Exogenous Glucocorticoid-induced Apoptosis In Rat Anterior Pituitary Cells And The Intervention Of Traditional Chinese Medicine

Background and ObjectGlucocorticoids (GC) is a kind of adrenal cortex hormones synthesized and secreted by fascicular zone, and it has physiological significance. At present, GC has been widely used clinically, and has anti-inflammatory, anti-allergic, anti-shock, immune suppression and other pharmacological effects. But long-term GC systemic application will cause a large number of serious adverse reactions, such as infection (viruses, bacteria, fungi, etc.), gastrointestinal ulceration or perforation, adrenocortical hyperfunction or insufficiency, electrolyte disturbance, osteoporosis or ischemic bone necrosis, as well as neuropsychiatric impact, but also aggravate the existing diabetes, high blood pressure. Inappropriate withdrawal or reduction of GC can also cause disease to rebound. The current consensus:release of GC regulated by the hypothalamus-pituitary-adrenal axis (HPAA), it has a strong negative feedback action on the neuroendocrine loop, mainly on anterior pituitary adrenocorticotropic hormone (ACTH) cell except for the hypothalamus and hippocampus structures. The corticotrophin releasing hormone (CRH) secreted by hypothalamic paraventricular nucleus can stimulate this cells to release ACTH, which is the main stimulus for GC. If any dose of GC inhibited the secretion of CRH and ACTH, and such a suppression has not been restored in the period of treatment, it will cause lack of central adrenal gland hormone and shrinkage of the peripheral adrenal. GC withdrawal after long-term treatment can reduce the HPAA reaction to endogenous or exogenous stimuli both in people and mice. This functional change reflects in the various levels of organs on the HPAA. Some people given rats dexamethasone sustainedly on 2 weeks after adrenalectomy, which resulted in very significant apoptotic activity in anterior pituitary cells, meanwhile mitotic activity was inhibited. While it is impossible to know what kind of pituitary cells decreased. but the total pituitary cells has been suggested that reduced to a level below the base at two weeks ago. LA Nolan believed GC withdrawal is accompanied by short-or long-term secretion damage of HPAA capacity. This anterior pituitary reaction maybe caused by cell-mediated apoptosis, damaged cells restoration, undifferentiated precursor cell differentiation and transformation of other types of secretory cells. This indicated that if there is sufficient time, full restoration of anterior pituitary cells is possible.Traditional Chinese medicine (TCM) believes that "kidney" function related to the pituitary-adrenal cortex system. HPAA is a research focus on the nature of kidney of combination of Chinese and Western medicine. Clinical use of TCM of tonifying kidney to treat kidney deficiency can regulate yin and yang, improve the symptoms and return to be healthy. TCM of tonifying kidney combined with the GC can reduce or eliminate the morphological changes of the brain anterior pituitary cells induced by long-term application of GC. At home and abroad, the nature of kidney deficiency and mechanism of TCM of tonifying have been made progress in many aspects in recent years, which concerned extensively the endocrine system, immune system, basic metabolism and so on. The purpose of TCM treatment based on syndrome differentiation is to begin to adjust the balance of kidney yin and yang, so that the HPAA function can be resumed. The Yougui Pill created by Zhang Jingyue is a typical "nourishing yin and tonifying yang" representative, which use a large number of tonifying yang tonic drugs to cure decline of vital gate fire, and use a small amount of warm yang and promote qi transformation drugs directly to warm and supplement kidney yang. Luo Hanwen et al. reported, Yougui Pill can increase the blood concentrations of cortisone, and the number of pituitary ACTH cells were more than control group.Therefore, based on the above theories, we choice Yougui Pill as protective drugs to cure HPAA disfunction experiment induced by GC, and explore its mechanism that may be related to reducing apoptosis of anterior pituitary cells.MethodsGiven rat exogenous glucocorticoid to build deficiency of kidney yang model, that is, animal model of glucocorticoid side effects, used as experiments in vivo, and primary cultured anterior pituitary cells used as experiments in vitro; in accordance with the principles of randomized controlled to group; application of Immunohistochemistry staining, TUNEL staining, Real-Time qPCR and flow cytometry to detect apoptosis in anterior pituitary cells in each group and the expression of apoptosis-related proteins, as well as changes in mitochondrial membrane potential; variance analysis to compare the differences in each group and draw the conclusion.ResultsIn vivo and in vitro experiments, the number of apoptotic cells in dexamethasone plus Yougui pill group was less than that in dexamethasone group (P＜0.05); apoptosis promoting protein and its mRNA expressions in dexamethasone plus Yougui pill group were significantly less than dexamethasone group (P＜0.05), while the anti-apoptotic protein and its mRNA expressions in dexamethasone plus Yougui pill group were significantly hihger than dexamethasone group(P＜0.05); the number of mitochondrial membrane potential decline cells in dexamethasone plus Yougui pill group was significantly less than dexamethasone group (P＜0.05).ConclusionThrough the results of this study combined with the current theoretical basis, we can conclude:Traditional Chinese medicine Yougui pill can reduce the glucocorticoid-induced apoptosis in rat anterior pituitary cells. Its mechanism may be involved in the mitochondrial pathway of apoptosis, positive regulation of apoptosis inhibitory protein and reverse effect on pro-apoptotic protein. This study provides a theoretical basis for using Yougui pill to treat the suppression of HPAA caused by exogenous glucocorticoid.