Administration of Kyung-Ok-Ko reduces stress-induced depressive behaviors in mice through inhibition of inflammation pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Kyung-Ok-Ko (KOK), a traditional medicinal formula composed of Rehmannia glutinosa (Gaertn.) DC, Poria cocos (Schw.) Wolf, Korean Red Panax ginseng C.A.Mey, and honey, has been used to treat amnesia and dementia. KOK has also been shown to ameliorate transient cerebral global ischemia-induced brain damage, but the antidepressant-like effect of KOK has not been examined. AIM OF THE STUDY: This study examined the antidepressant-like effect of KOK in an immobilization-induced stress mouse and its mechanisms of action. MATERIALS AND METHODS: The animals in the stress group were immobilized for two hours a day for two weeks. KOK at a dose of 1 g/kg/day was administered orally to the stressed mice for two weeks in advance of their immobilization. A forced swimming test was performed to analyze their depressive behaviors. To examine the anti-inflammatory or antioxidative effects of KOK, the murine macrophage cell line, RAW 264.7 cells and human neuroblastoma cell, SH-SY5Y cells, were treated with lipopolysaccharide (LPS) and hydrogen peroxide, respectively. RESULT: The KOK extract showed no significant toxicity when the cells were treated with a KOK extract at 5, 10, 25, 50, and 100 µg/mL. The KOK ethanol extract reduced LPS-induced TNF-α production, inducible nitric oxide (iNOS) mRNA level, and the levels of MAPK and p38 phosphorylation in RAW 264.7 cells. KOK also suppressed H2O2-induced cell death and the production of reactive oxygen species (ROS) in SH-SY5Y cells. In the forced swimming test, KOK induced a decrease in immobility and an increase in climbing activity. Finally, the administration of KOK reversed the up-regulation of IkB-α phosphorylation in the stressed mouse cortex. CONCLUSION: KOK might be useful for the treatment of depression caused by environmental and lifestyle-related stress.

Electromagnetic acupuncture to enhance the effects of manual acupuncture on recovery from muscle fatigue of the quadriceps.

The aim of this study was to investigate a new method of manual acupuncture that used a magnetic field to stimulate only one acupoint vertically. We developed an eight-channel electromagnetic acupuncture (EMA) system that uses a solenoid-type electrode to insert the manual acupuncture needle into a hole in an electrode. We used a manual acupuncture needle for magnetic induction in order to penetrate vertically and deeply into tissues. In order to confirm the usefulness of EMA, we investigated the effects of treatment on muscle fatigue after strenuous knee extension/flexion exercises that had been performed by three groups: the nonstimulation, the manual acupuncture, and the EMA groups. Electromyograms showed that the median frequency (MF) in the EMA group had rapidly recovered after 4 minutes (p = 0.608), but that the peak torque had not recovered to the normal state (p < 0.05). Thus, we confirmed that compared with manual acupuncture, EMA resulted in better recovery from muscle fatigue.

Comparison of 2 methods of non-invasive treatment between transcutaneous electrical stimulation and pulsed electromagnetic field stimulation as replacement of invasive manual acupuncture.

The aim of this study was to find the non-invasive optimal alternative method for Manual Acupuncture. Existing researches had reported that Transcutaneous Electrical Acupoint Stimulation (TEAS) was an effective treatment method instead of manual acupuncture. In place of the TEAS, we suggested the Pulsed Electromagnetic Fields (PEMFs). Thus, we designed the PEMFs system which can stimulate only an acupoint. There have been no researches which reported therapeutic effect when stimulating at an identical acupoint by TEAS and PEMFs. Hence, this study investigated the therapeutic effect on the muscle fatigue after the strenuous knee extension/flexion exercise by two stimulations. We selected the stimulation method of both TEAS and PEMFs by using 2Hz biphasic rectangular wave pulse and pulse width 0.2ms. The magnetic flux was the 30.92mT (309.2gauss) at 2 Hz. The electromyogram (EMG) and the maximal voluntary contraction (MVC) at rectus femoris were measured. The Median Frequency (MF) at TEAS group was significantly effective at 6 minutes (p=0.499). The PEMFs group was recovered to the MF rapidly after 4 minutes (p=0.166). The results of the peak torque indicated that both non-stimulation group and TEAS group did not recover to the peak torque at pre-exercise during the recovery period (p<0.05). In contrast, the significant treatment effect of PEMFs group was found after 14 minutes (p=0.135). The results of this study demonstrated that PEMFs were better than TEAS as a non-invasive method to replace the manual acupuncture.

Anti-apoptotic effect of melatonin on preimplantation development of porcine parthenogenetic embryos.

In the present study, we investigated the effect of melatonin on the preimplantation development of porcine parthenogenetic and somatic cell nuclear transfer (SCNT) embryos. Parthenogenetic embryos were cultured in mNCSU-23 supplemented with various concentrations of melatonin for 7 days. The results revealed that 100 pM was the optimal concentration, which resulted in significantly increased cleavage and blastocyst formation rates. Additionally, 100 pM melatonin provided the highest increase in total cell number of blastocysts. Therefore, the subsequent experiments were performed with 100 pM melatonin. ROS level in 2-8 cell stage embryos in the presence or absence of melatonin was evaluated. Embryos cultured with melatonin showed significantly decreased ROS. Blastocysts cultured with melatonin for 7 days were analyzed by the TUNEL assay. It was observed that melatonin not only increased (P < 0.05) the total cell number but also decreased (P < 0.05) the rate of apoptotic nuclei. Blastocysts cultured with melatonin were assessed for the expression of apoptosis-related genes Bcl-xl and Bax, and of pluripotency marker gene Oct-4 by real-time quantitative PCR. Analysis of data showed that the expression of Bcl-xl was higher (1.7-fold) compared to the control while the expression of Bax was significantly decreased relative to the control (0.7-fold) (P < 0.05). Moreover, the expression of Oct-4 was 1.7-fold higher than the control. These results indicated that melatonin had beneficial effects on the development of porcine parthenogenetic embryos. Based on the findings of parthenogenetic embryos, we investigated the effect of melatonin on the development of porcine SCNT embryos. The results also demonstrated increased cleavage and blastocyst formation rates, and the total cell numbers in blastocysts were significantly higher when the embryos were cultured with melatonin. Therefore, these data suggested that melatonin may have important implications for improving porcine preimplantation SCNT embryo development.

Effects of insulin-transferrin-selenium in defined and porcine follicular fluid supplemented IVM media on porcine IVF and SCNT embryo production.

Insulin-transferrin-selenium (ITS) together has been used in different in vitro maturation system to support in vitro maturation of oocytes. The present study was designed to evaluate the effects of ITS in defined (0.1% PVA) and porcine follicular fluid (10% pFF) supplemented IVM media on the developmental competence of porcine oocytes. Three combinations of ITS, 10 mg/L insulin (Ins), 5.5mg/L transferrin (Tf) and 5 microg/L selenium (Se), 20mg/L Ins, 11 mg/L Tf and 10 microg/L Se, and 30 mg/L Ins, 16.5 mg/L TF and 15 microg/L Se, were used. The data were analyzed by one-way ANOVA and Tukey was used as the post hoc test. Both in the defined and pFF supplemented media, higher concentration of intracellular glutathione was observed in presence of ITS (4.6-4.8, and 6.9-7.1 picomole/oocyte for defined and pFF groups, respectively) compared to the respective control (2.1 and 4.3 picomole/oocyte for defined and pFF group, respectively). ITS decreased polyspermy and increased male pronucleus formation in both the defined and pFF supplemented medium. There was no difference in different treatment groups. The highest frequency of blastocyst formation rate and number of cells in blastocyst following IVF and SCNT was observed in pFF+ITS group (p<0.05). In conclusion, ITS addition during IVM improved the developmental competence of porcine oocytes in both the defined and pFF supplemented groups. Thus, we recommended to supplement porcine IVM medium with 10 microg/mL insulin, 5.5 microg/mL transferrin and 5 microg/mL selenium.

Temporal effects of alpha-tocopherol and L-ascorbic acid on in vitro fertilized porcine embryo development.

The susceptibility of embryos to reactive oxygen species (ROS) varies in different stages of embryo development. The present study evaluated temporal effects of alpha-tocopherol and L-ascorbic acid on the porcine embryo development, and investigated whether a single or twice supplements of these two antioxidants at a divided concentrations favors the embryo development. In order to determine temporal effects of alpha-tocopherol and/or L-ascorbic acid, 100 microM alpha-tocopherol or 200 microM L-ascorbic acid were supplemented to the North Carolina State University (NCSU)-23 embryo culture media at 0, 48, 96 and 120 h of culture. In another set of experiments, the concentration was divided into two equal halves, i.e., 50 microM alpha-tocopherol and 100 microM L-ascorbic acid, and supplemented twice at 0 and 48, 0 and 96, or 48 and 96 h of culture. Supplementing culture media with 100 microM alpha-tocopherol for the entire culture period of 168 h or starting from the 48 h of culture yielded higher blastocyst percentage compared with the control or starting from the 96 or 120 h of culture. L-Ascorbic acid (200 microM) alone or together with alpha-tocopherol (100 microM) with a single supplement did not affect the frequency of blastocyst formation or number of cells in blastocyst. L-ascorbic acid with a divided supplements yielded higher blastocyst percentage compared with the control. No synergistic effect was observed on embryo development at a single supplement of these antioxidants. Although, at divided supplements higher blastocyst percentage was observed compared with control group, no further beneficial effect was observed compared with alpha-tocopherol or L-ascorbic acid alone. Our results demonstrated that the embryotrophic effects of alpha-tocopherol and/or L-ascorbic acid, in terms of frequency of blastocyst formation and number of cells in blastocyst, depends on the concentration and supplementation timing.

Antiapoptotic and embryotrophic effects of alpha-tocopherol and L-ascorbic acid on porcine embryos derived from in vitro fertilization and somatic cell nuclear transfer.

The objective was to determine optimal concentrations of alpha-tocopherol and l-ascorbic acid for development of porcine embryos derived from in vitro-fertilization (IVF) or somatic cell nuclear transfer (SCNT). The frequency of blastocyst formation in IVF embryos was 17.6, 28.6, 32.4 and 21.4% for control, 50, 100 and 200microM alpha-tocopherol, respectively, whereas in SCNT embryos, the frequency was 12.8, 19.0, 24.8 and 17.7% for corresponding concentrations of alpha-tocopherol. For both IVF and SCNT embryos, there were significantly more cells in blastocysts and the embryos had greater developmental competence when the embryo culture medium was supplemented with 100microM alpha-tocopherol. Although either alpha-tocopherol or l-ascorbic acid reduced the proportion of apoptotic cells in blastocysts, in combination they resulted in rates of apoptosis that were similar to the control group. For IVF embryos, the apoptotic index was 0.09 and 0.11 for alpha-tocopherol or l-ascorbic acid at a concentration of 100microM, respectively. Conversely, when these antioxidants were supplemented together, the apoptotic index increased significantly and was similar to the control group (i.e., 0.17 and 0.21 for combined and control group). For SCNT embryos, the apoptotic index was 0.10 for 100microM for both alpha-tocopherol and l-ascorbic acid, whereas the index was 0.23 and 0.17 for control and combined group. In conclusion, we recommend supplementing porcine embryo culture medium with 100microM alpha-tocopherol or 100microM l-ascorbic (as a second choice).