Minocycline protects PC12 cells against cadmium- induced neurotoxicity by modulating apoptosis

doi:10.21203/rs.3.rs-1536960/v1

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Minocycline protects PC12 cells against cadmium- induced neurotoxicity by modulating apoptosis

https://doi.org/10.21203/rs.3.rs-1536960/v1

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Cadmium (Cd) is a well-known heavy metal and an environmental toxic agent. Upon human exposure, severe outcomes, such as neurotoxicity, can be anticipated. Minocycline (Mino) is an anti-microbial agent with a lipophilic structure that crosses the blood-brain barrier and enters the cerebral tissue. Based on recent studies, Mino has been introduced as an anti-oxidative 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 hours 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 hours (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 anti-oxidant and anti-apoptotic activity and can protect cells against Cd-induced oxidative stress and prevent apoptotic cell death.

Cadmium

Neurotoxicity

Minocycline

Apoptosis

Oxidative stress

Neuroprotection

Cadmium (Cd), a highly toxic heavy metal and a substantial environmental pollutant, is found in batteries, cadmium pigments, electroplating, and plastic stabilizers. 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 be the cause of renal dysfunction, initial tubular damage followed by glomerular damage; cardiovascular and reproductive toxicity [5, 6]; decreased 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 then lead to DNA damage and apoptotic cell death. 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 [10–13]. Within the nervous system (both central and peripheral (CNS and PNS)), by disruptions in Na⁺, K⁺-ATPase, and calcium (Ca2+) 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 befall [10, 11]. Since humans can be exposed to Cd daily and considering all the severe outcomes as well, 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 cause toxicity. Rosemary (Rosmarinus officinalis), Ginkgo biloba L., and Black Seed (Nigella Sativa) have shown protective activity against Cd-induced toxicity [14–16].

Minocycline (Mino), is a tetracycline antibiotic that beyond its use in infectious diseases, possesses immunomodulatory effects, suppresses proinflammatory cytokine release, and exerts antiapoptotic properties [17–19]. 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 caspase-3-cleaved, both inducible and endothelial nitric oxide synthase, and inhibit the MAPK-p38 [20]. Mino's ameliorative effect has been studied in disorders like Parkinson’s disease, Multiple Sclerosis, Huntington’s disease, Alzheimer’s disease, and rheumatoid arthritis. Promising studies have recognized Mino as a preventive candidate for the progression of the aforementioned disorders [19, 21–24]. 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].

Regarding the neuroprotective properties of Mino, in the current study, we evaluated the protective effect of Mino against Cd-induced neurotoxicity using the PC12 cell line. Given that Cd causes toxicity mainly through oxidative imbalance and apoptotic cell death, we chose to investigate the anti-oxidant and anti-apoptotic effect of Mino on Cd-induced neurotoxicity.

Materials

Fluorescent probe 2,7-dichlorofluorescein diacetate (DCF-DA) and (4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium (MTT) were purchased from Sigma. RPMI 1640 was purchased from Bioidea Company and FBS was purchased from Gibco. Rabbit monoclonal anti-serums against procaspase-3, caspase-3-cleaved, Bcl-2, caspase-8, and ß-actin, rabbit polyclonal anti-serum against Bax, and anti-mouse and anti-rabbit IgG HRP-linked antibodies were purchased from Cell signaling. Cadmium and minocycline were purchased from Tinab Shimi. Polyvinylidene fluoride (PVDF) membrane was purchased from Bio-Rad.

Cell culture

PC12 (pheochromocytoma cell line of rat) cells were obtained from the Pasteur Institute of Iran (Tehran). Cells were cultured in RPMI-1640 medium and incubated at 37˚C with 5% CO₂. 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 hours. The next day, cells were treated with (1 µM, 10 µM, 25 µM, 50 µM, and 100 µM) of cadmium chloride for 24 hours to determine the IC₅₀. The same protocol was performed for defining the safe limitation for Mino concentration. Cells were treated with (1 nM- 100 µM) of Mino for 24 hours. After incubation, MTT solution with the final concentration of 0.5 mg/mL, was added to each well, and cells were incubated for 3 hours. MTT acts as an indicator of mitochondrial function. Viable cells can exchange MTT into insoluble formazan crystals [30]. 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.

Meaning 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 hours. Afterward, the chosen IC₅₀ of Cd (35 µM) was added and cells were incubated for 24 hours. 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 hours. The next day, pre-treatment with Mino (50 nM, 100 nM, 500 nM, and 1 µM) was done for 2 hours and then the IC₅₀ of Cd (35 µM) was added, just the same as the MTT test. Following the treatment day, PC12 cells were washed with PBS and then incubated with CM-H₂DCFDA for 30 minutes at 37˚C. CM-H₂DCFDA is hydrolyzed by intracellular esterases, turns into 2′,7′-dichlorofluorescein‐diacetate (DCFH) which can become DCF (a highly fluorescent compound) in the presence of ROS [30]. 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 cm² flasks with RPMI-1640. After 24 hours, they were treated with Mino (100 nM, 2h 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-second periods. 15-minute centrifugation at 10000 rpm 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 hours at room temperature. Afterward, the membranes were washed three times with TBST and incubated with primary antibodies for 2 hours 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), caspase-3-cleaved (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 hours at room temperature. Finally, after washing the membranes three times, 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 horseradish-peroxidase conjugated anti-mouse (#7076 Cell Signaling) were added at room temperature for 2 and 1.5 hours, respectively.

Statistical analysis

Results are expressed as mean ± SD. The IC₅₀ value was 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 hours. 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 IC₅₀ 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 – 100 µM) separately for 24 hours. 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 hours; and 6 hours pre-treatment with Mino before adding 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. 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). Therefore, from here on out, 2 hours of pre-treatment was performed in all the tests before adding Cd to the wells. 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 step, cells were initially exposed to different concentrations of Mino for 2 hours and then Cd at the final concentration of 35 µM was added to each well. The next day, ROS production of 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 & 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 caspase-8-cleaved (p < 0.001) were increased significantly after Cd exposure compared with the control group. Pre-treating cells with Mino (100 nM) for 2 hours was effective in reducing the level of procaspase-8 and caspase-8-cleaved proteins significantly compared with the Cd-treated cells (p < 0.0001). It’s worth noting that Mino alone (100 nM) did not induce any alteration in the level of procaspase-8 or caspase-8-cleaved 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 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 hours 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 & cleaved) proteins

Exposing PC12 cells to Cd (35 µM) increased the apoptotic protein, caspase-3-cleaved significantly (p < 0.01) compared with control; yet exposure of cells to Mino (100 nM) for 2 hours before Cd, significantly reduced the level of caspase-3-cleaved protein in comparison to Cd-treated cells (p < 0.01). Mino itself (100 nM) did not lead to any changes in the level of caspase-3-cleaved protein compared with the control group. The procaspase-3 level remained the same between all the experimental groups (Fig. 5A & D).

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. Both acute and chronic forms of Cd toxicity can lead to severe outcomes, one of which is neurotoxicity [6, 9, 31]. Mino is a broad-spectrum antibiotic with a lipophilic structure that can cross the blood-brain barrier, then act as a radical scavenger, an anti-apoptotic agent, and an anti-inflammatory factor. The neuroprotective effect of Mino has been assessed in both pre-clinical and clinical studies. Interestingly, promising results have been reported from great anti-oxidant 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, 32].

In the current study, the protective effect of Mino against Cd-induced neurotoxicity was evaluated. It’s been reported beforehand in the same cell line, Cd can reduce cell viability in a time and dose-dependent manner [33–35]. Cd also increased the intracellular ROS generation in cells and induce oxidative stress [36, 37].

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 hours reduced the level of malondialdehyde, and increased the activity of superoxide dismutase and catalase in PC12 cells; the changes that occurred after exposure to 6-hydroxydopamine [38].

In our study, Cd exposure led to reduced cell viability and caused ROS generation and oxidative stress in PC12 cells; yet pre-treating cells with Mino for 2 hours 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 [39–41].

As it’s 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 [42–44]. 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 [45].

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 [46]. Moreover, Mino decreased the level of Bax and caspase-3-cleaved, in Sprague-Dawley rats and ICR mice respectively [47, 48].

In our study, Cd activated extrinsic and intrinsic pathways of apoptosis and increased caspase-8, Bax/Bcl-2, and caspase-3 proteins. Pre-treating cells with Mino for 2 hours alleviated this effect and reduced caspase-dependent apoptosis through both pathways. The underlying mechanism of Cd toxicity and Mino neuroprotective effect are shown in Fig. 6.

In this study, we looked into the underlying mechanism of Cd-induced neurotoxicity and perused Mino’s potential protection against this matter. Concluding from our observations, Cd-induced oxidative stress ultimately led to apoptotic cell death. Nevertheless, pre-treating cells with Mino inhibited ROS production and apoptosis, through both intrinsic and extrinsic pathways and protected PC12 cells against Cd-induced neurotoxicity due to its anti-oxidant and anti-apoptotic activity.

Acknowledgments

The authors are thankful to the Vice-Chancellor of Research, Mashhad University of Medical Sciences for financial support. The results presented in this paper are part of a Pharm.D thesis.

Author Contribution

MSH did the research, analyzed the data, and wrote the paper. SM supervised, designed the experiments, verified the analytical methods, and checked the whole procedure and paper. BMR designed the experiments. HH supervised, conceived the original idea, and checked the whole procedure and paper.

Funding

This study was financially supported by a grant from the Vice-Chancellor of Research, Mashhad University of Medical Sciences (Grant number: 981440).

Competing Interests

The authors have no financial or non-financial interest to disclose.

Data Availability

All collected data and experiments are available in the article.

Ethics Approval

This article does not include any experiments on animals or human participants.

Consent to participate

Not applicable.

Consent to publish

Not applicable.

Conflict of interest

The authors declare no conflict of interest.

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No competing interests reported.

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Editorial decision: Major revision
09 May, 2022
Reviews received at journal
29 Apr, 2022
Reviewers agreed at journal
16 Apr, 2022
Reviewers invited by journal
13 Apr, 2022
Editor assigned by journal
13 Apr, 2022
Submission checks completed at journal
13 Apr, 2022
First submitted to journal
08 Apr, 2022

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Minocycline protects PC12 cells against cadmium- induced neurotoxicity by modulating apoptosis

Status:

Version 1

Abstract

Figures

Introduction

Experimental

Materials

Cell culture

Cell viability

Measurement of the intracellular ROS production

Western blot analysis for determination of protein expression

Statistical analysis

Results

Effect of Cd or Mino on cell viability

Effect of Mino on Cd-induced cytotoxicity

The effect of Mino on Cd-induced ROS generation

Effect of Mino and/or Cd on the level of Caspase-8 (Pro & cleaved) protein

Effect of Mino and/or Cd on Bax/Bcl-2 ratio

Effect of Mino and/or Cd on the level of Caspase-3 (Pro & cleaved) proteins

Discussion

Conclusion

Declarations

References

Additional Declarations

Status:

Version 1