Minocycline Protects PC12 Cells Against Cadmium-Induced Neurotoxicity by Modulating Apoptosis

Cadmium (Cd) is a well-known heavy metal and a neurotoxic agent. Minocycline (Mino) is an anti-microbial agent with a lipophilic structure that crosses the blood–brain barrier and enters the cerebral tissue. In recent studies, Mino has been introduced as an antioxidant and anti-apoptotic chemical compound, and therefore, it was examined as a protective candidate against Cd-induced neurotoxicity. In this study, PC12 cells were exposed to Cd alone, or after being pre-treated with Mino. Initially, the cell viability and oxidative stress were analyzed using the MTT assay and fluorimetry, respectively. Then, Cd-induced apoptosis and Mino anti-apoptotic effect were evaluated in both intrinsic and extrinsic pathways using western blot analysis. Exposing PC12 cells to Cd for 24 h decreased cell viability and increased production of reactive oxygen species in comparison with the control group. Cd (35 μM) also elevated the level of caspase-8, Bax/Bcl-2, and caspase-3 proteins in the cells. Mino pre-treatment for 2 h (100 nM) increased the number of viable cells and decreased the production of reactive oxygen species, and the level of all apoptotic markers in comparison to Cd-treated cells. Considering all the evidence, it appears that Mino holds promising antioxidant and anti-apoptotic activity and can protect cells against Cd-induced oxidative stress and prevent apoptotic cell death.


Introduction
Cadmium (Cd), a highly toxic heavy metal and a significant environmental pollutant, is found in batteries, cadmium pigments, electroplating, and plastic stabilizers. The high concentrations of Cd are normally associated with industrial emission sources such as mining and smelting operations. Other important sources of Cd exposure are linked with the ecosystem (soil or water) or associated with our daily diet [1,2]. Smoking is also a major source of airborne Cd exposure. Lungs can absorb approximately 5% of the Cd content in each cigarette (50-100 ng). Once Cd enters the body, its effects can last for up to 10 years due to its long half-life [3,4].
Human beings are at risk of chronic and repetitive exposure to Cd which can consequently induce renal dysfunction, initial tubular damage followed by glomerular damage, cardiovascular and reproductive toxicities [5,6], reduction in bone mineral density, osteomalacia, diabetes, cancer, and neurotoxicity [7].
Over the past decades, Cd has been recognized as an important neurotoxic agent. Cd can be the principal cause of cognitive decline, neurodegenerative disorders including Parkinson's disease or dementia, and even a significant risk factor for depression and mental disorders [8,9].
When Cd enters the body, it increases the production of free radicals. Increased reactive oxygen species (ROS) generation, oxidative stress, lipid peroxidation, cerebral edema, and cellular dysfunction can all occur and lead to DNA damage and apoptosis. Cd also interferes with the endothelium reticulum permeability and integrity and stimulates mitogen-activated protein kinase (MAPK)-p38 which once again can lead to cellular death [10][11][12][13]. Within the nervous system (both central and peripheral (CNS and PNS), by disruptions in Na + , K + -ATPase, and calcium (Ca 2+ ) channels functions, Cd can harm neurotransmitter signaling. Monoaminergic, glutaminergic, cholinergic, and GABAergic systems can all be affected; thus, deficiencies in motor activity, learning, memory, and possibly emotion may occur [10,11]. Since humans can be exposed to Cd daily and considering all the severe outcomes, the search for a reliable remedy has always been encouraged. Based on published reports, oxidative stress and induced apoptosis are the two most important mechanisms through which Cd can harm the human body. Rosemary (Rosmarinus officinalis), Ginkgo biloba L., and Black Seed (Nigella Sativa) have shown protective activities against Cd-induced toxicity [14][15][16].
Minocycline (Mino) is a tetracycline antibiotic that aside from its use in infectious diseases, possesses immunomodulatory effects, suppresses proinflammatory cytokine release, and exerts antiapoptotic properties [17][18][19]. Owing to its lipophilic structure, Mino can cross the blood-brain barrier and act as a potential neuroprotective agent. Mounting evidence suggests that Mino can decrease the level of cleaved-caspase-3, both inducible and endothelial nitric oxide synthase, and inhibit the MAPK-p38 [20]. Mino's ameliorative effect has been studied in disorders such as Parkinson's disease, multiple sclerosis, Huntington's disease, Alzheimer's disease, and rheumatoid arthritis, and it has been recognized as a preventive candidate for the progression of the aforementioned disorders [19,[21][22][23][24]. In animal models, Mino has also shown anti-nociceptive effects in chronic pain including diabetic peripheral neuropathy [25,26], inflammatory pain [27,28], and visceral pain [29]. As for metal-induced neurotoxicity, Mino has mitigated oxidative stress and neurobehavioral impairments caused by zinc in male Wistar rats [30], reduced iron overload, brain cell death and edema, and neurological damage due to Fe 2+ or Fe 3+ exposure [31,32].
In this study, inspired by the neuroprotective properties of Mino, we aimed to elucidate its potential against Cd-induced neurotoxicity. The PC12 cell line was used as a neuron cell model [33,34]. Given that Cd causes toxicity mainly through oxidative imbalance and apoptosis, we investigated the antioxidant and antiapoptotic effect of Mino on Cd-induced neurotoxicity.

Cell Culture
PC12 (pheochromocytoma cell line of rat) cells were obtained from the Pasteur Institute (Tehran, Iran). Cells were cultured in RPMI-1640 medium and incubated at 37 °C with 5% CO 2 . The medium contained 10% fetal bovine serum and 5% antibiotic mixture (100 U/mL penicillin and 100 μg/mL streptomycin) and was changed every 2-3 days.

Cell Viability
Cell viability was analyzed with the MTT assay. PC12 cells were seeded in a 96-well microtiter plate (6000 cells/well, 100μL) and incubated for 24 h. The next day, cells were treated with (1 μM, 10 μM, 25 μM, 50 μM, and 100 μM) of cadmium chloride for 24 h to determine the IC 50 values. The same protocol was performed for defining the safe limitation for Mino concentration. Cells were incubated with (1 nM to 100 μM) of Mino for 24 h. After incubation, MTT solution with the final concentration of 0.5 mg/mL was added to each well, and cells were incubated for 3 h. MTT acts as an indicator of mitochondrial function. Viable cells can exchange MTT into insoluble formazan crystals [35]. After the incubation period, the remaining medium in each well was emptied and 150 μL of DMSO was added to dissolve the crystals. Absorbance was measured at 570 nm and 630 nm using a microplate reader.
To evaluate the protective effect of Mino on Cd-induced cytotoxicity, cells were pre-treated with Mino (50 nM, 100 nM, 500 nM, and 1 μM) for 2 h. Afterward, the chosen IC 50 value of Cd (35 μM) was added and cells were incubated for 24 h. The next day, the MTT assay was performed and the effect of Cd, with or without the presence of Mino, on cell viability was studied.

Measurement of the Intracellular ROS Production
PC12 cells (6000 cells/well, 100 μL) were seeded in a 96-well plate and incubated for 24 h. The next day, just the same as the MTT treatment protocol, there was a 2-h incubation with Mino (50 nM, 100 nM, 500 nM, and 1 μM), followed by a 24-h exposure to the IC 50 value of Cd (35 μM). After the treatment day, PC12 cells were washed with PBS and then incubated with DCF-DA for 30 min at 37 °C. DCF-DA is hydrolyzed by intracellular esterase and turned into 2′,7′-dichlorofluoresceindiacetate (DCFH) which in the presence of ROS, becomes DCF (a highly fluorescent compound) [35]. The intensity of fluorescence was analyzed via the Bio Tek Synergy H4 plate reader (485 nm excitation, 530 nm emission).

Western Blot Analysis for Determination of Protein Expression
PC12 cells were cultured in 75-cm 2 flasks with RPMI-1640. After 24 h, they were treated with Mino (100 nM, 2 h pre-treatment) and/or Cd (35 μM). The next day, cells were harvested. After removing the supernatant, cells were resuspended in 200 μL homogenization buffer (50 mM Tris-HCl (pH: 7.4), 2 mM EDTA, 2 mM EGTA, 10 mM NaF, 1 mM sodium orthovanadate (Na3VO4), 10 mM ß-glycerophosphate, 0.2% w/v sodium deoxycholate, 1 mM phenylmethylsulfonyl fluoride (PMSF), and complete protease inhibitor cocktail) and sonicated for four 10-s periods. Fifteen-minute centrifugation at 8000 g and 4 °C was performed and the precipitates were collected. The protein concentration was determined by the Bradford protein assay. For the western blot analysis, equal amounts of samples were loaded on and separated by polyacrylamide gel (12-15%) and the electrophoresed samples were transferred into PVDF membranes. The membranes were then incubated in 5% skimmed milk for 2 h at room temperature. Afterward, the membranes were washed three times with Tris buffered saline tween 20 (TBST) and incubated with primary antibodies for 2 h at room temperature including Bax (Cell Signaling, #2772) and procaspase-3 (Cell Signaling, # 9665) or over-night at 4 °C including caspase-8 (Cell Signaling, # 4790), Bcl-2 (Cell signaling, #2870), cleavedcaspase-3 (Cell Signaling, #9664). After washing them again three times, the secondary antibody, horseradish-peroxidase conjugated anti-rabbit antibody (#7074 Cell Signaling), was added and incubation was done for 2 h at room temperature. To make the bands visible, enhanced luminol-based chemiluminescent (ECL) solution in the presence of hydrogen peroxide was used. Finally, the bands were visualized via Gel Doc UV Alliance and the band intensity was analyzed via UVtec software (UK). ß-actin was used as the loading control; primary ß-actin (Cell Signaling, Cat#3700) and secondary antibody horseradishperoxidase conjugated anti-mouse (#7076 Cell Signaling) were added at room temperature for 2 and 1.5 h, respectively.

Statistical Analysis
Results are expressed as mean ± SD. The IC 50 values were calculated using Prism (version 8) and statistical analyses were done with ANOVA followed by Tukey-Kramer test to compare the differences between groups. Differences were considered statistically significant when p < 0.05.

Effect of Cd or Mino on Cell Viability
PC12 cells were exposed to Cd (1 μM, 10 μM, 25 μM, 50 μM, and 100 μM) for 24 h. Cell viability reduced significantly in a concentration-dependent manner compared with the control group (p < 0.01 for 1 μM and p < 0.0001 for 10 μM, 25 μM, 50 μM, and 100 μM). The chosen IC 50 value for the following tests was 35 ± 0.77 μM. The result of Cd exposure in PC12 cells is shown in Fig. 1.
Cells were also treated with different concentrations of Mino (1 nM to 100 μM) separately for 24 h. The groups that were exposed to 5 μM and higher concentrations of Mino showed a lower percentage of viable cells compared with the control group (p < 0.0001), yet 50 nM, 100 nM, 500 nM, and 1 μM were appeared to be safe concentrations of Mino and were used for the following tests. The response of the cells to each concentration of Mino is shown in Fig. 2.

Effect of Mino on Cd-Induced Cytotoxicity
Firstly, to discover whether or not Mino can protect the cells against Cd, three different treatment regimens with the same concentrations of Mino and Cd were designed: adding Mino (50 nM, 100 nM, 500 nM, and 1 μM) and Cd (35 μM) at the same time (no pre-treatment); 2 h; and 6 h pre-treatment with Mino before adding Fig. 1 The effect of Cd on the viability of PC12 cells. Cell viability was determined using the MTT test. Data are presented as Mean ± SD of four separate experiments; ANOVA and Tukey-Kramer post-test were used for statistical analysis. **p < 0.01 and ****p < 0.0001 in comparison with the control group Cd to the wells. After completing the MTT tests for all groups, the second regimen was the only one that significantly improved cell viability compared with the Cd-treated cells. Therefore, from here on out, 2 h of pre-treatment was performed in all the tests before adding Cd to the wells. The viability of cells that were only exposed to Cd was reduced significantly compared with the control cells (p < 0.0001). This effect was significantly ameliorated following Mino pre-treatment (50 nM and 100 nM) compared with the Cd-treated cells (p < 0.01). The effect of Cd, with or without the presence of Mino, is shown in Fig. 3.

The Effect of Mino on Cd-Induced ROS Generation
Similar to the previous test, cells were initially exposed to different concentrations of Mino for 2 h and then Cd at the final concentration of 35 μM was added to each well. The next day, ROS production in different experimental groups was measured using a fluorimeter. Cd exposure (35 μM) significantly increased the amount of ROS compared with the control group (p < 0.01); through pre-treatment with Mino, oxidative stress was significantly attenuated, and ROS production was reduced compared with the Cd treated cells (p < 0.01 for 1 μM and p < 0.0001 for 50 nM, 100 nM, and 500 nM). The cells that were exposed to Mino alone (1 μM) did not show a different level of ROS production compared with the control group. As shown in Fig. 4, all the experimental concentrations of Mino pre-treatment appeared to be effective in the reduction of ROS.

Effect of Mino and/or Cd on the Level of Caspase-8 (Pro and Cleaved) Protein
The level of the apoptotic protein, caspase-8, was investigated after exposure to Cd alone, or exposure to Cd after pre-treatment with Mino. Both procaspase-8 (p < 0.0001) and cleaved-caspase-8 (p < 0.001) were increased significantly after Cd exposure compared with the control group. Pre-treating cells with Mino (100 nM) for 2 h was effective in reducing the level of procaspase-8 and cleaved-caspase-8 proteins significantly compared with the Cd-treated cells (p < 0.0001). It is worth noting that Mino alone (100 nM) did not induce any alteration in the level of procaspase-8 or cleaved-caspase-8 proteins in comparison to the control group (Fig. 5A, B).

Effect of Mino and/or Cd on Bax/Bcl-2 Ratio
The level of the apoptotic protein, Bax, and the level of anti-apoptotic protein, Bcl-2, were both analyzed after exposing cells to Cd alone or Cd with Mino. Bax/Bcl-2 was significantly increased in the Cd group (35 μM) (p < 0.01) compared with the control. Pre-treating cells with Mino (100 nM) for 2 h significantly decreased Bax/Bcl-2 compared with the cells that received Cd alone (p < 0.01). Exposing cells to Mino alone (100 nM) did not lead to any changes in Bax/Bcl-2 compared with the control group (Fig. 5A, C).

Effect of Mino and/or Cd on the Level of Caspase-3 (Pro and Cleaved) Proteins
Exposing PC12 cells to Cd (35 μM) increased the apoptotic protein, cleaved-caspase-3 significantly (p < 0.01) compared Bcl-2 and caspase-3 (pro and cleaved) proteins, respectively. Data are presented as mean ± SD of four separate experiments; ANOVA and Tukey-Kramer post-test were used for statistical analysis. ****p < 0.0001, ***p < 0.001, and **p < 0.01 compared with control, ####p < 0.0001, and ##p < 0.01 compared with Cd group with control, yet exposure of cells to Mino (100 nM) for 2 h before Cd significantly reduced the level of cleaved-caspase-3 protein in comparison to Cd-treated cells (p < 0.01). Mino itself (100 nM) did not lead to any changes in the level of cleaved-caspase-3 protein compared with the control group. The procaspase-3 level remained the same between all the experimental groups (Fig. 5A, D).

Discussion
Heavy metals such as Cd are one of today's main environmental concerns. Human beings are exposed to Cd through smoking, occupation, and dietary routes. Cd can induce both acute and chronic neurotoxicity [6,9,36]. Mino is a broad-spectrum antibiotic with a lipophilic structure that can cross the blood-brain barrier and has radical scavenging, anti-apoptotic, and anti-inflammatory properties in nervous systems. The neuroprotective effect of Mino has been assessed in both pre-clinical and clinical studies. Interestingly, promising results have been reported from great antioxidant and anti-apoptotic properties in cellular or animal studies to ameliorating chronic pain and improving the symptoms of Parkinson's disease or multiple sclerosis [17,20,26,37]. Moreover, Mino has shown a protective effect against metal-induced neurological damage, namely zinc and iron [30][31][32].
In the current study, the protective effect of Mino against Cd-induced neurotoxicity was evaluated. It has been reported beforehand in the same cell line, Cd can reduce cell viability in a time and dose-dependent manner [38][39][40]. Cd also increased the intracellular ROS generation in cells and induce oxidative stress [41,42].
In both in vitro and in vivo studies, Mino exhibited cytoprotective and anti-oxidative properties which led to a higher percentage of viable cells and a lower percentage of ROS in comparison to the groups that did not receive Mino. Additionally, Mino pre-treatment for 24 h reduced the level of malondialdehyde and increased the activity of superoxide dismutase and catalase in PC12 cells; the changes occurred after exposure to 6-hydroxydopamine [43].
In our study, Cd exposure reduced cell viability and caused ROS generation and oxidative stress in PC12 cells, yet pre-treating cells with Mino for 2 h attenuated these effects.
As mentioned previously, oxidative stress leads to the activation of other damaging pathways such as apoptosis. Apoptosis is a programmed form of death that is triggered by intracellular signals and eventually dismantles the cells. Caspases are a family of cysteine proteases that play a crucial role in apoptosis. Apoptotic pathways can be twosome: extrinsic pathway which is activated by the recruitment and cleavage of caspase-8 to the death-inducing signaling complex (DISC) and intrinsic (mitochondrial) pathway which initiates through an increase in the pro-apoptotic proteins (Bax, Bak, Bok, and Bim) as compared to the anti-apoptotic proteins (Bcl-2, Bcl-X L , and Bcl-W ). Where both extrinsic and intrinsic pathways meet, other members of the caspase family (caspase-3, caspase-7, and caspase-6) are activated [44][45][46].
As it has been previously reported, Cd can induce apoptosis and increase the level of caspase-8, Fas, caspase-3 proteins, and Bax/Bcl-2 ratio in different cell lines such as PC12, astrocytes, and Na2 cells in a time and concentration-dependent manner [47][48][49]. A similar outcome was described after male Wistar rats were exposed to Cd (1, 2, or 4 mg/kg) for 15 days. The mRNA expression was studied via the real-time PCR method; Cd led to an increased Bax/Bcl-2 ratio in rats [50]. Furthermore, in a recent study, the chronic low dose exposure of Cd (17 μg/kg/day) was studied in the rat striatum and hippocampus. As was anticipated, Cd, even at low doses, significantly increased oxidative stress and potentiated apoptotic markers, to be specific caspase-8, caspase-9, Bax/Bcl-2, and cytochrome c [51]. To elucidate Cd-induced neuronal death, primary cortical neurons obtained from Sprague-Dawley rats were transfected with Fas siRNA, then subsequently treated with Cd (10 μM) for 12 h. The Fas-knockdown cells showed lower levels of annexin V-positive cells, Fas, cleavedcaspase-8, and phosphorylated Jun N-terminal kinase (JNK). Fas knockdown also inhibited the damage done to the mitochondrial membrane and blocked the cleavage of caspase-3 and 9. Once compared with Cd alone, the role of caspase-8 and JNK pathways in Cd-induced neurotoxicity was inferred [52].
On the other hand, Mino has shown promising results as a neuroprotective agent in numerous studies. In a Huntington cellular model, Mino (45 mg/kg, intraperitoneal) reduced the level of caspase-8, caspase-9, caspase-3, and Bid proteins [53]. Moreover, Mino decreased the level of Bax and cleaved-caspase-3, in Sprague-Dawley rats and ICR mice respectively [54,55].
In a different study in 2021, the neuroprotective effect of Mino was elucidated using a traumatic brain injury mice model. Aside from inhibiting apoptosis (caspase-3), Mino repaired the blood-brain barrier, recovered neurobehavioral signs, and diminished inflammation (tumor necrosis factor-α and interleukin-6). In vitro, Mino increased the levels of adherens junction and tight junction proteins and therefore raised the number of viable cells [56].
In our study, Cd activated extrinsic and intrinsic pathways of apoptosis and increased the levels of caspase-8, Bax/ Bcl-2, and caspase-3 proteins. Pre-treating cells with Mino for 2 h alleviated this effect and reduced caspasedependent apoptosis through both pathways. The underlying mechanism of Cd toxicity and Mino neuroprotective effect are shown in Fig. 6.

Conclusion
In this study, we looked into the underlying mechanisms of Cd-induced neurotoxicity and perused Mino's potential protection against this matter. Concluding from our observations, Cd-induced oxidative stress ultimately leads to apoptotic cell death. Nevertheless, pre-treating cells with Mino inhibit ROS production and apoptosis, through both intrinsic and extrinsic pathways, and protect PC12 cells against Cd-induced neurotoxicity due to its antioxidant and anti-apoptotic activities.