Chi-Mei Hsueh

Molecular mechanisms responsible for microglia-derived protection of Sprague–Dawley rat brain cells during in vitro ischemia

Microglia-derived protection of brain cells (microglia, astrocytes, and neurons) during in vitro ischemic stress (deprivation of glucose, oxygen, and serum) was determined. Trypan blue exclusion assay, immunoblocking assay, Western blot analysis, and ELISA assay were used to determine the molecular mechanisms responsible for the microglia-derived protection. Results demonstrated that supernatants from the ischemic microglia protected all three cell-types from ischemia-induced damage by releasing the transforming growth factor-beta1 (TGF-beta1) and glial cell line-derived neurotrophic factor (GDNF). The protection of microglia was TGF-beta1 related, whereas astrocytes protection was GDNF-dependent. The protection of neurons was TGF-beta1 and GDNF independent, and the molecular nature responsible for their protection remains to be determined. These results indicate contribution from the surrounding cells and the types of receptors expressed on different brain cells probably also play an important role in determining their fate against ischemia.

Berberine inhibits platelet-derived growth factor-induced growth and migration partly through an AMPK-dependent pathway in vascular smooth muscle cells

Platelet-derived growth factor (PDGF) is released from vascular smooth muscle cells (VSMCs), endothelial cells, or macrophages after percutaneous coronary intervention and is related with neointimal proliferation and restenosis. Berberine is a well-known component of the Chinese herb medicine Huanglian (Coptis chinensis), and is capable of inhibiting growth and endogenous PDGF synthesis in VSMCs after in vitro mechanical injury. We analyzed the effects of berberine on VSMC growth, migration, and signaling events after exogenous PDGF stimulation in vitro in order to mimic a post-angioplasty PDGF shedding condition. Pretreatment of VSMCs with berberine inhibited PDGF-induced proliferation. Berberine significantly suppressed PDGF-stimulated Cyclin D1/D3 and Cyclin-dependent kinase (Cdk) gene expression. Moreover, berberine increased the activity of AMP-activated protein kinase (AMPK), which led to phosphorylation activation of p53 and increased protein levels of the Cdk inhibitor p21(Cip1). Compound C, an AMPK inhibitor, partly but significantly attenuated berberine-elicited growth inhibition. In addition, stimulation of VSMCs with PDGF led to a transient increase in GTP-bound, active form of Ras, Cdc42 and Rac1, as well as VSMC migration. However, pretreatment with berberine significantly inhibited PDGF-induced Ras, Cdc42 and Rac1 activation and cell migration. Co-treatment with farnesyl pyrophosphate and geranylgeranyl pyrophosphate drastically reversed berberine-mediated anti-proliferative and migratory effects in VSMCs. Based on these findings, we conclude that berberine inhibited PDGF-induced VSMC growth via activation of AMPK/p53/p21(Cip1) signaling while inactivating Ras/Rac1/Cyclin D/Cdks and suppressing PDGF-stimulated migration via inhibition of Rac1 and Cdc42. These observations offer a molecular explanation for the anti-proliferative and anti-migratory properties of berberine.

Protection of ischemic brain cells is dependent on astrocyte-derived growth factors and their receptors

An in vitro ischemia model (oxygen, glucose, and serum deprivation) is used to investigate the possible cellular and molecular mechanisms responsible for cerebral ischemia. We have previously demonstrated that supernatants derived from ischemic microglia can protect ischemic brain cells by releasing GDNF and TGF-beta1. In the present study, we investigate whether products of ischemic astrocytes can also protect ischemic microglia, astrocytes, and neurons in a similar manner. Supernatants from ischemic astrocytes were collected after various periods of ischemia and incubated with microglia, astrocytes, or neurons individually, under in vitro ischemic conditions. The components responsible for the protective effects of astrocyte-derived supernatants were then identified by Western blot, ELISA, trypan blue dye exclusion, and immunoblocking assays. Results showed that under conditions of in vitro ischemia the number of surviving microglia, astrocytes, and neurons was significantly increased by the incorporation of the astrocyte-derived supernatants. Astrocyte supernatant-mediated protection of ischemic microglia was dependent on TGF-beta1 and NT-3, ischemic astrocytes were protected by GDNF, and ischemic neurons were protected by NT-3. In addition, protein expression of TGF-beta1 and NT-3 receptors in microglia, GDNF receptors in astrocytes, and NT-3 receptors in neurons was increased by in vitro ischemia. These results suggest that astrocyte-derived protection of ischemic brain cells is dependent not only on factors released from the ischemic astrocytes, but also on the type of receptor present on the responding cells. Therapeutic potential of TGF-beta1, GDNF, and NT-3 in the control of cerebral ischemia is further suggested.

Attenuation of lipopolysaccharide-induced acute lung injury by treatment with IL-10.

Background and objective:  The aim of this study was to characterize the changes in neutrophils and cytokines in BAL fluid following acute lung injury (ALI), and to determine the protective effect of post‐injury treatment with IL‐10.

XIAP-mediated protection of H460 lung cancer cells against cisplatin.

Molecular mechanism(s) responsible for drug resistance of non-small cell lung cancer (NSCLC) cells to cisplatin was investigated. Results showed that cisplatin (50muM)-induced cell death (apoptosis) was more significant in CH27 and A549 cell lines than in H460. The high protein levels of X-linked inhibitor-of-apoptosis protein (XIAP) observed in H460 cells appeared to play a key role in the regulation of cisplatin resistance of H460 cells. XIAP can bind to and suppress the activities of caspase 3 in H460 cells and lead to apoptosis inhibition of these cells. Blockade of XIAP activity by Embelin (XIAP inhibitor) or siRNA has increased caspase 3 activities and promoted cisplatin-induced cell death of H460 cells. The results indicate a therapeutic value of Embelin and/or XIAP siRNA in the control of cisplatin-resistant NSCLC cells (H460).

Expression of the conditioned NK cell activity is β-endorphin dependent

We are interested in identifying the pathways which are responsible for triggering the conditioned enhancement of natural killer (NK) cell activity. Earlier studies have suggested that central opioid(s) are involved in eliciting the expression of the conditioned NK cell activity. The purpose of this study was to identify the central opioid peptides that allow the central nervous system (CNS) to communicate with the immune system. Mediators that activate the efferent pathway of communication between the CNS and immune system was examined by injection of the mediator via the cisterna magna (CM). Conditioning was used as a tool to show that the bi-directional communication between the CNS and the immune system does take place. We found that beta-endorphin but not dynorphin could stimulate NK cell activity, when beta-endorphin or dynorphin was injected into the CM. In addition, when anti-beta-endorphin or anti-dynorphin antibody was injected into the conditioned animals via CM the conditioned response was blocked by anti-beta-endorphin but not by anti-dynorphin antibody. These observations suggest that beta-endorphin appears to be one of the signals that is induced in the brain at the CS recall step of the conditioned response to trigger the elevation of NK cell activity.

The involvement of glutamate in recall of the conditioned NK cell response

The molecular mechanisms responsible for the conditioned enhancement of natural killer (NK) cell activity were investigated. The primary goal of the study was to examine the roles of glutamate and gamma-aminobutyric acid (GABA) in recall of the conditioned NK cell response. Both neurochemical blocking assay and high performance liquid chromatography (HPLC) technique were used in the study. Results from the neurochemical blocking assay demonstrated that glutamate but not GABA was required in recall of the conditioned NK cell response. NMDA but not the kainate/AMPA receptors, are believed to be involved. The levels of glutamate that were released and/or taken up also appeared to be critical in that interruption of glutamate release and/or uptake blocked the conditioned NK cell response. Results from the HPLC analysis, however, did not show any significant difference in the glutamate content between the conditioned and control brains.

Identification of specific pathways of communication between the CNS and NK cell system

The specific signals and pathways utilized by the natural killer (NK) cell system and the central nervous system (CNS) that results in the conditioned response (CR) is not clearly understood. Single trial conditioning of the NK cell activity provides us with a model to probe the mechanisms of communication between two major systems (Immune and CNS) which are involved in the health and disease of the individual. The studies show that the IFN-beta molecules possess the properties attributed to the unconditioned stimulus (US). IFN-beta can penetrate the CNS and evoke the elevation of NK cell activity in the spleen. This unconditioned response (UR) can be linked to a specific conditioned stimulus (CS). Specific odors such as camphor provide a neural pathway for the CS to associate with the US. Evidence is presented that in conditioning there are two locations where memory develops. The CS/US association is made centrally and its memory is stored at a central location, but the memory for the specificity of the odor is presumably stored in the olfactory bulbs. The CS recalls the CR by triggering the olfactory neural pathway which, in turn, signals the hypothalamic-pituitary axis to release mediators that modulate the activity of NK cells in the spleen. These results imply that through conditioning one has direct input into the regulatory hypothalamus that controls the internal environment of the organism and the health and disease of the individual. Consequently, it is not inconceivable that through this approach we might be able to alter the course of a disease process.

Ischemic brain cell-derived conditioned medium protects astrocytes against ischemia through GDNF/ERK/NF-kB signaling pathway.

Conditioned medium (CM) collected from cultures of ischemic microglia, astrocytes, and neurons were protective to astrocytes under the in vitro ischemic condition (deprivation of oxygen, glucose and serum). Molecular and signaling pathway(s) responsible for the CMs protective activity were investigated. Results showed that CMs from the ischemic microglia (MCM), astrocytes (ACM) and neurons (NCM) contained glial cell line-derived neurotrophic factor (GDNF), which protects astrocytes against the in vitro ischemia. Expression of extra cellular signal-regulated kinase (ERK1/2) and nuclear factor-kappa B (NF-kB) by GDNF led to the inhibition of apoptosis of the ischemic astrocytes in a caspase 3-independent manner. However, CMs- and GDNF-mediated protection of the ischemic astrocytes was protein kinase B (Akt) independent. These results provided mechanistic data regarding how GDNF- and CMs-mediated protection of the ischemic astrocytes is taking place. These observations provide information for the use of GDNF and GDNF containing CMs in the control of cerebral ischemia.

The central effect of methionine-enkephalin on NK cell activity.

The central effect of opioid peptide on natural killer (NK) cell activity in BALB/c mice was investigated. Injection of methionine-enkephalin (Met-Enk), 0.02 microgram/mouse or 1 microgram/kg, directly into the cisterna magna (CM) of the brain, resulted in a significant enhancement of NK cell activity. This enhancement was blocked by opiate antagonists, naltrexone and quaternary naltrexone. The same dose of Met-Enk had no effect on NK cell activity when given to the mouse intraperitoneally or intravenously. Moreover, des-tyrosine-methionine-enkephalin injected into the CM at 1 microgram/kg, had no effect on NK cell activity. The results indicate that activation of an opioid-mediated pathway in the central nervous system is capable of activating the pathways that stimulate the NK cell response in the periphery.