Abstract: Objective: A large number of clinical and epidemiological data showed that decreased estrogen levels in women during menopause may induce Alzheimer's disease (AD). Estrogen can Protect neurons by increasing neuronal activity and reducing Aβ aggregation in a variety of ways. However, clinical trial data of postmenopausal women showed that estrogen therapy has many adverse reactions, which limits its clinical application. Phytoestrogens, similar to the chemical structure of estrogens, are known as selective estrogen receptor modulators. And they have estrogen-like neuroprotective effects while reducing the adverse reactions of estrogen replacement therapy. Studies have shown that Quercetin (Que), as a phytoestrogens, has a protective effect on cerebral ischemia-reperfusion, and neuronal damage induced by β-amyloid and heavy metal. Our previous study also confirmed that Que can exhibit estrogen-like effects by binding to estrogen alpha receptors and promote the proliferation of human breast cancer MCF-7 cell line. This study continues the previous works and deeply explore the protective effects of Que estrogen-like effects on brain neurons and its mechanisms. Methods: Primary cultured rat hippocampal neurons, cortical neurons, and PC12 cells treated by Aβ25-35 were used to observe the effects of different concentrations of Que for 24 and 48 hours. The effects of Que on the activity of neurons and PC12 cells were detected by MTT assay. The effects of Que on synaptic morphology of hippocampus and cortical neurons were compared by immunofluorescence staining. It was examined by Western blotting that the changes in the expression of related receptors and proteins in the PC12 cell model. And we studied in-depth on the mechanisms of estrogen-like neuroprotection of Que by using the estrogen receptor antagonist, such as ICI182,780, and the estrogen receptor alpha antagonist MPP, and the estrogen receptor beta antagonist PHTPP. Results: The results of MTT assay showed that Que could increase the activity of rat hippocampal neurons (P<0.05), and the protective effect of Que on hippocampal neurons increased in a dose-dependent manner. In low-density hippocampal neurons, 25μmol•L-1 Que had protective effects on hippocampal neurons (P<0.05). After estrogen receptor antagonism, 50μmol•L-1 Que could still increase the activity of hippocampal neurons (P<0.05). After prolonging the action time, three different concentrations of Que could increase the activity of hippocampal neurons. In the test of PC12 cells, with the increase of Aβ25-35 concentration, the inhibition of cell viability was gradually enhanced. Compared with the control group, Aβ25-3510μmol•L-1 could significantly reduce cell viability after 24h treatment (P<0.01). Compared with the model group, the cell viability of the three different concentrations of Que groups was significantly increased (P<0.05). The results of immunofluorescence staining test showed that Que could increase the number and length of synapses in hippocampal neurons (P<0.05) and promote synapse development. The Que (50, 100 μmol•L-1) groups significantly promoted synapse formation in cortical neurons. Most of the neurons were full of cells, and the synapses of the neurons were significantly thickened, which grew and connected into a dense neural network. After the estrogen receptor was completely antagonized, Que could still increase the number and length of synapses in hippocampal neurons (P<0.05). Que could increase the number and length of synapses in hippocampal neurons after estrogen receptor β antagonism, which was higher than that of estrogen receptor α (P<0.05). Western blot results showed that Que could increase the expression of estrogen receptor α protein. Compared with the model group, Que increased the expression of ERα and p-ERK1/2 protein (P<0.05), but there was no significant difference in the expression of ERβ protein (P>0.05). In the PC12 cell experiment, the results of Western blot showed that the expression ratio of apoptosis-related protein Bcl-2/Bax was increased and the expression of Caspase-3 protein was decreased (P<0.05). When Que estrogen receptor was inhibited by ICI182,780, p-ERK1/2 protein expression decreased (P<0.05), Bcl-2/Bax ratio decreased and Caspase-3 protein expression increased (P<0.05). When U0126 was applied to Que, the ratio of Bcl-2/Bax decreased and the expression of Caspase-3 protein increased (P<0.05). Conclusion: It was further confirmed that Que coulld up-regulate the expression of estrogen receptor α protein, increase the number and length of neuronal synapses, promote the growth and occurrence of synapses, and thus improve the growth rate and survival rate of neuronal cells. Then Que played a protective role of neurons in the hippocampus and cortex in rats. The Que also protected the AD model of PC12 cell injury induced by Aβ25-35, mainly enhancing the expression of estrogen receptor α protein. It exerted its estrogen- like neuroprotective effect through the classical estrogen receptor pathway. And it can attenuate apoptosis through ERα-mediated activation of the MAPK signaling pathway. In a word, Estrogen-like effect of Que has protective effects on brain neurons and AD-like injury. The main mechanism is related to the regulation of estrogen receptor α and the ERα-mediated MAPK signaling pathway.

Key words: estrogen, quercetin, neuron, Alzheimer's disease, estrogen receptor