Vitamin D confers neuroprotective effects in traumatic brain injury by activating Nrf2 signaling through an autophagy-mediated mechanism

Background: Traumatic brain injury (TBI) initiates an oxidative cascade that contributes to the delayed progressive damage, whereas autophagy is critical in maintaining homeostasis during stressful challenge. We previously demonstrated that vitamin D (VitD) shows strong neuroprotective and anti-oxidative properties in the animal models of TBI. Therefore, the present study aimed to further explore the potential interrelationship between oxidative stress and autophagy in the progression of TBI and therapeutic mechanism of VitD. Methods: Neuroprotective effects of calcitriol, the active form of VitD, were examined following TBI. We further evaluated the impacts of TBI and VitD treatment on autophagic process and nuclear factor E2-related factor 2 (Nrf2) signaling. To conrm the mechanism, chloroquine (CQ) treatment and Nrf2 −/− mice were used to block autophagy and Nrf2 pathway, respectively. Results: We found that treatment of calcitriol markedly ameliorated the neurological decits and histopathological changes following TBI. The brain damage impaired autophagic ux and impeded Nrf2 signaling, the major regulator in antioxidant response, consequently leading to uncontrolled and excessive oxidative stress. Meanwhile, calcitriol promoted autophagic process and activated Nrf2 signaling as evidenced by the reduced Keap1 expression and enhanced Nrf2 translocation, thereby mitigating TBI-induced oxidative damage. To further conrm whether autophagy was responsible for Keap1 degradation and Nrf2 activation, the lysosomal inhibitor, CQ, was used to block autophagy. Our data suggested that CQ treatment abrogated calcitriol-induced autophagy and compromised Nrf2 activation with increased Keap1 accumulation and reduced expression of Nrf2-targeted genes. Additionally, both CQ treatment and Nrf2 genetic knockout abolished the protective effects of VitD against both TBI-induced neurological decits and neuronal apoptosis. Conclusions: Therefore, our work demonstrated a neuroprotective role of VitD in TBI by triggering Nrf2 activation, which might be mediated by autophagy. oxygen species; SOD, superoxide dismutase; TBI, Traumatic TUNEL, deoxynucleotidyl transferase-mediated cyanine–dUTP


Introduction
Traumatic brain injury (TBI) is one of the most common causes of death and long-term impairment that affects all ages (Stout et al., 2018). The brain damage is induced by both primary and secondary injury processes. The primary injury is the mechanical disruption of brain tissue that occurs immediately, whereas secondary injury subsequently develops over time via intricate processes, including excitotoxicity, neuroin ammation and oxidative stress that ultimately contribute to neuronal loss (Ma et al., 2018). Because of its complexity, effective therapies for the treatment of TBI are still inadequate.
Autophagy is a major proteolytic system that senses intracellular stressful conditions and rapidly mounts a molecular response to deal with the damage through sequestration and degradation of dysfunctional organelles and compromised proteins . The autophagic process is orchestrated through a series of autophagy-related genes (ATG genes), such as LC3-II (derived from LC3-I upon lipidation) and Beclin-1, which are veri ed biomarkers for evaluating autophagy. In addition, the adaptor protein p62 serves as a cargo receptor responsible for recognizing and loading ubiquitinated proteins into autophagosomes for degradation in selective autophagy (Su et al., 2019). Notably, p62 also activates nuclear factor E2-related factor 2 (Nrf2) signaling, a well-characterized cellular defense mechanism against oxidative stress, by interacting with Kelch-like ECH-associated protein 1 (Keap1) (Saito et al., 2020). In the cytoplasm, Nrf2 is bound to its partner, Keap1, and is rapidly degraded through the ubiquitinproteasome pathway . It has been demonstrated that p62 directly interacts with Keap1, thereby dissociating it from Nrf2 and directing it toward autophagic degradation (Deng et al., 2020;Dodson et al., 2015).
Vitamin D (VitD) now is recognized as a novel neuroactive steroid, which has antioxidant and neuroprotective activities in addition to its classical function in bone metabolism and calcium-phosphate homeostasis (Cui et al., 2019). We previously demonstrated that 1,25-dihydroxyvitamin D 3 (calcitriol), the active form of VitD, is effective in preventing TBI-induced behavioral abnormalities and oxidative stress . Except that, calcitriol treatment also promotes autophagic ux, preventing autophagosome accumulation following TBI . Emerging evidence has indicated that the antioxidant activity of VitD is tightly related to Keap1/Nrf2 signaling Nachliely et al., 2019), suggesting that vitamin D receptor (VDR) activation may mediate the interaction between autophagy and the Keap1/Nrf2 system to facilitate the neuroprotective functions of VitD. Therefore, the present study aims to test the protective actions of VitD following TBI and its impacts on autophagy and Nrf2 signaling. Moreover, through blocking autophagic process by using chloroquine (CQ) and genetic knockout of Nrf2, we further sought to con rm the hypothesis that autophagy may interact with Keap1/Nrf2 system to contribute to the neuroprotective effects of VitD. Moreover, to con rm the neuroprotective mechanisms of VitD, CQ (30 mg/kg/day) was co-treated with calcitriol by intraperitoneal injection to block autophagy ux.

Materials And Methods
Mouse model of TBI skull. TBI was induced by hitting the brain surface at the center of the craniotomy with a 2.5-mm-diameter rounded metal tip at a velocity of 4 m/s and a deformation depth of 2 mm using a controlled cortical impact (CCI) device (CCI Model 6.3; Custom Design, USA). The bone ap was immediately replaced and sealed, and the scalp was sutured.
Neurological score evaluation At 1-14 days following TBI, the neurological scores were determined as Neurological Severity Scores, a composite of motor, sensory, re ex, and balance tests (normal score: 2-3; maximal de cit score: 18; Additional le 1).

The Morris water maze (MWM) test
The apparatus consisted of a circular black water tank (180 cm in diameter, 50 cm high) lled with water (26 °C) to a depth of 30 cm. An escape platform (diameter 12 cm, height 28 cm, painted opaque) submerged 2 cm below the water surface was placed in the middle of one of the quadrants equidistant from the tank wall and the center of the pool. All the mice were trained to nd the platform before the sham operation or the induction of TBI. For each trial, each mouse was randomly placed into a quadrant start point (N, S, E, or W) facing the wall of the pool and was allowed a maximum of 60 s to nd the escape platform. The mice that failed to escape within 60 s were placed on the platform for a maximum of 20 s and returned to their cage to await a new trial (intertrial interval, 10 min). Probe trials were conducted at 11-14 days following the induction of TBI or the sham operation. The time spent in the target quadrant and the swim speeds were evaluated on the last day of the test after the platform was removed.

Histopathological staining
After xation, the brains were embedded in para n and sliced into 4-µm coronal sections at the level of the bregma and stained with hematoxylin and eosin (H&E). Apoptosis was assessed using terminal deoxynucleotidyl transferase-mediated cyanine-dUTP nick-end labeling (TUNEL) following the manufacturer's protocol. Nuclei were counterstained with DAPI (Beyotime Biotechnology, China). For each group, sections from three different mice were used for quanti cation.

Transmission electron microscopy (TEM)
Tissues were immersed in 2% glutaraldehyde and 1% osmium tetroxide (Sigma-Aldrich; Merck KGaA) for 2 h at 4 °C, and then dehydrated via a graded ethanol series. Following the displacement of ethanol with propylene oxide, the tissues were embedded in Epon (both from Sigma-Aldrich) and sectioned along the coronal plane with a diamond knife (FernAnclez-hIorln 1953; Ivan Sorvall, Inc., New York, NY, USA) at a thickness of 60 nm. The sections were stained with lead citrate and observed using a CM-120 electron microscope (Philips, Eindhoven, Netherlands). In order to quantify the alteration of the number of the autolysosomes, the area of the cell cytoplasm was measured by using Image-Pro Plus 6.0.

Real-time PCR analysis
Total RNA was extracted using Trizol reagent (Invitrogen, USA) following the manufacturer's instructions. Quantitative PCR was performed on a Bio-Rad Cx96 Detection System (Bio-Rad, USA) using a SYBR green PCR kit (Applied Biosystems, USA) and gene-speci c primers (Additional le 2).

Detection of oxidative parameters
The generation of reactive oxygen species (ROS) was determined by uorescence-labeled dihydroethidium (DHE). Frozen cross-sections (15 µm) were incubated in DHE for 30 min at 37 °C in a dark humidi ed chamber. The sections were rinsed three times in PBS and observed using an inverted uorescence microscope (Olympus, Japan). Malondialdehyde (MDA) levels were measured using the thiobarbituric acid reactive substances (TBARS) assay. The activities of superoxide dismutase (SOD), catalase (CAT), and glutathione (GSH) were determined using SOD, CAT, and GSH assay kits, respectively (Nanjing Jiancheng Bioengineering Institute, China).
Primary cortical neuron culture The skull, blood and meninges were carefully removed from fetal mouse brains. After the cortical tissue was digested in 0.25% trypsin (BI, Israel) for 5 min at 37 °C, the suspensions, containing fetal bovine serum (BI, Israel), were passed through lters with a 0.22-μm pore size (Millipore, USA) and then centrifuged at 1,500 rpm for 5 min. The cells were distributed in poly-D-lysine-coated plates. The medium was replaced with neurobasal medium supplemented with streptomycin, penicillin, HEPES, glutamate, and B27 (BI, Israel). The cells were exposed to different doses of calcitriol (1nM, 10nM, 100nM, 500nM) for 24 h. The cells were pre-treated for 6h with 25 μM CQ and then treated with the indicated doses of calcitriol.

Cell viability analysis
MTT was added to each well of a 24-well plate followed by incubation at 37 °C for 1 h. The purple formazan crystals formed through the reduction of MTT were then dissolved in 500 µL DMSO, and the absorbance of the wells was recorded at 590 nm. Cell viability was calculated by the absorbance ratio of the treated group to that of the control.

Autophagic ux analysis
Autophagic ux was detected by using the RFP-GFP-LC3 adenovirus (Hanbio, China). After plating the cells in a 24-well plate at a density of 1 × 10 4 cells/ dish and incubating with mRFP-GFP-LC3 adenovirus for 24 h. Autophagic ux was observed under an inverted uorescent microscope (Olympus, Japan). The yellow puncta indicated autophagosomes, and the red puncta indicated autolysosomes.

Statistical analysis
The results were expressed as means ± SD. All the analyses were performed using SPSS 17.0 software.
Statistical signi cance was determined using one-way analysis of variance (ANOVA), and the Student-Newman-Keuls post hoc test was used to determine differences among different groups. A P-value < 0.05 was considered statistically signi cant.

Results
The effects of calcitriol on TBI-induced neurological de cits The neurological severity scores were assessed at 1-14 days after TBI (Additional le 3). At 14 days, the level of neurological injury was signi cantly increased in the TBI group compared with that in shamoperated animals (P < 0.01). In addition, compared with the TBI-only group, all calcitriol treatment groups showed improved neurological de cit scores (P < 0.01). No signi cant differences were found among the mice treated with different calcitriol doses (Fig. 1A).
The effects of calcitriol on TBI-induced learning and memory ability impairment The MWM hidden platform task was used to investigate whether calcitriol could improve the spatial memory de cits at 11-14 days after TBI (Additional le 4A). As shown in Fig. 1B, compared with the sham operation group, mice with TBI spent longer searching for the hidden platform at 14 days postsurgery (P < 0.01). In contrast, calcitriol-treated mice displayed a markedly shorter latency time compared with that in the TBI-only treatment group (P < 0.01 for the low-dose and medium-dose groups; P < 0.05 for the high-dose group). In the probe trials (Fig. 1C), mice with TBI spent less time than their sham-operated counterparts swimming toward the goal quadrant that previously contained the platform (P < 0.01). Nevertheless, mice in the low-dose and medium-dose calcitriol treatment groups displayed improved learned bia (P < 0.01). Representative traces at 14 days are depicted in Fig. 1D and 1E. No signi cant differences in swimming speeds were found among the groups, indicating that the observed differences were not a result of an inability to execute the swimming task (Additional le 4B).
The effects of calcitriol on neuronal cell death after TBI As depicted in Fig. 2, the cortical neurons in the sham operation group were arranged in an orderly manner, the cytoplasm was transparent, the cell nuclei showed a round or oval shape, the chromatin was evenly distributed, and the nucleoli were clear. After TBI, the opposite morphology was observed. In the calcitriol treatment groups, the amount and level of neuronal degeneration, necrosis, and loss were signi cantly attenuated. Likewise, only a small number of TUNEL-positive neurons were found in the sham operation group. However, the neuronal cells in the TBI-only group were arranged in a disorderly manner, and a greater number of apoptotic neurons were observed (P < 0.01). After calcitriol treatment, the number of apoptotic cortical neurons was signi cantly decreased (P < 0.01).
The effects of calcitriol on TBI-induced autophagy impairment TEM analysis demonstrated that in the TBI-only group, there was a markedly increased accumulation of autophagosomes around the nucleus than in the sham operation group (P < 0.01) (Fig. 3A). However, in calcitriol-treated mice, the autophagosome abundance was reduced, whereas the autolysosomes were increased (P < 0.01 versus the TBI-only group). Moreover, we found that LC3 and beclin 1 mRNA levels were unchanged after TBI, whereas that of p62 was markedly increased (P < 0.01). The mRNA expression levels of LC3, p62, and beclin 1 were all markedly higher in the calcitriol treatment groups than in the TBIonly treatment group (P < 0.01 for LC3 and p62; P < 0.05 for beclin 1) (Fig. 3C). Intriguingly, TBI induced the protein expression of LC3-II and p62 (P < 0.01), but this effect was partly reversed following repeated calcitriol treatment (P < 0.01). However, no signi cant changes in beclin 1 protein expression were observed in the TBI-only treatment group, whereas beclin 1 was upregulated after calcitriol treatment (P < 0.01) (Fig. 3D, E). In addition, as shown in Fig. 3F and Fig. 3G, blocking autophagosome-lysosome fusion with CQ did not lead to an additional increase in LC3-II protein levels in TBI-only-treated mice. These results suggested that TBI induced autophagy dysfunction and impaired autophagosome clearance, which was mitigated by calcitriol treatment.
The effects of calcitriol on the Keap1-Nrf2 pathway As we found that calcitriol treatment could rescue TBI-induced autophagic ux dysfunction, we further investigated whether autophagic process affected the Keap1-Nrf2 pathway. The expression levels of Keap1 and Nrf2 were measured by western blotting (Fig. 4A, B). The level of Keap1 was higher in the TBIonly group than in the sham operation group (P < 0.01); however, calcitriol treatment partially reduced the Keap1 expression level (P < 0.01). Immuno uorescent staining revealed similar changes in the protein levels of p62 and Keap1 (Fig. 4C and Fig. 4D). TBI led to a signi cant reduction in the expression of Nrf2 in the nucleus (P < 0.01), while calcitriol treatment increased the nuclear translocation of Nrf2 (P < 0.01  (Fig. 4G, H, I). In line with the expression levels of Nrf2 in the nucleus, the mRNA levels of Nqo1, HO1, and Gclc were signi cantly decreased in the TBI-only treatment group (P < 0.01), whereas the expression of these genes was signi cantly increased in the calcitriol treatment groups (P < 0.01).
The effects of calcitriol on TBI-induced oxidative damage As shown in Fig. 5, calcitriol treatment markedly enhanced the activities of the antioxidant enzymes SOD, CAT, and GSH, while also reducing lipid peroxidation product (MDA) levels and ROS production.
The effects of inhibiting autophagy and knocking out Nrf2 on the neuroprotective activity of calcitriol Our data demonstrated that both autophagy ux and the Keap1-Nrf2 pathway were activated by calcitriol treatment. Consequently, we assessed whether a noncanonical signaling network that includes both autophagy ux and the Keap1-Nrf2 pathway is induced by calcitriol. First, mice in the calcitriol groups were treated with CQ, a selective autophagy inhibitor that prevents autophagosome-lysosome fusion. As shown in Fig. 6, CQ treatment markedly suppressed the Keap1-Nrf2 pathway, inducing Keap1 protein expression and decreasing that of Nrf2 in the nucleus when compared with the calcitriol-only treatment groups (P < 0.01). The levels of Nrf2 target genes were also examined. The mRNA levels of Nqo1 and Gclc were signi cantly decreased with CQ treatment, whereas ROS production was signi cantly increased (P < 0.01 versus the calcitriol-only group). Interestingly, CQ treatment blocked the ameliorative effect of calcitriol on neurological behavior and suppressed neuronal apoptosis (Fig. 7), highlighting the critical role of autophagy ux in the neuroprotective effects of calcitriol. Additionally, Nrf2 −/− mice exhibited lower Nqo1 and Gclc mRNA levels after calcitriol treatment (P < 0.01 versus the calcitriol-only treatment groups). As expected, the neuroprotection effect of calcitriol on TBI was abrogated in Nrf2 knockout mice, which displayed markedly impaired neurological functions and neuronal apoptosis (Fig. 7). The neurological score evaluation and MWM hidden platform task data are depicted in Additional le 5 and 6.

Effect of calcitriol on neuronal autophagy and Keap1-Nrf2 pathway in vitro
As shown in Fig. 8A, the results from the MTT assay demonstrated that while calcitriol at concentration of 1nM and 10nM had no toxic effect, high concentration of calcitriol exposure (100nM and 500nM) signi cantly reduced cell viability when compared to the control group (P < 0.01). Therefore, the impact of calcitriol on Nrf2 signaling was assessed. It was observed that calcitriol (1nM-1000nM) signi cantly increased nucleus Nrf2 expression compared to the control group (P < 0.01 for the 10 nM-500nM groups; P < 0.05 for the 1nM group) (Fig. 8B, 8C). Combining the results of the above experiments, we chose calcitriol with 10nM concentration for further experiments to demonstrate the mechanism between autophagy and Nrf2 pathway in response to VitD in vitro. In order to show the autophagy ux, a tandem RFP-GFP-LC3 reporter was used. This assay takes advantage of differential pH sensitivity of GFP (acid labile) and RFP (acid resistant) uorophores to assess acidi cation of autophagosomes (yellow) upon fusion with lysosomes (red only). As shown in Fig. 8D and Fig. 8E, in cells treated with calcitriol, we observed accumulation of red autolysosomes compared to controls (P < 0.01). However, inhibition of autophagy ux by CQ caused impairment in autophagosome-lysosome fusion, displayed an increased accumulation of yellow autophagosomes (P < 0.01). These data con rmed that VitD treatment induced the activation of neuronal autophagic ux. Additionally, the expression levels of autophagy-related proteins were measured by western blot. The level of p62 was higher in the calcitriol group than in the control group (P < 0.01). However, inhibition of autophagy ux by CQ induced the expression of LC3-II and p62 (P < 0.01). The expression of Keap1 was attenuated and the nuclear expression of Nrf2 was higher in the calcitriol treatment group than in control group (P < 0.01). However, inhibition of autophagy ux by CQ after calcitriol treatment markedly suppressed the Keap1-Nrf2 pathway, inducing Keap1 protein expression and decreasing Nrf2 status in the nucleus (P < 0.01).

Discussion
Oxidative stress is the major cause of TBI-induced secondary injury, whereas Nrf2 is an important oxidative stress regulator in the protection of various cell types and organ systems. Recent studies have also shown that impairment of Nrf2 signaling in the injured brain may aggravate ROS production and In line with these ndings, the present research also found that treated with calcitriol, at doses ranging from 0.5µg/kg to 3µg/kg, was effective in potentiating the recovery process in mice following TBI by mitigating neurological de cits, improving behavioral performance in the MWM test and retarding neuronal apoptosis. These ndings support the bene cial effects of VitD in TBI and further demonstrate that the sustained treatment of calcitriol immediately after TBI may enhance the recovery process and prevent potential secondary injury.
Nrf2 and autophagy are two critical stress responsive signaling pathways that are associated with redox balance. Nrf2 is a basic leucine zipper (CNC bZip) redox sensitive transcription factor that activates antioxidant response elements (AREs). The genes transcriptionally regulated by the AREs encode detoxi cation enzymes and antioxidant proteins thereby playing a central role in the oxidative stress modulation (Bhowmick,D'Mello 2019). Notably, oxidative stress and autophagy are also intricately connected. In the past few years, autophagy has been proposed as a potential survival mechanism in the context of exaggerated ROS generation by timely removal of damaged and redundant substance, serving as a cytoprotective mechanism to restrain oxidative injury (Filomeni et al., 2015). Moreover, p62dependent clearance of Keap1 has been found to regulate Nrf2 signaling and constitutes an important defense system against oxidative stress. p62 can bind and sequester Keap1, allowing the release of Nrf2 from Keap1 and Nrf2 translocation to the nucleus, thereby resulting in the activation of antioxidant genes (Dodson Redmann 2015). As previously reported (Cui Cui 2017), we found impaired autophagic ux in the injured brain with increased autophagosome accumulation. By using the lysosomal inhibitor, CQ, we con rmed the inhibited autophagic process following TBI as evidenced by the signi cant increase of LC3-II expression in Sham group but unchanged in TBI group following CQ treatment. Although autophagy was reported to be enhanced soon after acute brain injury to cope with the stress, it seems that prolonged time after TBI is likely to induce exhaustion of autophagy (Sarkar et al., 2014). Concomitantly, we observed a marked increase of Keap1 expression and a signi cant decrease of Nrf2 translocation, resulting in attenuated downstream antioxidant signaling and exaggerated oxidative stress following TBI.
In parallel, VitD accelerated the recovery process and activated autophagic ux following TBI. The increased transcriptional level of ATG genes but decreased protein expression of LC3-II and p62 compared with TBI group indicates that calcitriol not only induced autophagosome formation but also facilitated its degradation. Meanwhile, calcitriol also triggered Nrf2 signaling, inducing the expression of targeted genes and protecting the brain from excessive oxidative stress. In support of our ndings, recent . Given that Keap-1 is targeted to autophagosomes for degradation, the reduction of Keap-1 expression and the decreased co-expression of p62 and Keap1 following calcitriol treatment suggest that Nrf2 might be activated by VitD through autophagy mediated by the interaction between p62 and Keap1. By using CQ to block autophagy, we found cotreatment with CQ abrogated calcitriol-induced Keap-1 degradation and compromised Nrf2 signaling, resulting in the decreased downstream antioxidant genes and increased ROS production. As expected, both CQ treatment and Nrf2 genetic knockout abolished the bene cial effects of VitD on neurological function as well as its anti-apoptotic activity following TBI, indicating that the neuroprotective effect of VitD is through the interaction between autophagy and Nrf2 signaling. Additionally, we also observed a dose-dependent increase of Nrf2 signaling in neuronal cells following calcitriol exposure. Using the tandem uorescent mRFP-GFP-LC3 adenovirus, we further proved that calcitriol can effectively trigger autophagic ux. Meanwhile, inhibiting autophagic process with CQ also blocked the calcitriol-induced Keap1 degradation and Nrf2 activation in vitro. These data collaboratively support the facilitating effect of calcitriol on autophagy, whereby to activate Nrf2 signaling. However, it should be noted that some researchers suggest that blocking lysosomal function would lead to autophagosome accumulation and thereby promote p62 to bind with Keap1, sequestering it from Nrf2 and resulting in Nrf2 translocation (Park et al., 2019;Menglin et al., 2018). On the other hand, there is also accumulating evidence which is in accordance with our ndings, demonstrating that autophagy-induced Keap1 degradation can be effectively abrogated by the lysosomal inhibitors, CQ and Ba lomycin A1, which would accelerate oxidative stress or compromised the protective effects of various interventions in the brain, heart and liver tissues (Deng et al., 2020;Tan et al., 2020;Lee et al., 2020). These discrepancies might be attributed to the different cell types or disease models that may have various basal autophagic condition, and because of the different doses and treatment duration that may cause diverse autophagic inhibitory status. Following TBI or other stimuli, the injury progression and ROS production last even for weeks. To this end, the calcitriol-induced continuously autophagic clearance of Keap-1 and constantly active Nrf2 signaling are critical to mitigate the oxidative damage. In this study, CQ was co-treated with VitD, which may induce persistent inhibition on this process and thereby exacerbate brain damage.

Conclusions
Taken together, the present study rstly demonstrated that autophagic ux serves as an upstream regulator required for calcitriol-induced Nrf2 activation, thereby alleviating oxidative damage and cell apoptosis following TBI (Fig. 9). Additionally, our data demonstrated that autophagy and Nrf2, the two critical pathways in maintaining homeostasis and redox balance, are intricately interacted and both are indispensable for the antioxidant and anti-apoptosis actions of VitD. These ndings would further enrich and shed novel insight on the neurological functions of VitD, providing an interesting target for the autophagic dysfunction and oxidative stress following TBI-induced neuronal damage.

Declarations
The variation in neurological de cits at 14 days after treatment was determined by neurological severity score tests. B, The time (seconds) spent nding the submerged platform at 14 days. C, The time (seconds) spent on exploring the quadrant that initially contained the platform at 14 days. D, Representative traces indicating the sample paths of the mice in the probe trials. E, Representative traces indicating the sample paths of the mice after the platform was removed. Data are presented as means ± SD (n=10). *P < 0.05 and **P < 0.01 versus the indicated groups.  western blots of autophagic markers. E, Graphs of LC3-II, p62, and beclin 1 protein expression levels. F, G, Chloroquine (CQ) was used to evaluate the effects of calcitriol treatment and TBI on autophagic ux.

Figure 4
The effects of calcitriol on Nrf2 signaling after TBI. A, Representative images of western blot staining for Keap1, cytoplasmic Nrf2, and nuclear Nrf2. B, Statistical graphs of Keap1 protein expression and Nrf2 translocation. C, D, Representative immuno uorescence images and statistical graphs of p62 and Keap1 co-expression. E, F, Representative immuno uorescence images and statistical graphs of Nrf2 translocation. G, The relative mRNA expression level of Nqo1. H, The relative mRNA expression level of Gclc. I, The relative mRNA expression level of HO1. Data are presented as means ± SD (n=5). *P < 0.05 and **P < 0.01 versus the indicated groups.    reporter. E, Statistical graphs of number of autophagosomes and autolysosomes per cell. F,