As a study novelty, luteolin’s neuroprotective properties in a model of brain ageing in rats and its potential molecular mechanisms have been demonstrated. This is the first study to implicate SIRT1 as a crucial upstream molecule that mediate luteolin’s modulation of GLO1/AGE/RAGE signalling and its associated neutotoxicity, involving OS, mitochondrial dysfunction, neuro-inflammation, apoptosis, and senescence.

According to our study, luteolin effectively ameliorated brain agieng as it enhanced cognative function and cholinergiac transmission and additionally improved the cytomorhological alterations in the hippocampal regions of the D-gal group. This came in line with He et al. [16] and Ahmad et al. [19], who validated the neuroprotective impact of luteolin.

In the present work, the MWM test revealed an evident impairment in both short and long-term memories among D-gal rats, which was in harmony with previous findings. [1] Conversely, luteolin treatment efficiently improved rats’ spatial memory, as indicated by the diminished escape latency and increment in the number of platform crossings. Furthermore, outcomes from the OFT demonstrated the notable competence of luteolin in enhancing the rats’ exploratory capabilities in a novel environment. Collectively, these findings suggested that luteolin has the potential to serve as a protective agent against D-gal-triggered memory impairment.

Certainly, Ach is a crucial neurotransmitter, implicated in learning and memory, and regulated by the AchE enzyme. Cholinergic neurotransmission has been improved by activating cholinergic receptors and elevating the amount of Ach in the neuronal synaptic cleft [1]. Dynamic alterations in Ach and AchE activity are linked to progressive cognitive deterioration [34]. The neurotrophic factor BDNF is a crucial molecular mediator in synaptic transmission, plasticity, and neuronal proliferation. Furthermore, BDNF enhances the viability and differentiation of cholinergic neurons and induces the release of Ach, thereby essential for learning and memory. [35] During ageing, the cholinergic neurons exhibit neurodegenerative changes, highlighted by decreased choline acetyltransferase and raised AchE activities, ultimately reducing Ach release. Likewise, BDNF levels decrease considerably in ageing, contributing to cognitive deficits. [36]

Consistently, chronic D-gal administration herein dramatically boosted AchE activity and substantially reduced Ach and BDNF levels in rats’ brains. The increased AchE enzymatic activity caused by D-gal-associated OS was shown to be strongly correlated with learning decline and memory deficits, as affirmed previously [1], which agreed with our findings. Importantly, luteolin treatment herein significantly restored the normal levels of BDNF and Ach and lowered the AchE activity, which in turn could enhance the cholinergic neurotransmission and cognitive capabilities. These data highlighted the neuroprotective impact of luteolin in this ageing model, which is highly consistent with Ryu et al. [37] and Ali et al. [38]

The physiological levels of GLO1 have been postulated to regulate organismal physiology and may offer novel targets for postponing ageing and associated illnesses. Our results revealed a significant diminution in hippocampal GLO1 activity with concomitant elevated AGEs levels and RAGE expression levels in the D-gal-treated rats. In harmony, Li et al. [13] showed that long-term D-gal administration reduced GLO1 and increased MG accumulation, the primary precursor to intracellular AGEs. The augmented AGEs enhanced the expression of their specific receptors, RAGE, which bound to AGEs in a dose-dependent manner [6, 39]. Moreover, Faruqui et al. [40] stated that the AGE-RAGE interactions and binding of RAGE with various other ligands not only contribute to the exacerbation of OS but also to the over-expression of RAGEs themselves. AGE/RAGE signalling activation triggered OS, mitochondrial dysfunction, chronic inflammation, and apoptosis, with subsequent brain senescence [8], all of which were proved and mentioned later herein by our findings.

Surprisingly, luteolin intervention induced the activities of the GLO1 enzyme, which could be attributed to its SIRT1 upregulation as delineated later by our findings. Activation GLO1 enzyme has been implicated in MG detoxification and counteracting D-gal-induced oxidative damage; this is supported herein by the notable reduction of AGEs and downregulation of RAGE upon luteolin co-treatment. In line, the AGEs/RAGE transduction axis was downregulated by luteolin in an earlier study. [19]

Based on previous reports, including our findings, the binding of AGEs to RAGE triggers OS, activating the RAGE signal pathway and contributing to the aetiopathogenesis of D-gal-induced oxidative brain damage [41]. A manifest elevated MDA level and XO activity, while reduced SOD activities were observed in D-gal rats, all of which were reversed upon luteolin intervention. This coincides with the results of Alekhya Sita et al. [15] and Liu Y et al. [42], who acknowledged luteolin’s antioxidant potential. Our data highlighted that luteolin could alleviate the D-gal-evoked OS, making it a promising candidate for neurodegenerative disease prevention and treatment.

Interestingly, D-gal-provoked OS could trigger mitochondrial damage and dysfunction, which is a major cause of neurodegenerative disorders. [43] In support, D-gal intervention significantly reduced the energetic function of mitochondria as delineated herein by the reduced hippocampal mitochondrial complex I and CS Activities, which was in consistent with Liu et al.‘s [41] previous results. On the other hand, luteolin could successfully reverse the D-gal-evoked mitochondrial dysfunction, which agreed with previous findings. [16] This could be attributed to its antioxidant potential, hindering mitochondrial oxidative damage and decreasing the AGEs and their related neurotoxicity [43]. Furthermore, Hu et al. [44] reported that luteolin raised the mitochondrial membrane potential in LPS-treated cardiomyocytes and improved the CS activity, ATP content, and mitochondrial complex I/II/III/IV/V activities, documenting our findings.

Concomitantly, OS can trigger inflammatory responses in D-gal-induced brain injury. [45] In harmony, an obvious escalation in IL-1β and TNF‐α levels was noted in the D-gal-treated rats, pointing to hippocampal neuroinflammation, which was substantially reversed by luteolin treatment, proving its anti-inflammatory effect, which has been formerly observed in various studies. [46] .

It is common practice to use the astrocyte activation biomarker GFAP as a measure of reactive gliosis, a characteristic of the ageing brain. [47] Thus, the relationship between soluble cytokine release and activated astrocytes/microglial cells raises the assumption that inflammatory processes are important in the pathophysiology of ageing. [48] Immunohistochemical findings elucidated that luteolin administration decreased the elevated GFAP expression in the hippocampal regions of D-gal-induced ageing rats, suggesting that luteolin may prevent hippocampal senescence as supported previously. [49]

Interestingly, compelling evidence postulated that mitochondrial dysfunction, OS, and inflammation mechanisms work in harmony to induce apoptotic neuronal cell death in D-gal-induced brain injury. [50] Consistently, the current investigation revealed dramatic up-regulation of caspase-3 relative gene expression, the executioner of apoptosis, in the hippocampal region. In contrast, luteolin exhibited anti-apoptotic properties revealed via significantly downregulating hippocampal caspase 3 gene expression, preventing downstream executioner protease production, which coincides with previous reports. [51, 52]

Matched with Ma et al. [8], we speculated that brain senescence is the convergence point for the activated AGE/RAGE signalling, and its associated OS, mitochondrial dysfunction, inflammation, and apoptosis were shown to be cardinal factors in evoking brain senescence. In parallel, the D-gal rats exhibited a significant escalation in senescence-associated biomarker P21, which was shown to be a critical mediator of P53-dependent cell cycle arrest. [53] Meanwhile, luteolin administration substantially downregulated the AGEs/RAGE/P21 signalling pathway, which could be mediated via its induction to GLO1 enzyme activities, counteracting the D-gal-induced oxidative brain senescence. In support, Zhu et al.‘s [5] study suggested that luteolin suppresses cellular senescence induced by H2O2 in an oxidant-challenged model. To the author’s knowledge, this is the first study to reveal that luteolin’s modulation on the GLO1/AGEs/RAGE/P21 signalling pathway is a cardinal mediator for its senolytic criteria in this brain ageing model.

The ageing elicited upregulation of the p53/p21 pathway was suggested to stop the cell cycle and proliferation, as demonstrated herein by a notable decline in Ki67 (cellular proliferation biomarker) immunoreactivity in ageing rats. [54] Interestingly, our findings observed that luteolin was able to reverse the decline in Ki67 in the ageing rats, indicating enhanced hippocampal neurogenesis, which was in line with the former study of Zhou et al. [55] Luteolin’s antioxidant effect, as recorded herein, could contribute to maintain balance in the redox state of neurons, which promotes cell proliferation, growth, and differentiation. [56]

Intriguingly, SIRT1 is involved in memory, learning, cognitive function, and neural differentiation [57]. Matched with previous studies [5], our data strongly suggested that luteolin could significantly upregulate the SIRT1 expression level that was downregulated in the ageing rats. SIRT1 has been proven beneficial in neurodegenerative diseases by regulating their pathogenic mediators, involving OS, inflammation, mitochondrial dysfunction, cellular apoptosis, and senescence. [58] SIRT1 confers cellular protection against OS through numerous mechanisms, such as modulating forkhead transcription factors, enhancing catalase activity, and inducing manganese SOD. [59] Interestingly, SIRT1 is one of the signalling pathways that control the expression of peroxisome proliferator-activated receptor-γ coactivator-1α, which is the main regulator of mitochondrial biogenesis. [8] Emerging research indicates that SIRT1 is a potent inhibitor of NF-κB signalling, contributing to its anti-inflammatory signature. Furthermore, SIRT1 downregulates p21 via deacetylation and inactivation of its upstream p53. [13]

Importantly, SIRT1 was documented for its upregulation of GLO1 activity [60, 61], consequently decreasing AGEs accumulation with ultimate suppression of the RAGE signalling pathway. In the same line, Zeng et al., [62] reported downregulation of RAGE signaling by SIRT1 via NF-kB blockage-dependant mechanism. These data hypothesize SIRT1 as a crucial upstream molecule that mediates luteolin’s modulation of the GLO1/AGE/RAGE signalling pathway and its associated neurodegenerative pathology, making SIRT1 a pivotal mediator of luteolin’s neuroprotective potential in our model.

This was further confirmed by our correlation analysis, in which SIRT1 relative expression was positively correlated with the hippocampal levels of Ach level and BDNF along with GLO1, SOD, mitochondrial complex I and CS activities, while exhibiting negative correlations with hippocampal AchE and XO activities besides MDA, TNF-α, IL-1β, AGEs, and P21 levels. Collectively, we could speculate that luteolin protects neuronal tissue against D-gal-provoked OS, mitochondrial dysfunction, neuro-inflammation, and senescence, potentially via SIRT1 upregulation, which showed great relevance. However, additional research is required to identify the upstream regulatory factors involved in luteolin’s SIRT1 upregulation. Figure 11 illustrates the proposed mechanisms that underlie luteolin’s neuroprotective signature against D-gal-induced brain ageing in rats.

The proposed mechanisms implicated in luteolin’s neuroprotective potency against D-gal-induced brain ageing in rats