The Experimental Study Of Acupuncture Combined With Epimedium Pubescens Flavonoids On The Corticosterone-Induced Damages Of Hippocampus And Associated Mechanisms In Rats

Stress activates the hypothalamic-pituitary-adrenal axis, ultimately causing the release of glucocorticoids from the adrenal cortex. These hormones, cortisol and corticosterone (CORT), function to maintain or restore homeostasis through energy mobilization, and regulation of immune responses to infection. Glucocorticoids target tissues in sense organs, internal organs, and in the peripheral and central nervous system. Within the central nervous system, the hippocampus contains a high concentration of glucocorticoid receptors and the activity of the hypothalamic-pituitary-adrenal (HPA) axis is regulated by the hippocampus via mediation of glucocorticoid receptors, and conversely, the activity of hippocampal neurons is greatly influenced by glucocorticoids. A large body of evidence indicates that glucocorticoids regulate the death of neurons by apoptosis in the dentate gyrus. The removal of circulating glucocorticoids by adrenalectomy selectively induces apoptosis in neurons of the dentate granule cell layer, which can be prevented by replacement with adrenal steroids. Prolonged exposure to glucocorticoids also has been shown to damage neurons in the hippocampus.Deficiency of kidney-Yang is a syndrome of Traditional Chinese Medicine (TCM). When kidney-Yang is weakened, there will be deficiency-cold manifestations, such as lassitude and coldness in the loins and knees, intolerance of cold, listlessness, diarrhea at dawn, impotence or sterility. Meanwhile, kidney-Yang deficiency can also cause symptoms due to disturbance of body fluid metabolism, i. e., oliguria, anuresis, frequent urination, enuresis, or edema. A large body of evidence indicates that disease of kidney-Yang deficiency is characterized by increasing level of glucocorticoids and dysfunction of hypothalamic-pituitary-adrenal axis (HPA). The epimedium pubescens flavonoids (EF) has effects on cardiovascular system , circulatory system , immune system , genital system and bone marrow system. Traditionally, acupuncture therapy has been applied in the treatment of various diseases in Oriental medicine. However, acupuncture been applied with great success to treatment syndrome of kidney-Yang deficiency in TCM. Studies have shown that electroacupuncture may be helpful for women sterility which TCM diagnosis was kidney-Yang deficiency. Also, other studies have reported that electroacupuncture has effect on diarrhea as a consequence of kidney-Yang deficiency. In vivo, to study the effects of electroacupuncture (EA) combined with epimedium pubescens flavonoids (EF) on the corticosterone-induced damages of hippocampus in rats. An experimental model of HPA axis inhibited by corticosterone was established to observe the changes ofrelevant of hippocampus. In vitro, considering the potent neuroprotective action of astrocytes and the deleterious effects of glucocorticoids particularly on hippocampal neurons, we were going to address the culture conditions that suppress apoptosis against CORT in the present study. And if so, to further investigate molecular mechanisms involved in CORT-induced apoptotic cell death in primary hippocampal cultured neurons, we would examine activation of two signal pathways, i.e. extracellular signal-regulated kinases 1 and 2 (ERK1/2) and phosphoinositide 3-kinase (PI3-K)/Akt signal pathways. Both signal pathways are known to be important in mediating neuronal apoptosis.In vivo, experiments were performed on adult male Sprague-Dawley rats (Experimental Animal Center, Shanghai Medical College of Fudan University, China) weighing 200 220g. Prior to experimental manipulation, rats were allowed to acclimate for 1 week and maintained on a 12h light/12 h dark cycle with free access to food and water. All rats in the study were used strictly in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals in order to minimize the number of animals used and their suffering. Five groups of animals were considered: control (Cont), corticosterone-treated (Cort), corticosterone + EA-treated (EA), corticosterone + EF-treated (EF) and corticosterone + EF + EA-treated (EF+EA). The corticosterone treatment group received a daily subcutaneous injection of corticosterone 10 mg/kg in 0.2 ml sesame oil. The subcutaneous injections were performed every day at 18:00 19:00 p.m. for 15 days. EF was given by intra-gastric perfusion at the same time. "Shenshu" and "Guanyuan" points were chosen. While the normal group rats received a daily subcutaneous injection of sesame oil. The results are as follows:1. Effects of Acupuncture Combined with Epimedium Pubescens Flavonoids on the Corticosterone-Induced Inhibition of HPA in RatsCompared with normal group, weight of pituitary, adrenal and thymus decreased. The levels of ACTH in plasma and CRH in hypothalamus decreased. Both EA and EA plus intra-gastric administration of EF could markedly increased levels of ACTH in plasma and CRH in hypothalamus; and the effects of EA plus intra-gastric administration of EF were better than those of EA alone (P<0.05) .2. Changes of expression of GFAP in hippocampus after different treatments.Expression of GFAP in hippocampus of the rats was examined by western blot analysis and immunohistochemistry. The results showed that expression of GFAP in hippocampus decreased after corticosterone but enhanced by EF or EA treatment (P<0.05). However, the expression of GFAP was significantly increased in the rats treated by EA and EF(P<0.05). RT-PCR was performed to determine the change of GFAP mRNA levels in hippocampus after different treatment. Similar to the change of GFAP protein, GFAP mRNA level in hippocampus decreased after CORT treatment, but EA or EF treatment resulted in a significantly increase with mRNA level (P<0.05). However, EF enhanced the effects of EA on the GFAP mRNA level in the hippocampus after corticosterone treatment.3. Effects of different treatments on Changes of expression of NGF in hippocampus Immunohistochemistry, western blot and RT-PCR were used to determine theChanges of expression of NGF in hippocampus after different treatments. The results showed that the expression of NGF was significantly decreased by corticosterone treatment, but the expression of NGF was enhanced by EF or EA treatment (P<0.05). However, the expression of NGF was significantly increased in the rats treated by EA and EF(P<0.05). RT-PCR was performed to determine the change of NGF mRNA levels in hippocampus after different treatment. Similar to the change of NGF protein, NGF mRNA level in hippocampus decreased after CORT treatment, but EA or EF treatment resulted in a significantly increase with mRNA level (P<0.05). However, EF enhanced the effects of EA on the NGF mRNA level in the hippocampus after corticosterone treatment (P<0.05).4. Effects of different treatments on TUNEL positive cells in the dentate gyrus of hippocampus.Effects of corticosterone and substitutive treatments on the changes of TUNEL-positive cells within the hippocampus. TUNEL labeling demonstrated that corticosterone administration induced an increase in the number of TUNEL-positive cells. EA or EF treatment dramatically reduced TUNEL-positive cells. The number of TUNEL-positive cells in the hippocampus were very few in the EA+EF group rats.5. Effects of different treatments on cell proliferation in the subgranular layer of the dentate gyrus.The effects of different treatments on cell proliferation were determined by comparing the numerical density of BrdU-labeled nuclei detected in the hippocampus of control rats with that detected in the same regions after different treatments. BrdU labeling demonstrated that corticosterone administration reduced the number of BrdU-positive cells, which was increased by EA or EF treatment. The number of the positive cells in the EA+EF group rats was more than EA or EF.6. Effects of different treatments on the expression of polysialylated neural cell adhesion molecule in the hippocampus.The relationships between PSA-NCAM, neurogenesis and synaptic plasticity prompted us to test the possibility that the corticosterone-induced changes in hippocampal cell proliferation could be accompanied by alterations of PSA-NCAM expression. PSA-NCAM immunostaining was associated with cell bodies located in the subgranular layer of the DG, dendritic processes extending throughout the granule cell layer and axonal fibers projecting throughout the mossy fiber region. PSA-NCAM labeling demonstrated that corticosterone administration reduced the number of PSA-NCAM-positive cells, which was prevented by treatment with the EA or EF. There was even greater PSA-NCAM-positive cells in the hippocampus of EA+EF rats.In vitro, we found that astrocyte-conditioned medium (ACM) protects neurons of primary hippocampal cultures against the apoptotic effect of corticosterone (CORT). CORT added for 24 hours to primary cultures of rat hippocampus resulted in significant apoptosis. Hippocampal neurons were grown for 10 days, initially in regular medium for 4 days, and then in ACM for an additional 6 days before adding CORT for 24 hours. Apoptosis was measured by TUNEL staining. CORT-induced apoptosis was suppressed to hippocampal neurons cultured in ACM. To delineate themolecular basis underlying the neuroprotection of ACM, we assessed the activation of two signal pathways, namely, the extracellular signal-regulated kinases 1/2 (ERK1/2) and phosphoinositide 3-kinase (PI3-K)/Akt ones in response to CORT insult and subsequent neuronal apoptosis. Western blot revealed that CORT significantly increased the phosphorylation of ERK1/2 and PI3-K/Akt in hippocampal neurons grown in Neurobasal medium supplemented with B27 and 500 μm L-glutamine (NBM~+). Interestingly, the increase of phospho-ERK1/2 and Akt levels were much pronounced and the time course of phosphorlytion was altered with ACM treatment, suggesting that both signaling ways may participate in ACM protection. Furthermore, we detected that LY294002, a selective inhibitor of Akt, rather than U0126, an ERK1/2 inhibitor, blocked the anti-apoptotic effect against CORT in ACM-cultured hippocampal neurons. In sum, our data show that astrocyte-conditioned medium has potent anti-apoptotic effect in cultured hippocamal neurons. The protective actions against the neurotoxic CORT insult may involve PI3-K/Akt signal pathway, but not ERK1/2.The present study suggested:1. EA or EF has the effects of normalizing the dysfunction of hypothalamus-pituitary- adrenal axis.2. EA or EF treatment resulted in significantly decreased expression of GFAP andNGF in the hippocampus of corticosterone-treated rats. The results suggesting that EA and EF may play a normalizing role and possesses the effects such as the promotion of homeostasis of hippocampus.3. EA or EF has the effects of normalizing the hippocampal structural plastic changes induced by prolonged corticosterone treatment.4. The combination of EA and EF produces the synergic effects in normalizing thedysfunction of hypothalamus-pituitary- adrenal axis and hippocampal induced by prolonged corticosterone treatment.5. Both ERK1/2 and (PI3-K)/Akt signal pathways were activated in hippocampalneurons against CORT under ACM-cultured medium. (PI3-K)/Akt pathway, but not ERK1/2 is involved in enhance resistance against neurotoxin of CORT in hippocampal neurons grown in ACM. The neuroprotective effect of (PI3-K)/Akt pathway is probably mediated by promoting Bcl-2 expression.