Protective effect of β-asarone on primary rat hippcampal neurons against hypoxia/hypoglycemia and reperfusion injury

AIM：To investigate the protective effect of β-asarone against hypoxia/hypoglycemia and reperfusion injury in primary rat hippocampal neurons. METHODS：Cell viability, the activity of caspase-3, the protein expression of p-JNK and Bcl-2, and the mRNA expression of Bcl-2 and caspase-3 were determined by MTT assay, spectrophoto-metry, Western blotting and real-time PCR. RESULTS：Compared with normal control group, the cell viability decreased and the activity of caspase-3 increased obviously, the expression of p-JNK protein and caspase-3 mRNA increased obviously, and the expression of Bcl-2 protein decreased obviously in model group (P<0.05). Compared with model group, different doses of β-asarone inhibited the changes of the above indexes (P<0.05). CONCLUSION：β-asarone inhibits JNK-mediated chondrosome signaling pathway, thereby attenuating the process of hippocampal neuron apoptosis after hypoxia/hypoglycemia and reperfusion.     

Effect of EZH2 regulating Wnt/β-catenin signaling pathway on apoptosis of brain glioma cells

AIM: To investigate the effect of enhancer of zeste homolog 2 (EZH2) regulating Wnt/β-catenin signaling pathway on the apoptosis of brain glioma cell lines. METHODS: The expression level of EZH2 in glioma cell lines U87, H4 and U251 and normal human astrocytes (NHA) was detected by RT-qPCR and Western blot. The EZH2 siRNA and siRNA control were transfected into the H4 cells. The cell viability was measured by MTT assay. The apoptosis was analyzed by flow cytometry. Caspase-3 activity was detected by spectrophotometry. The expression levels of the key protein β-catenin of the Wnt/β-catenin signaling pathway and the downstream target molecule c-Myc were determined by Western blot. After the H4 cells transfected with EZH2 siRNA were treated with an activator of Wnt/β-catenin signaling pathway, the apoptosis rate was measured by flow cytometry, and the expression of β-catenin and c-Myc was determined by Western blot. RESULTS: The mRNA and protein expression levels of EZH2 in the glioma cell lines U87, H4 and U251 were significantly higher than those in NHA (P<0.05). The expression of EZH2 at mRNA and protein levels in the H4 cells was higher than that in U87 cells and U251 cells (P<0.05). EZH2 siRNA obviously inhibited the expression of EZH2 at mRNA and protein levels in the H4 cells. Knockdown of EZH2 expression decreased the viability of H4 cells, the apoptotic rate was significantly increased, and the activity of caspase-3 was significantly increased in the cells (P<0.05). Knockdown of EZH2 expression also inhibited the expression of β-catenin and c-Myc. The activator of Wnt/β-catenin signaling pathway reduced the apoptosis rate of H4 cells induced by down-regulation of EZH2, and reduced the activity of caspase-3 in the cells. CONCLUSION: EZH2 is over-expressed in glioma cells. Down-regulation of EZH2 expression induces apoptosis of glioma cells by inhibiting the activation of Wnt/β-catenin signaling pathway.     

Kaempferol inhibits cell proliferation,invasion and migration via Wnt/β-catenin signaling in HBx-HepG2 cells

AIM: To explore the effects of kaempferol on the proliferation, invasion and migration abilities of HBx-HepG2 cells and to examine the underlying molecular mechanisms. METHODS: The expression levels of related genes at mRNA and protein levels were determined by RT-qPCR and Western blot. The cell apoptotic rate was analyzed by flow cytometry. The cell proliferation, growth, invasion and migration abilities were measured by MTT assay, colony formation assay, Transwell invasion assay and wound healing assay, respectively. RESULTS: Kaemferol inhibited HBx-HepG2 cell proliferation in a concentration-and time-dependent manner. Kaempferol at 100 μmol/L significantly inhibited the colony formation, invasion and migration abilities of the HBx-HepG2 cells. Kaemferol at 100 μmol/L also increased cell apoptotic rate, increased the protein levels of cleaved caspase-3, cleaved caspase-9 and Bax, and decreased the expression level of Bcl-2. In addition, kaemferol at 100 μmol/L suppressed the mRNA and protein expression levels of β-catenin, c-Myc and cyclin D1 in the HBx-HepG2 cells. Kaemferol at 100 μmol/L also suppressed the protein level of p-GSK-3β and the β-catenin protein levels in both cytoplasm and nucleus. LiCl treatment reversed the inhibitory effect of kaempferol on the growth, invasion and migration of the HBx-HepG2 cells. CONCLUSION: Kaempferol inhibits cell proliferation, invasion and migration via activating Wnt/β-catenin signaling in HBx-HepG2 cells.     

Effects of GSK-3β inhibitor on proliferation and apoptosis of SW480 cells

AIM: To investigate the mechanism and the effect of glycogen synthase kinase 3β (GSK-3β) inhibitor (2’Z,3'E)-6-bromoindirubin-3'-oxime (BIO) on the protein expression of β-catenin and Bcl-2, and proliferation and apoptosis in colon carcinoma SW480 cells.METHODS: The immunohistochemical staining and Western blotting were performed to detect the protein expression of β-catenin, cyclin D1 and Bcl-2. The cell cycle distribution and apoptotic rate were detected by flow cytometry. The morphologic features of SW480 cells before and 24 h after BIO exposure at different concentrations were observed under microscope with HE staining.RESULTS: Compared with the untreated SW480 cells, the protein expression of β-catenin significantly increased and some β-catenin positive nuclear staining positive cells appeared in BIO treated cells. and The cells exposed to BIO showed that the cyclin D1 protein and the cells in S stage and G₂/M stage moderately increased, the protein level of Bcl-2 moderately decreased, and the cell apoptosis rate was significantly lower than those in control cells. Furthermore, the morphological changes of the SW480 cells were observed 24 h after BIO treatment. CONCLUSION: Our results indicate that GSK-3β inhibitor BIO participates in the cellular processes of promoting proliferation and inhibiting apoptosis in colon carcinoma cells. The mechanisms are mainly associated with activating the β-catenin pathway and regulating the balance of Bcl-2 pathway, and the up-regulation of β-catenin is most likely the possible factor for SW480 cell regression.     

Role of β-catenin in apoptosis of pancreatic acinar cells induced by cae-rulein

AIM: To study the role of β-catenin in the apoptosis of pancreatic acinar cells induced by cae-rulein. METHODS: Rat pancreatic acinar AR42J cells were treated with caerulein. The expression of β-catenin at mRNA and protein levels in the AR42J cells was determined by real-time PCR and Western blot. The β-catenin over-expression vector was transfected into AR42J cells. After treatment with caerulein, the over-expression effect was evaluated by real-time PCR and Western blot. The changes of cell viability were measured by MTT assay. The leakage rates of lactate dehydrogenase (LDH) and amylase (AMY) were measured by binitrophenyl hydrazine method and iodine starch colorimetry, respectively. The apoptosis was analyzed by flow cytometry. The protein levels of endoplasmic reticulum stress protein CHOP and cleaved caspase-12 in the AR42J cells were determined by Western blot. RESULTS: The expression of β-catenin at mRNA and protein levels in the AR42J cells was decreased after treatment with caerulein (P<0.05). The expression of β-catenin in the AR42J cells was significantly increased by transfection with β-catenin over-expression vector. The viability of AR42J cells after treatment with caerulein was reduced, while the leakage rates of LDH and AMY, the apoptotic rate and the protein levels of CHOP and cleaved caspase-12 in the cells were increased (P<0.05). Over-expression of β-catenin enhanced the viability of AR42J cells after treatment with caerulein, reduced the leakage rates of LDH and AMY, and decreased the apoptotic rate and the protein levels of CHOP and cleaved caspase-12 in the AR42J cells. CONCLUSION: β-Catenin significantly inhibits the apoptosis of pancreatic acinar cells induced by caerulein. The mechanism is related to the reduction of endoplasmic reticulum stress.     

Effect of integrin β1 on multidrug resistance in gastric cancer SGC-7901 cells

AIM: To study the effect of integrin β1 on multidrug resistance in gastric cancer and its possible mechanisms. METHODS: The expression of integrin β1 at mRNA and protein levels in the SGC-7901 cells and SGC-7901/DDP cells was determined by qPCR and Western blot. The expression of integrin β1 in the SGC-7901/DDP cells was silenced by antisense oligodeoxynucleotide. The cell viability was detected by the CCK-8 assay, the cell apoptosis were analyzed by flow cytometry, and the protein levels of integrin β1, Bcl-2/Bax, cleaved caspase-3/caspase-3, cytochrome C (Cyt-C) and p-AKT/AKT were determined by Western blot.RESULTS: The expression of integrin β1 at both mRNA and protein levels was significantly upregulated in SGC-7901/DDP cells. The expression of integrin β1 was increased in SGC-7901 cells treated with chemotherapeutic agents such as cisplatin, paclitaxel and 5-fluorouracil. Knockdown of integrin β1 induced apoptosis of SGC-7901/DDP cells with an increased sensitivity to the chemotherapeutic agents. Meanwhile, knockdown of integrin β1 downregulated the protein levels of Bcl-2/Bax, p-AKT^Ser473 and p-AKT^Thr308, while promoted the release of Cyt-C and upregulated the protein level of cleaved caspase-3. CONCLUSION: Knockdown of integrin β1 increases the sensitivity of SGC-7901/DDP cells to the chemotherapeutic agents, and promotes the cell apoptosis via mitochondrial apoptosis pathway. The mechanism may be related to the attenuation of AKT pathway by inhibiting phosphorylations of AKT at Ser473 and Thr308.     

Effect of Wnt/β-catenin signaling pathway regulated by HDAC1 on apoptosis of breast cancer cells

AIM: To study the effect of histone deacetylase 1 (HDAC1) on the apoptosis of breast cancer cells.METHODS: The expression of HDAC1 at mRNA and protein levels in normal mammary epithelial cell line MCF-10A and breast cancer cell lines BT549, MCF-7 and MDA-MB-231 was measured by RT-qPCR and Western blot. HDAC1 siRNA was transfected into MDA-MB-231 cells, and then RT-qPCR and Western blot were used to determine the expression level of HDAC1. The cell viability was measured by MTT assay, and apoptosis was analyzed by flow cytometry. The protein levels of β-catenin, c-Myc, cyclin D1 and cleaved caspase-3 were determined by Western blot. Breast cancer cells with HDAC1 knockdown were treated with Wnt/β-catenin signaling pathway activator, and then the cell viability and apoptosis were measured.RESULTS: The expression of HDAC1 at mRNA and protein levels in BT549, MCF-7 and MDA-MB-231 cells was significantly higher than that in normal mammary epithelial cell line MCF-10A, and the highest expression level of HDAC1 was observed in MDA-MB-231 cells (P<0.05). HDAC1 siRNA reduced the expression of HDAC1 at mRNA and protein levels in the breast cancer cells. The viability of MDA-MB-231 cells was decreased after knockdown of HDAC1 expression, the apoptotic rate was increased, the protein level of cleaved caspase-3 in the cells was elevated, and the protein levels of β-catenin, c-Myc and cyclin D1 were decreased (P<0.05). Wnt/β-catenin signaling pathway activator reversed HDAC1 knockdown-induced apoptosis and decrease in viability of MDA-MB-231 cells, and reduced the protein level of cleaved caspase-3.CONCLUSION: Knockdown of HDAC1 expression induces apoptosis of breast cancer cells by inhibiting the activation of Wnt/β-catenin signaling pathway.     

Ginsenoside Rg1 promotes growth and extracellular matrix synthesis in degenerative human lumbar nucleus pulposus cells by inhibiting Wnt/β-catenin pathway

AIM: To explore the role of ginsenoside Rg1 in the growth of degenerative human lumbar nucleus pulposus cells (HNPCs). METHODS: Cultured HNPCs were subjected to oxygen-glucose deprivation (OGD) to mimic the micro-environment of degenerative HNPCs. The morphological changes of the cells in control group and OGD group were observed under optical microscope. The cells were treated with ginsenoside Rg1 at concentrations of 25, 50 and 100 μmol/L. The expression of collagen Ⅱ and aggrecan at mRNA and protein levels was determined by real-time PCR and Western blot analysis. The cell viability was measured by CCK-8 assay. The mRNA level of Ki67 was detected by real-time PCR. The apoptosis was analyzed by flow cytometry. The activity of caspase-3 was measured by a caspase-3 kit. The expression of Wnt/β-catenin pathway-related proteins was determined by Western blot. Furthermore, the expression of Wnt/β-catenin pathway-related proteins, the cell viability and apoptosis, and the expression of extracellular matrix synthesis proteins were assessed after the cells were co-treated with LiCl and 100 μmol/L ginsenoside Rg1. RESULTS: Normal HNPCs attached on the cell culture plate faster, and were almost round with rich cytoplasm. However, the cell adherence was slower, and the cells were long fusiform with decreased cytoplasm after OGD treatment, indicating that the model of degenerative HNPCs was successfully established. Compared with normal HNPCs, the expression of collagen Ⅱ and aggrecan at mRNA and protein levels was decreased in OGD group (P<0.05), which was then increased after the cells were treated with ginsenoside Rg1 at 25, 50 and 100 μmol/L (P<0.05). Compared with normal HNPCs, the cell viability and Ki67 expression were decreased in OGD group (P<0.05), which were increased after treatment with ginsenoside Rg1 (P<0.05). Meanwhile, the apoptotic rate and caspase-3 activity were significantly increased in OGD-treated cells (P<0.05), which were decreased after treatment with ginsenoside Rg1 (P<0.05). In addition, the activation of Wnt/β-catenin pathway was also inhibited by ginsenoside Rg1 treatment at dose of 100 μmol/L (P<0.05). LiCl, a Wnt/β-catenin pathway agonist, obviously decreased the protective effects of ginenoside Rg1 on OGD-induced cells (P<0.05), indicating that the Wnt/β-catenin pathway was involved in the protective effects of ginenoside Rg1 on degenerative HNPCs. CONCLUSION: Ginsenoside Rg1 promotes growth and extracellular matrix synthesis of degenerative HNPCs through inhibiting Wnt/β-catenin pathway. This study will provide a new idea for prevention and treatment of degenerative HNPCs.     

Resveratrol inhibits chondrosarcoma via mitochondrial and PI3K/Akt signaling pathways

AIM：To investigate the inhibitory effects of resveratrol on chondrosarcoma and the relation with mitochondrial and PI3K/Akt pathways. METHODS：Chondrosarcoma SW1353 cells were treated with resveratrol at concentrations of 25, 50 and 100 μmol/L for the time intervals of 24 h, 48 h and 72 h. The viability and apoptosis of the SW1353 cells in the presence or absence of resveratrol were analyzed by CCK8 assay and Hoechst 33258 staining, respectively. The protein levels of Bcl-2, Bax, activated caspase-3, Akt and p-Akt were detected by Western blotting. The cell migration ability was determined by wound scratch assay. RESULTS：Exposure of the cells to resveratrol resulted in a decrease in the cell viability in a dose- and time-dependent manner (P<0.05). visible nuclei with apoptotic characteristics in resveratrol group were observed. The protein levels of activated caspase-3 and Bax were increased, and Bcl-2 and p-Akt were decreased compared with control group. The total Akt were not significantly changed. Resveratrol also significantly reduced the migration of tumor cells. CONCLUSION：Resveratrol induces apoptosis of chondrosarcoma, which plays a role of part through mitochondrial and PI3K/Akt signaling pathways.     

Endoplasmic reticulum stress is involved in cardiomyocyte apoptosis induced by β1-adrenoceptor autoantibody

AIM: To investigate the role of endoplasmic reticulum (ES) stress in cardiomyocyte apoptosis induced by β₁-adrenoceptor autoantibody (β₁-AA). METHODS: The rat model of active immunization with the second extracellular loop of β₁-adrenoceptor was established, and SA-ELISA was applied to detect the level of β₁-AA in serum of actively immunized rats. The apoptosis of cardiomyocytes was detected by TUNEL staining, and the protein expression levels of glucose-regulated protein 78 (GRP78), C/EBP homologous protein (CHOP) and caspase-12 in rat heart tissues were determined by Western blot and immunohistochemistry. After purified β₁-AA obtained by affinity chromatography was used to treat H9c2 myocardial cells, the cell viability was measured by CCK-8 assay and the apoptosis was analyzed by flow cytometry with Annexin V-FITC/PI double staining. The H9c2 cells were treated with ER stress inhibitor 4-phenoxybutyric acid (4-PBA) before interfered with β₁-AA, and the changes of cell viability and apoptosis were determined by CCK-8 assay and flow cytometry. RESULTS: Compared with vehicle group, the level of β₁-AA in the serum of rats was significantly increased after active immunization for 2 weeks and further rised in 8 weeks, and increased apoptosis was observed in cardiomyocytes after active immunization for 2 weeks, lasting till 8 weeks. Compared with vehicle group, the protein expression of GRP78, CHOP and caspase-12 increased after active immunization for 4 weeks and 8 weeks. Continuous reduction of cell viability and increased apoptosis of H9c2 cells were induced by β₁-AA. ER stress inhibitor 4-PBA pretreatment in H9c2 cells reversed the increased apoptosis and decreased cell viability induced by β₁-AA, indicating that suppression of ER stress effectively reduced cardiomyocyte apoptosis. CONCLUSION: β₁-AA induces increased apoptosis in cardiomyocytes by activating ER stress.     

Hypoxia promotes apoptosis of neural stem cells and down-regulates miR-26a

AIM: To investigate the effect of cobalt chloride (CoCl₂) on the apoptosis of neural stem cells (NSCs) and the expression of microRNA-26a (miR-26a) in vitro, and to explore the mechanisms of NSC apoptosis induced by CoCl₂. METHODS: NSCs were exposed to CoCl₂ at different doses (200~600 μmol/L) for 24 h. The cell viability and apoptosis were measured by CCK-8 assay and TUNEL method. The expression of miR-26a-3p, miR-26a-5p, GSK-3β, caspase-3, Bcl-2 and Bax was examined by real-time PCR. The protein levels of Bcl-2 and Bax were detected by Western blotting. RESULTS: The cell viability was inhibited and the apoptosis of NSCs was increased significantly by CoCl₂ in a dose-dependent manner (P<0.05). CoCl₂ at concentration of 400 μmol/L for 24 h was used to induce apoptosis and the expression of miR-26a was down-regulated compared with control (P<0.05). Exposure to CoCl₂ at concentration of 400 μmol/L up-regulated the expression of GSK-3β, caspase-3 and Bax, down-regulated the expression of Bcl-2 and Bcl-2/Bax (P<0.05). CONCLUSION: CoCl₂ at concentration of 400 μmol/L induces the apoptosis of NSCs obviously. CoCl₂ may induce the NSC apoptosis by mitochondrial apoptotic pathway. Declining miR-26a may be related to NSC apoptosis.     

Over-expression of SCUBE2 suppresses epithelial-mesenchymal transition through Wnt/β-catenin signaling pathway in colorectal cancer cells

AIM: To study the effect of SCUBE2 on epithelial-mesenchymal transition (EMT) in colorectal cancer cells and its mechanism. METHODS: The expression of SCUBE2 in human colorectal cancer cell line HCT116 and normal colonic cell line FHC was detected by real-time PCR and Western blot. HCT116 cells were transfected with GV144-SCUBE2 to over-express SCUBE2, and then the cell viability, migration, and apoptosis were determined by MTT assay, Transwell assay and flow cytometry, respectively. The expression of EMT markers (E-cadherin, vimentin, and Snail), β-catenin, c-Myc and cyclin D1 in the HCT116 cells was analyzed by real-time PCR or Western blot after transfection with GV144-SCUBE2 for 6 h, followed by the stimulation of 10 μg/L recombinant TGF-β1 protein for 48 h. Additionally, the EMT process of HCT116 cells, which were stimulated by TGF-β1, over-expressed SCUBE2, and treated with Wnt/β-catenin pathway activator lithium chloride (LiCl) or inhibitor XAV93920, was analyzed by Western blot. RESULTS: Compared with FHC cells, the expression of SCUBE2 in the HCT116 cells was significantly decreased. The viability and migration ability of the HCT116 cells were suppressed by SCUBE2 over-expression, but the apoptosis was not markedly changed. Elevated expression of SCUBE2 increased E-cadherin expression, and decreased the expression of vimentin, Snail, β-catenin, c-Myc and cyclin D1 induced by TGF-β1. Treatment with LiCl blocked but treatment with XAV93920 enhanced the effects of SCUBE2 on EMT. CONCLUSION: Over-expression of SCUBE2 may inhibit the cell growth and migration, and suppress EMT through Wnt/β-catenin signaling pathway.     

Effect of component II of broccoli polypeptide on glioma cell apoptosis

AIM：To explore the effect of component II of broccoli polypeptide on the apoptosis in glioma cells. METHODS：Human glioma SHG-44 cells were cultured and divided into control group and 3, 10, 30 and 100 mg/L component II of broccoli polypeptide groups. Cell viability was detected by MTT assay. The apoptotic rates were examined by Annexin V/PI staining. The morphological changes of the cells were observed under inverted microscope. The protein expression of Bax and Bcl-2 was detected by immunocytochemistry and Western blotting. The protein level of caspase-3 was also examined by Western blotting. RESULTS：Treatment with component II of broccoli polypeptide for 24 h, 48 h or 72 h induced significant inhibition of viability of SHG-44 cells in a time- and dose-dependent manner. The results of Annexin V/PI staining showed that the apoptotic rates were increased in treatment groups in a dose-dependent manner. The density of glioma cells was decreased after treated with increasing concentrations of the drug, and the apoptotic bodies were observed under inverted microscope at 72 h. The results of immunocytochemistry and Western blotting showed that the expression of Bax protein was increased but Bcl-2 protein expression was decreased, and the ratio of Bax/Bcl-2 was increased significantly compared with control group (P<0.05 or P<0.01). The level of caspase-3 protein was increased in 30 and 100 mg/L component II of broccoli polypeptide groups compared with control group (P<0.01). CONCLUSION：The component II of broccoli polypeptide increases the ratio of Bax/Bcl-2 and activates caspase-3 protein, thus inducing the apoptosis of glioma cells.     

Therapeutic effect of neuregulin-1β on pressure overload-induced myocardial hypertrophy in rats

AIM：To investigate the therapeutic effect and the mechanism of neuregulin-1β (NRG-1β) on the rat model of myocardial hypertrophy induced by pressure overload．METHODS：Eight weeks after coarctation of abdominal aorta, the Wistar rats were randomly divided into 4 groups: myocardial hypertrophy (model) group, sham operation (sham) group, NRG-1β treatment group (intravenous injection of NRG-1β at dose of 10 μg/kg daily for 7 d) and NRG-1β+Herceptin (HERCE) treatment group ［intravenous injection of NRG-1β (10 μg/kg) plus HERCE (10 μg/kg) daily for 7 d］. The characteristics of heart functions were evaluated by the methods of hemodynamics and echocardiography. Masson staining was employed to observe the pathological changes of myocardial tissues. The concentration of angiotensin II (Ang II) in myocardial tissues was measured by radioimmunoassay. The level of tumor necrosis factor α (TNF-α) in myocardial tissues was detected by ELISA. The mRNA expression of B-cell lymphoma/leukemia-2 (bcl-2) and bcl-2-associated X protein (bax) in the myocardium was determined by RT-PCR. RESULTS：The left ventricular ejection fraction (LVEF) and left ventricular fraction shortening (LVFS) were higher, while the left ventricular end-systolic diameter (LVESD) and left ventricular end-diastolic diameter (LVEDD) were smaller in NRG-1β group than those in model group. The left ventricular end-systolic pressure (LVESP) and maximal rate of increase/decrease in left ventricular pressure (±dp/dtmax) were higher, and left ventricular end-diastolic pressure (LVEDP) was significantly lower in NRG-1β group than those in model group. Compared with model group, treatment with NRG-1β decreased collagen volume fraction (CVF), reduced the Ang II and TNF-α, increased bcl-2 mRNA expression, and decreased bax mRNA expression in myocardial tissues. No difference of the above parameters between model group and NRG-1β+HERCE treatment group was observed. CONCLUSION：NRG-1 reduces the expression of Ang II and TNF-α in myocardial tissues in pressure-overload rats, thus reducing Ang II and TNF-α mediated myocardial interstitial remodeling. Increase in the mRNA expression of bcl-2 and decrease in the mRNA expression of bax by NRG-1 inhibit myocardial cell apoptosis, which is responsible for its role of improving cardiac function of myocardial hypertrophy induced by pressure overload.     

Effect of lysophosphatidic acid on expression of integrin β6 in epithelial cells

AIM: To investigate the effect of lysophosphatidic acid (LPA) on the expression of integrin β₆ (ITGB6) for determining the role of transforming growth factor β (TGF-β) activation induced by LPA in this process. METHODS: Normal human bronchial epithelial (NHBE) cells were primarily cultured in 6 well plate and stimulated with LPA. The mRNA expression of ITGB6 and the level of cell surface ITGB6 protein were detected by RT-PCR and flow cytometry,respectively. The activity of active TGF-β induced by LPA was measured by the method of transformed mink lung epithelial cells (TMLC) transfected with TGF-β responsive plasminogen activator inhibitor 1(PAI-1) promoter fused with firefly luciferase reporter gene. RESULTS: After stimulated with LPA at concentration of 10 μmol/L for 2 h, the mRNA expression of ITGB6 in epithelial cells was significantly increased in a time-dependent manner. The results of flow cytometry showed that the protein level of ITGB6 on cell surface was obviously increased after treated with LPA at concentration of 10 μmol/L for 4 h. The active TGF-β induced by LPA in epithelial cells was blocked by an α_Vβ₆ blocking antibody. However, α_Vβ₆ blocking antibody failed to inhibit the mRNA expression of ITGB6 induced by LPA. CONCLUSION: LPA induces the mRNA and cell surface protein of ITGB6 in epithelial cells. The up-regulated ITGB6 expression by LPA is independent on LPA-induced TGF-β activation.     

Effect of Vaccinium vitis procyanidin on regulation of glioma cell growth

AIM: To explore the effect of Vaccinium vitis procyanidin on the growth of glioma cells. METHODS: Glioma C6 cells were cultured and divided into control and 10, 20 and 40 μg/L Vaccinium vitis procyanidin groups. The influence of Vaccinium vitis procyanidin on the growth of C6 cells was measured by MTT assay and the observation under inverted microscope. The apoptotic rate was detected by Annexin V/PI staining. The protein expression of Bcl-2 and Bax was determined by immunocytochemistry. The protein levels of Bcl-2, Bax and caspase-3 were also examined by Western blotting. RESULTS: The growth of C6 glioma cells was inhibited by Vaccinium vitis procyanidin at concentrations of 10, 20 and 40 μg/L. The growth was significantly inhibited in 40 μg/L Vaccinium vitis procyanidin group at 24 h and 48 h, and in 20 and 40 μg/L Vaccinium vitis procyanidin groups at 72 h (P<0.01). The density of the cells was decreased when the concentration of Vaccinium vitis procyanidin increased. The apoptotic rate was increased when the concentration of Vaccinium vitis procyanidin increased either. The expression of Bcl-2 was decreased and Bax was increased after 10, 20 and 40 μg/L Vaccinium vitis procyanidin treatments. The ratio of Bax/Bcl-2 was increased when the dose of Vaccinium vitis procyanidin increased (P<0.05 or P<0.01). The expression of Bcl-2 was decreased (P<0.01), and Bax and caspase-3 were increased after 10, 20 and 40 μg/L Vaccinium vitis procyanidin treatments. The ratio of Bax/Bcl-2 was increased when the dose of Vaccinium vitis procyanidin increased (P<0.01). CONCLUSION: Vaccinium vitis procyanidin inhibits the growth of glioma cells by down-regulating Bcl-2 protein and up-regulating Bax protein to activate caspase-3, thus inducing apoptosis.     

Bone marrow-derived mesenchymal stem cells protect against hypoxia-induced injury and apoptosis of PC12 cells via up-regulation of erythropoietin expression

AIM： To investigate the effects of bone marrow-derived mesenchymal stem cells (BM-MSCs) on cobalt chloride (CoCl2)-induced injury and apoptosis of PC12 cells. METHODS：PC12 cells were divided into control group, CoCl2 group, BM-MSCs-siCTL+CoCl2 group and BM-MSCs-siEPO+CoCl2 group. The viability of the PC12 cells was measured by MTT assay. Flow cytometry and Hoechst 33258 staining were used to determine the apoptotic rate and the changes of chromatin distribution in PC12 cells. The expression of erythropoietin (EPO) in BM-MSCs was measured by RT-PCR and Western blotting. The mRNA expression of Bcl-2 and Bax in PC12 cells was detected by RT-PCR. Caspase-9 and caspase-3 assay kits were used to detect the activity of caspase-9 and caspase-3. RESULTS：The viability of PC12 cells treated with CoCl2 for 24 h and 48 h decreased to （43.0±6.4）% and （33.8±5.7）%, respectively, while 1∶15 ratio of BM-MSCs co-culture increased the cell viability to （77.9±3.8）% and （75.2±9.7）%,respectively. The expression of EPO in BM-MSCs was up-regulated after treated with 0.6 mmol/L CoCl2 for 24 h and 48 h, while EPO siRNA significantly abrogated the EPO expression in BM-MSCs. BM-MSCs-siCTL co-culture significantly inhibited the apoptosis of PC12 cells induced by CoCl2. However, EPO siRNA the protective effect of BM-MSCs. Compared with CoCl2 treatment group, BM-MSCs co-culture induced remarkable increase in the expression of Bcl-2 and decrease in the expression of Bax in PC12 cells, which was reversed by EPO siRNA. BM-MSCs-siCTL co-culture remarkably abrogated the CoCl2 induced up-regulation of caspase-9 and -3, while BM-MSCs-siEPO co-culture significantly reversed the down-regulation of caspase-9 and -3 induced by BM-MSCs-siCTL co-culture. CONCLUSION：BM-MSCs protect PC12 cells from apoptosis induced by CoCl2. The protective effect of BM-MSCs might be executed by up-regulating the expression of EPO.     

Pancreatic cancer cell-secreted exosomes promote apoptosis of β cells via activation of mitochondrial apoptotic pathways

AIM: To investigate the effects of exosomes secreted by pancreatic cancer cells on the viability and function of β cells and the possible mechanism. METHODS: ExoQuick-TC kit was used to extract exosomes in the supernatants of mouse pancreatic cancer Pan02 and MPC-83 cells, and the extracted exosomes were identified by transmission electron microscopy. Fluorescence-labeled exosomes were incubated with mouse insulinoma MIN6 cells for 48 h to detect whether exosomes secreted by pancreatic cancer cells were uptaken by MIN6 cells. MTT and glucose-stimulated insulin secretion (GSIS) assays were conducted to examine cell viability and insulin secretion of MIN6 cells after incubating with exosomes. The expression of miR-204 and Bcl-2 mRNA in MIN6 cells was detected by qPCR. The protein expression of Bcl-2, Bax, caspase-3 and cytochrome C (Cyt-C) in MIN6 cells was determined by Western blot. RESULTS: The results of transmission electron microscopy showed that both Pan02 cells and MPC-83 cells secreted exosomes, and Pan02 cells secreted more. The co-incubation results of fluorescence-labeled exosomes and MIN6 cells confirmed that MIN6 cells were able to ingest large amounts of exosomes secreted by pancreatic cancer cells. The results of MTT and GSIS assays showed that the viability and the level of high glucose-stimulated insulin secretion of MIN6 cells in exosome treatment group significantly decreased compared with nontreatment group (P<0.01). The results of qPCR showed that the exosomes secreted by pancreatic cancer cells were rich in miR-204, and the mRNA expression of Bcl-2 in MIN6 cells was significantly down-regulated by exosome incubation (P<0.01). The results of Western blot showed that the protein expression of Bcl-2 in the MIN6 cells treated with exosomes was significantly down-regulated (P<0.05), and the protein levels of Bax, cleaved caspase-3 and Cyt-C in exosomes treatment group were significantly up-regulated (P<0.01). CONCLUSION: Pancreatic cancer cells secrete exosomes. The exosomes secreted by pancreatic cancer cells are ingested by β cells, and reduce the viability and insulin secretion of β cells. The mechanism may be related to the increase in exosomal miR-204 in the β cells. Increasing miR-204 may inhibit the expression of Bcl-2 and promote the activation of mitochondrial apoptosis in β cells.     

Immunosuppressive and protective effects of human α1-antitrypsin on pancreatic β-cell transplantation

AIM：To study the immunosuppressive and protective effects of human α1-antitrypsin (hAAT) on pancreatic β-cell transplantation. METHODS： An NIT-1 cell line (NIT-hAAT) was constructed, which can stably express the protein of hAAT. The BALB/c mice were intraperitoneally injected with NIT-1 and NIT-hAAT cell lines twice to induce cytotoxic T-lymphocytes (CTL). The apoptotic situation, the cytokine expression, and the mRNA expression of inflammatory factors were examined after mixed culture of CTL with NIT-1or NIT-hAAT cell line pretreated with mitomycin. Both cell lines were transferred into the left renal capsule of the diabetic mice to dynamically observe the changes of blood sugar and body weight, the serum levels of insulin and C-peptide, and the pathological changes of the transplanted sites. RESULTS： The results of extended CTL killing assay showed that the cytotoxic effect on NIT-hAAT cell acceptor mice was significantly reduced compared with NIT-1 cell acceptor mice. hAAT effectively reduced apoptosis, inhibited the mRNA expression of inflammatory factors IL-1β and IL-6, and adjusted the balance of Th1/Th2 cytokine expression. After NIT-hAAT was transplanted into the diabetic mice, blood glucose decreased obviously and maintained for 28 d. The serum levels of insulin and C-peptide increased obviously. The infiltration of the inflammatory cells in the transplanted sites significantly reduced. CONCLUSION：hAAT has the abilities of reducing cytotoxic effect of CTL on the β-cells, inhibiting inflammatory factor expression, and stopping short-term immunological rejection of the acceptor. hAAT has obvious immunosuppressive and protective effects on pancreatic β-cell transplantation for treatment of diabetes.