Abstract
Background:
Fine particulate matter (particulate matter 2.5, PM2.5) is considered one of the harmful factors to neuronal functions. Apoptosis is one of the mechanisms of neuronal injury induced by PM2.5. Methylcobalamine (MeCbl) has been shown to have anti-apoptotic and neuroprotective effects.
Objective:
The current work tried to explore the neuroprotective effects and mechanisms that MeCbl protects mice against cognitive impairment and neuronal apoptosis induced by chronic real-time PM2.5 exposure.
Methods:
Twenty-four 6-week-old male C57BL/6 mice were exposed to ambient PM2.5 and fed with MeCbl for 6 months. Morris water maze was used to evaluate the changes of spatial learning and memory ability in mice. PC12 cells and primary hippocampal neurons were applied as the in vitro model. Cell viability, cellular reactive oxygen species (ROS) and the expressions of apoptosis-related proteins were examined. And cells were stained with JC-1 and mitochondrial membrane potential was evaluated.
Results:
In C57BL/6 mice, MeCbl supplementation alleviated cognitive impairment and apoptosis-related protein expression induced by PM2.5 exposure. In in vitro cell model, MeCbl supplementation could effectively rescue the downregulation of cell viability induced by PM2.5, and inhibited the increased levels of ROS, cellular apoptosis, and the expressions of apoptosis related proteins related to PM2.5 treatment, which may be associated with modulation of mitochondrial function.
Conclusion:
MeCbl treatment alleviated cognitive impairment and neuronal apoptosis induced by PM2.5 both in vivo and in vitro. The mechanism for the neuroprotective effects of MeCbl may at least be partially dependent on the regulation of mitochondrial apoptosis.
INTRODUCTION
With the rapid development of society and industry, the threat of air pollution to global public health has received widespread attention. Air pollution is a complex mixture consisting of particulate matter (PM), carbon monoxide, lead, nitrogen dioxide, sulfur dioxide, etc. Among them, fine particulate matter (PM2.5, aerodynamic diameter≤2.5μm) is considered to be one of the most harmful factors to human health [1]. Many studies have proved that air pollution is closely related to the onset of cardiovascular and respiratory diseases [2, 3]. Besides, some studies have even shown that PM2.5 can enter the central nervous system (CNS) through the blood-brain barrier (BBB), thereby inducing inflammatory changes, neuronal dysfunction and finally causing cognitive impairment [4].
The mechanisms of PM2.5 exposure-induced neu-ronal injury may include: oxidative stress, neuroinflammatory, mitochondrial dysfunction, and cellular apoptosis [5]. Apoptosis is a cellular physiological process that is involved in both the development of the nervous system and the pathogenesis of many diseases [6]. Earlier studies have shown that PM2.5 can invade into the smallest respiratory tract, induce the infiltration of inflammatory cells, and then change the cell cycle of alveolar epithelial cells and induce cell apoptosis [7–9]. Other studies have reported that PM2.5 exposure may also induce apoptosis and inflammation in endothelial cells and disrupt the function of autonomic nerve and vascular endothelium [10]. Reduced behavioral cognitive ability and abnormally expressed apoptosis-related proteins have been observed in mice exposed to PM2.5 [11]. Our previous study has demonstrated that neuronal apoptosis could be the crucial event in PM2.5 exposure-induced neuronal injury [12]. Other in vitro studies also showed that PM2.5 exposure may result to abnormal expression of apoptotic protein in cortical cells [13, 14]. Besides, inhibition of apoptosis has been proven to alleviate the neurological and cognitive damage in neurodegenerative diseases [15, 16]. Therefore, inhibition of cell apoptosis may be one of the effective means to prevent and control nerve injury caused by air pollution.
Vitamin B12 (VitB12), also known as cobalamin, is a polycyclic compound containing 3 valent cobalt and belongs to the family of tetrapyrrolic-derived macrocyclic compounds. VitB12 can be obtained by both food intake (meat and milk) and intestinal bacteria synthesis. VitB12 has some analogs, including cyanocobalamin, methylcobalamine (MeCbl), hydroxycobalamin, and adenosylcobalamin. MeCbl is a kind of VitB12 coenzyme which is present in the cytoplasm, mainly in blood and other body fluids [17]. MeCbl is very important in the development of the nervous system [18]. As an adjuvant, MeCbl has been applied in the therapy of many diseases, such as Alzheimer’s disease (AD) related cognitive decline [19]. MeCbl treatment may improve the memory and emotional function of AD patients, mainly dependent on promoting the regeneration of injured axons [20]. In animal models, MeCbl can promote peripheral neuronal regeneration in rats by up-regulating the expression of brain-derived neurotrophic factor [21]. In vitro, MeCbl has been shown to have neuroprotective effects by inhibiting amyloid misfolding [22]. In addition, MeCbl has been shown to protect neuronal cells by inhibiting cellular apoptosis induced by homocysteine [23].
To pile up all the evidence, PM2.5 pollutants may trigger neuronal apoptosis and subsequently cognitive impairment in mice. MeCbl is believed to be beneficial to neuronal function, which may be related to its anti-apoptosis ability. Thus, it would be reasonable to speculate that MeCbl may somehow be protective to neuronal injury induced by PM2.5 exposure in mice. In the current work, both in vitro and in vivo experimental models of PM2.5 exposure were established, the neuroprotective effects of MeCbl treatment were evaluated and the possible anti-apoptosis mechanisms were investigated.
METHODS AND MATERIALS
PM2.5 and MeCbl treatment and behavioral test in mice
Twenty-four 6-week-old C57BL/6 mice were randomly divided into 3 groups: filtered air (FA) group, PM2.5 exposure (PM) group, and PM + MeCbl treatment (PM + MeCbl) group. The mice were randomly assigned to ambient PM2.5 chamber (PM) or filtered air chamber (FA) in Ambient PM2.5 real-time Exposure System (APES) (Jukang, China). APES is a whole-body aerosol exposure device which can carry out research on animal models to simulate personal long-term real-time exposure to ambient PM2.5. It was configured with two humidified, temperature-controlled chambers. The PM chamber was directly connected to the outside air and the cyclone separator was used as a substitute for a PM2.5 filter. The FA chamber equipped with high efficiency particulate air (HEPA) filter can remove all the PM2.5 from the air. Mice were exposed for 12 h a day, 6 days a week, for a total of 6 months. The MeCbl treatment group was fed sterilized water containing MeCbl at a concentration of 1.25 mg/L every day.
After feeding for 6 months, the cognitive function of mice was examined. Following the previous protocol, the spatial learning and memory ability of mice was evaluated by Morris water maze (MWM) [25]. The MWM equipment (China Zhiduobao) consists of a platform (5 cm in diameter) and a circular water tank (100 cm in diameter and 60 cm in height). The temperature of the water tank is 23±1°C. The water tank is evenly divided into four quadrants, and the platform is immersed 2 cm below the water surface of the target quadrant (quadrant III). At the end of the PM2.5 exposure, the mice received 4 consecutive days of maze training, and performed 4 tests a day to search for hidden platforms before the test. On the test day (day 6), the probe test was carried out by removing the platform, and each mouse was given 120 s to find the platform. The incubation period in the target quadrant was recorded, and the frequency and duration of platform formation were used to evaluate the learning and memory ability of mice.
All animal procedures are conducted in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals and approved by the Experimental Animal Ethics Committee of Hangzhou Normal University.
Brain slice preparation and TUNEL test
At the end of exposure, mice were sacrificed by cervical dislocation under pentobarbital anesthesia (80 mg/kg body weight), and cardiac perfusion with normal saline. Then brain tissues were cut and weighed. Hemisphere brain from each mouse was fixed in 4% paraformaldehyde, dehydrated by alcohol, transparent in xylene, embedded in paraffin and sectioned into a thickness of 4μm. The other half was stored at –80°C for immunoblotting analysis and other experiments. TdT-mediated dUTP -End Labeling (TUNEL) staining was used for neural apoptosis detection and performed according to the manufacturer’s instructions for the TUNEL assay kit (Roche, USA). The prepared brain tissues were dehydrated with gradient alcohol after being embedded using conventional paraffin. After permeabilization, the tissue is transparent, and the endogenous peroxidase is blocked. the brain slice was labeled with fluorescein-dUTP and peroxidase-labeled anti-fluorescein antibody, peroxidase-labeled anti-fluorescein antibody and visualized using DAB chromogen. Finally, seal the sheet with neutral gum and observe under an optical microscope. Three pathologists counted 200 cells in 5 areas of each experiment, determined and calculated the percentage of apoptotic cells.
Primary neuron preparation and PM2.5 treatment
Before the primary hippocampal neurons were cultured, a 12-well cell culture plate was coated with L-polylysine (0.1 mg/mL), incubated in an oven at 37°C for 4 h, and then L-polylysine was recovered. It was rinsed twice with D-PBS solution and dried under UV light for later use. The C57BL/6 mice on the 0 day of the newborn were immersed in a 75% ice ethanol solution, the brain tissue was removed in a sterile environment and then placed in an ice D-PBS buffer solution. The meninges and blood vessels were removed, and the hippocampus separated. The hippocampus was cut into 1 mm3 pieces, added 0.25% trypsin and digested at 37°C for 30 min. It was pipetted every 10 min. Subsequently, 500μL FBS was used to terminate the digestion, centrifuged at 1800 r/min for 5 min, and the supernatant was discarded. The cells were suspended again with the seeding solution, the cell density was adjusted, and they were planted in a 12-well cell culture plate. Eight hours after the cells adhered, the medium was replaced with maintenance medium. After that, the medium was changed every 3 and a half days, and the cells were cultured to the 10th day for follow-up experiments. The identification of cultured primary neurons was performed by MAP-2 and NeuN staining (Supplementary Figure 1).
PM2.5 samples were collected on Teflon filter membranes with pore size of 2μm (Pall Life Sciences, USA), which was mounted on the air samplers connected with exposure chambers in Hangzhou, Zhejiang Province, China. The sampling flow rate was set at 5 L/min for consecutively 48 hr. Then, the membranes were weighed before and after sampling using an ultramicro electronic balance (Mettler Toledo, Schweiz) to calculate the PM2.5 concentration in a constant temperature and humidity room. PM2.5 samples were extracted following previous reports [24]. Briefly, Soxhlet extractor were applied for extraction by acetone for 24 hr. Samples were then dried by rotary evaporation, weighted, dissolved in DMSO, and stored at –80°C. After 24 h of pretreatment with MeCbl (100μM), PC12 cells or primary hippocampal neurons were exposed to extracted PM2.5 (100μg/mL) for another 24 h. At the end of exposure, the medium was discarded and 10μl/well CCK-8 (Biosharp, China) was added and incubated at 37°C for 2 h. The absorbance at 450 nm was detected with a microplate reader (Molecular Devices, USA).
Immunoblotting analysis
The total protein from hippocampus or cells was extracted with RIPA lysis buffer (Solarbio, China) containing phosphatase inhibitor and protease, and quantified according to our previous research [25]. Each well was filled with 40μg total protein and electrophoresed on SDS-PAGE, then transferred to PVDF membrane (Merck, Germany), and blocked with 5% bovine serum albumin (BSA) at room temperature for 1 h. The membrane was combined with the primary antibody β-actin, Bax, Bcl-2 (1 : 2000; Abcam, UK), cytochrome C (Cyt C) (1 : 1000; Proteintech, USA), caspase-9, caspase-3, caspase-8 (1 : 1000; Cell Signaling Technology, USA) and incubated overnight at 4°C. After washing 3 times with TBST, the membrane was incubated with the HRP-conjugated secondary antibody for 1 h and observed with ECL reagent (Millipore, USA). Quantity One software (Bio-Rad, USA) was used to analyze the density of each band and standardize it by β-actin.
Detection of reactive oxygen species (ROS), cell apoptosis, and mitochondrial membrane potential (MMP)
Cells were seeded in 96-well plates; after treatment, the cells were sonicated and centrifuged to collect the supernatant. ROS test kit (meilunbio, China) was used to detect the ROS levels. ROS concentrations were evaluated using the oxidant-sensitive probe 2, 7-dichlorofluorescein diacetate (DCFH-DA) according to the manufacturer’s instructions. A fluorometric microplate reader (FilterMax F5, Molecular Devices, Sunnyvale, USA) with excitation and emission at 488 and 525 nm, respectively, was used to measure the fluorescence intensity. All operations were according to the manufacturer’s instructions. The results were shown as a multiple relative to the control group. The experiment was replicated thrice.
Apoptosis was detected by using an Annexin V-FITC Apoptosis Detection Kit (Beyotime Biotechnology, China). Cells were seeded in 6-well plates at a density of 1×104 cells, according to the supplier’s instructions; after treatment, the cells were collected by adding trypsin without EDTA, followed by washing twice with phosphate-buffered saline and centrifugation for 5 min at 2000 rpm. The cells were suspended in binding buffer and incubated with FITC-labeled Annexin V and propidium iodide (PI) at room temperature in the dark for 15 min. The apoptotic rate was measured by flow cytometry within 1 h. The experiment was performed in triplicate.
PC12 cells and primary hippocampal neurons were taken for preparation of mitochondrial membrane potential detection. Cells were planted 1×105 in a 6-well plate containing built-in climbing sheet in advance. After treatment, cells were centrifuged at 1000 rpm for 5 min, the supernatant was discarded, and resuspended in 0.5 mL of cell culture medium, 0.5 mL of JC-1 staining working solution was added. And incubation was carried out at 37°C for 20 min, and then centrifuged at 1200 rpm and 4°C for 3 min. The mitochondrial membrane potential assays were measured by flow cytometers (FCM, BD Biosciences, USA) with JC-1 Detection Kit (Beyotime Biotechnology, China) according to the instruction of manufacturer. In the light incubation box, observe and collect images under a fluorescence microscope.
Statistical analysis
After all variables were tested for normality by SPSS 20.0 software, independent sample t-test and one-way analysis of variance were used to detect the average difference between the treatment group and the control group. The result was expressed as mean±standard deviation (
RESULTS
MeCbl ameliorated cognitive impairment and neuronal apoptosis induced by PM2.5 exposure in mice
During the 6-month period of real-time ambient PM2.5 exposure in mice (2017/09-2018/03), we monitored the concentration of PM2.5 in the experimental chambers. The PM2.5 concentration in the chamber varied between 17–57μg/m3 (Supplementary Figure 2), the monthly average concentration of PM2.5 was 38.0μg/m3. MeCbl was fed by drinking water containing MeCbl (1.25 mg/L). In general, by calculating the average water intake (5.5 mL/d) and body weight (30 g) of mice, the average MeCbl dosage would be 0.25μg/g. After 6-month exposure, behavioral evaluation from the MWM showed that mice in PM group took longer time to reach the target platform, had shorter time and distance in the platform quadrant and lower frequency of mice crossing the target platform than mice in FA group, indicating the impaired learning and memory function in mice triggered by air pollution. After treatment with MeCbl, mice in the PM+MeCbl group had obvious better cognitive performance than PM group (*p < 0.05) (Fig. 1A), experiments showed that MeCbl could restore the impaired learning and memory function caused by PM2.5 exposure in mice.
MeCbl alleviates cognitive decline and neuronal apoptosis induced by PM2.5 exposure in mice. Mice were kept in FA or PM chamber for 6 months, and with or without treatment of 1.25 mg/L MeCbl. A) The learning and memory function of mice was evaluated by MWM. The time spent in the duration in quadrant, the frequency to the platform, and the latency to platform were recorded. B) Hippocampus tissue sections were stained with TUNEL kit and neuronal apoptosis was evaluated. 5 non-overlapping pictures were randomly selected from the brain slices for each mouse (scale bar = 200 or 50μm). C) Expression levels of apoptosis-related proteins cleaved-caspase 3 was detected by immunoblotting; intensity quantification of cleaved-caspase 3 was carried out by data normalization to β-actin level. (n = 3, *p < 0.05, **p < 0.01).
In order to better understand the molecular mechanism for cognitive dysfunction, neuronal apoptosis in the hippocampus was determined by TUNEL assays in mice. Results showed that the average number of apoptotic cells in hippocampus CA1 area of mice from PM group were significantly higher than those from FA group, PM + MeCbl group were significantly lower than those from PM group (*p < 0.05) (Fig. 1B). The expression levels of apoptosis-related proteins were also detected in brain tissue from mice. Results showed that after PM2.5 exposure, the expression level of cleaved-caspase 3 was significantly increased compared with the FA group, while MeCbl treatment significantly reduced PM2.5-induced apoptosis (*p < 0.05) (Fig. 1C). Those results indicated that MeCbl relieved cognitive injury induced by air pollution and cellular apoptosis may be the key pathway.
MeCbl inhibits cell apoptosis induced by PM2.5 treatment
To further explore the cellular mechanisms in the process of PM2.5-induced neuronal injury, in vitro cell experiments were then performed. PM2.5 samples applied in the cell experiments were collected from 2017/09-2018/03. The components analysis was performed in our previous reports, which showed that the highest inorganic and organic components were sulphur and benzoate (B), respectively [11]. PM2.5 samples were then extracted, dissolved in DMSO and later applied in in vitro cell experiments.
First of all, PC12 cells were treated with diffe-rent concentrations of MeCbl for 24 h, and cell viability was evaluated. Results showed that MeCbl treatment did not influence cell growth in PC12 cells (Fig. 2A). Cell viability by different concentrations of PM2.5 treatment in PC12 cells was also detected. Results showed that 100μM MeCbl treatment showed significant cellular protection in PC12 cells (Fig. 2B). After that, Annexin-V/FITC apoptosis staining and flow cytometry were applied to evaluate the apoptosis rate in PC12 cells treated with PM2.5. Results showed that PM2.5 exposure increased the cell apoptotic rate to 11.4% compared to 0.9% in control group; while MeCbl treatment could effectively reduce apoptosis rate to 5.9% (Fig. 2C). Besides, immunoblotting results showed that after PM2.5 stimulation, the expression levels of cleaved-caspase 3 were significantly increased (*p < 0.05) (Fig. 2D), which could be obviously inhibited by MeCbl treatment.
Cell viability and neuronal apoptosis by MeCbl or PM2.5 treatment in PC12 cells. CCK-8 kit was used to evaluate the cell viability. PC12 cells were pre-treated with 100μM MeCbl for 24 h, and then exposed to PM2.5 for another 24 h. A) PC12 cells were treated with 0, 12.5, 25, 50, 100, and 200μM MeCbl for 24 h (n = 3, *p < 0.05, compared with FA and PM; # #p < 0.01, compared with PM and PM + MeCbl). B) PC12 cells were pretreated with 0, 12.5, 25, 50, 100, and 200μM MeCbl for 24 h, and then exposed to 100μg/ml PM2.5 exposure for 24 h. C) Cells were then stained with Annexin-V/PI, and flow cytometry was applied to detect apoptotic rate (n = 3, *p < 0.05). D) Expression levels of apoptosis-related proteins cleaved-caspase 3 was detected by immunoblotting; intensity quantification of cleaved-caspase 3 was carried out by data normalization to β-actin level (n = 3, *p < 0.05).
Besides, primary hippocampal neurons were also studied to further investigate PM2.5-induced neuronal injury. Primary hippocampal neurons were treated with different concentrations of MeCbl for 24 h, and cell viability was evaluated. Results showed that MeCbl treatment did not influence cell growth in primary hippocampal neurons (Fig. 3A). Cell viability by different concentrations of PM2.5 treatment in primary hippocampal neurons were also detected. Results showed that 100μg/mL PM2.5 treatment showed significant cellular toxicity in primary hippocampal neurons (Fig. 3B). After that, immuno-blotting results showed that after PM2.5 stimulation, the expression levels of cleaved-caspase 3 were significantly increased (*p < 0.05) (Fig. 3C), which could be obviously inhibited by MeCbl treatment. Those data demonstrated that MeCbl reduced PM2.5 exposure triggered neuronal apoptosis, by mediating expressions of some apoptosis-related proteins.
Cell viability and neuronal apoptosis by MeCbl or PM2.5 treatment in primary hippocampal neurons. CCK-8 kit was used to evaluate the cell viability. Primary hippocampal neurons were pre-treated with 100μM MeCbl for 24 h, and then exposed to PM2.5 for another 24 h. A) Primary hippocampal neurons were treated with 0, 12.5, 25, 50, 100, and 200μM MeCbl for 24 h (n = 3, *p < 0.05, compared with FA and PM; # #p < 0.01, compared with PM and PM + MeCbl). B) Primary hippocampal neurons were pretreated with 0, 12.5, 25, 50, 100, and 200μM MeCbl for 24 h and then exposed to 100μg/ml PM2.5 exposure for 24 h. C) Expression levels of apoptosis-related proteins cleaved-caspase 3 was detected by immunoblotting; intensity quantification of cleaved-caspase 3 was carried out by data normalization to β-actin level (n = 3, *p < 0.05).
MeCbl inhibits cell apoptosis through the mitochondrial pathway
Mitochondria are important cytoplasmic organ for intracellular respiration. Mitochondria-mediated cellular apoptosis is one of most crucial pathways for neuronal injury. Thus, proteins closely related to mitochondrial apoptosis were detected by immunoblotting from brain tissues in mice. The results showed that the ratio of Bax/Bcl2, and expression levels of Cyt C and cleaved-caspase 9 were significantly increased by PM2.5 treatment in mice (*p < 0.05), while the expression levels of Bax/Bcl2, and expression levels of Cyt C and cleaved-caspase 9 were obviously inhibited by MeCbl treatment in mice (Fig. 4). Those data demonstrated that MeCbl reduced PM2.5 exposure triggered neuronal apoptosis in mice, at least partially dependent on mitochondrial pathway.
MeCbl protected mitochondrial apoptosis pathway injured by PM2.5 treatment in mice. Mice were kept in FA or PM chamber for 6 months, and with or without treatment of 1.25 mg/L MeCbl. Expression levels of apoptosis-related proteins Bax, Bcl-2, Cyt-C, and cleaved-caspase 9 were detected by immunoblotting; intensity quantification of Bax, Bcl-2, Cyt-C, and cleaved-caspase 9 were carried out by data normalization to β-actin level. (n = 3, *p < 0.05).
In order to further verify the molecular mediators of apoptosis induced by PM2.5 exposure and the effect of adding MeCbl on apoptosis in vitro, we evaluated the level of ROS and some mitochondrial apoptosis-related proteins in hippocampal neurons and PC12 cells. Results in Fig. 5A showed that cellular active oxygen by PM2.5 treatment was significantly higher than that in the control group, while MeCbl treatment could significantly reduce the intracellular ROS level in PC12 cells (*p < 0.05). Thus, proteins closely related to mitochondrial apoptosis were detected by immunoblotting. The results showed that the ratio of Bax/Bcl2, and expression levels of Cyt C and cleaved-caspase 9 were significantly increased by PM2.5 treatment in PC12 cells (*p < 0.05), while the expression levels of Bax/Bcl2, and expression levels of Cyt C and cleaved-caspase 9 were obviously inhibited by MeCbl treatment in PC12 cells (Fig. 5B). Subsequently, PC12 cells were stained with JC-1 and MMP was found down-regulated after PM2.5 treatment in PC12 cells, which can be effectively blocked by MeCbl treatment (Fig. 5C).
MeCbl protected mitochondrial apoptosis pathway injured by PM2.5 treatment in PC12 cells. PC12 cells were pre-treated with 100μM MeCbl for 24 h, and then exposed to PM2.5 for another 24 h. A) The ROS levels were detected by ROS kit (n = 3, *p < 0.05). B) Expression levels of apoptosis-related proteins Bax, Bcl-2, Cyt-c, and cleaved-caspase 9 were detected by immunoblotting; intensity quantification of Bax, Bcl-2, Cyt-c, and cleaved-caspase 9 were carried out by data normalization to β-actin level (n = 3, *p < 0.05). C) PC12 cells were stained with JC-1 and DAPI, and fluorescence microscope was applied to detect the MMP (scale bar = 100μm).
Subsequently, mitochondrial apoptosis related Cyt C, cleaved-caspase 9, and Bax/Bcl2 were also detected in primary hippocampal neurons (Fig. 6A). And then primary hippocampal neurons were stained with JC-1 and MMP was evaluated after PM2.5 treatment (Fig. 6B). MeCbl treatment could in both tests ameliorated PM2.5 treatment-induced apoptotic reactions. Those data demonstrated that MeCbl reduced PM2.5 exposure triggered neuronal apoptosis, at least partially dependent on mitochondrial pathway.
MeCbl protected mitochondrial apoptosis pathway injured by PM2.5 treatment in primary hippocampal neurons. Primary hippocampal neurons were pre-treated with 100μM MeCbl for 24 h, and then exposed to PM2.5 for another 24 h. A) Expression levels of apoptosis-related proteins Bax, Bcl-2, Cyt-c, and cleaved-caspase 9 were detected by immunoblotting; intensity quantification of Bax, Bcl-2, Cyt-c, and cleaved-caspase 9 were carried out by data normalization to β-actin level (n = 3, *p < 0.05). B) Primary hippocampal neurons were stained with JC-1 and DAPI, and fluorescence microscope was applied to detect the MMP (scale bar = 100μm).
DISCUSSION
With the development of modern cities, air pollution has gradually deteriorated. The burden of air pollution on human health has attracted worldwide attention. A large number of epidemiological investigations have found that PM2.5 in the air may lead to increased morbidity and mortality. In EU countries, PM2.5 exposure shortens the average lifespan of human being by 8.6 months [26]. Long-term exposure to air pollution will have a negative impact on respiratory and cardiovascular systems [27, 28]. Furthermore, PM2.5 can enter the olfactory bulb through the nose and interrupt the integrity of the BBB [29–31]. In addition, our previous study has shown that PM2.5 may reduce the expression levels of tight junction proteins, thereby penetrating the BBB into the brain [11, 32]. In the CNS, PM2.5 may induce neuroinflammation and oxidative stress, followed by synaptic injury and the development of neurodegenerative diseases [13, 33–36]. Many studies have shown that PM2.5 can cause cognitive impairment [11]. In the current work, it was found that exposure to PM2.5 for 6 months can cause a decline of spatial memory function in mice (Fig. 1A), which is consistent with previous findings.
Many studies have shown that PM2.5 exposure may lead to cell apoptosis in various cell type and organs. In macrophage foam cells, long-term exposure to PM2.5 was shown resulting to impaired MMP and mitochondrial function, and then cell apoptosis [37]. Other studies showed that PM2.5 exposure may deteriorate MMP and then resulting to the apoptosis and destruction of morphology and function in human alveolar epithelial cells, and eventually lung damage [38]. Recent studies suggested that exposure to PM2.5 during pregnancy may disrupt the neurogenesis by inducing neuronal apoptosis, leading to depression-like behavior in offsprings [39]. Our previous study found that long-term PM2.5 exposure could lead to neuronal apoptosis in the brain tissue of mice, which happened earlier than PM2.5-induced cognitive damage [12]. In the current study, PM2.5 exposure led to the increased expression level of cleavage of caspase 3 in the hippocampus in mice. Apoptotic cells in hippocampus CA1 area were also observed in mice from PM group. The experimental data prove that PM2.5 exposure can induce cognitive impairment and neuronal cell apoptosis in mice, consistent with the previously reported evidence. The finding implied that neuronal injury induced by PM2.5 is partly dependent on abnormal neuronal apoptosis. In in vitro testing, PM2.5 exposure can cause neuronal apoptosis and increase the expression of apoptosis-related protein cleavage-caspase 3 in primary hippocampal neurons and PC12 cells (Figs. 2 3), indicating that apoptosis is an important mechanism of PM2.5-induced neuronal damage. Therefore, substances that inhibit neuronal apoptosis will be one of the promising therapeutic strategies against PM2.5 exposure-induced neuronal injury.
Recent studies have shown that MeCbl plays a key role in the normal function in the nervous system and brain [18]. Clinical evidence suggests that MeCbl may exert neuronal protective effects by promoting regeneration of injured nerves and antagonizing glutamate-induced neurotoxicity, thereby inhibiting neuropathic pain associated with diabetic neuropathy and the cognitive impairment in AD [19]. In animal models, MeCbl, as a neuroprotective agent, plays a neuroprotective role against neuronal apoptosis after peripheral nerve injury [39]. MeCbl has also been verified to go through the BBB and play a neuroprotective effect in the CNS. Studies have shown that in an animal model of ethanol-induced neuronal injury, oral administration of MeCbl could restore DNA and histone methylation capacity, promote the neurite growth of cerebellar, and finally achieve brain recovery [40]. In in vitro models, MeCbl was also shown to promote cell proliferation and migration, inhibit apoptosis, and protect against neuronal injury [23]. This evidence suggests that MeCbl may contribute to neuroprotection by inhibiting apoptosis-related signaling. In the current work, experiments showed that treatment with MeCbl could restore the impaired learning and memory function injured by PM2.5 exposure in mice (Fig. 1A). MeCbl can also effectively reduce the neuronal apoptosis induced by PM2.5 exposure both in vivo and in vitro (Figs. 1–3). Besides, MeCbl reduced the expression of apoptosis-related proteins induced by PM2.5 exposure in PC12 and primary neurons (Figs. 5 6), which could be also observed from brain tissues in mice. Both in vitro and in vivo data showed that MeCbl can effectively protect cell apoptosis induced by PM2.5 exposure, which could be the main mechanism involved in mediating process of cognitive function in mice.
Apoptosis refers to the orderly death of cells autonomously controlled by genes in order to maintain the stability of the internal environment [41]. Among them, mitochondrial pathway is an important pathway in cell apoptosis. When receiving an apoptotic signal, Bax relocates to the mitochondrial surface, which forms cross-mitochondrial membrane pores, resulting to the reduction of MMP and the increase of membrane permeability, thereby releasing apoptotic factors. A large number of studies have shown that mitochondrial damage can lead to mitochondrial dysfunction and eventually apoptosis [42]. Much evidence also has shown that MeCbl also protects mitochondria and inhibits apoptosis. For example, NADPH oxidase and downstream NF-κB pathways are inhibited by MeCbl, resulting in resulting to the rebalance of anti-inflammatory cytokines and repair of damaged mitochondria by MeCbl [40, 43]. Here in the current work, by in vivo experiments, we found that MeCbl treatment could significantly reduce the expression of mitochondrial pathway apoptotic proteins induced by PM2.5 exposure in mice (Fig. 4). After MeCbl treatment, mitochondrial apoptosis-related proteins, cleaved-caspase 9, Cyt C, and Bax/Bcl2 in PC12 cells and primary hippocampal neurons exposed to PM2.5 decreased obviously (Figs. 5 6). Furthermore, MeCbl treatment recovered the reduced MMP level by PM2.5 exposure in cell experiments (Figs. 5 6). All the above data implies that MeCbl may protect against cognitive dysfunction induced by PM2.5 exposure in mice by inhibiting mitochondrial apoptosis pathways (Fig. 7).
Schematic diagram for molecular mechanisms of MeCbl inhibiting neuronal apoptosis.
Conclusion
To pile all the evidence up, the current work dem-onstrates that MeCbl administration could ameliorate cognitive impairment and PM2.5-induced neuronal apoptosis in mice. Furthermore, PM2.5 exposure led to reduced cell viability and the activation of apoptosis-related signaling both in PC12 and primary hippocampal neurons. MeCbl treatment could partially protect against PM2.5-induced neurotoxicity by regulating mitochondrial apoptosis signaling pathway. This study provides new clues and possible therapeutic targets for MeCbl to treat neuronal injury induced by PM2.5. The current finding may provide a promising target for the treatment or prevention of air pollution-related neurodegenerative diseases.
