双歧杆菌脂磷壁酸对胃癌SGC7901细胞SphK1表达的影响

SphK1 mRNA水平采用半定量RT-PCR检测;SphK1蛋白表达水平采用Western blot测定.研究双歧杆菌脂磷壁酸(LTA)对胃癌SGC7901细胞内鞘氨醇激酶1(SphK1)基因和蛋白表达的影响.结果表明,经200、500 μg· mL-1 LTA处理72 h后,SGC7901细胞内SphK1 mRNA...     

Effects of Astragalus polysaccharide on expression of HSP70, PKB and P53 in rat cerebral cortex with cerebral ischemia and reperfusion

AIM:To explore the molecular effects of Astragalus polysaccharide(AP) on improving nervous functions and preventing neuronal apoptosis in rat cerebral cortex with cerebral ischemia and reperfusion. METHODS:One hundred and twenty male Wister rats were randomly divided into sham operation group(SOG), model groups(MG-1 d, 3 d and 7 d), low-dose AP treatment groups(L-APTG-1 d, 3 d and 7 d), and high-dose AP treatment groups(H-APTG-1 d, 3 d and 7 d). The right middle cerebral artery of the rats in MG and AGTG was intercepted by operation to induce ischemic brain injury. The rats in L-APTG and H-APTG were treated with AP at the doses of 5 mg/kg and 15 mg/kg by intraperitoneal injection, respectively. On the 1st day, 3rd day and 7th day after operation, those animals were sacrificed to collect the brain specimens for the study after cerebral blood flow reperfusion and determination of neurological deficit scores. The structural changes of the neurons were observed under electron microscope. Apoptosis was analyzed by flow cytometry. The protein levels of heat-shock protein 70(HSP70), protein kinase B(PKB) and P53 in cerebral corical neurons were determined by immunohistochemical staining and Western blotting. RESULTS:The neurological deficit scores and the apoptotic rate of cerebral cortical neurons in H-APTG were significantly lower than those in MG and L-APTG(P<0.05). The structures of the neurons in H-APTG, such as ribosome endoplasmic reticulum, nucleolus, Golgi complex, mitochondria, etc, were better than those in MG and L-APTG. On the 1st day, 3rd day and 7th day, the protein levels of HSP70 and PKB in cerebral cortical neurons in H-APTG were significantly higher than those in L-APTG, which were significantly higher than those in MG(P<0.05). However, the P53 protein level in H-APTG was significantly lower than that in L-APTG, which was significantly lower than that in MG(P<0.05). CONCLUSION:AP improves nervous functions and inhibits neuronal apoptosis during ischemia and reperfusion. The molecular mechanisms are associated with variations of protein expression in cerebral cortical neurons, such as promotion of HSP70 and PKB and inhibition of P53.     

Effects of Dryk1A,ASF and alternative splicing of CaMKⅡδ on myocardial hypertrophy in renovascular hypertensive rats

AIM: To investigate the role of dual-specificity tyrosine phosporylation-regulated kinase 1A (Dyrk1A)-alternative splicing factor (ASF)-calcium/calmodulin-dependent protein kinase Ⅱδ (CaMK Ⅱδ) pathway in the progression of myocardial hypertrophy in renovascular hypertensive rats. METHODS: The renovascular hypertension was induced by two-kidney one-clip (2K1C) method. The changes of blood pressure and myocardial hypertrophy were measured. The techniques of RT-PCR and Western blotting were used to detect CaMKⅡδ alternative splicing and the protein expression of Dyrk1A and ASF, respectively. RESULTS: Eight weeks after operation, systolic blood pressure (SBP) and diastolic blood pressure (DBP) in 2K1C rats increased (P<0.05). The increases in left ventricular weight (LVW), the ratio of LVW to body weight (BW) and the area of myocardial cells indicated that the hypertensive rats developed significant cardiac hypertrophy. The protein expression of Dyrk1A and mRNA expression of CaMKⅡδA and δB were significantly increased, while the protein expression of ASF and mRNA expression of CaMKⅡδC were decreased compared with sham-operated control rats (P<0.05). Treatment with Dryk1A inhibitor epigallocatechin gallate (EGCG) or harmine effectively attenuated cardiac hypertrophy and reversed the changes in the protein expression of Dyrk1A, ASF and alternative splicing of CaMKⅡδ (all P<0.05). CONCLUSION: Dyrk1A-ASF-CaMKⅡδ pathway plays a role in the development of myocardial hypertrophy in renovascular hypertensive rats.     

Up-regulation of Snail1 expression in renal tubular epithelial cells through Akt/GSK-3β pathway under high-glucose condition

AIM: To examine the effects of high glucose (HG) on the expression of Snail1 and protein kinase B (Akt)/glycogen synthase kinase 3β (GSK-3β) in primary renal tubular epithelial cells (RTECs). METHODS: The primary RTECs were randomly treated with normal glucose, high glucose or D-mannitol for 30 min~72 h. RT-PCR and Western blotting were used to observe the expression of Snail1, Akt and GSK-3β at mRNA and protein levels in these cells. The primary cultured RTECs were pretreated with LY294002 (a PI3K inhibitor, 25 μmol/L) to observe the specific inhibitory effects of phosphatidylinositol 3-kinase (PI3K) on HG-induced expression of Snail1 protein. RESULTS: Treatment of RTECs with HG resulted in increased mRNA and protein levels of Snail1, Akt1, and phosphorylation of Akt and GSK-3β. LY294002 blocked the HG-induced up-regulation of p-Akt, p-GSK-3β and Snail1 expression at protein level, but no effect of LY294002 was seen on the total protein expression of Akt1 and GSK-3β. HG did not affect the expression of GSK-3β at mRNA and protein levels. CONCLUSION: HG-induced up-regulation of Snail1 may be regulated by Akt/GSK-3β pathway in RTECs.     

Artesunate negatively controls expression of cyclin D1 and activity of AP-1 by inhibiting the activation of ERK1/2 protein in HSC-T6 cells

AIM: To investigate the effects of artesunate(Art) on the expression of ERK1/2, AP-1 and cyclin D1 in rat hepatic stellate cells (HSCs), and to elucidate the molecular mechanism of Art against hepatic fibrosis. METHODS: HSC-T6 cells were treated with platelet-derived growth factor BB(PDGF-BB) to induce cell proliferation. The cells were divided into control group, PDGF-BB group, PDGF-BB+Art groups (with 6.25 mg稬^-1, 25 mg稬^-1or 50 mg稬^-1 of Art) and PDGF-BB+PD98059 group. The level of collagen type I in the supernatant was detected by enzyme-linked immunosorbent assay (ELISA). The mRNA expression levels of ERK1/2 and cyclin D1 were measured by RT-PCR. The protein levels of p-ERK1/2 and cyclin D1 in HSC-T6 cells were detected by Western blotting. The activity of AP-1 was analyzed by electrophoretic mobility shift assay. RESULTS: The concentration of collagen type I was significantly higher in PDGF-BB group than that in control group (P<0.05), and decreased in PDGF-BB+Art group and PDGF-BB+PD98059 group in comparison with that in PDGF-BB group (P<0.05, P<0.01). The protein level of ERK1/2 in PDGF-BB+Art group (50 mg稬^-1) was lower than that in PDGF-BB group (P<0.05), and was even lower in PDGF-BB+PD98059 group (P<0.01). The mRNA expression of cyclin D1 in PDGF-BB+Art groups (25 mg稬^-1and 50 mg稬^-1) and PDGF-BB+PD98059 group were significantly lower than that in PDGF-BB group (P<0.05). The protein levels of p-ERK1/2 and cyclinD1 were the highest in PDGF-BB group, and significantly lower in PDGF-BB+Art groups (6.25 mg稬^-1, 25 mg稬^-1 and 50 mg稬^-1) and PDGF-BB+PD98059 group (P<0.05, P<0.01). The AP-1 binding activity in HSC-T6 cells was down-regulated by Art. CONCLUSION: Artesunate inhibits the proliferation of HSC-T6 cells in vitro by inhibiting the activation of ERK1/2, thus down-regulating the activity of AP-1 and expression of cyclin D1.     

Astragalus polysaccharide attenuates free fatty acid-induced toxicity by activating AMPK in C2C12 myoblasts

AIM: To examine the effects of Astragalus polysaccharide (APS) on the toxicity of free fatty acids (FFAs) in C2C12 myoblasts. METHODS: C2C12 cells were randomly divided into 5 groups: control group, APS group, 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR)group,FFAs group and FFAs+APS group. MTT assay was used to observe the viability of C2C12 cells. C2C12 cells pretreated with FFAs at concentration of 0.25 mmol/L for 24 h were exposed to APS at dose of 200 mg/L for 24 h. The expression of total AMP-activated protein kinase (AMPK), phosphorylated AMPK (p-AMPK) and phosphorylated acetyl-CoA carboxylase (p-ACC) was examined by Western blotting. The content of AMP and ATP was determined by HPLC. The structural changes of the mitochondria were examined by transmission electron microscopy. RESULTS: The results of MTT assay indicated that APS improved the viability of C2C12 cells pretreated with FFAs. In FFAs+APS group, the ratio of AMP/ATP was increased after treatment with APS. No difference of total AMPK expression in C2C12 cells between APS group and FFAs group was observed. However, the expression of p-AMPK and p-ACC increased in APS group as compared with FFAs group. The results of transmission electron microscopy indicated that APS improved the vacuolar degeneration of mitochondria resulted from treatment with FFAs in C2C12 cells. CONCLUSION: In C2C12 cells, APS attenuates FFA-induced lipotoxity via a mitochondria-and AMPK-dependent mechanism.     

Clinical significance and prognostic value of extracellular signal-regulated kinases in epithelial ovarian cancer

AIM: To investigate the expression of phosphorylated extracellular signal-regulated kinase (p-ERK) in epithelial ovarian cancer and to analyze the correlation between the expression of p-ERK and prognosis of epithelial ovarian cancer patients. METHODS: The samples of normal ovarian tissues, benign ovarian tissues and ovarian carcinoma were collected. The expression of p-ERK in each sample was detected by Western blotting. Assemble clinical data and survival were analyzed with Kaplan-Meier method and Cox regression. RESULTS: The expression level of p-ERK in ovarian carcinoma was significantly higher than that in benign ovarian tissues and in normal ovarian tissues. The overexpression of p-ERK protein was significantly higher in stage Ⅲ/Ⅳ disease than that in stage I/II disease. No difference of p-ERK expression level between the benign ovarian tissues and the normal ovarian tissues was observed. The overexpression of p-ERK was found more often in poorly-differentiated ovarian cancer. Compared with serous carcinoma of ovary, the expression level of p-ERK was similar in undifferentiated adenocarcinoma. The expression level of p-ERK was low in endometrioid carcinoma of ovary and mucinous carcinoma of ovary. The expression level of p-ERK was negatively correlated with the survival time. Increased p-ERK expression was strongly correlated to the poor survival. Shorter survival time was significantly associated with p-ERK expression, residual tumor, stage, grade and chemotherapy course. Shorter survival time was not significantly correlated with tumorous pathology. CONCLUSION: p-ERK is a key regulation kinase involved in epithelial ovarian cancer. The expression level of p-ERK seems to have a prognosis value in epithelial ovarian cancer.     

Expression of ERK5 in colorectal cancer tissues and its correlation with distant metastasis in colorectal cancer patients

AIM: To investigate the expression of extracellular signal-regulated kinase 5 (ERK5) in primary colorectal cancer (CRC) and adjacent normal mucosa, and to analyze the relationship between ERK5 expression and clinicopathological parameters for exploring the functions of ERK5 in the occurrence and development of CRC. METHODS: The expression of ERK5 in carcinoma tissues and normal mucosa was examined by a set of tissue microarrays and the method of immunohistochemistry. The potential relationship between ERK5 expression and clinicopathological features was also analyzed. RESULTS: ERK5 expression was significantly higher in CRC tissues (134/338, 39.6%) than that in normal tissues (21/80, 26.2%; P<0.05). Overexpression of ERK5 in CRC tissues was significantly correlated with distant metastasis (P<0.05). However, no correlation between ERK5 expression and age at surgery, sex, tumor location, the depth of invasion, lymph node metastasis, TNM staging or differentiation grade was found (P>0.05). According to the Kaplan-Meier analysis, there is no significant difference in 5-year overall survival between the patients with ERK5 expression at high level and at low level. CONCLUSION: ERK5 protein is highly expressed in CRC with distant metastasis. This may be a promotive factor in the process of distant metastasis.     

Effects of ATP-competitive GSK-3 inhibitor on multidrug resistance of human esophageal squamous-cell carcinoma cells

AIM: To study the changes and correlations of β-catenin, multidrug resistance-associated protein 2 (MRP2), P-glycoprotein (P-gp), Bcl-2 and the free ATP level in esophageal squamous-cell carcinoma (ESCC) cell line EC-109 induced by ATP-competitive glycogen synthase kinase(GSK-3) inhibitor 6-bromoindirubin-3'-oxime (BIO). METHODS: The methods of flow cytometry, immunofluorescence, cytochemistry and ATP assay were used to study the expression levels of MRP2, P-gp, β-catenin and Bcl-2, the efflux capability of ATP-binding cassette (ABC) transporters, and the free ATP level in ESCC cells. RESULTS: After induced by BIO, up-regulation of β-catenin and Bcl-2 expression in cytoplasm and accumulation of β-catenin in nucleus were found in ESCC cells. The expression of MRP2 was up-regulated in cytoplasm and cell membrane. On the contrary, the expression of P-gp was down-regulated in cytoplasm and cell membrane. The free ATP level and the efflux capability of ABC transporters increased in ESCC cells. CONCLUSION: The multidrug resistance of ESCC cells in enhanced by BIO treatment.