Parkinson’s disease (PD) is a progressive age-related neurodegenerative disorder that affects 12% of the population over 65 years. Its incidence is expected to double by 2030 due to the global increase in the age and lifespan (Hegarty et al., 2020).

The exact mechanism of PD has not yet been fully explained. However, PD is considered a multifactorial disease that includes a variety of genetic and environmental factors by which oxidative stress, neuroinflammation, and mitochondrial dysfunction play an essential role in the pathogenesis of PD (Hattingen et al., 2009). The available therapeutic approaches for PD are mainly symptomatic and can only improve the clinical symptoms; however, they cannot stop the PD progression (Zhao et al., 2019).

Recently, various studies have shown the protective role of Sirtuin-1 (Sirt1), one of the seven Sirtuins, in neuroinflammation. Sirt1 exerts an important effect in modulating multiple physiological processes, including programmed cell death, DNA repair, inflammation, and oxidative stress (Jiao and Gong, 2020). Several observations suggest that Sirt1 could induce a beneficial effect in PD models. Numerous in vivo and in vitro studies have found that upregulation of Sirt1 provided a protective role in various experimental models of PD (Zhang et al., 2018).

In addition, PD is correlated with protein aggregation in dopaminergic neurons (DA) (Prasad and Hung, 2020). As autophagy eliminates protein aggregation and accumulation of dysfunctional cell organelles, a defective autophagy-lysosome system is considered a key pathomechanism in PD (Monaco and Fraldi, 2020). Therefore, it is suggested that autophagy activation is one of the important therapeutic strategies in PD (Lin et al., 2019).

Thus, Sirtuins and functional autophagy appear to play a protective role against environmental stresses, including nutrient stress (Kroemer et al., 2010; Kim et al., 2013). Under cellular stress, Sirt1 activity is stimulated, autophagy-related proteins are deacetylated, and consequently, autophagy is enhanced. The characteristic feature of autophagy in mammals is increased microtubule-associated protein 1A/1B light chain 3 (LC3) expression. Importantly, increased Sirt1 expression is convenient for induced autophagy by deacetylation of LC3 (Lee et al., 2008).

Nuclear factor-κB (NF-κB) transcription factor is a central regulator of inflammation and apoptosis. NF-κB is involved in the aging and pathogenesis of a wide variety of neurodegenerative disorders. Recent studies highlight the role of NF-κB subunits in PD and indicate that dysregulation of this transcription factor may be associated with the onset of PD (Bellucci et al., 2020).

The relationship between calcitriol, the active form of vitamin D (VD), and PD has attracted consideration (Newmark and Newmark, 2007). Muscular and motor impairments, which could induce detrimental effects on motor behavior, were reported in VDR-knockout mice (Burne et al., 2005). Interestingly, Wang et al. (2016) found that decreased serum calcitriol levels could increase the risk of PD. Furthermore, patients with PD with lower calcitriol levels may exhibit more severe symptoms compared to patients with normal calcitriol levels (Sleeman et al., 2017). Recently, limited studies have discussed the neuroprotective effects of VD in experimentally-induced PD models. VD was reported to reverse behavioral changes and improve the decreased DA content in the striatum of 6-OHDA induced PD model. Furthermore, VD significantly improved the expression of tyrosine hydroxylase (TH), dopamine transporter (DAT), as well as brain-derived neurotrophic factor (BDNF) and reduced the expression of mitochondrial injury, oxidative stress (nitrite contents, H₂O₂, and lipid peroxidation) and inflammatory markers (TNF-alpha, IL-Iβ) markers (Lima et al., 2018; Araújo de Lima et al., 2022; Bayo-Olugbami et al., 2022). However, none of the previous studies discussed the effect of VD on Sirt1 and autophagy in the experimentally-induced PD models.

The present study was designed to investigate for the first time, the association of Sirt1/NF-κB molecular pathway and autophagy in the midbrain of the rotenone-induced PD model. The study also aimed to explore and compare the protective and restorative effects of calcitriol on the induced PD model.

Current dopamine replacement therapies for PD provide relief from some disease symptoms; however, their effectiveness wears off over time and their long-term use results in serious side-effects for PD patients (Hegarty et al., 2016). Therefore, the establishment of novel therapeutic approaches is urgently required. Although calcitriol is initially known for its importance in calcium homeostasis and metabolism, it is recently associated with multiple pathological disorders, including PD (Fullard and Duda, 2020). Current evidence suggests a transposed correlation between calcitriol levels and the risk of developing PD (Lv et al., 2020). In addition, the vitamin D receptor and 1α-hydroxylase, the enzyme responsible for converting vitamin D to its active form (calcitriol), are strongly expressed in SN (Eyles et al., 2005). These findings support the assumption that impaired vitamin D levels may result in malfunction or increased cell death within the SN (Fullard and Duda, 2020). The pathophysiology of PD is affected by calcitriol through genetic and non-genetic routes (Ebert et al., 2006). Calcitriol can enhance or decrease the expression of several genes, thus affecting intracellular signaling pathways (Lv et al., 2020). Our study reported that calcitriol significantly decreased ROT-induced motor impairment, neuroinflammation, and autophagy dysfunction in the midbrain of the rat model of PD presumably due to calcitriol-upregulated Sirt1 expression that could in turn with subsequent stimulation of autophagy and inhibition of NF-κB.

In the current study, ROT treated rats showed a significant dysfunction in locomotion and coordination. Consistent with our findings, ROT-treated animals showed motor abnormalities in the open field test (Zaitone et al., 2012). Our results of open field tests suggested calcitriol supplementation improves motor performance in rats, in agreement with Sakai et al. (2015) who showed that 1,25-dihydroxyvitamin D enhanced locomotor performance in mice. Calcitriol supplementation improved muscle strength and physical activity (Girgis et al., 2014; Wyon et al., 2016). Our results revealed that calcitriol affected balance and coordination as assessed by the rotarod test. We demonstrated an improvement in rotarod and immobile latency. These results were in agreement with Seldeen et al. (2020) who confirmed that mice showed better rotarod performance after three months of calcitriol treatment.

Parkinsonian rats injected with ROT displayed significant dopaminergic neurodegeneration in SN represented by decreased TH-immunopositive neurons. Consistent with our findings, Betarbet et al. (2002) reported that ROT-treated animals caused nigrostriatal neuronal loss. Furthermore, Zhang et al. (2019) showed that the number of TH-positive neurons in the normal brain hemisphere was twice the number of TH-positive neurons in the damaged brain hemisphere in a 6-hydroxy-dopamine Parkinsonian mouse model. TH is a rate-limiting enzyme for dopamine synthesis and, interestingly, may be directly regulated by calcitriol, which is also consistent with our observation by immune-histochemical staining. In line with this, Lima et al. (2018) found that calcitriol increased immunostaining of TH in hemi-parkinsonian. Cui et al. (2015) showed that application of calcitriol, before or after local 6-hydroxydopamine injury, relatively restores TH protein expression levels and TH-immunoreactive fibers in the striatum and SN. Although it is a matter of debate whether dopaminergic neurogenesis occurs in the adult midbrain, some reports showed that dopaminergic neurons are capable of regeneration in the salamander model of PD, probably by activating stem cells along the ependyma of the third ventricle and/or reprogramming of differentiated cells. Consequently, this restoration of dopaminergic cells could contribute to the behavioral recovery (Parish et al., 2007). However, further future studies are required to address this possibility in mammals.

Sirt1 is strongly expressed in both neurons and glial cells in the human brain (Koronowski and Perez-Pinzon, 2015), as well as in the adult mouse brain (Zakhary et al., 2010). It is also expressed in the nucleus and cytoplasm of dopaminergic neurons and microglial cells in SN of rats (Diaz-Ruiz et al., 2015). Its expression is reduced due to aging and some neuropathological changes (Quintas et al., 2012). Although there are very limited studies correlating PD with Sirt1 expression, α-synuclein protein aggregation reduces Sirt1 expression (Manjula et al., 2020), which may explain why patients with PD are particularly susceptible to neurotoxin-induced neuronal damage (Li et al., 2020). Our study demonstrates that ROT down-regulates Sirt1 expression, which is in agreement with Tao et al. (2020) who showed that Sirt1 levels were decreased in ROT-treated animals and in vitro models of PD.

However, previous reports have already documented that in vitro calcitriol upregulated Sirt1 and reversed Sirt1 down-regulation in human umbilical vein endothelial cells exposed to H₂O₂ oxidative stress (Polidoro et al., 2013). Chang and Kim (2019) revealed that vitamin D insufficiency significantly reduced Sirt1 mRNA expression in induced obese rats, while vitamin D supplementation restored Sirt1 transcription levels. Consistently, our results reveal that although the application of calcitriol in healthy rats had no effect on Sirt1, calcitriol supplementation in combination with ROT for 4 weeks rescued the expression of Sirt1, although it was lower than the control group. This may be explained by the long-lasting inhibitory effect of ROT on Sirt1 expressions. In addition, calcitriol administration for eight days after PD induction also up-regulates Sirt1. However, our data limit to gene expression level, while Sirt1 protein level as well as Sirt1-related activation markers of mitochondrial biogenesis, remain to be examined in future studies.

Dysregulation of autophagy has been involved in a wide range of environmental chemical-induced neurotoxicity (Zhou et al., 2017). Furthermore, strong evidence indicates that dysfunctional autophagy leads to the accumulation of abnormal proteins, including α-synuclein (Ghavami et al., 2014), or accumulation of damaged cell organelles in PD models (Xiong et al., 2013). Impairment of autophagy has been demonstrated in patients with Lewy body disease and in a model of α-synucleinopathy. It has been reported that α-syn can interfere with the early stages of autophagosome formation. Thus, accumulation of α-syn is cytotoxic for neurons and can also cause a disruption of the autophagy process, leading to even faster protein accumulation (Rakowski et al., 2022). The present study employed ROT as an environmental chemical neurotoxin and is capable of inducing PD in rats (Betarbet et al., 2000).

LC3 is widely recognized as an autophagic indicator to assess autophagy activity of autophagy as it is present in the membrane of the autophagosome (Kabeya et al., 2000). Furthermore, P62 is an autophagy receptor that interacts with ubiquitinated cargo and LC3 (Rogov et al., 2014) and is considered a hallmark of autophagic flux, during which P62 is constantly degraded (Pankiv et al., 2007). Thus, a high LC3/low P62 suggests activated and intact autophagy, while a low LC3/p62 may show low basal autophagy (Liu et al., 2014).

Here, we demonstrate down-regulation of LC3 gene and protein expressions, along with an increase in P62 level in the midbrain of ROT-treated rats signifying a suppressive effect of ROT on autophagy pathways. Our results were in agreement with the previous finding showing that ROT administration resulted in decreased LC3 and Beclin-1 expression, and increased P62 protein level (Zeng et al., 2019).

Recent reports have shown that vitamin D supplementation elevates basal levels of autophagy and reduces the parameters of oxidative stress (Sepidarkish et al., 2019). Serum levels of vitamin D in PD patients have been found to be approximately two times lower than in healthy subjects, suggesting that there may be a correlation between vitamin D level and autophagy in PD (Ogura et al., 2021). Interestingly, vitamin D modulates autophagy at several levels through different mechanisms, including the regulation of intracellular calcium levels and its downstream pathways (Abdel-Mohsen et al., 2018). Tavera-Mendoza et al. (2017) reported that the vitamin D receptor (VDR) acts as a master transcriptional regulator of autophagy. They observed that vitamin D supplementation induces autophagy in the normal murine mammary gland through up-regulation of the LC3 protein level accompanied by increased autolysosome volume. Our results added evidence that calcitriol administration induced both an increase in LC3 expression and a decrease in P62 expression in ROT-treated rats. Surprisingly, the expression of the LC3 gene was remarkably upregulated when vitamin D was administered post-ROT than the control rats. This may indicate persistent compensatory up-regulation of the LC3 gene. This increase was not reflected at the functional protein level, probably due to post-translational modification (Lindner and Demarez, 2009). However, this hypothesis needs to be tested experimentally.

Thus, to further study the effect of calcitriol on autophagic activity in PD, we measured the conversion of the soluble cytosolic form of LC3 (LC3I) to the lipidated and autophagosome-associated form (LC3II) (Tanida et al., 2008) by Western blot. We found that the LC3II/LC3I ratio was significantly increased upon administration of calcitriol to rats treated with ROT, indicating intact autophagy and increased autophagosome abundance. Our findings are consistent with previous study of Jang et al. (2014) that demonstrated that calcitriol protects against ROT-induced neurotoxicity in SH-SY5Y cells by improving autophagy signaling pathways such as those involving LC3 and beclin-1.

Importantly, calcitriol-induced Sirt1 up-regulation observed here may support activated autophagy. This is in line with previous studies that demonstrate that Sirt1 plays a central role in the promotion of autophagy through the deacetylation of autophagy-related proteins, including LC3 and by stimulating the conversion of LC3-I to LC3-II through proteolytic cleavage and lipidation (Lee et al., 2008). Furthermore, Sirt1 activation causes translocation of LC3 from the nucleus into the cytoplasm, which is conveyed by increased expression of LC3-II and of α-synuclein and P62 degradation in dopaminergic neurons (Guo et al., 2016).

Taken together, these findings suggest that the calcitriol effect is related to the induction of autophagy and the restoration of autophagy flux and, therefore, the inhibition of autophagy dysfunction in the midbrain of the PD rat model.

Although neuroinflammation might exert a protective effect on the CNS (Wee Yong, 2010), several studies advocate the hypothesis that neuroinflammation plays an essential role in the pathogenesis of PD (Anusha et al., 2017). Dysfunctional mitochondria that accumulate due to impaired autophagy may play a role in initiating this neuroinflammation (Berridge, 2017). NF-κB is a key player in inflammation and regulates several other inflammatory markers (Hajiluian et al., 2018). Activation of NF-κB leads to its translocation to the nucleus and stimulates the transcription of genes that encode multiple pro-inflammatory factors (Zhang et al., 2010). Up-regulation of NF-κB is generally associated with neurodegeneration (Anusha et al., 2017) and promotes apoptosis in patients with PD (Holmes et al., 2016). In the present study, we found that the NF-κB was activated in the midbrain of rats treated with ROT, which is in agreement with previous observation (Thakur and Nehru, 2015).

Interestingly, vitamin D may modulate inflammation through down-regulation of pro-inflammatory factors and, presumably, could improve cognition (Vanoirbeek et al., 2011). Furthermore, the expression of vitamin D receptors, 1α and 24α-hydroxylase in various cells of the CNS advocate its pivotal role in central inflammation (Erbaş et al., 2014). Calcitriol ameliorates pro-inflammatory effects by modulating cytokine production or release, which might lead to restoration of cellular homeostasis to prevent subsequent organ damage (Tsai et al., 2016). Consistent with our findings, calcitriol administration to ROT-treated rats significantly diminished NF-κB expression, with a stronger effect when calcitriol was administered after stopping ROT treatment. This can be explained by rotenone stimulation of intracellular reactive oxygen species generation, which then promotes the NF-κB activation and translocation to the nucleus (Yuan et al., 2013). This inhibition of NF-κB shows that calcitriol has anti-inflammatory activity, which may slow the progression of PD. Previous investigations, in line with our findings, revealed that vitamin D may down-regulate NF-κB expression in inflammatory diseases, for example, multiple sclerosis (Cantorna et al., 1996), type 1 diabetes (Mathieu et al., 1995), and inflammatory bowel disease (Lim et al., 2005).

In addition, calcitriol is capable of modulating NF-êB and its related inflammatory pathways in dendritic cells (D’Ambrosio et al., 1998), lymphocytes (Yu et al., 1995), fibroblasts (Harant et al., 1998), and keratinocytes (Riis et al., 2004). Furthermore, a vitamin D analog is shown to strongly suppress the production of pro-inflammatory chemokine in pancreatic islet cells, which is accompanied by an enhancement of IκBα transcription and a stop of NF-κB P65 nuclear translocation (Giarratana et al., 2004). It is noteworthy that pro-inflammatory cytokines, which are closely linked to PD including IL-1ß, IL-6, and TNF, remain to be investigated to better elucidate the potential role of calcitriol in modulation of neuroinflammation in PD models (Nagatsu and Sawada, 2005).

Importantly, calcitriol might exert its anti-inflammatory effect through the Sirt1/NF-κB signaling pathway (Li et al., 2020). Vitamin D supplementation upregulated Sirt1expression and decreased NF-κB phosphorylation (Safarpour et al., 2020). In addition, NF-κB has emerged as a negative regulator of autophagy (Djavaheri-Mergny et al., 2006). Taken together, these results suggest that calcitriol administration leads to decreased neuroinflammation in the midbrain of PD rat model. Interestingly, we showed improvement in some aspects including higher TH neurons, increased Sirt1 and LC3 expression, and decreased NF-κB cells when calcitriol was administered after ROT administration. This may indicate that the calcitriol may have a stronger therapeutic effect than its role in neuroprotection, and the beneficial effect of calcitriol could be maintained even after the onset of the pathology.

In conclusion, our data point to a prophylactic and restorative effect of calcitriol on motor and exploratory behaviors, as well as neuronal architecture, autophagy, and neuroinflammation in the ROT-induced rat model of PD, presumably through Sirt1-dependent mechanisms. Given this association between calcitriol and Sirt1 expression, calcitriol could be a target that protects against or improves the manifestation of PD at both the functional and structural levels. However, it is of great importance to elucidate whether calcitriol treatment decreases the α-Syn protein aggregation in PD, affects other Sirt1-related pathways, or modulates neuroinflammatory endpoints, including cytokines, in future studies.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The animal study was reviewed and approved by Ethical committee, Faculty of Medicine, Mansoura University, Egypt.

AM, EF, SH, ZA, and ME-k performed the material preparation, data collection, and analysis. AM contributed to the project administration. EE and MA acquired the funding and contributed to the study conception and design. AM, EF, AA, and ME-k performed the visualization and wrote the first draft of the manuscript. AM, EF, SH, ZA, EE, MA, AA, and ME-k commented on previous versions of the manuscript and read and approved the final manuscript.