Reproductive and Developmental Medicine

ORIGINAL ARTICLE
Year
: 2018  |  Volume : 2  |  Issue : 2  |  Page : 74--80

Vitamin K3 Regulates Reactive Oxygen Species and Extracellular-Regulated Protein Kinase in Differentiated PC-12 Cells within a Safe Dose Range


Jing Yang1, Jun-Ya Ke1, Yu-Ling Lian2, Yang Zhang1, Jin-Feng Huang1, Li Wang1,  
1 Shanghai Obstetrics and Gynecology Hospital, Fudan University; Shanghai Key Laboratory of Female Reproductive Endocrine-Related Diseases, Shanghai 200011; Department of Obstetrics and Gynecology, Shanghai Medical College, Fudan University, Shanghai 200032, China
2 Shanghai Obstetrics and Gynecology Hospital, Fudan University; Shanghai Key Laboratory of Female Reproductive Endocrine-Related Diseases, Shanghai 200011, China

Correspondence Address:
Li Wang
No. 419, Fangxie Road, Shanghai 200011
China

Abstract

Objective: Acupuncture can relieve pain by acting on the mitogen-activated protein kinase (MAPK) signal pathway, which plays a critical role in the balance between hyperalgesia and inflammation. Our previous studies have suggested that acupoint injection of Vitamin K3 (Vit K3) had an intensive analgesic effect on primary dyspareunia. However, the mechanism by which Vit K3 worked on nerve cells has not been elucidated. Methods: Cell apoptosis, mitochondrial membrane potential (MMP), and reactive oxygen species (ROS) changes of PC-12 cells with Vit K3 treatment, for which the concentration gradient was 0, 5, 10, 20, 40, and 60 μmol/L, were quantified by flow cytometry. The expression and phosphorylation of c-Jun N-terminal kinase, p38, and extracellular-regulated protein kinase (ERK), the three critical molecules of the MAPK pathway, were further assessed using Western blotting. Results: The level of ROS first decreased and then increased with Vit K3 at 20 μmol/L, but no change in neither apoptosis nor MMP was evident. In addition, only ERK level decreased at 20 μmol/L and the relative phosphorylation level increased. Changes in ROS were negatively correlated with the expression of ERK. Conclusions: The rapid analgesic effect of Vit K3 acupoint injection may be through the reduction of ROS in nerve cells with a small dose of Vit K3 or by influencing the expression of ERK but without damaging the nerve cells themselves.



How to cite this article:
Yang J, Ke JY, Lian YL, Zhang Y, Huang JF, Wang L. Vitamin K3 Regulates Reactive Oxygen Species and Extracellular-Regulated Protein Kinase in Differentiated PC-12 Cells within a Safe Dose Range.Reprod Dev Med 2018;2:74-80


How to cite this URL:
Yang J, Ke JY, Lian YL, Zhang Y, Huang JF, Wang L. Vitamin K3 Regulates Reactive Oxygen Species and Extracellular-Regulated Protein Kinase in Differentiated PC-12 Cells within a Safe Dose Range. Reprod Dev Med [serial online] 2018 [cited 2019 Dec 13 ];2:74-80
Available from: http://www.repdevmed.org/text.asp?2018/2/2/74/242757


Full Text



 Introduction



Dysmenorrhea is a symptom that is primary or secondary to other gynecological diseases, such as endometriosis and adenomyosis. Currently, the pathogenesis of dysmenorrhea caused by endometriosis remains unclear. In addition to pelvic inflammatory adhesion, endometriosis pain is now regarded as nociceptive (including inflammatory), neuropathic, or a combination of the two.[1] Inflammation-activated noxious stimuli are transduced to the dorsal root ganglia (DRG)[2] and spinal cord dorsal horn (SCDH).[3] The integrated signal in DRG and SCDH is then transduced to the cerebrum. These long-term nociceptive inputs can increase neuron response in the peripheral or central sensory nervous system, causing peripheral or central sensitization, respectively, thus reducing the pain threshold. Peripheral and central sensitization may be associated with refractory pain in patients with endometriosis, primary dysmenorrhea, and chronic pelvic pain.

Different treatments have been used to reduce dysmenorrhea pain, including medical and nonmedical treatment, such as administration of nonsteroidal anti-inflammatory drugs (NSAIDs), steroids, herbal therapies, acupuncture, and dietary therapies. However, NSAIDs and corticosteroids have the potential to cause side effects and are rarely effective. NSAIDs often lead to gastrointestinal symptoms and are ineffective in 25% patients.[4] Steroids, such as progestin and combined oral contraceptive pills, may cause breakthrough bleeding, amenorrhea, nausea, vomiting, headache, bloating, carcinogenesis, and so on.[5] Moreover, pain symptoms in approximately 17% of patients are not reduced after treatment;[6] approximately 5%–59% still report pain at the end of treatment; and during follow-up, 17%–34% experience a recurrence of pain symptoms after ectopic lesion excision.[7]

Acupoint injection is a reinforced analgesia method, which can effectively suppress neuropathic sensitization. In our previous clinical study, we found that Vitamin K3 (Vit K3) acupoint injection once significantly alleviated acute menstrual pain in dysmenorrhea patients within 30 min;[8],[9] menstrual distress was reduced after a single injection in 6 months.[10] It was observed the phenomenon of analgesia effect of Vit K3, but little is known about its mechanism. We therefore aimed to explore how Vit K3 influences nerve cells and their mechanisms.

Vit K3, also called menadione, was an inexpensive artificial chemical. Although we still use Vit K clinically (including Vit K3), there was much evidence in vitro that Vit K, especially Vit K3, had a broad-spectrum pro-apoptotic effect on cells. In addition to tumor cells,[11],[12] Vit K3 also caused damage to nerve cells,[13] optic nerve,[14] cardiomyocytes, hepatocytes,[15] and parasite,[16] etc., which may cause Parkinson's disease, amyotrophic lateral sclerosis, and hair growth inhibition through producing a mass of reactive oxygen species (ROS),[17] which is associated with cell viability and neuropathic sensitization. Therefore, the safety of Vit K3 is to be verified first in our experiments.

ROS interacts with critical signaling molecules directly to initiate signaling in a broad variety of cellular processes, such as mitogen-activated protein kinases (MAPKs).[18] MAPKs are a critical pathway for nociception and neural plasticity. Activation of p38, c-Jun N-terminal kinase (JNK), and extracellular-regulated protein kinases (ERKs) after tissue and nerve injury contribute primarily to pain sensitization in sensory neurons, DRG or SCDH.[19] We assumed that Vit K3 may reinforce the inhibited effects on ROS in nerve cells through MAPK pathway.

PC-12 is a cell line derived from a pheochromocytoma of the rat adrenal medulla, which has characters that a mixture of neuroblastic and sympathetic cells. It is round and suspension cell in undifferentiated condition, and it has potential to differentiate into a neuron-like adherent cell when treated with nerve growth factor (NGF), which has long neurite outgrowth. Because of this characteristic, PC-12 serves as a cell model system for neuropharmacology. According to the results of Barcena de Arellano et al.,[20] PC-12 cells incubated with the peritoneal fluid of endometriosis women showed a higher proliferation rate, a stronger neurite outgrowth, and differentiation. Moreover, sympathetic nerves can cause contraction of uterine smooth muscle, and in our previous experiments, Vit K3 inhibited contraction of uterine smooth muscle.[21] To determine whether it has similar inhibited effect on sympathetic nerves, we chose differentiated PC-12 as the cell model to research Vit K3 neurotoxicity.

Vit K3 often has lethal effect in vitro experiment, but it is safe in clinical application, indicating that the dose plays a decisive role in experiments in vitro. To understand the role of Vit K3 for acupoint injection to neurocyte, safe dose range for PC-12, and changes of MAPK signaling pathway in the safe dose range is the purpose of this experiment.

 Methods



Materials

Rat pheochromocytoma-differentiated PC-12 cell line was obtained from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences (Shanghai, China). Menadione sodium bisulfite (Vit K3) (purity ≥95%) and DMSO were purchased from Sigma-Aldrich (Shanghai, China). Dulbecco's Modified Eagle Medium (DMEM), fetal bovine serum (FBS), trypsin, and phosphate-buffered saline (PBS) were purchased from Gibco Life Technologies (Shanghai, China). PE Annexin V Apoptosis Detection Kit was obtained from BD Pharmingen (Shanghai, China). MAPK Family Antibody Sampler Kit, Phospho-MAPK Family Antibody Sampler Kit, and GAPDH (D4C6R) Mouse mAb were purchased from Cell Signaling Technology (CST, Shanghai, China). RIPA Lysis Buffer, SDS-PAGE Sample Loading Buffer, SDS-PAGE Gel Quick Preparation Kit, and ROS Assay Kit were purchased from Beyotime in Shanghai. Protein ladder (26616) and colorimetric protein assays were purchased from Thermo Fisher (Shanghai, China). Tetramethylrhodamine Ethyl Ester (TMRE)-Mitochondrial Membrane Potential (MMP) Assay Kit was purchased from Abcam (Shanghai, China).

Cell culture

Differentiated PC-12 cells were cultured in DMEM medium containing FBS (10%), penicillin (100 units/mL), and streptomycin (100 μg/mL) within T75 culture flasks under a humidified incubator with CO2 (5%) and air (95%) at 37°C. Every 3–4 days, the adherent cells were suspended after treatment with 1 mL of 0.25% trypsin-EDTA solution for 1–2 min at 37°C and then were subcultured at a 1:3 split ratio. The culture medium was changed every 2 days. Stocks of cells were routinely frozen and stored in liquid nitrogen. Cells with 15–20 passages were used for experiments to ensure cell line stability.

Cell apoptosis assay using flow cytometry

Differentiated PC-12 cells were plated in a 12-well plate at a density of 1.5 × 105 cells per well. Cells were allowed to adhere and proliferate for 24 h before being exposed to different concentration solutions of menadione sodium bisulfite (Vit K3) within DMSO. After incubation, cells were exposed to varying concentrations of Vit K3 within DMSO and DMEM (0, 5, 10, 20, 40, or 60 μmol/L) for an additional 24 h. Then, trypsinized adherent cells collected from each well were transferred into a centrifuge tube, with the supernatant medium. Supernatant was discarded after centrifuging at 168 g. Cells were washed three times using PBS via centrifuging and re-suspending. Then, all cells were incubated in a solution containing Annexin V and 7-AAD dyes and incubated for 15 min at 25°C before flow cytometry analysis.

Mitochondrial membrane potential measurement

Levels of MMP were measured with TMRE. Cell culture protocol was the same as that of cell apoptosis assay. After 24 h treatment, we replaced the supernatant and washed cells twice with PBS, then incubated them with DMEM and 200 μmol/L TMRE for 20 min at 37°C in darkness, washed the cells twice with PBS, and trypsinized for flow cytometry analysis.

Reactive oxygen species measurement

Levels of intracellular ROS were measured with oxidation-sensitive fluoroprobe 2′,7′-dichlorofluorescin diacetate (DCF-DA). In this part, cell cultivation was performed in a manner similar to that for the cell apoptosis assay. After 24 h treatment, we replaced the supernatant, washed the cells with PBS, and then incubated them with DMEM and 10 μmol/L DCF-DA for 30 min at 37°C in darkness. We then washed the cells twice with PBS and trypsinized for flow cytometry analysis.

Western blotting

Protein was completely extracted from the T75 flasks, using RIPA Lysis Buffer with 1% protease and phosphatase inhibitor cocktail. Soluble protein from the cells was separated using 10% SDS-PAGE and then transferred to PVDF membranes. The membranes were then blocked in 5% BSA with TBST buffer for 1 h at 25°C. The membranes were incubated overnight at 4°C with different primary antibodies and then with the corresponding secondary antibodies for 1 h at 25°C. Primary antibodies were as follows: p44/42 MAPK (Erk1/2) antibody (4695, 1:1,000, CST, China), p38 MAPK antibody (8690, 1:1,000, CST, China), SAPK/JNK antibody (9252, 1:1,000, CST, China), and GAPDH antibody (97166, 1:1,000, CST, China). Secondary antibodies were as follows: HRP goat anti-rabbit IgG (H + L) antibody (7074, 1:5,000, CST, China), anti-mouse IgG antibody, and HRP-linked antibody (7076, 1:5,000, CST, China). The density of the bands was then determined using an imaging densitometer, and the gray value of the bands was quantified by an ImageJ analysis software (https://imagej.nih.gov/ij/).

Statistical analysis

The above experiments were repeated 5 times. Data were shown by mean ± standard error of the mean. The statistical significance of differences was established using the Student's t-test or the Pearson r-test to compare the means, where appropriate. Differences with P = 0.05 or less were considered statistically significant.

 Results



Different Vitamin K3 concentrations had different effects on cell apoptosis

Cell apoptosis was evaluated using flow cytometry in PC-12 cells by increasing Vit K3 from 0 to 60 μmol/L after 24 h incubation. As shown in [Figure 1], Vit K3 treatment did not cause significant apoptosis; among the concentration of 0 μmol/L (9.523 ± 3.279), 5 μmol/L (6.790 ± 1.631), 10 μmol/L (6.213 ± 2.018), and 20 μmol/L (10.82 ± 1.619), however, apoptosis increased sharply when concentration was 40 μmol/L (93.72 ± 0.7285, t-test, P < 0.0001) and 60 μmol/L (92.90 ± 0.6228, t-test, P < 0.0001) when each group was compared with 0 μmol/L. Interestingly, the trend detected that apoptosis first decreased and then increased with growing concentration from 0 to 20 μmol/L, but there was no statistical significance. The threshold in this experiment between cell survival and apoptosis was 20 μmol/L. We defined “safe dose range” as 0 to 20 μmol/L in our experiments.{Figure 1}

Reactive oxygen species changes of PC-12 within Vitamin K3 safe dose range

ROS was quantified by flow cytometry with the same Vit K3 concentration gradient with 24 h incubation that was used in apoptosis experiment. ROS was presented as mean fluorescence intensity of DCF in all cells. It suggested that ROS first decreased at 0 μmol/L (30.13 ± 0.8252) to 5 μmol/L (26.03 ± 0.4699) and then increased with concentration growing from 5 to 10 μmol/L (29.51 ± 1.412), and ROS increased at 20 μmol/L (35.72 ± 2.290) Vit K3 treated in PC-12 cells [Figure 2]a and [Figure 2]b, whereas MMP, which was detected by mean fluorescence intensity (TMRE), did not change in any group [Figure 2]c and [Figure 2]d.{Figure 2}

Safe doses of Vitamin K3 effects on PC-12 via mitogen-activated protein kinase signal pathway

The expression and phosphorylation levels of the three critical members of MAPK pathway were measured using Western blot. JNK, P38, and their absolute and relative phosphorylation did not change after Vit K3 treatment in a safe dose range [Figure 3]a, [Figure 3]b, [Figure 3]c, [Figure 3]d, [Figure 3]e, [Figure 3]f. ERK expression had a negative trend that increased first and then significantly decreased at 20 μmol/L [Figure 3]g. As shown in [Figure 3]i, p-ERK did not change within 20 μmol/L [Figure 3]h, but relative phosphorylation levels – (p-ERK/(ERK) – increased significantly at 20 μmol/L (P < 0.05). [Figure 3]j shows that ERK expression in PC-12 following Vit K3 treatment under 20 μmol/L Vit K3 exposure at 0 h, 4 h, 8 h, 12 h, 16 h, and 24 h had a tendency to first increase (P > 0.05) and then decline after 8 h incubation. It finally significantly reduced at 24 h (P < 0.01).{Figure 3}

Correlation analysis of reactive oxygen species and other variations

Pearson r-test analysis suggested that there was no obvious correlation between ROS and apoptosis or MMP [Figure 4]a and [Figure 4]b. However, correlation analysis indicated that ERK expression was related to ROS, and the line regression showed that both had negative correlations [Figure 4]c.{Figure 4}

 Discussion



Although there were evidence that menadione (Vit K3) can damage cell viability in vitro, our experiments have shown that the damage does not occur within a certain concentration range, providing experimental basis for safety of Vit K3 injection to Sanyinjiao acupoint (3 cun directly above the tip of the medial malleolus on the posterior border of the tibia). In addition, it was reported that the plasma concentration of Vit K was usually very low in its conventional application against hemorrhagic disease. The peak median concentration was 3.95 μmol/L in neonates after intramuscular injection of 1 mg Vit K1,[22] while other research suggested that it was 0.035–0.25 μmol/L after 10 mg Vit K1 intramuscular injection to one adult[23] and 0.04–0.425 μmol/L after 10 mg Vit K1 acupoint injection over a 20–48 h period[24] as same as our previous clinical study. In a word, it followed that the peak value of Vit K plasma concentration was lower than the 20 μmol/L “safe value” in our experiments, when used within the usual dose [Figure 2]a, which could not induce apoptosis.

In some new research, increased dietary Vit K intake helped serious subjective memory complaints[25] and even promoted behavior and cognition in the elderly (over 65 years old) and may be the source of antioxidant nutrients in brain ischemia.[26] These studies have shown that normal intake or supplementation of Vit K did not cause damage to the nervous system and may even be beneficial.

Vitamin K, with a double bond in quinone ring, changes to oxidized Vit K (quinone epoxide) by the posttranslational modification of γ-glutamyl carboxylase (Gla), which required calcium ions (Ca2+) for biological activity in the endoplasmic reticulum,[27] and oxidized Vit K is catalyzed by Vit K epoxide reductase to reduced Vit K.[28] ROS can lead to intracellular Ca2+ elevation in DRG.[29] This additional intracellular Ca2+ active Gla catalyzes menadione to the oxidized state by absorbing radicals. The increase of ROS in hyperalgesic DRG and the decrease of MMP can induce the upregulation of JNK. Our previous study suggested that Vit K3 inhibited mouse uterine contraction, explained by Vit K3-weakened Ca2+ influx.[21] The results of this experiment showed that ROS decreased after 5 μmol/L Vit K3 treatment, as suggested by Li et al. that low doses of Vit K act as free radical scavengers that cleared accumulated ROS in oligodendrocytes and neurons in vitro.[30] Although the ROS level significantly increased between a concentration of 10 μmol/L and 20 μmol/L, it did not impact MMP. Moreover, the ROS only caused a decrease in ERK. Li et al. suggested that ERK was upregulated in DRG neurons of endometriosis rats.[31] Moreover, acupuncture may inhibit the P38 and ERK activation to initiate analgesia in local.[32],[33],[34] For the mechanism of MAPK pathway in neurodegeneration of PC-12 cells, Xia et al. found that NGF-activated ERK and stress-activated JNK were in dynamic balance and that the co-occurrence of activation of JNK and P38 and inhibition of ERK was a key factor in neuronal apoptosis.[35] From this, it was speculated that only the downregulation of ERK caused subsequent suppression of some cellular function without damage of the nerve cell itself. Electroacupuncture can induce ERK to be phosphorylated in the cerebral cortex of rats.[36] We found that p-ERK was increased after 20 μmol/L Vit K3 treatment, which suggested that Vit K3 acupoint injection has analgesia effect. Therefore, considering that different kinds of nerve cells may have different tolerance thresholds for Vit K3, we speculated that Vit K3 may exert an analgesic effect by reducing the intracellular ROS at low doses within the safe range or changing the ERK expression at high doses within the safe range.

It was speculated that for the administration of Vit K3 or Vit K within the usual dose range, low doses of Vit K3 could clear ROS in nerve cells, and high doses of Vit K3 could reduce the expression level of ERK in neurons. These may be mechanisms underlying the rapid and long-lasting analgesic effects of Vit K3 injection, respectively, and these results provide the basis for in vivo studies of the neuromechanism of the analgesic effects of Vit K3 acupoint injection in the future. In conclusion, a low dose of VitK3 was safe for PC-12, and intracellular ROS and ERK had a negative correlation within the safe concentration range for Vit K3 in this experiment.

Financial support and sponsorship

This work was financially supported by the National Natural Science Foundation of China (grant no. 81473459).

Conflicts of interest

There are no conflicts of interest.

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