Effect of lumbrokinase on the synthesis of Src and FAK induced by TGF-β1 in human renal tubular epithelial cells / 国际中医中药杂志

Objective To observe the regulation of lumbrukinase on the expression of α-smooth muscle actin,fibronectin,focal adhesion kinase and Src kinase induced by transforming growth factor β1 in human renal tubular epithelial cells.Methods According to random number table method,the human renal tubular epithelial cells were divided into the normal group,TGF-β1 model group,benazepril group,lumbrukinase low,medium and high dose group.Except the normal group,the other groups were treated with TGF-β1 10 μg/ml.After 30 minutes,the benazepril group added benazepril 10 μmol/L,and the low,medium,high dose groups of lumbrukinase were respectively treated with lumbrokinase 30,60,120 U/ml to intervene.After 24 hours of cultivation.Western blotting and real-time PCR were used to detect the expression of α-SMA,FN,FAK and Src.Results Compared with the model group,the expressions of α-SMA (0.84 ± 0.14,0.72 ± 0.08,0.69 ± 0.05 vs.1.24 ± 0.03) and FN (0.59 ± 0.09,0.55 ± 0.11,0.44 ± 0.08 vs.0.83 ± 0.18) and FAK (0.94 ± 0.04,0.79 ± 0.05,0.70 ± 0.02 vs.1.29 ± 0.07) and Src (0.87 ± 0.20,0.78 ± 0.15,0.71 ± 0.11 vs.1.23 ± 0.01) proteins in the high doses of lumbrical kinase group were significantly lower than those in the model group (P＜0.05),the expressions of α-SMA (3.13 ± 0.62,2.76 ± 0.14,2.15 ± 0.33 vs.4.12 ± 0.32) and FN (3.08 ± 0.34,2.78 ± 0.17,2.49 ± 0.11 vs.4.34 ± 0.06) and FAK (1.73 ± 0.23,1.63 ± 0.36,1.57 ± 0.27 vs.2.61 ± 0.59) and Src (2.11 ± 0.17,1.78 ± 0.25,1.71 ± 0.22 vs.2.78 ± 0.47) mRNA in the high doses of lumbrical kinase group decreased significantly (P＜0.05).Conclusions Lumbrokinase may prevent the development of renal fibrosis by regulating the expression ofFN,FAK and and reducing the production of α-SMA,FN.

Roles and mechanisms of connexin 43 in cerebral ischemia-reperfusion / 国际脑血管病杂志

In the central nervous system, gap junctions exist between neurons and glial cells. Among them, connexin 43 (Cx43) is one of the most abundant connexin proteins in the central nervous system,involved in the metabolic coupling of intercellular substance exchange and electrical coupling of electrical signaling. It plays an important role in regulating cell metabolism, homeostasis, and cell differentiation. After cerebral ischemia, the uncoupling of gap junctions and abnormal hemichannel activity cause a steady-state imbalance of the internal and external environment of the cells, eventually leading to brain tissue damage.Therefore, maintaining the normal function of Cx43 is essential for protecting brain tissue from neuronal damage induced by cerebral ischemia-reperfusion.

Effect of dexmedetomidine on Src-mediated NR2A tyrosine phosphorylation in hippocampal neurons subjected to hypoxia-reoxygenation in mice / 中华麻醉学杂志

Objective To evaluate the effect of dexmedetomidine on Src-mediated NR2A tyrosine phosphorylation in hippocampal neurons subjected to hypoxia-reoxygenation (H/R) in mice.Methods C57 mice at 18 days of gestation were sacrificed by pulling neck,fetal mice were obtained by caesarean section,and hippocampal neurons were isolated.Neurons were cultured in culture medium for 7 days and then divided into 3 groups (n =30 each) using a random number table method:control group (group C),H/R group and dexmedetomidine group (D group).Hippocampal neurons were exposed to 95％ N2-5％ CO2 in an incubator at 37 ℃ for 4 h followed by reoxygenation with 95％ O2-5％ CO2 for 24 h to establish the model of H/R.Dexmedetomidine was given at a final concentration of 1 μ mol/L,and neurons were incubated for 4 h before establishing the model.The viability of hippocampal neurons was measured by MTT assay,TUNEL staining was used to observe apoptosis in hippocampal neurons,and the expression of c-Src,phosphorylated Src Y416 (p-Src Y416),NR2A,phosphorylated NR2A Y1325 (p-NR2A Y1325) and cleaved caspase-3.was determined by Western blot.Apoptosis index was calculated.Results Compared with group C,the viability of hippocampal neurons was significantly decreased,apoptosis index was increased,and the expression of p-Src Y416,p-NR2A Y1325 and cleaved caspase-3 was up-regulated in group H/R (P＜0.05).Compared with group H/R,the viability of hippocampal neurons was significantly increased,apoptosis index was decreased,and the expression of p-Src Y416,p-NR2A Y1325 and cleaved caspase-3 was down-regulated in group D (P＜0.05).There was no significant difference in the expression of c-Src and NR2A among three groups (P＞0.05).Conclusion Dexmedetomidine can inhibit apoptosis in hippocampal neurons subjected to H/R,and the mechanism may be associated with decreasing Src-mediated NR2A tyrosine phosphorylation in mice.

Inhibition of Src kinase can ameliorate renal interstitial fibrosis in unilateral ureteral obstruction mice / 中华肾脏病杂志

Objective To investigate the effect and mechanism of Src kinase in renal interstitial fibrosis of unilateral ureteral obstruction (UUO) mice.Methods Male C57BL/6J mice were randomly divided into 4 groups,including sham operation group (n=8),sham operation+PP2 group (n=8),UUO operation group (n=8) and UUO operation+PP2 group (n=8).The mice were injected 2 mg/kg PP2 by intraperitoneal everyday after surgery in sham+PP2 group and UUO+PP2 group.PP2 dissolved in 1％ DMSO (formulated with normal saline).Sham and UUO group were given equal 1％ DMSO.The mice were sacrificed at 7th day.Renal collagen was observed with Sirius red stain.The activities of Src,protein kinase B (PKB,AKT),p38 mitogen-activated protein kinase (p38 MAPK),extracellular signal-regulated kinase (ERK) and the protein expressions of α-smooth muscle actin (α-SMA) and fibronectin (FN) were detected by Western blotting.The expression of collagen I (COL [) was detected by immunohistochemistry and the expressions of matrix metalloprotein 9 (MMP-9),tissue inhibitor of metalloproteinase 1 (TIMP-1),transforming growth factor-β31 (TGF-β31),monocyte chemotactic protein-1(MCP-1),interleukin-6 (IL-6) were measured by ELISA.Resuts Compared with sham mice,UUO mice on 7th day displayed obvious renal fibrosis.Meanwhile,UUO mice had increased expressions of COL Ⅰ and FN,and activities of AKT,ERK and p38 MAPK (all P ＜ 0.05).Their renal expressions of α-SMA,TGF-β1,MMP-9,TIMP-1,MCP-1 and IL-6 were also raised (all P ＜ 0.05).Compared with those in UUO group,in UUO + PP2 group the activities of Src,AKT,p38 MAPK and ERK,and expressions of TGF-β1,MCP-1 and IL-6 decreased (all P ＜ 0.05).Additionally,expressions of COL Ⅰ,FN and α-SMA,collagen deposition and renal fibrosis receded in UUO + PP2 group (all P ＜ 0.05).However,the expressions of MMP-9 and TIMP-1 were not influenced by PP2 treatment.Conclusions Src kinase promotes myofibroblasts accumulation and inflammatory reaction through activating its downstream signaling pathway in the progressing of renal interstitial fibrosis.

The roles of spinal microglial Src-family kinases in diabetic neuropathic pain / 实用医学杂志

Objective To explore the roles of spinal microglial Src-family kinases (SFKs) in diabetic neuropathic pain. Methods Male Sprague-Dawley rats, weighing 200 ~ 220 g, were used in the experiments. The level of p-SFKs in spinal dorsal horn was detected by single immunofluorescence staining on day 7th , 14th and 28th after intraperitoneally injection of Streptozotocin (STZ) and its location was detected by double immunofluorescence staining. The changes of 50% paw-withdrawal thresholds of rat were detected by behavioral tests when PP2 , a specific inhibitor of SFKs , was intrathecally administered before intraperitoneally injection of STZ. Results Compared with vehicle group, the blood glucose level increased on day 1 (P < 0.001) and the hyperglycemia persisted at least for 28 days (P < 0.001) after intraperitoneally administered of STZ (50 mg/kg). Paw-withdrawal threshold (PWT) decreased gradually from day 1 (P < 0.05) and reached the minimum on day 28 (P < 0.001) after STZ administration. Meanwhile, the expression of p-SFKs in spinal dorsal horn markedly increased on day 7 (P < 0.01), day 14 (P < 0.01) and day 28 (P < 0.01). The p-SFKs was mainly co-localized only with microglia , but not with neurons or with astrocytes. Intrathecally administered of PP2 before STZ reversed STZ-induced mechanical hyperalgesia. Conclusion Microglial SFKs in spinal dorsal horn maybe play a pivotal role in diabetic neuropathic pain.

Role of Src kinase in liver injury in endotoxemic mice / 中华麻醉学杂志

Objective To evaluate the role of Src kinase in liver injury in endotoxemic mice.Methods Forty-eight female BABL/c mice,aged 3-4 months,weighing 15-20 g,were randomly divided into 3 groups (n =16 each) using a random number table:control group (C group),endotoxemia group (lipopolysaccharide (LPS) group) and Src kinase inhibitor PP2 group (PP2 group).Endotoxemia was induced by intraperitoneal LPS 20 mg/kg in LPS and PP2 groups,while the equal volume of PBS was given in group C.In PP2 group,PP2 1 mg/kg was injected intraperitoneally at 2 h after LPS administration.At 6 h after LPS or PBS injection,8 mice in each group were chosen,and blood samples were collected from the abdominal aorta for determination of the serum levels of alkaline phosphatase (ALP).The mice were then sacrificed and livers were removed for determination of nuclear factor E2-related factor 2 (Nrf2) level,superoxide dismutase (SOD) activity,malondialdehyde (MDA) content and myeloperoxidase (MPO) activity in liver tissues.The other 8 mice in each group were sacrificed at 24 h after LPS or PBS injection,and the livers were harvested for examination of pathological changes.Results Compared with C group,the serum levels of ALP and MDA content and MPO activity in liver tissues were significantly increased,and SOD activity and Nrf2 levels in liver tissues were decreased in LPS and PP2 groups.Compared with LPS group,the serum levels of ALP and MDA content and MPO activity in liver tissues were significantly decreased,and SOD activity and Nrf2 levels in liver tissues were increased in PP2 group.The pathological changes of liver tissues were significantly attenuated in PP2 group as compared with LPS group.Conclusion Src kinase is involved in endotoxemia-induced liver injury in mice.

Role of C-terminal Src kinase in angiotensin Ⅱ-induced cytoskeletal rearrangement in glomerular podocytes / 中华肾脏病杂志

Objective To investigate the effects of angiotensin Ⅱ (Ang Ⅱ) on the expression of C-terminal Src kinase (Csk) in Ang Ⅱ-infused rat model and cultured podocytes,and to explore the role of Csk in Ang Ⅱ-induced cytoskeletal rearrangement of podocytes.Methods Twenty-four Wista rats were randomly subjected to normal saline infusion,or Ang Ⅱ infusion at 400 ng · kg1 · min-1 (via subcutaneous osmotic minipumps) for 2 or 4 weeks.Renal histomorphology was evaluated through electron microscopy.The expression of glomerular Csk was analyzed by immunofluorescence and Western blotting.In vitro,conditionally immortalized mouse podocytes were cultured and treated with Ang Ⅱ doses ranging from 10-9 mol/L to 10-5 mol/L and for different hours.The expression of podocytes Csk was assessed by Western blotting.After transfection to podocytes with Csk siRNA,FITC-conjugated phalloidin was used to stain F-actin,to investigate the role of Csk in Ang Ⅱ-induced or cytochalasin D-induced cytoskeletal rearrangement.Results (1) Examination of Ang Ⅱ infusion rats glomerular and podocyte ultrastructure by electron microscopy revealed foot process effacement and fusion; (2) In Ang Ⅱ infusion rats,the expression of glomerular Csk was increased (P ＜ 0.05); (3) In vitro,Ang Ⅱ-stimuli up-regulated the expression of Csk (P ＜ 0.05),and the effects of Ang Ⅱ were on dose-dependent and time-dependent manner; (4) Ang Ⅱ-induced disruption of F-actin was alleviated by Csk siRNA transfection in cultured podocytes; furthermore,cytochalasin D depolymerized the F-actin cytoskeleton,while Csk siRNA stabilized the actin filaments.Conclusion The enhanced expression of Csk may be involved in Ang II-induced podocytes cytoskeletal rearrangement and foot process fusion.

Role of Src tyrosine kinase in damage to alveolar epithelial cells caused by mechanical stretch / 中华麻醉学杂志

Objective To evaluate the role of Src tyrosine kinase in damage to alveolar epithelial cells caused by mechanical stretch.Methods MLE-12 cells cultured in vitro were randomly divided into 3 groups using a random number table:mechanical stretch group (group S),dimethyl sulfoxide control group (group D),and Src tyrosine kinase inhibitor PP2 group (group P).In D and P groups,dimethyl sulfoxide 30 μl/ml and PP2 100 μmol/L were added to the culture medium,respectively,and the cells were then cultured for 30 min.The cells underwent mechanical stretch for 8 h with frequency of0.5 Hz and amplitude of 20％ in the three groups.At 0,2,4 and 8 h of mechanical stretch,MLE-12 cells in 3 wells of each group were collected for determination of cell apoptosis with flow cytometry and expression of occludin using Western blot.The apoptosis rate was calculated.Results Compared with S group,no significant changes were found in the apoptosis rate and expression of occludin at each time point in group D,and the apoptosis rate was significantly decreased,and the expression of occludin was up-regulated at 2,4 and 8 h of mechanical stretch in group P.Conclusion The activation of Src tyrosine kinase is involved in damage to alveolar epithelial cells caused by mechanical stretch.

Src kinases and cerebrovascular diseases / 国际脑血管病杂志

Src family kinases (SFKs) are the largest non-receptor tyrosine kinase family and play key roles in the regulation of cell morphology,proliferation,growth,adhesion,and motility.The activation of src kinases couples with many signals input on cell surface,including growth factor,cytokine,immune cell receptor,G protein-coupled receptor,integrin,and other cell adhesion molecules.In addition,as an important molecular switch connecting many extracellular and intracellular important signaling pathways,src kinases also play an important role in the occurrence of cerebrovascular diseases.

Effects of Src tyrosine kinase inhibitor dasatinib on the proliferation and apoptosis of human esophageal squamous cell carcinoma cell line KYSE180 / 肿瘤

Objective: To investigate the effects of Src tyrosine kinase inhibitor dasatinib on the cell proliferation and apoptosis of human esophageal squamous cell carcinoma and the related mechanism. Methods: Western blotting was used to detect the expressions of total Src and phos-Src tyrosine kinase in esophageal squamous-cell cancer cell lines KYSE180, EC109 and KYSE30 as well as human immortalized esophageal epithelial cell line SHEE. The inhibition effect of different concentrations of Src tyrosine kinase inhibitor dasatinib on Src kinase and the effects of dasatinib on proliferation, cell-cycle and apoptosis of KYSE180 cells as well as the growth of subcutaneous xenograft of KYSE180 cells in nude mice were examined by MTT method, flow cytometry (FCM), Western blotting and an experimental xenograft mouse model. Results: The activation level of Src tyrosine kinase was significantly increased in KYSE180, EC109 and KYSE30 cells, and this effect was not observed in SHEE cells. Dasatinib could significantly inhibit the proliferation of KYSE180 cells, and also inhibit G1/S transition, induce apoptosis and upregulate the expression levels of apoptosis-related caspase 3, cytochrome C and Bax proteins in KYSE180 cells. Furthermore, the growth of subcutaneous xenograft of KYSE180 cells in nude mice was obviously inhibited by dasatinib treatment. Conclusion: Dasatinib can inhibit the growth of subcutaneous xenograft of KYSE180 cells in nude mice by inhibiting cell proliferation, inducing apoptosis and influencing the distribution of cell cycle, and it may be considered as an effective agent for the treatment of esophageal squamous cell carcinoma. Copyright © 2012 by TUMOR.

Effects of SFKs in microglia on ATP-induced long-term potentiation in spinal dorsal horn / 中国病理生理杂志

AIM: To investigate the effects of Src family kinases (SFKs) on adenosine 5'-triphosphate (ATP)-induced long-term potentiation (LTP) in the spinal dorsal horn. METHODS: Male Sprague-Dawley rats (250-280 g) were used in the experiments. Western blotting, electrophysiological recording in spinal dorsal horn in vivo and immunohistochemistry were used in the study. The C-fiber-evoked field potentials were recorded at the superficial layers of spinal dorsal horn at the lumbar enlargement and the phosphorylation level and location of SFKs in spinal dorsal horn were examined by Western blotting and immunohistochemistry. RESULTS: Thirty min and 60 min after ATP application, the levels of phosphorylated SFKs (p-SFKs) were significantly increased.The p-SFKs were expressed in microglia, but not in astrocytes or neurons. Spinal application of SFK inhibitors prevented ATP-induced LTP. CONCLUSION: Microglial SFKs may play an important role in ATP-induced LTP of C-fiber evoked field potentials in the spinal dorsal horn.

Metastasis-suppressor KAI1/CD82 induces homotypic aggregation of human prostate cancer cells through Src-dependent pathway

To investigate the functional role of KAI1/CD82, a metastasis suppressor for human prostate cancer, in the regulation of homotypic cell adhesion, we transfected KAI1 cDNA into DU 145 human prostate cancer cells and established stable transfectant clones with high KAI1/CD82 expression. The KAI1 transfectant cells exhibited significantly increased homotypic cell aggregation in comparison with the control transfectant cells. This aggregation of the KAI1 transfectants was further enhanced upon exposure to anti-CD82 antibody, suggesting that KAI1/CD82 may be involved in the intracellular signaling for the cell adhesion. Among several signal pathway inhibitors tested, PP1, an inhibitor of Src family kinases, significantly suppressed homotypic aggregation of the KAI1 transfectant cells. Ligation of KAI1/CD82 with anti-CD82 antibody increased endogenous Src kinase activity of the KAI1 transfectant cells. When different types of src expression constructs were retransfected into the KAI1-transfected DU 145 cells, kinase-negative mutant src transfectant cells exhibited much lower homotypic aggregation than the mock cells transfected with an empty vector. Moreover, homotypic aggregation of the mutant src transfectant cells was not enhanced by KAI1/CD82 ligation with anti- CD82 antibody. These results suggest that Src mediates the intracellular signaling pathway of KAI1/CD82 for the induction of homotypic adhesion of human prostate cancer cells.