In the present study, we demonstrated the learning and memory functions were impaired in APP/PS1 mice by SDA and MWM behavioral experiments. Subsequently, we inquired the TCMSP database to ascertain that most of the Chinese medicines used to treat AD contained AST. Based on this discovery, we intraperitoneal injection of AST in APP/PS1 mice for one month noticed that AST treatment productively accelerated the clearance of Aβ plaque deposits in the brains of APP/PS1 mice and further restored their cognitive dysfunction. Importantly, we revealed AST could active autophagy and keep autophagic flow smooth of APP/PS1 mice hippocampal neurons for neuroprotective effect by inhibiting PI3K/Akt-mTOR pathway (Fig. 8). The above findings indicate AST might be a potential trerapeutic agent for future clinical AD treatment.
AD, as a common progressively neurodegenerative disorder, endangers the memory and cognitive abilities of people around the world [34, 35]. As the universal animal model of AD, APP/PS1 mice stably display mouse/human APP located in neurons and mutant human PS1 and there are senile plaque deposits in the brain at the age of 6 months, and memory ability decline with age [36]. Our data in this study manifested that APP/PS1 mice had severe memory deterioration, which is consistent with several related researches [37–39]. The brains in AD showing excessive senile plaque depositions are extensively regarded as one of the earliest changes [40]. Concurrently, we observed that, dissimilar to WT mice, there were numerous Aβ plaque deposits in the brain of APP/PS1 mice. Interestingly, we found the contents of Aβ and Aβ1-42 which were the most common and toxic [41, 42] in the blood of APP/PS1 mice were much higher than those of WT mice. Hence, inhibiting the aggregation of Aβ and reducing the content of Aβ and Aβ1-42 in the serum of mice may be a hopeful treatment strategy for AD.
Currently, traditional Chinese medicines play an indispensable performance in the prevention and treatment of AD in domestic and international studies. Thus, we screened out AST, a potential new natural flavonoid isolating from traditional Chinese medicines for AD treatment [43], by consulting a large amount of literatures and operating the TCMSP database. In the central nervous system, AST further exerted a good hypnotic effect by prolonging the convulsion latency and diminishing the convulsion rate of mice [44]. Concurrently, AST showed neuroprotective impact on PC12 cells and SH-SY5Y by enhancing cell viability and weakening intracellular oxidative stress [45]. Additionally, the experimental evidence of Chung et al. proved that AST could prevent H2O2-induced cell death by inhibiting JNK, p38 and ERK 1/2 pathways in SK-N-SH cell [46]. However, studies on the function of AST in neurodegenerative diseases such as AD are comparatively less frequent and solely concentrated on the cell levels in vitro. In the previous investigation of AD in vitro, it has been reported that AST could shorten the intracellular tau protein level and alleviate the cell damage through pharmacological analysis [47]. Here, we administered AST to APP/PS1 mice and primarily confirmed that AST was validly in recovering impaired cognitive functions in APP/PS1 mice, including learning and memory and spatial navigation. Subsequently, we were surprised to discover that abundant and widespread Aβ plaques in the brains of APP/PS1 mice were productively abolished, as well as Aβ and Aβ1-42 levels in the serum of mice. Notably, the therapeutic effect of 40 mg/kg AST treatment was the most visible in this process. In line with those, we further discussed the relevant mechanisms of AST on the neuroprotective infection of APP/PS1 mice.
From early to late AD, the autophagy of hippocampal neurons is continuously disrupted and the process of autophagic flow is blocked, bringing about the inefficiency to eliminate the pathogenic Aβ plaques accumulated in the cytoplasm, thus exhibiting high toxicity to cells [48–50]. Revamping hippocampal neuronal autophagy functional impairment may be of indispensable significance in postponing aging and hampering neurodegenerative diseases such as AD [51]. In the present research, we revealed NeuN, a labeled neuronal protein, was co-expression with autophagy-related proteins involved in the whole autophagic flow by immunofluorescent multiple staining experiments, which further suggested the presence of autophagy in mouse hippocampal neurons. LC3B presents in autophagosomes in the form of LC3B-Ⅰ and LC3B-Ⅱ, while the level of the lapidated form of LC3B-Ⅱ is a factual measure of autophagic flux relative to the unprocessed LC3B-Ⅰ [52, 53]. SQSTM1/p62 is a ubiquitin-binding protein that can be efficiently degraded by autophagy, and thus, the level of p62 is another indicator of autophagic flux [54, 55]. The WB analysis demonstrated that LC3B-II levels in hippocampal neurons of APP/PS1 mice were much lower than those of WT mice, whereas p62 levels showed the opposite trend, which could be the result of blocked autophagic flow pathways. Importantly, the two changes were reversed again with treatment of 20 and 40 mg/kg AST, indicating that autophagy was activated. Owing to autophagic flow is a dynamic process, as a meaningful protein for autophagy initiation, Beclin-1 mediates the positioning of other autophagic proteins, and its increased levels indicate autophagic activation [56]. Here, we determined AST predominantly enhanced the decreased Beclin-1 levels coincided with the ability of AST to ensure that the autophagic flow initiation phase was normalized again in the hippocampus of APP/PS1 mice. ATG5 gene exerts an imperatively character in the extension of autophagosomal membrane as well as autophagosome formation [57]. Meanwhile, the role of ATG12, an essential gene of the ATG12-ATG5 coupling system, can not be underestimated [58]. In this study, ATG5 and ATG12 in the hippocampus of APP/PS1 mice shifted expeditiously from lower to higher after AST treatment, in a sense revealing that AST established the proper functioning of the intermediate link of autophagic flow (autophagosome formation phase). The inability of autophagosomes to fuse with lysosomes to further form autophagic lysosomes, which in turn break down harmful substances, can also lead to serious consequences [59]. The reduction of LAMP-1 (a lysosomal membrane-bound protein that works in autophagic lysosome fusion) in our study indicated that autophagic lysosome formation was arrested in the hippocampus of APP/PS1 mice [60–62]. Novertheless, AST bolstered the fusion of autophagosomes with lysosomes by extending LAMP-1 level, thereby accelerating the clearance of toxic substances in the brain of APP/PS1 mice. The above results indicated in the APP/PS1 mice hippocampal neurons AST could active autophagy and keep autophagic flow smooth by stimulating the degradation of p62, augmenting the formation of LC3B and Beclin-1, up-regulating ATG5, ATG12 and LAMP-I.
We hypothesized that AST upregulated the level of autophagy and controlled autophagic flow in the hippocampus of APP/PS1 mice by restraining the PI3K/Akt-mTOR pathway. mTOR as known to be the major negative regulator of autophagy, is monitored by PI3K/Akt kinase cascade response, and when it is inhibited, the autophagic activity is enhanced [63, 64]. Numerous researches have shown that superfluous activation of the PI3K/Akt-mTOR signaling pathway made for the inhibition of neuronal autophagy level and the inability to clear intracellular accumulation of Aβ and tau proteins in a timely manner, which to some extent aggravated amyloid plaque production and neurofibrillary tangles in the AD brains [33, 65]. In contrast, inhibition of PI3K/Akt-mTOR pathway was able to scale down Aβ aggregation and improve cognitive function of AD mice [66, 67]. In our study, equivalently, above mentioned pathway was aberrantly mobilized in the hippocampus of APP/PS1 mice compared to normal mice, whereas AST stimulated autophagy and sustained either process of autophagic flow unimpeded by inhibiting the PI3K/Akt-mTOR pathway.
In conclusion, our study demonstrated that AST could exert the partial neuroprotective effect on APP/PS1 mice by facilitating PI3K/Akt-mTOR-mediated autophagy flux pathway. And our data provides more theoretical support for the application of AST to clinical AD effective drug candidates. However, in the current study we didn't clearly clarify which key link autophagic flux was regulated by AST to exert neuroprotective effects. Therefore, we need to explore it further in the future.