| Objective: Under the pathological condition of hyperuricemia(HUA),try to use the network pharmacology method to build a regulatory network combining "hypouricemic compound-hyperuricemia" based on the traditional Chinese medicine compound of "Liuwei goufeng drink",and predict the effective components/groups of hypouricemia in Liuwei goufeng drink compound;Preliminary screening was carried out for compounds that predicted the effect of reducing uric acid by network pharmacology.The inhibition activity of xanthine oxidase of single compound was screened through the screening platform of xanthine oxidase inhibition activity in vitro,and the screened compounds were combined to verify whether they had synergistic effect;According to the component-target-disease network of Liuwei Tongfeng Drink in the treatment of hyperuricemia,PI3K-Akt signal pathway is the pathway with the highest correlation to reduce uric acid.This pathway Plays a certain regulatory role in various physiological activities of cells,For example,it regulates cell proliferation and apoptosis,and has a close relationship with the development of a number of diseases.Therefore,based on the HK-2 cell model,the signal pathways PI3K-Akt were experimentally verified;It is expected to predict the effective ingredients/groups in the compound formula for reducing uric acid,and provide theoretical basis and new treatment ideas for clinical treatment of hyperuricemia.Methods:1.The active compounds in Liuwei Tongfeng drink were collected by using TCMSP and Web of Science database to obtain the compound library.Collect target protein data of active compounds based on TCMSP and Swiss Target Prediction database.Search the Gene Cards database and OMIM database,screen the targets related to hyperuricemia,take the intersection,and construct the protein-protein interaction network diagram.Through the analysis of GO function and KEGG pathway enrichment,the potential pathway of Liuwei Tongfeng Drink in reducing uric acid was predicted,and the synergy combination was found by analyzing the pathway.2.A total of 31 compounds were collected from 54 compounds predicted by the network pharmacology to have the inhibitory activity of xanthine oxidase.With allopurinol as the positive control,the IC50 value is calculated according to the concentration and average inhibition rate of each compound,and the appropriate combination of synergetic action of reducing uric acid is found according to the corresponding mass ratio of IC50.The combination with better inhibition activity was optimized,and the combination was re-combined according to the ratio of compound A:compound B=1:2,2:1,and the inhibition activity of xanthine oxidase was determined.3.The HK-2 cells were cultured: Western Blot test was performed after resuscitation,passage and cryopreservation;The combination of compounds predicted by PI3K-Akt pathway was experimentally verified.The HK-2 cells were inoculated on a six-well plate,and 80% of them were given different combinations of compounds.Western blot was used to detect the protein expression of URAT1 and GLUT9.Results:1.Through TCMSP and Web of Science database,54 compounds with uric acid lowering activity and 1247 related targets of Liuwei Tongfeng Drink were collected.2007 disease targets were obtained in Gene Cards and OMIM databases.Combine compound target and disease target to obtain 172 cross genes and import them into STRING database.The protein-protein interaction network has 172 nodes in total.GO function and KEGG pathway enrichment analysis showed that the PI3K-Akt signaling pathway inhibited uric acid reabsorption and promoted uric acid excretion in the body by inhibiting the expression of uric acid transporters URAT1 and GLUT9 in the kidney.2.According to the study of components that exert purine oxidase inhibitory activity by single action,apigenin has the best inhibitory activity,and the inhibitory activity of each compound is apigenin>luteolin>kaempferol>quercetin>aesculetin>hesperidin.The compounds were combined in pairs,and the concentration corresponding to the IC50 value was 1:1 for the next study.The results of the study on the components of purine oxidase inhibitory activity by synergism showed that apigenin,quercetin and aesculetin showed additive effect;The combination of luteolin and aesculetin is additive;Kaempferol-quercetin,aesculetin and hesperidin showed additive effect.These results show that the combination of single compound with other compounds with strong activity may be a simple additive effect.The study of synergetic components adjusted the proportion of compounds in the combination.The proportion of 1:2 and 2:1 was investigated respectively.Six combinations showed additive effects,namely,apigenin:kaempferol(1:2),apigenin: quercetin(1:2),apigenin: quercetin(2:1),apigenin:hesperidin(2:1),luteolin: kaempferol(2:1),aesculetin: hesperidin(2:1).As the proportion of luteolin increased,the combination changed from antagonistic effect to additive effect.To sum up,different combinations of compounds have different effects.3.In the experimental validation of PI3K-Akt pathway,the research results of resveratrol+naringin on the expression level of URAT1 and GLUT9 proteins in HK-2cells showed that compared with the blank group,the concentration of resveratrol/naringin was less than 2 μg/m L,the protein expression of URAT1 and GLUT9 could not be down-regulated,and there was no significant difference(p>0.05),when the concentration was 4 μg/m L,resveratrol/naringin could significantly inhibit the up-regulation of URAT1 by UA(p<0.01).It is suggested that the predicted drug combination can affect the expression of URAT1 protein mediated by UA,and may achieve the purpose of reducing uric acid.The study of resveratrol+apigenin on the protein expression level of URAT1 and GLUT9 in HK-2 cells showed that compared with the blank group,resveratrol/apigenin had no effect on URAT1,GLUT9 expression in different concentrations,and the difference was not statistically significant(p>0.05).Conclusion: 1.Network pharmacological studies have shown that the PI3K-Akt signaling pathway can inhibit the reabsorption of uric acid and promote the excretion of uric acid in the body by inhibiting the expression of uric acid transporters URAT1 and GLUT9 in the kidney,thus achieving the purpose of treating hyperuricemia.2.According to the study of components that exert purine oxidase inhibitory activity by single action,apigenin has the best inhibitory activity,and the inhibitory activity of each compound is apigenin>luteolin>kaempferol>quercetin>aesculetin>hesperidin.The compound was combined in pairs according to 1:1.The results showed that the combination of single compound and other compounds with strong activity might be a simple additive effect.According to the study of synergetic components,the proportion of compound combinations was increased to 2:1.The results showed that 6combinations showed additive effect,and some compounds changed from antagonistic effect to additive effect with the increase of the proportion.3.Based on the results of Western Blot experiment,in the experimental verification of PI3K-Akt pathway,when the concentration is 4μg/m L,resveratrol/naringin could significantly inhibit the up-regulation of URAT1 by UA(p<0.01).Compared with the blank group,resveratrol/apigenin could not down-regulate the protein expression of URAT1 and GLUT9 at different concentrations,and the difference was not statistically significant(p>0.05). |
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