Effects of TGF-β1 on the expression and secretion of cytokines induced by Aβ in hippocampal neuron and microglia co-cultures / 中国应用生理学杂志

OBJECTIVE@#To investigate the neuroprotective effects of transforming growth factor beta 1(TGF-β1) on the expression and secretion of cytokines induced by Aβ in hippocampal neurons and microglial co-cultures.@*METHODS@#Hippocampal neurons and microglia obtained from SD rat were co-cultured. TGF-β1 was applied on day 5 after the neurons and microglia co-cultures were incubated at the concentrations of 5 or 20 ng/ml, Aβ was added 1 h following TGF-β1 application at a concentration of 5 μmol/L. They were incubated for 72 h and then assessed for further studies. Western blot analyses were employed to examine the expression of inducible nitric oxide synthase (iNOS); Real-time PCR and ELISA were used to detect the mRNA expression and secretion of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and insulin-like growth factor-1 (IGF-1).@*RESULTS@#In the hippocampal neuron-microglia co-cultures, Aβ induced upregulation of iNOS, TNF-α and IL-1β, downregulation of IGF-1. TGF-β1 pretreatment ameliorated the pro-inflammatory effects caused by Aβ.@*CONCLUSIONS@#TGF-β1 significantly inhibits the increase in inflammatory cytokines and the decrease in neurotrophic factor which are caused by Aβ-induced microglia activation.

Acetylcholine suppresses microglial inflammatory response in rats via acting on microglial α7nAChR / 生理学报

Microglia are the main immune cells in the central nervous system. In the present study, the mechanism for acetylcholine (ACh) inhibiting microglial inflammatory response was investigated. Primary culture of microglia was isolated from cerebral cortex of Sprague-Dawley (SD) rats. Lipopolysaccharide (LPS) was used to activate the microglia to induce inflammatory response, and then the microglia were treated with ACh for 24 h. Protein expressions of several inflammatory factors, insulin-like growth factor 1 (IGF-1) and α7 nicotinic acetylcholine receptor (α7nAChR) were detected by Western blot. Release of inflammatory factors and IGF-1 into media was detected by ELISA. After α7nAChR gene silence was achieved by lentivirus-transfection of α7nAChR-shRNA, the change of ACh effect was observed. The results showed that LPS induced microglial activation, up-regulated inducible nitric oxide synthase (iNOS) protein expression, increased the expressions and release of IL-1β and TNF-α, and decreased the expression and release of the neurotrophic factor, IGF-1. ACh could reverse these effects of LPS. Meanwhile, LPS reduced the protein expression of α7nAChR on the microglial cells, whereas ACh could reverse the effect. Silencing of α7nAChR gene in microglia abolished the ability of ACh to inhibit LPS-induced inflammatory responses. These results suggest that ACh exerts its protection against LPS-induced microglial inflammation via acting on α7nAChR on microglia, which may provide a novel target for the treatment of neuro-inflammatory diseases.

Roles of tyrosine hydroxylase expressed by CD4+ T cell subsets in collagen type II-induced arthritis / 中国应用生理学杂志

<p><b>OBJECTIVE</b>We used an animal model of collagen-induced arthritis (CIA) to study changes and roles of tyrosine hydroxylase (TH) expressed by CD4+ T cell subsets, and then explore the relationship between CD4+ T cell subset-derived catecholamines and inflammatory responses in CIA.</p><p><b>METHODS</b>Thirty-six male DBA/1 mice were randomly divided into control group, CIA model group (day 35) and CIA model group (day 55) (n = 12). CIA model was induced by type II collagen (CII) in DBA/1 mice. On the 35th and 55th day following primary immunization, the joints of the mice were observed for clinical score of swelling and the level of anti-CII IgG antibody in serum was examined. Expression of specific transcription factors and cytokines of Th1, Th17, Th2 and Treg cells and TH in mesenteric lymph nodes was measured by means of Western blot. The changes of TH expressed by CD4+ T cell subsets in mesenteric lymph nodes were determined by flow cytometry.</p><p><b>RESULTS</b>Clinical score and anti-CII antibody level increased in CIA compared with that in intact mice. Specific transcription factors and cytokines expressed by Th1 and Th17 cells were upregulated and cytokines expressed by Th2 and Treg cells were downregulated in mesenteric lymph nodes in CIA mice. Expression of TH was upregulated and the increased expression of TH in CD4+ T cells was attributed to Th1 and Th17 cells in mesenteric lymph nodes of CIA.</p><p><b>CONCLUSION</b>The increase in catecholamines from CD4+ T cell subsets in mesenteric lymph nodes of CIA may be related to inflammatory alleviation in CIA progression.</p>

A dysfunction of CD4+ T lymphocytes in peripheral immune system of Parkinson's disease model mice / 中国应用生理学杂志

<p><b>OBJECTIVE</b>Parkinson's disease (PD), a neurodegenerative disorder, has been reported to be associated with brain neuroinflammation in its pathogenesis. Herein, changes in peripheral immune system were determined to better understand PD pathogenesis and provide possible target for treatment of PD through improvement of immune disorder.</p><p><b>METHODS</b>1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) was intraperitoneally injected into mice to prepare PD model. Expression levels of pro-inflammatory and anti-inflammatory cytokines and transcription factors of CD4+ T lymphocyte subsets in spleen and mesenteric lymph nodes and concentrations of the cytokines in serum were examined on day 7 after MPTP injection. Percentages of CD4+ T lymphocyte subsets were measured by flow cytometry.</p><p><b>RESULTS</b>MPTP induced PD-like changes such as motor and behavioral deficits and nigrostriatal impairment. Expression levels of the pro-inflammatory cytokines including interferon (IFN)-γ, interleukin (IL)-2, IL-17 and IL-22, in spleen and mesenteric lymph nodes were upregulated and their concentrations in serum were elevated in PD progression. But, the concentrations of the anti-inflammatory cytokines including IL-4, IL-10 and transforming growth factor (TGF)-β were not altered in the two lymphoid tissues or serum of PD mice. In addition, expression of T-box in T cells (T-bet), the specific transcription factor of helper T (Th) 1 cells, was downregulated, but expression of transcription factor forkhead box p3 (Foxp3), the transcription factor of regulatory T (Treg) cells, was upregulated. In support of the results, the numbers of IFN-γ-producing CD4+ cells (Th1 cells) were reduced but CD4+CD25+ cells (Treg cells) were elevated in both the lymphoid tissues of PD mice.</p><p><b>CONCLUSION</b>PD has a dysfunction of peripheral immune system. It manifests enhancement of proinflammatory response and CD4+ T cell differentiation bias towards Treg cells away from Th1 cells.</p>

Changes in expression of tyrosine hydroxylase by CD4+ T lymphocytes in lymphoid tissues of DBA/1 mice with collagen-induced arthritis / 中国应用生理学杂志

<p><b>OBJECTIVE</b>To show the involvement of lymphocyte-derived catecholamines in the pathogenesis of rheumatoid arthritis (RA), we investigated the change in expression of tyrosine hydroxylase (TH), a rate-limiting enzyme of catecholamine synthesis, by CD4+ T lymphocytes in lymphoid tissues of DBA/1 mice with collagen-induced arthritis (CIA).</p><p><b>METHODS</b>CIA model was induced by chicken type II collagen in DBA/1 mice. The joints of the mice were observed for clinical score of swelling on and after the 22nd day of primary immunization. Pathological changes of ankles were examined by staining of tissue sections with hematoxylin and eosin on the 35th and 55th day following primary immunization. Immunofluorescent histochemistry was used to identify the number of TH-positive, CD4-positive, and double-labeled cells in the mesenteric lymph nodes and the spleen.</p><p><b>RESULTS</b>Paw-swelling onset was on days 29 - 32 after the first immunization in DBA/1 mice. Clinical score for swelling of the paws reached peak on day 46 after the first immunization. Compared with the ankles of intact or vehicle mice, the joints of CIA mice had these characteristics: increased inflammatory cells in the synovial tissues, proliferated synoviocytes in the multilayers, narrowed articular space, and destructed articular cartilages. Simultaneously, the number of TH-positive, CD4-positive, and double-labeled cells in the mesenteric lymph nodes and the spleen was significantly increased on days 35 and 55 following the first immunization. Between day 35 and day 55 post-immunization, there was no significant difference in the number of these positive cells.</p><p><b>CONCLUSION</b>CD4+ T lymphocytes up-regulate TH expression in the process of CIA and therefore, it is suggested that endogenous catecholamines of lymphocytes involve in the pathogenesis of RA.</p>

Inhibitory effect of interleukin-6 on NMDA-stimulated neuronal firing activity and possible mechanism involved in the effect / 中国应用生理学杂志

<p><b>OBJECTIVE</b>To study the effect and the possible mechanism of IL-6 on NMDA-excited neuronal discharges of rats in vitro.</p><p><b>METHODS</b>The cerebellar slices were prepared and spontaneous discharges of single cerebellar interposed nuclear (IN) neurons were recorded by extracellular recordings. The cerebellar slices were perfused with artificial cerebral spinal fluid (ACSF) containing N-methyl-D-aspartate (NMDA), IL-6, JAK inhibitor AG490. The changes in firing activities of the neurons treated with the drugs were recorded. The levels of phosphorylation at serine 897 site of NMDA receptor subunit 1 (NR1) in the neurons treated with various drugs mentioned above were detected by Western blot.</p><p><b>RESULTS</b>The discharge rates of the neurons that were treated with IL-6 together with NMDA were significantly lower than those of the neurons treated with NMDA alone. AG490 partially blocked the inhibitory effect of IL-6 on the NMDA-stimulated neuronal firing activity. The treatment of the neurons with IL6 and NMDA led to a concentration-dependent suppression of the phospho-NR1 expression relative to those neurons treated with NMDA alone. AG490 blocked the effect of the IL-6-induced depression of phospho-NR1 expression.</p><p><b>CONCLUSION</b>IL-6 inhibits NMDA-stimulated neuronal firing activity, and simultaneously down-regulates the phosphorylation of NR1 at serine 897 site.</p>

Effect of the endogenous catecholamines synthesized by lymphocytes on T cell proliferation / 中国应用生理学杂志

<p><b>AIM</b>To provide further evidence for the synthesis of catecholamines (CAs) in lymphocytes and to investigate the effect of the endogenous CAs synthesized by lymphocytes on function of the lymphocytes themselves and the receptor mechanisms involved in the effect.</p><p><b>METHODS</b>RT-PCR was performed to detect the expression of TH mRNA in the lymphocytes from the mesenteric lymph nodes of rats. Different concentrations of pargyline, an inhibitor of monoamine oxydase, and antagonists of alpha1-, alpha2-, beta1-, and beta2-adrenergic receptor (AR) were added to the lymphocyte cultures, and then proliferative response of the lymphocytes to mitogen concanavalin A (Con A) were measured via methyl-thiazole-tetrazolium (MTT) assay.</p><p><b>RESULTS</b>The lymphocytes could express TH mRNA, and the expression of TH mRNA was significantly higher in the Con A-activated lymphocytes than in the resting ones. The treatment of pargyline of 10(-6) and 10(-5) mol/L (not 10(-7) mol/L) notably attenuated Con A-induced lymphocyte proliferation. Beta2-AR antagonist ICI118551 (10(-7) and 10(-6) mol/L) completely blocked, but alpha1-AR antagonist corynanthine and alpha2-AR antagonist yohimbine (10(-7) and 10(-6) mol/L) partly blocked the suppressive effect of pargyline on the Con A-induced lymphocyte proliferation. Nevertheless, atenolol, an antagonist of beta1-AR, had no blocking effect on pargyline inhibition of lymphocyte proliferation.</p><p><b>CONCLUSION</b>Lymphocytes have the ability to synthesize CAs and the ability is enhanced in the activated lymphocytes. The endogenous CAs synthesized by lymphocytes can inhibit T cell proliferation and the inhibition of T cells by the CAs is mediated predominantly by beta2-AR on the lymphocytes.</p>

Effect of cerebellar interposed nuclei on lymphocyte function / 中国应用生理学杂志

<p><b>AIM</b>To increase the cognition of cerebellar functions and the knowledge of neuroimmunology, the effect of cerebellar interposed nuclei (IN), one of three deep nuclei in cerebellum, on lymphocyte function was investigated.</p><p><b>METHODS</b>Kainic acid (KA) was microinjected into bilateral IN for lesions of neuronal bodies in the IN. Control rats was microinjected with saline into their IN. On days 8, 16 and 32 following the IN lesions, the lymphocyte number in the peripheral blood was measured by blood corpuscle counter. Meanwhile, lymphocyte proliferation induced by concanavalin A (Con A), cytotoxicity of natural killer (NK) cells against YAC-1 cells, and anti-SRBC IgM antibody in the serum were examined respectively by methyl-thiazole-tetrazolium (MTT) assay, flow cytometry and ELISA assay.</p><p><b>RESULTS</b>The lymphocyte number in the peripheral blood was significantly reduced on days 8, 16 and 32 following the effective lesions of the bilateral IN in comparison with that of control. The Con A-induced lymphocyte proliferation, the NK cell cytotoxicity to YAC-1 cells, and the titer of anti-SRBC IgM antibody in the serum, were all significantly attenuated on days 8, 16 and 32 following the effective lesions of the bilateral IN in comparison with those of control. There were not remarkable differences between the days 8, 16 and 32 in the decreased lymphocyte number and functions induced by the lesions of the bilateral IN.</p><p><b>CONCLUSION</b>Effective lesions of the cerebellar bilateral IN of rats cause an inhibition in lymphocyte number and functions of T, B and NK cells, strongly showing that the cerebellar IN can modulate lymphocyte functions.</p>

Interleukin-6 protects cerebellar granule neurons from NMDA-induced neurotoxicity / 生理学报

Interleukin-6 (IL-6) is an important cytokine that participates in inflammation reaction and cell growth and differentiation in the immune and nervous systems. However, the neuroprotection of IL-6 against N-methyl-D-aspartate (NMDA)-induced neurotoxicity and the related underlying mechanisms are still not identified. In the present study, the cultured cerebellar granule neurons (CGNs) from postnatal (8-day) infant rats were chronically exposed to IL-6 for 8 d, and then NMDA (100 micromol/L) was applied to the cultured CGNs for 30 min. Methyl-thiazole-tetrazolium (MTT) assay, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) method and confocal laser scanning microscope (CLSM) were used to detect neuronal vitality, apoptosis and dynamic changes of intracellular Ca(2+) levels in the neurons, respectively. Anti-gp130 monoclonal antibody (75 ng/mL) was employed to the cultured CGNs with IL-6 to inhibit IL-6 activity so as to evaluate the role of gp130 (a 130 kDa glucoprotein transducing IL-6 signal) in mediating IL-6 neuroprotection. Western blot was used to measure the expressions of phospho-signal transducer and activator of transcription 3 (STAT3) and phospho-extracellular signal regulated kinase 1/2 (ERK1/2) in the cultured CGNs. The NMDA stimulation of the cultured CGNs without IL-6 pretreatment resulted in a significant reduction of the neuronal vitality, notable enhancement of the neuronal apoptosis and intracellular Ca(2+) overload in the neurons. The NMDA stimulation of the CGNs chronically pretreated with IL-6 caused a remarkable increase in the neuronal vitality, marked suppression of neuronal apoptosis and intracellular Ca(2+) overload in the neurons, compared with that in the control neurons without IL-6 pretreatment. Furthermore, anti-gp130 antibody blocked the inhibitory effect of IL-6 on NMDA-induced intracellular Ca(2+) overload in the neurons. The levels of phospho-STAT3 and phospho-ERK1/2 were significantly higher in IL-6-pretreated CGNs than those in IL-6-untreated neurons. The results suggest that chronic IL-6 pretreatment of CGNs protects the neurons against NMDA-induced neurotoxicity. The neuroprotective effect of IL-6 is closely related to its suppression of NMDA-induced intracellular Ca(2+) overload and is possibly mediated by gp130/JAK-STAT3 and gp130/RAS-ERK1/2 transduction pathways.

Immunoregulatory role of endogenous catecholamines synthesized by immune cells / 生理学报

It has been well known that catecholamines (CAs) in the body, including norepinephrine (NE), epinephrine (E) and dopamine (DA), are synthesized and secreted by neurons and endocrine cells and mainly modulate visceral activities such as cardiovascular, respiratory and digestive functions. The studies over the past nearly 30 years have shown that CAs can also regulate immune function. The immunomodulation of CAs is generally considered as a role mediating the regulation of nervous and endocrine systems. However, recent studies reveal that immune cells can also synthesize CAs, which is an update of traditional concept. A classical metabolic pathway of CAs shared by the nervous and endocrine systems is present in the immune cells, i.e., the immunocytes have the enzymes for synthesis of CAs [e.g. tyrosine hydroxylase (TH)] and the enzymes for degradation of CAs [e.g. monoamine oxidase (MAO) and catechol-O-methyl transferase (COMT)]. The endogenous CAs synthesized by immune cells can regulate many immune functions, including cellular proliferation, differentiation, apoptosis and cytokine production. These roles of the endogenous CAs may be mediated by an autocrine/paracrine pathway via relevant receptors on the immunocytes and intracellular cAMP. Intracellular oxidative mechanism may also be involved in immunoregulation of endogenous CAs in immune cells. In addition, some metabolic abnormalities of CAs in the immune cells probably induce some autoimmune diseases, such as multiple sclerosis (MS) and rheumatoid arthritis. These findings not only provide evidence for the new concept that the immune system is possible to become the third CA system other than the nervous and endocrine systems, but also extend our comprehension on functional significance of the endogenous CAs synthesized by immune cells.

Interleukin-6 prevents cultured cerebellar granule neurons from glutamate-induced neurotoxicity / 中国应用生理学杂志

<p><b>AIM</b>To explore IL-6 neuroprotection against glutamate-induced neurotoxicity and primary mechanisms involved in this neuroprotection.</p><p><b>METHODS</b>The cerebellar granule neurons from postnatal 8-day infant rats were chronically exposed to IL-6 for 8 days, and then glutamate stimulated the cultured cerebellar granule neurons for 15 min. Methyl-thiazole-tetrazolium (MTT) assay and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) method were used to observe the changes of neuronal vitality and apoptosis, respectively. Laser scanning confocal microscope (LSCM) and reverse transcription-polymerase chain reaction (RT-PCR) were respectively employed to measure dynamic changes of intracellular Ca2+ levels and expression of gp130 mRNA, a 130-kDa intracellular IL-6 signal-transduction protein, in the neurons.</p><p><b>RESULTS</b>The chronic IL-6 (2.5, 5 and 10 ng/ml) pretreatment of the cultured cerebellar granule neurons remarkably improved the decreased neuronal vitality by glutamate in a concentration-dependent manner. The neuronal apoptosis induced by glutamate was significantly attenuated by the chronic IL-6 pretreatment. The intracellular Ca2+ overload evoked by glutamate was also inhibited by the chronic IL-6 pretreatment. The expression of gp130 mRNA was dramatically lower in the IL-6-pretreated cerebellar granule neurons than in the IL-6-untreated neurons.</p><p><b>CONCLUSION</b>IL-6 can protect neurons against glutamate-induced exciting neurotoxicity. The mechanism of IL-6 neuroprotection may be closely related to the suppression of glutamate-induced intracellular Ca2+ overload and mediated by gp130 intracellular signal transduction pathways.</p>

Effect of cerebellar fastigial nucleus lesions on lymphocyte function / 中国应用生理学杂志

<p><b>AIM</b>To investigate effect of cerebellar fastigial nuclei (FN), one of three deep nuclei in cerebellum, on lymphocyte function, and possible central pathway involved in the effect.</p><p><b>METHODS</b>Kainic acid (KA) was microinjected into bilateral FN of rats. On the eighth day after the surgery, lymphocytes from the mesenteric lymph nodes were incubated to measure their proliferative reaction to concanavalin A (Con A) by means of colorimetric assay of methyl thiazole tetrazolium (MTT), and natural killer (NK) cells from the spleen were cultured to evaluate their cytotoxicity to YAC-1 cells with the aid of flow cytometric assay. Simultaneously, glutamate content in the hypothalamus was determined by high performance liquid chromatography (HPLC). As control, 0.9% saline was microinjected into the bilateral FN of rats. When these experiments ended, cerebellar sections and Nissl stain for each rat were made to observe the location and extent of the lesions. If the lesion areas were not in the bilateral FN or not limited in the FN, the results were discarded.</p><p><b>RESULTS</b>On day 8 following the KA injection of FN, the Nissl-stained cerebellar sections showed the neuronal bodies in the FN were effectively damaged by KA. Simultaneously, the lymphocyte proliferation induced by Con A was significantly increased and the NK cell cytotoxicity to YAC-1 target cells was remarkably enhanced when compared with those of the control animals microinjected with saline in their bilateral FN. At the same time as these changes of lymphocyte functions occurred, glutamate content in the hypothalamus was markedly reduced relative to that in the control hypothalamus.</p><p><b>CONCLUSION</b>Effective lesions of cerebellar bilateral FN of rats can cause an enhancement of lymphocyte functions, including increase of proliferation of T cells and cytotoxicity of NK cells. The cerebello hypothalamic glutamatergic projections may be involved in the pathway of cerebellar FN immunomodulation.</p>

Effect of acetylcholine on the cytotoxicity of natural killer cells / 中国应用生理学杂志

<p><b>AIM</b>To investigate the effect of acetylcholine (ACh) on the cytotoxicity of natural killer (NK) cells and to explore the receptor mechanisms involved in the effect.</p><p><b>METHODS</b>The effector cells (i. e. NK cells) from the spleens of rats were collected and cultured with the target cells (Yac-1 cells). The various concentrations of ACh, cholinergic receptor agonists or antagonists were added to the cultures, respectively according to distinct experimental purposes. Lactate dehydrogenase (LDH) release assay was used to evaluate NK cell cytotoxicity.</p><p><b>RESULTS</b>NK-cell-mediated lysis of Yac-1 lymphoma cells was reduced by 10(-10) - 10(-6) mol/L ACh. The inhibitory effect of ACh on NK cell cytotoxicity was mimicked by pilocarpine, an agonist of muscarinic receptor, and by nicotine, an agonist of nicotinic receptor, at all applied concentrations (10(-10) - 10(-6) mol/L). Muscarinic receptor antagonist atropine blocked the inhibitory effect of ACh on the cytotoxicity of NK cells. Nevertheless, tubocurarine, an antagonist of nicotinic receptor, had no blocking effect on the suppression of NK cell cytotoxicity by ACh.</p><p><b>CONCLUSION</b>ACh results in an inhibition of the cytotoxicity of NK cells, and this inhibition is realized mainly through M and N1 cholinergic receptor.</p>

Inhibitory effect of triptolide on proliferation of PC12 cells / 中国应用生理学杂志

<p><b>AIM</b>To investigate the effect of triptolide on proliferation of PC12 cells and the mechanism involved in the effect, and provide evidence for clinical use of triptolide in treatment of tumor.</p><p><b>METHODS</b>By means of morphological observation, MTT assay, flow cytometry (FCM) and RT-PCR, the effect of triptolide on the proliferation of PC12 cells was observed in vitro.</p><p><b>RESULTS</b>The proliferation inhibition was found on PC12 cells incubated with triptolide (5 x 10(3) or 25 x 10(3) g/L) for 24 h, 48 h and 72 h, and with the higher concentration of triptolide, the inhibition was stronger. Low concentration of triptolide (1 x 10(3) g/L) showed no significant effect on proliferation of PC12 cells. After treatment of PC12 cells with triptolide (5 x 10(3) g/L) for 24 h, increased percentage of G0-G1 phase and decreased percentage of S phase were found. Expression of translational elongation factor 2A3-2 was reduced after treatment of PC12 cells with triptolide (5 x 10(3) g/L). The expression of 2A3-2 was weaker in PC12 cells treated with triptolide for 48 h than for 24 h.</p><p><b>CONCLUSION</b>Triptolide inhibits the proliferation of PC12 cells. The inhibition may be realized by changing the expression of 2A3-2 and preventing the transition from G0-G1 phase to S phase.</p>