Zheng Xiang-yi (郑湘宜), Du Yan-jun (杜艳军)

Acupuncture-moxibustion and Orthopedic College, Hubei University of Chinese Medicine, Wuhan 430061, China

Abstract

Objective: To evaluate the prevention and treatment effects of acupuncture-moxibustion for Alzheimer disease (AD)based on various AD mouse models.

Methods: Several representative types of mouse models were selected according to the pathophysiological causes of AD, including senescence accelerated mouse/prone (SAMP) mice, soluble amyloid-β protein (Aβ) injection mice/rats,amyloid precursor protein (APP) transgenic mice, and APP/PS1 double transgenic mice. Through the observation of behavioral changes and analysis of core items, the possible mechanisms of acupuncture-moxibustion in preventing and treating AD were explored.

Results: Acupuncture-moxibusiton therapy can improve AD mice’s cognitive dysfunction; the major action mechanisms including increasing cerebral blood flow, improving the expressions of vital proteins in the hippocampus, preventing neuron cell apoptosis, promoting the clearance of Aβ deposition, activating autophagy pathway to reduce memory deficits and regulating the metabolisms of brain-derived neurotrophic factor, tyrosine kinase receptor B, Nacetylaspartate and glutamic acid.

Conclusion: Although the optimal mouse model is not determined, it is sure that acupuncture-moxibustion therapy can improve cognitive function. A more suitable AD animal model should be duplicated in order to better explore the inherent action mechanism of acupuncture-moxibustion in preventing and treating AD.

Keywords: Acupuncture-moxibustion Therapy; Acupuncture Therapy; Moxibustion Therapy; Alzheimer Disease; Models,Animal; Mice

Alzheimer disease (AD), first mentioned by Alois Alzheimer in 1907, is a neurodegenerative brain disease,and progressively cognitive deterioration is one of its most obvious clinical manifestations[1]. The number of AD patients in the USA was estimated to be 5.4 million in 2016, and may probably reach nearly 13.8 million by 2050[2]. AD is the most common underlying factor of dementia, accounting for 60 to 80 percent of cases according to statistics[3]. The incidence of AD in China is comparable to that in Western countries[4]. The prominent pathological features of AD include the abnormal deposition of amyloid beta peptide and the hyperphosphorylated tau protein in neurofibrillary tangles (NFTs)[5], accompanied by dystrophic neurites,neuropil threads, associated astrogliosis and microglial activation[6]. The progression of AD has been categorized into three stages, namely preclinical periods with subtle behavioral symptoms, mild cognitive impairment, and dementia due to AD, which makes it clear that the pathological changes occur earlier than typical symptoms[7]. The clinical manifestations of AD are most frequently seen among the elderly, affecting several cognitive domains such as memory, executive function and language, which subsequently impacts the routine life and degrades the quality of life.Unfortunately, the existing drugs only exert symptomatic relief while no disease-modifying drugs are validated to reverse the condition, suggesting a serious socioeconomic burden on individuals, families and medical care systems[8]. AD has been acknowledged by the World Health Organization and the United Nations health agency as an intractable medical issue.Appropriate adoption of AD experimental models,mainly animal models and cell-culture models, is indispensable in the translation of preclinical studies to clinical trials[9]. Acupuncture has been a traditional medical treatment in China for over 2 000 years based on the meridian theory[10]. The good news is that it has gradually been applied to treat patients in America as a major complementary therapy since the hearing held by the National Institutes of Health in 1997[11]. It has been considered as a useful complementary treatment in serious conditions[12-16], such as nervous system diseases[17]. In view of a large number of studies regarding neurological diseases, the alleviation of cognitive loss in daily life via electroacupuncture (EA)may attribute to the onset of anti-oxidation pathway,the modulation of local inflammation, the prevention of nerve cell apoptosis and the promotion of neuron cell growth[18], which suggests the scientific and systematic utility of acupuncture in AD treatment. Additionally,researchers found that acupuncture may be more effective than medicine in improving the clinical syndromes of AD, not to mention about the high level of safety in clinical practice[19]. In the following section,we will review the effects of acupuncture in various mouse models of AD.

Selection of animal models for AD is critical to help assess the potential mechanisms and effects of acupuncture in the process, and explore innovative therapeutic alternatives for the global population. Quite a few animals have been confirmed to fit the criteria for replicating AD models, including mice, rats, dogs, cats,sheep, goats, Octodon degu, non-human primates(NHPs), and even drosophila[20-21]. It has also uncovered the role of β-amyloid precursor protein (β-APP) in the process of complex pathology[22]. Rodent models, such as rats and mice, serve commonly as AD models nowadays. The majority of animal models used in the studies of neurodegenerative processes are transgenic mice, though the mouse models fail to mimic the disease in all directions[20,23]. Rats may have closer physiological connection with human than mouse models, but in terms of genetics, morphology and the embryonic stem cells, mouse models show more priorities than rat models in solving technical problems.Apart from that, breeding mice is less expensive and time-consuming[24-25]. In this article, we focus on the mouse models to fully explore the effects and mechanisms of acupuncture in treating AD.

1 Commonly Used Experimental Models

Experimental models targeting AD play a significant role to determine the pathology and seek effective drugs. With regard to pathophysiology, four types of AD models were widely mentioned: physiological animal models, transgenic animals, interventional modelings,and human cell-based experimental models[20,26].

Researchers favor aging models such as NHPs in conducting cognitive deterioration researches related to human clinical studies for their considerable similarities with human in both overall brain structures and functional connections which play a critical role in the translational researches[27]. However, the wide adoption of aging animals is hindered by easy accessibility,financial limitations, and ethical factors[28]. The NHPs have long been criticized for their short supply in some species, resulting in high cost and long waiting time to get access to the animals.

Interventional animal modelings serve as the outcomes of surgery, drug efficacy researches, or application of environmental lesions. Amyloid-β protein(Aβ) injection animal models are widely adopted to impair learning ability and memory[29]. However, some scholars doubt that they have not shown obvious characteristics in AD proceedings, and these modelings fail to cover a wide range of signal patterns[26,30].Chronic D-galactose (D-gal) injection-induced subacute aging animal models are commonly used due to its cognitive deficits[31-32]. Yet, there are insufficient data to demonstrate that D-gal can reproduce the typical pathological features of AD[26]. Genetic engineering has accelerated the growth of genetic modified animals[33].Animals which are transgenic and express human protein mutants account for most familial AD models[18].Among the high-capacity genetic modified models,rodent models especially mouse models tend to have priorities over others for their short lifespan, easy breeding, low cost, and most importantly, their well-qualified genome to be modified and utilized[34].We must make it clear that none of the existing transgenic animal models can cover all aspects of AD pathology, but the multi-transgenic animal models can better mimic the proceedings of AD.

The application of AD neuronal cell culture models plays a vital role in neurodegenerative disorders.Human induced pluripotent stem cells (hiPSCs)[35-36]stand out as a powerful tool for searching underlying novel therapeutic strategies and deepening the study of pathogenesis of AD. Firstly, it avoids the species differences between human and animals in neuroanatomy[37]. Secondly, compared with human embryonic stem cells (hESCs), hiPSCs exist no ethical issues and little rejection between donator and donee[38]. However, some problems cannot be ignored towards the application of hiPSCs, including optimizing hiPSCs lines, determining the observation targets and mimicking patients’ brain[37](Table 1) .

Table 1. Merits and demerits of the four commonly used experimental models regarding AD

2 Commonly Used Experimental Models for Traditional Chinese Medicine (TCM)

It is widely accepted that pharmacological treatments,such as acetylcholinesterase inhibitors, can only relieve AD to some degree. Since AD is a complex and multifactorial disease, it’s crucial to combine other treatments with the efforts of professionals, clinicians and carers to attenuate clinical course[44]. Western nonpharmacological methods related to lifestyle and diet are usually mentioned in the guidelines. The TCM therapies cannot be ignored for their unique effects in treating AD. Having a good command of the experimental models related to TCM will benefit more patients. In terms of Chinese herbs, Zhang JH,et al[46]have found that Huatuo Zaizao pill, examined via six-month-old APP/PS1 transgenic mice, can reduce memory deterioration and strengthen synaptic function.With regard to Huannao Yicong recipe, early application to APP transgenic mice can reduce Aβ deposition related factors such as APP, BACE1, and PS1[45]. Besides,as for the moxibustion treatment, a systematic review targeting the underlying mechanisms of moxibustion from Korean experts summarized that all the related studies were based on the Aβ injection mice AD models[47]. Measuring the preventive effect of moxibustion for AD with Aβ1-42 Wistar rats, Du Y,et al[48]confirmed that pre-moxibustion could significantly reinforce the kidney essence, conforming with the theory of ‘preventive treatment of diseases’.The application of acupuncture in treating AD is not rare,and four types of AD models have been widely used to study the efficacy of manual acupuncture (MA) and EA[49-57]. In summary, TCM, especially acupuncture therapy, has been acknowledged as an effective method in laboratory experiments (Table 2).

Table 2. Commonly used experimental models in TCM studies concerning AD

3 Effects of Acupuncture-moxibustion on Various Mice AD Models

There are many methods for researchers to replicate AD mouse model. In view of the real situation and the integrity of the article, we mainly focus on three commonly used experimental mouse models, and several interventional rat models. By and large, all the AD models could fall into two categories, namely nontransgenic models and transgenic models.Unfortunately, there exist no alternatives for optimal experimental design. As for non-transgenic models,featured by the senescence accelerated mouse (SAM)models and soluble Aβ injection mice, most experiments target specific aspects of the disease,relatively easy to conduct, but failed to mimic the complex neurodegenerative processes. Transgenic models, widely acquired like APP/PS1 double-transgenic mice, remain a hotspot nowadays, making it possible to elucidate the pathological changes in the development of AD. Recently, a large number of experts have focused on the validation of acupuncture therapy to ease AD on the basis of mouse models altered in ethology,neuropathology, neurochemistry and other assessment indicators (Table 3).

Table 3. Widely accepted AD models for acupuncture

3.1 Non-transgenic mice

3.1.1 SAMP mice

The SAM models, which exhibit obvious symptoms of rapid aging naturally and spontaneously, were initially bred by professor Toshio Takeda at Kyoto University,stemming from AKR/J strain mice[64]. The sublines of SAM models contain SAM prone (SAMP) and SAM resistant (SAMR) strain mice, with the former including nine strains, well-known for SAMP8 and SAMP10, while the latter including three strains[65]. Neuropathological alterations, with resemblance to human AD, such as amyloid deposition and disrupted blood-brain barrier in the vessels[66], AD-like cortical network activity[67],increased oxidative stress[68], and microglial activation[69], have been confirmed in SAMP8 models.SAMP10 mice have already been widely applied to detect the etiology and pathogenesis of AD. SAMP8 and SAMP10 mice are both ideal animal models for studying brain-aging and dementia[70]. Both of them have cognitive impairments, close to the progressive development of clinical dementia[71], but they are still quite different in the pathological changes. SAMP8 mice have obvious inflammatory characteristics, with abundant Aβ in the brain[72-73], significant reduction in the density of hippocampal pyramidal cells in dendritic spines, and increasingly aging astrocyte hyperplasia and Periodic acid-Schiff staining positive granular structures in the hippocampus[74]. SAMP10 mice are a brain-aging model developed after SAMP8 mice, whose significant pathological features are extensive brain atrophy,reduced brain weight and aging of hippocampal capillaries[75-76]. Therefore, it is believed that SAMP8 mice seem to be more suitable concerning the pathological mechanism of AD at the level of genes and proteins[77]. These models contribute to figuring out the effects and mechanisms of acupuncture in the treatment of AD.

In terms of SAMP8 mice, manual stimulation to Baihui (GV 20) and Yintang (GV 29) once a day for 15 consecutive days demonstrated obvious decline in the escape latency and higher platform crossover frequency in the hidden platform and probe trial compared with the AD group[49]. It is worth mentioning that this study has first confirmed that MA can increase cerebral blood flow in the prefrontal lobe and hippocampus, which is quite important in the control of the memory organization, with no significant differences between the effect of MA and donepezil[50]. In another study,researchers stimulated Qihai (CV 6), Zhongwan (CV 12),Danzhong (CV 17), bilateral Zusanli (ST 36) and Xuehai(SP 10) once a day for 14 consecutive days to investigate whether acupuncture can reduce dendritic damage in the brain of SAMP8 mice[51]. The Golgi staining turned out that the total dendritic length and the amount of dendritic branches increased after the acupuncture treatment, compared with those in the SAMP8 group.In a recent study, He XL,et al[52]stimulated Baihui(GV 20) and Yintang (GV 29) in a similar way and ascertained that EA helped generate more terminaldeoxynucleoitidyl transferase mediated nick end labeling-positive cells in the brain and alleviate neuronal apoptosis in the SAMP8 mice. Additionally, the intensity of Aβ production was significantly decreased. In SAMP10 mice, manual stimuli to Danzhong (CV 17),Zhongwan (CV 12), Qihai (CV 6), Zusanli (ST 36) and Xuehai (SP 10) can reverse some AD-related gene expressions and also affect gene expressions not related to AD[60]. In summary, these findings emphasize that acupuncture stimulation to certain acupoints of SAMP mice can effectively mitigate the cognitive dysfunction,increase cerebral blood flow and the expressions of vital proteins in the prefrontal lobe and hippocampus, as well as prevent the neuron cells from apoptosis and reverse some genes related to AD.3.1.2 Soluble Aβ injection mice

For a long time, it was widely accepted that AD attributed to insoluble senile plaques of Aβ peptide and the NFTs, namely the amyloid cascade hypothesis (ACH).However, studies targeting the amyloid plagues for treatments only received few outcomes. In the recent years, the Aβ oligomer hypothesis (AβOH), derived from the original ACH, has ignited widespread curiosity[78-79].For the healthy individuals, Aβ is generated in the brain via a normal process, while for the AD patients, the balance between its formation and clearance is broken,leading to the abnormal formation of aggregates. There are several different types of aggregation state in the brain suffering AD, but soluble Aβ oligomers are confirmed to play a dominant role in affecting hippocampal synaptic function and impairing memory recall, and most strikingly, they are reversible[80-81]. The aim of injection of Aβ into mouse model brains is to induce cognitive deterioration, replicating the manifestation of AD. Three types of Aβ are commonly used, namely Aβ1-40 oligomer, Aβ1-42 oligomer and Aβ25-35 oligomer, in which Aβ1-42 oligomer is given priorities to the experiment concerning neurotoxicity[82].The Aβ1-42 oligomer, derived from the β-APP, is quite a highly hydrophobic aggregation compared with Aβ1-40 oligomer[83-84]. The intrahippocampal injection of Aβ1-42 has ascended to one of the most helpful animal AD models, involving the direct infusion of oligomeric Aβ1-42 into certain regions of the brain[85]. In this part,we will examine the function of acupuncture methods on AD in a novel way in Aβ injection mice.

Based on the bilateral hippocampal Aβ1-42 injection AD model, Tang SH,et al[61]applied acupuncture and moxibustion to Baihui (GV 20) and Shenshu (BL 23)once a day for 12 consecutive days, and determined that acupuncture plus moxibustion could not only improve the basic observation items of Morris water maze test compared with the model group, but also increase the transthyretin, lipoprotein lipase, alpha 2 macroglobulin, as well as apolipoprotein E contents in the serum, which may help the clearance of Aβ in the brain. With an Aβ1-40 injection model, researchers conducted immunohistochemistry and Western blotting to find that EA at Baihui (GV 20) and Shenshu (BL 23)played a crucial role in the up-regulation of PPAR-γ and down-regulation of p-p38MAPK expression which AD plays a detrimental impact[53]. Besides, other researchers confirmed the learning/memory progress via EA in Aβ1-40 rats by upregulating the autophagy pathway[86]. The Aβ25-35 injection models are commonly conducted in rats[59,87-88]. Early application of acupuncture and moxibustion to both Baihui (GV 20)and Shenshu (BL 23) prevented neuronal cytoplasmic edema in AD rats with the downregulation of axin and upregulation of β-catenin expression. And, the neuroprotective effects have close connection with the axin and β-catenin expressions. The experimental studies elucidated that acupuncture may promote the clearance of Aβ through the expression of certain proteins and activate the autophagy pathway to reduce the memory deficits.

3.2 Transgenic mice

Genetics have made great contributions to AD, and the discovery of genes related to AD has underpinned the fundamental development of mouse models. Hall AM,et al[89]harbored the view that all genes related to AD could be categorized into two type: mutated autosomal genes and polymorphic genes. According to the previous experiments, transgenic mouse models targeting the mechanisms and pathology of AD have received wide acknowledgment. The mutations of three genes, the APP, and the presenilin genes (PSEN1 and PSEN2) have been confirmed to cause autosomal dominant AD, though these mutations comprise a tiny propotion of AD patients[90]. Taken the lack of pathological changes into consideration, transgenic mice of tau mutations fail to be selected as they are more optimized for frontotemporal dementia mouse models[66,90]. It is important to realize that none of the existing transgenic mouse models can completely reproduce every aspect of AD, but multi-transgenic mouse models are prone to unravel both the clinical and pathological features of AD. A multidisciplinary method is needed to help select higher-qualified mouse models to meet the demand[54]. In this article, we select several commonly used transgenic mouse models to identify the effects and mechanisms of acupuncture in treating AD.

3.2.1 APP transgenic mice

Transgenic mouse models have been an ideal tool to determine the mechanisms and pathology of AD and contribute to conducting high-efficiency drug discoveries. Transgenic human APP accounts for the most widely accepted mouse models mimicking AD. EA at Baihui (GV 20) and Yongquan (KI 1) of APP 695 V 717 I transgenic mice with 2 Hz/100 Hz, 1-2 mA for three months revealed the down-regulation of Aβ expression and the decreasing number of neurons prone to apoptosis[81]. In another study targeting the cognitive levels, the expressions of Aβ and low density lipoprotein receptor-related protein 1 (LRP 1) for the same transgenic mouse strain, Xue WG,et al[62]found that great effects had been made by the EA group in the Lashley Ⅲ water maze test in comparison with the model group, and the results showed that the expression of hippocampal LRP1 increased while the Aβ deposition suffered a downward tendency. These experiments obviously indicated that EA can enhance the cognitive function of AD-affected APP transgenic mice via reducing the Aβ abnormal deposition,accelerating the expression of hippocampal LRP1 as well as slowing down the path of neural apoptosis.3.2.2 APP/PS1 double transgenic mice

Although transgenic mouse models remain one of the most useful methods replicating AD, most models merely focus on limited pathophysiological or behavioral characteristics. As known to all, there are varied factors accounting for the ongoing of AD, so it is of great importance to make preparations for the multiple transgenic mouse models. Various APP/PS1 double transgenic mice have been produced by AD researches via excessive replication of human APP and PS1 gene together[91]. What’s more, it has been confirmed by APP/PS1 mice that the nucleotide-binding oligomerization domain (Nod)-like receptor protein 3(NLRP3) inflammasome advanced the AD process[56].These multiple transgenic mouse models make it more convincible to determine the acupuncture effects.

Concentrating on the EA stimulation to single acupoint Baihui (GV 20), researchers found the improvement of cognitive function in APP/PS1 mice.Besides, the Western blot markedly illustrated the rising expression of brain-derived neurotrophic factor (BDNF)and proBDNF in the hippocampus[57]. Measured by magnetic resonance spectroscopy, the material metabolisms including N-acetylaspartate (NAA) and glutamate (Glu) metabolisms have witnessed significantly up-regulation in APP/PS1 mice after EA therapy was applied to Baihui (GV 20)[92]. Moxibustion at Baihui (GV 20), Fengfu (GV 16) and Dazhui (GV 14) for 20 min once a day has shown improvements in APP/PS1 double-transgenic AD mice[63]. Compared with the normal control group, the moxibustion group had lower expression levels of Aβ1-42, p-P70S6K, LC3-Ⅰand p62 proteins in the brain and enhanced autophagy. In terms of the grain-sized moxibustion, moxa applied to Xinshu(BL 15) and Shenshu (BL 23) showed significant effects on the memory deficit and the reduction of Aβ1-42 in the hippocampus[93]. The present studies demonstrated that the neuroprotective amelioration effect of EA may derive from the modulation of BDNF and tyrosine kinase receptor B (TrkB), the up-regulation of NAA and Glu metabolisms, and the inhibition of neurons apoptosis in APP/PS1 double transgenic mice, while the moxibustion therapy is vital in the up-regulation of autophagy, lower expressions of AD-related proteins and clearance of Aβ1-42.

4 Conclusion

To sum up, the results of acupuncture-moxibustion therapies regarding AD indicate that MA, EA,moxibustion, or even acupuncture plus moxibustion mitigate several aspects of cognitive dysfunction via increasing the cerebral blood flow, reinforcing the expressions of vital proteins in the hippocampus,preventing neuron cells from apoptosis, promoting the clearance of Aβ, activating autophapy pathway to reduce memory deficits and modulating BDNF, TrkB,NAA and Glu metabolisms. Although a number of animal models studies still lack in-depth exploration of the mechanisms of acupuncture therapy, they have set good examples for the future experiments. We should never stop our persistent striving in the pursuit of a distinct and reasonable explanation for the role of acupuncture- moxibustion in the treatment of AD with the help of various animal models.

Conflict of Interest

The authors declare that there is no potential conflict of interest in this article.

Acknowledgments

This work was supported by National Natural Science Foundation of China (国家自然科学基金项目, No.81473786, No. 81873380, No. 82074566).

Received: 6 December 2019/Accepted: 23 April 2020