Aluminum has been examined for its broad neurotoxic effects and close relationship with AD, which promote tau hyperphosphorylation, aggregation, and neurofibrillary tangle formation in AD brain (via activating tau kinases CDK5 and GSK3β), accumulate in microglia and induce proinflammatory cytokines, bind to Aβ and induce its aggregation, stimulate iron-induced membrane lipid peroxidation and oxidative damage, decrease the activity of antioxidant enzymes, interact with AChE on γ-peripheral site to enhance enzymatic activity resulting in reduced neurotransmission [12, 58, 59]. Furthermore, activated AChE can deteriorate Aβ aggregation, decrease BDNF expression [58], and further promote oxidative stress and neuroinflammation through a ‘cholinergic anti-inflammatory pathway’ (CAIP) via α7 nicotinic acetylcholine receptors [59, 60]. Besides, in many reports on Aluminum induced AD or toxicity models, the alteration of host gut microbiota had been observed [61, 62].
The present study has established the zebrafish model of subchronic inflammation induced by acute i.p. AlCl3 administration, resulting in subchronic peripheral and central inflammatory responses and enhanced oxidative stress and AChE activity in the brain. On behavioral level, administration of AlCl3 strongly impaired spatial and contextual memory of zebrafish in the T-maze test. On gut microbiota level, high-throughput sequencing results showed that the intestinal flora of zebrafish was dramatically disturbed by acute AlCl3 administration. Collectively, these findings are generally consistent with previous evidence that AlCl3 induce memory deficits in both humans and animals, including zebrafish, and change intestinal flora [61–63]. In contrast, BTL-Ⅰ co-administration reversed these induced memory deficits and microbiota imbalance, indicating potential neuroprotective role of this drug.
In the present study, acute central and peripheral inflammation was characterized by release of pro-inflammatory cytokines IL-1β and TNF-α following AlCl3 administration. Supplementation with BTL-Ⅰ potently inhibited acute central and peripheral inflammation in the AlCl3-treated zebrafish. Mounting evidence implicates BTL-Ⅰ in multi-targeted neuroprotective activity against oxidative stress, neuroinflammation and neuronal apoptosis, as well as in nerve growth without inducing cytotoxicity [22–26]. Here, we found that BTL-Ⅰ also prevents cognitive deficits (induced in zebrafish by AlCl3) and exerts neuroprotective effects in this zebrafish model.
Previously studies have shown that hepatotoxicity and liver injury potentially induce inflammation [64, 65] and lead to increased levels of inflammatory markers such as IL-1β and TNF-α [66, 67] In this study, we noted that peripheral TNF-α levels were elevated after BTL-I administration, presumably due to BTL-I's potential hepatotoxicity and liver injury effects. This is suggested by ADMET analysis (Table 1). But It remains unclear why peripheral IL-1β and TNF-α behave differently after BTL-I administration, which may be attributable to the different mechanisms, and this needs further investigations.
The inhibition of brain AChE elevates ACh levels, and hence positively affects cognitive function in rats [59, 68]. Subchronic exposure of zebrafish to AlCl3 or i.p. injection of AlCl3 in mice enhance brain AChE activity [37]. In line with this, our results show that i.p. injection of AlCl3 also elevate AChE activity in zebrafish brain, whereas BTL-Ⅰ evokes neuroprotection and lowers AChE activity (Fig. 4e). Because the thin-layer chromatography bioautography shows that BTL-Ⅰ does not inhibit AChE catalytic activity (data not shown), this compound seems to indirectly decrease zebrafish AChE activity here, likely involving other molecular pathways.
Furthermore, oxidative stress involves the excessive production of ROS and reactive nitrogen species (RNS) [69], and may result in tissue damage. GSH is the most important nonenzymatic antioxidant, whose neuroprotective role in the brain is critical against oxidative damage caused by catecholamine oxidation or lipid peroxidation [70]. In the present study, GSH levels markedly decreased, 24 h after the AlCl3 administration. With the pretreatment of BTL-I at the dose of 25 mg/kg and 50 mg/kg, the GSH levels of the zebrafish were even lower than the model group. But, when the dose rose to 100 mg/kg, the GSH content increased to the same level as model group though it was still lower than the control group. BTL-I seemed to display doubtful antioxidant effect via GSH. The lack of significant antioxidant effects of BTL-I relative to the model group may be due to the limited sample size and the resulting low statistical power. Although it may also suggest that the antioxidant mechanism of action of BTL-I may exist elsewhere. Further study with larger sample sizes and better designs are warranted to testify and explain this complicated phenomenon for a solid conclusion.
Intestinal flora plays a crucial role in the stability and balance of intestinal microecological environment, and the composition of human intestinal microbial community remains basically stable after the age of 3 years [71]. In recent years, a growing number of studies had shown that specific intestinal flora play important role in neuroprotection and their disorders were closely related to neurodegenerative diseases including AD [15, 16, 72]. Some reports indicated that, in the gut of healthy human or animals, there are higher population of Gram-positive (G+) bacteria including Firmicutes and Actinobacteria and lower Gram-negative (G−) bacteria like Bacteroidetes on phylum level [15, 16]. At family or genus level, Some G+ taxa like Bacillus, Eubacterium, Clostridiaceace in Firmicutes and Bifidobacterium in Actinobacteria show higher abundance in healthy individuals and benefit their hosts through different mechanisms including reducing leakage of gut by the protection of biofilm, inhibiting inflammation, anti-oxidation, reducing Aβ deposition and transferation from gut to brain, etc [15, 16, 73]. On the contrary, some G− taxa like Bacteriodes, Blautia, Escherichia coli, Shigella, Chlamydia, Fusobacterium, etc., are closely and positively correlated with AD mainly involving the activation of systematic inflammation by their enriched LPS in cell wall, the invasion of proinflammatory cytokines, LPS, and even bacteria into blood circulation system and brain, inducing Aβ deposition and tau phosphorylation [15, 16, 72, 73].
In the present study, the control group zebrafishes host higher G+ bacteria (Firmicutes on phylum level and predominantly Bacillus on genus level) than AlCl3 injured model group with memory impairment, while the model group zebrafishes have much less G+ bacteria than the control group but significantly more G− bacteria including Cetobacterium (in family of Fusobacteriaceae) and Chlamydiales (on order level). This is highly consistent with the previous studies especially the report on the benefits of Bacillus subtilis in delaying neurodegeneration and behavior impairment in the AD model Caenorhabditis elegans and the report on the negative effect of G− bacteria including Fusobacterium and Chlamydia [72–74].
Generally, the pretreatment of BTL-I maintained the dominance of G+ bacteria VS G− ones in the gut of zebrafishes when facing the impact of Aluminum. However, the dose levels exerted different influence. In low and high dose groups, the abundances of Bacillus have kept at high levels close to that of control group and the abundances of G- bacteria were much lower. Additionally, in low dose group, another G+ bacteria Micromonospora in phylum of Actinobacteria was recognized as a biomarker, hinting its possible positive role, when compared with a report [16] about Actinobacteria. Likewise, in high dose group, G+ bacteria Lutispora in family of Gracilibacteriaceae and order of Clostridiales was also biomarker. Considering the report on the strongly negative correlation of Clostridiaceae [16] with AD biomarkers in cerebrospinal fluid, we speculate that Lutispora may also have some benefit in neuroprotection.
It’s intriguing that the middle dose group did not possess high abundance of Bacillus. This discrepancy may be attributable to a non-linear relationship of BTL-I and Bacillus, more concentration gradients of BTL-I will be set in the further study to explore this relationship. However, G+ bacteria Clostridiaceae was found to be the key biomarker of this group; besides, G− bactereia Chloroflexi bacteria was its another key biomarker but bacteria in this phylum have no LPS in cell walls [75]. These may help to explain the behavioral improvement of this group.
Our study suggests that administration of marine fungal metabolite BTL-I prior to AlCl3 injection may be able to maintain the predominance of beneficial G+ bacteria in the gut of zebrafish to resist the acute injury of Aluminum, the related inflammation and AD pathology. The detailed mechanisms of intestinal flora regulation and the treatment effect on AlCl3 induced chronic AD model need to be further investigated for BTL-I in future.
The early evaluation of ADMET and drug-likeness properties of drug candidates are highly significant, as many drugs have been withdrawn in clinical trials and even in the marketing process due to unacceptable pharmacokinetics properties [76–78]. In this sense, the prediction of ADMET and drug-likeness properties of drug candidates have received extensive attention. And numerous tools have been developed such as ADMETlab [79] admetSAR [80] and SwissADME [81] In this study, the in silico prediction with ADMETlab suggested that BTL-I caters to the majority of the ADMET properties, drug-likeness profiles such as the typical Lipinski rule with 0 violation, and possess good properties in crossing BBB. Such features render it a promising drug candidate for NDs, since overcome BBB penetration is essential for the drugs of NDs [82, 83] While it should be noted that BTL-I was predicted to be at high risk of liver injury, which requires further confirmation and assessment and may provide clues for structural optimization.