overexpression of TLR4 and FOXO3a,the expression of FOXO3a increased,while the protein expression of TLR4,Wnt1 andβ-catenin did not change.In addition,the overexpression of FOXO3a reversed the effect of overexpression of TLR4 on the proliferation and apoptosis of cancer cells,and the results in vivo showed that 6-Shogaol could significantly inhibit the size of hepatoma in mice,while the overexpression of TLR4 reversed this phenomenon.6-Shogaol could significantly inhibit the expression of Ki-67 in mouse hepatoma cells,and could be partially reversed by TLR4 overexpression.In addition,6-Shogaol could significantly inhibit the expression of TLR4,WNT1 andβ-catenin protein and up-regulate the level of FOXO3a protein in mouse hepatocellular carcinoma,but it could be reversed by the overexpression of TLR4.3.The effects of 6-Shogaol combined with 5-FU on the proliferation,cycle and apoptosis of HepG2 and LI-7 hepatoma cells were studied.the results showed that 6-Shogaol and 5-FU significantly inhibited the viability of hepatoma cells,and the concentration of 50μM 6-Shogaol further enhanced the inhibitory effect of 5-FU on cell viability.The CI values of the combined action of 6-Shogaol and 5-FU at different concentration ratios were less than 1,indicating that the two drugs had synergistic effect.Compared with 5-FU,6-Shogaol combined with 5-FU could significantly increase the percentage of cells in G0/G1 phase and decrease the percentage of cells in S and G2/M phase.Both 6-Shogaol and 5-FU can down-regulate the expression of cell cycle-related proteins（Cyclin A2,Cyclin D1 and Cyclin E1）,and 6-Shogaol can enhance the inhibitory effect of 5-FU on the cell cycle of hepatocellular carcinoma.In addition,the results also showed that 6-Shogaol further enhanced the promoting effect of 5-FU on apoptosis of hepatocellular carcinoma cells,and the effects of 6-Shogaol combined with 5-FU on proliferation,cycle and apoptosis of hepatocellular carcinoma cells through AKT/mTOR pathway showed that the combined action of 6-Shogaol and5-FU further down-regulated the phosphorylation levels of AKT and mTOR.The combined treatment of 6-Shogaol and 5-FU significantly decreased the phosphorylation level of AKT and mTOR in hepatoma cells,and the activation of AKT could partially reverse this effect.Cell viability analysis showed that the inhibition of hepatoma cell viability by 6-Shogaol combined with 5-FU was significantly reversed by AKT activation.The combination of 6-Shogaol and 5-FU could significantly increase the proportion of cells in G0/G1 phase and down-regulate the proportion of cells in S phase and G2/M phase,but the activation of AKT reversed this change.SC79 could partially reverse the inhibitory effect of 6-Shogaol combined with 5-FU on the expression of Cyclin A2,Cyclin D1 and Cyclin E1.In addition,6-Shogaol combined with5-FU can effectively promote apoptosis of cancer cells,and the activation of AKT can partially reverse this effect;the effects of 6-Shogaol combined with5-FU on proliferation,cycle and apoptosis of hepatoma cells through MRP1pathway showed that the m RNA and protein levels of MRP1 in 6-Shogaol and5-FU treated cells were lower than those in the control group,respectively,and the combined use of 6-Shogaol and 5-FU could further reduce the level of MRP1.The treatment of activated AKT/mTOR signal with SC79 can effectively reverse the decrease of MRP1 level caused by the combined action of 6-Shogaol and 5-FU.After transfection with MRP1 overexpression vector,the m RNA and protein expression levels of MRP1 were significantly increased.The combination of 6-Shogaol and 5-FU could significantly inhibit the activity of hepatoma cells,which could be partially reversed by increasing MRP1.6-Shogaol combined with 5-FU induced cell cycle arrest in G0/G1phase,which could be reversed by increasing the expression of MRP1.Overexpression of MRP1 could partially reverse the inhibitory effect of6-Sho Goal combined with 5-FU on the expression of Cyclin A2,Cyclin D1 and Cyclin E1.The combined effect of 6-Shogaol and 5-FU can significantly promote the apoptosis of hepatocellular carcinoma cells,while the combined effect can be partially reversed by increasing the expression of MRP1.6-Shogaol combined with 5-FU regulated the effects of AKT/mTOR/MRP1axis on proliferation,cycle and apoptosis of hepatocellular carcinoma cells.The results showed that the combined action of 6-Shogaol and 5-FU could significantly reduce the expression of MRP1m RNA in hepatocellular carcinoma cells,which could be reversed by the activation of AKT and consolidated by silencing MRP1.In hepatoma cells co-treated with 6-Shogaol and 5-FU,silencing MRP1 could effectively reverse the increase of MRP1m RNA induced by AKT activation.Combined application of 6-Shogaol and 5-FU could reduce the phosphorylation level of AKT and mTOR and the expression of MRP1 protein.In addition,after hepatoma cells were co-treated with 6-Shogaol and 5-FU,silencing MRP1 could weaken the promoting effect of SC79 on the expression of MRP1 in hepatoma cells.Cell proliferation assay showed that the activation of AKT could significantly promote the proliferation of hepatoma cells treated with 6-Shogaol and 5-FU,while MRP1gene knockout inhibited the cell proliferation.Cell cycle analysis by flow cytometry showed that the activation of AKT significantly reduced the G0/G1phase arrest induced by the combination of 6-Shogaol and 5-FU,which was aggravated by MRP1 silencing.After AKT activation,the expression of Cyclin A2,Cyclin D1 and Cyclin E1 in hepatoma cells increased,and MRP1silencing could partially reverse their expression.In addition,the combination of 6-Shogaol and 5-FU can significantly promote the apoptosis of cancer cells,but the activation of AKT can reduce the apoptosis of cancer cells.However,silencing MRP1 effectively limited the effect of SC79 and promoted the apoptosis of cancer cells.The results of in vivo experiments showed that the tumor volume of liver cancer in 6-Shogaol group and 5-FU group was smaller than that in control group.Compared with other groups of mice,the tumor volume and tumor weight of mice treated with 6-Shogaol and 5-FU were the smallest.The efficacy of 6-Shogaol combined··with 5-FU was significantly better than that of single drug,and showed a synergistic effect in inhibiting the formation of liver cancer.The level of phosphorylated AKT,the level of phosphorylated mTOR and the expression of MRP1 in the tumor of mice treated with 6-Shogaol and 5-FU were lower than those in the control group.The combination of 6-Shogaol and 5-FU further reduced the phosphorylation of AKT and mTOR and the expression of MRP1 in tumors.The expression of MRP1 in tumor tissue was detected by immunohistochemical method.4.The results of single factor investigation on the preparation process of blank nanoemulsion show that,in addition to ultrasonic time,the amount of oil phase,mixed emulsifier and ultrasonic power all have significant influence on the particle size of blank nanoemulsion in a certain range.According to the star design-response surface method,the optimized process conditions are as follows:oil phase dosage（10%）,mixed emulsifier dosage（20%）,ultrasonic power（300W）.The optimal prescription is verified,and the results show that the deviation between the predicted value and the real value is small,indicating that the star design-effect surface optimization has a good prediction effect,according to the optimal prescription,it is determined that the theoretical drug loading of the nano-emulsion to 6-Shogaol is 36.30 mg·g-1,and considering the stability of the whole drug loading system,the actual drug loading is 30 mg·g-1At room temperature,the prepared 6-Shogaol nanoemulsion is a yellowish liquid with good fluidity.However,the fluidity of6-Shogaol nanoemulsion decreased at 4℃,and the other changes were not obvious.The particle size and Zeta potential of 6-Shogaol nanoemulsion were measured by Malvern laser particle sizer,the results showed that the average particle size of 6-Shogaol nanoemulsion was 38.03±1.92 nm,and the Zeta potential was-19.06±0.25 MV.Under transmission electron microscope,the morphology of 6-Shogaol nanoemulsion was spherical,the size and distribution were uniform,and the particle size was between shogaol.The6-Shogaol nano-emulsion was identified as O/W type by staining method;the entrapment efficiency of 6-Shogaol nano-emulsion was determined by centrifugation,and the average entrapment efficiency of 6-Shogaol nano-emulsion was 97.26%.The in vitro drug release of Shogaol nano-emulsion showed that the cumulative drug release of 6-Shogaol nano-emulsion and Shogaol nano-emulsion were 38.12%and 18.33%,respectively.At 4 h,the cumulative drug release of 6-Shogaol nanoemulsion reached more than 85%,while the final cumulative drug release of 6-Shogaol bulk drug at 24 h was only 60.42%.After fitting the drug release curve,it was found that the in vitro release of 6-Shogaol conformed to the first-order kinetic equation,while the in vitro release of 6-Shogaol nanoemulsion conformed to the Higuchi equation.The results of stability study showed that in 30 days,the fluidity of 6-Shogaol nanoemulsion in 4℃refrigerator became worse than that at 25℃and 40℃,but there was no significant change.Compared with day 0,the particle size changed obviously at 40℃,but not at 4℃and 25℃.After 30days,the drug content decreased slightly at three temperatures,and the overall stability was good.The results of anti-hepatoma effect of 6-Shogaol nano-emulsion showed that compared with the control group,6-Shogaol group and 6-Shogaol nano-emulsion combined with 5-FU group could significantly inhibit the tumor growth of mice,while the anti-hepatoma effect of 6-Shogaol nano-emulsion group was stronger than that of 6-Shogaol group.Conclusion:In this study,high purity 6-Shogaol was prepared from dried ginger by flash extraction technology combined with high speed countercurrent chromatography.In vitro and in vivo studies,6-Shogaol can down-regulate the expression of TLR4 in hepatocellular carcinoma cells,mediate Wnt/β-catenin signal pathway,and up-regulate FOXO3a to inhibit the development of hepatocellular carcinoma.There is a synergistic effect between 6-Shogaol and5-FU.6-Shogaol can effectively improve the efficacy of 5-FU in the treatment of liver cancer by inhibiting AKT/mTOR/MRP1 signal pathway.The preparation process of blank nano-emulsion was optimized by single factor experiment and star design-effect surface method.6-Shogaol nano-emulsion with high drug loading,high entrapment efficiency,fast drug release rate,smaller particle size than 100nm,good appearance property and stability was successfully prepared.Compared with 6-Shogaol raw material drug,6-Shogaol nano-emulsion has stronger anti-liver cancer effect. |
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