Analysis Of Blood Components Of Yougui Yin In Normal And Kidney-deficiency Rats And Its Mechanism Of Improvement Of Kidney-deficiency Osteoporosis

BackgroundYougui yin is composed of rehmanniaglutinosa,wolfberry,cornel,fried yam,aconite,cinnamon,ginger and eucommia,and roasted licorice.89 characteristic peaks of Yougui Yining water decoction were detected by UPLCQ-TOF/MS mass spectrometry in earlier studies,and 85 of them were identified with clear structures.It is also proved that Yougui yin has a significant improvement effect on osteoporosis rats with kidney deficiency.However,which components can be absorbed into the blood and exert the effect of Yougui Yin’s "Essence and Marrow" is not yet known.In this paper,through the combination of serum medicinal chemistry,network pharmacology and serum pharmacology,we intend to study the blood components of Yougui Yin and its mechanism to improve osteoporosis due to kidney deficiency.This research was supported by the Municipal Science and Technology Commission-Family Planning Commission Joint Chinese Medicine Science and Technology Key Project(zy201801001).Purpose1.To study the blood components of Yougui Yin in normal rats and adenineinduced kidney deficiency model rats.2.To further verify the mechanism of blood components of Yougui Yin on kidney deficiency osteoporosis.Methods1.The analysis method of blood components of Yougui Yin in normal rats:This study is based on the theoretical basis of serum medicinal chemistry,combined with ultra-high liquid phase-mass spectrometry and multi-reaction monitoring technology to analyze the plasma of rats after giving Yougui yin by gavage.The ingredients in it were analyzed.(1)The liquid quality conditions of UPLC-Q-TOF/MS:The column is Waters ACQUITY UPLC BEH-C18(2.1×100mm,1.7 μm);the mobile phase is 0.1% formic acid-acetonitrile solution(A);0.1% formic acid-water solution(B);gradient elution,column temperature: 45 ℃;injection volume 6 μl.The conditions of mass spectrometry were electrospray ionization ion source(ESI),and data were collected in positive and negative ion modes.(2)MRM monitoring chromatographic conditions: Liquid chromatographic conditions: Column: ACQUITY UPLC BEH C18(2.1×100 mm,1.7 μm)Mobile phase: 0.1% formic acid acetonitrile(A)-0.1% formic acid aqueous solution(B);gradient elution,flow rate : 0.4 m L·min-1;column temperature: 35 ℃;sample room temperature: 30 ℃;injection volume: 6 μL.2.Reproduction of kidney deficiency rat model caused by adenine and analysis of blood components:(1)Model reproduction: 6 male SD rats were placed in a breeding cage and adapted for 7 days to build a model.Adenine(150 mg·kg-1)was given by intragastric gavage every day for 4 weeks.(2)Model success criteria:Compared with the normal group,the contents of renal function indexes creatinine(CREA)and urea(UREA)increased significantly,and serum testosterone(T)and triiodothyronine(T3),thyroxine(T4),adrenocorticotropic hormone(ACTH),corticosterone(CORT)and other indicators decreased significantly.(3)UPLC-QTOF/MS liquid quality conditions: the column is Waters ACQUITY UPLC BEH-C18(2.1×100 mm,1.7 μm);the mobile phase is 0.1% formic acid-acetonitrile solution(A);0.1% formic acid-Aqueous solution(B);gradient elution,column temperature: 45 ℃;injection volume 6 μl.The conditions of mass spectrometry were electrospray ionization ion source(ESI),and data were collected in positive and negative ion modes.(4)MRM monitoring chromatographic conditions: Liquid chromatographic conditions:Column: ACQUITY UPLC BEH C18(2.1×100 mm,1.7 μm)Mobile phase: 0.1%formic acid acetonitrile(A)-0.1% formic acid aqueous solution(B);gradient elution,flow rate :0.4 m L·min-1;column temperature: 35 ℃;sample room temperature: 30 ℃;injection volume: 6 μL.3.Comparison method of blood components of Yougui Yin in normal rats and kidney-deficiency rats: Analyze the blood components of Yougui yin in normal rats and kidney-deficiency rats,and compare the similarities and differences of components determined by the reference substance or inferred from the literature between two groups.4.Network pharmacology method to screen the target and mechanism of Yougui Yin in improving various diseases of kidney deficiency syndrome: use the blood prototype components of Yougui Yin as the entry point to predicts the target point of the blood component through online databases,including sea,TCMSP,Swiss Target Prediction,and Pharm Mapper.Four online databases,including four online databases OMIM,GENECARDS,TTD,and CTD,were used to predict targets of nine disease related to kidney deficiency.The Venn diagram was used to determine the potential targets of Yougui yin in treating kidney deficiency-related diseases.On this basis,the string platform is used to analyze the protein-protein interaction between its compounds and disease targets;the cytoscape software is used for topological analysis to screen out key targets;the data is visualized,and the string online database is used for GO biological analysis and KEGG pathway analysis,to find out the relevant mechanism of Yougui yin in treating various diseases of kidney deficiency syndrome,and construct a network relationship diagram through cytoscape software.5.Network pharmacology method to predict the target and mechanism of Yougui Yin in improving kidney deficiency osteoporosis: use the blood prototype components of Yougui Yin as the entry point through four online databases,including sea,TCMSP,Swiss Target Prediction,and Pharm Mapper.Four online databases,the targets of kidney-deficiency osteoporosis disease were predicted through OMIM,GENECARDS,TTD,and CTD,and use the Venn diagram to determine the potential targets of Yougui yin in the treatment of kidney-deficiency osteoporosis.On this basis,the string platform is used to analyze the protein-protein interaction between its compounds and disease targets;the cytoscape software is used for topological analysis to screen out key targets;the data is visualized,and the string online database is used for GO biological analysis and KEGG pathway analysis predicts the main signal pathways for the treatment of kidney deficiency osteoporosis;the experiment finally uses cytoscape software to build a network relationship diagram.6.Serum pharmacology method to verify the target points and pathways of Yougui yin’s medicinal components in improving kidney deficiency osteoporosis:(1)The MTT method was used to detect the effect of Yougui yin-containing serum on the proliferation activity of bone marrow mesenchymal stem cells.(2)Alkaline phosphatase(ALP)and alizarin red staining methods were used to identify the effect of Yougui yin’s drug-containing serum on the osteogenic differentiation of BMSCs in normal rats and adenine-induced kidney deficiency rats.(3)Western Blot was used to detect the expression of PI3 K,AKT,AKT1,P-AKT,BAD,BCL-2 and BAX protein in the cell signaling pathway.Results1.The detection results of the blood components of Yougui Yin in normal rats:42 characteristic peaks were detected in the plasma of normal rats,13 chemical components were identified and inferred,and 29 metabolites were not identified.Six chemical components,including verbascoside,heterocohoside,cohodoside,pinoresinol diglucoside,loganin,and morroniside,were identified by multiple reaction monitoring(MRM)technology.2.The replication of adenine-induced kidney deficiency rat model and the test results of blood components:(1)The replication of the adenine-induced kidney deficiency rat model was successful;Rats in the control group were larger,full of energy and active.With shiny hair.In comparison with the control group,rats in the model group were thin,hunched and sluggish,with dull hair.Compared with the control group,the kidneys of rats in the model group were significantly larger,swollen,pale in color;the control group rats showed that the kidney tissue structure was intact,the renal tubules were tightly arranged,and the glomeruli were full and intact.In the model group,more vacuoles were seen in the renal tissues,the renal tubules were enlarged and brown adenine precipitates were seen in the tubes,and the renal tissues had obvious inflammatory cell infiltration.Compared with the control group rats,the contents of CREA,UREA,and T in the serum of model rats were significantly increased,and the contents of T3,T4,ACTH,and CORT in the serum were significantly decreased(all **P<0.01).(2)Detection results of blood components of Yougui yin in kidney deficiency model rats;22 characteristic peaks were detected in the plasma of model rats,12 chemical components were identified and detected,and 10 metabolites were not identified.Six components,including verbascum glycosides,isophylloside,cohodoside,pinoresinol diglucoside,loganin,and morroniside were identified by multiple reaction monitoring(MRM)technology.3.Comparison of blood components of Yougui Yin between normal rats and kidney-deficiency rats: UPLC-Q-TOF/MS technology was used to detect 42 characteristic peaks in the plasma of normal rats,and 13 chemical components were identified,including 6 alkaloids,2 flavonoids,2 triterpene saponins,1 iridoid,1phenylpropanoid,and 1 monoterpene.There are 22 characteristic peaks in the plasma of rats with kidney deficiency,and 12 chemical components are identified,including 2iridoids,6 alkaloids,2 flavonoids,monoterpenes,triterpene saponins Ingredients 1 each.Using MRM technology,verbascosides,isophylloside,cohodoside,pinoresinol diglucoside,loganin,and morroniside were detected in the plasma of normal group and kidney-deficiency rats.Studies have shown that there are 17 common prototype components in the plasma of normal rats and kidney-deficiency rats.They are verbasc glycosides,heterocymicidosides,cohodosides,and radix rehmanniae aglycones derived from Radix Rehmanniae,pinoresinol diglucoside and geniposide derived from Eucommia ulmoides,and loganin derived from Cornus officinalis,Morroniside,neoaconitine,benzoyl neoaconitine,benzoyl aconitine,benzoyl hypoaconitine,neoaconitine,and aconitine derived from aconite,Glycyrrhizin,isoliquiritin,and glycyrrhizic acid derived from licorice.31 metabolites of medicinal ingredients that were not in the plasma of the kidney-deficiency model rats caused by adenine were also detected in the plasma of normal rats,and 12 medicinal materials that were not in the plasma of the normal rats were also detected in the plasma of the kidney-deficiency model rats Metabolites of ingredients.4.Network pharmacology predicted the targets and pathways of the blood prototype components of Yougui Yin that improve 9 disease of kidney deficiency syndrome: 194 common targets and 168 related pathways have been screened out for the blood prototype components of Yougui Yin and 9 diseases of kidney deficiency syndrome.The blood prototype components of Yougui yin can improve the 9 diseases of kidney deficiency syndrome through AKT1,STAT3,VEGFA,HRAS,SRC and other targets.The main pathways for the prototype components of Yougui yin to improve the 9 diseases of kidney deficiency syndrome include PI3K/Akt,MAPK,osteoclast differentiation,HIF-1,VEGF and other signaling pathways.The interaction network diagram of "yougui Yin into the blood prototype component-target-9 diseases of kidney deficiency syndrome-pathway" was constructed.5.Network pharmacology predicted the targets and pathways of the blood prototype components of Yougui Yin that improve of kidney deficiency osteoporosis disease: The blood prototype components of Yougui Yin and kidney deficiency osteoporosis have screened out 84 common targets,131 related pathways.The blood prototype components of Yougui Yin can improve kidney-deficiency osteoporosis disease through STAT3,MAPK1,AKT1,VEGFA,SRC,TNF and other targets.The main pathways for the blood-based components of Yougui Yin to improve kidney-deficiency osteoporosis include PI3 K /Akt,MAPK,osteoclast differentiation,HIF-1,VEGF and other signaling pathways.The interaction network diagram of XII "yougui Yin into the blood prototype component-target-osteoporosis-pathway" was constructed.6.Yougui yin medicated serum has a therapeutic effect on kidney-deficiency osteoporosis:(1)The medicated serum of Yougui yin in normal rats and kidneydeficiency model rats have proliferative activity on BMSCs at 24 h,48 Both h and 72 h in a concentration range of 5%-20%.(2)The medicated serum of Yougui yin in normal rats and kidney-deficiency model rats can promote the osteogenic differentiation of bone marrow mesenchymal stem cells,increase the expression of osteoblast alkaline phosphatase(ALP)and the formation of mineralize calcium nodules.(3)The medicated serum of Yougui yin in normal rats and kidney-deficiency model rats up-regulated the expression of PI3 K,AKT,AKT1,P-AKT and BCL-2 protein in the PI3K/AKT pathway and down-regulated the protein expression of BAD and BAX expression.Conclusions1.A total of 16 prototype components of Yougui yin in normal rat plasma were identified through the reference substance,including 6 alkaloids,4 phenylpropanoids and their triterpene saponins,and 3 iridoids.Terpenoids,3 flavonoids,1 lignans.One flavonoid prototype compound,two flavonoid metabolites,and 29 metabolites were speculated from the literature.2.A total of 16 prototype components of Yougui yin in the plasma of rats with kidney deficiency caused by adenine were identified through the reference substance,including 6 alkaloids,4 phenylpropanoids and their triterpene saponins,3 One iridoid compound,three flavonoid compounds,and one lignan compound.One flavonoid prototype compound,one iridoid metabolite,and ten metabolites were speculated from the literature.3.A total of 16 identical prototype components in normal rats and kidneydeficiency rats were identified through the reference substance.They are verbascosides,heterocymicidosides,and cohodosides derived from Rehmannia glutinosa;they are derived from Eucommia ulmoides Pinoresinol diglucoside and geniposide acid;loganin and morroniside from cornus;neoaconitine,benzoyl neoaconitine and benzoyl aconitine from aconite Alkali,benzoyl hypoaconitine,neoaconitine,aconitine;glycyrrhizin,isoliquiritin and glycyrrhizic acid derived from licorice.These components are the main effective ingredients of various medicinal materials and may be used to treat diseases Medicinal substances.Through literature comparison,it was inferred that a same prototype ingredient was derived from Radix Rehmanniae Radix rehmanniae aglycone.In normal rats,two metabolites derived from licorice,phloretic acid and glycyrrhizin glucuronide,were speculated and 29 metabolites were not identified.In kidneydeficiency rats,it is speculated that one metabolite from Eucommia ulmoides genipin glucuronic acid is ring-opened and 10 metabolites have not been identified.4.Yougui yin can improve 9 diseases of kidney deficiency syndrome through multiple components,multiple targets,and multiple pathways.Its mechanism of action provides a scientific basis for further mechanism research.5.Yougui yin medicated serum can improve kidney deficiency osteoporosis through multiple components,multiple targets,and multiple pathways.The mechanism of action may be related to the activation of PI3K/AKT pathway to inhibit osteoblast apoptosis.