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Objective To evaluate the effect of Annexin-A1 mimetic peptide Ac2-26 on activation of astrocytes in rats.Methods The primarily cultured astrocytes from the cortex of fetal Sprague-Dawley rats after 4 passages were divined into 4 groups (n =24 each) using a random number table method:control group (C group),LPS group,LPS+scramble peptide group (LPS+Src group) and LPS+Ac2-26 group.LPS was added to LPS group with the final concentration of 1 mg/ml.LPS at the final concentration of 1 mg/ml and scramble peptide at the final concentration of 3.3 mmol/L were added to LPS+Src group.LPS at the final concentration of 1 mg/ml and Ac2-26 at the final concentration of 3.3 mmol/L were added to LPS+Ac2-26 group.After 24-h incubation,the cell survival rate was measured by CCK-8 assay,the migration was determined by Transwell assay,the concentrations of tumor necrosis factor-alpha (TNF-α),interleukin-1 beta (IL-1β),monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-1 a (MIP-1a) in the supernatant were measured (by enzyme-linked immunosorbent assay),and the expression of glial fibrillary acidic protein (GFAP),extracellular signal-regulated kinase (ERK),phosphorylated ERK (p-ERK),c-Jun N-terminal kinase (JNK),phosphorylated JNK (p-JNK),p38 mitogen-activated protein kinase (p38MAPK),and phosphorylated p38MAPK (p-p38MAPK) in astrocytes was detected by Western blot.Results Compared with group C,the expression of GFAP was significantly up-regulated,and the cell mobility,concentrations of TNF-α,IL-1β,MCP-1 and MIP-1α in the supernatant,p-ERK/ERK ratio,p-JNK/JNK ratio and p-p38MAPK/p38MAPK ratio were increased (P＜0.05),and no siguificant change was found in the cell survival rate in group LPS (P＞0.05).Compared with group LPS,the expression of GFAP was significantly down-regulated,and the cell mobility,concentrations of TNF-α,IL-1β,MCP-1 and MIP-1α in the supernatant,p-JNK/JNK ratio and p-p38MAPK/p38MAPK ratio were decreased in group LPS+Ac2-26 (P＜0.05),and no significant change was found in the parameters mentioned above in group LPS+Src (P＞0.05).Conclusion Ac2-26 can inhibit activation of astrocytes and produces anti-inflammatory effect in rats.
ABSTRACT
Objective@#To evaluate the effect of Annexin-A1 mimetic peptide Ac2-26 on activation of astrocytes in rats.@*Methods@#The primarily cultured astrocytes from the cortex of fetal Sprague-Dawley rats after 4 passages were divined into 4 groups (n=24 each) using a random number table method: control group (C group), LPS group, LPS+ scramble peptide group (LPS+ Src group) and LPS+ Ac2-26 group.LPS was added to LPS group with the final concentration of 1 mg/ml.LPS at the final concentration of 1 mg/ml and scramble peptide at the final concentration of 3.3 mmol/L were added to LPS+ Src group.LPS at the final concentration of 1 mg/ml and Ac2-26 at the final concentration of 3.3 mmol/L were added to LPS+ Ac2-26 group.After 24-h incubation, the cell survival rate was measured by CCK-8 assay, the migration was determined by Transwell assay, the concentrations of tumor necrosis factor-alpha (TNF-α), interleukin-1beta (IL-1β), monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-1a (MIP-1a) in the supernatant were measured (by enzyme-linked immunosorbent assay), and the expression of glial fibrillary acidic protein (GFAP), extracellular signal-regulated kinase (ERK), phosphorylated ERK (p-ERK), c-Jun N-terminal kinase (JNK), phosphorylated JNK (p-JNK), p38 mitogen-activated protein kinase (p38MAPK), and phosphorylated p38MAPK (p-p38MAPK) in astrocytes was detected by Western blot.@*Results@#Compared with group C, the expression of GFAP was significantly up-regulated, and the cell mobility, concentrations of TNF-α, IL-1β, MCP-1 and MIP-1α in the supernatant, p-ERK/ERK ratio, p-JNK/JNK ratio and p-p38MAPK/p38MAPK ratio were increased (P<0.05), and no significant change was found in the cell survival rate in group LPS (P>0.05). Compared with group LPS, the expression of GFAP was significantly down-regulated, and the cell mobility, concentrations of TNF-α, IL-1β, MCP-1 and MIP-1α in the supernatant, p-JNK/JNK ratio and p-p38MAPK/p38MAPK ratio were decreased in group LPS+ Ac2-26 (P<0.05), and no significant change was found in the parameters mentioned above in group LPS+ Src (P>0.05).@*Conclusion@#Ac2-26 can inhibit activation of astrocytes and produces anti-inflammatory effect in rats.
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Objective To investigate the role of OMA1 in acute kidney injury (AKI) induced by lipopolysaccharide (LPS).Methods OMA1 wild-type and knocked out mice (8 week old) were injected with 10 mg/kg body weight of LPS.The model was confirmed by testing mouse serum creatinine and blood urea nitrogen.The apoptosis in mouse kidney cortex was examined by TUNEL staining and cleaved caspase 3.In vitro,in humam kidney proximal tubular cells (HK2) were knocked down OMA1 by transfecting OMA1 shRNA,with the scramble shRNA being used as negative control of transfection.HK2 cells were cultured with 5 μg/ml of LPS for 24 hours to induce apoptosis.DAPI staining of cells and caspase-3 activity were applied to test apoptosis.The images of mitochondria in cells were obtained by transfection of mito-green plasmid and OMA1 shRNA.Western blotting was used to exam the OMA1 and Cytochrome C expressions.Resudts Compared with OMA1 KO mice,LPS induced more severe AKI of WT mice with higher Scr [(97.2±26.5) μmol/L vs (53.0±17.7) μmol/L,P ＜ 0.05] and BUN [(43.3± 13.7) mmol/L vs (29.7±7.7) mmol/L,P ＜ 0.05].Moreover,there were more apoptosis cells in kidney cortex in WT mice than in OMA1 KO mice [(75.4± 26.1)/ram2 vs (38.3± 14.4)/mm2,P＜ 0.05].About 46％ of OMA1 expressions in HK2 cells were inhibited by OMA1 shRNA transfection (P ＜ 0.05).Further,OMA1 shRNA cells with LPS stimulation had decreased mitochondria fragmentation [(29.8±10.9)％ vs (43.2±6.8)％,P ＜ 0.05],Cytochrome C release [(37.0±12.3)％ vs (76.0±26.2)％,P ＜ 0.05],and cell apoptosis [(13.2±3.9)％ vs (25.0±7.1)％,P ＜ 0.05] as compared with control cells.Conclusion Knockdown of OMA1 alleviated septic AKI through inhibition of cell apoptosis,mitochondria fragmentation,and Cytochrome C release.
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The methods of 1H Nuclear Magnetic Resonance (NMR) and mass spectroscopy were used in detecting the metabolites of brodimoprim (BDP) in rat plasma. The endogenic compounds in the plasma were removed with solid phase extraction (SPE) column firstly, then the mixture of metabolites was identified with 1H NMR and mass spectroscopy (MS). Two metabolites of BDP in the plasma at 20h were detected, they were demethyl-brodimorpim glucuronide and brodimoprim sulfurate. The study proved that the method of SPE coupled with NMR and MS can be applied to the analysis of metbolites in plasma quickly and conveniently.