Neuroprotective activities of icariin in rodent models of Alzheimer’s disease: a systematic review and meta-analysis

Introduction

Alzheimer’s disease (AD) is a neurodegenerative disease, which is irreversible and considered to be the most common cause of dementia, accounting for 50–70% of dementia patients worldwide (1). The clinical symptoms of AD are mainly short-term memory loss, spatial orientation disorders, and personality and behavior changes. In 2020, the official website of the World Health Organization (WHO) published that there were about 50 million people with dementia all over the world, and the number of patients is expected to rise to 152 million in 30 years.

The pathogenesis of AD is complex, which is generally thought to be related to environmental and genetic factors. The typical pathological features are the deposition of amyloid beta (Aβ) plaques, the accumulation of neurofibrillary tangles (NFTs), and the death of neurons accompanied by loss of synapses (2). At present, a variety of hypotheses have been proposed to clarify the pathogenesis of AD, among which the most widely accepted hypotheses cite the Aβ-cascade, tau hyperphosphorylation, oxidative stress, inflammatory response, and metabolic disorders (3,4). Despite much research, the commonly used therapeutic drugs, such as donepezil and memantine, can only alleviate the symptoms of AD patients, but cannot reverse or prevent the development of the disease, and also have certain side effects. Therefore, research and development of new effective drugs is a hot topic in AD research.

Icariin is a major flavonoid component extracted from the Chinese herb Epimedium brevicornum Maxim. (Berberidaceae; name in English, Shorthorn Barrenwort; name in Chinese, Yinyanghuo). Pharmacological studies have shown that icariin has protective effects on the cardiovascular system, as well as immunoregulatory, anti-oxidant, anti-aging and anti-inflammatory properties (5-7). In recent years, many studies have found that icariin also has significant neuroprotective effects. For example, in APP transgenic mice, icariin alleviated the impairment of learning and memory by decreasing the expressions of APP and β-secretase expression (BACE1) (8). In APP/PS1 mice, icariin improved cognitive function by inhibiting the endoplasmic reticulum (ER) stress signal pathway (9).

There have been many studies to evaluate icariin in AD models. No single study can provide sufficient, comprehensive information on the efficacy and mechanism of icariin in the treatment of AD, yet there has been no review of these studies to summarize and systematically evaluate their findings. Therefore, this review took a scientific approach to eliminate bias against each study, comprehensively evaluated the effects of icariin in different rodent models of AD, and summarized the neuroprotective mechanisms of icariin. It is hoped that this summary evaluation can provide reliable evidence and insight for developing icariin into a clinical drug.

We present the following article in accordance with the PRISMA reporting checklist (available at https://lcm.amegroups.com/article/view/10.21037/lcm-21-50/rc).

Methods

Search strategy

To study the neuroprotective effect of icariin in rodent models of AD, we carefully searched the literature in three independent literature databases, PubMed, Web of Science and Google Scholar from inception to July 2021. The keywords of the search were “icariin (Title/Abstract)” and “Alzheimer’s disease (Title/Abstract)” OR “Alzheimer disease (Title/Abstract)”. The study was limited to preclinical studies in rodent models. The reviewers (Yu-Xuan Kan and Cheng-Fu Su) screened articles independently by reading the title and full-text according to eligibility criteria.

Eligibility criteria

Inclusion criteria

• Study on evaluating the effect of icariin on AD in vivo;
• AD rodent models of any gender, age, or weight;
• Any dosage, treatment time and mode of administration;
• Inclusion of at least one icariin treatment group and a control group with placebo.

Exclusion criteria

• Repetitive studies, review articles, articles without access rights;
• In vitro studies;
• Icariin was used in combination with other drugs;
• Failure to evaluate the effects of icariin on AD rodent models.

Data extraction

Two investigators separately screened the studies and performed data collection procedures. Disputes during the process were resolved after discussion by a third party. According to the inclusion criteria, the basic information of the selected studies is as follows (Table 1): (I) authors and publication year; (II) animal information, including gender, age, species, weight and modeling methods; (III) icariin treatment, including administration methods, dosage and duration time; (IV) evaluation indicators; (V) pharmacological activity/mechanisms. Image J software was used to extract the data of the result graph.

Quality assessment

According to the characteristics of AD experimental study, the methodological quality assessment form was established by adjusting the basis of the CAMARADES study quality checklist (27,28). The assessment criteria included: (I) peer-reviewed publication; (II) sample size calculation; (III) random allocation sequence; (IV) appropriate animal models of AD; (V) blinded intervention; (VI) blinded evaluation of outcome; (VII) compliance with animal welfare regulations; (VIII) statement of conflict of interest. Each study had a quality score of no more than 7 points. “Y”, “N”, and “U” were used to indicate low, high and uncertain risk of bias, respectively.

Statistical analysis

RevMan 5.3 software were used for statistical analysis. Each outcome was continuous data, represented by mean difference (MD) and 95% confidence interval (CI). Heterogeneity in included studies was evaluated by I-square (I²) statistical tests. I²>50 was considered that there is significantly heterogeneity.

Results

Study selection

Preliminary searches of the data resources were carried out according to the above method, and a total of 139 articles were identified (114 articles from Web of Science and PubMed databases, 25 articles from Google Scholar). By browsing titles and abstracts, we selected 25 articles on the effects of icariin on AD. After reading the full text, we determined that, 19 of these articles qualified for meta-analysis (Figure 1).

Figure 1 Research methodology for selection process. AD, Alzheimer’s disease.

Of the 19 articles selected, 14 studies chose mice and 5 studies chose rats. Among the studies on the effects of icariin on mice models, 8 studies used male mice, 2 studies used female mice, and 4 studies used both male and female. In the rat models, 4 studies used male rats and the other one study did not mention gender.

Study characteristics

Different animal models

In this review, five kinds of rodent models were used in the 19 studies. They were: Aβ-induced and ibotenic acid (IBO)-induced animal models; APP/PS1/tau transgenic models; senescence-accelerated mouse prone 8 (SAMP8) model; and the permanent bilateral common carotid artery occlusion (BCCAO) model. We summarized the different animal models in Table 2.

The transgenic mice model is an ideal tool to study the pathogenesis of AD. Currently, Tg 2576, APPV717I, APP/PS1, 3xTg-AD, and 5xFAD transgenic mice have been widely used in experimental studies. Tg 2576 mice, the first transgenic mice model of AD showing cognitive impairment, express the 695 aa long isoform of human APP containing the K670N/671L double mutation. They have amyloid deposits in the cortex and limbic areas of the brain at 9 months, along with behavioral deficits (16,29).The APPV717I transgenic mice carry the V717I mutation, resulting in the increased level of Aβ₄₂ and AD-like pathological features (30,31).The APP/PS1 mice model has Aβ deposition and neurocognitive impairment due to APP and PS1 overexpression. Aβ can be detected in this model at 6 months old, and after 3 months, extracellular Aβ deposition is evident in the cortex, accompanied by significant impairments in synaptic transmission and long-term potentiation (32,33).3xTg-AD mice harboring the PS1_M146V, APP_Swe, and tau_P301L transgenes mimic both amyloid and tau AD neuropathologies. Extracellular Aβ deposition is increased significantly and mainly distributed in the hippocampus and frontal cortex (34).The 5xFAD transgenic mice carry five mutated neuron-specific genes, including Swedish (K670N and M671L), Florida (I716V) and London (V717I) mutations in APP, and M146L and L286V mutations in PS1. They have similar pathological characteristics to AD, especially the aggregation of Aβ in neurons at 1.5 months of age, indicating that this model is particularly relevant for the study of the early stage of AD (19,32,35).

Besides the transgenic mice models mentioned above, chemical-induced AD models such as IBO-induced and Aβ-induced are also used in AD studies. IBO is a neurotoxin, which can cause neuron degeneration, apoptosis, and mild cognitive impairment. Injecting IBO into the brains of animals, such as monkeys and rats, can cause pathological features similar to human AD patients (36,37). Aβ deposition is an important pathological mechanism of AD. The Aβ-induced model is generally established by multiple injections of Aβ fragments into the hippocampal CA1 area or lateral ventricle. It is considered to be a non-genetic model of AD. Many studies have shown that injection of Aβ can cause Aβ deposition, tau hyperphosphorylation, Ca²⁺ influx, ROS production (38). Excessive deposition of Aβ may lead to high production of senile plaques and NFTs, while hyperphosphorylated tau and Aβ can further exacerbate neurotoxicity and lead to dementia (39,40).

In addition to the transgenic mice models and chemical-induced models, SAMP8 mice and BCCAO model are also used. The SAMP8 mice model is a spontaneous animal model of accelerated senescence. These mice are characterized by hyperphosphorylation of tau-like NFTs with age, and they develop learning and memory deficits at about 8 months of age (41-43). The decrease of cerebral blood flow is another important factor of cognitive dysfunction, which is common in the elderly (44,45). Previous studies have shown that the BCCAO model induces cognitive impairment and neuron loss (46,47). Therefore, this model is considered to be a chronic cerebral perfusion model that can better simulate the pathological development of human AD (48).

Behavioral test analysis

Behavioral test analysis is an important research method in AD research. By observing the behavior of experimental animals in a specific environment, we can analyze the neurological changes in mice. Morris water maze (MWM) was used in most of the included studies. Also, Y maze, open field test, and object recognition test were used in some of the studies.

MWM was proposed and applied by the British psychologist Morris in the 1980s (49). It is an important detection method for analyzing the spatial sense and learning ability of experimental animals. By comparing and analyzing the changes in spatial cognition, learning and memory of AD model animals treated or untreated with drugs, we can evaluate the effectiveness of the intervention, in this case, icariin, to alleviate AD (50). In a typical MWM, escape latency of experimental animals is recorded during the training period, and then a probe test is performed by removing the platform. For each animal, the time spent in the target quadrant and the number of times crossing the platform are recorded. The same test is performed again after administration of icariin. According to the changes in the indexes of each group, the effect of icariin on cognitive function of the AD animals was evaluated.

In this review, 13 of the 19 studies performed the MWM test, and only 5 of them recorded the number of times crossing the platform. A total of 52 animals (SAMP8, n=10; Aβ-induced rats, n=12; transgenic mice, n=30) were enrolled and given different doses of icariin, while 51 vehicle-treated animals were added as controls. Image J and RevMan 5.3 software were used to extract and analyze the number of times crossing the platform in these five studies. The results are shown in the forest plot in Figure 2. Compared with vehicle-treated animals, icariin significantly increased the number of times crossing the platform in these three different AD animal models (MD =2.41, 1.72 and 1.28). Furthermore, there was no significant heterogeneity among the subgroups of transgenic mice (P=0.20; I²=39%). The analysis of combined subgroups showed that icariin treatment significantly improved cognitive function in AD rodent models (MD =1.70; 95% CI: 1.46 to 1.94; P<0.00001), with significant heterogeneity (P=0.0007; I²=79%). The reason for the elevated heterogeneity may be related to different AD animal models.

Figure 2 Forest plot of studies investigating the effect of icariin on the number of times crossing the platform in MWM compared with the control group. MWM, Morris water maze; CI, confidence interval; SAMP8, senescence-accelerated mouse prone 8; Aβ, amyloid beta.

Neuroprotective mechanisms analysis

We summarized the neuroprotective mechanisms of icariin in the included studies (Figure 3). There are four main mechanisms, namely, reduction of Aβ deposition, anti-inflammation, anti-oxidant activity, and regulation of autophagy. Furthermore, some studies reported that icariin inhibited apoptosis and regulated synaptic plasticity.

Figure 3 Neuroprotective mechanisms of icariin. “↑” means up-regulation, “↓” means down-regulation. Aβ, amyloid beta.

Thirteen studies demonstrated that icariin inhibited the increase of Aβ levels in AD rodent models. Li et al. (11) reported that treatment with icariin decreased the number of plaques (P<0.05) and Aβ immune-reactivity areas (P<0.05) in the hippocampus and cortex compared with the control group. Jiang et al. (12) found that the level of Aβ₄₂ declined in icariin-treated AD model rats (P<0.01). They performed immunohistochemical studies using a 6E10 antibody bound to amino acids 1–16 of Aβ to assess the distribution of Aβ clearly. The results showed that icariin reduced 6E10 positive staining (P<0.05). Zhang et al. (13) and Wang et al. (14) demonstrated that icariin administration decreased the number of Aβ plaques in AD models. Li et al. (9) showed that treatment with icariin reduced the Aβ burden (P<0.01) and increased the expression of neuroprotective ADAM10 protein (P<0.01) in the hippocampus of APP/PS1 mice. Li et al. (22) reported that, in BCCAO rats, icariin administration decreased the level of Aβ through multiple targets, such as by reducing the production of Aβ and by increasing the degradation of Aβ, which may be related to the TGFβ1 signaling pathway. Jin et al. (23) showed a significant decrease in the levels of Aβ and PDE5 mRNA or protein in icariin-treated transgenic mice. Li et al. (16) and Zhu et al. (24) reported that icariin lowered the insoluble Aβ₄₀ and Aβ₄₂ levels in the brain, but had no significant effect on soluble Aβ. Nie et al. (26) showed that icariin inhibited Aβ_1–40 and mRNA levels of β-secretase in Aβ_25–35-induced rats in a dose-dependent manner. They hypothesized that icariin may reduce the production of Aβ peptide by inhibiting β-secretase, thus improving the cognitive function in AD rats. Using APP transgenic mice or SAMP8 mice, Zhang et al. (8), Wu et al. (18) and Chen et al. (25) all found that icariin alleviated Aβ deposition by decreasing the expression of BACE1, thus improving cognitive function.

Four of the included studies assessed the level of Aβ in the brains of rodent animals by ELISA. A total of 33 rodent animals (mice and rats, n=23 and n=10) were treated with different doses of icariin, while 33 vehicle-treated animals were added as controls (mice and rats, n=23 and n=10). The level of Aβ was analyzed using a forest plot in RevMan 5.3 software (Figure 4). After icariin treatment, Aβ in the brain was significantly reduced compared with the model group (MD =−2.75; 95% CI: −3.29 to −2.22; P<0.00001). There was high heterogeneity among the subgroups (I²=99%), which may be related to the different species of experimental animals, modeling methods and detection methods, as well as differences in the dose and time of icariin administration.

Figure 4 Forest plot of studies investigating the effect of icariin on the level of Aβ in AD rodent models detected by ELISA. Aβ, amyloid beta; AD, Alzheimer’s disease; CI, confidence interval. ELISA, enzyme-linked immunosorbent assay.

In recent years, more attention has been paid to the role of neuroinflammation in AD. Microglia appear to be important. Activated microglia can be divided into M1 and M2 phenotypes. M1 phenotype releases a large number of pro-inflammatory cytokines to cause neurotoxicity, while M2 phenotype releases mediators such as IL-4 and IL-1 to inhibit the inflammatory response (51). Therefore, inhibiting the activation of microglia and the massive production of inflammatory cytokines may be an effective strategy to alleviate AD. Four studies have indicated that icariin has an anti-inflammatory effect. Zhang et al. (10) showed that the levels of TNF-α, IL-1 and IL-6 were decreased in icariin-treated transgenic mice compared with the model group (P<0.05). Zhang et al. (13) reported that icariin inhibited the activation of microglial (P<0.05) and the level of TGF-β1 (P<0.05) in the hippocampus and cortex, and alleviated behavioral deficits. Studies confirmed that PPAR-γ can attenuate M1 activation of microglia and mediate its transformation to the M2 phenotype (52,53). Wang et al. (14) found that treatment with icariin decreased the levels of IL-1β, TNF-α and IL-6, and increased the levels of IL-4, IL-10 and TGF-β in the hippocampus and prefrontal cortex of APP/PS1 mice (P<0.05). These changes may be related to PPAR-γ inhibiting the activation of M1 microglia. Zhu et al. (24) reported that icariin regulated the differentiation of CD4⁺ T cells and inhibited the release of pro-inflammatory cytokines in plasma and brain tissue.

Oxidative stress is one of the earliest events in the pathological development of AD. When the body is stimulated by certain conditions, excessive oxygen free radicals are produced, which greatly exceed the capacity of the anti-oxidant system, resulting in oxidative stress and neuron damage. Superoxide dismutase (SOD) and glutathione peroxidase (GSH), which can remove excessive free radicals, are the first line of defense against oxidation in the body. Malondialdehyde (MDA) is the product of lipid peroxidation, which indirectly reflects the level of oxidative stress. Three studies have shown the anti-oxidant function of icariin in AD rodent models. Zhang et al. (10) reported that icariin decreased MDA (P<0.01) and increased the activities of SOD (P<0.01) and GSH (P<0.01) in the brains of APP/PS1 transgenic mice, thus alleviating the tissue damage caused by iron overload. Nie et al. (26) and Jiang et al. (12) demonstrated that treatment with icariin inhibited oxidative stress by increasing SOD-2 mRNA expression (P<0.05) or regulating SOD (P<0.05) and MDA (P<0.05) levels in Aβ-induced AD rat models respectively.

Two studies indicated that the alleviation of icariin on cognitive impairment is related to autophagy. Jiang et al. (12) reported that treatment with icariin reduced the levels of LC3II (P<0.01), Beclin1(P<0.01) and Cathepsin D (P<0.05), and increased the phosphorylation of p70S6K (P<0.01) in Aβ-induced rats, suggesting that it reversed Aβ-induced autophagy activation. Meanwhile, icariin inhibited apoptosis by increasing the ratio of pS473AKT/AKT. Chen et al. (17) showed that icariin administration reduced the number of autophagosomes in hippocampal neurons of SAMP8 mice, and inhibited the expressions of LC3-II and p62 in the hippocampus and cortex (P<0.05), suggesting that the cognitive function of SAMP8 mice could be improved by regulating autophagy.

The neuroprotective mechanisms of icariin also include anti-apoptosis and regulation of synaptic plasticity. Wu et al. (18) reported that icariin treatment increased the level of anti-apoptotic protein Bcl-2 and reduced the level of pro-apoptotic protein Bax, thus inhibiting neuronal apoptosis. Sheng et al. (15) found that icariin restored normal synaptic morphology and increased the expression of postsynaptic density protein 95 (PSD95), which helped improve cognitive function in AD mice.

Methodological quality

In assessing the methodological quality of studies, it is necessary to evaluate the various risks of bias that may arise in the design, implementation, and result analysis of individual studies. According to the characteristics of AD experimental study, the methodological quality assessment form was established by adjusting the basis of the CAMARADES study quality checklist. The results were shown in Table 3, the methodological quality of each study ranged from 3 to 5 (highest possible score, 8). All included studies were published in peer-reviewed journals. Random allocation sequence was reported by 17 publications (89.5%). Compliance with animal welfare regulations and conflict of interest were mentioned in 18 publications (94.7%) and 16 publications (84%), respectively. All studies failed to mention whether blind intervention and blind evaluation of outcome were used, and no study performed sample size calculation. Appropriate sample size is important for animal experiments, and the number of animals selected should be sufficient for statistical analysis while avoiding unnecessary sacrifices.

Discussion

In recent years, many studies have focused on the treatment of neurodegenerative diseases with Chinese herbal extracts. Icariin is a flavonoid in Epimedium, a traditional Chinese herbal medicine. Many studies have shown that icariin has neuroprotective effects and can improve cognitive function in AD animal models (14,15). Meta-analysis is used to evaluate multiple trials of a single intervention because it can eliminate bias in individual studies. This is of great significance for evaluating the efficacy of drugs in preclinical trials. This review used meta-analysis to systematically evaluate the neuroprotective effects of icariin in different rodent models of AD, providing some reference for future clinical studies.

In this review, 19 studies of icariin in rodent models of AD were selected from three databases and systematically evaluated. Among them, 13 studies all used the MWM test to assess the effects of icariin on learning and spatial memory abilities in AD animals. This review conducted a meta-analysis on the number of times crossing the platform by forest plot. The results showed that icariin treatment significantly increased crossing times in animals compared with the control groups, suggesting that icariin improves cognitive function in AD rodent models.

A dependable animal model plays an essential role in exploring the pathogenesis and drug development of AD. In the included studies, 12 studies used AD transgenic mice, 3 studies used Aβ-induced AD models, 2 studies used SAMP8 mice, one study used an IBO-induced model, and one study used a BCCAO model. Different models have their own characteristics. For example, the Aβ-induced model is an acute model, which may cause some damage to the brain tissue of animals during the modeling process. This model is inconsistent with the pathological changes of slow Aβ deposition in the brains of human AD patients. SAMP8 mice are considered as a model of sporadic AD. Since over 95% of human AD patients are sporadic, it could be argued that these mice are a much more relevant pre-clinical model. But it is relatively expensive. Meanwhile, transgenic mice also commonly used AD animal model at present. They carry multiple AD-related pathogenic genes and can more fully simulate the pathological characteristics of Aβ deposition, plaque formation and neuron loss in the brain of AD patients.

In this review, the neuroprotective mechanisms of icariin are systematically introduced, mainly include reducing Aβ deposition, anti-inflammation, anti-oxidation and autophagy regulation. In 13 studies using transgenic mice, the neuroprotective mechanisms of icariin mainly concentrate on anti-Aβ deposition (4 studies) and anti-inflammation (4 studies). In addition, one study showed that icariin played a neuroprotective role by inhibiting neurons apoptosis, and one study showed that icariin regulated synaptic plasticity through BDNF/TrkB/Akt pathway.

This review also has some limitations. By assessing the methodological quality of each study for the various risks of bias that may occur during the design, implementation, and analysis of results, we found that: The quality of the studies is inconsistent and there is a general lack of high-quality evidence. All studies lacked the calculation of sample size. The number of animals in each group may affect the significance of the differences between groups. In addition, we did not conduct subgroup analysis of icariin dosage, so the effect of dosage on the efficacy of icariin needs further study.

In conclusion, we systematically reviewed 19 studies of the effects of icariin on AD rodent models, and summarized the mechanisms. Icariin has a significant neuroprotective effect on different AD models, mainly through reducing Aβ deposition, anti-inflammation, anti-oxidation and regulating autophagy. The results of this review will provide useful information for the design of future experiments related to the neuroprotective effects of icariin and provide a basis for the further development of icariin as an anti-AD drug.

Acknowledgments

We would like to thank Dr. Martha Dahlen for her English editing of this manuscript.

Funding: This work was supported by the Health and Medical Research Fund (HMRF/17182541, HMRF/17182551) from the Food and Health Bureau of Hong Kong Government and the General Research Fund (HKBU 12101417, HKBU12100618) from the University Grants Committee of Hong Kong Government. The work was partly supported by the Research Fund from Hong Kong Baptist University (RC-IRCs/17-18/03, IRCMS/19-20/H02), and also supported by the National Natural Science Foundation of China (NSFC 81703487, NSFC 81773926) and Basic Research Fund from Shenzhen Science and Technology Program (JCYJ20180507184656626, JCYJ20180302174028790).

Footnote

Provenance and Peer Review: This article was commissioned by the Guest Editors (Zhi-Xiu Lin, Yanfang Xian and Hongxi Xu) for the series “Pharmacology of Chinese Herbal Medicine” published in Longhua Chinese Medicine. The article has undergone external peer review.

Reporting Checklist: The authors have completed the PRISMA reporting checklist. Available at https://lcm.amegroups.com/article/view/10.21037/lcm-21-50/rc

Peer Review File: Available at https://lcm.amegroups.com/article/view/10.21037/lcm-21-50/prf

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://lcm.amegroups.com/article/view/10.21037/lcm-21-50/coif). The series “Pharmacology of Chinese Herbal Medicine” was commissioned by the editorial office without any funding or sponsorship. The authors have no other conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

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