HO has been reported to improve cognitive deficits in several animal models of AD via clearing Aβ deposition, inhibiting AChE activity and suppressing neuroinflammation [14, 16, 17, 37, 38], but the poor water solubility badly limited its bioavailability and potential medicinal application. Nano-DDS is beneficial for prolonging exposure time, increasing drug efficacy and overcoming poor bioavailability of drugs, which makes it appealing as a universal vehicle for lipophilic drugs. It is well-known that droplet size of nanoparticles (10–50 nm) is a critical factor since it is closely associated with the rate, extent and absorption of drug release. Our results showed that Nano-HO could form nano-sized microemulsion droplets (23.30 ± 0.46 nm) when diluted with distilled water (Fig. 1B). Meanwhile, low PDI reflects the uniformity of particle size. The closer the PDI value is to zero, the more homogeneous the droplets are [39]. Additionally, stability of nanoparticles partially depends on the surface zeta potential, a parameter that gives the magnitude of the electrostatic repulsive interactions between particles [40]. A higher value of zeta potential usually hinders the probability of coalescence, thereby maintaining homogeneity of droplet size [41]. Our results indicated that Nano-HO formulation exhibited a relatively high negative average zeta potential and a low PDI value, suggesting that it met the required zeta potential prerequisite for a stable microemulsion. In addition, the accumulative release rate of HO from Nano-HO (86.3%) in PBS (pH 7.4) was significantly higher than that from regular HO (27.0%) over a period of 24 h (Fig. 2A). Possible reasons may include that small droplet size of Nano-HO provided a large surface area for drug release into the aqueous phase. On the other hand, the pharmacokinetics study was investigated in rats to compare the bioavailability of Nano-HO with that of regular HO. The results demonstrated that the t1/2 and MRT0 − 12 were both prolonged in Nano-HO group than in the regular HO group, suggesting that the oral bioavailability of Nano-HO was greatly improved as compared with regular suspension. Moreover, the AUC0 − 12 h of Nano-HO (2.20 ± 0.06 µg·h/ mL) was significantly increased when compared with HO (1.18 ± 0.05 µg·h/ mL), resulting in a relative bioavailability of 186.44% to HO. These findings indicated that the improved bioavailability of Nano-HO was predominantly owing to the increased solubility. Moreover, Nano-HO exerted better improving effects on cognitive deficits in TgCRND8 mice than HO, and these findings were believed to be related to the improved oral bioavailability of Nano-HO.
Neuroinflammation is widely considered as one of the major pathological factors of AD. Microglias are the primary inflammatory cells in the brain. Astrocytes, the most abundant glial subtype in the central nervous system, also play a critical role in the pathogenesis of AD. Growing lines of evidence have demonstrated that Aβ accumulation in AD causes microglia activation and astrocyte recruitment, thereby inducing the release of pro-inflammatory cytokines including TNF-α, IL-6 and IL-1β [42–44]. Meanwhile, inflammation could induce the expression of BACE-1, promote Aβ deposition, and exacerbate tau protein hyperphosphorylation and neurons loss. Therefore, inflammation is the core driver of AD pathogenesis. In this study, we found that both Nano-HO and regular HO could prevent the microgliosis, astrogliosis and Aβ deposits in the hippocampus and cortex of TgCRND8 mice, as well as suppress the release of TNF-α, IL-1β and IL-6 in the brain tissues of TgCRND8 mice (Fig. 4 and Fig. 5). Interestingly, Nano-HO inhibited astrogliosis in both hippocampus and cortex of TgCRND8 mice in a more potent manner than HO (Fig. 5B). These findings indicated that the amelioration of Nano-HO on hippocampal-dependent memory function was attributable to its anti-inflammatory property.
It is well-known that Aβ deposition is a key pathogenic hallmark in AD pathogenesis. Increased production of Aβ peptides and formation of Aβ plaques through sequential cleavage of APP by the β- and γ-secretases contribute to the pathological basis of AD [45]. Specifically, p-APP (Thr 668), as observed near the plaques, may increase the Aβ levels by facilitating the exposure and cleavage by β-secretase BACE-1 [46]. PS-1 and APH-1 are vital catalytic submits of γ-secretase responsible for APP cleavage to Aβ [47, 48]. Increasing evidence revealed that proteolytic degradation is a particularly important determinant of cerebral Aβ levels, and Aβ-degrading enzymes including IDE and NEP play critical roles in Aβ degradation [49]. Therefore, inhibition of β- or γ-secretase or enhancement of Aβ-degrading enzymes could help to reduce the Aβ production. Our results demonstrated that Nano-HO showed similar effect in inhibiting the protein expressions of APH-1 and PS-1 as HO (Fig. 6). Interestingly, Nano-HO showed better effect on inhibiting the protein expressions of p-APP (Thr 688) and BACE-1, and enhancing the protein expressions of IDE and NEP than that of HO. These results suggested that Nano-HO may modulate APP processing and phosphorylation through suppressing the activities of β- and γ-secretases and enhancing the activities of Aβ-degrading enzymes to clear the Aβ deposition in the brains of TgCRND8 mice. Furthermore, our molecular docking results demonstrated that HO was well docked with BACE-1 at three active sites including Lys 107, Asp 216 and VAL 170 (Fig. 6E), suggesting that HO may be a BACE-1 inhibitor.
Abnormally high level of hyperphosphorylated tau protein is another typical pathological hallmark of AD, which also leads to oxidative stress via increasing the reactive oxygen species (ROS) production. Increased ROS could promote inflammatory response, then induce neuronal apoptosis or loss, ultimately resulting in learning and memory impairments [50, 51]. It has been reported that the phosphorylation of tau protein is abnormally accentuated at different sites of Thr 205 (7.61 times increase), Ser 396 (4.95 times increase) and Ser 404 (2.97 times increase) in the postmortem brain tissues of AD patients [52]. In addition, up-regulation of caspase-3 is directly responsible for cellular apoptosis in AD [53]. Thus, inhibition of tau protein hyperphosphorylation and neuronal apoptosis may be potential therapeutic targets for AD. Our results revealed that Nano-HO could inhibit tau protein hyperphosphorylation at Thr 205 and Ser 404 sites, as well as the protein expression of caspase-3, but enhance the protein expression of Bcl-2 in the brain tissues of TgCRND8 mice (Fig. 7), indicating that the inhibitory effect of Nano-HO on specific hyperphosphorylation of tau protein and apoptosis may be the underlying molecular mechanisms of its cognitive function improving effects.
Activation of JNK pathway has been consistently found in the surrounding area of the Aβ plaques in AD patients and transgenic mice via facilitating p-APP (Thr 668) in culture cell lines [54–56] and exaggerating p-tau (Thr 205) [57]. In our present study, both Nano-HO and HO significantly down-regulated the ratio of protein expressions of p-JNK/JNK in brain tissues of TgCRND8 mice (Fig. 8A). In addition, JNK pathway is also closely involved in the activation of GSK-3β, which is considered to be a key kinase responsible for APP phosphorylation in neuronal cells and intimately associated with AD progression [58, 59]. Hyperactivation of GSK-3β has been found in the brains of AD patients [60]. Suppressing GSK-3β activity has been demonstrated to decrease the generation and accumulation of Aβ in APP transgenic mice of AD [61]. Moreover, GSK-3β is also a major kinase associated with the aberrant phosphorylation of tau [62], which could be inactivated by phosphorylation at Ser 9 site, suggesting that agents with ability to up-regulate p-GSK-3β (Ser 9) maybe potential candidates for the treatment or prevention of AD [63, 64]. Our results indicated that Nano-HO had better efficacy than HO on enhancing the ratio of p-GSK-3β (Ser9)/GSK-3β in the brain tissues of TgCRND8 mice (Fig. 8C).
Meanwhile, CDK5 plays a crucial role in the development of central nerve system and AD progression [65]. Under pathological conditions, CDK5 was activated via direct binding to its neuronal specific activators p-35, and then aggravate tau hyperphosphorylation by enhancing GSK-3β, exacerbate neuronal loss and subsequently lead to neurodegeneration [66–71]. Therefore, agents that suppress the CDK5 activity may ameliorate plaque pathology, neurofibrillary and neuronal loss in AD. Our results indicated that Nano-HO suppressed the ratio of p-35/CDK5 in the brain tissues of TgCRND8 mice, suggesting that the cognitive deficits improving effects of Nano-HO were associated with its ability to inhibit the CDK5 activity, and the finding was also consistent with the improvement on kinase activity GSK-3β of Nano-HO.
The bacteria community in the gut can directly reflect the health status of the host by maintaining a certain proportion to protect the bacterial flora balance. The changes in bacterial diversity and richness can lead to the dysfunctions of the bacterial community, and trigger brain-gut axis dysbiosis, contributing to the occurrence of neurodegenerative disorders like AD [72]. In our study, the decreased Shannon index and increased Simpson index suggested that TgCRND8 mice were associated with the diversity and evenness deduction of the bacterial community, as compared with the WT mice (Fig. 9B-C), and the observation was consistent with the similar decline of bacterial diversity in AD patients [73, 74]. The structural variability or similarity among different treatment groups was assessed by system clustering tree, PCA and PLS-DA in our study (Fig. 9B and 9E-F). These results showed that the mice in Nano-HO group clumped visibly far away from the TgCRND8 mice, emphasizing that the bacteria community tended to recover to normal. This observation was consistent with the finding of the changed intestinal bacteria in AD patients as reported before [75].
Several studies have demonstrated an essential role of gastrointestinal microbes in the development of cerebral Aβ amyloidosis along with a peripheral inflammatory state [76, 77]. Bacteria living in the intestinal tract adhere to the intestinal mucosal surface of epithelial cells, forming bacterial flora, thereby affecting the intestinal integrity and permeability [78]. When the harmful bacteria destroyed the integrity of intestinal epithelial cells, the inflammatory reaction was triggered or aggravated accompanied with an increase in inflammatory cytokine (e.g., IL-6 and TNF-α) levels [79]. Our study showed that the bacterial community altered, which coincided with the productions of TNF-α, IL-1β and IL-6, along with increase of Aβ plaques in brains. These results implied that TgCRND8 mice might cause the damage of the brain via changing the bacteria condition in the gut.
Reduction of given beneficial bacteria increased the inflammation, which can be harmful to the intestinal structure. Such reduction can be characterized in Firmicutes spp. and Bifidobacteria spp. [80–82]. Metabolites secreted by Firmicutes spp. decreased the production of pro-inflammatory factors such as TNF-α, thus suppressed the occurrence of inflammation [83]. Probiotics such as Lactobacillales spp. and Bifidobacteriales spp. improved the conditions of inflammation and intestinal epithelial barrier function impairment [80, 82]. In AD mouse model, acetate (a metabolite of Bifidobacterium breve strain A1) has been reported to ameliorate cognitive disturbances [84]. It is worth noting that when compared to the WT mice (53.6%, in phylum level, 1.18% in order level, and 2.61% in genus level,), there was a decline of Firmicutes, Bifidobacteria and Lactobacillus by 42.4%, 87.3%, and 69.7%, respectively, in TgCRND8 mice (Fig. 11A, C and E), revealing that the reduction of beneficial bacteria was a potential cause of intestinal inflammation in TgCRND8 mice.
Additionally, fewer Actinobacteria, but more Bacteroidetes and Proteobacteria were found in the intestinal microbiota of AD patients or APP/PS1 transgenic mice when compared to healthy controls [74, 77], suggesting that bacterial dysbiosis was positively associated with the progression of AD. In our study, we noticed that the relative abundance of Firmicutes, Proteobacteria and Bacteroidetes were major community at the phylum level, which account to almost 90%, followed by Actinobacteria and Cyanobacteria. The relative abundance of Actinobacteria had an 88% decrease in TgCRND8 mice, as compared with the WT mice, while the proportion of the Proteobacteria, Bacteroidetes and Cyanobacteria visibly increased by 235.3%, 99.7% and 125% respectively in TgCRND8 mice, as compared to the WT mice. Those alterations were in accordance with the previous reports [85, 86]. Nano-HO inhibited the relative abundance of the Firmicutes, Proteobacteria, Bacteroidetes and Cyanobacteria in TgCRND8 mice as similar to that of regular HO. Interestingly, Nano-HO enhanced the relative abundance of Actinobacteria in TgCRND8 mice in a more potent manner than HO (Fig. 11A).
Recently, the effect of chronic Helicobacter pylori infection on AD has been demonstrated by the release of massive inflammatory mediators [87]. Helicobacter pylori filtrate could cause tau protein hyperphosphorylation in mouse neuroblastoma N2a cells and brains of rats via activation of GSK-3β [88]. Our results demonstrated that the relative abundance of Helicobacteraceae (at family level, Fig. 11D) in TgCRND8 group was augmented by 532.3% as compared to the WT group. Interestingly, Nano-HO reversed this change in TgCRND8 mice in a more potent manner than HO.
Mucin-degrading bacteria are identified as microbial drivers. Among them, Prevotella degrades mucin and Desulfovibrio enhances the rate-limiting sulfatase step by hydrolyzing glycosyl sulfate esters. Ruminococcus is also able to degrade mucins [89]. As probiotics strains, Akkermansia can secrete immunoglobulin A (IgA) and antibacterial peptides by immunological rejection to resist pathogen damage to the intestine, thereby possessing anti-inflammatory and barrier-improving properties [90, 91]. Our results showed that the relative abundance of Desulfovibrionales (at order level, Fig. 11C), Prevotellaceae (at family level, Fig. 11D) and Ruminococcaceae (at family level, Fig. 11D) drastically increased to 457.6%, 325.6% and 139.3%, respectively, in TgCRND8 mice, as compared with the WT group, and the changes may be of relevance to the increased transmembrane permeability. The relative abundance of Akkermansia (at genus level, Fig. 11E) significantly decreased in TgCRND8 group, as compared with the WT group. Nano-HO and HO reversed these changes in TgCRND8 mice. Figure 12 schematically summarized the molecular mechanisms underlying the cognitive deficits ameliorating actions of Nano-HO and HO in TgCRND8 mice.