Screening Of Targets For Tatal Flavonoids Of Hippophae Rhamnoides Against Excessive Erythrocytosis In HAPC Mice Based On Network Pharmacology And Protemics

Objective:1.Network pharmacology methods were used to predict the targets and related pathways of total flavonoids of Hippophae rhamnoides against HAPC;2.To study the pharmacological effects of total flavonoids of Hippophae rhamnoides on HAPC model mice;3.Screening and verifying the target proteins of total flavonoids of Hippophae rhamnoides inhibiting excessive proliferation of erythroid Cells in HAPC mice based on 4D-label-free quantitative proteomics and PRM protein quantitative technology.Methods:1.The active components of total flavonoids of Hippophae rhamnoides were obtained from TCMSPW database.The targets of active components were predicted by Pharm Mapper server.The targets related to HAPC were searched in Drug Bank database and OMIM database.The overlapping targets were the targets of total flavonoids of Hippophae rhamnoides against HAPC.The targets were input into David6.8 database for GO enrichment and KEGG pathway analysis;2.Establishing the HAPC model mice.Mice were randomly divided in:Plain group,HAPC model group,Duoxuexukang group(8.120mg/g),total flavonoids of Hippophae rhamnoides low-dose group(0.085mg/g),total flavonoids of Hippophae rhamnoides medium-dose group(0.170mg/g),total flavonoids of Hippophae rhamnoides high-dose group(0.340mg/g),Standard mixture low-dose group(0.383mg/g),Standard mixture medium-dose group(0.574mg/g),Standard mixture high-dose group(0.767mg/g),isorhamnetin group low-dose group(0.112mg/g),isorhamnetin medium-dose group(0.169mg/g),Isorhamnetin high-dose group(0.224mg/g).Drug groups were given the corresponding dose of drug intragastric administration,plain group and HAPC model group were given the same amount of normal saline intragastric administration.Detecting the hematological indexes of mice in each group.Detecting the serum and kidney EPO,SOD,MDA.The pathological changes of heart and spleen were observed under light microscope.The ultrastructural damage of renal EPO-producing cells was observed by projective electron microscopy;3.The 4D-label-free quantitative proteomics technique was applied to identify and quantitatively to screen out the common significantly different proteins in each group of mice kidney tissue samples.According to the results of bioinformatics analysis,the significant difference proteins related to the occurrence and development of HAPC were screened and quantitatively analyzed by PRM.Results:1.6 active components of total flavonoids of Hippophae rhamnoides were obtained by network pharmacology method.Targets of these active components were predicted by Pharm Mapper database,and a total of 116 potential targets were obtained,18 of which were related to total flavonoids of Hippophae rhamnoides against HAPC.A total of 58 pathways were identified by bioinformatics analysis(P<0.05).Among them,PI3K-Akt signaling pathway HIF-1 signaling pathway and VEGF signaling pathway were related to the occurrence and development of HAPC;2.Compared with HAPC model group,haematological index in total flavonoids of Hippophae rhamnoides high-dose,standard mixture high-dose and isorhamnetin high-dose group were lower than HAPC model group(P<0.05).Compared with HAPC model group,serum EPO in total flavonoids of Hippophae rhamnoides high-dose,standard mixture high-dose and isorhamnetin high-dose group were lower than HAPC model group(P<0.05).Compared with HAPC model group,SOD and MDA in kidney and serum in total flavonoids of Hippophae rhamnoides high-dose,standard mixture high-dose and isorhamnetin high-dose group were significantly different from HAPC model group(P<0.05).Compared with HAPC model group,total flavonoids of Hippophae rhamnoides high-dose,standard mixture high-dose and isorhamnetin high-dose group could significantly reduce the damage of spleen,heart and renal EPO-producing cells in HAPC mice;3.148 differential proteins were obtained by proteomics.20 targets of inhibitory effect of total flavonoids of Hippophae rhamnoides on the excessive proliferation of erythroid cells in HAPC mice were screened out.PRM results of 5 proteins were consistent with proteomics.Conclusion:1.Total flavonoids of Hippophae rhamnoides prevent and cure HAPC through PI3K-Akt signaling pathway,HIF-1 signaling pathway and VEGF signaling pathway;2.Total flavonoids of Hippophae rhamnoides can significantly reduce hematological indexes of HAPC mice,inhibitting oxidative stress,improving tissue and cell damage,and reducing the concentration of EPO;3.CAT,STAT3,CP,HSCB and UQCRB were related to the inhibitory effect of total flavonoids of Hippophae rhamnoides on excessive proliferation of erythroid cells in HAPC mice.