Different Effects of Berberine Delivery to Mitochondria on Cells Derived from the Neural Crest

Abstract

Energy metabolism is crucial for cell polarity and pathogenesis. Mitochondria, which are essential for maintaining energy homeostasis within cells, can be targeted by drug delivery to regulate energy metabolism. However, there is a lack of research comparing how mitochondria control energy metabolism in different cell types derived from the neural crest. Understanding the effects of berberine (BBR), a compound that acts on mitochondria, on energy metabolism in neural crest-derived cells is important. This study reports how MITO-Porter, a mitochondria-targeted liposome, affects neuroblasts (Neuro2a cells) and normal human epidermal melanocytes (NHEMs) when loaded with BBR. We found that treatment with MITO-Porter containing BBR reduced mitochondrial respiration in Neuro2a cells, while it caused a slight increase in NHEMs. Additionally, the treatment shifted the ATP production pathway in Neuro2a cells to rely more on glycolysis, while in NHEMs, there was a slight decrease in the reliance on glycolysis. We also observed a significant decrease in ATP production in Neuro2a cells, while NHEMs showed a tendency to increase ATP production. Importantly, on the basis of the results of the Premix WST-1 assay, the study found that BBR treatment was not toxic to either cell type. It is important to take note of the varied effects of BBR treatment on different cell types derived from the neural crest. These findings necessitate attention when utilizing NHEMs as a cell model in the development of therapeutic strategies for neurodegenerative diseases, including the use of BBR for metabolic control.

INTRODUCTION

Neurons and melanocytes originate from neural crest cells. They share similar developmental and morphological features and signaling pathways with nervous system cells.^1,2) Melanocytes could serve as a promising model for studying the normal and pathological behavior of neurons. Currently, the mechanisms associated with neurodegenerative diseases are being elucidated using melanocyte culture systems.³⁾ However, there are few reports comparing the morphological changes and energy metabolism responses, which are crucial in disease progression, induced by drug administration in cells derived from different neural crest cells.

Berberine (BBR) is an alkaloid extracted from Coptis or Berberis aristata and possesses various biochemical and pharmacological activities. Over the past several decades, studies have focused on BBR as a potential drug for treating neurodegenerative diseases, cancer, and diabetes by controlling intracellular metabolism, with its main point of action in the mitochondria.^4–6) BBR could be beneficial for evaluating energy metabolism in various neural crest cells; however, there are no reported studies on the changes in energy metabolism induced by BBR in these cells. It is commonly thought that intracellular BBR is mainly located in mitochondria, the nucleus, and the cytoplasm, depending on the concentration of BBR.⁷⁾ Efficient delivery of BBR to the mitochondria is crucial for enhancing its pharmacological effectiveness. However, the issue is that the intracellular kinetics of BBR are not regulated.

Mitochondria can be a significant target for regulating cellular energy metabolism by drug delivery. We developed MITO-Porter, which is a mitochondria-targeted liposome, and have successfully delivered various compounds to mitochondria.^8,9) Furthermore, we have improved mitochondrial function by using MITO-Porter to encapsulate BBR (MITO-Porter (BBR)).^10,11) The efficient delivery of BBR to mitochondria can be a valuable tool for evaluating mitochondria-mediated energy metabolism.

The aim of this study is to investigate the metabolic changes in neural crest cells resulting from BBR delivery. These findings are crucial for understanding the metabolic alterations in melanocytes, which are currently utilized as a valuable model for neurodegenerative diseases. This study involved the preparation and assessment of MITO-Porter (BBR) to determine its physicochemical properties. We then assessed the respiratory activity by introducing naked BBR and MITO-Porter (BBR) into these cells. Additionally, we evaluated the rates of glycolysis and mitochondrial respiration as they relate to energy metabolism. Finally, we measured the intracellular ATP production and cell viability following drug administration.

We hypothesized that using MITO-Porter (BBR) would have a strong effect on the metabolic changes in cell types that rely heavily on the mitochondrial energy production pathway compared with those when naked BBR is used. Furthermore, we aimed to clarify the effect of drug administration on metabolic changes by conducting a comparative study with melanocytes. In these experiments, we used the neuroblastoma Neuro2a cells and normal human epidermal melanocytes (NHEMs). Our findings showed that the pharmacological effects differed between neurons and melanocytes (Fig. 1). The MITO-Porter system proved to be a valuable tool for elucidating drug administration-induced metabolic changes in cells derived from different neural crest cells. It also revealed the differential pharmacological effects of BBR on Neuro2a cells and NHEMs.

Fig. 1. The Schematic Diagram Illustrates the Various Pharmacological Effects of BBR on Neural Crest-Derived Cell Types Using the MITO-Porter System

It was observed that mitochondrial oxygen consumption rate differed between the two cell types when MITO-Porter (BBR) was used. These differences were attributed to alterations in mitochondrial respiratory function affecting ATP production, consequently leading to changes in intracellular ATP levels.

MATERIALS AND METHODS

Materials

1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and methoxy polyethene glycol 2000 (DMG-PEG-2k) were obtained from NOF Corporation (Tokyo, Japan). Sphingomyelin (SM) was obtained from Avanti Polar Lipids (Alabaster, AL, U.S.A.). Stearylated R8 (STR-R8) was obtained from TORAY Research Center, Inc. (Tokyo, Japan). All other chemicals used were commercially available, reagent grade products.

Preparation of the MITO-Porter (BBR)

The MITO-Porter was prepared using the lipid film hydration method. First, 900 nmol of lipids (DOPE/SM = 9/2 molar ratio) was dissolved in a mixture of ethanol and chloroform (1 : 1 per volume) in a glass tube. The organic solvents were then allowed to evaporate over a period of more than 2 h to form a thin lipid film. After that, 10 mM N-(2-hydroxyethyl)piperazine-N′-2-ethanesulfonic acid (HEPES) buffer at pH 7.4 containing BBR (1 mM) was added to the tube, followed by sonication for 1 min in a bath-type sonicator. Finally, a solution of STR-R8 (10 mol% of total lipids) was added to the resulting suspension to attach the STR-R8 to the surface of the liposomes (LPs), followed by a 30 min incubation at room temperature. The properties of the resulting LPs were measured using a Zetasizer Nano ZS (Malvern Instruments, Worcestershire, U.K.). The lipid compositions of these LPs are summarized in Table 1. After storing the prepared particles for 1 d at 4, 25, 37, and 42 °C, respectively, the recovery of BBR was calculated using the previously described method.¹⁰⁾

Table 1. Characteristics of the LPs Used in This Study

LP type	Diameters (nm)	Polydispersity index (PdI)	ζ Potential (mV)
MITO-Porter	96.6±6.7	0.266±0.01	29.8±6.7
MITO-Porter (BBR)	97.1±6.8	0.283±0.02	35.l±4.7

Lipid composition of prepared LPs are DOPE and SM (9 : 2, molar ratio). Data are the mean ± S.D. (n = 10).

Assessment of Cellular Uptake by a Fluorescence Activated Cell Sorter (FACS)

NHEMs were cultured in complete medium for 24 h under an atmosphere of 5% CO₂/air at 37 °C. DiD, a carbocyanine dye, which was integrated into lipid membranes as an indicator of particle uptake, was present at a concentration of 0.5 mol% of total lipids. The cells were then transfected with LPs and incubated in complete medium for 30 min under an atmosphere of 5% CO₂/air at 37 °C. Subsequently, the cells were washed twice with phosphate buffered saline (PBS) (−) containing heparin (20 U/mL) and collected using trypsin 0.25% ethylenediaminetetraacetic acid (EDTA). After centrifugation at 700 × g, at 4 °C for 3 min, the supernatant was removed, and the collected cells were suspended in FACS buffer, consisting of bovine serum albumin (5 mg/mL) and sodium azide (1 mg/mL) in PBS (−). The cells were then filtered through a nylon mesh and analyzed using a CytoFLEX Flow Cytometer (Beckman Coulter Inc., Brea, CA, U.S.A.). DiD was excited by a 638 nm light, and the band pass filter for the fluorescence detection was set to 660 nm. The cellular uptake value of LPs was expressed as the mean fluorescent intensity (MFI), the integrated fluorescence intensity, and the cell counts.

Cell Cultures and Transfection Study

Neuro2a cells, a mouse neuroblastoma cell line, and Eagle’s minimum essential medium (EMEM) were obtained from the ATCC (Manassas, VA, U.S.A.). Fetal bovine serum (FBS), penicillin, and streptomycin were obtained from Sigma-Aldrich (St. Louis, MO, U.S.A.). The cells were maintained in complete medium, which is EMEM supplemented with 10% FBS, penicillin (100 U/mL), and streptomycin (100 µg/mL). NHEMs (KM-4009) and the DermaLife M Medium Complete kit were obtained from KURABO Industries Ltd. (Osaka, Japan). These cells were cultured under an atmosphere of 5% CO₂/air at 37 °C. One day prior to treatment with the samples, these cells were seeded on plates or dishes for each experiment. Transfection was performed by the following method: cells were washed with phosphate buffered saline without calcium chloride (PBS (−)), the media were replaced with the LPs (final lipid concentration: 0.36 µM) in EMEM without serum, and the resulting suspension was then incubated for 30 min under an atmosphere of 5% CO₂/air at 37 °C.

Assessment of Mitochondrial Respiratory Function

The mitochondrial respiratory function was assessed using a Seahorse XF HS Mini Analyzer (Agilent Technologies Inc., Santa Clara, CA, U.S.A.). Neuro2a cells and NHEMs were cultured in complete medium for 24 h in an atmosphere of 5% CO₂/air at 37 °C. These cells were then transfected with LPs and incubated in complete medium for 3.5 h under the same conditions. After that, the medium was replaced with Seahorse XF assay culture medium (Agilent Technologies Inc.), which contains glucose, pyruvate, and glutamine. The plate was incubated for 1 h at 37 °C without CO₂. The respiratory capacity was measured using the Seahorse XF Cell Mito Stress Test Kit (Agilent Technologies Inc.). The rate of ATP production from glycolysis and mitochondria was quantified using the Seahorse XF Real-Time ATP Rate Assay Kit. These experiments were conducted in accordance with the product manual. Following the determination of the basal oxygen consumption rates (OCRs), the cells were treated with oligomycin A (2 µM), carbonyl cyanide 4-trifluoromethoxy phenylhydrazone (FCCP, 1.5 µM), and rotenone/antimycin A (0.5 µM) as necessary. Viable cell numbers were counted and used to normalize the values.

Assessment of Intracellular ATP Production

ATP production was assessed using a colorimetric/fluorometric ATP Assay Kit (Abcam, U.K.) following the manufacturer's instructions. Neuro2a cells and NHEMs were cultured in complete medium for 24 h at 37 °C in an atmosphere of 5% CO₂/air. The cells were then transfected with LPs. After replacing the medium with complete medium and incubating for 3.5 h at 37 °C in an atmosphere of 5% CO₂/air, the cells were collected, washed with PBS, and resuspended in ATP assay buffer. The cells were homogenized, and the resulting homogenate was centrifuged at 13000 × g at 4 °C for 5 min to remove any insoluble material. The supernatants were then incubated with the ATP probe and the reaction mix for 30 min. Absorbance readings were taken at 535/587 nm using a microplate reader (EnSpire^® Multimode PlateReader, PerkinElmer, Inc., Waltham, MA, U.S.A.). The ATP concentration was determined using a calibration curve generated with the ATP standard solution.

Assessment of Cell Viability

Neuro2a cells and NHEMs were cultured in complete medium for 24 h at 37 °C in an atmosphere of 5% CO₂/air. The cells were then transfected with the LPs, and the medium was replaced with complete medium, followed by 1 h of further incubation under the same conditions. After that, the medium was replaced with complete medium containing Premix WST-1 reagent (TaKaRa Bio Inc., Shiga, Japan) and incubated for 2 h under the same conditions. The absorbance was then measured at 440 and 650 nm using a microplate reader. The relative cell viability was calculated by normalizing the treated cell viability to the control cell viability using the following formula:

  

where V_t and V_u represent the cell viability of the treated and untreated cells, respectively.

Statistical Analysis

The data are presented as the mean ± standard deviation (S.D.) for the specified number of experiments. We used the Excel Statistical Program File (ystat2013) for statistical analysis. For multiple comparisons, we conducted a one-way ANOVA followed by the Student–Newman–Keuls test. We considered p-values less than 0.05 to be significant.

RESULTS

Preparation of the MITO-Porter Using the Lipid Film Hydration Method and Cellular Uptake

The MITO-Porter was prepared using the lipid film hydration method. It was composed of DOPE and SM in a 9 : 2 molar ratio. The physical properties of the LPs are summarized in Table 1. Our data showed that the MITO-Porter was positively charged with a diameter of approximately 100 nm. Furthermore, our findings suggested that the physical properties of MITO-Porter were not changed by the inclusion of BBR. As previously mentioned,¹⁰⁾ the physical properties of LPs remained unchanged in the culture medium, and the recovery rate of BBR was approximately 10% (Fig. 2(A)). Cellular uptake of LPs in NHEMs may be higher than in N2a cells¹⁰⁾ (Fig. 2(B)).

Fig. 2. Assessment of the Recovery Rate of BBR and Cellular Uptake

After storing the LPs under various conditions for one day, the recovery rate was measured (A) (n = 3). The cellular uptake of LPs into NHEMs was evaluated based on MFI using FACS. The LPs were labeled with DiD, a fluorescent dye, and the cells were analyzed after the LPs treatment. The data are represented as the mean ± standard deviation (S.D.) (B) (n = 3). Statistical analysis showed ** p < 0.01, using one-way ANOVA followed by the Student–Newman–Keuls test.

Assessment of Mitochondrial Respiratory Function after Treatment with MITO-Porter (BBR)

To clarify the metabolic changes in cells, we assessed the mitochondrial respiratory capacity as a measure of mitochondrial function following treatment with BBR. When energy production relies on mitochondrial respiration, it is expected that the respiratory capacity will be significantly affected, as BBR acts on mitochondrial respiratory chain complex I. We measured the mitochondrial respiratory capacity by analyzing the oxygen consumption rates (OCRs) using the Seahorse XF Analyzer assay. For our control groups, we used non-treatment and MITO-Porter alone treatment. The changes in OCRs in Neuro2a cells and NHEMs after treatment with these samples represented basal respiration, maximum respiration, and spare respiration (Figs. 3(A), (B)). Treatment with BBR resulted in a 30% decrease in basal respiration (naked BBR) or a 40% decrease (MITO-Porter (BBR)) in Neuro2a cells compared with the non-treatment group (Fig. 3(C)). Additionally, treatment with MITO-Porter (BBR) also reduced basal respiration by 30% compared with treatment with MITO-Porter alone (Fig. 3(C)). Furthermore, treatment with MITO-Porter (BBR) reduced the maximal respiration by 30% compared with that of the negative controls (Fig. 3(E)). Conversely, treatment with BBR caused a slight decrease in basal respiration in NHEMs (Fig. 3(D)). However, treatment with MITO-Porter (BBR) led to a slight increase in maximal respiration (Fig. 3(F)). These results suggest that the impact of BBR treatment on mitochondrial respiratory activity differs between Neuro2a cells and NHEMs.

Fig. 3. The Analysis Focused on the Change in Cellular Respiration Activity in Neuro2a Cells or NHEMs When Treated with BBR

The OCR of Neuro2a cells(A) or NHEMs (B) in different groups (non-treatment, naked BBR, MITO-Porter, MITO-Porter (BBR)) was measured over time (in minutes). The injection order of oligomycin, FCCP, and rotenone/antimycin A is provided. The OCR was calculated by normalizing based on cell numbers. Basal respiration of the Neuro2a cells (C) (n = 9) or NHEMs (D) (n = 5) in each group was also calculated. Maximum respiration of the Neuro2a cells (E) (n = 9) or NHEMs (F) (n = 5) in each group was recorded. The data is presented as the mean ± S.D. with * p < 0.05, ** p < 0.01 indicating significance.

Assessment of the ATP Production Rate after Treatment with MITO-Porter (BBR)

Cells typically maintain a consistent intracellular ATP level by adjusting the rate of ATP production in response to changes in mitochondrial respiratory capacity. To assess the ratio of ATP production by mitochondria and glycolysis, we conducted real-time quantitative analysis of ATP production. We used the Seahorse XF Analyzer assay to measure the ATP production ratio by determining the mitochondrial OCR and glycolytic extracellular acidification rate (ECAR), which represent the mitochondrial respiratory, glycolytic, and total ATP production rates. In our experiments, we used non-treatment and MITO-Porter alone as negative controls. When Neuro2a cells were treated with BBR, the mitochondrial ATP production rate decreased by 30%, while the glycolytic ATP production rate increased by 30% compared with those of the negative controls (Fig. 4(A)). The ratio of the ATP production rate by mitochondrial respiration and glycolysis significantly decreased with BBR treatment (Fig. 4(B)). Conversely, after treating NHEMs with MITO-Porter (BBR), the mitochondrial ATP production rate increased by 14% and the glycolytic ATP production rate decreased by 6% compared with those of the non-treatment group (Fig. 4(C)). Additionally, no significant change was observed in the overall rate of ATP production (Fig. 4(D)). These results suggest that the rate of ATP production by mitochondrial respiration differs between Neuro2a cells and NHEMs under normal physiological conditions. Furthermore, the pharmacological effect of BBR treatment was notably influenced in Neuro2a cells, likely because of the higher rate of ATP production by mitochondrial respiration in these cells.

Fig. 4. Measuring the Rate of ATP Production Dependent on Mitochondria or Glycolysis

Seahorse XFp metabolic flux analysis was conducted to measure mitochondrial ATP production rates and glycolytic ATP production rates of Neuro2a cells (A) (n = 5) or NHEMs (C) (n = 3) in each group (non-treatment, naked BBR, MITO-Porter, MITO-Porter (BBR)). The ATP production rate was calculated by normalizing based on the number of cells. (B, D) The XFp ATP rate index was then calculated from the data in each panel. The data are presented as the mean ± S.D. with ** p < 0.01 indicating significance.

Assessment of the Intracellular ATP Amounts after Treatment with MITO-Porter (BBR)

It was suggested that the amount of intracellular ATP production may change because of alterations in the ATP production ratio. An ATP assay kit was used to assess the amount of intracellular ATP necessary for cell survival. For our control groups, we used non-treatment and MITO-Porter alone. When Neuro2a cells were treated with MITO-Porter (BBR), intracellular ATP levels decreased by approximately 50% compared with levels in the other groups (Fig. 5(A)). In contrast, treatment with MITO-Porter (BBR) in NHEMs led to an approximately 40% increase in intracellular ATP compared with that in the non-treatment group (Fig. 5(B)). These findings suggest that effective delivery of BBR to mitochondria via MITO-Porter enhances its pharmacological effects. The study also indicated that BBR treatment largely contributes to the amount of ATP production through mitochondrial respiration in both Neuro2a cells and NHEMs.

Fig. 5. Assessment of Intracellular ATP Production

The intracellular ATP levels were measured using an ATP assay kit. The intracellular ATP concentrations in Neuro2a cells (A) (n = 6) and NHEMs (B) (n = 3) were determined for each group (non-treatment, naked BBR, MITO-Porter, MITO-Porter (BBR)). The data are presented as the mean ± S.D. and with * p < 0.05 and ** p < 0.01 indicating significance.

Assessment of Cell Viability after Treatment with R8-MITO-Porter (BBR)

Cell viability was assessed using the WST-1 reagent, which measures mitochondrial enzyme activity. The samples were treated, and the results were compared with non-treatment and MITO-Porter alone, which were used as negative controls. The values with MITO-Porter (BBR) treatment were similar to those of the other samples in Neuro2a cells and NHEMs (Figs. 6(A), (B)). These findings suggests that treatment with MITO-Porter (BBR) does not have highly toxic effects on these cells.

Fig. 6. Assessment of Cell Viability

Cell viability was assessed using PremixWST-1 reagent. The survival rate of Neuro2a cells (A) (n = 4) or NHEMs (B) (n = 3) in each group (non-treatment, naked BBR, MITO-Porter, MITO-Porter (BBR)) was determined. Relative cell viability was calculated by normalizing the cell viability to the non-treatment group. The data are presented as the mean ± S.D. (n = 3). No significant difference was observed.

DISCUSSION

In this study, we used MITO-Porter (BBR) to confirm if there were variations in the effects of drug actions on different cell types derived from the neural crest. BBR was enclosed in MITO-Porter using the lipid film hydration method. The diameter of the MITO-Porter (BBR) was approximately 100 nm. Transporting BBR to mitochondria using MITO-Porter (BBR) in Neuro2a cells led to a decrease in both basal and maximal respiration (Figs. 3(C), (E)). Conversely, the delivery of BBR to NHEMs slightly affected their respiratory activity (Figs. 3(D), (F)). This difference might be due to the changed balance between mitochondrial respiration and glycolysis in the production of ATP for each cell type (Fig. 4). These findings indicate a reduction in intracellular ATP production in Neuro2a cells but an increase in ATP production in NHEMs (Fig. 5).

Cells derived from the neural crest have been used to study neurodegenerative disease mechanisms. However, it is uncertain if they can be used to test the effects of drug administration. To address this issue, we assessed the changes in the respiratory capacity of mitochondria, which are the cells' power source. When we treated these cells with MITO-Porter (BBR), a drug that affects energy metabolism by acting on mitochondrial respiratory chain complex I,^12,13) their mitochondrial respiratory capacity and ATP production changed compared to treatment with naked BBR (Figs. 3, 5). These results indicate that the effects on energy metabolism may differ in cells derived from the neural crest and that caution should be taken when using these models. In addition, similar experiments should be conducted in other neural crest-derived cells, as changes in OCR may impact the effects of BBR. BBR taken up by cells accumulates in mitochondria, nucleus, and endoplasmic reticulum (ER).¹⁴⁾ BBR accumulated in mitochondria act on the mitochondrial respiratory chain complex I, reducing energy production and inducing activation of energy regulators through feedback regulation. Additionally, by regulating metabolic pathways such as mitochondria and lipids, BBR ultimately increase energy production and contribute to neuroprotective effects.¹⁵⁾ Conversely, BBR accumulated in the nucleus inhibit DNA damage repair through direct interaction with DNA and interference with the DNA replication process.¹⁶⁾ Hence, the accumulation of BBRs outside of mitochondria may bring about cell-damaging effects. The direct intracellular kinetics of BBR should be examined to determine if it is possible to evaluate the intracellular kinetics of BBR in particles using fluorescent labeling of BBR.¹⁷⁾

It has been suggested that the change in mitochondrial respiratory capacity due to treatment with BBR may be attributed to the ratio of mitochondrial respiration to glycolysis for energy production. Therefore, the mitochondrial OCR and glycolytic ECAR were measured using the Seahorse assay. The results indicated that in the control group, Neuro2a cells and NHEMs depended on mitochondrial respiration for approximately 70 and 60% of ATP production, respectively (Fig. 4). Furthermore, treatment with BBR significantly inhibited mitochondrial respiration in Neuro2a cells but not in NHEMs (Fig. 4). Previous reports have indicated that epidermal cells are in a mildly hypoxic state, which may limit mitochondrial function.^18,19) Therefore, NHEMs showed more resistance to hypoxia compared with Neuro2a cells, suggesting that BBR treatment may have enhanced mitochondrial function. It has been reported that cells with low levels of Peroxisome proliferator-activated receptor (PPAR)-γ coactivator-1α (PGC1α) exhibit reduced mitochondrial oxidative metabolism but higher rates of glycolysis and lactate production.^20,21) Conversely, cells with high levels of PGC1α demonstrate an opposite metabolic phenotype characterized by elevated cellular energy status.^20,21) It may be important to assess PGC1 expression levels, as the amount of ATP production rate by BBR varies among cells in Fig. 4.

From Fig. 6, it was observed that there was no cytotoxicity in these cells when treated with MITO-Porter (BBR). Previous reports have indicated that the viability of neurons is not affected when their respiratory activity is reduced by drugs.²²⁾ It has also been reported that cell viability is not affected at different drug concentrations.²³⁾ Additionally, BBR reportedly does not show cytotoxicity in controlling cell metabolism,²⁴⁾ and it may contribute to stress resistance by activating AMPK via ATP control. BBR administration may lead to cell dormancy without significant changes in cell death, even with reduced ATP production.⁷⁾ Therefore, evaluating cell cycle changes and different cell death indices is necessary.

BBR has been reported to suppress neurodegenerative diseases, aging, and oxidative stress.^25–28) Systemic hypoxia occurs in many pathological conditions, such as stroke, ischemic heart disease, infection, inflammation, and cancer.²⁹⁾ Therefore, using BBR for pharmacological preconditioning and exercise for non-pharmacological preconditioning are promising strategies to treat central nervous system diseases.^30–32) This study suggests that the effects of BBR on the mitochondrial respiratory capacity may vary significantly depending on the cell type, with more pronounced changes in neurons. Therefore, precise control during pharmacological preconditioning is necessary. The MITO-Porter system may be a useful tool for enhancing pharmacological effects and preconditioning. However, a limitation of this study is that the pharmacological effects of BBR were examined only in vitro. Further experiments are needed to elucidate the mechanisms from the perspective of factors related to cellular metabolism and hypoxia. Additionally, in vivo experiments need to be conducted for practical application in the treatment of neurodegenerative diseases.

CONCLUSION

We have successfully demonstrated that the MITO-Porter system enhances the pharmacological effects of N2a cells as well as NHEMs. The MITO-Porter system could be a useful tool in treating neurodegenerative diseases and in wound care. It's important to note that N2a cells and NHEMs have different cellular characteristics, so caution should be exercised when using them as disease models.

Acknowledgments

This work was supported, in part, by a Grant-in-Aid for Challenging Exploratory Research (22K19928 to Y.Y.) from the Ministry of Education, Culture, Sports, Science and Technology, the Japanese Government (MEXT); a Grant from the Special Education and Research Expenses of the MEXT [to H.H.]; and JST FOREST [Grant Number: JPMJFR203X to Y.Y., I.H.]. We thank Jenna MacArthur, PhD, for editing a draft of this manuscript.

Conflict of Interest

The authors declare no conflict of interest.

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