Min-Hee Yi

Ethanol extract of Astragali Radix and Salviae Miltiorrhizae Radix, Myelophil, exerts anti-amnesic effect in a mouse model of scopolamine-induced memory deficits.

ETHNOPHARMACOLOGICAL RELEVANCE Myelophil, a combination of extracts taken from Astragali Radix and Salviae Miltiorrhizae Radix, is a traditional Chinese medicine used for the treatment of chronic fatigue-associated disorders. Here we examined the ability of Myelophil to alleviate memory impairment in a mouse model. We aimed to investigate whether Myelophil has the pharmacological effects on memory deficits associated with brain dysfunctions using an animal model. MATERIALS AND METHODS Ten week-old male C57BL/6N mice were pretreated with Myelophil (50, 100, or 200 mg/kg), or tacrine (10 mg/kg) for 7 days, and then intraperitoneally injected with scopolamine (1 mg/kg). Memory-related behaviors were evaluated using the Morris water maze for 5 days. Levels of biomarkers of oxidative stress, antioxidant activity, acetylcholinesterase (AChE) activity, and extracellular signal-regulated kinase (ERK) were measured in brain tissues. RESULTS Scopolamine treatment increased the escape latency time and shortened time spent in the target quadrant; these effects were ameliorated by pretreatment with Myelophil. Scopolamine-induced changes in reactive oxygen species (ROS), malondialehyde (MDA), and AChE activity were significantly attenuated in mice pretreated with Myelophil. Recovery of antioxidant capacities, including total glutathione (GSH) content, and the activities of GSH-reductase, GSH-S-transferase, and catalase was also evident in Myelophil-treated mice. The strongest effects were seen for ERK and muscarinic acetylcholine receptor 1 (mAChR1) at both the protein and gene expression levels, with significant amelioration of expression levels in the Myelophil pretreatment group. CONCLUSIONS These results suggest that Myelophil confers anti-amnesic properties in a mouse model of memory impairment, driven in part by the modulation of cholinergic activity.

Expression of CD200 in alternative activation of microglia following an excitotoxic lesion in the mouse hippocampus.

CD200 is a glycoprotein that is expressed on the surfaces of neurons and other cells. It interacts with its receptor, CD200R, which is expressed on cells of the myeloid lineage, including microglia. The interaction of CD200 with its receptor plays a significant role in maintaining microglia in a quiescent state; thus, a decrease in CD200 expression in the brain is associated with evidence of microglial activation. However, their roles in pathological progression remain unclear. We examined the expression of CD200 in kainic acid (KA)-induced neurodegeneration of the mouse hippocampus. Our quantitative analysis revealed that CD200 was constitutively expressed in the normal brain and transiently upregulated by KA treatment. At the cellular level, CD200 was expressed in neurons in control, and was upregulated primarily in the microglia of KA-treated mouse hippocampi. We examined the contribution of CD200 to both the classical and alternative activation of microglia in vitro using an adult microglia culture, which was exposed to interleukin-4 (IL-4) with and without lipopolysaccharide (LPS). CD200 expression was increased after exposure to IL-4, but not to LPS. These in vivo experiments demonstrated that CD200 was transiently expressed in microglia in a process mediated by the inflammatory response. Based on CD200R expression in microglia, it suggests that microglia is maintained in an activated state with autocrine signaling by interactions between microglial CD200 and its CD200R. Moreover, we suggest that CD200 may be expressed in the alternative activation of microglia and play a beneficial role in neuroinflammation.

Roles of endoplasmic reticulum stress-mediated apoptosis in M1-polarized macrophages during mycobacterial infections

Alteration of macrophage function has an important regulatory impact on the survival of intracellular mycobacteria. We found that macrophages infected with attenuated Mycobacterium tuberculosis (Mtb) strain H37Ra had elevated expression of M1-related molecules, whereas the M2 phenotype was dominant in macrophages infected with virulent Mtb H37Rv. Further, the TLR signalling pathway played an important role in modulating macrophage polarization against Mtb infection. Interestingly, endoplasmic reticulum (ER) stress was significantly increased in M1 polarized macrophages and these macrophages effectively removed intracellular Mtb, indicating that ER stress may be an important component of the host immune response to Mtb in M1 macrophages. This improved understanding of the mechanisms that regulate macrophage polarization could provide new therapeutic strategies for tuberculosis.

The effect of botulinum neurotoxin type A on capsule formation around silicone implants: the in vivo and in vitro study.

This study confirms that botulinum neurotoxin type A (BoNT‐A) decreases capsular contracture and elucidates a possible mechanism. Silicone blocks were implanted subcutaneously in 20 mice. The experimental groups received BoNT‐A (1, 2·5 or 5 U) instilled into the subcutaneous pocket. After 30 days, periprosthetic capsules were harvested and evaluated. The effect of BoNT‐A on the differentiation of human dermal fibroblasts to myofibroblasts in culture was examined by Western blot analysis. Changes in transforming growth factor‐beta1 (TGF‐β1) expression in cultured fibroblasts were determined by enzyme‐linked immunosorbent assay (ELISA). In in vivo study, the thickness of capsules (P < 0·05) and the number of alpha‐smooth muscle actin (α‐SMA)+ cells in capsules (P < 0·05) were significantly decreased in the experimental groups. TGF‐β1 was significantly underexpressed in the experimental groups (P < 0·05). In in vitro study, BoNT‐A did not significantly affect fibroblast viability. Western blot analysis showed that α‐SMA protein levels were significantly decreased in the experimental groups (P < 0·05). Based on ELISA, the amount of TGF‐β1 was significantly decreased in the experimental groups (P < 0·05), especially cells treated with a high dose of BoNT‐A (P < 0·001). This study confirms that BoNT‐A prevents capsular formation around silicone implants, possibly by blocking TGF‐β1 signalling and interrupting the differentiation of fibroblasts to myofibroblasts.

Expression of LC3 and Beclin 1 in the spinal dorsal horn following spinal nerve ligation-induced neuropathic pain.

Impaired spinal GABAergic inhibitory function is known to be pivotal in neuropathic pain (NPP). At present, data concerning time-dependent alterations in cell type and cell death in the spinal dorsal horn are highly controversial, likely related to the experimental NPP model used. In this study, we examined the expression of autophagy using a L5 spinal nerve ligation (SNL)-induced neuropathic pain rat model. Following ligation of the spinal nerve, neuropathic pain behavior, such as mechanical allodynia, was induced rapidly and maintained for 14 days. After testing for mechanical allodynia, we assessed the changes in expression of LC3 and Beclin 1 in the spinal cord following SNL. Immunohistochemical analysis showed that the levels of LC3 and Beclin 1 protein in the ipsilateral L5 spinal dorsal horn were significantly elevated on day 14 following SNL. Double immunohistochemical analysis further confirmed increases in LC3 and Beclin 1 in mostly neurons and a few astrocytes following SNL. LC3 and Beclin 1 expressions were upregulated in GABAergic interneurons of spinal dorsal horn after SNL, while the loss of GABAergic interneurons did not change significantly. Our results suggest that autophagic disruption in GABAergic interneurons and astrocytes following peripheral nerve injury might be involved in the induction and maintenance of neuropathic pain.

Effect of Botulinum Toxin Type A on Tgf-β/smad Pathway Signaling: Implications for Silicone-induced Capsule Formation.

Background: One of the most serious complications of breast surgery using implants is capsular contracture. Several preventive treatments have been introduced; however, the mechanism of capsule formation has not been resolved completely. The authors previously identified negative effects of botulinum toxin type A on capsule formation, expression of transforming growth factor (TGF)-&bgr;1, and differentiation of fibroblasts into myofibroblasts. Thus, the authors investigated how to prevent capsule formation by using botulinum toxin type A, particularly by means of TGF-&bgr;1 signaling, in human fibroblasts. Methods: In vitro, cultured human fibroblasts were treated with TGF-&bgr;1 and/or botulinum toxin type A. Expression of collagen, matrix metalloproteinase, and Smad was examined by Western blotting. The activation of matrix metalloproteinase was observed by gelatin zymography. In vivo, the effect of botulinum toxin type A on the phosphorylation of Smad2 in silicone-induced capsule formation was evaluated by immunocytochemistry. Results: In vitro, the phosphorylation of Smad2 was inhibited by botulinum toxin type A treatment. The expression levels of collagen types 1 and 3 were inhibited by botulinum toxin type A treatment, whereas those of matrix metalloproteinase-2 and matrix metalloproteinase-9 were enhanced. Gelatin zymography experiments confirmed enhanced matrix metalloproteinase-2 activity in collagen degradation. In vivo, botulinum toxin type A treatment reduced capsule thickness and Smad2 phosphorylation in silicone-induced capsules. Conclusion: This study suggests that botulinum toxin type A plays an important role in the inhibition of capsule formation through the TGF-&bgr;/Smad signaling pathway. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, V.

Endoplasmic reticulum stress impairment in the spinal dorsal horn of a neuropathic pain model

Endoplasmic reticulum (ER) stress has been implicated in neurodegenerative diseases, but its role in neuropathic pain remains unclear. In this study, we examined the ER stress and the unfolded protein response (UPR) activation in a L5 spinal nerve ligation (SNL)-induced rat neuropathic pain model. SNL-induced neuropathic pain was assessed behaviorally using the CatWalk system, and histologically with microglial activation in the dorsal spinal horn. L5 SNL induced BIP upregulation in the neuron of superficial laminae of dorsal spinal horn. It also increased the level of ATF6 and intracellular localization into the nuclei in the neurons. Moreover, spliced XBP1 was also markedly elevated in the ipsilateral spinal dorsal horn. The PERK-elF2 pathway was activated in astrocytes of the spinal dorsal horn in the SNL model. In addition, electron microscopy revealed the presence of swollen cisternae in the dorsal spinal cord after SNL. Additionally, inhibition of the ATF6 pathway by intrathecal treatment with ATF6 siRNA reduced pain behaviors and BIP expression in the dorsal horn. The results suggest that ER stress might be involved in the induction and maintenance of neuropathic pain. Furthermore, a disturbance in UPR signaling may render the spinal neurons vulnerable to peripheral nerve injury or neuropathic pain stimuli.

High ω3-polyunsaturated fatty acids in fat-1 mice prevent streptozotocin-induced Purkinje cell degeneration through BDNF-mediated autophagy

Loss of Purkinje cells has been implicated in the development of diabetic neuropathy, and this degeneration is characterized by impairment of autophagic processes. We evaluated whether fat-1 transgenic mice, a well-established animal model that endogenously synthesizes ω3 polyunsaturated fatty acids (ω3-PUFA), are protected from Purkinje cell degeneration in streptozotocin (STZ)-treated model with fat-1 mice. STZ-treated fat-1 mice did not develop hyperglycemia, motor deficits, or Purkinje cell loss. The expression of LC3 I, II, Beclin-1 and p62 were increased in the cerebellum of STZ-treated wild-type mice, and these expressions were more increased in STZ-treated fat-1 mice, but not of p62. Moreover, cerebellar Rab7, Cathepsin D, and ATP6E were increased in STZ-treated fat-1 mice. There was also increased BDNF expression in Purkinje cells without any changes in TrkB, and phosphorylation of Akt and CREB in the cerebellums of fat-1 mice. Collectively, these findings indicate that STZ-treated fat-1 mice were protected from Purkinje cell loss and exhibited increased BDNF signaling, enhancing autophagic flux activity in cerebellar Purkinje neurons. These processes may underlie Purkinje cell survival and may be potential therapeutic targets for treatment of motor deficits related to diabetic neuropathy.

Growth Differentiation Factor 15 Expression in Astrocytes After Excitotoxic Lesion in the Mouse Hippocampus

Growth differentiation factor 15 (GDF15) is, a member of the transforming growth factor β (TGF-β) superfamily of proteins. Although GDF15 is well established as a potent neurotrophic factor for neurons, little is known about its role in glial cells under neuropathological conditions. We monitored GDF15 expression in astrocyte activation after a kainic acid (KA)-induced neurodegeneration in the ICR mice hippocampus. In control, GDF15 immunoreactivity (IR) was evident in the neuronal layer of the hippocampus; however, GDF15 expression had increased in activated astrocytes throughout the hippocampal region at day 3 after the treatment with KA. LPS treatment in astrocytes dramatically increased GDF15 expression in primary astrocytes. In addition, LPS treatment resulted in the decrease of the IκB-α degradation and increase of the phosphorylation level of RelA/p65. These results indicate that GDF15 has a potential link to NF-κB activation, making GDF15 a valuable target for modulating inflammatory conditions.

Uwhangchungsimwon, a traditional herbal medicine, protects brain against oxidative injury via modulation of hypothalamus-pituitary-adrenal (HPA) response in a chronic restraint mice model.

ETHNOPHARMACOLOGICAL RELEVANCE Uwhangchungsimwon (UCW) is a representative traditional herbal medicine for central nervous system disorders in East Asia countries over thousand years. To evaluate the pharmacological effects of UCW against oxidative brain injury in a chronic restraint stress mice model. METHODS AND MATERIALS C57BL/6 male mice underwent daily oral administration of distilled water, UCW or ascorbic acid 1h before induction of restraint stress (5h of immobilization daily for 14 days). Nitric oxide (NO), total reactive oxygen species (ROS) levels, malondialdehyde, protein carbonyl contents, and activities of antioxidant enzymes, and concentrations of corticosterone, adrenaline, noradrenaline, and dopamine, were measured in brain tissues or sera. RESULTS Restraint stress notably increased NO and ROS levels, malondialdehyde and protein carbonyl contents in brain tissues, but decreased activities of catalase, glutathione reductase and glutathione peroxidase. These alterations were significantly ameliorated by UCW. UCW significantly attenuated the elevated serum concentrations of corticosterone, adrenaline and noradrenaline. UCW also significantly normalized the gene expressions in brain tissues altered by restraint stress; up-regulation of phenylethanolamine N-methyltransferase (PNMT) and N-methyl-d-aspartate type 1 receptor (NMDAR1), and down-regulation of gamma-Aminobutyric acid type A receptor (GABAAR), glutamate decarboxylase 1 (GAD 67), and glutamate decarboxylase 2 (GAD 65), respectively. Moreover, UCW considerably restored neurogenesis in the hippocampal regions which was disturbed by chronic restraint stress. CONCLUSIONS These results evidenced that UCW has pharmacological properties for brain protection and neurogenesis in status of stress-associated oxidative damage, and the underlying mechanisms involve the regulation of HPA axis in stress responses.