| Background:Prolonged application of supraphysiological glucocorticoids results in various side effects including inhibition of hypothalamus-pituitary- adrenal (HPA)axis, even eventually atrophy of adrenal. Sudden discontinuance of glucocorticoids causes adrenal insufficiency, which could be a latent menace to internal environment, even to life. So far, in order to avoid diseases recurrence and restore the inadequate adrenal function, dose-tapering of glucocorticoids is the only widely applied practice in clinics, but which takes at least several months even up to more than one year. Thus novel methods and drugs to relieve the inhibition of glucocorticoids on HPA axis and more quickly recover the adrenal function are an important mission for clinic medicine and still remained to be found. No effective drug to protect the adrenal function from inhibition of glucocorticoids has been discovered so far aboard. But according to our previous clinical and experimental studies, Kidney-tonifying complex prescription, Epimedium herb chosen from the Kidney-tonifying complex prescription and Epimedium flavonoids(EF) extracted from Epimedium herb can all protect HPA axis from inhibition of glucocorticoids.Recovery of adrenal function depends on steroidogenenic activity and number of adrenocortical cells. It has been reported that In the outer part of adrenocortical region stem cells exist, cell proliferation, migration inwards and differentiation of which constitute adrenal regeneration. Adrenocortical regeneration and another cellular biological process of apoptosis of adrenocortical cortex cells both regulate the number and function of adrenocortical cortex. Thus two objectives will be achieved in this study. Objectives:The effects of EF on (1) apoptosis of adrenocortical cells and (2) adrenocortical regeneration in rats with inhibition of HPA axis by corticosterone and respective corresponding mechanisms will be investigated. Materials and methods:Animals: 4 month-old male Sprague Dawley rats were employed,Drug preparation: EF was obtained from Korean epimedium herb raised in Liaoling province China. Animals were divided into three groups including normal, model and treatment group. Rats in model group were injected subcutaneously with corticosterone by dose of 5 mg/Kg per day for 14 days. Rats in treatment group were infused orally with EF by dose of 0.06 g/Kg per day 5 days before injection of corticosterone till the end of the experiment. Rats in normal group were administrated with the equal volume of distilled water. (1) Using competitive binding immunoassay, levels of plasma corticosterone of rats were detected to evaluate the protection of adrenocortical function by EF. (2) Adrenal weight and histology of adrenal were analysised to demonstrate the effect of prevention of atrophy of adrenal by EF. (3) Using two technologies to analysis cell apoptosis, Flow cytometry was employed to detect apoptosis ratio of isolated adrenocytes. Another is Terminal transferase mediated X-dUTP nick end labeling (TUNEL) assay, by which cell apoptosis in situ were demonstrated. (4) Two methods employed to cell proliferation, cell cycle distribution was analysised by flow cytometry. BrdU immunochemistry was used to demonstrate the cells in DNA synthesis in rats injected with BrdU one day before death. (5) Migration of BrdU-labeled cells were revealed by BrdU immunochemistry in rats injected with BrdU one day before injection of corticosterone. (6) Using DNA microarrays and real time quantitative PCR, the underlying molecular mechanisms of EF on the cellular process above were investigated. Results:(1) Plasma corticosterone Plasma corticosterone in nomal, model and treatment group were 96.03±30.95ng/ml, 30.72±21.73ng/ml, 53.79±25.63 ng/ml respectively. When compared to that in normal group, plasma corticosterone in model group decreased significantly (P<0.01). But compared to that in model group, the plasma corticosterone in treatment group increased significantly (P<0.05). Results showed a protection role for adrenocortex function in corticosterone-treated rats.(2) Arenal weight The adrenal weight in nomal, model and treatment group is 49.63±6.00, 28.88±9.45, 34.97±4.84 mg respectively. Adrenal weight of rats in model group decreased signicantly compared with that of rats in nomal group (p<0.01), and that of rats in treatment group increased significantly compared with that of rats in moedel group (p<0.05). These results showed a protection of adrenal from atrophy.(3) Cell apoptosis Flow cytometry analysis revealed that apoptosis ratio in nomal, model and treatment group was 7.91±6.063%, 14.42±4.851%, 8.62±5.59% respectively. Compared to that of the normal group, the ratio signicantly increased in model group (p<0.05), and compared to the model group, the ratio in treatment group decreased signicantly (p<0.05). TUNEL showed mean apoptosis cell number in each slide was 4.67±1.53, 70.67±9.29, 18.67±7.64 respectively. The number increased signicantly (p<0.05) in model group compared that in nomal group, and decreased signicantly (p<0.05) in treatment group compared to that in the model group. The data revealed EF can relieve the excessive cell apoptosis induced by corticosterone of adrenocortical cells.(4) Cell proliferation It was demonstrated by flow cytometry that cell cycle distribution of adrenocytes from rats in nomal, model and treatment group in phase G0/G1 was 76.36±1.93%, 93.40±1.70%, 85.70±2.62 respectively, when compared to that of normal group, the percentage in model group increased significantly (p<0.01), and compared to that of model group, the percentage in treatment group decreased significantly ((p<0.01). While the proportion of cells in G2/M phase in nomal, model and treatment group was 20.28±1.50%, 2.86±1.73%, 9.53±5.15% respectively, when compared to that of normal group, the percentage in model group decreased significantly (p<0.01), and compared to that of model group, the percentage in treatment group increased significantly ((p<0.01). Data from two methods showed EF can promote cell proliferation occurring in the outer part region of the adrenocortex.(5) Migration of BrdU-labeled cells Cells in proliferation can be labeled by injection of BrdU one day before starting injection of corticosterone, which can migrate inwards as time goes. Thus we can compare the migration speed among groups 14 days later. We found BrdU positive staining cells were all in zona glomerulosa in normal group and in model group, but in treatment group, most BrdU-labeled cells were in zona fasculata and zona reticularis. These revealed EF can accelerate migration of labeled cells.(6) The underlying molecular mechanisms of cytocycle-promoting, apoptosis-relieveing and steroid secretion-promoting action of EF Using Affymetrix Ltd.Co(USA) gene chip Rat expression 230 2.0, more than 2-fold up- or down-regulation of gene expression was seclected to analysis further. Results demonstrated that 29 genes were up-regulated while 785 down-regulated whencompared to that in model group. Three of them are apoptosis-related including AMP-activated protein kinase alpha 1 catalytic subunit, inhibitor of apoptosis protein 1, baculoviral IAP repeat-containing 4, which are all of anti-apoptosis activity. There are 6 genes associated with cell cycle among them, which all promote cell cycle including neuroblastoma RAS viral (v-ras) oncogene homolog, nuclear factor I/A,forkhead box ,v-jun sarcoma virus 17 oncogene homolog (avian), fibroblast growth factor 7, interleukin 1 alpha and insulin-like growth factor II. While 237 genes were up-regulated and 122 down-regulated after treatment with EF. 6 of 237 genes of upregulation is cytocycle-related, including mismatch repair protein, ret proto-oncogene, insulin-like growth factor II, v-jun sarcoma virus 17 oncogene homolog (avian), fibroblast growth factor 7, prostaglandin- endoperoxide synthase 2, which all promote cell cycle. Two of 237 genes of upregulation is apoptosis-related which has activity of anti-apoptolsis, and 7 of them were steroid sythesis related genes including mevalonate kinase, mevalonate pyrophosphate decarboxylase, farnesyl diphosphate farnesyl transferase 1 , isopentenyl-diphosphate delta isomerase, 2,3-oxidosqualene anosterol cyclase, cytochrome P450 subfamily 51, farensyl diphosphate synthase. These results are well cinsistent with the action of anti-apoptosis of EF at cellular level, also suggest that EF can promote the adrenocortex regeration due to proliferation and differentiation-promoting action of EF. According to reports, IGF II and FGF7 play important role in all the proliferation, differentiation and steroid production, and act through binding to membrane receptor, thus can be regarded as upstream factor involved in regeneration. We further analysised IGF II and FGF7 mRNA expression in vitro using real time quantitative PCR. Results showed that after coculture with adrenocytes from nomal rats, IGF II transcripts were increased by EF (p<0.05). FGF7 expression was no change after treatment with EF (p>0.05). So both IGF II and FGF7 mediate the regeneration-promoting action of EF, but IGF- II can be transactivated directly by EF and perhaps more important. Conlusions:EF has clear and dominant effects on protection of adrenocortex function in HPA-inhibited rats by two discrete cellular mechanisms below.(1) EF can relieve excessive apoptosis induce by corticosterone in rats with inhibited HPA axis function.(2) EF can promote adrnocortex regeneration in rats with inhibited HPA axisfunction by exogenous corticosterone. According to a huge amount of reports, BrdU-labeled cells can be regarded as stem cells. By the essentially same method, we discovered the BrdU-labeled cells proliferated more actively, more cells returned to cell cycle, migrate more quickly and enzymes that catalyse steroid production were up-regulated. Baed on these evidences, we conclude intervention of proliferation, migration, differentiation by EF results in active adrenocortex regeneration, in which two important growth factors IGF II and FGF7 mediate the actions of EF.It is an important question of relieving the inhibition of HPA axis by glucocorticoids. According to our series of studies, EF has clear efficacy due to apoptosis-reducing and regeration-promoting mechanisms, consequence of which is to reconstruct adrenocortex function in essence.
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