Study On The Anti-hepatocellular Carcinoma Pharmacodynamics Substances And Mechanism Of Gansui Banxia Decoction

The incidence of hepatocellular carcinoma（HCC）ranks sixth in the world and the mortality rate ranks third.In the next five years,the incidence of liver cancer will exceed one million,and the proportion of HCC will be as high as 90%.Traditional Chinese medicine（TCM）is a treasure of Chinese medicine.TCM has played an important role in the prevention and treatment of liver diseases,especially in immune activation,inflammation improvement,and metabolic regulation.Gansui-Banxia Decoction（GSBXD）is a classic formula of traditional Chinese medicine form"Synopsis of the Golden Chamber".It has five ingredients,including Euphorbia kansui T.N.Liou ex T.P.Wang（Gansui）,Pinellia ternata（Thunb.）Breit.（Banxia）,Glycyrrhiza uralensis Fisch.（Gancao）,Paeonia lactiflora Pall.（Baishao）and Mel（Fengmi）.It is mainly used to treat cancerous ascites.The research of GSBXD mainly focuses on the evaluation of the"toxicity-effect"of the herbal pair of Gansui and Gancao,but the mechanism of immune regulation and its substances remain unknown,especially,the study of GSBXD on the anti-hepatocellular carcinoma immunity is rare.Therefore,we systematically researched the anti-hepatic carcinoma pharmacodynamics and the possible mechanisms.First,we evaluated the efficacy of GSBXD water and alcohol extracts by establishing the H22 solid and ascites mice models.The results showed that in the solid tumor model,the inhibition rate of ethanol extract on tumor volume was 63.00±1.24%to 70.50±0.70%,but the inhibition rate of water extract was 20.50±0.70%to 69.50±2.12%.In cancerous ascites model,the inhibition rate of high-dose ethanol extract on ascites volume was 53.57±4.84%,and the inhibition rate of high-dose water extract was 71.43±4.87%.The results means that the effect of ethanol extract was better than that of water extract in solid tumor models,conversely,the effect of water extract on ascites volume was better than that of ethanol extract.In addition,we found that the spleen index of tumor-bearing mice correlated with the model types.In the solid tumor model,the spleen index positively correlated with tumor size,and in the ascites tumor model,the spleen index negatively correlated with ascites volume.The extracts could maintain the normal size and morphology of the spleen,and spleen index ranged from 6.21±2.64 mg/g to 7.65±4.01 mg/g.The extracts could significantly inhibit the release of inflammatory cytokines,such as interleukin-1β（IL-1β）and interleukin-6（IL-6）in lipopolysaccharide（LPS）-stimulated RAW264.7cells.Additionally,the extracts significantly down-regulated the activation of nuclear factorκB（NF-κB）and inhibited its translocation into the nucleus,thereby exerting anti-inflammatory effect.Herein,ethanol extract showed better activity than that of the water extract.Ethanol extract mainly contained terpenoids and flavonoids by UHPLC-Q/TOF-MS analysis.A total of 44 ion peaks were identified from National Library of Medicine,Pub Med,Science Direct of Elsevier and CNKI that based on their chemical names,molecular formulas,accurate molecular mass,chemical structures and relevant fragments,including 19 terpenoids,13 flavonoids,1 chalcone,2 steroids,2 tannins,1 hormone and other types.Terpenoids were mainly from Gansui,flavonoids were mainly from Gancao.Ethanol extract could inhibit the tumor growth and regulate the frequencies of immune cells in tumor and spleen tissues.It significantly inhibited the decreasing of T cells and B cells,inhibited the accumulation of CD11b⁺F4/80⁺tumor-associated macrophages,increased the infiltration of NK1.1⁺cells,and inhibited the recruitment of Gr-1⁺-Ly6C⁺/Ly6G⁺myeloid-derived immunosuppressive cells（MDSCs）.Based on the results of network pharmacology analysis and mechanism verification,the antitumor mechanism of ethanol extract was inhibition the signal transducer and activator of transcription 3（STAT3）/protein kinase B（AKT）/extracellular regulated protein kinase（ERK）signaling pathways.Further study showed that ethanol extract could inhibit the proliferation and accumulation of MDSCs via downregulating the STAT3/AKT/ERK signaling pathway in vitro.Based on the pharmacodynamics of kansui and the chemical components of Gansui Banxia decoction,we systematically studied the chemical components of kansui,and obtained 91 compounds from kansui,including 21 triterpenoids,49diterpenoids,12 steroids,and 9 other types of compounds.We detected the anti-Hep G2 activity and anti-inflammatory activity of 12 steroids and 58 terpenoids;we found that 5 C21 steroids and 17 terpenoids had anti-Hep G2 activity.Cynsaccatol L（5）with the best activity was studied the mechanism.The results showed that it could significantly inhibit the proliferation of Hep G2 cells and induce autophagy-related apoptosis.The expression of Bax,cleaved caspase 3,cleaved caspase 8 were significantly upregulated,and decreased the expression of Bcl-2.Based on the molecular configuration of 5,we focused its effect on the regulation of Na+,K+-ATPaseα1 subunit（ATP1A1）.The results showed that 5 could promote the endolysosomal degradation of ATP1A1 protein,and promoted the ubiquitination of ATP1A1 protein.Besides,5 could bind to theα1-4 chain amino acid residues of ATP1A1 active pocket,to inhibit the activity of sodium-potassium pump,thereby exerting the anti-tumor activity through down-regulating the activation of ATP1A1-related AKT and ERK signaling pathways.Simultaneously,5 inhibited macrophages to polarize to M2 subtype,enhanced its phagocytic ability and regulated cytokines secretion,thereby further promoting the apoptosis of Hep G2 cells in co-culture medium.5 inhibited the proliferation of MDSCs and down-regulated the intracellular STAT3/AKT/ERK signaling pathway.Based on the immunomodulatory activity of Gansui Banxia decoction,we detected 17 terpenoids with anti-Hep G2 activity for anti-inflammatory activity,and found that 11 compounds had both anti-tumor and anti-inflammatory activities.Herein,we selected the most active compound,20-O-Acetylingenol-3-angelate（AAI）,to study the mechanism.The half-maximal inhibitory concentration(IC₅₀)of AAI on the production of nitric oxide was 0.015μM.In LPS-stimulated RAW264.7 cells,AAI could inhibit the high expression of IL-6 and IL-1βwith a dose-dependent,and inhibited the expression of i NOS and the nucleus translocation of NF-κB.In IL-4stimulated RAW264.7 cells,AAI significantly down-regulated the expression of ARG-1,CD163,TGF-βand CD206,inhibited the activation of STAT6 and PKC-δ/ERK and AKT1 signaling pathways,and inhibited the macrophage M2 type polarization.At together,AAI maintained the balance of M1 and M2 ratio.In co-culture condition,AAI promoted the expression of apoptotic proteins of Hep G2cells,inhibited the polarization of macrophages to M2 type,enhanced the phagocytic ability of macrophages,and exhibited inhibitory effects on the proliferation of MDSCs by regulating mitogen-activated protein kinase signaling pathways.AAI inhibited the proliferation of MDSCs and downregulated the intracellular PKC-δ/MAPK signaling pathways.In conclusion,（1）Gansui Banxia decoction had the effect on inhibiting solid tumor growth and cancerous ascites volume in H22-bearing mice,and showed the different efficacies.（2）The material basis of ethanol extract was mainly terpenoids and flavonoids,terpenoids were mainly from Kansui,and flavonoids were mainly from Gancao.（3）Ethanol extract could down-regulate STAT3/AKT/ERK signal pathways of tumors and MDSCs.（4）5 C21 steroids had anti-Hep G2 and anti-inflammation.Cynsaccatol L,as C21 steroidal glysides could bind to ATP1A1 for inhibition of pump activity,and downregulated ATP1A1 expression to inhibit ATP1A1-related AKT-ERK signaling pathways,thereby promoting autophagy-related apoptosis of Hep G2 cells.（5）Ingenane-type diterpene AAI regulated the polarization of M1 and M2 macrophages by multiple signaling pathways,such as PKC-δ/ERK,NF-κB,and STAT6.In co-culture conditions,AAI could inhibit the macrophages M2polarization,reduced the release of cytokines（such as IL-6,IL-10）,and enhanced the phagocytic ability of macrophages,thereby further promoted the apoptosis of Hep G2cells.The research provided the new ideas and new methods for the study of the immune and anti-tumor effects of traditional Chinese medicine fumula,which provided a certain scientific basis for clinical application and product development.