Study on the neuroprotective effects of Genistein on Alzheimer’s disease

Abstract Along with the aging of the world population, the incidence rate of Alzheimer's disease (AD) has been increasing. At present, AD has become one of the most serious problems faced by modern medicine. Studies have shown that estrogen has a positive effect on AD, but estrogen has the side effect of leading to tumors. Recent in vivo studies have shown that genistein, one of the selective estrogen receptor modulators (SERMs), can improve brain function through the blood–brain barrier (BBB), antagonize the toxicity of amyloid β‐protein (Aβ), that is, to inhibit neurotoxicity due to aggregation of beta amyloid protein, and have neuroprotective effects. In addition, the use of Gen can avoid the risk of endometrial cancer and breast cancer caused by estrogen therapy while exerting an estrogen‐like effect, which has some potential for the delay and treatment of AD.


| INTRODUC TI ON
Alzheimer's disease (AD), also known as senile dementia, is a chronic degenerative disease of the central nervous system. Its main clinical manifestation is progressive dementia, and it is characterized by mental symptoms, such as decreased cognitive function, memory and language dysfunction, and behavioral disorders of varying degrees. Epidemiological statistics show that the majority of patients with this disease are female, and the incidence rate in females over 65 years old is 15%, which is three times that of males of the same age.  compared the activity of cytochrome c oxidase (COX) and the level of mitochondrial adenosine triphosphate (ATP) in the hippocampi of young and middle-aged ovariectomized (OVX) rats. The results showed that the activity of COX and the level of mitochondrial ATP in the hippocampus of young OVX rats changed for a short time, while the activity of COX and the level of mitochondrial ATP in the hippocampus of middle-aged OVX rats decreased for an extended period of time. This suggests that the onset of AD is related to an increase in age, especially to a decrease in ovarian function and a decrease in estrogen levels, and to a decrease in mitochondrial ATP and a decrease in mitochondrial ATP synthesis in the hippocampus .
At present, experimental studies and clinical observations have shown that estrogen has a protective effect on nerve cells. However, estrogen also has a proliferative and carcinogenic effect on non-neuronal cells (mammary gland cells and endometrial cells; Beresford et al., 1997), greatly increasing the risks of using estrogen. Thus, phytoestrogen, which is similar in structure to estrogen and can combine with and activate the estrogen receptors of both nonhuman mammals and humans, is attracting much attention.
Genistein (Gen) is the most active molecule in soybean isoflavones and can protect different kinds of cells from various toxic substances. It has been shown in in vivo studies that Gen can improve brain function through the blood-brain barrier (BBB), antagonize the toxicity of amyloid β-protein, and have a neuroprotective effect. In addition, use of Gen, one of the selective estrogen receptor modulators (SERMs), can avoid the risk of endometrial cancer and breast cancer caused by estrogen therapy, while exerting an estrogen-like effect, which has potential for delaying and treating AD. Therefore, Gen is an encouraging candidate to become an estrogen substitute for the prevention and treatment of AD. The effect of Gen on AD is summarized below.

| The effect of Genistein on amyloid β-protein
Amyloid β-protein (Aβ) is hydrolyzed by β-amyloid precursor protein (APP). In normal physiological conditions, APP secretes soluble Aβ, which can promote the growth of neurites, improve the survival rate of neurons, and protect the activity of neurons. However, in pathological conditions, APP secretes insoluble Aβ under the action of Beta-site (βsite) APP cleaving enzyme 1 (BACE1). The insoluble Aβ has a strong neurotoxic effect after deposition and aggregation, leading to the apoptosis and dysfunction of AD neurons. It has been shown that Gen can reduce the production of Aβ by inhibiting BACE1 (Li et al., 2013;Youn et al., 2018). Kim et al. (2002) found that Gen can block the stimulating effect of platelet-derived growth factor (PDGF) on APP and so reduce the secretion of APP. Hirohata fragments. The data produced by Yu et al. (2010) showed that Gen can reduce the neurotoxicity induced by Aβ 42 and Aβ accumulation by inhibiting Inhibition of kinesin AP180T (AP180) and Ras homolog family member A (RhoA).

| Genistein's mediation of the antiinflammatory effect
Microglia is the first and most important line of defense in the central nervous system. The initial neurologic change in AD patients is the activation of microglia around the Aβ deposition site, which increases proinflammatory cytokine levels. In addition, nuclear factor-B is an important transcription factor; inflammation caused by activation of the enhancement of κ-light chain in nuclear factoractivated B cells(NF-κB) signal path is also the key to the onset of neurodegenerative diseases. Zhou et al. (2014) found that the expression of the Toll-like receptor (TLR4) increases in microglia BV-2 with Aβ 25-35 accumulation. After TLR4 is activated, it can activate the NF-kB signal path by signal transduction. NF-kB can cause inflammatory reactions by regulating the expression of several inflammatory mediators and cytokines. The results showed that the expression of TLR4 was inhibited by the microglia BV-2 after pretreatment with 50 µM Gen for two hours, indicating that Gen can inhibit the activity of NF-kB by blocking the NF-kB signal path mediated by TLR4 to exert an anti-inflammatory effect. This is consistent with the report from Seo et al. (2018) that Gen can block the binding of NF-kB to target DNA that antagonizes the neuro-inflammatory effect of AD. Jantaratnotai et al. (2013) reported that Gen reduced the production of nitric oxide(NO) in rat microglia induced by lipopolysaccharide (LPS) and lowered the level of interferon regulatory factor-1 (IRF-1) and STAT1, achieving anti-inflammatory effects.
The anti-inflammatory effect of Gen is also reflected in the inhibition of inflammatory factor levels. When the microglia cell line HAPI was pretreated with Gen, it decreased the monocyte chemoattractant protein-1, interleukin-6 mRNA, and proinflammatory chemokines produced by the HAPI, stimulated by LPS. Valles et al. (2010) also demonstrated that levels of interleukin-1β (IL-1β) and tumor necrosis factorα (TNFα) were also controlled after astrocytes pretreated by Gen were induced by Aβ.

| The effect of Genistein on cholinergic neurons
Dysfunction of the central cholinergic nervous system (CNS) and lack of neurotransmitter acetylcholine (ACH) are the key mechanisms of cognitive decline in AD patients (Pedersen et al., 1996;Rajput & Sarkar, 2017). It has been shown that Gen can improve cognitive disorder in diabetic mice by inhibiting the activity of acetylcholinesterase (AChE) because Amyloid β-protein reduces acetylcholine synthesis in cell lines of basal forebrain cholinergic neurons (Pedersen et al., 1996). Amyloid β-protein can induce Ca 2+ to enter cholinergic neurons in the basal forebrain cortex-a region closely related to learning, memory, and cognition-through voltage-gated channels, eventually leading to the death of cholinergic neurons. (Weiss et al., 1994). In the presence of the tyrosine kinase inhibi-  (Guo J, 2004). Some Gen derivatives also show a good inhibitory effect on AChE (Qiang et al., 2014;Shi et al., 2012), thus improving cognitive disorder and dysmnesia in mice.

| Genistein's inhibition of phosphorylation of tau protein
The abnormal hyperphosphorylation of microtubule-associated protein tau leads to neuronal fiber entanglement (NFT), which is one of the pathological features of AD. The brain of an AD patient is extensively destroyed by the microtubule structure of the neurons. The normal axonal transport is damaged, the function of the synapticloss neurons is damaged, and neurodegeneration occurs. Tau protein can be phosphorylated by CAMK4, and its hyperphosphorylation leads to the formation of NFTs (Miyano et al., 1992). Ye et al. found that both CAMK4 and tau protein increased significantly in the hippocampi of AD model rats (Ye et al., 2017). Before construction of the AD rat model, the level of CAMK4 and tau protein decreased after seven days of pretreatment (1 ml/100 g bodyweight) with Gen.
Therefore, Gen may decrease the hyperphosphorylation of tau protein by regulating CAMK4 in AD model rats.

| Genistein's regulation of the expression of Apolipoprotein E
Apolipoprotein E (ApoE) participates in many aspects of the pathogenesis of AD. It has an endocytosis effect on soluble starch precursor protein APP and has a strong affinity with Aβ. There are a large number of ApoE receptors in the brain. The receptors combine with ApoE and ingest Aβ, thus reducing the extracellular level of Aβ and reducing nerve injury (Pappolla et al., 1997). Apolipoprotein E promotes axonal growth. The basal cholinergic neurons in the forebrain, in particular, are more dependent on ApoE because the basal cholinergic synapses in the forebrain significantly reduce while the other cerebral cholinergic neurons remain unchanged in ApoE-deficient mice (Bonet-Costa et al., 2016;Kleifeld et al., 1998). Apolipoprotein E in the brain participates in the normal metabolism of lipids, which may lead to transport deficiency of cholesterol and phospholipid and lead to insufficient acetylcholine synthesis. In the brain, ApoE is mainly synthesized by astrocytes and is activated by RXR/PPARγ.

Genistein can increase (up-regulate) PPARγ induced by Aβ to in-
crease the release of ApoE and decrease the deposition of Aβ.

| The effect of Genistein on mitochondria and oxidative stress
The destruction of mitochondrial structures increases the production of oxygen free radicals. The active oxygen free radicals produced by mitochondrial destruction also act as a signal to initiate the mitochondrial apoptosis pathway and attack the mitochondrial membrane, which causes a change in the mitochondrial matrix volume, a decrease of ATP production, and eventually cell death (Wei et al., 2010;Zhang et al., 2007).
Shi et al. observed that Gen could restore the synthetic ratio of mitochondrial ATP in the hippocampi of OVX rats .
Genistein also inhibited neuronal oxidant hydrogen peroxide levels induced by Aβ and decreased the release of cytochrome C, preventing mitochondrial toxicity induced by Aβ . 8-Hydroxy-deoxyguanosine (8-OHdG) is currently the most common and reliable biomarker of mitochondrial DNA (mtDNA) damage (Fenga et al., 2017).  (Wang et al., 2016) found that Gen can reduce hippocampal nerve injury by decreasing the expression levels of the cytochrome C, Bax, and caspase-3 , suggesting that Gen may protect the hippocampal neurons of AD rats through the mitochondrial apoptosis pathway.
Manganese superoxide dismutase can eliminate superoxide free radicals and play a key defensive role in mitochondrial dysfunction.
The absence of MnSOD also increases the formation of plaques and NFTs in the AD brain (Esposito et al., 2006;Melov et al., 2007).
The decrease of impaired mitochondrial ATP in the AD brain results in a decrease in Na, K-ATPase, and neurological function (Hattori et al., 1998;Kairane et al., 2002). Manganese superoxide dismutase is an important prerequisite for maintaining the normal function of Na and K-ATPase. Kairane et al. compared the effects of three antioxidants on the activity of MnSOD, Na, and K-ATPase in the AD brain (Kairane et al., 2014). The results showed that Gen can directly eliminate free radicals and increase the expression of MnSOD.
Genistein also increased the activity of Na and K-ATPase in the cerebral cortex of the AD brain, and there was a correlation between the present concentration and time.
Genistein has strong antioxidant properties, which can eliminate reactive oxygen species (ROS) and free radicals (Vallés et al., 2008) and enhance antioxidant enzyme activity (Cai & Wei, 1996). Genistein also shows a good antioxidative effect in the prevention and treatment of AD (Polkowski & Mazurek, 2000). Genistein can inhibit the pro-oxygenic agent action of 24-hydroxylatedcholesterol (24-hydroxycholesterol), prevent neuronal necrosis and apoptosis induced by Aβ (Gamba et al., 2011), inhibit overproduction of ROS induced by Aβ 25-35 (Andersen et al., 2003) in rat brain synapses, and inhibit the influx of intracellular Ca 2+ and the production of free radicals (Zeng et al., 2004). In addition, Gen also inhibited the oxidation of hemoglobin and myoglobin induced by hydrogen peroxide in AD rats (Boadi & Johnson, 2014;Occhiuto et al., 2009), indicating that Gen can play a role in neuroprotection through antioxidant activity.
Alzheimer's disease is a common and destructive neurodegenerative disease. In China, the number of AD patients is the highest in the world. The prevention and treatment of AD has become a topic of worldwide concern. Genistein is a natural isoflavone compound found in plants, which not only selectively combines with estrogen receptor ERβ and exerts an estrogen-like effect but also has unique antitumor effects (Radzikowski et al., 2004;Yu et al., 2005). Gen has strong antioxidant properties, which can eliminate reactive oxygen species (ROS) and free radicals (Vallés et al., 2008), improve the activity of antioxidant enzymes (Cai & Wei, 1996), and also play a good antioxidant stress role in the prevention and treatment of AD (Polkowski & Mazurek, 2000). In this study, through a literature review, we have found that Gen can delay and improve the harmful effects of AD through many mechanisms and targets both in in vivo and on the intracellular and F I G U R E 1 Gen's proposed mechanisms of action extracellular molecular level (Figure 1), suggesting that Gen is a good alternative to substitute for estrogen in the treatment of AD.

ACK N OWLED G M ENTS
We would like to acknowledge the hard and dedicated work of all the staff that implemented the intervention and evaluation components of the study.

CO N FLI C T O F I NTE R E S T
All of the authors confirm they have no conflict of interest to declare.

AUTH O R CO NTR I B UTI O N
Xiaoying Duan conceived the idea and conceptualized the study.

PE E R R E V I E W
The peer review history for this article is available at https://publo ns.com/publo n/10.1002/brb3.2100.
[Correction added on March 20, 2021, after first online publication: Peer review history statement has been added.]