The Study On The Therapeutic Effects And Mechanism Of Euphorbia Pekinensis And Glycyrrhiza Glabra On Hepatocellular Carcinoma Ascites Based On Network Pharmacology

ObjectiveTo clarify unknown rationalities of herbaceous compatibility of Euphorbia Pekinensis(DJ)and Glycyrrhiza glabra(GC)acting on hepatocellular carcinoma(HCC)ascites,so as to provide a scientific basis for eighteen incompatible medicaments DJ and GC according to theories of traditional Chinese medicine.MethodsThe therapeutic effect and mechanism of Euphorbia Pekinensis and Glycyrrhiza glabra on hepatocellular carcinoma ascites was detected by HCC ascites mice.1.The DJ/GC-synergistic group and DJ/GC-antagonist group were screened by pharmacodynamic results:120 Kunming mice were randomly divided into normal control group(Con),HCC ascites mouse model(Mod),DJ/GC(1:0.5,1:1,1:2 and 1:4),DJ-alone group and GC-alone groups,each group of 15.After the mice were cultured for 3 days,the mice were inoculating H22 cells intraperitoneally except the control group.To construct the H22 HCC ascites mouse model,the needle was inserted into the left lower abdomen,and H22 cells were inoculated intraperitoneally.Each mouse was inoculated with 1×107 H22 cells.DJ/GC 1:0.5 group,1:1 group,1:2 group,1:4 group were given DJ/GC co-decoction 0.579,0.772,1.158,1.931 g/Kg/d;DJ/GC-alone decoction 0.386 g/Kg/d;GC-alone 1.546 g/Kg/d.At this dose administered,the dose of DJ was 3 g of the human equivalent dose.The Con group and the Model group were given the same amount of normal saline every day for 12 days.After 7 d,the mouse who still no significant increase in abdominal circumference were removed.Modeling success rate was 70 to 80%.At the 12 d,the therapeutic effects of herbal pair DJ and GC acting on HCC acites was analyzed in terms of body weight,ascites volume,abdominal circumference.The serum was centrifugated at 3000 rpm for 15 min for serum biochemical analyses.Serum biochemical analyses included alanine aminotransferase(ALT,related to liver),aspartate amino transferase(AST,related to liver),creatinine(Cre,related to kidney),and blood urea nitrogen(BUN,related to kidney).The peritoneal tissue were stored in Trizol and dry ice for preservation for the he global gene expression profile spectrum detection.2.To explore the mechanism of DJ and GC on hepatocellular carcinoma ascites based on the global gene expression profile and network pharmacology:(1)The gene set of HCC ascites-related gene(Con vs Mod);(2)DJ/GC combination-related gene;(3)Known therapeutic target gene for ascites.The network of HCC ascites-related gene-DJ/GC combination-related gene-known therapeutic target gene for ascites was constructed based on the links among HCC ascites-related genes,DJ/GC combination-related genes and known therapeutic targets for ascites,to shed light on the combination principles of herbal pair DJ and GC on the HCC ascites.3.The target protein was validated by experiment:the renal tissue obtained by the previous experiment was validated by Western Blotting,RT-PCR and M-1 cell experiment.Results1.Synergistic and antagonistic effects of DJ and GC with different combinational ratios acting on HCC malignant ascitesCompared to normal mice,the ascites volumes,body weights and abdominal circumferences were significantly increased in HCC ascites mice,which was all markedly reversed by the treatment of DJ-alone and DJ/GC-synergy combination.This reduction trend was more obvious in DJ/GC-synergy group.However,no differences with statistical significance were observed between DJ/GC-antagonism and model groups.Compared to DJ-alone treatment group,DJ/GC-synergy combination could enhance the therapeutic effects of DJ on the ascites volumes,body weights and abdominal circumferences,while DJ/GC-antagonism combination aggravated the malignant phenotypes of HCC ascites mice.2.Toxicities of DJ and GC with different combinational ratios to the liver and kidney tissues of mice with HCC malignant ascitesThe liver index in HCC ascites mice were significant higher than that in normal mice,which were reversed by the treatment of both DJ and DJ/GC synergy combination,although without significant differences.In addition,the kidney index in HCC ascites mice were obviously reduced when compared with normal mice.Both DJ and DJ/GC-synergy combination had a tendency to increase the kidney index in HCC ascites.Moreover,the serum levels of ALT,AST,Cr and BUN in HCC ascites mice were all markedly increased compared to normal mice.However,the administrations of DJ-alone and DJ/GC-combinations could not influence the changes of ALT,AST,Cr and BUN levels in sera of HCC ascites mice in their response to DJ-alone or any combination designs of DJ and GC in comparison with model groups.Moreover,histopathological examination was performed to confirm the evidence from biochemical analyses.The normal hepatic structure of normal mice characterized with normal lobular architecture and radiating hepatic cords.Severe hepatocytes swelling,diffused and enlarged vacant spaces in cells were observed in HCC ascites mice.Regarding to the kidney tissues,abnormal alterations were shown in the glomerulus and renal tubes in HCC ascites mice.Notably,the treatment of DJ and DJ/GC combinations could ameliorate the damage of HCC malignant ascites to the liver and kidney tissues.3.The mechanism of DJ-GC against hepatocellular carcinoma ascitesAfter comparing the gene expression profiles of peritoneal tissue among DJ/GC-synergy,DJ/GC-antagonism,DJ-alone treatment groups with that of HCC ascites mouse model,we obtained the DJ/GC combination-related gene.The network of HCC ascites-related gene-DJ/GC combination-related gene-known therapeutic target gene for ascites was constructed based on the links among HCC ascites-related genes,DJ/GC combination-related genes and known therapeutic targets for ascites,to shed light on the combination principles of herbal pair DJ and GC on the HCC ascites.Thus,we hypothesized that the therapeutic effect of the herb pair DJ and GC might be exerted by targeting the Frk-Arhgdib-Inpp5d-Avpr2-Aqp4 signal axis.4.To validate the above hypothesis,we performed WB、RT-PCR and M-1 collecting duct cell based on HCC ascites mouse modelOur data from Western blot analysis found that the expression levels of Frk,Arhgdib,Inpp5d,Avpr2 and Aqp4 protein in kidney tissues of HCC ascites mice were all significantly higher than those in normal controls.After the administrations of DJ and DJ/GC-synergy combination,their expression was effectively reduced.In contrast,the treatment of DJ/GC-antagonismcombination did not exert this regulatory effects.These findings wereconsistent with the results of quantitative PCR analysis at mRNA levels.ConclusionDJ has a certain anti-hepatocarcinoma ascites effect,and GC in different proportions compatibility with DJ has different effects:When DJ-GC to 1:2 compatibility,GC can coordinate with DJ’ anti-hepatocarcinoma cancer ascites,when the DJ-GC 1:4 compatibility,GC will antagonize the effect.And its anti-hepatocarcinoma ascites function may be through the regulation of Frk-Arhgdib-Inpp5d-Avpr2-Aqp4 signal axis.