Investigation The Compatibility Between Euphorbia Kansui And Glycyrrhiza Based On The Pathway Of Water Metabolism Regulation

Objective:This study aims to investigate the underlying mechanism of synergistic or antagonistic effects between the herbal pair of Euphorbia kansui (GS) and Glycyrrhiza (GC) using the uniform-design method for screening the synergistic or antagonistic doses, along with the study on network pharmacology and experimental validation.Methods:1. Screen the synergistic or antagonistic doses between GS and GC based on the uniform-design method:1) Confirm the effective dose of GS against a H22 hepatocellular carcinoma (HCC) ascites model; 2) Observe the pharmacodynamics of the seven doses of GS/GC herb pair on HCC ascites mice based on uniform-design principle of two factors and seven levels; 3) Make multiple stepwise regression analysis towards the results of uniform design using MATLAB 7.8 software, to screen the doses of GS/GC herb pair that will cause antagonistic or synergetic effects.2. Predict the compatibility mechanism of GS/GC herb pair based on the research strategy of network pharmacology:1) Predict the putative targets for GS and GC based on their containing compounds that possess the same structure; 2) Constructe a drug target network using the Protein-protein interaction (PPI) information of the putative targets of GS and GC, known therapeutic targets for ascites and other human proteins, to shed light on the combination principles of the actions of herbal pair GS and GC against HCC ascites.3. Experimental validation based on the regulatory network of antidiuretic hormone secretion.1) The H22 HCC ascites model was prepared according to the methods described in 2.1, all H22 HCC ascites models were divided randomly into 7 groups: ①the normal control group; ②the model control group;③ the GS/GC antagonism group;④ the GS/GC synergy group; ⑤ the high dose of GS alone group;⑥ the low dose of GS alone group;⑦ the furosemide group. The H22 HCC ascites mice were intragastrically administered the respective drugs one time per day, and for consecutive seven days.2) All mice were weighed and the abdominal circumference was measured.To measure the ascites volume, ascites fluid was aspirated via syringe from the opened abdominal wall following cervical dislocation. 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), and were determined using routine kinetic and fixed rate colorimetric methods.3) Observe the pathological section of liver and kidney by using HE staining.4) The ascites and sera were diluted to different concentrations and analyzed using mouse Beta-1 adrenergic recepter (ADRB1), Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit gamma isoform (PIK3CG), Arginine Vasopressin receptor (AVPR2), Aquaporin 2 (AQP2) ELISA kits; expression patterns and subcellular localizations of ADRB1, PIK3CG and AVPR2 proteins were examined by immunohisto- chemical staining; protein and gene expression of ADRB1、PIK3CG、AVPR2、AQP2 in kidney were measured by Western blot and qRT-PCR, respectively.Results:1. To screen the doses of GS/GC herb pair that will cause antagonistic or synergetic effects based on uniform design:1) At a dose of GS< 0.70g/kg with GC:GS≤0.39:1, the combination of GS and GC reduced the ascites volume in H22 HCC ascites mice; 2) At a GS dose exceeding 0.70 g/kg but less than 0.93g/kg combined with GC<1.03 g/kg, GC had no obvious impact on the diuretic function of GS; 3) When GS≥0.93 g/kg was combined with GC≥1.03 g/kg, and GC:GS≥1.11:1, GC exerted an antagonistic effect to GS.2. Predict the compatibility mechanism of GS/GC herb pair based on the research strategy of network pharmacology:1) ADRB1 and PIK3CG were the putative targets for GS and GC, respectively, which directly related to the target AVPR2 of anti-ascites; 2) The interaction of ADRB1, PIK3CG, AVPR2 could be the underlying mechanism about reduction of the GS on HCC ascites caused by GC.3. Experimental validation based on the regulatory network of antidiuretic hormone secretion: 1) The underlying mechanism in combination therapy of the GS-GC herb pair against HCC ascites may be related to the regulatory network of antidiuretic hormone secretion; 2) When GC exerted an antagonistic effect to GS, GS inhibited the regulations of ADRB 1 expression by GS, increased the AVPR2-AQP2 expression in kidney, promoting the reabsorption of water by kidney, thereby aggravating HCC ascites; 3) When GC exerted an synergistic effect to GS, GS and GC down-regulated their targets ADRB1 and PIK3CG, respectively, increased the AVPR2-AQP2 expression in kidney, inhibited the antidiuretic hormone secretion, thereby promoting the excretion of HCC ascites.4. The administration of GS and GC at different combination ratios did not aggravate hepatic or renal pathological changes in HCC ascites mice.Conclusion:In the current study, we illuminated the effective dose range of GS-GC herb pair based on its antagonistic or synergetic function against HCC ascites, and confirmed that the underlying mechanism in combination therapy of the GS-GC herb pair against HCC ascites was related to the regulatory network of antidiuretic hormone secretion. This study is the first to introduce the network pharmacology analysis into the compatibility mechanism exploration towards "eighteen antagonistic medicaments", thereby providing a validated methodology for revealing the new concept of "eighteen antagonistic medicaments".