Effects of ICG-001 on autistic behaviors and the development of hippocampal dendritic spine morphology in rats / 中华实用儿科临床杂志

Objective:To explore the effects of the compound ICG-001 on autism-like behaviors and the morphological development of dendritic spines in hippocampal pyramidal neurons of rats.Methods:Healthy Wistar rats were mated.The offspring were divided into the saline-treated group, ICG-001 control group, Sodium valproate (VPA) group and ICG-001 treatment group by using the random number table method.Each group had 12 rats.Social interaction, repetitive, compulsive and anxiety-like behaviors in rodents were assessed by three-chambered social approach, marble burying, open-field and elevated plus maze tests.The number of neuronal nuclei (NeuN)-positive neurons in the hippocampal CA1 region was calculated by the immunofluorescence method.Golgi staining was carried out to detect the density and morphological changes of dendritic spines in hippocampal pyramidal neurons of rats.The expression of phosphorylated LIM kinase 1(LIMK1), phosphorylated actin binding protein(Cofilin), fibros actin (F-actin) and developmentally-regulated brain protein A (Drebrin A) was examined by Western blot.The univariate analysis was made to examine whether the difference was statistically significant, and the data between groups were compared by the Tukey method. Results:(1) In the three-chambered social approach test, the rats in the saline-treated group, ICG-001 control group, VPA group and ICG-001 treatment group spent (219.42±5.38) s, (218.67±10.12) s, (126.58±5.02) s, and (218.58±6.63) s in the chamber, respectively.The corresponding preference score of the said 4 groups were 0.43±0.05, 0.43±0.04, 0.22±0.01 and 0.42±0.04, respectively.Compared with the VPA group, the ICG-001 treatment group spent longer time in the chamber and had a higher preference score (all P<0.05). (2) In the marble burying experiment, the number of marbles buried in said 4 groups were 9.13±0.52, 9.08±0.64, 15.13±0.82 and 9.42±0.86, respectively.ICG-001-treated rats buried markedly less marbles than VPA-exposed rats ( P<0.05). (3) In the open-field test, the rats in the said 4 groups spent (82.33±1.83) s, (81.32±4.19) s, (45.51±3.02) s and (81.44±3.19) s in the center area, respectively.Administration of ICG-001 significantly increased the time that VPA-exposed rats spent in the center area ( P<0.05). (4)In the elevated plus maze trial, the rats in the said 4 groups spent (107.75±7.23) s, (106.08±7.50) s, (63.42±1.91) s and (106.67±7.07) s in open arms, respectively.ICG-001 treatment notably increased the time that VPA-exposed rats spent in open arms ( P<0.05). (5) Immunofluorescence analysis results revealed that the number of NeuN-positive cells in the hippocampal CA1 region of said 4 groups was (41.83±1.17)×10 4/μm 2, (41.00±0.77)×10 4/μm 2, (27.17±0.95)×10 4/μm 2 and (40.00±0.90)×10 4/μm 2, respectively.ICG-001 treatment normalized the alteration in the number of NeuN-containing neurons in VPA-exposed rats ( P<0.05). (6) Golgi staining showed that the density of dendritic spines in hippocampal CA1 pyramidal neurons of said 4 groups was (0.74±0.04)/μm, (0.73±0.03)/μm, (0.49±0.03)/μm and (0.70±0.02) /μm, respectively.Of all types of dendritic spines, mushroom spines accounted for (0.49±0.02)%, (0.49±0.02)%, (0.33±0.02)% and (0.43±0.02) % in said 4 groups.Thin spines accounted for (0.27±0.02)%, (0.26±0.02)%, (0.34±0.01)% and (0.26±0.01) % in said 4 groups, respectively.Compared with the VPA group, the ICG-001 treatment group showed a significant increase in the density of dendritic spines in hippocampal CA1 pyramidal neurons ( P<0.05). After ICG-001 treatment, the number of mushroom spines greatly increased and the number of thin spines sharply decreased in VPA-exposed rats (all P<0.05). (7) According to Western blot test results, the phosphorylated LIMK1/LIMK1 ratio of the hippocampus in said 4 groups were 100.33±2.30, 99.34±2.28, 57.76±4.10 and 99.13±1.90, respectively.The phosphorylated Cofilin /Cofilin ratio were 100.18±2.43, 100.18±1.70, 57.12±1.88 and 99.53±1.69, respectively.The F-actin/globular actin(G-actin) ratio were 100.07±0.86, 99.99±1.72, 51.19±1.23 and 99.28±3.17, respectively.The expression level of Drebrin A were 100.79±1.19, 100.12±2.04, 52.86±3.26 and 99.97±2.44, respectively.Administration of ICG-001 effectively prevented the decrease of phosphorylated LIMK1, phosphorylated Cofilin, F-actin and Drebrin A in the hippocampus of VPA-exposed rats (all P<0.05). Conclusions:ICG-001 regulates the LIMK1/Cofilin signaling pathway, promotes the generation of F-actin, increases the expression of Drebrin A, and thereby alleviates autistic-associated symptoms.

Effects of ganoderma triterpenoids combined with exogenous GM1 on cognitive function and hippocampal synaptic structure in rats with epilepsy / 中华行为医学与脑科学杂志

Objective:To study the intervention effect of ganoderma triterpenoids combined with exogenous monosialotetrahexosyl ganglioside(GM1) on cognitive dysfunction and synaptic ultrastructure of hippocampal neurons in rats with epilepsy caused by pentylenetetrazol(PTZ).Methods:A total of 40 Sprague-Dawley rats were divided into blank control group, epileptic model group, ganoderma triterpenoids group, GM1 group and GM1 combined with ganoderma triterpenoids group according to the random number table method( n=8 in each group). The rats were intraperitoneally injected with PTZ subconvulsant dose (35 mg·kg -1·d -1) once a day for 28 days to replicate the models of chronic epilepsy. And the rats in different medication groups were given corresponding administration based on daily intraperitoneal injection of PTZ(GM1: intraperitoneal injection of 30 mg·kg -1·d -1, ganoderma triterpenoids: gavage 1 000 mg·kg -1·d -1). Morris water maze was used to test the spatial exploration and learning and memory ability of epileptic rats.Transmission electron microscopy was used to observe the ultrastructure of hippocampal neurons in epileptic rats.Immunofluorescence staining was used to observe expression levels of cofilin and SYN protein in hippocampus CA1 of rats. In addition, Western blot was used to detect the expression levels of cofilin, p-cofilin and synaptophysin(SYN) protein in hippocampus of rats. SPSS 17.0 software was used for statistical analysis. Repeated one-way ANOVA was used for comparing among groups, LSD test was used for pairwise comparisons. Results:Morris water maze results showed that there were statistically significant differences in escape latency, times of crossing the platform and time spent in the target quadrant among the groups( F=5.259, 8.240, 5.961, all P<0.05). Compared with the epilepsy model group, the escape latencies((20.31±7.39) s, (21.81±6.05) s, (17.66±4.76) s) of the ganoderma triterpenoids group, GM1 group and GM1 combined with ganoderma triterpenoids group were shorter (all P<0.05), the numbers of crossing the platform ((4.63±1.41) times, (4.50±1.93) times, (5.50±1.77) times) were more (all P<0.05), the residence time in target quadrant ((31.91±5.00) s, (30.49±5.72) s, (35.70±5.34) s) were longer (all P<0.05). And the most obvious change was found in the GM1 combined with ganoderma triterpenoids group ( P<0.01). The results of transmission electron microscope showed that there were significant differences in the numbers of hippocampal neurons synapses, the synaptic gap, the density of postsynaptic membrane and length of active area of postsynaptic membrane among the groups( F=3.693, 7.201, 5.012, 4.033, all P<0.05). Compared with the epilepsy model group, the numbers of synapses ((8.00±1.79), (7.83±1.84), (8.50±1.87)) in the ganoderma triterpenoids group, GM1 group and GM1 combined with ganoderma triterpenoids group were all more (all P<0.05), synaptic gap ((33.83±3.81)nm, (32.43±4.14)nm, (30.23±3.08)nm)were narrower, and the postsynaptic dense substances ((57.50±6.03)nm, (58.10±2.40)nm, (60.73±3.81)nm) were all thicker (all P<0.05). The length of active region of postsynaptic membrane ((271.66±11.80) nm, (279.06±13.58) nm) in ganoderma triterpenoid group and GM1 combined with ganoderma triterpenoids group were longer than that in epilepsy model group (both P<0.05). Immunofluorescence results showed that the average fluorescence intensity of cofilin in the epilepsy model group was higher than that in the blank control group, and the average fluorescence intensity of SYN was lower than that in the blank control group (both P<0.05). The average fluorescence intensity of cofilin in GM1 group and GM1 combined with ganoderma triterpenoids group were lower than that in epilepsy model group (both P<0.05), and the average fluorescence intensity of SYN in ganoderma lucidum triterpenoids combined with GM1 group was higher than that in epilepsy model group ( P<0.05). Western blot showed that the expression levels of cofilin protein in the epilepsy model group was higher than that in the blank control group ((1.454±0.080), (1.092±0.099), P<0.05), and the expression of p-cofilin and SYN were lower than those in the blank control group ((1.103±0.120) vs (1.420±0.934), (1.650±0.062) vs (1.958±0.062), both P<0.05). The expression of cofilin protein ((1.227±0.071), (1.262±0.078), (1.162±0.129), P<0.05) in ganoderma triterpenoids group, GM1 group and GM1 combined with ganoderma triterpenoids group were lower than that in epilepsy model group, and the expression levels of p-cofilin(1.357±0.199) and SYN protein(1.873±0.010) in ganoderma triterpenoids combined with GM1 group were higher than that in epilepsy model group (both P<0.05). Compared with ganoderma lucidum triterpenoids group and GM1 group, there was no significant difference in each index of GM1 combined with ganoderma triterpenoids group (all P>0.05). Conclusion:GM1 combined with ganoderma triterpenoids may promote the synaptic plasticity of neurons, improve the learning and memory ability of epileptic rats.Combination medication is better than single medication in some observed indicators.

Electroacupuncture Attenuates Ischemic Brain Injury and Cellular Apoptosis via Mitochondrial Translocation of Cofilin / 中国结合医学杂志

OBJECTIVE@#To investigate the potential mechanisms of electroacupuncture (EA) to prevent ischemic stroke.@*METHODS@#The method of middle cerebral artery occlusion (MCAO) was employed to establish a rat model of ischemic stroke. Seventy-eight Sprague-Dawley rats were divided into the sham group, MCAO + EA control (EC) group, and MCAO + EA (EA) group according to a random number table (n=26 per group). EA was applied to the acupoints of Baihui (DU 20) and Shenting (DU 24) 5 min and 6 h, respectively after the onset of MCAO. Rats in the sham and EC groups received only light isoflurane anesthesia for 30 min after MCAO. The neuroprotective effects of EA were evaluated by rota-rod test, neurological deficit scores and infarct volumes. Additionally, Nissl staining and immunostaining were performed to examine brain damage, rod formation, cellular apoptosis, and neuronal loss induced by ischemia. The activities of caspase-3, and expression levels of cofilin and p-cofilin in mitochondria and cytoplasm after ischemic injury were determined by Western blot.@*RESULTS@#Compared with the EC group, EA significantly improved neuromotor function and cognitive ability after ischemic stroke (P<0.05 or P<0.01). Therapeutic use of EA also resulted in a significant decrease of cofilin rod formation and microtubule-associated protein-2 (MAP2) degradation in the cortical penumbra area compared with the EC rats (P<0.01). Furthermore, Western blot analysis showed that EA stimulation significantly inhibited mitochondrial translocation of cofilin and caspase-3 cleavage (P<0.05 or P<0.01). Additionally, brain damage (infarct volume and neuropathy), cellular apoptosis and neuronal loss induced by ischemia were remarkably suppressed by EA in the cortical penumbra of rats (P<0.05 or P<0.01).@*CONCLUSION@#EA treatment after ischemic stroke may attenuate ischemic brain injury and cellular apoptosis through the regulation of mitochondrial translocation of cofilin, a novel mechanism of EA therapy.

Phosphorylation of Cofilin-1 Enhances Paclitaxel Resistance of Epithelial Ovarian Cancer Cells by Inhibiting Apoptosis / 生物医学与环境科学（英文）

Objective@#To investigate the molecular mechanism of high phosphorylation levels of cofilin-1 (p-CFL-1) associated with paclitaxel resistance in epithelial ovarian cancer (EOC) cells.@*Methods@#Cells displaying varying levels of p-CFL-1 and CFL-1 were created by plasmid transfection and shRNA interference. Cell inhibition rate indicating paclitaxel efficacy was assessed by Cell Counting Kit-8 (CCK-8) assay. Apoptosis was assessed by flow cytometry and protein levels were detected by western blotting. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to measure the expression levels of phosphokinases and phosphatases of CFL-1. Survival analysis evaluated the correlation between the prognosis of EOC patients and the levels of p-CFL-1 and slingshot-1 (SSH-1).@*Results@#High levels of p-CFL-1 were observed in EOC cells that survived treatment with high doses of paclitaxel. SKOV3 cell mutants with upregulated p-CFL-1 showed impaired paclitaxel efficacy, as well as decreased apoptosis rates and pro-survival patterns of apoptosis-specific protein expression. Cytoplasmic accumulation of p-CFL-1 inhibited paclitaxel-induced mitochondrial apoptosis. SSH-1 silencing mediated CFL-1 phosphorylation in paclitaxel-resistant SKOV3 cells. Clinically, the high level of p-CFL-1 and the low level of SSH-1 in EOC tissues were closely related to chemotherapy resistance and poor prognosis in EOC patients.@*Conclusion@#The SSH-1/p-CFL-1 signaling pathway mediates paclitaxel resistance by apoptosis inhibition in EOC and is expected to be a potential prognostic predictor.

Effects of Triptolide on Expression of Drebrin and Cofilin in Hippocampus of Rats with Alzheimer's Disease / 中国康复理论与实践

@#Objective To observe the effects of triptolide on drebrin and cofilin expression in the hippocampus of rats with Alzheim-er's disease (AD). Methods Sixty male Sprague-Dawley rats were equally divided into control group, model group and triptolide-treated group with 20 cases in each group. The AD model was established with unilateral injection of beta amyloid 1-40 (Aβ1- 40) into hippocampus in rats. The control group was established with unilateral injection of normal saline with the same volume into hippocampus in rats. The triptolide-treated group was administered triptolide intraperi-toneally, 0.4 mg/kg, once a day, for 15 days after modeling. Spine density of hippocampal neurons was assayed by Golgi staining. Drebrin and cofilin expression of hippocampal neurons was assayed by immunohistochemical staining and reverse transcription polymerase chain reaction (RT-PCR). Results The spine density of hippocampal neurons was higher in the triptolide-treated group than in the model group (P<0.05). The average optical density of drebrin was higher in the triptolide-treated group than in the the model group (P<0.01), while the cell number and average optical density of cofilin were lower (P<0.05). The drebrin mRNA expression was higher in the triptolide-treated group than in the model group (P<0.05), and the cofilin mRNA expression was lower (P<0.01). Conclusion Triptolide may delay the degeneration of dendritic spines in hippocampal neurons of AD rats by regulating the expression of drebrin and cofilin.

Detection of sputum cofilin-1 as indicator of malignancy

Cofilin-1 (CFL1), a small protein of 18 kDa, has been studied as a biomarker due to its involvement in tumor cell migration and invasion. Our aim was to evaluate CFL1 as an indicator of malignancy and aggressiveness in sputum samples. CFL1 was analyzed by ELISA immunoassay in the sputum of 73 lung cancer patients, 13 cancer-free patients, and 6 healthy volunteers. Statistical analyses included ANOVA, ROC curves, Spearman correlation, and logistic regression. Sputum CFL1 levels were increased in cancer patients compared to cancer-free patients and volunteers (P<0.05). High expression of sputum CFL1 was correlated to T4 stage (P=0.01) and N stage (P=0.03), tobacco history (P=0.01), and squamous cell carcinoma histologic type (P=0.04). The accuracy of sputum CFL1 in discriminating cancer patients from cancer-free patients and healthy volunteers were 0.78 and 0.69, respectively. CFL1 at a cut-off value of 415.25 pg/mL showed sensitivity/specificity of 0.80/0.70 in differentiating between healthy volunteers and cancer patients. Sputum CFL1 was also able to identify cancer-free patients from patients with lung cancer. The AUC was 0.70 and, at a cut-off point ≥662.63 pg/mL, we obtained 60% sensitivity and 54% specificity. Logistic regression analysis controlled for tobacco history, histologic types, and N stage showed that cancer cell-associated CFL1 was an independent predictor of death. Smoker patients with squamous cell carcinoma, lymph node metastasis and sputum CFL1>1.475 pg/mL showed augmented chance of death, suggesting lung cancer aggressiveness. CFL1 presented diagnostic value in detecting lung cancer and was associated to tumor aggressiveness.

Effects of cucurbitacin Ⅱa on apoptosis of humanlung cancer cell lines NCI-H460 and A549 and its mechanism / 中国药理学通报

Aim To study the apoptosis effect of cucurbitacin Ⅱa on non-small cell lung cancer cell lines NCI-H460 and A549 and its underlying mechanism.Methods Cell viability was assessed by CCK-8 assay.The apoptosis effect and cell cycle arrest were detected by Flow cytometry.Western blot was employed to detect the related protein.Results The proliferation of lung cancer cell lines NCI-H460 and A549 was inhibited by CuⅡa, which showed cytotoxic activity with IC50 values of 224.9 nmol·L-1 and 108.3 nmol·L-1 against NCI-H460 and A549 respectively.CuⅡa induced the cells apoptosis and cell cycle arrest at G2/M phase.The results of Western blot showed CuⅡa inhibited the phosphorylation of STAT3 and Cofilin in a dose-dependent manner.Further, CuⅡa inhibited the phosphorylation of Aurora A, in line with the important characteristics of anti-tumor effect of Aurora A kinase inhibitor with blocking cells in the G2/M phase.Conclusion CuⅡa has obvious anti-tumor effect against non-small cell lung cancer, which suggests its value as a lead compound for lung cell carcinoma.

Relationship between the expression of Cofilin in alveolar and peripheral blood mononuclear cells in COPD patientsand the function of phagocytic aspergillus / 实用医学杂志

Objective To investigate the expression of Cofilin in alveolar and peripheral blood mononuclear cells in COPD patients and the correlation with the function of phagocytic aspergillus. Methods 20 COPD pa-tients were selected from July 2015 to May 2016 in the outpatient department of respiratory medicine of Zhujiang Hospital of Southern Medical University ,then divided into the experimental group 1(group Aand B in 2011 version of GOLD),group 2(test group C and group D in 2011 version of GOLD),and the healthy control group in 10 people. The AM and MDM in the peripheral bloodwere extracted respectively in the 3 groups by bronchoalveolar lavage and,and the ability of AM and MDM in each group were detected. The expression of Cofilin protein was measured by real-time fluorescence quantitative(qRT-PCR) and Western blotting ,and the differenceswere compared among the three groups. Results The colony numbers of MDM/AM in the 3 groups were 17 ± 3,16 ± 2, 42 ± 3(F = 73.446 ,P < 0.001),and the colony numbers of MDM/AM in the two test groupshad significantly different from the healthy group ,while no significant difference was found in the two test groups. The results of fluorescence quantitative reverse transcription polymerase chain reaction and Western blot analysis showed that the expression of Cofilin in the two test groups was significantly higher than that in the healthy group. Conclusion The decreased function of phagocytic aspergillus in the alveolar and peripheral blood mononuclear cells in COPD patients may relates to the increase of cofilin.

The relationship between phosphorylation at Ser3 of cofilin1 and Taxol resistance of ovarian cancer in elderly female patients / 中华老年医学杂志

Objectives To analyze the relationship between phosphorylation of serine-3 of cofilin1 and Taxol resistance of ovarian cancer and to evaluate the prognostic value of cofilin1 phosphorylation in estimating ovarian cancer survival using clinical samples.Methods Wild type (WT) plasmids with over-expression of wild cofilin1,S3L plasmids with over-expression of cofilin1 and inhibited phosphorylation at serine-3,and siRNA-C plasmids with down-regulation of cofilin1 were constructed using molecular biology techniques.After the SKOV3 and SK-TR30 cell-lines were transfected with these plasmids,changes in biological characteristics and drug resistance after instant transfection were compared.Fifty-one elderly female patients with microscopically confirmed ovarian cancer,who had received chemotherapy with Taxol and cis-platinum (TC)after surgery,including 30 chemo-sensitive and 21 chemo-resistant cases,were recruited in this study.Immunohistochemical methods were used to detect the expression of cofilinl and phosphorylated cofilin in tissue sections from the two groups.Progression free survival(PFS)was analyzed.Results Compared with the control group,the proportion of cells increased at the G0/G1 phase(P=0.034)and decreased at the S phase (P=0.031),and the apoptosis rate decreased(P=0.020),in SKOV3 cells with high expression of cofilin1.However,these characteristics disappeared when the wild type was replaced with the inactive mutant S3L.When cofilin1 expression was down-regulated in SK-TR30 cells,the proportion of cells at the G0/G1 phase decreased(P=0.020).The expression of cofilin1 was detected in 56.7 ％ (17/30)and 57.1％ (12/21),respectively,sections of ovarian tumor tissues of the chemo-sensitive and chemoresistant groups,and there was no statistic difference (x2=0.332,P =0.943)between the two groups.The expression of phosphorylated cofilin was much higher in the chemo-resistant group (85.7％ or 18/21)than in the chemo-sensitive group (53.3％ or 16/30),and the difference was statistically significant(x2=5.829,P=0.016).The higher expression of phosphorylated cofilin was also correlated with shorter PFS(x2 =21.440,P＜0.01,95％ CI:0.068-0.883),especially in the chemo-sensitive group.Conclusions Serine-3 phosphorylation status of cofilin 1 is associated with paclitaxel resistance in ovarian cancer,but the underlying mechanisms of regulation need further investigation.

Rhein inhibits the movement and invasion of human ovarian carcinoma cells through Rac1/LIMK1/cofilin signaling pathway / 中国药理学通报

Aim To investigate the effect of Rhein on the movement and invasiveness of human ovarian carci-noma cells with directional high lymphatic metastasis SKOV3-PM4 cells and explore the role of Rac1/LIMK1/cofilin signaling pathway. Methods Migration assay and invasion assay were used to observe the effect of Rhein on the metastatic and invasive ability of SK-OV3-PM4 cells in vitro. The effect of Rhein on the morphology and cytoskeleton ultrastructure of ovarian cancer cells was observed by laser scanning confocal microscope and scanning electron microscope. The protein expression level of Rac1,LIMK1,PAK1 and co-filin were detected by Western blot, respectively. Re-sults Rhein inhibited the abilities of cell invasion and migration of SKOV3-PM4 cells,and the inhibitory rate increased along with the increase of the concentration and treatment duration. After treated with 8. 80 μmol· L-1 ,17. 60 μmol · L-1 , 26. 40 μmol · L-1 of Rhein for 24 h,the abilities of migration and invasion of SK-OV3-PM4 cells were inhibited ( P <0. 05 ); the mor-phology and cytoskeleton ultrastructure of SKOV3-PM4 cells were changed, cellular pseudopod reduced, cell microfilament fractured and its distribution disordered, plasma membrane was uneven and cell gap widened . After treatment of Rhein and Rac1 inhibitor , Rac1 protein expression and the expression of P-LIMK1 , P-PAK1 and P-cofilin notably decreased in a dose-de-pendent manner compared with the control group ( P<0. 05 ) . After Rhein and Rhein plus Rac1 inhibitor treatment ,P-LIMK1, P-cofilin, P-PAK1 protein levels of SKOV3-PM4 cells significantly decreased compared with the control group , and the group of Rac1 inhibi-tor plus Rhein treatment, the phosphorylated protein decreased more significantly ( P <0. 05 ) . After Rac1 activator plus Rhein treatment, phosphorylated protein expression of P-LIMK1 ,P-PAK1 and P-cofilin upregu-lated significantly ( P <0. 05 ) . Conclusions Rhein may be a potential inhibitor of Rac1 and can inhibit the migrating and invasive capabilities of directional high lymphatic metastasis SKOV3-PM4 cells through down-regulating the phosphorylation of Rac1/LIMK1 /cofilin pathway associated protein.

Screening Molecular Chaperones Similar to Small Heat Shock Proteins in Schizosaccharomyces pombe

To screen molecular chaperones similar to small heat shock proteins (sHsps), but without alpha-crystalline domain, heat-stable proteins from Schizosaccharomyces pombe were analyzed by 2-dimensional electrophoresis and matrix assisted laser desorption/ionization time-of-flight mass spectrometry. Sixteen proteins were identified, and four recombinant proteins, including cofilin, NTF2, pyridoxin biosynthesis protein (Snz1) and Wos2 that has an alpha-crystalline domain, were purified. Among these proteins, only Snz1 showed the anti-aggregation activity against thermal denaturation of citrate synthase. However, pre-heating of NTF2 and Wos2 at 70degrees C for 30 min, efficiently prevented thermal aggregation of citrate synthase. These results indicate that Snz1 and NTF2 possess molecular chaperone activity similar to sHsps, even though there is no alpha-crystalline domain in their sequences.

Effects of RNAi-mediated Cofilin-1 Gene Silencing on proliferation and invasiveness in hepatocellular carci-noma Huh-7 cells / 医学研究生学报

[Abstract ] Objective Cofilin-1 is involved in the pathogenesis of various tumours .However, the expression and effect of Cofilin-1 in hepatocellular carcinoma is not clear .The aim of this study is to observe the Cofilin-1 gene expression in human hepatocel-lular carcinoma (HCC) tissues, and to explore the effect of Cofilin-1 gene expression on invasion and metastasis of HCC HuH-7 cells. Methods Real-time quantitative PCR was used to assess the Cofilin-1 gene expression in human HCC tissues and normal tumor-ad-jacent tissues.The specific small interfering RNA ( siRNA) of Cofilin-1 sequence was synthetized in vitro , and was transfected into HCC HuH-7 cells using liposome transfection.The experiment was divided into Cofilin-1-siRNA group, Ctrl-siRNA group and un-transfected group.Western blot assay was used to detect the protein expression of Cofilin-1.Migration and invasion experiments in vitro were used to investigate the invasive ability of transfected cells. Results Compared with the adjacent liver tissue , Cofilin-1 gene ex-pression in human liver cancer tissue was significantly increased (0.698 ±0.156 vs 3.523 ±0.412, P and in untransfected group (0.961 ±0.020) (P<0.05).The results of migration and invasion experiments in vitro showed that the amount of migration and invasion cells in Cofilin-1-siRNA group were significantly lower than Ctrl-siRNA group or untransfected group (58.50 ±1.78 vs 79.00 ±1.33, 74.50 ±1.35,P<0.05; 36.50 ±0.83 vs 60.20 ±1.60, 51.50 ±1.14, P<0.05). Conclusion Cofilin-1 is highly expressed in HCC, and the invasion and metastasis of HCC HuH-7 cells is suppressed by inhibiting the Cofilin-1 gene expression.

Chronic ethanol exposure and withdrawal alter the expression of actin-binding protein and cyclin-dependent kinase-5 in rat brain / 中华行为医学与脑科学杂志

Objective To investigate the effects of chronic ethanol exposure and withdrawal on the expression of actin-binding protein cofilin,p-cofilin and cyclin-dependent kinase-5 (cdk5) in the nucleus accumbens and striatum in rat brain.Methods Twenty-four male SD rats were randomly divided into one control group and three experimental groups.In the experimental groups,ethanol was administered in drinking water at the concentration of 6％ (V/V) for two months.Rats in control group drank normal drinking water.After two months ethanol was removed and ethanol withdrawal syndromes were evaluated.Rats were sacrificed on withdrawal 0 h,withdrawal 6 h and withdrawal 2 d.The expression levels of cofilin,p-cofilin(ser3)and cdk5 in the rat brain were measured by immunohistochemistry methods.Results Withdrawal syndrome scores of ethanol fed rats were obviously higher than those of control rats after ethanol was removed,the highest score occurred at 6 h after ethanol withdrawal.In the nucleus accumbens area of rat brain,the levels of cofilin on withdrawal 0 h significantly decreased compared with control group ((0.31±0.05),(0.39± 0.05),P＜ 0.05).The levels of cdk5 on withdrawal 0 h and withdrawal 6 h significantly increased compared with control group((0.36±0.07),(0.34±0.07),(0.25±0.05),P＜0.05).In the striatum of rat brain,the levels of cofilin on withdrawal 0 h significantly decreased compared with control group ((0.26±0.04),(0.34±0.05),P＜0.05).The levels of p-cofilin on withdrawal 6 h significantly increased compared with control group((0.43±0.06),(0.30±0.06),P＜0.01).The levels of cdk5 on withdrawal 0 h significantly increased compared with control group((0.35±0.06),(0.26±0.05),P＜0.05),and the levels of cofilin on withdrawal 6 h significantly decreased compared with control group((0.37±0.06),(0.26±0.05),P＜0.01).Conclusion Chronic ethanol exposure can induce the development of ethanol dependence,and it accompanies with changes in the expression of actin-binding protein and cdk5 in the brain of rats.

Effect of baicalin on proliferation and migration activity in human skin SCC cells / 中国药理学通报

Aim To investigate the effect of baicalin on cell proliferation and cell migration in human skin SCC A431 cell line. Methods The A431 cells were incu- bated with 50 mg·L-1 baicalin. The protein level of cofilin-1 was assayed by Western blot. Cofilin-1 specific siRNA fragment was designed , synthesized and trans- fected into A431 cells. The proliferative activity and migration ability of cells were assessed by CCK8 assay and scratch wound healing assay separately. ResultsWestern blot results showed that baicalin treatment in-hibited the cofilin-1 protein expression to 49.3% com-pared with the control group. Single baicalin treatment and cofilin-1 silencing could drease the A431 cell growth and migration. And cofilin-1 silencing signifi- cantly enhanced the efficacy of baicalin. Conclusions Baicalin could significantly inhibit the tumor cell's growth and migration in the A431 cell line. And cofi-lin-1 might become the potential target gene to enhance the effect of anticancer drugs.

Diallyl disulfide inhibits migration and invasion in human colon cancer SW480 cells through Rac1-ADF/cofilin1 pathway / 中国肿瘤临床

Objective:This work aims to investigate diallyl disulfide (DADS) inhibition of cell migration and invasion in human colon cancer SW480 cells through the Rac1-ADF/cofilin1 pathway. Methods:The potential of cell migration and invasion was examined by scratch healing assay and transwell membrane assay. The expression of Rac1-ADF/cofilin1 pathway was detected by RT-PCR and Western blot. Results:After the SW480 cells were treated with 40 and 50 mg·L-1 of DADS for 24 h, the number of transmembrane cells through the Matrigel obviously decreased by 57.12%and 64.59%, respectively (P0.05). After the treatment with 45 mg·L-1 of DADS for 6, 12, 24, and 48 h, the expression of Rac1, Rock1, PAK1, LIMK1, and Destrin proteins respectively decreased in a time-dependent manner compared with the control group (P0.05). Moreover, the expression of p-LIMK1 and p-cofilin1 notably decreased in a time-dependent manner (P<0.05). Conclusion:DADS inhibits cell migration and invasion, which is related to the down-regulation of Rac1, Rock1, PAK1, LIMK1, p-LIMK1, p-cofilin1, and destrin through the Rac1-ADF/cofilin1 pathway.

Pak1/LIMK1/Cofilin Pathway Contributes to Tumor Migration and Invasion in Human Non-Small Cell Lung Carcinomas and Cell Lines

Squamous cell carcinoma (SCC) and adenocarcinoma (AC) are the major histological types of non-small cell lung carcinoma (NSCLC). Although both SCCs and ACs have been characterized histologically and clinically, the precise mechanisms underlying their migration and invasion are not yet known. Here, we address the involvement in NSCLC of the p21-associated kinase1 (Pak1)/LIM kinase1 (LIMK1)/cofilin pathway, which recently has been reported to play a critical role in tumor migration and invasion. The Pak1/LIMK1/cofilin pathway was evaluated in tumors from SCC (n=35) and AC (n=35) patients and in SCC- and AC-type cell lines by western blotting, immunohistochemistry, and in vitro migration and invasion assays. The levels of phosphorylated Pak1, LIMK1, and cofilin in lung tumor tissues from SCC patients were increased as compared to normal tissues. In addition, immunohistochemistry showed greater expression of phosphorylated cofilin in SCC tissues. Expression of phosphorylated Pak1 and LIMK1 proteins was also significantly higher in SCC-type cells than in AC-type cells. Moreover, migration and invasion assays revealed that a higher percentage of SCC type cells exhibited migration and invasion compared to AC type cells. Migration was also decreased in LIMK1 knockdown SK-MES-1 cells. These findings suggest that the activation of the Pak1/LIMK1/cofilin pathway could preferentially contribute to greater tumor migration and invasion in SCC, relative to that in AC.

The Interface between Cytoskeletal Aberrations and Mitochondrial Dysfunction in Alzheimer's Disease and Related Disorders

The major defining pathological hallmarks of Alzheimer's disease (AD) are the accumulations of Abeta in senile plaques and hyperphosphorylated tau in neurofibrillary tangles and neuropil threads. Recent studies indicate that rather than these insoluble lesions, the soluble Abeta oligomers and hyperphosphorylated tau are the toxic agents of AD pathology. Such pathological protein species are accompanied by cytoskeletal changes, mitochondrial dysfunction, Ca2+ dysregulation, and oxidative stress. In this review, we discuss how the binding of Abeta to various integrins, defects in downstream focal adhesion signaling, and activation of cofilin can impact mitochondrial dysfunction, cytoskeletal changes, and tau pathology induced by Abeta oligomers. Such pathological consequences can also feedback to further activate cofilin to promote cofilin pathology. We also suggest that the mechanism of Abeta generation by the endocytosis of APP is mechanistically linked with perturbations in integrin-based focal adhesion signaling, as APP, LRP, and beta-integrins are physically associated with each other.

Relationship between cofilin-1 expression and implantation capacity in eutopic endometrium of patient with endometriosis / 中华妇产科杂志

Objective To investigate the relationship between expression of eofilin-1 and in vivo implantation capacity of eutopic endometrium of endometriosis.Methods Eutopic endometrium of 20 cases with stage Ⅲ or Ⅳ endometriosis were obtained by laparoscopic or laparotomy surgery (endometriosis group) matched with 20 cases of eutopic endometrium from patients with cervical cancer in situ (control group) in Department of Obstetrics and Gynecology,ShengJing Hospital.All cases' eutopic endometrium were collected and injected into abdominal cavity of nude mice to establish endometriosis animal model, then the successful rate of animal model and volume of endometriosis lesion were calculated.The expression and positive rate of eofilin-1 protein were measured by western blot and immunohistochemistry staining.Results The mean volume of endometriosis lesions (2.38 ± 0.22)mm~3 in endometriosis group was significantly bigger than (0.36 ± 0.08) mm~3 in control group (P < 0.05).The successful rate of establishing endometriosis model was 95% (19/20) in endometriosis group and 5% (1/20)in control group, which reached statistical difference(P <0.05).The expression and positive rate of cofilin-1 protein in eutopic endometrium and the expression of cofilin-1 protein in endometriosis lesion of animal model were 0.82±0.06, 90% (18/20), 0.85 ± 0.03 and 0.21 ± 0.03, 20% (4/20), 0.22 ± 0.02 in control group, which reached statistical difference(P <0.05).The successful rate of establishing endometriosis model with positive cofilin-1 in endometrium 86% (19/22) was significantly higher than 6% (1/16) of model's endometrium with negative cofilin-1 expression(P <0.05).Conclusions Implanting capacity and cofilin-1 expression level of eutopic endometrium of endometriosis were more intensive than that of normal endometrium.High cofilin-1 expression was probably related with implanting capacity of eutopic endometrium of endometriosis.

Propofol Inhibits Platelet-derived Growth Factor-stimulated Migration in Rat Aortic Smooth Muscle Cells / 대한마취과학회지

BACKGROUND: Propofol is the extensively used general anesthetic-sedative agent.Although propofol is known to be involved in migration of various cells, migration response to it in vascular smooth muscle cells is not investigated. This study was carried out to determine the role of propofol in migration of rat aortic smooth muscle cells (RASMCs). METHODS: A7r5 RASMCs were used.Cell migration was examined by the analysis of 5 ng/ml of platelet-derived growth factor (PDGF)-induced RASMC response after treatment of cells with propofol (1-100micrometer) in the Boyden chamber.The activity of cofilin by propofol in RASMCs was measured by the Western blot analysis for the change of cofilin dephosphorylaton in cells treated with 10micrometer propofol for 5, 10, 15 and 20 min, for the effect of propofol (1, 10 and 100micrometer) on cofilin phosphorylation, and for the effects of ethylene glycol-bis (beta-aminoethyl ether)-N,N,N',N'-tetra acetic acid (2 mM; EGTA), Na3VO4 (200micrometer), and calyculin A (10 nM) on 10micrometer propofol-induced cofilin dephosphorylation. RESULTS: PDGF increased RASMC migration and this response was dose-dependently inhibited by treatment with propofol. Propofol attenuated the cofilin phosphorylation in RASMCs in a dose- and time-dependent manner.Propofol-induced dephosphorylation of cofilin in RASMCs was abolished by calyculin A, a protein phosphatase 2A inhibitor, but not by EGTA, a Ca2+ chelating agent, or Na3VO4, a protein tyrosine phosphatase inhibitor. CONCLUSIONS: The present results suggest that propofol induces the diminution of PDGF-stimulated RASMC migration and this response may be associated with dephosphorylation of cofilin mediated by the protein phosphatase 2A-dependent pathway.