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.

Objective To investigate the effects of Annexin-A1 ( Anxa1 ) gene silencing induced by siRNA on the growth and migration of microglial BV-2 cells and its possible mechanisms.Methods A synthesized siRNA duplex targeting Anxa1 gene was transfected into BV-2 cells.The efficiency of siRNA-in-duced Anxa1 gene silencing was evaluated on both mRNA and protein levels by using reverse-transcription PCR and Western blot assay.MTT assay was performed to measure the proliferation of BV-2 cells with si-lenced expression of Anxa1 gene.Flow cytometry with Annexin V-FITC/PI double staining was used to de-tect the apoptosis rate of BV-2 cells.Transwell chambers were used to analyze the effects of siRNA-induced Anxa1 gene silencing on the migration of BV-2 cells.Western blot assay was performed to detect the expres-sion of signaling proteins related to cell cycle and migration.Results Compared with the siRNA negative control ( siRNA-NC) group, the inhibitory rates of siRNA-induced Anxa1 gene silencing on the proliferation of BV-2 cells were significantly increased at the time points of 24 h, 48 h and 72 h after intervention [(16.9 ±2.1)%, (23.1±3.6)%and (42.4±1.7)%vs (1.35±0.5)%, (2.06±0.7)% and (8.65±0.9)%, P<0.05 ].The apoptosis rate of BV-2 cells transfected with Anxa1 siRNA was (18.4±2.1)%, which was significantly elevated as compared with that of the siRNA-NC group (5.2±0.3)%and control group (4.3±0.2)%.Cell migration of the Anxa1 siRNA transfected BV-2 cells was inhibited remarkably at 48 h as com-pared with that of the siRNA-NC group (28.7±5.2 vs 173.4±11.4, P<0.01).Moreover, the suppressed expression of Cyclin D1 protein and activation of p38 and JNK signaling pathways were induced by silenced expression of Anxa1 gene in BV-2 cells.Conclusion The growth and migration of BV-2 cells were signifi-cantly inhibited by silencing the expression of Anxa1 gene with siRNA, the possible mechanisms might be associated with the suppressed expression of Cyclin D1protein and the activation of p38 and JNK signaling pathways.

Objective To explore the changes of serum miR-145 and miR-497 levels and their correlations with high sensitive C-reactive protein (Hs-CRP) and matrix metalloproteinase-9 (MMP-9) in patients with ischemic stroke.Methods One hundred and twenty-six patients with ischemic stroke,including 28 mild neurologic impairment (NI) patients,61 moderate NI patients and 37 severe NI patients,and 107 normal controls,were collected in our hospital from January 2013 to October 2013.Expressions of serum miR-145 and miR-497 were measured by real-time fluorescent quantitative PCR,protein markers Hs-CRP and MMP-9 were determined by immune turbidimetry and ELISA assay,respectively,correlations between microRNAs (miRNAs) and protein markers were analyzied by partial correlation analysis.Results Both expressions of serum miR-145 and miR-497,and Hs-CRP and MMP-9 levels in ischemic stroke patients were significantly elevated as compared with those in the normal controls (P＜0.05).Positive correlations were noted between of serum miR-145 expression and both serum miR-497 expression and expressions of serum Hs-CRP and MMP-9 (r=0.718,P=0.000;r=0.658,P=0.000;r=0.455,P=0.000);meanwhile,serum miR-497 expression and expressions of serum Hs-CRP and MMP-9 were also positively correlated (r=0.845,P=0.000;r=0.787,P=0.000).Severe NI patients had significantly higher expressions of serum miR-145 and miR-497,and Hs-CRP and MMP-9 levels as compared with mild NI patients and moderate NI patients (P＜0.05),and these levels in the moderate NI patients were significantly higher than those in the mild NI patients (P＜ 0.05);these miRNAs and protein markers showed a different correlation strength depended on the different severities of neurologic impairment.Conclusion Both of serum miR-145 and miR-497 levels may be useful biomarkers for screening ofischemic stroke;and miR-145 and miR-497 may have synergistic effect on regulating the pathogenesis of ischemic stroke.

Objective: To investigate the effects of formyl peptide receptor 2 (FPR2) silencing on the proliferation, migration and invasion of human glioma U87 cells and its possible mechanisms. Methods: The expression of FPR2 was detected in normal glial cells, glioma cells, normal brain tissues and glioma tissues using Western blot and immunohistochemistry staining. A synthesized siRNA duplex was employed to inhibit FPR2 in human glioma cells (U87). The knockdown efficiency was evaluated by real-time reverse transcription-polymerase chain reaction (RT-qPCR) and Western blot. MTT, transwell assays and flow cytometry analyses were used to determine the cell proliferation, migration, invasion and apoptotic rates of U87 cells, respectively. Mice xenograft experiments were used to observe the effect of FPR2 silencing on the tumorigenesis of U87 cells in vito. Western blot and enzyme-linked immunosorbent assays were performed to detect the expression and release of cell cycle and migration-related proteins. Results: The expression of FPR2 was significantly higher in glioma cell lines and glioma tissues than that in normal glial cells and brain tissues. Compared with blank control and negative control, FPR2 mRNA and protein levels in siRNA group were significantly downregulated. The cell proliferation inhibitory rates in FPR2 siRNA group were (23.1±5.1)%, (39.6±5.6)% and (44.4±6.7)% at 24 h, 48 h and 72 h, respectively, which were significantly increased than those in negative control group [(3.2±0.6)%, (5.7±0.8)% and (7.9±0.9)%, respectively; P<0.05]. The apoptosis rate in FPR2 siRNA group was (17.4±2.1)%, which was significantly elevated than that in the negative control group with (5.4±0.5)% and blank control group with (3.8±0.3)% (all P<0.05). In addition, the numbers of migrated cells were 108.7±9.5 in FPR2 siRNA group, which was significantly lower than that in blank control group 312.9±17.5 and negative control group (304.4±15.7, all P<0.05). Likewise, the numbers of invaded cells were 19.3±3.2 in FPR2 siRNA group, which was significantly lower than that in blank control group 106.9±8.5 and negative control group (102.4±7.4, all P<0.05). Moreover, the growth of FPR2 siRNA transfected U87 cells in vivo was remarkably decreased comparing with the negative group (P<0.05). Furthermore, the expression of cyclin D1 and VEGF in FPR2 silencing U87 cells was suppressed mainly through β-catenin signaling pathway. Conclusions FPR2 silencing by siRNA can inhibit the growth, migration and invasion ability, but promote the apoptosis of U87 cells. The possible mechanisms might be associated with the inhibitory expression of cyclin D1 and VEGF.

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.