Electroacupuncture inhibits dendritic spine remodeling through the srGAP3-Rac1 signaling pathway in rats with SNL

Previous studies have shown that peripheral nerve injury can lead to abnormal dendritic spine remodeling in spinal dorsal horn neurons. Inhibition of abnormal dendritic spine remodeling can relieve neuropathic pain. Electroacupuncture (EA) has a beneficial effect on the treatment of neuropathic pain, but the specific mechanism remains unclear. Evidence has shown that slit-robo GTPase activating protein 3 (srGAP3) and Rho GTPase (Rac1) play very important roles in dendritic spine remodeling. Here, we used srGAP3 siRNA and Rac1 activator CN04 to confirm the relationship between SrGAP3 and Rac1 and their roles in improving neuropathic pain with EA. Spinal nerve ligation (SNL) was used as the experimental model, and thermal withdrawal latency (TWL), mechanical withdrawal threshold (MWT), Western blotting, immunohistochemistry and Golgi-Cox staining were used to examine changes in behavioral performance, protein expression and dendritic spines. More dendritic spines and higher expression levels of srGAP3 were found in the initial phase of neuropathic pain. During the maintenance phase, dendritic spines were more mature, which was consistent with lower expression levels of srGAP3 and higher expression levels of Rac1-GTP. EA during the maintenance phase reduced the density and maturity of dendritic spines of rats with SNL, increased the levels of srGAP3 and reduced the levels of Rac1-GTP, while srGAP3 siRNA and CN04 reversed the therapeutic effects of EA. These results suggest that dendritic spines have different manifestations in different stages of neuropathic pain and that EA may inhibit the abnormal dendritic spine remodeling by regulating the srGAP3/Rac1 signaling pathway to alleviate neuropathic pain.

Relationship between long-term learning and memory impairment induced by sevoflurane anesthesia and PSD-95/Kalirin-7/Rac1 signaling pathway in neonatal rats / 中华麻醉学杂志

Objective:To evaluate the relationship between long-term learning and memory impairment induced by sevoflurane anesthesia and postsynaptic density protein-95 (PSD-95)/Kalirin-7/Ras-related C3 botulinum toxin substrate 1 (Rac1) signaling pathway in neonatal rats.Methods:Sixty SPF male Wistar rats, aged 7 days, weighing 12-18 g, were divided into 5 groups ( n=12 each) using a random number table method: control group (group C), 1% sevoflurane anesthesia for 2 h group (group S 1), 1% sevoflurane anesthesia for 4 h group (group S 2), 2% sevoflurane anesthesia for 2 h group (group S 3) and 2% sevoflurane anesthesia for 4 h group (group S 4). Morris water maze test was performed at 4, 8 and 12 weeks after anesthesia.The rats were sacrificed after the last Morris water maze test, and the hippocampal tissues were obtained for microscopic examination of the pathological changes (using HE staining), neuron apoptosis (by TUNEL staining), and expression of PSD-95, Kalirin-7 and Rac1 protein and mRNA (by Western blot and quantitative real-time polymerase chain reaction). The apoptosis rate was calculated. Results:Compared with group C, the escape latency was significantly prolonged, the number of crossing the original platform was reduced, the time of stay in the target quadrant was shortened, and the apoptosis rate of hippocampal neurons was increased at 4th, 8th and 12th weeks after anesthesia, phosphorylated Rac1/Rac1 ratio was decreased, and the expression of PSD-95 and Kalirin-7 protein and mRNA was down-regulated in S 1, S 2, S 3 and S 4 groups ( P<0.05). Compared with group S 4, the escape latency was significantly shortened, the number of crossing the original platform was increased, the time of stay in the target quadrant was prolonged, and the apoptosis rate of hippocampal neurons was decreased, phosphorylated Rac1/Rac1 ratio was increased, the expression of PSD-95 and Kalirin-7 protein and mRNA was up-regulated, and the histopathological changes of hippocampal tissues were attenuated in S 1, S 2 and S 3 groups ( P<0.05). Conclusions:The mechanism by which sevoflurane anesthesia induces long-term learning and memory impairment may be related to inhibition of activity of PSD-95/Kalirin-7/Rac1 signaling pathway in hippocampi of neonatal rats.

Piperine suppresses growth and migration of human breast cancer cells through attenuation of Rac1 expression

Objective: To investigate the effect of piperine on human breast cancer cells. Methods: The effect of piperine on proliferation and migration of human breast cancer cells, MCF-7 and MDA-MB-231, was investigated using colony formation assays, wound healing assays, Matrigel migration assays, flow cytometry, RT-qPCR, and Western blotting assays. Results: Piperine inhibited the growth of MCF-7 and MDA-MB-231 cells and suppressed colony formation. Cell reduction at the G 0 / G 1 phase and cell arrest at the G 2 /M phase were observed in breast cancer cells. However, the significant effect was only demonstrated in MDA-MB-231 cells. Moreover, cancer cell migration was suppressed by piperine at low concentration. RT-qPCR and Western blotting assays showed that piperine downregulated Rac1 gene and protein expression. Conclusions: Piperine could inhibit growth and migration of breast cancer cells by reducing Rac1 gene and protein expression.

Rac1 Signaling in Amygdala Astrocytes Regulates Fear Memory Acquisition and Retrieval / 神经科学通报·英文版

The importance of astrocytes in behavior control is increasingly appreciated, but little is known about the effects of their dynamic activity in regulating learning and memory. In the present study, we constructed AAVs of photoactivatable and photoinactivatable Ras-related C3 botulinum toxin substrate 1 (Rac1) under the mGFAP promoter, which enabled the manipulation of Rac1 activity in astrocytes by optical stimulation in free-moving mice. We found that both up-regulation and down-regulation of astrocytic Rac1 activity in the basolateral amygdala (BLA) attenuated memory acquisition in a fear conditioning mouse model. Meanwhile, neuronal activation in the BLA induced by memory acquisition was inhibited under both the up- and down-regulation of astrocytic Rac1 activity during training. In terms of the impact on fear memory retrieval, we found both up- and down-regulation of BLA astrocytic Rac1 activity impaired memory retrieval of fear conditioning and memory retrieval-induced neuronal activation. Notably, the effect of astrocytic Rac1 on memory retrieval was reversible. Our results demonstrate that the normal activity of astrocytic Rac1 is necessary for the activation of neurons and memory formation. Both activation and inactivation of astrocytic Rac1 activity in the BLA reduced the excitability of neurons, and thereby impaired fear memory acquisition and retrieval.

Afzelin inhibits migration of MDA-MB-231 cells by suppressing FAK expression and Rac1 activation

Triple-negative breast cancer (TNBC) has the worst prognosis and the highest rate of metastasis among other types ofbreast cancer. These characteristics are supported by the dysregulation of focal adhesion kinase (FAK) and Rac1 whichare the key players of mesenchymal cell migration on TNBC. Afzelin is a secondary metabolite that is contained ina variety of plants. This study explored the anti-migration effect of afzelin and its interaction with FAK and Rac1 onthe highly invasive TNBC cell line, MDA-MB-231. Cell viability was assessed by 3-(4,5-dimethyl 2-thiazolyl)-2,5-diphenyltetrazolium bromide assay, and cell migration was evaluated using in vitro scratch assay. Rac1 activation wasanalyzed using the colorimetric assay, while vinculin and actin filaments were stained through immunofluorescence. Thequantity of total FAK and phosphorylated FAK tyr397 was detected by Western blotting. Afzelin decreased cell viabilityand inhibited two-dimensional cell migration in a dose-dependent manner. Under confocal laser scanning microscopy,vinculin localization at the cell edge demonstrated a reduction of focal adhesion formation by afzelin. Further explorationshowed that afzelin decreased FAK expression but did not affect FAK phosphorylation at tyr397. In addition, afzelindecreased Rac1-GTPase activation, which is a downstream effector of FAK. Taken together, these results suggest thatafzelin suppresses TNBC cell migration, through inhibition of FAK expression and Rac1-GTPase activation.

Inhibition of chemotherapy-related breast tumor EMT by application of redox-sensitive siRNA delivery system CSO-ss-SA/siRNA along with doxorubicin treatment / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Metastasis is one of the main reasons causing death in cancer patients. It was reported that chemotherapy might induce metastasis. In order to uncover the mechanism of chemotherapy-induced metastasis and find solutions to inhibit treatment-induced metastasis, the relationship between epithelial-mesenchymal transition (EMT) and doxorubicin (DOX) treatment was investigated and a redox-sensitive small interfering RNA (siRNA) delivery system was designed. DOX-related reactive oxygen species (ROS) were found to be responsible for the invasiveness of tumor cells in vitro, causing enhanced EMT and cytoskeleton reconstruction regulated by Ras-related C3 botulinum toxin substrate 1 (RAC1). In order to decrease RAC1, a redox-sensitive glycolipid drug delivery system (chitosan-ss-stearylamine conjugate (CSO-ss-SA)) was designed to carry siRNA, forming a gene delivery system (CSO-ss-SA/siRNA) downregulating RAC1. CSO-ss-SA/siRNA exhibited an enhanced redox sensitivity compared to nonresponsive complexes in 10 mmol/L glutathione (GSH) and showed a significant safety. CSO-ss-SA/siRNA could effectively transmit siRNA into tumor cells, reducing the expression of RAC1 protein by 38.2% and decreasing the number of tumor-induced invasion cells by 42.5%. When combined with DOX, CSO-ss-SA/siRNA remarkably inhibited the chemotherapy-induced EMT in vivo and enhanced therapeutic efficiency. The present study indicates that RAC1 protein is a key regulator of chemotherapy-induced EMT and CSO-ss-SA/siRNA silencing RAC1 could efficiently decrease the tumor metastasis risk after chemotherapy.

ARHGEF16 variants screening and mutation function analysis for children with total anomalous pulmonary venous connection / 上海交通大学学报（医学版）

Objective:To investigate the pathogenesis of total anomalous pulmonary venous connection (TAPVC), and to identify ARHGEF16 gene through full exon sequencing screening and analyze its mutation function. Methods:The blood, clinical data and auxiliary examination results of 78 children with TAPVC and 100 healthy controls were collected, and the genomic DNA was extracted for ARHGEF16 mutation screening. ARHGEF16 wild-type and mutant expression plasmids were constructed and transfected into 293T cells. mRNA and protein expression levels were detected by quantitative real-time PCR (RT-qPCR) and Western blotting, respectively. Protein-protein interaction exploration was performed by Cytoscape software. Results:Two novel variants c.C236>T (A79V) and c.G619>C (G207R) were found in TAPVC patients and were not found in healthy controls. Compared with the wild type, the mutants ARHGEF16 were up-regulated in both mRNA and protein expression levels. Protein interaction analysis showed that ARHGEF16 and RAC1 were directly associated; RAC1 expression was up-regulated in HEK293 cells with ARHGEF16 overexpression through RT-qPCR. Conclusion:The missense mutations of ARHGEF16 affect the mRNA and protein expression levels of ARHGEF16. Overexpression of ARHGEF16 up-regulates the expression of RAC1, suggesting that it may participate in the development and formation of TAPVC by regulating RAC1.

Dose-and Time-Dependent Suppression of Rac1 and STIM1 in Acute Myeloid Leukaemia Cell Line Model

@#Introduction: Rac1 and STIM1 genes are emerging therapeutic targets for cancers. However, their roles in acute myeloid leukaemia (AML) are not well understood. The goal of this study was to evaluate the effects of dose and time on Rac1 and STIM1 knockdown in the AML cell line model (THP-1 cells). Methods: THP-1 cells were transfected with siRac1 at doses of 50, 100, and 200 nM or dsiSTIM1 at doses of 2, 5, and 10 nM. Expression level of Rac1 and STIM1 then were assessed at time points between 12 and 72 h post-transfection using real-time reverse transcription polymerase chain reaction. Results: Compared to the control, 87% Rac1 knockdown was attained with 50 nM siRac1 at 24 h post-transfection, and 70% STIM1 knockdown was achieved with 10 nM dsiSTIM1 at 48 h post-transfection. Conclusion: These results show that effective knockdown of Rac1 and STIM1 is possible, and therapy that includes Rac1 and STIM1 inhibitors eventually could provide a new and highly effective strategy for AML treatment.

Inhibition of chemotherapy-related breast tumor EMT by application of redox-sensitive siRNA delivery system CSO-ss-SA/siRNA along with doxorubicin treatment / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Metastasis is one of the main reasons causing death in cancer patients. It was reported that chemotherapy might induce metastasis. In order to uncover the mechanism of chemotherapy-induced metastasis and find solutions to inhibit treatment-induced metastasis, the relationship between epithelial-mesenchymal transition (EMT) and doxorubicin (DOX) treatment was investigated and a redox-sensitive small interfering RNA (siRNA) delivery system was designed. DOX-related reactive oxygen species (ROS) were found to be responsible for the invasiveness of tumor cells in vitro, causing enhanced EMT and cytoskeleton reconstruction regulated by Ras-related C3 botulinum toxin substrate 1 (RAC1). In order to decrease RAC1, a redox-sensitive glycolipid drug delivery system (chitosan-ss-stearylamine conjugate (CSO-ss-SA)) was designed to carry siRNA, forming a gene delivery system (CSO-ss-SA/siRNA) down-regulating RAC1. CSO-ss-SA/siRNA exhibited an enhanced redox sensitivity compared to nonresponsive complexes in 10 mmol/L glutathione (GSH) and showed a significant safety. CSO-ss-SA/siRNA could effectively transmit siRNA into tumor cells, reducing the expression of RAC1 protein by 38.2% and decreasing the number of tumor-induced invasion cells by 42.5%. When combined with DOX, CSO-ss-SA/siRNA remarkably inhibited the chemotherapy-induced EMT in vivo and enhanced therapeutic efficiency. The present study indicates that RAC1 protein is a key regulator of chemotherapy-induced EMT and CSO-ss-SA/siRNA silencing RAC1 could efficiently decrease the tumor metastasis risk after chemotherapy.

miR-223-3p regulates proliferation and apoptosis of hepatocellular carcinoma SMMC-7721 cells by targeting RAC1 / 中国肿瘤生物治疗杂志

@#[Abstract] Objective: To investigate the effects of miR-223-3p on the proliferation and apoptosis of hepatocellular carcinoma (HCC) cells by regulating Ras-related C3 botulinum toxin substrate 1 (RAC1) and its possible mechanism. Methods: Thirty pairs of HCC and corresponding para-cancer tissues resected in Jilin Central Hospital from August 2016 to August 2018 were collected for this study; in addition, human HCC cell lines SMMC-7721, BEL-7402, HepG2 and human normal hepatocyte QSG-7701 were also collected. The expression level of miR-223-3p in HCC tissue and cell lines was detected by qPCR. miR-223-3p mimics, miR-223-3p inhibitor and siRAC1 were transfected into SMMC-7221 cells, respectively. CCK-8 assay, Colony formation assay and Annexin V-FITC/PI staining Flow cytometry were used to detect the proliferation, clone formation and apoptosis of SMMC-7721 cells, respectively. The relationship between miR-223-3p and RAC1 was confirmed by Dual luciferase reporter gene assay. The protein level of RAC1 in SMMC-7721 cells was detected by Western blotting. Results: The expression of miR-223-3p in HCC tissues was significantly lower than that in paracaner tissues (P<0.01), and had significant correlation with pathological characteristics, such as tumor size, TNM stage, EdmondsonSteiner grade (all P<0.05 or P<0.01). miR-223-3p expression in HCC cell lines was significantly lower than that in QSG-7701 cells with the lowest expression in SMMC-7721 cells. Dual luciferase reporter gene assay confirmed that RAC1 was a target gene of miR-223-3p, and miR-223-3p negatively regulated RAC1 expression. Over-expression of miR-223-3p significantly inhibited the proliferation and colony formation (P<0.05 or P<0.01) of SMMC-7721 cells and promoted cell apoptosis (P<0.01). Contrarily, knockdown of miR-223-3p reversed the inhibitory effect of miR-223-3p mimics on cells. Conclusion: miR-223-3p over-expression inhibits proliferation and colony formation and promotes apoptosis of HCC cells, the mechanism of which may be related with its targeted down-regulation of RAC1.

MicroRNA-34a promotes the senescence and apoptosis of human lens epithelial cells by up-regulating Cdc42 and Rac1 / 国际眼科杂志(Guoji Yanke Zazhi)

@#AIM: To observe the effect and mechanism of MicroRNA-34a on senescence and apoptosis of human lens epithelial cell line SRA01/04.<p>METHODS: MicroRNA-34a expression levels in ARC lens and transparent lens epithelial cells were detected by qRT-PCR. MicroRNA-34a mimics, MicroRNA-34a inhibitors and empty liposome(control group)were transfected into SRA01/04 cells by liposome transfection kit. Annexin V-FITC/PI double staining was used to detect the effect of MicroRNA-34a on the apoptosis of human lens cell line SRA01/04. The expression of Cdc42 and Rac1 protein was detected by western blot. <p>RESULTS: The expression level of MicroRNA-34a in anterior capsular tissue of transparent lens was significantly lower than that in ARC anterior capsular tissue(<i>P</i><0.05). The positive rates of SA-β-gal in the MicroRNA-34a mimics group, the control group and the MicroRNA-34a inhibitors group were(87.56±2.34)%,(12.22±2.74)% and(3.45±0.45)%, respectively. The positive rates of SA-β-gal in the MicroRNA-34a mimics group was significantly higher than the control group, while the SA-β-gal positive rate in the MicroRNA-34a inhibitors group was significantly lower than that in the control group(<i>P</i><0.05). The apoptosis rate of the MicroRNA-34a inhibitors group, control group and MicroRNA-34a mimics group were(5.87±1.22)%,(12.26±2.14)% and(29.45±3.12)%, respectively. The apoptosis rate of the MicroRNA-34a mimics group was significantly higher than that of the control group, while that of the MicroRNA-34a inhibitors group was significantly lower than that of the control group(<i>P</i><0.05). The expressions of Cdc42 and Rac1 in the MicroRNA-34a mimics group were significantly higher than those in the control group(<i>P</i><0.05), while the expressions of Cdc42 and Rac1 in the MicroRNA-34a inhibitors group were significantly lower than those in the control group(<i>P</i><0.05).<p>CONCLUSION: MicroRNA-34a may promote the senescence and apoptosis of human lens epithelial cells by up-regulating Cdc42 and Rac1.

Construction of luciferase reporter gene cell model and its application in screening rhein derivatives targeting RAC1 / 中国药理学通报

Aim To establish luciferase reporter gene expression cell models of CNEi-RACI-Luc2 based on the target of RAC1 promoter, and explore the application of screening anti-tumor active of rhein derivatives targeted regulating RAC1 at transcriptional level. Methods The lentiviral carrying luciferase reporter vector was designed and synthesized using RAC1 promoter se-quence, and CNE1 cells were infected with recombinant plasmid lentiviral to obtain cell lines that stably expressed firefly luciferase. Luciferase reporter assay was used to detect the luciferase luminescence value after stimulating with RAC1 activator PMA and inhibitor NSC23766 that targeted regulating the RAC1 promoter activities in cells, and RAC 1 expression was verified by Western blot. The effect of series of rhein derivatives on the lu-cifcrase activity of RAC1 promoter was observed, and RAC1 expression was determined by Western blot. Results The identification result of double enzyme digestion showed that a lentiviral expression vector carrying luciferase reporter vector rc-combined with RAC1 promoter was successfully constructed. Lcntivirus-infcctcd CNE1 cells were screened by puromycin, the CNE1-RAC1-Luc2 cells stably expressing firefly luciferase were obtained, and the Iransfection efficiency was over 90%. The RAC1 luciferase reporter assay system was sensitive to FMA and NSC23766 and consistent with the result of RAC1 protein expression by Western blot. The regulation of series of rhein derivatives to RAC1 luciferase activity of CNE1-RAC1-Luc2 cells was consistent with the results of Western blot. Conclusions The cell model of luciferase reporting system containing RAC1 promoter can be successfully constructed, which provides a practical platform for high throughput screening of RAC1-targeted drugs.

Rac1 Modulates Excitatory Synaptic Transmission in Mouse Retinal Ganglion Cells / 神经科学通报·英文版

Ras-related C3 botulinum toxin substrate 1 (Rac1), a member of the Rho GTPase family which plays important roles in dendritic spine morphology and plasticity, is a key regulator of cytoskeletal reorganization in dendrites and spines. Here, we investigated whether and how Rac1 modulates synaptic transmission in mouse retinal ganglion cells (RGCs) using selective conditional knockout of Rac1 (Rac1-cKO). Rac1-cKO significantly reduced the frequency of AMPA receptor-mediated miniature excitatory postsynaptic currents, while glycine/GABA receptor-mediated miniature inhibitory postsynaptic currents were not affected. Although the total GluA1 protein level was increased in Rac1-cKO mice, its expression in the membrane component was unchanged. Rac1-cKO did not affect spine-like branch density in single dendrites, but significantly reduced the dendritic complexity, which resulted in a decrease in the total number of dendritic spine-like branches. These results suggest that Rac1 selectively affects excitatory synaptic transmission in RGCs by modulating dendritic complexity.

Inhibition of Rac1-dependent forgetting alleviates memory deficits in animal models of Alzheimer's disease

Accelerated forgetting has been identified as a feature of Alzheimer's disease (AD), but the therapeutic efficacy of the manipulation of biological mechanisms of forgetting has not been assessed in AD animal models. Ras-related C3 botulinum toxin substrate 1 (Rac1), a small GTPase, has been shown to regulate active forgetting in Drosophila and mice. Here, we showed that Rac1 activity is aberrantly elevated in the hippocampal tissues of AD patients and AD animal models. Moreover, amyloid-beta 42 could induce Rac1 activation in cultured cells. The elevation of Rac1 activity not only accelerated 6-hour spatial memory decay in 3-month-old APP/PS1 mice, but also significantly contributed to severe memory loss in aged APP/PS1 mice. A similar age-dependent Rac1 activity-based memory loss was also observed in an AD fly model. Moreover, inhibition of Rac1 activity could ameliorate cognitive defects and synaptic plasticity in AD animal models. Finally, two novel compounds, identified through behavioral screening of a randomly selected pool of brain permeable small molecules for their positive effect in rescuing memory loss in both fly and mouse models, were found to be capable of inhibiting Rac1 activity. Thus, multiple lines of evidence corroborate in supporting the idea that inhibition of Rac1 activity is effective for treating AD-related memory loss.

Research progress of relevant signals and mechanisms in clearance of apopto-sis cells / 中国免疫学杂志

Our bodies produce a lot of apoptotic cells every day,and the timely removal of these apoptotic cells is essential to maintain the immune balance of the body. In the removal of apoptotic cells,macrophages play a major role,and their removal process is divided into three stages:recruitment,identification and phagocytosis. In the recruitment stage,apoptotic cells secrete′find me′signals, and phagocytes respond and are recruited to apoptotic cells. At the identification stage, the ′eat-me′ signal of apoptotic cells was identified with the phagocytic receptor on the surface of phagocytes. Phagocytic phase, the ′eat-me′ signal transmits signals to macrophages,such as activating the small GTPase Rac1,which leads to actin polymerization and cytoskeleton rearrangement to promote the phagocytosis of apoptotic cells. If the removal mechanism of apoptotic cells is obstructive,the apoptotic cells that have not been cleared in time will enter the secondary necrotic state and release the self-antigens. These self-antigens may stimulate the body′s immune system to produce autoantibodies,leading to autoimmune diseases such as SLE. The research progress of the macrophage on the removal mechanism of apoptosis cells is reviewed in this paper.

Role of Rac1 signaling pathway of azathioprine and peptidoglycan in the regulation of monocyte-macrophage apoptosis in Crohn's disease / 中华内科杂志

Objective To evaluate the changes of macrophages and expression of Rac1 in the inflammatory site of Crohn's disease,and to investigate the effects of 6-thioguanine (6-TG) and peptidoglycan on apoptosis of human peripheral blood monocyte-macrophage by regulating Rac1 signaling pathway.Methods Ten patients with Crohn's disease and eight healthy controls diagnosed were enrolled at Department of Gastroenterology and Hepatology,Tianjin Medical University General Hospital from January 2013 to January 2014.The number of macrophages,apoptosis and expression of Rac1 in the inflammation sites and non-inflammation sites of intestinal mucosa were detected in both patients and controls.Peripheral blood mononuclear cells (PBMCs) were sorted by CD14 immunomagnetic beads.The apoptosis of monocytes,expression of Rac1 and related apoptosis signaling molecules were detected in patients treated with peptidoglycan,6-TG and Rac1 inhibitor NSC23766 and another 15 healthy donors.Results The number of macrophages and apoptotic cells significantly increased in the inflammatory group of Crohn's disease patients compared with the non-inflammatory group.The expression of PAK1,downstream molecular of Rac1 signaling pathway of macrophages was also significantly higher in the inflammatory group of Crohn's disease patients than that in healthy controls and non-inflammatory group.Compared with control group,anti-apoptotic signals (NF-κB,Bcl-xL and STAT-3) in PBMCs increased in the peptidoglycan group,while slightly decreased in 6-TG group.6-TG and NSC23766 significantly promoted peptidoglycan-related anti-apoptosis [peptidoglycan group (8.6±3.7)％,peptidoglycan + 6-TG group (42.0±2.7)％,peptidoglycan + NSC23766 group (58.5±6.9)％,P＜0.05].Conclusions Peptidoglycan plays a role in the pathogenesis of Crohn's disease by recruiting macrophages.However,6-TG inhibits peptidoglycan-induced activation of Rac 1 signaling pathway leading to macrophage apoptosis.

Expression ofβ1 integrin, Rac1, and RhoA in invasive micropapillary breast carcinoma / 中国肿瘤临床

Objective:To investigate the expression and significance ofβ1 integrin, Rac1, and RhoA in invasive micropapillary breast carcinoma (IMPC). Methods:Immunohistochemical staining was performed to detect the expression ofβ1 integrin, Rac1, and RhoA in 89 patients with IMPC and 90 patients with invasive ductal carcinoma-not otherwise specified (IDC-NOS) who were treated between January 2007 and December 2008 in Tianjin Medical University Cancer Institute and Hospital. The relationship among the three proteins and the expression ofβ1 integrin, Rac1, and RhoA with clinicopathological features were determined. Results:β1 integrin (78.7%) and Rac1 (76.4%) were highly expressed in patients with IMPC. This expression was significantly higher than that in patients with IDC-NOS (63.3%and 54.4%). Statistical difference was found between the two groups (P0.05). Conclusion:Thus,β1 integrin, Rac1, and RhoA were overexpressed and might play an important role in the high frequency of metastasis in patients with IMPC. These proteins could be considered as biomarkers for the prognosis and new targets for IMPC therapy.

Effects of Wnt5A gene overexpression on cytoskeletal proteins of melanocytes / 中华皮肤科杂志

Objective To evaluate the effect of Wnt5A gene overexpression on cytoskeletal proteins of melanocytes after the plasmid containing the Wnt5A gene is transfected into primary melanocytes.Methods In vitro cultured primary human melanocytes were divided into three groups:blank control group receiving no treatment,negative control group transfected with endotoxin-free pcDNA3.1 (+)empty vector by Lipo3000 in Opti-MEM medium,Wnt5A plasnid group transfected with endotoxin-free pcDNA3.1 (+) vector containing the Wnt5A gene by Lipo3000 in Opti-MEM medium.After the transfection,quantitative PCR (qPCR) was performed to measure the mRNA expression of Wnt5A,ras-related C3 botulinum toxin substrate 1 (Rac1),filamentous actin (F-actin) and β-tubulin,Western blot analysis to determine the protein expression of Wnt5A,receptor tyrosine kinase like orphan receptor 2 (ROR2),Rac1,F-actin and β-tubulin,and an immunofluorescence assay (IFA) to observe the expression of cytoskeletal proteins.Results qPCR showed significant differences in the mRNA expression of the Wnt5A gene and its downstream genes Rac1 and F-actin among the Wnt5A plasmid group,negative control group and blank control group (F =1374.179,112.576,66.458,respectively,all P ＜ 0.01),but there was no significant difference in the mRNA expression of β-tubulin among the three groups (P ＞ 0.05).Additionally,the Wnt5A plasmid group showed significantly higher mRNA expression of Wnt5A,Rac1 and F-actin compared with the blank control group and negative control group (all P ＜ 0.05).As Western blot analysis revealed,compared with the blank control group and negative control group,the Wnt5A plasmid group showed significantly higher Wnt5A protein expression (both P ＜ 0.05),but significantly lower protein expression of Rac 1,ROR2 and F-actin (all P ＜ 0.05).However,no significant difference in β-tubulin protein expression was observed among the three groups (P ＞ 0.05).IFA showed no obvious difference in the fluorescence intensity of β-tubulin or F-actin between the Wnt5A group and the two control groups,but melanocytes showed larger size and increased number of dendrites,and the cytoskeleton changed dramatically with varying fluorescence intensity of F-actin,fuzzy texture,fractured or locally clustered tonofilaments in the Wnt5A group.Conclusion The overexpression of the Wnt5A gene in melanocytes can regulate the mRNA and protein expression of cytoskeletal proteins,nake melanocytes larger and more dendritic,and cause changes in the cytoskeleton,which may facilitate the transportation of melanosomes,and participate in the occurrence of hyperpigmented diseases.

Study on expression and correlation of Rac1 and E-cadherin protein in oral squamous cell carcinoma / 天津医药

Objective To investigate the expression levels of Rac1 and E-cadherin and their correlation in oral squamous cell carcinoma, and to explore their relations to clinical pathological parameters. Methods The expression levels of Rac1 and E-cadherin were detected in 22 samples of oral squamous cell carcinoma and 8 samples of para-carcinoma tissue by using immunohistochemistry technique (SP). The patients were divided into two groups according to the clinicopathological parameters. The differences of Rac1 and E-cadherin expression levels were analyzed between the groups. Results The positive expression rates of Rac1 were 1/8 and 77.3%(17/22) in para-carcinoma tissue and oral squamous cell carcinoma, respectively. The difference was statistically significant (P=0.003). The positive expression rates of E-cadherin were 8/8 and 31.8%(7/22), and there was a difference between them (P=0.002). The Rac1 protein expression rate was increased and E-cadherin was decreased in oral squamous cell carcinoma. There were no significant differences in Rac 1 and E-cadherin between different age and gender groups. There was significant difference in Rac1 expression between patients with metastasis and patients without metastasis (P=0.021). While there was no difference in E-cadherin expression between patients with metastasis and patients without metastasis. No correlation was found between Rac 1 and E-cadherin expressions in oral squamous cell carcinoma. Conclusion The high expression of Rac1 and low expression of E-cadherin may play an important role in the tumorigenesis and progression of oral squamous cell carcinoma.

Role of phosphorylated Moesin in the injury of pulmonary microvascular endothelial cells of rats and its mechanism / 中华危重病急救医学

Objective To investigate the role of phosphorylated Moesin (p-Moesin) in the injury of pulmonary microvascular endothelial cells (PMVECs) of rats induced by tumor necrosis factor-α (TNF-α), and to approach the impact of Rac1 signal pathway on Moesin phosphorylation.Methods PMVECs of rats were culturedin vitroand passed on to the third generation, and the TNF-α time-effect experiment, dose-effect experiment and Rac1 signaling pathway intervention experiment were performed respectively. ① Time-effect experiment: PMVECs were stimulated with 10μg/L TNF-α for 0, 15, 30 minutes and 1, 3, 6, 12 hours, and the protein expressions of Moesin and p-Moesin were determined by Western Blot. ② Dose-effect experiment: PMVECs were stimulated with 0, 0.1, 1, 10μg/L TNF-α for 6 hours, and the protein expressions of Moesin and p-Moesin were determined by Western Blot. ③ Rac1 signaling pathway intervention experiment: PMVECs were divided into two parts, which were pretreated with 3 mL Rac1 specific inhibitor NSC23766 (200μmol/L) for 0.5 hour or Rac1 specific agonist O-Me-cAMP (200μmol/L) for 1 hour, respectively, and then incubated with 10μg/L TNF-α for 6 hours. The PMVECs without treatment were served as blank control group, andthose were treated with only O-Me-cAMP, NSC23766 or TNF-α were served as corresponding groups. The protein expressions of Moesin and p-Moesin were determined by Western Blot.Results ① Time-effect experiment results: the expression of Moesin showed no change among all time points after 10μg/L TNF-α stimulated PMVECs. But the expression of p-Moesin was sharply up-regulated at 15 minutes after TNF-α stimulation as compared with 0 minute (p-Moesin/Moesin: 4.399±0.523 vs. 1.000±0.195), peaked at 30 minutes (6.069±0.557), and then gradually decreased after 1 hour (5.005±0.544, 4.599±0.478, 1.742±0.288, 1.503±0.352 at 1, 3, 6, 12 hours, respectively) with significant difference among all time points (F = 15.397,P = 0.002). ② Dose-effect experiment results: no significant change in expression of Moesin was found among all doses of TNF-α incubated with PMVECs for 6 hours. But the expression of p-Moesin was significantly up-regulated after TNF-α stimulation with 0.1μg/L as compared with0μg/L (p-Moesin/Moesin: 2.194±0.430 vs. 1.000±0.273), which showed an upward trend with dose increase in TNF-α (3.201±0.688 and 4.413±0.296 with 1μg/L and 10μg/L TNF-α, respectively) with significant difference among all doses (F = 92.513,P < 0.001). ③ Rac1 signaling pathway intervention experiment results: there was no significant difference in Moesin expression among all the groups. Compared with blank control group, Rac1 specific agonist O-Me-cAMP or Rac1 specific inhibitor NSC23766 alone could not change the expression of p-Moesin, while TNF-α could induce p-Moesin expression. Compared with TNF-α group, the expression of p-Moesin induced by TNF-α was up-regulated by NSC23766 (p-Moesin/Moesin: 2.612±0.355 vs. 1.911±0.297,P < 0.05), and it was attenuated by O-Me-cAMP (p-Moesin/Moesin: 1.928±0.331 vs. 3.030±0.353,P < 0.05).Conclusion The phosphorylation of Moesin is involved in the damage of TNF-α-induced PMVECs in rats, and PMVECs damage could be alleviated by modulating Moesin phosphorylation in the Rac1 signaling pathway.