Electroacupuncture at GB34 modulates neurogenesis and BDNF-ERK signaling in a mouse model of Parkinson's disease.

Background and aim: It has been reported that acupuncture at GB34 can enhance neurogenesis in the subventricular zone (SVZ) of mice treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). However, the signaling pathway that plays a critical role in neurogenesis needs to be established. Herein, we investigated the neurogenesis-promoting pathway mediated by acupuncture, focusing on extracellular signal-regulated kinase (ERK) signaling. Experimental procedure: Male 10-week-old C57BL/6 mice were intraperitoneally injected with 30 mg/kg MPTP once daily for 5 days. Subsequently, mice were intraperitoneally injected with 50 mg/kg bromodeoxyuridine (BrdU), and electroacupuncture (EA) was performed at GB34 and BL60 for 3 weeks. The survival of dopaminergic neurons in the nigrostriatal pathway, cell proliferation in the SVZ, and expression levels of brain-derived neurotrophic factor (BDNF) and phosphorylated ERK (pERK) were evaluated. Results and conclusion: MPTP induced dopaminergic neuronal death in the nigrostriatal pathway, and reduced the number of BrdU-positive and BrdU/doublecortin double-positive cells in the SVZ; these parameters were restored by EA. Moreover, EA prevented MPTP-induced reduction in striatal expression of BDNF and pERK. These results indicate that EA could prevent dopaminergic neuronal death in the nigrostriatal pathway and restore neurogenesis in the SVZ, which may be attributed to the activation of the BDNF-ERK pathway.

Korean red ginseng decreases 1-methyl-4-phenylpyridinium-induced mitophagy in SH-SY5Y cells.

OBJECTIVE: Mitophagy is known to contribute towards progression of Parkinson's disease. Korean red ginseng (KRG) is a widely used medicinal herb in East Asia, and recent studies have reported that KRG prevents 1-methyl-4-phenylpyridinium ion (MPP+)-induced cell death. This study was undertaken to investigate whether KRG suppresses MPP+-induced apoptosis and mitophagy. METHODS: SH-SY5Y cells were incubated with KRG for 24 h, and subsequently exposed to MPP+. The MPP+-induced cell death was confirmed with the 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay, and the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling assay. Changes in the structure and function of mitochondria were confirmed using mitotracker, MitoSOX red mitochondrial superoxide indicator, parkin, and phosphatase and tensin homolog deleted on chromosome ten-induced putative kinase 1 (PINK1) immunofluorescent staining. Western blotting was performed to evaluate the expression of apoptosis-related factors in whole cells, including Bax, Bcl-2 and cleaved caspase-3, and mitophagy-related factors in the mitochondrial fraction, including cytochrome c, parkin, PINK1, translocase of the outer membrane 20 (TOM20), p62 and Beclin 1. RESULTS: MPP+ induced cell death by cytochrome c release and caspase-3 activation; however, this effect was suppressed by KRG's regulation of the expressions of Bcl-2 and Bax. Moreover, MPP+ exposure increased the mitochondrial expressions of parkin, PINK1, Beclin 1 and p62, and decreased TOM20, cytochrome c and Bcl-2 expressions. These MPP+-induced changes in the mitochondrial fraction were attenuated by treatment with KRG. CONCLUSION: KRG effectively prevents MPP+-induced SH-SY5Y cell death by regulating cytochrome c release from mitochondria and PINK1/parkin-mediated mitophagy, through regulation of the Bcl-2 family.

Korean red ginseng promotes hippocampal neurogenesis in mice.

Neurogenesis in the adult hippocampus plays a major role in cognitive ability of animals including learning and memory. Korean red ginseng (KRG) has long been known as a medicinal herb with the potential to improve learning and memory; however, the mechanisms are still elusive. Therefore, we evaluated whether KRG can promote cognitive function and enhance neurogenesis in the hippocampus. Eight-week-old male C57BL/6 mice received 50 mg/kg of 5-bromo-2'-deoxyuridine (BrdU) intraperitoneally and 100 mg/kg of KRG or vehicle orally once a day for 14 days. Pole, Rotarod and Morris water maze tests were performed and the brains were collected after the last behavioral test. Changes in the numbers of BrdU- and BrdU/doublecortin (DCX; a marker for neuronal precursor cells and immature neurons)-positive cells in the dentate gyrus and the gene expression of proliferating cell nuclear antigen (a marker for cell differentiation), cerebral dopamine neurotrophic factor and ciliary neurotrophic factor in the hippocampus were then investigated. KRG-treated mice came down the pole significantly faster and stood on the rotarod longer than vehicle-treated mice. The Morris water maze test showed that KRG administration enhanced the learning and memory abilities significantly. KRG also significantly increased BrdU- and BrdU/DCX-positive cells in the dentate gyrus as well as the proliferating cell nuclear antigen, cerebral dopamine neurotrophic factor and ciliary neurotrophic factor mRNA expression levels in the hippocampus compared to vehicle. Administration of KRG promotes learning and memory abilities, possibly by enhancing hippocampal neurogenesis. This study was approved by the Pusan National University Institutional Animal Care and Use Committee (approval No. PNU-2016-1071) on January 19, 2016.

Acupuncture stimulation at GB34 suppresses 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced oxidative stress in the striatum of mice.

Recent studies have suggested that increased oxidative stress is a potential etiology in Parkinson's disease (PD). In this study, we investigated whether acupuncture regulates antioxidants in the striatum (ST) of a PD mouse model. Male C57BL/6 mice were administered 30 mg/kg of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) intraperitoneally once a day for 5 days and given acupuncture stimulation at SI3 or GB34 (Yanglingquan) was for 12 consecutive days. Dopaminergic neuronal survival in the nigrostriatal pathway and DJ-1 expression in the ST was evaluated by immunostaining, and the activities of superoxide dismutase (SOD) and catalase (CAT) in the ST was by enzyme-linked immunosorbent assay. MPTP administration induced dopaminergic neuronal death in the nigrostriatal pathway, which was suppressed by acupuncture stimulation at GB34. MPTP administration also suppressed DJ-1 expression and SOD and CAT activities in the ST, which were restored by acupuncture stimulation at GB34. These results indicate that the neuroprotective effect of acupuncture stimulation is due to regulation of the antioxidants.

Acupuncture alters pro-inflammatory cytokines in the plasma of maternally separated rat pups.

OBJECTIVE: To investigate the potential alleviating effects of acupuncture on maternal separation (MS)-induced changes in plasma pro-inflammatory cytokine levels of rat pups. METHODS: On postnatal day 15, rat pups were randomly assigned to 4 groups (n=6 per group) using a random number table: normal, MS, MS with acupuncture stimulation at Shenmen (HT 7) acupoint (MS+HT 7), and MS with acupuncture stimulation at Chengshan (BL 57) acupoint (MS+BL 57) groups. Rat pups in the normal group were housed with their mothers under standard conditions; those in the MS, MS+HT 7 and MS+BL 57 groups were maternally separated and individually maintained. Acupuncture stimulation was performed at HT 7 or BL 57 acupoints once a day for 7 consecutive days. A tail suspension test was performed to measure immobility time of rats and the plasma was collected on postnatal day 21, then levels of corticosterone (CORT), interleukin (IL)-1ß, IL-6 and glial cell-derived neurotrophic factor (GDNF) in plasma were measured. RESULTS: Compared with the normal group, the immobility time and the plasma levels of CORT, IL-1ß, IL-6 and GDNF in the MS group were significantly increased (P<0.05 or P<0.01). Compared with the MS group, the immobility time and the plasma levels of CORT, IL-1ß, IL-6 and GDNF were significantly reduced in the MS+HT 7 group (P<0.05 or P<0.01). Moreover, the immobility time and plasma levels of IL-1ß and IL-6 in the MS+HT 7 group were significantly lower than those in the MS+BL 57 group (P<0.05). CONCLUSION: Acupuncture stimulation at HT 7 can alleviate the behavioral impairment and changes of the cytokines by MS, indicating that acupuncture can help to relieve MS-induced depression.

Acupuncture Stimulation at GB34 Restores MPTP-Induced Neurogenesis Impairment in the Subventricular Zone of Mice.

Adult neurogenesis has recently been considered a new therapeutic paradigm of Parkinson's disease. In this study, we investigated whether acupuncture restores 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine- (MPTP-) induced impaired neurogenesis in the subventricular zone (SVZ). Male C57BL/6 mice were given 30 mg/kg of MPTP intraperitoneally once a day for 5 days, after which they were intraperitoneally injected with 50 mg/kg of bromodeoxyuridine (BrdU) and given acupuncture stimulation at HT7 or GB34 for 12 consecutive days. Dopaminergic neuronal survival in the nigrostriatal pathway and cell proliferation in the SVZ was then evaluated by immunostaining. MPTP administration induced dopaminergic neuronal death in the nigrostriatal pathway, which was suppressed by acupuncture stimulation at GB34. MPTP administration also suppressed the number of BrdU-positive cells and glial fibrillary acidic protein/BrdU-positive cells and increased the number of doublecortin/BrdU-positive cells in the SVZ, which were restored by acupuncture stimulation at GB34. These results indicate that acupuncture stimulation at GB34 restores MPTP-induced neurogenesis impairment.

Proteomic Analysis of the Effect of Korean Red Ginseng in the Striatum of a Parkinson's Disease Mouse Model.

Recent studies have shown that Korean Red Ginseng (KRG) suppresses dopaminergic neuronal death in the brain of a Parkinson's disease (PD) mouse model, but the mechanism is still elusive. Using a 2-dimensional electrophoresis technique, we investigated whether KRG can restore the changes in protein expressions in the striatum (ST) of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-injected mice. Male C57BL/6 mice (9 weeks old) were injected with 20 mg/kg MPTP intraperitoneally four times at 2-h intervals. KRG (100 mg/kg) was orally administered once a day for 3 days from one hour after the first MPTP injection. Two hours after the third KRG administration a pole test was performed to evaluate motor function, after which the brains were immediately harvested. Survival of dopaminergic neurons in the nigrostriatal pathway and protein expression in the ST were measured by immunohistochemistry and 2-dimensional electrophoresis. KRG suppressed MPTP-induced behavioral dysfunction and neuronal death in the nigrostriatal pathway. Moreover, 30 proteins changed by MPTP and KRG in the ST were identified and shown to be related to glycolysis/gluconeogenesis and neurodegenerative diseases including Alzheimer's disease and PD. KRG has neuroprotective effects against MPTP toxicity and alleviates protein expression profiles related to enhancing energy metabolism in the ST of MPTP-treated mice.

Electroacupuncture Enhances the Antiallodynic and Antihyperalgesic Effects of Milnacipran in Neuropathic Rats.

BACKGROUND: Milnacipran, a selective serotonin/norepinephrine-reuptake inhibitor, has been shown to elicit a beneficial effect in various models of neuropathic pain. Previously, we reported that repetitive electroacupuncture (EA) significantly ameliorates neuropathic pain induced by L5 spinal nerve ligation (SNL). In the present study, we sought to determine whether a single treatment with EA produces analgesia and whether EA in combination with a subeffective dosage of milnacipran exhibits an additive effect in SNL rats. METHODS: Mechanical allodynia and thermal hyperalgesia were assessed by measuring paw withdrawal thresholds and latencies in response to mechanical and thermal stimuli, respectively, 1 day before and 5 days after neuropathic surgery. In addition, on day 5 post-SNL, time courses of behaviors were assessed at 0, 1, 2, 4, 6, and 8 hours after intrathecal (i.t.) milnacipran (1, 5, and 20 µg) administration. EA (10 Hz/1 mA) was administered at the ST36 and GB34 acupoints for 30 minutes on day 5 and the time courses of behaviors were also assessed at 0, 1, 2, 4, 6, and 8 hours later. Similarly, when treated in combination (milnacipran [5 µg, i.t.] and EA [10 Hz/1 mA]), time courses of behaviors were assessed at the same time points. RESULTS: Intrathecal injection of milnacipran at 5 or 20 µg exerted dose-dependent effects on thermal hyperalgesia but had similar efficacies on mechanical allodynia. Furthermore, EA itself detectably attenuated hyperalgesia at 4 hours after the application, but no statistically significant difference was found in mechanical allodynia. Importantly, cotreatment with EA and milnacipran (5 µL) produced more potent antiallodynic and antihyperalgesic effects than those obtained from EA or milnacipran alone at 1, 2, and 4 hours after treatment, indicating an additive effect. In addition, the analgesic effect of EA plus milnacipran was almost completely abolished by the catecholamine neurotoxin 6-hydroxydopamine hydrobromide (25 µg), which depletes spinal norepinephrine, and by yohimbine (an α2-adrenoceptor antagonist, 30 µg, i.t.). Somewhat surprisingly, the analgesic effect of milnacipran plus EA lasted for 6 hours. CONCLUSIONS: The study shows that, in male rats with SNL, spinal administration of milnacipran effectively alleviates mechanical allodynia and thermal hyperalgesia, and that a single treatment of EA has an antihyperalgesic effect. Furthermore, our findings suggest that coapplication of EA and milnacipran enhanced antiallodynia and antihyperalgesia by activating spinal noradrenergic systems coupled with spinal α2-adrenoceptors and prolongs the duration of analgesia.

Inhibitory Effects of Scolopendra Pharmacopuncture on the Development and Maintenance of Neuropathic Pain in Rats: Possible Involvement of Spinal Glial Cells.

Scolopendra extracts were used for pharmacopuncture at the Kidney 1 acupoint to investigate the role of Scolopendra pharmacopuncture (SPP) in both the development and maintenance of neuropathic pain induced by L5 spinal nerve ligation in rats and the contribution of spinal glial cells. A single treatment and five once-daily treatments with SPP were given to evaluate its effects on the development and maintenance stages of neuropathic pain, respectively, which was followed by behavioral tests. Immunohistochemistry and Western blotting tests were also carried out. A single treatment of SPP delayed spinal nerve ligation-induced mechanical allodynia and thermal hyperalgesia and induced a profound decrease in the expression of ionized calcium binding adaptor protein in the lumbar spinal cord. Repeated SPP treatments reliably suppressed mechanical allodynia and thermal hyperalgesia at later time points, and these results correlated mainly with decreases in glial fibrillary acidic protein. Intriguingly, ionized calcium binding adaptor protein expression was also reduced after repeated SPP. These results illustrate that neuropathic pain in the development and maintenance stages is alleviated by SPP treatment, which may be ascribed principally to deactivations of microglia and astroglia, respectively. Additionally, microglial inactivation seems to be partially involved in preventing neuropathic pain in the maintenance stage.

Comparison of electroacupuncture frequency-related effects on heart rate variability in healthy volunteers: a randomized clinical trial.

This study aimed to compare the effects of high frequency electroacupuncture (EA) and low-frequency EA on the autonomic nervous system by using a heart rate variability measuring device in normal individuals. Fourteen participants were recruited and each participated in the high-frequency and low-frequency sessions (crossover design). The order of sessions was randomized and the interval between the two sessions was over 2 weeks. Participants received needle insertion with 120-Hz stimulation during the high-frequency session (high-frequency EA group), and with 2-Hz stimulation during the low-frequency session (low-frequency EA group). Acupuncture needles were directly inserted perpendicularly to LI 4 and LI 11 acupoints followed by delivery of electric pulses to these points for 15 minutes. Heart rate variability was measured 5 minutes before and after EA stimulation by a heart rate variability measuring system. We found a significant increase in the standard deviation of the normal-to-normal interval in the high-frequency EA group, with no change in the low-frequency EA group. Both the high-frequency and low-frequency EA groups showed no significant differences in other parameters including high-frequency power, low-frequency power, and the ratio of low-frequency power to high-frequency power. Based on these findings, we concluded that high-frequency EA stimulation is more effective than low-frequency EA stimulation in increasing autonomic nervous activity and there is no difference between the two EA frequencies in enhancing sympathovagal balance.

Synergistic antinociceptive effects of N-methyl-D-aspartate receptor antagonist and electroacupuncture in the complete Freund's adjuvant-induced pain model.

This study examined the synergistic antinociceptive effects associated with signaling pathway proteins of the spinal cord in a complete Freund's adjuvant (CFA)-induced pain model when electroacupuncture (EA) and a N-methyl-D-aspartate receptor (NMDAR) antagonist were administered in combination. EA stimulation (2 Hz, 1 mA) was needle-delivered for 20 min once daily at acupoints corresponding to Zusanli and Sanyinjiao with intrathecal injection of the NMDAR antagonist dizocilpine (MK801). Thermal sensitivity of the hindpaw induced by CFA was strongly inhibited by dizocilpine injection and EA stimulation. Co-treatment with EA and dizocilpine showed a synergistic antinociceptive effect against inflammatory pain. On day two of the experiment, we examined the phosphorylation of the NMDAR NR2B subunit, of the extracellular signal-regulated kinase (ERK), p38 and of the cAMP response element-binding protein (CREB) in the ipsilateral dorsal horn of L4-5 segments by Western blot analysis. Phosphorylation of the NMDAR NR2B subunit induced by CFA was markedly inhibited by co-treatment with dizocilpine and EA, but not by dizocilpine or EA treatment alone. CFA-induced phosphorylation of the ERK was inhibited by both dizocilpine and EA, but that of p38 was inhibited by EA only. CFA-induced phosphorylation of CREB was inhibited by dizocilpine, but did not show marked changes. Immunohistochemical analyses confirmed that there was a significant difference in the NMDAR NR2B subunit and ERK phosphorylation. It is possible that the combined treatment with EA and the NMDAR antagonist dizocilpine resulted in synergistic antinociceptive effects in an inflammatory pain model via the inactivation of both the NMDAR NR2B subunit and ERK of the spinal cord.

A Parametric Study on the Immunomodulatory Effects of Electroacupuncture in DNP-KLH Immunized Mice.

This study was conducted to compare the effects of low frequency electroacupuncture (EA) and high frequency EA at acupoint ST36 on the production of IgE and Th1/Th2 cytokines in BALB/c mice that had been immunized with 2,4-dinitrophenylated keyhole limpet protein (DNP-KLH), as well as to investigate the difference in the immunomodulatory effects exerted by EA stimulations at acupoint ST36 and at a non-acupoint (tail). Female BALB/c mice were divided into seven groups: normal (no treatments), IM (immunization only), ST36-PA (IM + plain acupuncture at ST36), ST36-LEA (IM + low frequency (1 Hz) EA at ST36), ST36-HEA (IM + high frequency (120 Hz) EA at ST36), NA-LEA (IM + low frequency (1 Hz) EA at non-acupoint) and NA-HEA (IM + high frequency (120 Hz) EA at non-acupoint). EA stimulation was performed daily for two weeks, and total IgE, DNP-KLH specific IgE, IL-4 and IFN-γ levels were measured at the end of the experiment. The results of this study showed that the IgE and IL-4 levels were significantly suppressed in the ST36-LEA and ST36-HEA groups, but not in the NA-LEA and NA-HEA groups. However, there was little difference in the immunomodulatory effects observed in the ST36-LEA and ST36-HEA groups. Taken together, these results suggest that EA stimulation-induced immunomodulation is not frequency dependent, but that it is acupoint specific.

Spinal cholinergic mechanism of the relieving effects of electroacupuncture on cold and warm allodynia in a rat model of neuropathic pain.

This study was performed to determine whether spinal cholinergic systems mediate the relieving effects of electroacupuncture (EA) on cold and warm allodynia in a rat model of neuropathic pain. For neuropathic surgery, the right superior caudal trunk was resected at the level between the S1 and S2 spinal nerves innervating the tail. Two weeks after the injury, the intrathecal (i.t.) catheter was implanted. Five days after the catheterization, the rats were injected with atropine (non-selective muscarinic antagonist, 30 microg), mecamylamine (non-selective nicotinic antagonist, 50 microg), pirenzepine (M(1) muscarinic antagonist, 10 microg), methoctramine (M(2) antagonist, 10 microg) or 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) (M(3) antagonist, 10 microg). Ten minutes after the injection, EA was applied to the ST36 acupoint for 30 min. The cold and warm allodynia were assessed by the tail immersion test [i.e., immersing the tail in cold (4 degrees C) or warm (40 degrees C) water and measuring the latency of an abrupt tail movement] before and after the treatments. The i.t. atropine, but not mecamylamine, blocked the relieving effects of EA on cold and warm allodynia. Furthermore, i.t. pirenzepine attenuated the antiallodynic effects of EA, whereas methoctramine and 4-DAMP did not. These results suggest that spinal muscarinic receptors, especially M(1) subtype, mediate the EA-induced antiallodynia in neuropathic rats.

Protective effects of electroacupuncture on acetylsalicylic acid-induced acute gastritis in rats.

AIM: To investigate the protective effects of electroacupuncture (EA) pretreatment on acetylsalicylic acid (ASA)-induced ulceration in rats. METHODS: We randomly divided 72 rats into three groups including control (administered with distilled water), ASA group (administered 100 mg/kg ASA) and EA group (administered EA + 100 mg/kg ASA). Each rat was fasted for 18 to 24 h before experimentation, and lesion scores, gastric acidity, cyclooxygenase (COX)-1 and -2 mRNA levels, and total nitric oxide (NO) concentration were measured. RESULTS: The lesion scores of the EA group were significantly lower than those of the ASA group. Gastric acidity of the ASA and EA groups was reduced compared to the control group. COX-1 and -2 mRNA levels were significantly increased in the EA group as compared to the control and ASA groups, and NO levels were also significantly increased in the EA group as compared to the ASA group. CONCLUSION: These results suggest that EA-mediated protection against ASA-induced ulceration in rats may occur via gastric defense components.

Electroacupuncture potentiates the antiallodynic effect of intrathecal neostigmine in a rat model of neuropathic pain.

This study was performed to examine whether electroacupuncture potentiates the neostigmine-induced antiallodynia in neuropathic pain rats. Although intrathecal neostigmine (0.05, 0.1, and 0.3 microg) dose-dependently relieved cold allodynia, 0.3 microg neostigmine caused side effects. The coapplication of 0.1 microg neostigmine and electroacupuncture, however, produced potent antiallodynia, which was parallel to the effect of 0.3 microg neostigmine, without side effects. These results indicate that electroacupuncture can enhance the antiallodynic action of intrathecal neostigmine.

Antinociception of heterotopic electro-acupuncture mediated by the dorsolateral funiculus.

We investigated the inhibitory pathways that mediate the antinociceptive effects of heterotopic electro-acupuncture (EA) on formalin injection-induced pain in rats. EA (2 ms, 10 Hz, 3 mA) was delivered to heterotopic acupoints HT(7) and PC(7) for 30 min; this was followed immediately by subcutaneous injection of formalin into the left hind paw of rats. Naltrexone (10 mg/kg, i.p.), an opioid receptor antagonist, was administered to evaluate the involvement of endogenous opioids. The dorsolateral funiculus (DLF), which is a descending pathway that inhibits pain, was transected at the ipsilateral T10-11 level of the thoracic spinal cord. EA inhibited behavioral responses to formalin injection-induced pain and prevented the pain-induced increase in cFos expression in the lumbar spinal cord. Pretreatment with naltrexone did not inhibit the antinociceptive effects of EA on formalin injection-induced pain. Transection of the DLF ipsilateral to the acupuncture site eliminated the antinociceptive effects of EA. These results suggest that the antinociceptive effects of heterotopic EA are mediated by the DLF and not by endogenous opioids.