Neuroprotective effects of berberine in animal models of Alzheimer’s disease: a systematic review of pre-clinical studies

Background Berberine is an isoquinoline alkaloid extracted from various Berberis species which is widely used in East Asia for a wide range of symptoms. Recently, neuroprotective effects of berberine in Alzheimer’s disease (AD) animal models are being extensively reported. So far, no clinical trial has been carried out on the neuroprotective effects of berberine. However, a review of the experimental data is needed before choosing berberine as a candidate drug for clinical experiments. We conducted a systematic review on AD rodent models to analyze the drug effects with minimal selection bias. Methods Five online literature databases were searched to find publications reporting studies of the effect of berberine treatment on animal models of AD. Up to March 2018, 15 papers were identified to describe the efficacy of berberine. Results The included 15 articles met our inclusion criteria with different quality ranging from 3 to 5. We analyzed data extracted from full texts with regard to pharmacological effects and potential anti-Alzheimer’s properties. Our analysis revealed that in multiple memory defects animal models, berberine showed significant memory-improving activities with multiple mechanisms, such as anti-inflammation, anti-oxidative stress, cholinesterase (ChE) inhibition and anti-amyloid effects. Conclusion AD is likely to be a complex disease driven by multiple factors. Yet, many therapeutic strategies based on lowering β-amyloid have failed in clinical trials. This suggest that the threapy should not base on a single cause of Alzheimer’s disease but rather a number of different pathways that lead to the disease. Overall we think that berberine can be a promising multipotent agent to combat Alzheimer’s disease.


Background
Alzheimer's disease (AD) is a progressive degenerative disease of the central nervous system. Its main clinical manifestations are progressive declines in memory and cognitive function, accompanied by psychiatric symptoms and abnormal behavior. AD mostly occurs in elderly persons over 65 years of age. According to 2017 statistics, there are nearly 46 million AD patients worldwide [1,2]. In the brain, senile plaques (SP) and neurofibrillary tangles (NFT) are the diagnostic hall markers of AD. Its other pathological features include diffuse atrophy of the cortex, widening of the sulcus, enlargement of the ventricles, loss of neurons and decreases in choline acetylase and acetylcholine levels. The etiology of AD is still elusive, and several hypotheses have been proposed to explain the pathogenesis of AD. The most prevalent hypotheses are the amyloid β-protein (Aβ) cascade hypothesis [3,4], hyper-phosphorylated Tau hypothesis [4], the free radical theory [5], the inflammatory theory [6] and cholinergic hypothesis [7]. The diversity and uncertainty of the pathogenesis of AD have caused difficulties in the development of effective treatment, and most of the clinical trials performed in recent decades have failed.
Berberine is an isoquinoline alkaloid that is widely present in several medicinal plants, especially in those belonging to the Berberis genus (e.g., Berberis vulgaris L., Berberidaceae). It also occurs, for example, in Coptis chinensis Franch. (Ranunculaceae), a plant which is used in traditional Chinese medicine as an anti-diarrheal, anti-bacterial, anti-fungal, and anti-protozoal agent, particularly in combination with other herbs [8][9][10]. The chemical structure of berberine is shown in Fig. 1. In several years, accumulating evidence has revealed a wide variety of bioactivities of berberine such as antiviral, antibacterial and anti-inflammatory [11,12].
The pharmacological effect of berberine on the nervous system was first reported in the 1970s as sedation-inducing [13]. The therapeutic activity of berberine has been widely examined in various neurological conditions including cerebral ischemic injury, AD, Parkinson's disease, depression, anxiety, Huntington's disease, epilepsy and convulsions. Several studies have shown that berberine can alleviate AD pathology through various mechanisms, including inhibition of hyper-phosphorylation of Tau protein and Aβ production. Berberine can reduce the hyper-phosphorylation of Tau protein, and this reduction may be related to the activation of the phosphatidylinositol 3-kinase/protein kinase/ glycogen synthase kinase 3 pathway to restore protein phosphatase 2A activity and reverse glycogen synthase kinase-3 (GSK-3) activation [14]. In addition, berberine can inhibit the expression of beta-secretase by activating the extracellular signal-regulated kinase 1/2 signaling pathway, thereby inhibiting the production of Aβ40/42 [15]. Moreover, researchers have recently revealed that, on a molecular basis, berberine exerts inhibitory effects on the four key enzymes in the pathogenesis of AD: acetylcholinesterase, butyrylcholinesterase, monoamine oxidase A, and monoa mine oxidase B [16].
Before this, several experiments have been performed to evaluate the anti-AD properties of berberine. However, these pre-clinical studies have not been systematically analyzed to provide a whole picture and un-biased understanding of the therapeutic potential of berberine for AD. The aim of this systematic review is to summarize the current evidence and analyze that evidence as to what it reveals about the underlying mechanism of the protective effects of berberine in animal AD models. We hope to provide more insightful information for future clinic trials.
To perform the systematic review, we searched the literatures and selected the studies passing our selection criteria for data extraction and analysis. Our search of electronic databases returned a total of 91 articles. After deleting 16 which contained duplicated experimental data, we had a total of 72 references. After reading the titles and abstracts, we deleted 57 papers for the following reasons: (1) Not including experiments on animal models; (2) Not directly administering berberine; (3) No experimental details provided. Thus, finally, we had 15 articles that reported the efficacy of berberine in AD animal models; this review is based on these articles (Fig. 2).

Literature search
A careful literature search was performed to find publications reporting studies of the effect of berberine treatment on animal models of AD. Online literature databases (PubMed, Google scholar, PsychINFO, Embase and Web of Science) were searched up to March 2018 using search terms for English or Chinese publications. The following search strategy was used for each database. (1) Berberine was administered alone.
(2) Experimental AD was induced in rodents (i.e., rats or mice). (3) AD treatment group was treated with a pharmacological agent, and a control group was administered a placebo after injury. (4) Article was published in English or Chinese.

Exclusion criteria
(1) Not an original paper (review or letter etc.); (2) Berberine was not administered alone.  Two investigators independently screened papers and listed them based on publication year, the first author's name and experimental models. Using a structured form, they extracted individual data on study characteristics, methods and outcome measures. The differences in papers selected were resolved through discussion. Finally, the methodological quality of the included basic research was assessed by applying six correction scales.

Results
The search strategy retrieved 91 papers through online literature databases (PubMed, Google scholar, Psy-chINFO, Embase and Web of Science), 15 papers met our selection criteria. These 15 studies evaluated in this review involved animals from two species and four varieties: TgCRND8 mice, APP/PS1 mice, Sprague Dawley rats and Wister rats. The scales of the studies varied, from 6 to 104 animals in a single study. Rat and mouse weights were 200-300 g and 20-55 g, respectively. Eleven studies used male animals, and 1 study used female rats. After selecting and classifying these 15 studies, 3 were diabetic rat models with memory-impairment, 2 were 3 × Tg-AD mice models, 2 were Aβ infused rats models, 1 was an APP/PS1 mice model, 1 was a (Pilo)-induced epilepsy rat model, 1 was an ibotenic acid (ibo)-induced rat model and 5 memory-impairment models induced by Scopolamine, ICV-STZ, ethanol and D-galactose respectively. The research parameters evaluated in the 15 studies included the Morris water maze, immunohistochemistry (IHC), Western blot, RT-PCR (reverse transcription-polymerase chain reaction) and ELISA. The Morris water maze, a behavior test, was used to evaluate memory function. The IHC method as a molecular biology technique was used to investigate neuroprotective effects. Western blot, ELISA and RT-PCR techniques were used to measure potential genetic and proteins markers involved in Alzheimer's disease. Table 1 lists the basic characteristics of the 15 studies.

Methodological quality
We assessed the scores of the quality according to these 6 points: A: peer reviewed publication; B: random allocation to group; C: blinded assessment of outcome; D: a sample size calculation; E: compliance with animal welfare regulations; F: a statement of a potential conflict of interest.
The quality items scored in the included studies ranged from 3 to 5 out of a total of 6 points as shown in the Table 2. Two of the studies (13.3%) achieved 3 points; seven studies (46.7%) achieved 4 points; and Six studies (40%) achieved 5 points. Table 2 shows the Methodological quality of the 15 reviewed studies. Table 3 shows the main outcomes and results of the included studies. Twelve studies investigated whether berberine improved cognitive abilities; four studies examined hippocampal cells of CA1 region and apoptosis of pyramidal neurons in the CA1 area. The changes in oxidative stress and acetylcholinesterase (AChE) activity were examined in 8 studies. Three studies tested NF-kB signaling. In addition, one study reported that berberine induced autophagy to reduce the APP and BACE1 levels. The above proposed neuroprotective mechanisms of berberine are summarized in Fig. 3.

Discussion
Potential mechanisms underlying anti-Alzheimer's disease properties of berberine The neuroprotective effects of berberine have been extensively studied in different animal experimental models and we summarized the studies which include a rat model of amyloid beta induced-Alzheimer's disease, a memory impairment model induced by ethanol in rats, a D-galactose-induced memory deficits model in rats, a pilocarpine (Pilo)induced epilepsy model in rats, a scopolamine and streptozotocin-induced memory impairment model in rats, a memory-deficient rat model induced by stereotaxic injection of ibotenic acid into entorhinal cortex (Ibo model), and the transgenic mouse model of Alzheimer's disease. Interestingly, berberine displayed significant effects in preventing memory impairment in these mechanistically different animal models, suggesting an over-all improvement of memory function by berberine. Indeed, mechanistic studies showed that berberine modulated a wide range of biological functions to exert neuroprotection and the detailed mechanisms are discussed in the following part.

(a) Antioxidant properties of berberine
Alzheimer's disease is characterized by extensive evidence of oxidative stress which is the result of uncontrolled production of reactive oxygen species (ROS) [35]. ROS has been regarded as a critical factor in the neuron dysfunction or death of neuronal cells that contribute to the pathogenesis of the disease [36]. Under normal conditions, the damage caused by oxygen free radicals can be controlled through a series of reactive antioxidant systems. However, under pathological conditions, the balance between oxidants and antioxidants is disturbed such that active oxygen production exceeds cellular antioxidant defenses. The antioxidant activity of berberine has been widely demonstrated [34,[37][38][39]. For instance, berberine displayed peroxynitrite (ONOO − ) scavenging activity and total ROS inhibitory capacities [37]. Bhutada et al. [27] showed that berberine treatment during training trials also improved learning and memory, lowered hyperglycemia, oxidative stress, and ChE activity in diabetic rats.

(b) Anti-inflammatory properties of berberine
In the brain of patients with Alzheimer's disease, chronic inflammation has been well described. On the histological level, this inflammation is characterized by activated microglia, reactive astrocytes and increased inflammatory cytokines release [33]. This observation has led to the hypothesis that brain inflammation is a cause of neuronal damage in AD and anti-inflammatory drugs may be used as protective agents. Chen et al. [18] studied the functions of berberine involved in anti-inflammation and the amelioration of insulin resistance in the prefrontal cortex of diabetic rats. They found that intragastric administration of berberine (187.5 mg/kg/d) inhibited inflammation mediator release and insulin resistance in the mPFC of diabetic rats. Finally, it relieved the impairment of cognitive function in diabetic rats. The promising effect of Phellodendron amurense (PA) and its major alkaloid compound, berberine, on memory dysfunction has also been studied in scopolamine-induced memory deficient rats [26]. A two-week administration of 20 mg/ kg of berberine improved memory impairment as measured by the passive avoidance test, and it reduced the escape latency for finding the platform in the Morris water maze test.

(c) Anti-cholinesterase activity of berberine
The cholinergic hypothesis was initially presented several years ago, then several studies demonstrated the adverse effects of anticholinergic drugs on memory [40], the low intracerebral cholinergic activity in patients with Alzheimer's disease (AD) [41,42] and the association of AD with cholinergic transmission disorders [43]. This hypothesis suggests that decreased cholinergic activity is associated with the AD symptoms and the improvement of cholinergic activity will relieve the AD symptoms. The cholinesterase (ChE) is the major enzyme for acetylcholine destruction and its inhibition results in increasing acetylcholine level in the brain. Therefore, many anti-AD pharmacological studies have focused on cholinesterase (ChE) inhibitors to ameliorate the cognitive symptoms [44]. Several studies have been performed to examine the effect of berberine on the ChE activity. For example, chronic treatment with berberine (25-100 mg/kg) lowered oxidative stress and ChE activity in ethanol treated rats [21]. A similar promising effect of one-month treatment with berberine on streptozotocin-induced memory impairment in rats has been reported [20]. In another set of experiments, berberine (100 mg/kg) treatment during training trials also improved learning and memory and lowered hyperglycemia, oxidative stress, and ChE activity [27].

(d) Anti-amyloid activity of berberine
The 42-amino acid amyloid beta (Aβ) is released from cleavage of the amyloid precursor protein by β-secretase and γ-secretase [45]. The Aβ sequenced from the meningeal blood vessels of AD patients and individuals with Downs' syndrome is highly aggregated, and spontaneously assumes the β-sheet conformation and polymerizes into oligomers, fibrils, fibrils and plaques [46]. Berberine has been shown to ameliorates β-amyloid pathology and cognitive impairment in an AD transgenic mouse model [19]. After berberine treatment, the levels of extracellular and intracellular Aβ1-42 were decreased, mediated by increased autophagy activity.
With advances in science, there is increasing interest in another constituent of neurofibrillary tangles(NFTs), hyper-phosphorylated Tau protein. He et al. found that berberine improved learning and memory in APP/PS1 mice, decreased hyper-phosphorylated Tau protein and lowered the activity of NF-kB signaling in the hippocampus of APP/PS1 mice [17]. Berberine administration promoted the activity of glutathione (GSH) and inhibited lipid peroxidation in the hippocampus of AD mice. They concluded that berberine attenuated cognitive deficits and limited hyper-phosphorylation of Tau via inhibiting the activation of the NF-kB signaling pathway and by retarding oxidative stress and neuro-inflammation.

Opportunities and challenges
Berberine is a natural product with a definite structure and a wide range of pharmacological effects. Berberine displays many biological functions and potential therapeutic applications in neurological diseases. Animal research is an essential early step toward evaluating and developing an intervention for clinical trials in humans [31]. This systematic review has examined high quality animal studies on the anti-AD effects of berberine and finds a consistent effect of berberine in improving the memory defects in multiple animal models, indicating the therapeutic potential of berberine for treating AD. While the effects are clear, the mechanism is not; further research is needed to determine the details of the biochemical mechanisms and specific drug target(s). Meanwhile, perhaps the greatest barrier to the pharmaceutical development of berberine is its naturally low bioavailability. More effort, for example, in structural modification and/or pharmaceutical processing, is needed for berberine to achieve its full potential in clinical use [32]. The following suggestions are worth considering: 1. The feasibility of targeted drug delivery should be explored. It is difficult to achieve effective concentrations, especially in the brain, by oral administration so targeted administration is worth considering; 2. The effects of berberine in combination with other drugs for AD treatment can be tested. 3. The possibility of toxic effects of berberine during long-term drug administration must be considered, and thoroughly studied.

Conclusions
In this paper, we have reviewed 15 high-quality animal studies on the neuroprotective effects of berberine against AD, with systematic evaluation of its efficacy and pharmacology mechanisms. Berberine has showed significant memory-improving activities in multiple memory defects animal models; common properties, including anti-oxidation, anti-inflammation Zhu et al. [29] Ameliorates the spatial memory impairment. Phenomenon research.
ChE, cholinesterase; IL-1β, Interleukin 1 beta; TNF-α, Tumor necrosis factor alpha; GFAP, Glial fibrillary acidic protein; CD45, CD45 Antigen; mPFC, The medial prefrontal cortex; BACE-1, β-site amyloid precursor protein cleaving enzyme 1; ICV-STZ, Intracerebroventricular streptozotocin; NF-kB, Nuclear factor kappa-lightchain-enhancer of activated B cells; MAPK, Mitogen-activated protein kinases; PKCη, Protein kinase C-eta type; PKCε, Protein kinase C epsilon type; fEPSP, Field excitatory postsynaptic potential; cAMP, Cyclic adenosine monophosphate and anti-ChE activity were revealed. So far, no clinical trial has been carried out on the neuroprotective effects of berberine. Considering the positive results from animal studies and the relatively low toxicity of berberine, the performance of clinical trials to evaluate the anti-AD effect of berberine on human patients appears justified.