Effect Of Ganlong Capsule On Proliferation And Hypoxia Microenvironment Of Hepatocellular

Objective:To investigate the effects of the marketed drug Ganlong Capsule on the proliferation and hypoxic microenvironment of hepatocellular carcinoma（HCC）and its mechanism.Methods:In this study,Human hepatocellular carcinoma sensitive cell line BEL-7402 and Balb/c-nude mice were used as the research subjects;Ganlong capsule,Periplaneta americana crude extract degreasing cream and Periplaneta americana crude extract CII-3were used as the test drugs for dual intervention in vivo and in vitro.Sorafenib was used as the positive control drug in this study.The effects of Ganlong Capsule on liver cancer,in vivo and in vitro were observed,and the effects were better than those of CII-3,degreasing cream and CII-3 Sorafeni et al.In vitro cell experiment:（1）MTT assay was used to detect the effect of Ganlong capsule on the proliferation of human hepatocellular carcinoma sensitive cell line BEL-7402.To further determine the best action time and the optimal concentration of Ganlong capsule in vitro;（2）The inhibitory effect of Ganlong Capsule on Bel-7402 hepatoma cell line was investigated by clonal colony experiment;（3）Morphological changes of Bel-7402 liver cancer cell lines were observed under different drug concentrations and different time.（4）The apoptosis degree of Bel-7402 liver cancer cell line was observed by laser confocal microscope after DAPI fluorescent dye staining.To determine the inhibitory effect of Ganlong capsule on BEL-7402 liver cancer cell line;（5）Flow cytometry was used to detect the ability of Ganlong capsule to induce apoptosis of Bel-7402 hepatoma cell line;（6）RT-q PCR was used to detect the m RNA relative expression levels of hypoxia-inducing factor HIF-1α,VEGF and VEGFR2 in Bel-7402 hepatoma cell lines to determine the effect of Ganlong capsule on hypoxia-induced microenvironment;In vivo animal experiment:（7）In Balb/c-nude nude mice,establish an axillary xenograft model of BEL-7402 cells,by detecting axillary transplanted tumor mice’s general status,immune organs,liver andkidney function,inhibit tumor growth and tumor tissue hypoxia factor HIF-1α,the changes of VEGF and VEGFR2 and tumor histopathology examination link,the influence of the drug in the body to determine Ganlong capsule on hepatocellular carcinoma（HCC）proliferation and the effect of hypoxic microenvironment and its mechanism of action;Results:In vitro cell experiment:（1）Determined by MTT method in detection of Ganlong capsule on liver cancer cell line BEL-7402 proliferation experiment,the influence of the drug after 36 h and 24 h,48 h,hepatocellular carcinoma cell line BEL-7402 for Ganlong capsule IC₅（mg/ml）values of4.12±1.65,6.56±2.30,4.07±1.10 after 24h,36h and 48h.IC₁₀（mg/m L）values were7.71±2.24,14.43±4.21,15.39±5.51;IC₂₀（mg/m L）values were 10.76±3.94,19.57±5.09,17.23±5.82;IC₅₀（mg/m L）values were 31.46±5.46,42.57±3.20,33.16±3.77;（2）MTT method was used to determine the dose of Ganlong capsule,and finally determined the dose of 48 h of IC₅,IC₁₀and IC₂₀on Bel-7402 liver cancer cell line,which were 4.07,15.39 and 17.23 mg/m L respectively,corresponding to low,medium and high dose.（3）Observed under inverted microscope under different drug concentration and time of hepatocellular carcinoma cell line BEL-7402 morphological changes,visible liver cancer cell lines after the treatment has had the obvious changes in number,number of rendering with the increase of drug concentration and gradually reduce,dead cells increased obviously,and cancer cells form also change the obvious apoptosis;（4）Clone colony experiment showed that the number of clone colonies of BEL-7402hepatoma cell line decreased obviously after the treatment with Ganlong capsule liquid,and showed a dose-dependent trend with the increase of drug dose;（5）The laser confocal experiments showed that Ganlong capsule could induce the apoptosis of Bel-7402 hepatoma cell line,and showed a dependent relationship with the increase of Ganlong capsule dose;（6）Flow cytometry results showed that Ganlong Capsules could induce apoptosis of liver cancer cell line obviously（P<0.05）,the effect of Ganlong Capsules high-dose group was second only to the best sorafenib group,and the effect of delipid-cream group was similar to that of CII-3 group（P>0.05）;（7）RT-q PCR results showed that Ganlong capsule,CII-3,delipid-cream and all drug groups could inhibit the expression level of VEGF,and showed a dose-dependent trend with the increase of the concentration of Ganlong capsule liquid.Sorafenib group and Ganlong capsule high-dose group showed better effect（P<0.05）and similar effect（P>0.05）.All of them inhibited HIF-1αexpression level,and Ganlong capsule medium and high dose groups and Periplaneta Americana extract groups showed better effect（P<0.05）and similar effect（P>0.05）.Except for sorafenib group,the expression level of VEGFR2 was inhibited in all groups.The effects of Ganlong capsule medium and high dose groups and Periplaneta Americana extract groups were better（P<0.05）and the effect was similar（P>0.05）;In vivo animal experiment:（8）In the tumor-forming experiments of BALB/C-nude mice,Ganlong capsule,CII-3,delipide and all the drug groups inhibited the growth of transplanted hepatocellular carcinoma tumor mass in nude mice,and increased organ indexes（except spleen indexes）,suggesting the safety of Ganlong capsule;（9）In pathological HE staining observation,tumor cell volume of BALB/C-nude mice after administration of Ganlong capsule was generally small,some necrotic areas were observed,and some morphologic changes of early apoptosis such as nuclear shrinkage,rupture and disappearance were observed,suggesting that Ganlong capsule still had a good anti-tumor effect in vivo;（10）RT-q PCR results showed that the expression levels of HIF-1α,VEGF and VEGFR2in most of the drug groups were higher than those in the sensitive group,indicating that the production of hypoxic microenvironment was related to the high expression of the above factors.Combined with the effects of Ganlong Capsules,sorafenib group and Ganlong Capsules medium dose group had the ability to significantly down-regulate the levels of three kinds of hypoxic microenvironment related proteins in vivo（P<0.05）;（11）The results of immunohistochemical staining showed that the expression levels of HIF-1α,VEGF,VEGFR2,Ki67 and CD31 in most of the drug administration groups were higher than those in the sensitive group,indicating that the production of hypoxic microenvironment was related to the high expression of the above factors.Comprehensive effects of Ganlong Capsules,Ganlong Capsules medium dose group and CII-3 group had significant ability to down-regulate the levels of these related proteins in vivo（P<0.05）;Conclusion:（1）Ganlong capsule can effectively inhibit the proliferation of BEL-7402 hepatoma cell line and can effectively promote the apoptosis of BEL-7402 hepatoma cell line.;also could down-regulate the expression level of hypoxic microenvironment related proteins in Bel-7402 liver cancer cell line;（2）Ganlong capsule can effectively inhibit the growth of transplanted axillary tumors in BALB/C-nude mice and induce tumor apoptosis in vivo and can effectively down-regulate the m RNA and protein expression levels of hypoxic microenvironment related proteins in the axillary xenograft of BALB/c-nude mice.（3）The mechanism of Ganlong Capsule in inhibiting HCC proliferation and improving hypoxic microenvironment in vivo and in vitro may be to down-regulate the expression levels of related proteins HIF-1α,VEGF,VEGFR2,Ki67 and CD31,reduce tumor angiogenesis,and thus achieve the anti-tumor effect;（4）The anti-tumor effect of Ganlong Capsules was better than that of Periplaneta Americana crude extract degrease and CII-3;...