Ramulus Cinnamomi extract attenuates neuroinflammatory responses via downregulating TLR4/My D88 signaling pathway in BV2 cells

Ramulus Cinnamomi(RC), a traditional Chinese herb, has been used to attenuate inflammatory responses. The purpose of this study was to investigate the effect of RC extract on lipopolysaccharide(LPS)-induced neuroinflammation in BV2 microglial cells and the underlying mechanisms involved. BV2 cells were incubated with normal medium(control group), LPS, LPS plus 30 μg/m L RC extract, or LPS plus 100 μg/m L RC extract. The BV2 cell morphology was observed under an optical microscope and cell viability was detected by MTT assay. Nitric oxide level in BV2 cells was detected using Griess regents, and the levels of interleukin-6, interleukin-1β, and tumor necrosis factor α in BV2 cells were determined by ELISA. The expression levels of cyclooxygenase-2, Toll-like receptor 4 and myeloid differentiation factor 88 proteins were detected by western blot assay. Compared with the LPS group, both 30 and 100 μg/mL RC extract had no significant effect on the viability of BV2 cells. The levels of nitric oxide, interleukin-6, interleukin-1β and tumor necrosis factor α in BV2 cells were all significantly increased after LPS induction, and the levels were significantly reversed after treatment with 30 and 100 μg/mL RC extract. Furthermore, RC extract significantly inhibited the protein expression levels of cyclooxygenase-2, Toll-like receptor 4 and myeloid differentiation factor 88 in LPS-induced BV2 cells. Our findings suggest that RC extract alleviates neuroinflammation by downregulating the TLR4/My D88 signaling pathway.     

Pathway for interferon-gamma to promote the differentiation of cholinergic neurons in rat embryonic basal forebrain/septal nuclei

BACKGROUND: The supernatant of interferon-gamma (IFNγ) co-cultured with neonatal rat cortical glia can promote the cells in embryonic basal forebrain/septal nuclei to differentiate into cholinergic neurons, but the mechanism is still unclear. OBJECTIVE: To analyze the pathways for IFNγ to promote the differentiation of primarily cultured cholinergic neurons in rat embryonic basal forebrain/septal nuclei through culture in different conditioned medium. DESIGN: A controlled experiment taking cells as the observational target. SETTINGS: Department of Biochemistry and Molecular Biology, Youjiang Medical College for Nationalities; Department of Cell Biology, Beijing University Health Science Center. MATERIALS: Sixty-four pregnant Wistar rats for 16 days (250–350 g) and 84 Wistar rats (either male or female, 5–7 g) of 0–1 day after birth were provided by the experimental animal department of Beijing University Health Science Center. Rat IFNγ were provided by Gibco Company; Glial fibrillary acidic protein by Huamei Company. METHODS: The experiments were carried out in the Department of Cell Biology, Beijing University Health Science Center and Daheng Image Company of Chinese Academy of Science from July 1995 to December 2002. ① Interventions: The nerve cells in the basal forebrain/septal nuclei of the pregnant Wistar rats for 16 days were primarily cultured, and then divided into four groups: Blank control group (not any supernatant and medium was added); Control group (added by mixed glial cell or astrocyte conditioned medium); IFNγ group (added by mixed glial cell or astrocyte conditioned medium+IFNγ). Antibody group (added by mixed glial cell or astrocyte conditioned medium+IFNγ+Ab-IFNγ). Mixed glial cell or astrocyte conditioned medium was prepared using cerebral cortex of Wistar rats of 0–1 day after birth. ② Evaluation: The immunohistochemical method was used to perform the choline acetyltransferase (ChAT) staining of cholinergic neurons. The ChAT positive cells were counted. MAIN OUTCOME MEASURES: Comparison of ChAT positive cells in rat basal forebrain and septal nuclei in different conditioned medium. RESULTS: ① ChAT positive cells in mixed glial cell conditioned medium: The ChAT positive cells in the IFNγ group and antibody group were significantly more than those in the control group (P < 0.01). ② ChAT positive cells in astrocyte conditioned medium: The ChAT positive cells in the IFNγ group were significantly more than those in the control group, but there was no significant difference between the antibody group and control group (P > 0.05). CONCLUSION: IFNγ cannot directly promote the differentiation of cholinergic neurons, but plays a role through activating glial cells (except astrocytes) to produce IFNγ like molecules.     

塞来昔布防护DA能神经元损伤实验研究

目的 观察非甾体类抗炎药物-塞来昔布对DA能(DA)神经元损伤的保护作用.方法 取新生SD大鼠进行中脑神经细胞的原代培养.采用脂多糖(LPS)制作体外PD炎症模型.实验分为对照组、LPS组、塞来昔布(CB)组.倒置显微镜下观察细胞形态变化,第12小时、第24小时测定中脑细胞培养液上清TNF-α浓度及进行酪氨酸羟化酶(TH)免疫细胞化学检测.结果 倒置显微镜下见,12h时LPS组细胞体积略有肿胀,折光性增强,突起变细、缩短,胶质细胞活化.24h时细胞数目明显减少,胞体皱缩,突起明显变细、缩短,部分细胞甚至崩解.对照组、CB组细胞形态变化不明显.12h LPS、CB组中脑神经细胞培养液上清TNF-α浓度与对照组相比均显著升高(P<0.01).组间比较,LPS组TNF-α浓度比CB组明显升高(P<0.01).24h时实验两组TNF-α浓度与12h时相比均显著增高(P<0.01).对照组TH阳性细胞数较多.LPS组部分神经元衰亡,12h TH阳性细胞数与对照组相比显著减少,24h减少更明显(P<0.01).CB组TH阳性细胞数与对照组相比亦显著减少(P<0.01).组闻比较,LPS组TH阳性细胞数与CB组相比明显减少.结论 塞来昔布可减轻LPS对体外培养DA能神经元的炎性损伤作用.

Abstract：

Objective To observe the neuroprotective effects of celecoxib against degeneration of dopaminergic neurons. Methods The primary mesencephalic neural cell deriving from SD newborn rat were cultured. Lipopolysaccharide (LPS) were used to establish inflammator reaction model of PD in vitro. Cultured cells were divided into control group,LPS and CB groups. The concentration of tumor necrosis factor-α ( TNF-α) in the culture medium were determined by using ELISA method, and tyrosine hydroxylase immunohistochmisry were performed after cutured for 12 hours and 24 hours. Results The concentration of TNF-α in control group was remarkably lower than in experiment groups at the same time point. ( P < 0. 01) . The concentration of TNF-a in LPS group was highest, respectively: in CB group. The amount of TH-positive cells in control group were more than in experimental groups (P <0. 01). The amount of TH-positive cells in LPS group were lowerest, respectively: in CB group. Conclusion Celecoxib have neuroprotective effect through lessening the degeneration of dopaminergic neurons caused by LPS in vitro.     

Lipopolysaccharide-activated microglial-induced neuroglial cell differentiation in bone marrow mesenchymal stem cells☆

BACKGROUND： Microglia are very sensitive to environmental changes, often becoming activated by pathological conditions. Activated microglia can exert a dual role in injury and repair in various diseases of the central nervous system, including cerebral ischemia, Parkinson＇s disease, and Alzheimer＇s disease. OBJECTIVE： An immortal microglial cell line, BV2, was treated with varying concentrations of lipopolysaccharide （LPS） to induce a pathological situation. Supernatant was harvested and incubated with bone marrow mesenchymal stem cells and, concomitantly, bone marrow mesenchymal stem cell differentiation was observed. DESIGN： A controlled observation, in vitro experiment. SETTING： Department of Neurology, First Affiliated Hospital of China Medical University. MATERIALS： Five male 2-3-week-old Sprague Dawley rats were purchased from Animal Laboratory Center of China Medical University and included in this study. The protocol was performed in accordance with ethical guidelines for the use and care of animals. The microglial cell line BV2 was produced by Cell Research Institute of Chinese Academy of Sciences. LPS was produced by Sigma Company, USA. METHODS： This study was performed in the Central Laboratory of China Medical University from September 2006 to March 2007. Rat femoral and tibial bone marrow was collected for separation and primary culture of bone marrow mesenchymal stem cells. Bone marrow mesenchymal stem cell cultures were divided into 5 groups： control group, non-activated group, as well as low-, medium-, and high-dose LPS groups. In the control group, bone marrow mesenchymal stem cells were cultured with Dulbecco＇s modified Eagle＇s medium （DMEM） supplemented with fetal bovine serum （volume fraction 0. 1）. In the non-activated group, bone marrow mesenchymal stem cells were incubated with non-activated BV2 supernatant. In the low-, medium-, and high-dose LPS groups, bone marrow mesenchymal stem cells were incubated with LPS （0.01,0.1 and 1 μg/L, respectively）-act     

Effects of increased human tumor necrosis factor-like molecule 1A expression in peripheral blood of children with acute Guillain-Barre syndrome on interferon-gamma secretion

BACKGROUND： Human tumor necrosis factor-like molecule 1A （hTL1A） is a strong T helper cell type 1 （Thl） co-stimulator. Guillain-Barre syndrome （GBS） is an autoimmune disorder of the nervous system, which is mediated by Thl cells. OBJECTIVE： To determine hTL1A expression in peripheral blood T lymphocytes of acute GBS children and the effects of hTL1A on secretion of interferon-γ. DESIGN, TIME AND SETTING： A randomized, controlled, neuroimmunological in vitro study was performed at the Central Laboratory of First Hospital of Jilin University, China from November 2005 to November 2007. MATERIALS： Venous blood samples were obtained from 6 healthy donors, aged 6-12 years （all routine blood examination items were normal）, and 6 additional children with acute GBS, aged 6-12 years. The GBS children fell ill within 1 week and were not treated with hormones or immunoglobulin Purified recombinant human soluble tumor necrosis factor-like molecule 1A （rhsTL1A, 1 mg/mL, relative molecular mass 22 000, 6× His tag, soluble form） was supplied by the Central Laboratory of First Hospital of Jilin University, China. METHODS： Peripheral blood mononuclear cells were isolated from healthy donors using the standard Ficoll gradient centrifugation and were incubated in 96-well culture plates. The cells were assigned to the following groups： control （2 μg/mL phytohemagglutinin）, 2μg/mL phytohemagglutinin ＋ 25, 100 and 400 ng/mL rhsTL1A. T cell proliferation was quantified using the tritiated thymidine （3H-TdR） method. Serum interferon-γ levels in acute GBS children were detected by enzyme-linked immunosorbent assay （ELISA）. The ratio of hTL1A-positive T cells to CD3-positive T cells in peripheral blood of acute GBS children was determined using flow cytometry. Following in vitro pre-activation of peripheral blood mononuclear cells by 2 μg/mL phytohemagglutinin, the peripheral blood mononuclear cells were treated with 400 ng/mL exogenous rhsTLIA. Finally, peripheral blood mononuclear cell-secreted interferon-γlevels were measured by ELISA. MAIN OUTCOME MEASURES： The following parameters were measured： rhsTLIA stimulation index to stimulate proliferation of T cells; the serum interferon-γ levels in acute GBS children; the ratio of hTL1A-positive cells to CD3-positive cells; the levels of interferon-γ secreted by peripheral blood mononuclear cells in acute GBS children, as well as rhsTL1A-stimulated interferon-γ levels. RESULTS： T cell proliferation assay revealed that the stimulation index in each rhsTL1A group was greater than the control group. The stimulation index of the 400 ng/mL rhsTL1A group was the greatest. Serum interferon-γ levels in acute GBS children were significantly greater than the control group （P 〈 0.05）. The ratio of hTLIA＋ CD3＋ T cells to CD3＋ T cells in acute GBS children was significantly greater than the control group （P 〈 0.01 ）. Phytohemagglutinin stimulated peripheral blood mononuclear cells to a greater extent than 400 ng/mL rhsTL1A in the acute GBS group, and the secreted interferon-γ levels were significantly increased （P 〈 0.05）. CONCLUSION： In T cells pre-activated with 2 μg/mL phytohemagglutinin, proliferation was effectively increased with 400 ng/mL rhsTL1A treatment. Expression of hTLIA was increased in activated T cells from peripheral blood of acute GBS children, followed by increased interferon-γ secretion. These mechanisms are considered to be part of the pathological process that induces the secretion of inflammatory cytokines in GBS syndrome.     

Effect of curcumin on nitric oxide synthase expression in Iipopolysaccharide-activated microglia cells and the anti-oxidative effect of curcumin

BACKGROUND: It has been demonstrated that curcumin can increase the activities of various anti-oxidase in blood and tissue, effectively eliminate various free radicals, reduce the production of peroxisome, and alleviate oxidative stress reaction. Whether it has the same effect on microglia? OBJECTIVE: To observe the effects of curcumin on the expressions of inducible nitric oxide synthase (iNOS), nuclear factor-κB (NF-κB), and superoxide dismutase (SOD) in microglial cell line BV stimulated by lipopolysaccharide (LPS). DESIGN: An observational comparative study. SETTING: Research Room of Biochemistry, Medical College of Nantong University. MATERIALS: Mice microglia cell line BV, iNOS and NF-κB reporter gene plasmids were presented by Dr. Bhat.NR. from the Medical University of South Carolina (USA). Curcumin was produced by the Xi'an Branch of China Chengdu Scholar Bio-Tech. Co.,Ltd.; LPS (E.Coli O26:B6), anti-mice iNOS monoclonal antibody, horseradish peroxidase labeled goat-anti-mice IgG were the products of Sigma Company (USA). METHODS: The experiments were carried out in the Research Room of Biochemistry, Medical College of Nantong University from May 2006 to April 2007. ① Detection of iNOS: The cells were seeded onto 24-well plate at the density of 1×105, After the cells had adhered to the cover glasses, the cells were grouped as negative control group (the primary antibody was replaced by phosphate buffered solution PBS); normal control group (the cells were normally cultured); LPS-treated group (the cells were treated with LPS for 24 hours); curcumin+LPS group (the cells were treated with curcumin for 1 hour and LPS for 24 hours). The expressions of iNOS protein were detected with immunocytochemical staining. ② Determination of iNOS and NF-κB gene activities: According to the introduction of the kit for transfection, iNOS or NF-κB report gene plasmids were transiently transfected with LipofectamineTM2000 liposomes into the cells in the 24-well plate for 24 hours. The cells were divided into normal control group (the cells were normally cultured after transfected with report gene plasmids); blank plasmid group (the cells were normally cultured after transfected with blank plasmids); LPS-treated group (the cells were treated with LPS for 4 hours after transfected with report gene plasmids); curcumin+LPS group (the cells were treated with curcumin for 1 hour and LPS for 24 hours after transfected with report gene plasmids). The content of luciferase in the cell lysis buffer was determined after cell lysis. ③ Determination of SOD activity: The cells were seeded into culture bottle at the density of 1×106, and the divided into four groups, including normal control group (the cells were normally cultured); LPS-treated group (the cells were treated with LPS for 24 hours); curcumin+LPS group (the cells were treated with curcumin for 1 hour and LPS for 24 hours); vitamin C+LPS group (the cells were treated with vitamin C for 1 hour and LPS for 24 hours). The SOD activity was determined with xanthine oxidase and quantitative colorimetric assay. MAIN OUTCOME MEASURES: The expressions of iNOS protein, iNOS and NF-κB, and the activity of SOD were observed. RESULTS: ① Expression of iNOS protein in microglia: The expression of iNOS protein in the LPS-treated group was obviously higher than that in the negative control group (P < 0.01); Those in the curcumin+LPS group were significantly decreased as compared with that in the LPS-treated group (P < 0.01). ② Expressions of iNOS and NF-κB genes: The expressions of iNOS and NF-κB genes in the LPS-treated group were significantly higher than those in the normal control group (P < 0.01); Those in the curcumin+LPS group were significantly lower than those in the LPS-treated group (P < 0.01). ③ SOD activity: The activity of SOD in the LPS-treated group was significantly lower than those in the normal control group (P < 0.01). It in the curcumin+LPS group and vitamin C +LPS group was significantly higher than that in the LPS-treated group (P < 0.01). CONCLUSION: Curcumin could inhibit the expression of iNOS in the activated microglia, and it also has the abilities in eliminating free radicals and antagonizing lipid peroxidation.     

口服免疫耐受治疗颅脑创伤的实验研究

目的 探讨口服免疫耐受对脑损伤的治疗作用.方法 58只兔随机分为对照组、脑脊液引流组、脑脊液引流+口服组和免疫佐剂组.收集第1、3、7、14、21天血清,以ELISA法检测IL-1、IFN-γ、IL-10、TGF-β和ABAb浓度,并进行对比.结果 脯脊液引流组IL-1、IFN-γ、ABAb较对照组降低,IL-10较对照组升高;脑脊液引流+口服纰IL-1、IFN-γ、ABAb较其他组明显降低,而IL-10、TGF-β明显升高;免疫佐剂组介于对照组和脑脊液引流+口服组之间.结论 脑脊液引流对颅腩创伤有治疗作用;口服免疫耐受对颅脑创伤有治疗作用;完全弗氏佐剂部分抑制了口服免疫耐受.

Abstract：

Objective To explore the cerebral protective effects of oral tolerance therapy in rabbit TBI-models established by lateral fluid percussion machine.Method 58 rabbits were divided into four groups randomly( 18 in control group, 15 in CSF drained group, 10 in CSF drained + feed group and 15 in CSF drained + feed + immunologic adjuvant group).Rabbit's CSF were drained and injected into cistern in control group; were drained only in CSF drained group;were drained and feed by mouth in drained + feed group;were drained and injected with 50 μg complete Freud's adjuvant under the skin of posterior foot and rabbits in drained + feed + immunologic adjuvant group.1 ml CSF was drained everyday until a week and phlebotomized and tested with ELISA to observe the changes of IL-1 ,IFN-γ,IL-10,TGF-β and ABAb at the time points of 1 d,3 d,7 d,14 d,21 d in each groups.Results Levels of IL-1,IFN-γ and ABAb in CSF drained group were lower and levels of IL-10 were higher than that in control group;levels of IL-1 ,IFN-γ, ABAb in CSF drained + feed group were lower and levels of IL-10,TGF-β were higher than that in any other groups significantly; levels of IL-1 , IFN-γ, ABAb in CSF drained + feed + immunologic adjuvant were lower than that in control group,while they were higher than that in CSF drained + feed group;levels of IL-10 and TGF-β in CSF drained + feed + immunologic adjuvant group were higher than that in control group,while they were lower than that in CSF drained +feed group.Statistic differences among groups were significant P < 0.05 ).The mortality rate and disability rate of CSF drained + feed group were the lowest, while the cure rate was the highest among four groups.The control group came to be the worst outcome.And the other two groups were in the middle.Conclusions CSF drainage can help to cure traumatic brain injury.CSF drainage and feed by mouth can result in oral tolerance and lighten the injury,so oral tolerance is a useful therapy to treat traumatic brain injury.     

JAK/STAT信号转导通路在IGF-1抑制左旋多巴诱导的多巴胺能神经元凋亡中的作用

Objective To explore the protective effect of insulin-like growth factor 1 (IGF-1) on apoptosis of dopaminergic neurons induced by L-dopa via JAK/STAT signaling pathway. Methods PC12 cells were induced to differentiate into dopaminergic neurons with 100 μg/L β-NGF; MTT assay was employed to identify the changes in the viability of PC12 cells following L-dopa treatment at 0, 10,20, 50, 100, 150 and 200 μmol/L, and the different concentrations of IGF-1 at 0, 10, 25, 50 and 100 nmol/L with the same concentration of L-dopa (150 μmol/L); Western blotting was used to detect the levels of P-JAK2/P-STAT3 in PC12 cells treated with PBS (controls), L-dopa, L-dopa+IGF-1 and L-dopa+IGF-1+AG490 for 24 h, and then the apoptosis rate was assessed by flow cytometry and Hchest33258 staining. Results Western blotting showed that the expressions of P-JAK2 and P-STAT3 were detected in the L-dopa+IGF-1 and L-dopa+IGF-1+AG490 treatment groups but not in the control group or L-dopa treatment group; the expression of P-STAT3 in the L-dopa+IGF-1+AG490 treatment group was obviously lower than that in the L-dopa+IGF-1 treatment group (P<0.05). Hchest33258 staining indicated that L-dopa treatment group had the most obvious karyopyknosis and karyorrhexis,much more apoptotic bodies than the L-dopa+IGF-1 and L-dopa+IGF-1+AG490 treatment groups. Flow cytometry showed that the apoptosis rate was significantly different among the 4 groups (F=180.991,P=0.000): as compared with the control group, the other 3 groups had a higher apoptosis rate (P<0.05);L-dopa treatment group (38.13 ±2.54 %) enjoyed the highest level, followed by L-dopa+IGF-1 +AG490treatment group (25.60±1.30 %) and L-dopa+IGF-1 treatment group (20.17±1.54 %). Conclusion L-dopa has toxic effect on PC12 cells; IGF-1 could protect the PC12 cells from the neurotoxic effect of L-dopa and JAK2/STAT3 signaling pathway is activated in this procedure.     

JAK/STAT信号转导通路在IGF-1抑制左旋多巴诱导的多巴胺能神经元凋亡中的作用

Objective To explore the protective effect of insulin-like growth factor 1 (IGF-1) on apoptosis of dopaminergic neurons induced by L-dopa via JAK/STAT signaling pathway. Methods PC12 cells were induced to differentiate into dopaminergic neurons with 100 μg/L β-NGF; MTT assay was employed to identify the changes in the viability of PC12 cells following L-dopa treatment at 0, 10,20, 50, 100, 150 and 200 μmol/L, and the different concentrations of IGF-1 at 0, 10, 25, 50 and 100 nmol/L with the same concentration of L-dopa (150 μmol/L); Western blotting was used to detect the levels of P-JAK2/P-STAT3 in PC12 cells treated with PBS (controls), L-dopa, L-dopa+IGF-1 and L-dopa+IGF-1+AG490 for 24 h, and then the apoptosis rate was assessed by flow cytometry and Hchest33258 staining. Results Western blotting showed that the expressions of P-JAK2 and P-STAT3 were detected in the L-dopa+IGF-1 and L-dopa+IGF-1+AG490 treatment groups but not in the control group or L-dopa treatment group; the expression of P-STAT3 in the L-dopa+IGF-1+AG490 treatment group was obviously lower than that in the L-dopa+IGF-1 treatment group (P<0.05). Hchest33258 staining indicated that L-dopa treatment group had the most obvious karyopyknosis and karyorrhexis,much more apoptotic bodies than the L-dopa+IGF-1 and L-dopa+IGF-1+AG490 treatment groups. Flow cytometry showed that the apoptosis rate was significantly different among the 4 groups (F=180.991,P=0.000): as compared with the control group, the other 3 groups had a higher apoptosis rate (P<0.05);L-dopa treatment group (38.13 ±2.54 %) enjoyed the highest level, followed by L-dopa+IGF-1 +AG490treatment group (25.60±1.30 %) and L-dopa+IGF-1 treatment group (20.17±1.54 %). Conclusion L-dopa has toxic effect on PC12 cells; IGF-1 could protect the PC12 cells from the neurotoxic effect of L-dopa and JAK2/STAT3 signaling pathway is activated in this procedure.     

Transcriptomics: mRNA and alternative splicing

Spinal glial activation has been implicated in sustained morphine-mediated paradoxical pain sensitization. Since activation of glial CB2 cannabinoid receptors attenuates spinal glial activation in neuropathies, we hypothesized that CB2 agonists may also attenuate sustained morphine-mediated spinal glial activation and pain sensitization. Our data indicate that co-administration of a CB2-selective agonist (AM 1241) attenuates morphine (intraperitoneal; twice daily; 6 days)-mediated thermal hyperalgesia and tactile allodynia in rats. A CB2 (AM 630) but not a CB1 (AM 251) antagonist mitigated this effect. AM 1241 co-treatment also attenuated spinal astrocyte and microglial marker and pro-inflammatory mediator (IL-1β, TNFα) immunoreactivities in morphine-treated rats, suggesting that CB2 agonists may be useful to prevent the neuroinflammatory consequences of sustained morphine treatment.     

Activation of the cannabinoid 2 (CB2) receptor inhibits murine mesenteric afferent nerve activity

Abstract Cannabinoid 2 (CB2) receptors have both antinociceptive and antihypersensitivity effects, although the precise mechanisms of action are still unclear. In this study, the modulatory role of CB2 receptors on the mesenteric afferent response to the endogenous immunogenic agent bradykinin (BK) was investigated. Mesenteric afferent recordings were obtained from anaesthetized wild-type and CB2(-/-) mice using conventional extracellular recording techniques. Control responses to BK were obtained in all experiments prior to administration of either CB2 receptor agonist AM1241, or AM1241 plus the CB2 receptor antagonist AM630. Bradykinin consistently evoked activation of mesenteric afferents (n = 32). AM1241 inhibited the BK response in a dose dependent manner. In the presence of AM630 (10 mg kg(-1)), however, AM1241 (10 mg kg(-)1) had no significant effect on the BK response. Moreover, AM1241 had also no significant effect on the BK response in CB2(-/-) mice. Activation of the CB2 receptor inhibits the BK response in mesenteric afferents, demonstrating that the CB2 receptor is an important regulator of neuroimmune function. This may be a mechanism of action for the antinociceptive and antihypersensitive effects of CB2 receptor agonists.     

Cannabinoids ablate release of TNFalpha in rat microglial cells stimulated with lypopolysaccharide

Upon activation, brain microglial cells release proinflammatory mediators, such as TNFalpha, which may play an important role in eliciting neuroinflammatory processes causing brain damage. As cannabinoids have been reported to exert anti-inflammatory and neuroprotective actions in the brain, we here examined the effect of both synthetic and endogenous cannabinoids on TNFalpha release elicited by bacterial endotoxin lypopolysaccharide (LPS) in cultured microglia. Exposure of primary cultures of rat cortical microglial cells to LPS significantly stimulated TNFalpha mRNA expression and release. The endogenous cannabinoids anandamide and 2-arachidonylglycerol (2-AG), as well as the synthetic cannabinoids (+)WIN 55,212-2, CP 55,940, and HU210, inhibited in a concentration-dependent manner (1-10 microM) the LPS-induced TNFalpha release. Unlike the high-affinity cannabinoid receptor agonist (+)WIN 55,212-2, the low-affinity stereoisomer (-)WIN 55,212-2 did not exert any significant inhibition on TNFalpha release. Given this stereoselectivity, the ability of (+)WIN 55,212-2 to inhibit LPS-induced TNFalpha release from microglia is most likely receptor-mediated. By RT-PCR we found that the two G(i/o) protein-coupled cannabinoid receptors (type 1 and 2) are both expressed in microglial cultures. However, selective antagonists of type 1 (SR141716A and AM251) and type 2 (SR144528) cannabinoid receptors did not affect the effect of (+)WIN 55,212-2. Consistent with this finding is the observation that the ablative effect of (+)WIN 55,212-2 on LPS-evoked release of TNFalpha was not sensitive to the G(i/o) protein inactivator pertussis toxin. In addition, the cAMP elevating agents dibutyryl cAMP and forskolin both abolished LPS-induced TNFalpha release, thus rendering unlikely the possibility that (+)WIN 55,212-2 could ablate TNFalpha release through the inhibition of adenylate cyclase via the G(i)-coupled cannabinoid receptors type 1 and 2. In summary, our data indicate that both synthetic and endogenous cannabinoids inhibit LPS-induced release of TNFalpha from microglial cells. By showing that such effect does not appear to be mediated by either CB receptor type 1 or 2, we provide evidence suggestive of the existence of yet unidentified cannabinoid receptor(s) in brain microglia.     

Cannabinoids inhibit LPS-inducible cytokine mRNA expression in rat microglial cells

The effect of cannabinoids on the induction of cytokine mRNA by rat microglial cells was examined. Exposure of neonatal rat cortical microglial cells to the exogenous cannabinoid delta(9)-tetrahydrocannabinol (THC) resulted in reduced amounts of lipopolysaccharide (LPS)-induced mRNAs for IL-1alpha, IL-1beta, IL-6, and TNF-alpha. Of these cytokine mRNAs, the response of that for IL-6 was exquisitely sensitive to THC. Similarly, exposure of microglial cells to the putative endogenous cannabinoid anandamide before LPS treatment resulted in a decrease in cytokine mRNA levels, but not to the same extent as that caused by THC; however, when methanandamide, the non-hydrolyzable analog of anandamide was tested, its ability to inhibit cytokine mRNA expression was comparable to that of THC. Exposure of microglial cells to either of the paired enantiomers CP55,940 or CP56,667 resulted in similar inhibition of LPS-induced cytokine mRNA expression. A comparable inhibitory outcome was obtained when the paired enantiomers levonantradol and dextronantradol were employed. Neither the CB(1)-selective antagonist SR141716A nor the CB(2)-selective antagonist SR144528 was able to reverse the inhibition of cytokine mRNA expression by levonantradol. The CB(2) antagonist, however, when administered alone augmented the production of cytokine mRNAs. Collectively, these studies demonstrate that cannabinoids can modulate levels of cytokine mRNA in rat microglial cells; however, the inhibition of cytokine mRNA expression is apparently not mediated through either the CB(1) or CB(2) cannabinoid receptors.     

IFN-γ和热干预对U251胶质瘤细胞的MHC-Ⅰ类分子和HSP70表达的影响

目的观察重组人干扰素-γ（IFN-γ）和热干预对人多形胶质母细胞瘤（GBM）U251细胞胞膜主要组织相容性抗原复合体-Ⅰ（MHC-Ⅰ）类分子和热休克蛋白70（HSP70）分子表达的影响。方法将U251细胞分为3个干预组：IFN干预组（IFN-γ500U/ml诱导48h）,热干预组（43℃热休克2h）,联合干预组（IFN-γ500U/ml诱导48h＋43℃热休克2h）,分别行相应诱导干预。流式细胞仪（FCM）检测不同干预因素处理后的U251细胞胞膜MHC-Ⅰ类分子和HSP70表达情况。结果IFN干预组、热干预组、联合干预组U251细胞胞膜MHC-Ⅰ及HSP70表达率分别为（89.92±3.45）%和（5.11±1.89）%、（78.89±2.02）%和（42.18±3.85）%、（96.00±2.63）%和（53.61±4.24）%。结论双因素（IFN-γ500U/ml诱导48h＋43℃热休克2h）联合干预是U251细胞胞膜MHC-Ⅰ类分子和HSP70双高表达的最佳体外诱导方案。     

Locomotor activity and occupancy of brain cannabinoid CB1 receptors by the antagonist/inverse agonist AM281

The goals of this study were to examine the relationship between intravenous doses of the cannabinoid CB1 receptor antagonist AM281 (N-(morpholin-4-yl)-5-(4-iodophenyl)-1-(2, 4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide) and the degree of occupancy of this receptor, and to relate occupancy to the ability of this compound to antagonize the sedative effects of the cannabinoid receptor agonist WIN 55,212-2. Occupancy was determined by measuring the ability of intravenous doses of AM281 to inhibit in vivo binding of [(131)I]AM281 in brain areas, and locomotor activity was assessed by measuring the rate of beam crossings in a photocell apparatus. As previously documented, WIN 55,212-2 (1 mg/kg, i.v.) significantly reduced locomotor activity at early times after administration. Co-injection of AM281 (0.3 mg/kg i/v) and WIN 55, 212-2 restored the rate of beam crossings to that seen on injection of vehicle. In addition, AM281 (0.3 mg/kg i/v) approximately doubled locomotor activity between 60-120 min when injected alone. The IC(50) value for displacement of [(131)I]AM281 by AM281 was 0.45 mg/kg. These observations confirm earlier indications that AM281 is a CB1 receptor antagonist or inverse agonist and suggest the existence of an endogenous cannabinoid tone that moderates exploratory locomotor activity.     

Participation of hypothalamic CB1 receptors in reproductive axis disruption during immune challenge

Immune challenge inhibits reproductive function and endocannabinoids (eCB) modulate sexual hormones. However, no studies have been performed to assess whether the eCB system mediates the inhibition of hormones that control reproduction as a result of immune system activation during systemic infections. For that reason, we evaluated the participation of the hypothalamic cannabinoid receptor CB1 on the hypothalamic‐pituitary‐gonadal (HPG) axis activity in rats submitted to immune challenge. Male adult rats were treated i.c.v. administration with a CB1 antagonist/inverse agonist (AM251) (500 ng/5 μL), followed by an i.p. injection of lipopolysaccharide (LPS) (5 mg/kg) 15 minutes later. Plasmatic, hypothalamic and adenohypophyseal pro‐inflammatory cytokines, hormones and neuropeptides were assessed 90 or 180 minutes post‐LPS. The plasma concentration of tumour necrosis factor α and adenohypophyseal mRNA expression of Tnfα and Il1β increased 90 and 180 minutes post i.p. administration of LPS. However, cytokine mRNA expression in the hypothalamus increased only 180 minutes post‐LPS, suggesting an inflammatory delay in this organ. CB1 receptor blockade with AM251 increased LPS inflammatory effects, particularly in the hypothalamus. LPS also inhibited the HPG axis by decreasing gonadotrophin‐releasing hormone hypothalamic content and plasma levels of luteinising hormone and testosterone. These disruptor effects were accompanied by decreased hypothalamic Kiss1 mRNA expression and prostaglandin E2 content, as well as by increased gonadotrophin‐inhibitory hormone (Rfrp3) mRNA expression. All these disruptive effects were prevented by the presence of AM251. In summary, our results suggest that, in male rats, eCB mediate immune challenge‐inhibitory effects on reproductive axis at least partially via hypothalamic CB1 activation. In addition, this receptor also participates in homeostasis recovery by modulating the inflammatory process taking place after LPS administration.     

Cannabinoid‐induced delayed gastric emptying is selectively increased upon intermittent administration in the rat: role of CB1 receptors

Background Cannabinoids acutely administered depress central, cardiovascular and gastrointestinal functions. These effects might be modified upon repeated administration. Compared to the effects induced by daily administration, those induced by intermittent administration are less known. The effect of intermittent treatment with the CB1/CB2 cannabinoid agonist WIN55,212‐2 (WIN) was studied in the rat. Methods Male rats received saline, vehicle or WIN at 0.5 (low‐WIN) or 5 (high‐WIN) mg kg^?1 week^?1 for 4 weeks. WIN effects on the central nervous system (cannabinoid tetrad tests), cardiovascular function and gastrointestinal motor function were evaluated after the first and last doses, and, where appropriate, 1 week after the last dose. To determine the involvement of CB1 receptors in the chronic effect of WIN, the CB1 receptor antagonist/inverse agonist AM251 (1 mg kg^?1) was used. Key Results High‐ (but not low‐) WIN induced the four signs of the cannabinoid tetrad, and reduced gastrointestinal motility, but did not alter cardiovascular parameters. Upon chronic intermittent administration, tolerance did not clearly develop to WIN effects. Quite the opposite, depression of gastric emptying was intensified. No effect was long‐lasting. Repeated administration of AM251 was more efficacious than single administration to block WIN chronic central effects, but the opposite occurred regarding lower intestinal motility. Conclusions & Inferences Upon intermittent administration, hypersensitization may develop to some effects (particularly delayed gastric emptying) induced by cannabinoid agonists. CB1 antagonists/inverse agonists may show different efficacy upon repeated or single administration to block cannabinoid‐induced central and gastrointestinal effects. Thus, cannabinoid effects are dependent on the pattern of drug administration.     

Species and strain differences in the expression of a novel glutamate-modulating cannabinoid receptor in the rodent hippocampus

A novel, non-CB1 cannabinoid receptor has been defined by the persistence of inhibition of glutamatergic EPSPs by the cannabinoid receptor agonist WIN55,212-2 in mice lacking the cloned CB1 receptor (CB1-/-) (Hajos et al., 2001). This novel receptor was also distinguished from CB1 by its sensitivity to the antagonist SR141716A and its insensitivity to the antagonist AM251 (Hajos & Freund, 2002). We have chosen to refer to this putative receptor as CBsc due to its identification on Schaffer collateral axon terminals in the hippocampus. We examined properties of CBsc receptors in Sprague Dawley (SD) rats and two strains of wild-type (WT) mice (C57BL/6J and CD1) used as backgrounds for two independent lines of CB1-/- mice (Ledent et al., 1999; Zimmer et al., 1999). The inhibition of synaptic glutamate release by WIN55,212-2 was observed in hippocampal slices from WT CD1 mice and SD rats but was absent in WT C57 mice. We also found that AM251 and SR141716A antagonized the effect of WIN55,212-2 in hippocampal slices from CD1 mice and SD rats demonstrating a lack of selectivity of these ligands for CB1 and CBsc receptors in these animals. The results indicate that the glutamate-modulating CBsc cannabinoid receptor is present in the hippocampi of CD1 mice and SD rats but not in C57BL/6J mice. Thus, we have identified animal models that may permit the study of cannabinoids independently of the novel CBsc receptor (C57CB1+/+), the CBsc receptor independently of the cloned CB1 receptor (CD1CB1-/-), or in the absence of both receptors (C57CB1-/-).     

Functional interaction between morphine and central amygdala cannabinoid CB1 receptors in the acquisition and expression of conditioned place preference

The present study was done to determine whether cannabinoid CB1 receptors of the central amygdala (CeA) are implicated in morphine-induced place preference. Using a 3-day schedule of conditioning, it was found that subcutaneous (s.c.) administration of morphine (2, 4 and 6 mg/kg) caused a significant dose-dependent conditioned place preference (CPP) in male Wistar rats. Intra-CeA microinjection of the cannabinoid CB1 receptor agonist arachidonylcyclopropylamide (ACPA; 0.5, 2.5 and 5 ng/rat) dose-dependently potentiated the morphine (2mg/kg)-induced CPP. Furthermore, the administration of ACPA (5 ng/rat, intra-CeA) alone induced a significant CPP. It should be considered that the higher dose of ACPA (5 ng/rat, intra-CeA) in combination with morphine decreased locomotor activity on the testing phase. On the other hand, intra-CeA microinjection of the cannabinoid CB1 receptor antagonist AM251 (120 ng/rat) alone induced a significant conditioned place aversion (CPA). Moreover, intra-CeA microinjection of AM251 (90 and 120 ng/rat) inhibited the morphine-induced place preference with a significant interaction. Intra-CeA microinjection of AM251 reversed the effect of ACPA on morphine response. Interestingly, microinjection of ACPA (2.5 and 5 ng/rat) or AM251 (60-120 ng/rat) into the CeA increased or decreased the expression of morphine (6 mg/kg)-induced place preference respectively. These observations provide evidence that cannabinoid CB1 receptors of the CeA are involved in mediating reward and these receptors are also implicated in the acquisition and expression of morphine-induced CPP.