Cerebral neuroinflammation models were established by injecting 10μg lipopolysaccharide into the hippocampus of male Sprague-Dawley rats. The rats were treated with an intraperitoneal injection of 120, 90, or 60 mg/k...Cerebral neuroinflammation models were established by injecting 10μg lipopolysaccharide into the hippocampus of male Sprague-Dawley rats. The rats were treated with an intraperitoneal injection of 120, 90, or 60 mg/kg oxymatrine daily for three days prior to the lipopolysaccharide injection. Twenty-four hours after model induction, the hippocampus was analyzed by real-time quantitative PCR, and the cerebral cortex was analyzed by enzyme-linked immunosorbent assay and western blot assay. The results of the enzyme-linked immunosorbent assay and the real-time quantitative PCR showed that the secretion and mRNA expression of the pro-inflammatory cytokines interleukin-113 and tumor necrosis factor-a were significantly decreased in the hippocampus and cerebral cortex of model rats treated with oxymatrine. Western blot assay and real-time quantitative PCR analysis indicated that toll-like receptor 4 mRNA and protein expression were significantly decreased in the groups receiving different doses of oxymatrine. Additionally, 120 and 90 mg/kg oxymatrine were shown to reduce protein levels of nuclear factor-KB p65 in the nucleus and of phosphorylated IKBa in the cytoplasm of brain cells, as detected by western blot assay. Experimental findings indicate that oxymatrine may inhibit neuroinflammation in rat brain via downregulating the expression of molecules in the toll-like receptor 4/nuclear factor-KB signaling Dathwav.展开更多
Lipopolysaccharide stimulates Toll-like receptor 4 on immune cells to produce immune mediators. Toll-like receptor 4 is also expressed by non-immune cells, which can be stimulated by lipopolysaccharide. However, wheth...Lipopolysaccharide stimulates Toll-like receptor 4 on immune cells to produce immune mediators. Toll-like receptor 4 is also expressed by non-immune cells, which can be stimulated by lipopolysaccharide. However, whether Toll-like receptor 4 is expressed by primary cultured hippocampal neurons and its specific role in lipopolysaccharide-induced neuroinflammation is currently undefined, in this study, Toll-like receptor 4 antibody blocking was used to analyze the Toll-like receptor 4 signaling pathway and changes in inflammation of lipopolysaccharide stimulated hippocampal neurons. Immunofluorescence showed that Toll-like receptor 4 protein was mainly located in the membrane of hippocampal neurons. Quantitative reverse transcription-PCR and western blot assay showed that after stimulation of lipopolysaccharide, the mRNA and protein levels of Toll-like receptor 4 and the mRNA levels of interleukin-ll3 and tumor necrosis factor-（] were significantly increased. In addition, there was increased phosphorylation and degradation of kappa B a inhibitor in the cytosol and increased nuclear factor-KB p65 expression in the nuclei. Pretreatment with Toll-like receptor 4 antibody could almost completely block this increase. These experimental findings indicate that lipopolysaccharide participates in neuroinflammation by stimulating Toll-like receptor 4/nuclear factor-KB pathway in hippocampal neurons, which may be both ＂passive victims＂ and ＂activators＂ of neuroinflammation.展开更多
BACKGROUND： The main components of the traditional Chinese medicine compound Nao Yikang have been shown to possibly alleviate neural damage. OBJECTIVE： To observe the effects of Nao Yikang on expression of choline a...BACKGROUND： The main components of the traditional Chinese medicine compound Nao Yikang have been shown to possibly alleviate neural damage. OBJECTIVE： To observe the effects of Nao Yikang on expression of choline acetyltransferase （CHAT） and caspase-3 in the rat brains of an experimental Alzheimer＇s disease （AD） model, and to investigate the mechanisms of potential neuroprotective effects. DESIGN, TIME AND SETTING： A randomized, controlled experiment was performed at the Department of Pathophysiology, Medical School of Nantong University between November 2006 and December 2007. MATERIALS： The main active components of Nao Yikang were as follows： prepared polygonum multiflorum, Rhizoma anemarrhenae, and Rhizoma acori tatarinowii. Nao Yikang granules were prepared by Nantong Hospital of Traditional Chinese Medicine. Ibotenic acid （IBO） was purchased from Sigma-Aldrich, USA, ChAT goat anti-rat antibody from Chemicon, USA, and cleaved caspase-3 rabbit anti-rat （Asp175） （5A1） antibody from Cell Signaling, USA. METHODS： A total of 60 male, Sprague Dawley rats （2 months old） were randomly assigned to 6 groups： sham-surgery, model, Nao Yikang 1.73, 3.45, 6.90 g/kg per day, and piracetam, with 10 rats in each group. Bilateral infusions of 5 pg IBO into the nucleus basalis of Meynert were performed with Hamilton syringe and stereotaxic apparatus for AD model establishment. For the sham-surgery group, rats received 1 μL saline in the identical stereotaxic position. From the second day, Nao Yikang groups were administrated 1.73, 3.45, and 6.90 g/kg per day Nao Yikang, respectively, while the piracetam group received 0.04 g/mL piracetam, the model group received 0.5% sodium carboxymethyl cellulose, and the sham-surgery group received normal saline. Rats were intragastrically administered 1 mL/100 g daily for 28 consecutive days. MAIN OUTCOME MEASURES： Following treatment of the various solutions for 28 days, Western blot was utilized to observe ChAT expression in the frontal cortex of AD rats, and immunohistochemistry was applied to quantify caspase-3-positive cells in the frontal cortex. RESULTS： ChAT protein expression significantly decreased in the model group （P 〈 0.01）, however caspase-3 expression was significantly elevated （P 〈 0.01） compared with the sham-surgery group. Compared with the model group, ChAT protein expression increased in the Nao Yikang 1.73 g/kg per day, 3.45 g/kg per day, 6.90 g/kg per day groups, and the piracetam group （P 〈 0.05 or P 〈 0.01） and the number of caspase-3-positive cells decreased in the Nao Yikang 3.45 g/kg per day and 6.90 g/kg per day groups （P 〈 0.01）. However, there was no change in the number of caspase-3-positive cells in the 3.45 g/kg per day group. CONCLUSION： The traditional Chinese medicine compound Nao Yikang increased ChAT protein expression and suppressed caspase-3 expression in the frontal cortex in a dose-dependent manner.展开更多
基金supported by a project of the Priority Academic Program Development of Jiangsu Higher Education InstitutionsApplied Research and Technology Plan of Nantong City, No. k2010036+2 种基金2011 Jiangsu Graduated Students' Research and Innovation Program, No. CX2211-0640Nantong University Graduated Students' Technological and Innovative Program, No. YKC11033Students' Practice Innovative Training Project of Nantong University
文摘Cerebral neuroinflammation models were established by injecting 10μg lipopolysaccharide into the hippocampus of male Sprague-Dawley rats. The rats were treated with an intraperitoneal injection of 120, 90, or 60 mg/kg oxymatrine daily for three days prior to the lipopolysaccharide injection. Twenty-four hours after model induction, the hippocampus was analyzed by real-time quantitative PCR, and the cerebral cortex was analyzed by enzyme-linked immunosorbent assay and western blot assay. The results of the enzyme-linked immunosorbent assay and the real-time quantitative PCR showed that the secretion and mRNA expression of the pro-inflammatory cytokines interleukin-113 and tumor necrosis factor-a were significantly decreased in the hippocampus and cerebral cortex of model rats treated with oxymatrine. Western blot assay and real-time quantitative PCR analysis indicated that toll-like receptor 4 mRNA and protein expression were significantly decreased in the groups receiving different doses of oxymatrine. Additionally, 120 and 90 mg/kg oxymatrine were shown to reduce protein levels of nuclear factor-KB p65 in the nucleus and of phosphorylated IKBa in the cytoplasm of brain cells, as detected by western blot assay. Experimental findings indicate that oxymatrine may inhibit neuroinflammation in rat brain via downregulating the expression of molecules in the toll-like receptor 4/nuclear factor-KB signaling Dathwav.
基金supported by the Priority Academic Program Development of Jiangsu Higher Education Institutionsthe Nantong Applied Research Program,No.k2010036+1 种基金the 2011 Jiangsu Graduated Students' Research and Innovation Program,No.CX2211-0640the Nantong University Graduated Students' Technological and Innovative Program,No.YKC11033
文摘Lipopolysaccharide stimulates Toll-like receptor 4 on immune cells to produce immune mediators. Toll-like receptor 4 is also expressed by non-immune cells, which can be stimulated by lipopolysaccharide. However, whether Toll-like receptor 4 is expressed by primary cultured hippocampal neurons and its specific role in lipopolysaccharide-induced neuroinflammation is currently undefined, in this study, Toll-like receptor 4 antibody blocking was used to analyze the Toll-like receptor 4 signaling pathway and changes in inflammation of lipopolysaccharide stimulated hippocampal neurons. Immunofluorescence showed that Toll-like receptor 4 protein was mainly located in the membrane of hippocampal neurons. Quantitative reverse transcription-PCR and western blot assay showed that after stimulation of lipopolysaccharide, the mRNA and protein levels of Toll-like receptor 4 and the mRNA levels of interleukin-ll3 and tumor necrosis factor-（] were significantly increased. In addition, there was increased phosphorylation and degradation of kappa B a inhibitor in the cytosol and increased nuclear factor-KB p65 expression in the nuclei. Pretreatment with Toll-like receptor 4 antibody could almost completely block this increase. These experimental findings indicate that lipopolysaccharide participates in neuroinflammation by stimulating Toll-like receptor 4/nuclear factor-KB pathway in hippocampal neurons, which may be both ＂passive victims＂ and ＂activators＂ of neuroinflammation.
基金Supported by: the Natural Science Foundation of Jiangsu Province, No. BK2004048Social Development and Technology Plan of Nantong City, No. K2008009
文摘BACKGROUND： The main components of the traditional Chinese medicine compound Nao Yikang have been shown to possibly alleviate neural damage. OBJECTIVE： To observe the effects of Nao Yikang on expression of choline acetyltransferase （CHAT） and caspase-3 in the rat brains of an experimental Alzheimer＇s disease （AD） model, and to investigate the mechanisms of potential neuroprotective effects. DESIGN, TIME AND SETTING： A randomized, controlled experiment was performed at the Department of Pathophysiology, Medical School of Nantong University between November 2006 and December 2007. MATERIALS： The main active components of Nao Yikang were as follows： prepared polygonum multiflorum, Rhizoma anemarrhenae, and Rhizoma acori tatarinowii. Nao Yikang granules were prepared by Nantong Hospital of Traditional Chinese Medicine. Ibotenic acid （IBO） was purchased from Sigma-Aldrich, USA, ChAT goat anti-rat antibody from Chemicon, USA, and cleaved caspase-3 rabbit anti-rat （Asp175） （5A1） antibody from Cell Signaling, USA. METHODS： A total of 60 male, Sprague Dawley rats （2 months old） were randomly assigned to 6 groups： sham-surgery, model, Nao Yikang 1.73, 3.45, 6.90 g/kg per day, and piracetam, with 10 rats in each group. Bilateral infusions of 5 pg IBO into the nucleus basalis of Meynert were performed with Hamilton syringe and stereotaxic apparatus for AD model establishment. For the sham-surgery group, rats received 1 μL saline in the identical stereotaxic position. From the second day, Nao Yikang groups were administrated 1.73, 3.45, and 6.90 g/kg per day Nao Yikang, respectively, while the piracetam group received 0.04 g/mL piracetam, the model group received 0.5% sodium carboxymethyl cellulose, and the sham-surgery group received normal saline. Rats were intragastrically administered 1 mL/100 g daily for 28 consecutive days. MAIN OUTCOME MEASURES： Following treatment of the various solutions for 28 days, Western blot was utilized to observe ChAT expression in the frontal cortex of AD rats, and immunohistochemistry was applied to quantify caspase-3-positive cells in the frontal cortex. RESULTS： ChAT protein expression significantly decreased in the model group （P 〈 0.01）, however caspase-3 expression was significantly elevated （P 〈 0.01） compared with the sham-surgery group. Compared with the model group, ChAT protein expression increased in the Nao Yikang 1.73 g/kg per day, 3.45 g/kg per day, 6.90 g/kg per day groups, and the piracetam group （P 〈 0.05 or P 〈 0.01） and the number of caspase-3-positive cells decreased in the Nao Yikang 3.45 g/kg per day and 6.90 g/kg per day groups （P 〈 0.01）. However, there was no change in the number of caspase-3-positive cells in the 3.45 g/kg per day group. CONCLUSION： The traditional Chinese medicine compound Nao Yikang increased ChAT protein expression and suppressed caspase-3 expression in the frontal cortex in a dose-dependent manner.
