Mild hypothermia alleviates oxygen-glucose deprivation/reperfusion-induced apoptosis by inhibiting ROS generation, improving mitochondrial dysfunction and regulating DNA damage repair pathway in PC12

doi:10.21203/rs.3.rs-2159170/v1

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Mild hypothermia alleviates oxygen-glucose deprivation/reperfusion-induced apoptosis by inhibiting ROS generation, improving mitochondrial dysfunction and regulating DNA damage repair pathway in PC12

https://doi.org/10.21203/rs.3.rs-2159170/v1

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The brain ischemia/reperfusion (I/R) injury have a great impact on human life and property safety, as far as we know, mild hypothermia (MH) is an effective measure which reduces neuronal injury. However, the precise mechanism is not extremely clear. The purpose of this study was to explore whether mild therapeutic hypothermia can play a protective role in nerve cells dealing with brain I/R injury and its specific mechanism in vitro. A flow cytometer, cell counting kit-8 (CCK-8) assay, lactate dehydrogenase (LDH) release assay were performed to detect apoptotic rate of cells, cell viability and cytotoxicity respectively, while reactive oxygen species (ROS) assay kit, JC-1 fluorescent methods, immunofluorescence, western blot were used to explore ROS, mitochondrial transmembrane potential (Δψm), mitochondrial permeability transition pore (MPTP), expression of proteins respectively. The result indicated that the activity was decreased, while the cytotoxicity and apoptosis rate were increased after treating with OGD/R in PC12, however, MH could antagonize this phenomenon. Strangely, treating with OGD/R increased the release of ROS and the transfer of Cytochrome C (Cyt-C) from mitochondria to cytoplasm, besides, it also upregulated the expression of γH2AX, Bax and Clv-caspase3 but downregulated the expression of PCNA, Rad51, Bcl-2 and inhibited the function of mitochondria in PC12, the opposite trend was observed after MH treatment. Therefore, our results suggest that MH alleviates PC12 against oxygen-glucose deprivation/ reoxygenation-induced injury with the mechanism of inhibiting cell apoptosis by reducing ROS production, improving mitochondrial function, reducing DNA damage, and enhancing DNA repair.

Mild hypothermia

OGD/R

ROS

Mitochondrial function

DNA damage repair

Ischemic stroke is a dangerous disease which causes of disability in adults, ischemia-reperfusion injury is the most critical part in the progress of ischemic stroke[1, 2]. At present, there are limited therapeutic methods for I/R injury. Mild hypothermia, A temperature of 32–35 ℃, was not only the valuable therapy method heart surgery in the previous, but also the first treatment with proven efficacy for postischemic neurological injury[3, 4], and was still an effective method to treat ischemic encephalopathy[5]. However, mild hypothermia involves many pathophysiological change, the exact mechanisms are not fully understood.

At present, mild hypothermia has been used in lots of neurologic emergencies with considerable success, such as ischemic stroke, subarachnoid hemorrhage, traumatic brain injury, hepatic encephalopathy, cardiopulmonary arrest and so on [6]. It is reported that this may play its therapeutic role in a number of ways, such as regulated lysosomal function, autophagic flux, caspase-3 activation and the Wnt/b-catenin signaling pathway in our previous study[7–9], there are also calcium influx, miR-122, IGF-1R/AKT pathway, IRAK2/NF-κB signaling pathway, etc[10–12]. In addition, DNA damage is defined as physical or chemical changes in DNA within cells that affect the interpretation and transmission of genetic information[13]. DNA repair is a cellular response mechanism to DNA damage to reduce its detrimental consequences on genomic integrity[14]. Research shows that DNA damage repair were related to I/R injury[15]. However, there are few studies on whether mild hypothermia can protect the nerve injury caused by cerebral I/R injury by regulating DNA damage repair pathway.

More and more researches revealed that the conduction of mitochondria get in touch with I/R injury. Studies have shown that inhibition of mitochondrial overfission, promotion of mitochondrial biogenesis and reduction of oxidative stress can alleviate liver I/R injury[16]. Targeting mitochondrial dynamics and biogenesis could accelerate recovery from renal I/R injury[17]. The apoptosis induced by I/R injury can be improved by inhibiting the activation of critical factors in mitochondrial pathway[18]. Therefore, mitochondria make a vast difference in the progress of I/R injury was shown in lots of programs, in addition, accumulating evidence suggests a close relationship between mitochondrial dysfunction, oxidant-antioxidant imbalance and DNA damage repair[19]. In conclusion, it is reasonable to assume that mild hypothermia may counteract I/R injury by altering generation of ROS, pattern of mitochondrial dysfunction, and expression of DNA damage repair proteins.

In the present study, the model of OGD/R injury in PC12 was built to perform experiment, and it has been widely used to simulate I/R injury in vitro. As a result, the activity, cytotoxicity and apoptosis rate of PC12 were altered significantly after treating with OGD/R. In the meantime, the morphology and transmembrane potential of mitochondrion, the level of ROS, the expression of DNA damage repair proteins, the release of Cyt-C and openness of MPTP channel had also changed accordingly, however, this change could be reversed by MH. We aim to demonstrate that mild hypothermia alleviates PC12 against OGD/R-induced injury by regulating ROS, mitochondrial function and DNA damage repair pathwy.

Cell culture

PC12 cells were purchased from the cell bank of the Chinese Academy of Science (Shanghai, China), and were cultured in Dulbecco's Modified Eagle's Medium (DMEM)/F12 medium (Gibco Company, U.S.) supplemented with 10% fetal bovine serum (Hyclone Company, USA). Then, cells were grown at 37°C in a humidified atmosphere that contained 5% CO2.

Oxygen Glucose Deprivation And Reoxygenation (Ogd/r)

In vitro, ischemia-reperfusion injury was simulated by oxygen-glucose deprivation/reperfusion injury generally. As in previous research, a glucose-free balanced salt solution(BSS) containing 130 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl2, 26 mM NaHCO3, 0.8 mM MgCl2, and 1.18 mM NaH2PO4 were used to incubate PC12 cells instead of normal DMEM medium. PC12 cells were equilibrated with a continuous flux of gas (95% N2/5% CO2) for 10 min using an anaerobic

chamber (PLAS & LABS, MI, USA), next, they were humidified at 37 ℃ for 2–6 h. Then, PC12 cells after OGD were incubated with DMEM medium with 10 mM glucose and 10% fetal bovine serum, and were reoxygenated in an incubator (74% N2/21% O2/5% CO2) at 33℃, 35℃ or 37℃ for 12 h or 24 h. The control groups cells were treated with normal medium in the condition of 74% N2/21% O2/5% CO2 at 37℃.

Cell Activity Assay

Cell viability was assessed by cell counting Kit-8(CCK-8). As shown below, the cells of PC12 was seeded into 96-well plates at 1×10⁴ cells/well, next day, cells in 96-well plates were treated with OGD/R 6h, OGD/R 6h + mild hypothermia (33℃for 12h), OGD/R 6h + mild hypothermia (33℃for 24h), OGD/R 6h + mild hypothermia (35℃for 12h), OGD/R 6h + mild hypothermia (35℃for 24h) respectively. Lastly, the cells of every well added 10 µl CCK-8 and detected optical density (OD) value after 2 hours and a Multiskan Ascent microplate photometer (EnSpire 2300 Multilabel Reader, PE, USA) was performed to determine absorbance of the solution at 450 nm.

Lactic Acid Dehydrogenase (Ldh) Assay

The LDH release assay kit was used to detect injury of PC12 cells. According to the manufacturer's protocol, 50 µl of the culture liquid groups of control, OGD/R 6h, OGD/R 6h + mild hypothermia (33℃for 12h), OGD/R 6h + mild hypothermia (33℃for 24h), OGD/R 6h + mild hypothermia (35℃for 12h), OGD/R 6h + mild hypothermia (35℃for 24h) was removed for the detection of extracellular LDH activity, the absorbance of the formazan was detected at 490 nm using a reader (EnSpire 2300 Multilabel Reader, PE, USA).

Intracellular Ros Detection

The level of ROS of PC12 cells after OGD/R and MH was detected using ROS assay kit (Beyotime, Beijing, China). After treatment of PC cells according to the product instructions, cells were incubated for 20 min at 37℃ in dark and were analyzed by the flow cytometer (America Becton Dickinson Company).

Flow Cytometry

The apoptotic rate of PC12 was measured using Annexin V-APC/7-AAD double staining kit (Keygen Biotechnology, Nanjing, china). Briefly different groups of cells treated with control, OGD/R 6h, OGD/R 6h + mild hypothermia (33℃for 12h), OGD/R 6h + mild hypothermia (33℃for 24h), OGD/R 6h + mild hypothermia (35℃for 12h), OGD/R 6h + mild hypothermia (35℃for 24h) were dealt with the above kits according to protocol. Lastly, the analysis of stained cells was performed using the flow cytometer (America Becton Dickinson Company).

Transmission Electron Microscopy

PC2 cells were collected after treating with different groups, then, washing with PBS before fixed in 3% glutaraldehyde at 4°C overnight. Subsequently, cells were fixed in 1% osmium tetroxide for 1 h. After embedding in Epon resin, uranyl acetate and lead citrate were performed to stain sections. Finally, the transmission electron microscope JEM-2010HR (Electronics Co. Ltd, Japan) was used to observe samples.

Immunofluorescence

The mitochondrial membrane potential was measured by JC-1 fluorescent probe (Beyotime Biotechnology, Jiangsu, China). Succinctly, PC12 of control, OGD/R 6h, OGD/R 6h + mild hypothermia(33℃for 24h) were harvested and incubated at 37℃ with JC-1 staining solution (5mg/ml) for 20 min. Following, the red fluorescence intensity of JC-1 aggregates (excitation wavelength: 525nm and emission wavelength: 590nm) and green intensity of JC-1 monomers (excitation wavelength: 490nm and emission wavelength: 530nm) were exported by FACScan flow Cytometry. Lastly, the FlowJo software version 9.3.2 (TOMY Digital Biology) was performed to detect the relative ratio of red to green fluorescent intensity and the proportion of mitochondrial depolarization.

Western Blot Analysis

Firstly, the total proteins were extracted with RIPA buffer (Beyotime, Shanghai, China) after treating with different groups. Cyt-C was extracted using Cell Mitochondria Isolation Kit (Beyotime, Shanghai, China). Secondly, SDS-PAGE was used to separate proteins, next, proteins would be transferred onto a PVDF membrane (EMD Millipore, Billerica, MA, USA) and blocked with 5% non-fat milk. Thirdly, the membrane was incubated with primary antibody at 4℃overnight, primary antibody of Bad (#9239), Bcl-2 (#4223S), Cyt-C (#4280S), caspase-3 (#9664S), γH2AX (#9718S), PCNA (#13110S), GAPDH (#5174S)( all Cell Signaling Technology, Inc., Danvers, MA, USA) and Rad51 (#133534, Abcam, UK) was diluted by primary antibody dilution buffer (Beyotime)(1:1000), in addition, it was incubated with anti-rabbit IgG HRP-linked antibody(Cell Signaling Technology, Inc., Danvers, MA, USA) for 1hour at room temperature. Lastly, the Image J software (NIH, Bethesda, MD, USA) was used to analyze densitometry on immunoblots.

Statistical analysis

All the data analysis was performed using GraphPad Prism 8.0 software (GraphPad Software, La Jolla, CA, USA). Unpaired Student’s t-test analysis was used to determine the significance. ^*p and ^∆ p < 0.05, ^**p and ^{∆ ∆} p < 0.01, ^***p and ^{∆ ∆ ∆} p < 0.001 were considered statistically significant. All experiments were performed in triplicate. Data were expressed as the means ± standard deviation.

The model of oxygen-glucose deprivation/reperfusion-induced injury (OGD/R) in PC12 cell

An OGD/R model was utilized with differentiated PC12 cells, we examined the apoptosis rate of OGR/R 2h, 4h and 6h. The results showed that the apoptosis rate was increasing significantly in groups of OGD/R 4h and OGD/R 6h (Fig. 2A and 2B), however, the apoptosis rate of PC12 cells increased more obviously after OGD/R 6h. Therefore, we selected OGD/R 6h for further experiments.

Mild Hypothermia Reversed Ogd/ R-induced Apoptosis By Decreasing Ros Production

To explore the effect of mild hypothermia in regulating PC12 cells growth after OGD/R. The cell viability, cytotoxicity and apoptotic rate of PC12 cells were determined by CCK-8 kit, LDH release assay kit and flow cytometry separately. The results indicated that the viability of cells treated with MH 33℃ for 24h increased individually after OGD/R 6h (Fig. 3A). In additon, both cytotoxicity (Fig. 3B) and apoptotic rate (Fig. 3D and 3E) of cells showed varying degrees of reduction, but the most significant reduction was observed at 33℃ 24h (Fig. 3B and 3D). Furthermore, the expression of pro-apoptotic proteins (Bax and Cas-pascase3) were up-regulated while anti-apoptotic proteins (Bcl-2) was down-regulated after OGD/R 6h, however, the opposite result was obtained after further MH 33℃ 24h (Fig. 7A).The level of ROS in PC12 cells was detected by ROS assay kit. The result revealed that the level of ROS up-regulated significantly after OGD/R 6h, while it down-regulated significantly after further MH 33℃ for 24h (Fig. 3C).

Mild Hypothermia Alleviates Ogd/r Induced Mitochondrial Dysfunction And Ultrastructure Of Mitochondria

To explore the effect of MH on mitochondrial function after OGD/R treatment, we detected the mitochondrial membrane potential using JC-1 fluorescent probe. The result showed that the membrane potential of mitochondria decreased significantly after OGD/R 6h, while it increased significantly after further MH 33℃ for 24h (Fig. 5). Furthermore, the ultrastructure of mitochondria was observed by transmission electron microscopy. The result revealed that the mitochondria of PC12 cells were severely damaged, most of mitochondria were swollen and the mitochondrial cristae network disappeared after OGD/R 6h. Inversely, the mitochondrial damage was improved as the mitochondrial cristae network was vaguely visible after further MH 33℃ 24h (Fig. 4).

Mild hypothermia inhibited mitochondrial permeability transition pore and suppressed the transfer of Cyt-C rom mitochondria to cytoplasm.

To detect how MH made its function in regulating growth and apoptosis in PC12 cells, the MPTP assay kit was performed to measure the openness of MPTP. The result showed that the openness of the MPTP increased after OGD/R 6h, while the openness of the channel decreased significantly after further MH 33℃ 24h (Fig. 6A). In addition, after mitochondria were extracted with cell mitochondria isolation kit, the expression of Cyt-C n mitochondrial and cytoplasm was measure by western. The result indicated that the expression of Cyt-C as decreased in mitochondria but increased in cytoplasm after OGD/R 6h, however, the opposite result was obtained after further MH 33℃ 24h (Fig. 6B).

Mild Hypothermia Inhibited Dna Damage And Promoted Dna Repair

To explore the possible mechanism of MH (33℃ 24h) reversing OGD/R (6h)-induced apoptosis in PC12 cells, western blot was used to detect the expression of DNA damage repair proteins. The result showed that the expression of DNA damage proteins (γH2AX) increased, while the expression of DNA repair proteins(PCNA and Rad51) decreased after OGD/R 6h, the results were reversed after MH 33℃ 24h (Fig. 7B).

The prognosis of cerebral ischemia-reperfusion injury caused by diseases such as cardiac arrest and stroke is always poor[20, 21]. Mild hypothermia (32°C-35°C), an effective neuroprotective agent, plays a neuroprotective role after ischemia reperfusion by inhibiting inflammatory response, apoptosis and necrosis, and prolongs the treatment time of other neuroprotective agents[22], whereas, keeping the body temperature below 34℃ can lead to serious systemic complications such as immunologic, hematologic, cardiac, and metabolic side effects[23]. Therefore, it is necessary to investigate the specific mechanism of mild hypothermia in the treatment of ischemia-reperfusion injury in order to further enhance the efficacy and reduce side effects. In this study, we aimed to investigate whether MH could reduce apoptosis of PC12 cells by reducing ROS release, regulating DNA damage repair pathway and improving mitochondrial function.

ROS, produced by all kinds of cells, are a set of unstable molecules including hydroxyl radical, hydrogen peroxide, superoxide and singlet oxygen[24]. ROS can participate in the regulation of cellular proliferation and apoptosis, migration, invasion, differentiation, vascular tone, cellular adhesion and so on by redox-responsive signaling pathways[25]. According to literature reports, excessive production of intracellular reactive oxygen species is a key factor in the occurrence of recurrent ischemia-reperfusion injury[26]. In our study, results consistent with reports suggest that the release of ROS was increased after OGD/R, it is confirmed again that ROS is associated with ischemia-reperfusion injury, in addition, this phenomenon can be reversed by MH. ROS is mainly produced by mitochondria, so mitochondria are susceptible to oxidative stress, which leads to their dysfunction, It is usually manifested as loss of mitochondrial membrane potential, release of Cyt-C, alteration of mitochondrial respiratory complexes, open of MPTP, increase in mitochondrial mass, decrease in mitochondrial transcription factor A and increase in mitochondrial DNA fragmentation, Collapse of the mitochondrial membrane[13, 27]. The results in our study indicated that OGD/R leaded to decreased mitochondrial membrane potential, excessive opening of MPTP, mitochondrial swelling and morphological variation while the opposite result was obtained after further MH. Therefore, it is reasonable to assume that mild hypothermia improves mitochondrial dysfunction by reducing ROS release and reverses the apoptosis of PC12 cells.

DNA damage is classified into two types: endogenous and exogenous. The hydrolysis and oxidation of chemically active DNA with intracellular water and ROS are referred to as endogenous DNA damage. On the other side, exogenous DNA damage refers to DNA damage caused by environmental, physical, and chemical factors[28]. Double-Strand DNA Breaks is one of the most severe types of DNA damage, γH2AX, produced by H2AX phosphorylation, is a member of the histone H2A family and is widely used as a biomarker for detecting Double-Strand DNA Breaks[29]. In our study, the expression of γH2AX was significantly upregulated after OGD/R, while it was downregulated after further MH, indicating that OGD exacerbated Double-Strand DNA Breaks, while MH repaired Double-Strand DNA Breaks. DNA repair mainly consists of sensors, sensors, and effectors with the aim of coordinating the repair of DNA damage and ensuring the fidelity of DNA replication[14]. PCNA, an important factor in DNA replication and repair, reflects the activity of intracellular DNA replication and repair[30], In addition, RAD51 is a key regulator of DNA fidelity and plays multiple roles in double-strand break repair, replication stress, and meiosis, enabling accurate and timely DNA repair[31]. Our study indicated that the expression of PCNA and Rad51 decreased after OGD/R, while the opposite results were observed after further MH. This suggests that DNA replication and repair are inhibited after OGD/R and enhanced after further MH. ROS aggravates cellular DNA damage and inhibits DNA repair, which further induces apoptosis through mitochondria-dependent pathways[32]. Our study showed that the expression of Bax and Clv-caspase3 were up-regulated while the expression of Bcl-2 was down-regulated. Therefore, MH may alleviate the apoptosis of PC12 cells through reducing the release of ROS, inhibiting DNA damage, improving DNA repair.

In summary, our study revealed that MH may inhibit apoptosis by reducing OGD/ R-induced ROS release, improving mitochondrial function and regulating DNA damage repair pathway. This further elucidates the mechanism of MH in the treatment of brain ischemia/reperfusion injury, and provides a new idea for reducing the side effects of MH in the treatment of brain ischemia/reperfusion injury and improving the treatment efficiency.

I/R

ischemia/reperfusion

mild hypothermia

OGD/R

Oxygen-glucose deprivation/reperfusion-induced injury

Cell counting Kit-8(CCK-8)

LDH

Lactate dehydrogenase

ROS

Reactive oxygen species

Δψm

Mitochondrial transmembrane potential

MPTP

Mitochondrial permeability transition pore

Cyt-C

Cytochrome C.

Acknowledgements

We are grateful to the anonymous reviewers for their critical insight into improving this manuscript.

Author Contributions

J J, Y F and TE Z designed the experiments. JR M, QH H and HF L participated in the experiments. WG X finished statistical analyses. TE Z and JR M wrote the article and performed figures. J J and Y F provided the supervision and the financial support. All authors reviewed and approved the manuscript.

Funding

This work was supported by the medical science and technology research project of Foshan (No. 2016AB002111) (to J J) and the 14th Five-Year high-level Medical Key Specialty Project of Foshan. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Data availability

The data that support the findings of this study are available.

Competing interests

The authors declare no competing interests.

Conflict of interest

All authors declare no conflict of interest.

Ethical approval

Not applicable.

Consent for publication

All authors have given their consent for the manuscript to be published.

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You are reading this latest preprint version

Mild hypothermia alleviates oxygen-glucose deprivation/reperfusion-induced apoptosis by inhibiting ROS generation, improving mitochondrial dysfunction and regulating DNA damage repair pathway in PC12

Status:

Version 1

Abstract

Figures

Introduction

Materials And Methods

Cell culture

Oxygen Glucose Deprivation And Reoxygenation (Ogd/r)

Cell Activity Assay

Lactic Acid Dehydrogenase (Ldh) Assay

Intracellular Ros Detection

Flow Cytometry

Transmission Electron Microscopy

Immunofluorescence

Western Blot Analysis

Statistical analysis

Results

The model of oxygen-glucose deprivation/reperfusion-induced injury (OGD/R) in PC12 cell

Mild Hypothermia Reversed Ogd/ R-induced Apoptosis By Decreasing Ros Production

Mild Hypothermia Alleviates Ogd/r Induced Mitochondrial Dysfunction And Ultrastructure Of Mitochondria

Mild Hypothermia Inhibited Dna Damage And Promoted Dna Repair

Discussion

Conclusion

Abbreviations

Declarations

References

Additional Declarations

Status:

Version 1