Study On The Mechanism Of The Commonly Used Chinese Medicine For Kidney Tonification Promoting The Secretion Of Corticosterone By Adrenal Y1 Cell

ObjectiveCompare the effects of different kidney-tonifying of Chinese medicines on Y1 cells,a type of adrenal cortex cell,and how Chinese medicines regulate the mechanism on the synthesis and secretion of corticosterone hormone.Methods1.The effect of commonly used kidney-tonifying Chinese medicines on the secretion of corticosterone in adrenal Y1 cells: MTT essay was used to detect the safe dose of each Chinese medicine extract on Y1 cells;use q PCR to detect the genes expression of Star,Cyp11a1,Cyp21a1,Cyp11b1,Hsd3b1 which play an important role in the process of steroid hormone synthesis;use Western blot to detect the expression of St AR,CYP11A1,3βHSD2,CYP11B1 and other genes which in the process of steroid hormone synthesis on protein level;ELISA method was used to detect the effect of different Chinese medicine extracts on the secretion of corticosterone.2.Establishment of Y1 cell model of drug-induced kidney deficiency syndrome and research on the efficacy of Epimedium and related Chinese medicines.On the basis of the above studies,hydrocortisone,H89,and mifepristone were selected to construct a Y1 cell model of kidney deficiency syndrome;48 hours after the intervention of the Y1 cell model with kidney-tonifying Chinese medicines and compound prescriptions,the MTT essay was used to detect the proliferation of Y1 cells;use q PCR to detect Star,Cyp11a1,Cyp21a1,Cyp11b1,Hsd3b1,Hsd3b2 and other m RNA expression;use Western blot to detect the protein expression of St AR,CYP11A1,CYP11B1,3β-HSD1,3β-HSD2;use ELISA essay to detect the content of corticosterone in cell culture fluid.3.Study on the comparison of the pharmacodynamics of the main components of the traditional Chinese medicine Epimedium and the effect of icariin on the gene expression of Y1 cells in the model of kidney deficiency syndrome.On the basis of the above research,CCK8 technology was used to detect the effect of icariin on cytotoxicity and proliferation in Y1 cells;cell apoptosis and cell cycle changes were detected by flow cytometry;use ELISA essay to detect corticosterone content in cell culture fluid;RTq PCR technology was used to verify the expression of important genes in the process of steroid hormone synthesis,such as Star,Cyp11a1,Cyp21a1,Cyp11b1,Hsd3b1,etc;Western blot was used to detect the expression of steroid hormone synthase proteins St AR,CYP11A1,3βHSD2,CYP11B1,etc;RNA-seq technology was used to detect the wide-ranging gene differential expression effect of icariin on Y1 cells and preliminary data analysis;based on the RNA-seq results,RNA interference technology was used to detect that icariin up-regulated Adcyap1 may have an effect on the important genes in the process of steroid hormone synthesis.Results1.The effect of extracts of Epimedium,Curculio,Phellodendron amurense and Radix Rehmanniae on the expression of steroid hormone synthesis genes in Y1 cells:Epimedium significantly promotes the expression of Star;Curculio vulgaris promotes the expression of Cyp11a1;Habitat promotes the expression of Hsd3b1,Hsd3b2 and Cyp11b1;Phellodendron amurense has a significant inhibitory effect on the proliferation of Y1 cells,and has an inhibitory trend on the expression of Hsd3b1 and other genes;Epimedium promotes the expression of St AR on protein level;Curculio and Habitat have no effect on St AR,3βHSD2,CYP11B1 and other proteins.Obviously,Phellodendron amurense inhibits the expression of proteins such as St AR,3βHSD2,and CYP11B1;Epimedium promotes the synthesis of corticosterone in Y1 cells;Habitat and Curculigo also have a certain promoting effect,while Phellodendron amurense inhibits the synthesis of corticosterone in Y1 cells.2.The combined application of 10μM hydrocortisone and 5μM H89 can simulate the ACTH inhibition induced by high-dose hydrocortisone in the clinic;the combined application of 10μM mifepristone and 5μM H89 can simulate the ACTH inhibition induced by high-dose hydrocortisone in the clinic and the phenomenon of low levels of glucocorticoids after ACTH suppression and withdrawal.Among the tested traditional Chinese medicines,Epimedium and Phellodendron chinense have outstanding pharmacodynamic effects on the expression of steroid hormone synthesis genes in Y1 model cells.Epimedium promotes the expression of Star in this model cell,and Phellodendron chinense inhibits the m RNA expression of Star and protein expression of St AR,3βHSD1,3βHSD2 and other proteins.3.Icariin has no cytotoxic effect on Y1 cells within a large dose range in the process of cell proliferation,apoptosis and cell cycle.Icariin has the ability to promote the expression of related genes and proteins during the synthesis of steroid hormones.Icariin also promotes the synthesis of corticosterone hormone.The effect is similar to that of Epimedium.RNA seq found that icariin has a regulatory effect on multi-gene transcription in Y1 cells,while the up-regulated Adcyap1 has no significant effect on the expression of Star.Conclusions1.As a commonly used kidney-tonifying Chinese medicine,Epimedium can promote the expression of St AR gene in Y1 cells,and promote the secretion of corticosterone;Curculio has a similar effect to Epimedium,but not as strong as Epimedium;Morinda officinalis,Tusizi,Jinkuishenqi Pills,Liuwei Dihuang Pills,Dihuang,and Zhimu have no effect on the expression of St AR genes,suggesting that the kidney-tonifying effect of these traditional Chinese medicines and compound prescriptions is achieved through other ways.At the same time,Phellodendron chinense can inhibit the proliferation of Y1 cells and the secretion of corticosterone.2.In this study,a drug-induced kidney deficiency syndrome model was established in Y1 cells.Epimedium could promote the expression of star gene in the drug-induced model and promote the secretion of corticosterone.3.Icariin is a representative medicinal substance of Epimedium.It can regulate the expression of a variety of genes in adrenal cells.Changes in the expression of these genes may directly or indirectly promote the expression of St AR and promote the synthesis of steroid hormones.