Study of Electrical Stimulation with Different Electric-Field Intensities in

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Study of Electrical Stimulation with Different Electric Field Intensities in Regulating Differentiation of PC12 Cells Wei Jing, Yifan Zhang, Qing Cai, Guoqiang Chen, Lin Wang, Xiaoping Yang, and Weihong Zhong ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.8b00286 • Publication Date (Web): 13 Sep 2018 Downloaded from http://pubs.acs.org on September 14, 2018

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ACS Chemical Neuroscience

Study of Electrical Stimulation with Different Electric Field Intensities in Regulating Differentiation of PC12 Cells Wei Jinga, Yifan Zhanga, Qing Caia,*, Guoqiang Chenb,*, Lin Wangb, Xiaoping Yanga and Weihong Zhongc a

State Key Laboratory of Organic-Inorganic Composites; Beijing Laboratory of

Biomedical Materials; Beijing University of Chemical Technology, Beijing 100029, P.R. China. b

Department of Neurosurgery, Aviation General Hospital of China Medical

University, Beijing 100012, P.R. China. c

School of Mechanical and Materials Engineering, Washington State University,

Pullman, Washington 99164, United States.

* Corresponding to: Prof. Qing Cai, Tel and Fax: (86)-10-64412084; E-mail: [email protected] (Q.Cai) Prof.

Guoqiang

Chen,

Tel

and

Fax:

(86)-10-59520008;

[email protected] (G.Q.Chen)

ACS Paragon Plus Environment

E-mail:

ACS Chemical Neuroscience 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

For Table of Contents Use Only Study of Electrical Stimulation with Different Electric Field Intensities in Regulating Differentiation of PC12 Cells Wei Jinga, Yifan Zhanga, Qing Caia,*, Guoqiang Chenb,*, Lin Wangb, Xiaoping Yanga and Weihong Zhongc


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ABSTRACT: The strategy of using electrical stimulation (ES) to promote neural differentiation and regeneration of injured nerves is proven feasible. The study on the possible molecular mechanisms in relation to this ES promotion effect should be helpful to understand the phenomenon. In this study, it was identified the neuronal differentiation of PC12 cells was enhanced when the electric field intensity was in the range of 30-80 mV/mm, lower or higher electric field intensity displayed inferior effect. Under ES, however, levels of intracellular reactive oxygen species (ROS), intracellular Ca2+ dynamics and expression of TREK-1 were measured gradually increasing alongside higher electric field intensity. Trying to understand the relationship between the ES enhancement on differentiation and these variations in cell activities, parallel experiments were conducted by introducing exogeneous H2O2 into culture systems at different concentrations. Similarly, the effects of H2O2 concentration on neuronal differentiation of PC12 cells, intracellular ROS and Ca2+ levels, and TREK-1 expression were systematically characterized. In comparative studies, it was found the two cases that ES of 50 mV/mm for 2 h/day and H2O2 of 5 µM in culture medium shared comparable results in intracellular ROS and Ca2+ levels, and TREK-1 expression. Higher H2O2 concentration (e.g. 10 µM and 20 µM) demonstrated adverse effect on cell differentiation and caused DNA damage. A stronger ES (e.g. 100 mV/mm), being associated with higher intracellular ROS level, also resulted in weaker enhancement on the neuronal differentiation of PC12 cells. These facts suggested that the intracellular ROS generated under ES might be an



intermediate signal transducer involved in cascade reactions relative to cell differentiation. KEYWORDS: neuronal differentiation; electrical stimulation; electric field intensity; reactive oxygen species


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INTRODUCTION Millions of individuals suffer some degree of nerve injury leading to reduced motor or sensory function each year, and nerve regeneration remains a challenging task in clinical therapy.1, 2 In nerve tissues, the existence of endogenous electric fields has been identified. These endogenously generated bioelectric fields, ranging from a few mV/mm to several hundred of mV/mm, play critical roles in important biological processes including organ development and tissue regeneration via transmitting electric signals impulses.3-6 To induce nerve regeneration, in view of these findings, researchers have applied the strategy of using conductive substrates in combination with electrical stimulation (ES).7, 8 A number of reports have shown that proper ES can significantly promote the neurite growth and neuronal differentiation of PC12 cells and retinal ganglion cells (RGCs) etc., as well as nerve regeneration.9-11 A generally accepted explanation is that ES can induce depolarization of cytomembrane, change membrane potential, affect membrane protein functions like enzyme activity, membrane-receptor complexes and ion-transporting channels by altering charge distributions on these biomolecules.12-16 However, the mechanism of how ES regulates neuronal differentiation still remains unclear. One possible mechanism is suggested that reactive oxygen species (ROS) like superoxide anions (O2-) and hydrogen peroxide (H2O2) are generated within cells under ES, which plays essential role in regulating cell activities.17



ROS is highly reactive molecules that can be generated during the normal metabolism of oxygen by NADPH oxidases or as side products of several enzymatic systems (e.g., cyclooxygenases, nitric oxide synthases, mitochondrial cytochromes).18 They may determine cell fate in relation to the particular intracellular redox state within cells.19, 20 Excessive concentration of ROS is considered destructive, resulting in inhibition of gene expression.21 Some reports point out that the generation of ROS by NADPH-oxidase can be regulated with physical cues.22-24 Since ES is identified able to affect activities of enzymes including NADPH-oxidase,25, 26 it is postulated that the activation and expression of ROS within cells can be modulated by ES. In some researches, actually, exogenous ES has been found able to increase intracellular ROS production in cells like mouse embryoid bodies (EBs), mesenchymal stromal cells (MSCs) and cardiomyocytes.18, 27-29 And thus, the level of intracellular ROS may also be an important intermediate signal transducer between the ES and cell activities in relation to neuronal differentiation. To clarify this point, in the present study, the role of ROS generated from ES in regulating neuronal differentiation of PC12 cells was systematically investigated by applying different ES parameters and using exogenous ROS (H2O2) for comparison. PC12 cells were chosen for the study because they had been widely used as model cells in nerve tissue engineering and reported able to be induced neuronal differentiation efficiently under proper ES.30-32 To carry out the study, PC12 cells were cultured on conductive fibers (termed as POP), which were fabricated by coating polypyrrole (PPY) onto parallel-aligned poly(L-lactide) (PLLA) fibers as previously


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reported.33 ROS levels within cells were determined for all the cases of ES being applied or H2O2 being introduced, at the meantime, the neuronal differentiation of PC12 cells was characterized by methods including immunofluorescent staining, western-blot and gene expression via reverse-transcriptase polymerase chain reaction (PCR). To deepen the understanding, intracellular Ca2+ concentration and K+ signaling pathway were investigated because of their vital roles in determining specific neurogenic cells fates under ES.34-37 For comparative studies, the influences of ES and H2O2 for these two events were both evaluated.

RESULTS POP Fibrous Mesh for Cell Culture. Parallel-aligned POP fibers were prepared by depositing PPY onto PLLA fibers, and their average diameter was 964 ± 360 nm (Figure S1a). The conductivity of POP fibrous mesh was measured as 0.094±0.026 S/cm by the 4-point probe method. As shown in Figure S1b, the surface of POP fibers was very rough, which was suitable for cell adhesion. In primary studies, PC12 cells were found able to adhere firmly on POP fibers and able to be differentiated with neurites extending along the fibers (Figure S1c and d), which confirmed the good biocompatibility and cell affinity of conductive POP fibers.



Figure 1. Median neurite lengths (A), neurite length distributions (B) and fluorescent phalloidin staining pictures (C) of PC12 cells cultured on POP fibrous mesh after being stimulated with different electric field intensities (10 - 100mV/mm) for 3 days (2 h per day) on POP fibrous meshes, using the group without ES treatment as control. Significant *p < 0.05; highly significant ** p < 0.01, n = 100.

Neuronal Differentiation under ES. ES could stimulate neuronal differentiation of PC12 cells, especially, when the cells were cultured on conductive substrates. The electrical electric field intensity and duration, however, had proper ranges to achieve


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the strongest stimulation effect. By quantitatively measuring lengths of outgrowing neurites under different ES parameters, optimized electric field intensity and duration were identified for the present study. When the ES duration was set from 1 h to 12 h every day at the electric field intensity of 50 mV/mm, no significant difference could be found between groups on cell morphology and neurite length if the ES duration was longer than 2 h (Figure S2). When the ES duration was increased from 0 h to 2 h, however, the lengths of neurites could be seen becoming longer gradually, indicating a duration of 2 h being proper to favor the neuronal differentiation of PC12 cells. Similarly, the strength of ES could also influence cell activities. When PC12 cells were simulated with electric field intensities of 0, 10, 30, 50, 80 or 100 mV/mm at a fixed duration time (2 h/day), average lengths of neurites extending from cell bodies were measured 24.23, 26.06, 30.48, 32.05, 30.98 and 27.92 µm, respectively (Figure 1). The fraction of longer neurites increased along with the electric field intensity increasing (Figure 1B and C). The longest neurites were found within the range of 30 - 80 mV/mm. From Figure S3, these tested electric field intensities were found having no significant adverse effect on cell viability. Expressions of three specific proteins including tubulin (TUBB3), neurofilament light polypeptide (NEFL) and neurofilament heavy polypeptide (NEFH), which are in relation to neuronal differentiation, were evaluated to confirm the aforementioned findings. Firstly, expressions of TUBB3 and NEFL were illustrated using immunofluorescent staining, in which, TUBB3-positive and NEFL-positive cells were stained green and shown in Figure 2A(a-f) and Figure S4(a-f), respectively. The



strength and area of the green fluorescent displayed a clear dependence on the electric field intensity of ES, that the most abundant expressions of TUBB3 and NEFL were found in the groups with 50 - 80 mV/mm being applied. Quantitative analysis on the expression of early neuronal marker TUBB3 was performed using western-blot, the results demonstrated that ES could effectively induce TUBB3 expression when the electric field intensity was set above 50 mV/mm (Figure 2B and C). Quantitative analysis on the expressions of mature neuronal markers NEFH and NEFL were conducted using reverse-transcriptase PCR, the results demonstrated that the highest average expressions of both the proteins were liable to be obtained around the electric field intensity of 50 mV/mm (Figure 3). From all these results, it was suggested that a proper ES (e.g. 50 mV/mm, 2 h) could be an efficient tool to accelerate the transformation of PC12 cells into neural-like cells.

Figure 2. Immunolabeling (A) and western-blot analysis (B, C) of neural specific protein TUBB3 for PC12 cells cultured on POP fibrous meshes after being treated with ES (2 h per day) or H2O2 for 3 days, using the group without any treatment as


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control. TUBB3 was stained green and nuclei were counterstained blue with Hochest. Significant *p < 0.05; highly significant ** p < 0.01, n = 4.

Figure 3. Reverse-transcriptase PCR analysis results of neural specific genes NEFH (A) and NEFL (B) for PC12 cells cultured on POP fibrous meshes after being treated with ES (2 h per day) or H2O2 for 3 days, using the group without any treatment as control. Significant *p < 0.05; highly significant ** p < 0.01, n = 4.

Neuronal Differentiation with Exogeneous H2O2. ROS is important in regulating cell proliferation and differentiation. Before detecting the effect of exogeneous ROS on neuronal differentiation of PC12 cells, H2O2 was introduced into culture medium at gradient concentrations to examine its influence on cell viability (Figure S5). Continuous cell growth was identified when the H2O2 concentrations was below 50 µM, while only 5 and 10 µM H2O2 had no significant cytotoxicity in comparison with the control group. When the concentration of H2O2 was in the range of 20-50 µM, cell proliferation was suppressed more significantly alongside the H2O2 concentration increasing. Thereby, 5, 10 and 20 µM of H2O2 were used as exogenous ROS sources



to study the neuronal differentiation of PC12 cells. From Figure 2 and Figure S6, ROS was identified able to promote TUBB3 expression and neurite outgrowth, in which, the 5 µM of H2O2 demonstrated enhancement on differentiation of PC12 cells significantly in comparison with the control group, while the other two concentrations (10, 20 µM) were not. PCR analysis results of NEFH revealed a similar finding but displaying no significant difference between the H2O2 groups and the control group (Figure 3A). The PCR results of NEFL, however, did not show a clear trend for the three H2O2 concentrations (Figure 3B and Figure S4). In considering all these facts, 5 µM of H2O2 was indicated having stronger ability in inducing the neuronal differentiation of PC12 cells than the two higher H2O2 concentrations. Noticeably, in general, it was found the promotion effect of 5µM H2O2 was inferior to those cases treated with ES at intensities 30-80 mV/mm. Intracellular ROS Evaluation. In comparing the effects of ES and H2O2 treatments on the neuronal differentiation of PC12 cells, it was interesting to know if there was a correlation between the two cases. Therefore, intracellular ROS accumulation was stained with DCFH-DA immediately after the cells being treated with ES or H2O2, and detected with flow cytometer. Ten thousand cells were detected for each group. In the control group, the percentage of ROS-positive cells was measured 86.5%, which was significantly lower than those in the groups with ES or H2O2 being applied (Figure S7A). Almost all cells were detected ROS-positive if the cells had been treated with ES or H2O2. The ROS levels generated inside PC12 cells increased along with the intensity of ES being increased. When the ES electric field


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intensity was above 50 mV/mm, the intracellular ROS level became significantly higher than that in the control group (Figure 4 and S7B). In the cases of H2O2 being applied, the increase in intracellular ROS level was in the ascending order of 5 µM < 10 µM < 20 µM H2O2. The intracellular ROS level detected at 20 µM of H2O2 was quite high, and this was consistent with the low cell viability under 20 µM of H2O2 (Figure S5). As aforementioned, strong promotions on the neuronal differentiation of PC12 cells was liable to be achieved at ES of 50 mV/mm or H2O2 of 5 µM. From Figure 4 and Figure S7B, it could be seen the intracellular ROS generated under ES was comparable with the 5 µM H2O2 group only when the ES electric field intensity was 50 mV/mm, and the ROS level increased under the stimulation of higher electric field intensities. The amounts of intracellular ROS generated under 80 and 100 mV/mm were comparable to that in the case as cells being treated with 10 µM H2O2. These comparative studies suggested that the level of intracellular ROS might be an importance factor in regulating cell behaviors, and ES might influence the neuronal differentiation of PC12 cells via its action in raising the intracellular ROS level. However, higher intracellular ROS level that was induced by stronger ES electric field intensity (e.g. 100 mV/mm) did not result in more significant enhancement on the neuronal differentiation of PC12 cells (Figure 2 and Figure 3). And a thing to be noted was that the promotion effect of H2O2 (e.g. 5 µM) was inferior to that of ES (e.g. 50 mV/mm), even they shared comparable intracellular ROS levels (Figure 4 and S7B).



Figure 4. Comparative analysis of ROS generated inside PC12 cells cultured on POP fibrous mesh after being stimulated with different electric field intensities or different concentrations of H2O2 for 3 days (2 h per day) via flow cytometric measurement on DCFH-DA stained cells using the cells without any treatment as control.

Intracellular Ca2+ Dynamics and Expression of TREK-1. External stimulation like ES and H2O2 might alter the conformation of ion channels gating on cytomembrane, which would change the state of ion transportation to cause ion fluctuant inside cells. Intracellular Ca2+ dynamics and expression of TREK-1 (a member of the subfamily of mechano-gated potassium channels) were thus detected for the purpose to better understand the possible regulators those were involved in triggering the differentiation of PC12 cells under ES or H2O2 treatment. After PC12 cells were treated with ES or H2O2, and loaded with Fluo-4-acetoxymethyl ester


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(Fluo-4 AM) dye, stronger fluorescent densities were observed along with higher electric field intensity or H2O2 concentration (Figure 5), indicating more Ca2+ ions having transported into the cells. In comparison with the control group, the increase in intracellular Ca2+ concentration was not quite significant when the electric field intensity was below 30 mV/mm.

Figure 5. PC12 cells being loaded with Fluo-4 AM and observed under laser confocal scanning microscope (LCSM) right after the cells being treated with ES (b-f) or H2O2 (g-i) for 2 h to evaluate intracellular Ca2+ levels using the cells without any treatment as control.

The expression of TREK-1 mRNA was detected increasing with ES or H2O2 treatment being applied (Figure 6). When the cells were treated with ES, only the



cases by applying electric field intensity higher than 50 mV/mm demonstrated significant expression of TREK-1 gene than the control group. The expression of TREK-1 was also induced by both 5 µM and 10 µM H2O2, and the levels were comparable with those in the cases as 50-100 mV/mm ES being applied. When the cells were treated with 20 µM H2O2, the expression of TREK-1 gene became remarkably higher than any other group, showing a 3.5-fold up-regulation in comparison with the control group. Since TREK-1 is a member of the subfamily of mechano-gated potassium channels, this abnormal increase in its expression might cause abnormal cell behaviors.

Figure 6. Reverse-transcriptase PCR analysis on expression of TREK-1 gene for PC12 cells after being treated with ES (2 h per day) or H2O2 for 3 days using the cells without any treatment as control. Significant *p < 0.05; highly significant ** p < 0.01, n = 4.

DNA damage. As aforementioned, intracellular ROS and Ca2+ levels, as well as


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expression of TREK-1 gene of PC12 cells would be up-regulated as ES or H2O2 treatment being applied, showing dependence on the intensity of the stimulation. These changes inside cells might cause DNA damage, even no abnormality was detected in cell proliferation. In the control group, normally, appearance of ~5 abasic sites (AP) in every 1,000,000 base pairs (bp) was unavoidable because of the experimental operation. The numbers of base sites missing for PC12 cells after being treated with ES or H2O2, from Figure 7, could be seen significantly increasing in comparison with the control group. H2O2 treatment would be quite harmful to cells when the concentration was higher than 5 µM. The average AP numbers were determined 12 and 18, respectively, at the 10 and 20 µM of H2O2 concentrations. When the ES electric field intensity had been raised to 80 mV/mm, the AP number was higher than that in the control group, especially in the case of 100 mV/mm, which was closely to the case of 10 µM H2O2. As for the two cases of 5 µM H2O2 and ES with 50 mV/mm being applied, the AP number of base site missing was found higher in the former case. The damage on DNA integrity caused by ES treatment was identified weaker than those in the H2O2 groups.



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Figure 7. DNA damage analysis of PC12 cells cultured on POP fibrous mesh after being treated with ES (2 h per day) or H2O2 for 3 days using the cells without any treatment as control. Significant *p < 0.05; highly significant ** p < 0.01, n = 4.

DISCUSSION ES is proven an effective strategy to induce cell differentiation in the researches of nerve regeneration.38-40 In many reports, conductive substrates are used for the culture of nerve-derived cells to take advantage of their ability in boosting electrical transmission and strengthening

local

ES,

which

is able

to

mimic

the

electrophysiological environment in native nerve tissues.41, 42 Parameters of ES like electric field intensity and duration are vital in regulating the neuronal differentiation of PC12 cells, Schwann cells and RGCs, etc.

11, 43, 44

. It had been proposed that the

increase in intracellular ROS production under exogenous ES might be responsible for the promotions in cell biological behaviors in the cases of mouse EBs, MSCs and cardiomyocytes,18,

27, 28

while no clear understanding between ES and neuronal

differentiation could be found in reports. Conductive parallel-aligned POP fibrous mesh was prepared and used in this study to provide the conductive microenvironment, which was able to guide neurites outgrowth of PC12 cells along the oriented micro-structure.33 The ES, being applied along the fiber direction, helped to further elongate cells to meet the oriented nerve morphology. Basing on these observations, proper electric field intensity and duration


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of ES for culturing PC12 cells were decided by evaluating cell viability and neuronal differentiation. Some articles pointed out that high electric field intensity would be harmful for cells, which could lead to cell membrane fragment.45 It was found that the proliferation of PC12 cells was comparable with that in the control group when the duration time was fixed at 2 h / day and the intensity was in the range of 10 - 100 mV/mm. As for the neuronal differentiation of PC12 cells, however, it turned out to be the electric field intensity of 50 mV/mm likely providing an efficient promotion which was confirmed by evidences of outgrowing neurite lengths and neural special protein (TUBB3, NEFL and NEFH) expressions. ROS was reported highly reactive molecule generated during the normal metabolism of oxygen by NADPH oxidases or as side products of several enzymatic systems,46 reasonably, its intracellular level should be fluctuant if exogenous stimulation was applied.24 For ES-treated PC12 cells, then, intracellular ROS levels were determined to judge the possible mechanism behind the enhancement of ES on neuronal differentiation. The ratio of cells with ROS increased from ~85% in the control group to over 95% in all the ES-treated groups, indicating the generation of ROS inside PC12 cells under ES. However, the level of the generated ROS displayed obvious dependence on the intensity of ES. Only when the electric field intensity was higher than 50 mV/mm, the intracellular ROS level would be remarkably higher in the ES group than that in the control group. Thus, it was proposed that the neuronal differentiation of PC12 cells being promoted under proper ES was ascribed to the induced intracellular ROS. Generally, these findings were in accordance with similar



phenomena observed in studies of other cells under ES.18, 47 To understand the role of ROS in regulating cell behaviors, in this study, further studies on neuronal differentiation of PC12 cells were conducted by treating the cells with H2O2 of different concentrations, and the results were systematically compared with the ES groups. Under the H2O2 stimulation, intracellular ROS level also increased alongside higher H2O2 concentration, and only a proper H2O2 concentration of 5 µM would achieve efficient promotion on the neuronal differentiation of PC12 cells. Higher H2O2 concentrations (10 and 20 µM) displayed adverse effects on cell differentiation. These studies were in accordance with other reports, that the increasing of intercellular ROS in relative to control cells could enhance PC12 cell differentiation48, 49, while excessive amount of ROS would induce oxidative stress and be harmful for neural cells to result in neurodegeneration.50 It was interesting to find that the 5 µM H2O2 and the 50 mV/mm ES shared comparable intracellular ROS levels, and both of them displayed efficient promotion in inducing the neuronal differentiation of PC12 cells. Further increase in the H2O2 concentration or the ES electric field intensity would cause DNA damage and be harmful to cell viability. This comparative study suggested that the intracellular ROS induced by ES should have been involved in the cascade reactions relative to cell differentiation, possibly, acted as a regulator in correlating the ES and cell activities. The different thing between the ES and the H2O2-treated groups, however, was found that the promotion efficiency on neuronal differentiation of PC12 cells was generally stronger in the former case, even if they shared comparable intracellular ROS levels.


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This suggested there was other cellular responses to the treatments, which also had contributions to the neuronal differentiation. ES was reported able to improve intercellular Ca2+ concentration of PC12 cells under high electric field intensity, the influx of Ca2+ was induced by the depolarizing current, which could activate the calmodulin-kinases to elicit neurites outgrowth and expedite neurites development.51-54 ES could also regulate potassium concentrations via

controlling

ion

channels

gating

on

cytomembrane,

which

induced

hyperpolarization of the cell membrane potential and cell differentiation.55,

56

Intercellular K+ was important for regulating membrane potential, and the mechano-gated potassium channel TREK-1 could improve the neural differentiation of neural stem cells and that was gated through both chemical and physical mechanisms.5,

57

In this study, both the intracellular Ca2+ concentration and the

TREK-1 expression were found up-regulated when H2O2 (5, 10 and 20 µM) and ES (higher than 50 mV/mm) treatments were applied. However, the extent of up-regulation was generally higher in groups being treated with H2O2 than those under ES. The abnormal increase in the expression of TREK-1 gene caused by 20 µM H2O2 might be the abnormal cytoprotection behavior of the cells.58, 59 It was easy to understand that the Ca2+ concentration and the TREK-1 expression should be in appropriate ranges to act positively on neuronal differentiation of PC12 cells. An uncertain issue here was that the up-regulations in intracellular Ca2+ concentration and TREK-1 expression were directly induced by the ES, or by the ROS generated under ES, since ROS could also take actions in raising them.60.61 However, it could not be



denied that the ROS generated by ES should have played an important role in regulating the neuronal differentiation of PC12 cells. Nevertheless, ES was believed able to play more roles in activating cell differentiation. There were possible redistributions of membrane proteins responding to electrical field/current, membrane depolarization of neurons, or changes in the transmembrane potential of neurons under ES.12-16 Therefore, the influence of ES was complex that other cellular responses in corresponding to ES also might have contributions if the ES parameters were set properly.

CONCLUSION It was identified that the differentiation of PC12 cells could be promoted efficiently under ES of appropriate electric field intensity and duration time. Generation of ROS was determined associating with the ES treatment, and the amount of generated intracellular ROS was closely related to the intensity of ES. As ROS itself was identified able to induce the neuronal differentiation of PC12 cells, therefore, it was reasonably to propose that ROS was a possible signal transducer in correlating the ES treatment and the cell differentiation. However, the ES demonstrated stronger ability in promoting cell differentiation than the exogeneous H2O2, even they sharing similar intracellular ROS level. This finding revealed that ES treatment could cause complex cell responses in addition to ROS generation, and regulate cell biological behaviors in many aspects.


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METHODS Preparation of Conductive POP Fibrous Mesh. Conductive POP fibrous mesh was prepared in referring to our previous report.33 Briefly, PLLA (Mw: 100,000, Shandong Pharmaceutical Sciences Pilot Plant, China) was dissolved

in

trifluoroethanol (Sigma-Aldrich) to get a homogeneous solution (10 wt.%) and electrospun at fixed parameters (flow rate: 0.6 mL/h; voltage: 20 kV; receiving distance: 20 cm) using a rotating cylinder as collector. The produced PLLA fibrous mesh was then immersed into an aqueous solution containing 14 mM pyrrole (Aladdin, USA) and 14 mM paratoluenesulfonic acid sodium salt (pTs, Aldrich, USA) at 4oC for 1 h, followed by 100 mL FeCl3 aqueous solution (38 mM) being added. Polymerization of pyrrole and its deposition onto PLLA fibers were completed at 4oC in the next 12 h. The obtained PPY-coated PLLA fibrous mesh was termed as POP. In vitro Cell Culture. PC12 cells (purchased from Beijing Xiehe Cell Resource Center, China) were cultured in RPMI-1640 (Gibco, USA) supplemented with 10% heat-inactivated horse serum (HS, Gibco, USA), 5% fetal bovine serum (FBS, Gibco, USA) and 1% penicillin/streptomycin solution (Hyclone, USA), and maintained in a humid incubator at 37oC with 5% CO2 supply. For the neural differentiation of PC12 cells, the cells were cultured in RPMI-1640 supplemented with 1% HS, 0.5% FBS, 1% penicillin/streptomycin solution and 100 ng/mL nerve growth factor (NGF, Sigma, USA). Before cell seeding, POP meshes were cut into circular pieces, and placed into



cell culture plates. To each well, 75% ethanol was added, and the plates were exposed to ultraviolet light for 4 h. After the sterilization, the materials were washed three times with PBS. Then the meshes were treated with a solution containing 0.1 mg/mL rat tail type I collagen (Sigma) for 24 h at 4oC, followed by PBS washing and being stored in PBS at 4oC overnight for further use. Cell Cultured with Exogenous ROS. Hydrogen peroxide was chosen as an exogenous ROS source to evaluate the ROS effect on neuronal differentiation of PC12 cells. To determine the optimal concentration of H2O2 for in vitro treatment, PC12 cells (1×104 cells/mL) were suspended in 200 µL of growth medium containing different concentrations of H2O2 (0, 5, 10, 20, 30, 40, 50 or 100 µM) and seeded into each well of 96-well plates. Cell count kit (CCK-8, Dojindo, Japan) was used to detect cell viability at 1, 3, 5 and 7 days after cell seeding. In order to keep the concentration of H2O2 steady, the culture medium was refreshed every day. Cell Cultured under ES. To perform cell culture under ES, a homebuilt electric field was set up as reported in our previous study.62 Briefly, two Pt-electrodes (ϕ = 0.5 mm) were placed apart across the circular fibrous meshes, and the direction of electric field was set along the fiber direction. To stimulate the neuronal differentiation of PC12 cells, different electric field intensities (10, 30, 50, 80 or 100 mV/mm) and durations (1, 2, 4, 8 or 12 h) were applied on the cells seeded on POP fibrous meshes from the second day of cell seeding (for simplification, the day the ES being applied was recorded the first day). The ES treatment was conducted one time per day, and cell proliferation was monitored using CCK-8 assay at 1, 3, 5 and 7 days. To avoid


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possible adverse effect of ES on culture medium, the medium was refreshed every day right after the ES treatment. The seeding density of PC12 cells was set as 1×104 cells/cm2 mesh. Intracellular ROS Measurement. To quantify the amounts of oxyradicals within cells induced by ES or H2O2, PC12 cells were seeded onto conductive POP fibrous meshes at a density of 5×104 cells/cm2 and cultured for 24 h with NGF-containing medium, followed by being treated 2 h with varied electric field intensities or H2O2 concentrations. Subsequently, the cells were detached using 0.05% trypsin-EDTA (Invitrogen), incubated with 10 mM 2',7'-dichlorofluorescin diacetate (DCFH-DA, Beyotime) for 30 min under dark condition, rinsed 2 times with PBS buffer. Ten thousand of cells were recorded per sample using flow cytometry (CytoFLEX, Beckman Coulter, USA), and the acquired data were analyzed with Summit 5.2. The CytoFLEX was set at an excitation wavelength of 488 nm and an emission wavelength of 512 nm. Intracellular Calcium Measurement. As aforementioned, PC12 cells were seeded onto POP fibrous meshes at a density of 1 ×104 cells/cm2, cultured at 37°C for 24 h with NGF-containing medium, followed by being treated 2 h with different electric field intensities or H2O2 concentrations. Then, the media were removed and the samples were washed 3 times with PBS buffer. Fluo-4 AM (5 mM, Dojindo), a Ca2+-sensitive dye, was pre-dissolved in dimethyl sulfoxide (DMSO, Sigma) and added into the systems, followed by 30 min of incubation. Fluorescent images of calcium signals were obtained with LCSM (Nikon Eclipse C1, Japan) by setting an



excitation wavelength of 494 nm and an emission wavelength of 516 nm. Neuronal Differentiation. To perform neuronal differentiation studies of PC12 cells under different stimulation, circular POP meshes (ϕ = 1 cm) were fitted into 24-well culture plates with PC12 cells being seeded at a density of 2 ×104 cells/cm2 mesh. After 24 h of incubation, cells were treated with different electric field intensities for 2 h per day and the NGF-containing medium was refreshed right after the treatment every day. Or, cells were treated by introducing different concentrations of H2O2 into medium, and the medium was refreshed every day to keep ROS level steady during the culture. For all these cases, the inductive culture was continued for 3 days and samples were then submitted to neuronal differentiation assays. Cell Morphology. Cell/mesh complexes were retrieved and fixed with 4% formaldehyde solution (Novon, USA) for 10 min, permeabilized in 0.1% Triton X-100 solution (Sigma, USA) for 6 min, and then blocked in 1% bovine serum albumin (BSA) solution (Sigma, USA) for 60 min. For fluorescent observation, the cells were labeled with Alexa Fluor 555 phalloidin (Life Technologies, USA) for F-actin staining and Hochest 33342 (Sigma, USA) for nuclei staining, and fluorescent images were captured using LCSM. Neurite length was measured as a linear distance between the cell junction and the tip of a neurite with Nano Measurer 1.2. Median neurite length was averaged from 100 measurements for each sample and statistically analyzed between samples. Immunofluorescence Staining. Expressions of selected neuronal proteins as


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TUBB3 and NEFL in PC12 cells were detected by immunofluorescent staining. After the cells were fixed, permeabilized and blocked as aforementioned, primary antibody labeling was performed in diluted PBS buffer (containing 1% BSA and 0.01% Triton X-100S) for 12 h at 4oC with corresponding antibodies as mouse anti-βIII tubulin antibody (1:200 dilution, Abcam) and mouse anti-neurofilament light polypeptide (NEFL) antibody (1:200 dilution, Abcam). The samples were rinsed intensively with PBS for 3×5 min, then the secondary antibody labeling was performed with goat anti-mouse IgG-FITC (1:200 dilution, Jackson ImmunoResearch). Fluorescent images were captured using LCSM. Western Blotting. Cells in the retrieved cell/mesh complexes were lysed with RIPA

lysis

buffer

(Applygen,

China)

supplemented

with

0.5

mM

phenylmethanesulfonyl fluoride (PMSF, Sigma, USA), and total proteins were quantified using a BCA protein assay Kit (Thermo, USA). Around 40 µg of total protein from each cell lysate was loaded onto 8% sodium dodecyl sulfonate containing polyacrylamide (SDS-PAGE) gel (Solarbio, China), and further blotted onto poly(vinylidene difluoride) (PVDF) membrane (Immobilon P, Millipore). After being incubated with a blocking buffer containing 5% skim milk powder in TBST (Tris buffered saline with Tween) for 2 h, samples were further incubated with primary antibodies at 4oC overnight, followed by being rinsed with TBST for 3×15 min. Then the samples were incubated with secondary antibody of HRP conjugated goat anti-mouse IgG (1:5000 dilution, Abcam) at room temperature for another 1.5 h. Finally, the samples were rinsed with TBST for 3 times and the signals were detected



by Molecular Imager Versa Doc MP 4000 System (Bio-Rad, USA). Band intensity was normalized. The primary antibodies used in this immunoblotting assay was mouse anti-βIII tubulin purified (1:1000 dilution, Abcam). Gene Expressions. The neuronal differentiation of PC12 cells in different inductive culture designs were further evaluated at the genetic level by performing real-time reverse-transcriptase PCR (RT-PCR) analysis. After 3 days of inductive culture, cells were harvested and total RNA was extracted by Trizol reagent (Invitrogen, USA) and reversely transcribed using SuperScript® III Reverse Transcriptase (Invitrogen, USA) according to the manufacturer’s instructions. TREK-1 gene and two neuron-specific genes (NEFH and NEFL) were detected along with the housekeeping gene GAPDH. Their specific primers were designed as listed in Table S1. RT-PCR analysis was performed using SYBR Green Master Mix (Toyobo, Osaka, Japan) and qTOWER RT-PCR system (Aanlytic Jena, Germany). All experiments were performed in triplicate, and the amplification signal from the target gene was calculated using a ∆∆Ct method by being normalized to the signal of the house-keeping gene GAPDH in the same reaction. DNA Damage. Oxidative stress induced by ROS overproduction would make DNA of cells damaged with base sites missing. Possible DNA damages of PC12 cells after various ES or H2O2 treatments were evaluated using the DNA damage kit (Dojindo, Japan). In detail, PC12 cells were seeded onto POP fibrous meshes in 24-well culture plates at a density of 5 ×104 cells/cm2 and incubated for 24 h. After that, the cells were treated with ES or H2O2 and the media were refreshed similarly as the neuronal


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differentiation study. Three days later, the cells were detached immediately using 0.05% trypsin-EDTA, DNA was extracted by DNA kit (Omega Bio-tek, USA), measured by Nanodrop (Thermo, USA) and tested by the DNA damage kit under the manufacturer’s instructions. Statistical Analysis. All numerical data are given as mean ± standard deviation for n ≥ 4. Differences among groups were analyzed by one-way analysis of variance (ANOVA) followed by using SPSS 22.0 software. Statistical difference was determined that *p < 0.05 were considered statistically significant and **p < 0.01 was considered highly significant.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Contains SEM images of POP fibers prepared in the present study, optimization of electrical voltages and duration times to carry out differentiation study under ES, cell viability study in the presence of exogeneous H2O2 at different concentrations, intracellular ROS analysis for PC12 cells after being treated with ES or H2O2, immunolabeling neural specific protein, NEFL, and primers designed for PCR analysis (PDF)



AUTNOR INFORMATION Corresponding Authors *E-mail: [email protected]; [email protected]. ORCID Qing Cai: 0000-0001-6618-0321 Author Contributions W.J. performed the preparation of POP fibers, cell culture and characterizations; Y.Z. provided help in setting up electric field and cell culture under ES; W.J. and Q.C designed the study and wrote the manuscript; G.C. and L.W. made contribution in analyzing data in relation to neuronal differentiation; X.Y. and W.Z. helped in material preparation. All the authors discussed the results, commented on the study and approved the final manuscript. Funding The authors acknowledged the financial support from the National Natural Science Foundation of China (51472068, 51473016). Notes The authors declare no competing financial interest.


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ACKNOWLEDGMENTS The authors thank Huilei Yu, Bo Ren and their organization of Institute of Sports Medicine, Peking University Third Hospital for their technical support.

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Study of Electrical Stimulation with Different Electric-Field Intensities in

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