Spatial Memory Impaired In Ovariectomized Rats Correlated To ERK1/2 Pathway Activity And The Related Effect Of Polygonum Multiflorum Thunb

Objective: Estrogen is the necessary endogenous active compound to maintain the normal physiological functions of women. In central nervous system, estrogen not only acts on genital circuit to affect sex hormone cycle, but also act on the circuit related with cognition (hippocampal circuit) to affect learning and memory. Hippocampus is important encephalic region related with various functions, such as learning and memory,emotion,behavior, and estrogen receptorαandβare both distributed in hippocampus, so it's also the main target of estrogen. Many animal experiment studies demonstrated that estrogen had neuroprotective effect and could promote learning and memory, but the mechanisms of these actions are very complicated.Studies indicated that postmenopausal women adopted estrogen replacement therapy (ERT) could delay and improve cognitive impairment. But ERT increased the attack rate of breast cancer and endometrial cancer. Thus, it is imperative to find a substitutive drug that could prevent cognitive impairment in post-menopause and also avoid those adverse reactions. Traditional Chinese medicine holds the theory that learning and memory have a close relation with pneuma of kidney. Polygonum multiflorum thunb is a traditional Chinese herb that has the efficacy of invigorating the kidney. Polygonum multiflorum thunb has extensive pharmacologic actions. Besides the effect on hyperlipemia,angiosclerosis,immune function,senescence, it also could improve learning and memory. But the pharmacologic mechanisms remain unclear.Extracellular regulated protein kinase1/2 (ERK1/2) pathway belongs to Mitogen-activated protein kinases (MAPK) signal transduction system. It is one of the most important signal pathway in this system. The studies in recent years showed that ERK1/2 pathway activity involved in long-term potentiation (LTP) and formation of memory. Large amount of ERK1/2 phosphorylated (p-ERK1/2) represent the activation of this pathway, then p-ERK1/2 act on many downstream substrates to conduct extracellular signal into nucleus. Raf kinase inhibitor protein (RKIP) is the endogenous inhibitor of MAPK signal transduction pathway. Its regulatory mechanism is to interact with Raf-1 kinase domain and inhibit p-ERK1/2 to affect the ERK1/2 pathway activity. Neuron cell culture in vitro showed that estrogen could cause phosphorylation of ERK1/2 by non-classics signal pathway. But in vivo animal experiment, it is unknown that whether defect of estrogen affect ERK1/2 pathway activity and RKIP expression.In this experiment, we selected the ovariectomized rats as the object to observe the effect of estrogen on learning and memory and on ERK1/2 pathway activity and RKIP expression to approaching their correlation. At the same time, to observe the related effect of polygonum multiflorum thunb.Methods1 Animals and groups30 female SD rats aged 4 months were randomly divided into 3 groups: sham operated group (Sham group), ovariectomized group (OVX group), ovariectomized and polygonum multiflorum thunb supplied group (OVX+He group). Sham group was ablated some adipose tissue above ovaries; OVX group was ablated two sides ovaries; OVX+He group was supplied water apozem of polygonum multiflorum thunb (2g/kg.d) after 2 weeks of ovariectomy. Postoperative rats were fed by routine method and then sacrificed 4 months later.2 Behavioral training and testThe ability of spatial learning and memory were measured by Morris water maze before the rats killed. The training in first three days was adapted. Then rats in each group were randomly divided into training group (n=5) and untraining group (n=5). Behavioral test was formally carried out in the fourth day. Training group was gave six consecutive times tests of hidden platform and the average time of escape latencies in the last three times was the index of spatial learning and memory in rats. Rats of untraining group swim 1 minute in pool without platform.3 Tissues and data determiningAfter behavioral test, rats were killed, and dorsal hippocampus was quickly separated and deepfreezed in liquid Nitrogen. Then tissues were stored at -70℃to extract protein.3.1 protein abstraction and quantitationThe protein of dorsal hippocampus was extracted. Protein quantity was measured by Bradford, expressed in mg/ml.3.2 Interest protein determiningThe relative protein content of p-ERK1/2 and RKIP were measured by western-blotting using ERK1/2 andβ-actin as inner standards respectively.Results1 The ability of spatial learning and memory of rats Through six times tests, the escape latency of each training group showed a trend of getting shorter. The average time of the last three escape latency showd that the escape latency of OVX group(26.798±6.215) was longer than Sham group(15.726±4.001) (P<0.05). It suggested that estrogen defect led to the impairment of learning and memory; There was no difference between OVX+He group (17.972±7.558) and Sham group(15.726±4.001) (P>0.05), suggested that polygonum multiflorum thunb could improve the impairment of learning and memory induced by estrogen defect.2 Activity of ERK1/2 pathway in hippocampus2.1 The relative level of p-ERK1/2 protein in the taining rats of Sham,OVX and OVX+HE group(Sham 0.696±0.074;OVX 0.442±0.054;OVX+HE 0.502±0.061) was higher than untraining rats(Sham 0.51±0.09;OVX 0.285±0.081;OVX+HE 0.394±0.067) (P<0.05), indicated the phosphorylation of ERK1/2 increased. It suggested that activation of ERK1/2 pathway in CA1/CA2 region of hippocampus involved in the formation of spatial memory.2.2 Compared the untraining rats in each group, the relative level of p-ERK1/2 protein in OVX group(0.285±0.081) was lower than Sham group(0.51±0.09) (P<0.05), indicated the basic activity of ERK1/2 pathway decreased after ovariectomy. Meanwhile, compared the training rats in each group, the relative level of p-ERK1/2 protein in OVX group(0.442±0.054) was lower than Sham group(0.696±0.074) (P<0.05), indicated estrogen defect after ovariectomy led to the decrease of the induced activity of ERK1/2 pathway induced by behavioral training (P<0.05). It suggested that estrogen could directly or indirectly affect ERK1/2 pathway activity.The relative level of p-ERK1/2 protein in training rats or untraining rats has no difference between OVX+He group and OVX group, indicated that polygonum multiflorum thunb had no effect on ERK1/2 pathway activity. It suggested that the positive effect of polygonum multiflorum thunb on learning and memory in ovariectomized rats was not through the ERK1/2 pathway.3 Expression of RKIP protein in hippocampusCompared with Sham group(0.461±0.067), the relative expression of RKIP(0.702±0.084) was higher than OVX group (P<0.05), indicated estrogen defect after ovariectomy led to the increase of RKIP expression. It suggested that estrogen maybe directly or indirectly regulated RKIP expression in CA1/CA2 region of hippocampus. But there was no difference between OVX+He group (0.662±0.078) and OVX group(0.702±0.084), indicated polygonum multiflorum thunb had no effect on RKIP expression in ovariectomized ratsConclusion1 Estrogen defect causes impairment of spatial learning and memory in ovariectomized rats.2 Both the basic phosphorylation level and the induced phosphorylation level of ERK1/2 in CA1/CA2 region of hippocampus are lower, but the expression of RKIP, the inhibitor of this pathway, is increased. The effect of the two aspects is one of the molecule mechanisms of spatial learning and memory impairment in ovariectomized rats.3 Polygonum multiflorum thunb improves the impairment of spatial learning and memory in ovariectomized rats. But it has no effect on basic phosphorylation and induced phosphorylation of ERK1/2 as well as the expression of RKIP protein. It indicates that the effect of polygonum multiflorum thunb on spatial learning and memory is not through the ERK1/2 signal pathway.