Preclinical Evidence and Possible Mechanisms of Extracts or Compounds from Cistanches for Alzheimer’s Disease

Currently, disease-modified strategies to prevent, halt or reverse the progress of Alzheimer’s disease (AD) are still lacking. Previous studies indicated extracts or compounds from Cistanches (ECC) exert a potential neuroprotective effect against AD. Thus, we conducted a preclinical systematic review to assess preclinical evidence and possible mechanisms of ECC in experimental AD. A systematical searching strategy was carried out across seven databases from their inceptions to July 2018. Twenty studies with 1696 rats or mice were involved. Neurobehavioral function indices as primary outcome measures were established by the Morris water maze test (n = 11), step-down test (n = 10), electrical Y-maze test (n = 4), step-through test (n = 3), open field test (n = 2) and passage water maze test (n = 1). Compared with controls, the results of the meta-analysis showed ECC exerted a significant effect in decreasing the escape latency, error times and wrong reaction latency in both the training test and the retention test, and in increasing the exact time and the percentage of time in the platform-quadrant and the number of platform crossings (all P<0.01). In conclusion, ECC exert potential neuroprotective effects in experimental AD, mainly through mechanisms involving antioxidant stress and antiapoptosic effects, inhibiting Aβ deposition and tau protein hyperphosphorylation and promoting synapse protection. Thus, ECC could be a candidate for AD treatment and further clinical trials.

bring undesirable side effects, such as headache, dizziness, nausea, vomiting, insomnia, other somatic symptoms and drug interactions [11][12][13]. Diseasemodified strategies to prevent, halt or reverse AD progress are urgently needed. There are rising numbers of AD patients seeking various kinds of complementary and alternative medicines worldwide, among which Chinese herbal medicines (CHMs) have high potential [14].
Herba Cistanches, a desert living Cistanche, Roucongrong, the dried fleshy stem of Cistanche deserticola Y. C. Ma, first recorded in Shennongbencaojing (Shennong's Classic of Materia Medica, written about 475 B.C.-220 A.D.), is known as the desert ginseng and is of high medicinal value [15,16]. Showing a high antioxidative and antiinflammatory activity, Cistanches possess broad medicinal functions in neuroprotection, immunomodulation, endocrine regulation, hepatoprotection and bone-formation promotion. Nowadays, Cistanches is widely used in CHM formulas for treating various kinds of disorders, including aging and dementia [17][18][19][20]. Extracts or compounds from Cistanches (ECC), containing or representing the major bioactive ingredients, include Cistanches deserticola polysaccharides (CDPS), glycosides of Cistanches (GCs), and phenylethanoid glycosides (PhGs) such as echinacoside (ECH), acteoside (AS) and tubuloside B [21,22]. Some preliminary clinical trials [23][24][25][26] indicated that ECC monotherapy for AD symptom control encouragingly received positive feedbacks. However, the effects of ECC and possible mechanisms behind these effects on AD remain uncertain. Furthermore, the clinical study is limited owing to various restrictions due to morality and methodology [27]. The systematic evaluation of preclinical researches is an essential method to integrate preclinical evidence and can be of high value in improving the quality of preclinical researches and guiding potential clinical translation and application [28,29]. Thus, in the present study, we aim to conduct a preclinical systematic review of the efficacy of ECC and the mechanisms involved in experimental AD.

Search strategies
Seven English and Chinese databases, including PubMed, the Cochrane Library, EMBASE, China National Knowledge Infrastructure (CNKI), VIP Journals Database, China Biology Medicine Database (CBM) and Wanfang Database, were electronically searched from their inceptions to July 2018. The following keywords were used: "Cistanche* OR Roucongrong" and "Alzheimer's disease OR dementia OR mild cognitive impairment". All studies were limited to animals.

Types of studies
Animal studies that assess the effectiveness of ECC for AD were included, regardless of blinding, publication status or language. Reviews, comments, cases, clinical experiences or trials were excluded.

Types of experimental animals
Animal models of AD were included, regardless of animal species, gender, age and methods of model establishment. Models of other kinds of dementia, such as vascular dementia or Parkinson's disease, were excluded.

Types of intervention and comparator
Intervention versus comparator was as follows: A, ECC versus non-functional liquid/normal saline/no treatment; B, ECC versus western conventional medicine (WCM); C, ECC plus WCM versus WCM. ECC included CDPS, GCs and PhGs such as ECH, AS and tubuloside B, regardless of dose, form, administration method, or duration. However, ECC plus acupuncture/other CHMs versus acupuncture/other CHMs were excluded.

Types of outcome measures
The primary outcome measures were neurobehavioral function indices (NFIs) such as the Morris water maze test and step-down test. The secondary outcome measures were neurobiochemical and neuropathologic changes.

Data extraction
Two independent authors extracted data from the qualified articles according to a standardized data extraction form. The data of the highest dose were included when the treatment groups included various doses of the drug. The result of the peak time point was included when the data were expressed at different times. If published outcome data were demonstrated graphically, we made an effort to contact the author for further information. Digital ruler software was applied when a response was not received.

Risk of bias in individual studies
The risk of bias was assessed by the nine-item scale [30] and our previous publications [31] with minor modifications. In Item G, we considered the involvement of aged or female animals. Each item was given one point. Two reviewers independently evaluated the study quality. Divergences were well settled through consulting with correspondence authors.

Statistical analysis
The pooled analyses were carried out using RevMan 5.3 software. Heterogeneity across the subgroups was assessed using the Cochrane Q-statistic test and the I 2 statistic test. A fixed effects model (I 2 < 50%) or a random effects model (I 2 > 50%) was used depending on the value of I 2 . We calculated the standard mean difference (SMD) with 95% confidence intervals (CIs). Sensitivity analyses omitting one study at a time from the original analysis were conducted to demonstrate our main results to be robust. Considering two-tailed statistical tests, results were considered statistically significant when P< 0.05.

Study quality
The quality of the 20 included studies ranged from 4 to 7, with a mean score of 5.05. All studies used random allocation and declared no potential conflict of interests. Nineteen studies were peer reviewed, while one study [32] was an online PhD thesis. The use of anesthetic without significant intrinsic neuroprotective activity was reported in 18 studies, compliance with animal welfare regulations in 10 studies, control of temperature in 8 studies and animal models with relevant comorbidities in 8 studies.
No study reported sample size calculation or blinded assessment of the model or outcome; see Table 2. Step-through test compared with control group.

Neurobehavioral function indices
The Morris water maze test, including the spatial test and the probe test, was conducted in 11 studies [32, 33, 38-41, 43, 45, 47-49]. All 11 studies reported the spatial test using the escape latency as an outcome measure, of which 3 studies [33,48,49] provided graphic data, and we failed to apply digital ruler software or to get in touch with the author for further information.  Fig. 2A). Four studies [33,41,48,49] showed an insignificant decrease in escape latency in ECC group compared with WCM control; however, meta-analysis was failed owing to unavailable data in 3 studies [33,48,49]. In the probe test, meta-analysis of 4  Supplementary Fig. 1A). Two studies [45,48] showed ECC significantly decreased the total swimming length (P<0.05), whereas two other studies [33,49] showed there were no significant difference in reducing the swimming velocity (P>0.05) compared with controls.    The step-down test, including the training test for learning score and retention test for memory score, was conducted in 10 studies [32, 34-37, 42, 46, 48, 50, 51]. Meta-analysis of 8 studies [32, 34-36, 37, 42, 48, 50] showed ECC were significant for decreasing the error times  4D) and 1 study [32] showed ECC significantly decreased the number of errors in both the training test and the retention test (P<0.05) compared with controls. The open field test was conducted in 2 studies [33,49] that both clearly showed ECC increased the frequency of visits and time spent in the hole compared with controls (P<0.05), while 1 study [40] indicated that ECC markedly reduced the escape latency and the number of errors (P<0.01) compared with controls. However, compared with WCM controls, ECC were statistically less effective in increasing the frequency (n = 64, MD = −3.  Supplementary Fig. 1E).

Summary of evidence
This is the first preclinical systematic review to assess the efficacy of ECC for experimental AD. Twenty studies with 1696 rodents were selected. The quality of the studies included ranged from 4 to 7. The evidence available from the present study showed that ECC improved cognitive function in experimental AD mainly through mechanisms involving antioxidant stress and antiapoptosic effects, inhibiting Aβ deposition and tau protein hyperphosphorylation and promoting synapse protection.

Limitations
First, we only searched English and Chinese studies, which may lead to a certain degree of selective reporting and publication bias. It is well known that negative findings are less likely to be published. In the present analysis, some primary studies didn't provide original data, and some information was inaccessible. Thus, the dominance of positive studies might lead to the efficacy of ECC being overestimated. Second, the study quality was considered moderate, ranging from 4 to 7 out of 9 with a mean score of 5.05, indicating that the results should be explained with caution. Third, heterogeneity may exist due to the variety of AD model selection and preparation. Also, people with AD are always of old age, and a gender difference is observed in AD prevalence. In the present analysis, most AD models used healthy male rodents, which may lead to some challenges in clinical application.

Implications for practice
Preclinical animal research is the foundation of understanding of human diseases [52][53][54]; however, original preclinical research is often conducted with a small sample size, leading to less solid conclusions and poor repeatability [55]. The systematic review can integrate comprehensive preclinical evidence efficiently and guide potential clinical translation [56,57]. The present study showed ECC could improve cognitive function and exert potential neuroprotective effects in experimental AD according to a large amount of experimental animal data, with 1696 rodents, indicating that ECC are candidates for AD treatment and can be used for further clinical trials. Besides, systematic review of animal researches is a more economical and ethical method to integrate preclinical evidence, helping to reduce unnecessary sacrifice of laboratory animals and preventing invalid or less informative researches [58,59]. Systematic review of preclinical researches can identify defects in study design and implementation, contributing to improvement of the quality of follow-up preclinical researches [60,61]. In the present analysis, the quality of included studies ranged from 4 to 7 out of 9 points. The main flaws were a lack of sample size calculation, poor blinding in model induction and outcome assessment and an establishment of AD models based on no comorbidities. Reporting guidelines, such as ARRIVE, can provide guidance on the complete and transparent reporting of in vivo animal researches regularly and scientifically, helping to improve the quality of further researches [62][63][64][65]. Thus, we suggest that further animal researches of AD should follow up the reporting guidelines, increasing the value of clinical trials and further application.
Animal models are essential for understanding the induction and pathogenesis of a disease and developing therapeutic strategies that limit disease progression and eventually lead to effective treatments for the disease [66,67]. An ideal AD model is essential for preclinical research and should include the following points: (1) correspondence to AD pathogenesis; (2) stable cognitive impairment; (3) low mortality; (4) simple to operate [68,69]. In the present study, various kinds of AD models were used, including Aβ cerebral ventricle infusion, Dgal, scopolamine, sodium nitrite, AlCl3 or quinolinic acid intraperitoneal injection, and using SAMP8 mice and APP/PS1 transgenic mice. The former two kinds of AD models are more cost-effective and accessible, and are widely used in experimental AD research currently [70]; however, they can only partly simulate the pathological features and memory impairment symptoms of AD. In addition, injection injury and ischemia or anoxia in multiple local organs is inevitable. Based on the aging comorbidity and pathology of AD, SAMP8 mice and APP/PSI double transgenic rats are better to mimic the characteristics of AD [71]; however, the weaknesses are a longer preparation time and a higher cost, which to a great extent limits their current use in practice. Further research on ideal AD model is urgently needed, which may also be of great importance in data analysis and preclinical evidence assessment.
The present study demonstrated ECC had neuroprotective effects in AD models according to the neurobehavioral, neurobiochemical and neuropathological observations. The mechanisms of ECC for AD are summarized as follows: (1) Antioxidant stress: ECC passed through the injured membrane, affecting the signal pathway of reactive oxygen species (ROS). ECC reduced the amount of excessive ROS in the mitochondrion, increased the activity of GSH-PX, SOD and sodium-potassium adenosine triphosphatase (NA + -K + ATPase) and decreased NO and MDA levels [39,72,73]. (2) Regulation of neuroinflammation: ECC decreased the level NO and down-regulated the over-activation of microglia, exerting potential inhibitory effects on microglia involving neuroinflammation [74,75]. In the included studies, the effects on neuroinflammation of ECC in AD are less pronounced, indicating modification in further research is needed. (3) Resisting Aβ deposition and tau protein hyperphosphorylation: ECC decreased Aβ deposition and tau protein hyperphosphorylation [33,43,49], which may have an effect on oxidant stress and neuroinflammation; however, evidence on how ECC inhibit Aβ deposition and tau protein hyperphosphorylation is lacking, and further preclinical researches in vitro are essential. (4) Synapse protection: ECC decreased the activity of AchE, maintained normal Ach and NE levels in cholinergic neurons and increased the level of DA in the hippocampus. ECC activated the NMDA receptor and ameliorated the loss of synapses [48,50], helping to regulate the proper function of synapses and guarantee essential intercellular contacts. However, evidence on the influence of ECC in increasing the level of 5-hydroxytryptamine (5-HT) in the brain is inadequate currently [41]. (5) Antiapoptosis: ECC can maintain the mitochondrial membrane potential and reduce the amount of excessive ROS, inhibiting the initiation of neural apoptosis [32,76]. ECC can up-regulate the expression of Bcl-2, decrease the ratio of Bax/Bcl2, down-regulate the expression levels of Caspase-3, P53, P65, SYN, PSD-95 and iNOS [45,77] and reduce neuron apoptosis eventually. (6) Maintaining neuronal calcium homeostasis: An abnormal calcium steady state is the final common pathway of neuron destruction and is connected to oxidant stress, neuroinflammation, Aβ deposition and tau protein hyperphosphorylation [78,79]. ECC can protect the neuronal membrane, regulate the opening of calcium channels and maintain neuronal calcium homeostasis [34,37,80]. Further researches on calcium homeostasis and possible signal pathways are of great importance. In concluding, ECC act through complex, multicompound, multitarget and multipathway mechanisms in AD and might prove to be of great value in further clinical trials.
Animal experiments have contributed to our understanding of disease mechanisms, but the translation of preclinical experiments, which results in a prediction of the effectiveness of treatment strategies, to clinical trials is still challenging [81]. AD patients always have other medical problems such as aging, diabetes, hypertension and hyper lipidemia [82], and a gender difference is observed in AD prevalence [83]. The present study mainly included healthy male rats/mice, which may lead to selection bias to some extent. Registration of animal research prior to its execution in a generally accessible database similar to human (drug) research (www.clinicaltrials.com) may help to provide a more informed view before proceeding to clinical trials and may reduce publication bias [69,81].

Conclusions
The present study showed ECC could improve cognitive function and exert potential neuroprotective effects in experimental AD, largely through mechanisms involving antioxidant stress and antiapoptosic effects, inhibiting Aβ deposition and tau protein hyperphosphorylation, and promoting synapse protection. Thus, ECC could be a candidate for further clinical trials of AD.