Repeated exposure of mouse dermal fibroblasts at a sub-cytotoxic dose of UVB leads to premature senescence: a robust model of cellular photoaging.

BACKGROUND: Photoaging skin is due to accumulative effect of UV irradiation that mainly imposes its damage on dermal fibroblasts. To mimic the specific cellular responses invoked by long term effect of UVB, it is preferable to develop a photo-damaged model in vitro based on repeated UVB exposure instead of a single exposure. OBJECTIVE: To develop a photo-damaged model of fibroblasts by repeated UVB exposure allowing for investigation of molecular mechanism underlying premature senescence and testing of potential anti-photoaging compounds. METHODS: Mouse dermal fibroblasts (MDFs) at early passages (passages 1-3) were exposed to a series of 4 sub-cytotoxic dose of UVB. The senescent phenotypes were detected at 24 or 48h after the last irradiation including cell viability, ROS generation, mitochondrial membrane potential, cell cycle, production and degradation of extracellular matrix. RESULTS: Repeated exposure of UVB resulted in remarkable features of senescence. It effectively avoided the disadvantages of single dose such as induction of cell death rather than senescence, inadequate stress resulting in cellular self-rehabilitation. CONCLUSION: Our work confirms the possibility of detecting cellular machinery that mediates UVB damage to fibroblasts in vitro by repeated exposure, while the potential molecular mechanisms including cell surface receptors, protein kinase signal transduction pathways, and transcription factors remain to be further evaluated.

Inhibition of beta-amyloid precursor protein gene in SK-N-SH cells by piperlonguminine/dihydropiperlonguminine components separated from Chinese herbal medicine Futokadsura stem.

Alzheimer disease (AD) is a progressive neurodegenerative disease characterized by progressive cognitive and memory decline. Amyloid precursor protein (APP) is a transmembrane protein, it has been known to play an important role in AD pathogenesis. Previous studies have shown that a Chinese herb Futokadsura stem can selectively inhibit the expression of amyloid precursor protein (APP) gene. We want to find the effective components in Futokadsura stem which have the inhibitory effect. Futokadsura stem was separated and purified with chemical methods, and then different separation components were added on SK-N-SH cells in different concentrations. Using MTT methods, we detected proliferation activity of SK-N-SH cells which were treated with different separation components of Futokadsura stem. Using RT-PCR, Western blot methods, we detected APP gene expression in SK-N-SH cells after they are treated with different Futokadsura stem separation components. We found that piperlonguminine/dihydropiperlonguminine components (1:0.8) separated from Futokadsura stem acetic ether extracts could selectively inhibit the expression of APP gene in SK-N-SH cells in mRNA and protein levels. This inhibition effect is concentration-dependent. Under experimental concentrations, the components did not affect the proliferation activity of SK-N-SH cells. These data suggest that piperlonguminine/dihydropiperlonguminine components are the effective components in Futokadsura stem which can inhibit the expression of APP gene.

[Involvement of Caspase-3 and p38 mitogen-activated protein kinase in MMT-induced apoptosis in PC-3M cells].

OBJECTIVE: To explore the function of Caspase-3 and p38 MAPK in MMT-induced apoptosis in PC-3M cells. METHODS: After incubation of PC-3M cells with 1 mmol/L MMT, the activity of Caspase-3 was examined. The influence on cells viability of Z-DEVD-FMK, a Caspase-3-specific peptide inhibitor, was also examined. Western blot was used to examine the change of p38 MAPK. The effect on cells viability and Caspase-3 activity of SB203580, a specific inhibitor of p38 MAPK, were also examined. RESULTS: The activity of Caspase-3 increased significantly in MMT-induced apoptosis in PC-3M cells /9P < 0.01), and Z-DEVD-FMK could protect cells from apoptosis (P < 0.01). In this course, the phosphorylation of p38 MAPK could be observed. SB203580 inhibited Caspase-3 activity (P < 0.05) and prevented PC-3M cells from MMT-induced apoptosis (P < 0.05). CONCLUSION: Caspase-3 and p38 MAPK are involved in MMT-induced PC-3M cells apoptosis.

Effects of scutellarin on apoptosis induced by cobalt chloride in PC12 cells.

The present study investigated the protective effects of scutellarin on cobalt chloride (CoCl2)-induced apoptosis in PC12 cells. Incubation of PC12 cells with 500 microM CoCl2 for 24 h resulted in significant apoptosis as evaluated by the crystal violet, electron microscopy and flow cytometry assays. The increase of caspase-3 activity, decrease of Bcl-XL expression, phosphorylation of p38 mitogen-activated protein kinase (MAPK) and accumulation of intracellular reactive oxygen species (ROS) were also seen in CoCl2-treated PC12 cells. Scutellarin at 0.1, 1 and 10 microM significantly protected against the apoptotic cell death induced by CoCl2. Scutellarin decreased caspase-3 activity, increased Bcl-XL expression, inhibited p38 phosphorylation and attenuated ROS production. These results demonstrate that scutellarin can protect PC12 cells from cobalt chloride induced apoptosis by scavenging ROS, inhibiting p38 phosphorylation, up-regulating Bcl-XL expression and decreasing caspase-3 activity, and may reduce the cellular damage in pathological conditions associated with hypoxia-mediated neuronal injury.

[Protective effect of NEP on Abeta-induced apoptosis in PC12 cells].

OBJECTIVE: To identify the effect of NEP on Abeta -induced apoptosis in PC12 cells. METHODS: PC12 cells that stably express NEP is generated and the effect of NEP on the process of apoptosis induced by Abeta is analyzed, including the viability of the cells, the production of LDH, ROS and ATP, the activity of Caspase-3. RESULTS: NEP could improve the viability of cells and the production of ATP, inhibit the release of LDH and ROS. In the same time, the activity of caspase-3 descended (P < 0.05). But iNEP had not significant effect on cells apoptosis (P > 0.05). CONCLUSION: NEP has the protective effect on Abeta-induced apoptosis in PC12 cells.

[siRNA inhibits efgl7 expression in human endothelial cell line HUVEC].

OBJECTIVE: To evaluate the effects of small interference RNA (siRNA) on epidermal growth factor-like domain 7 (egfl7) gene expression in human endothelial cell line HUVEC. METHODS: siRNA targeting egfl7 (siRNA1, siRNA2, siRNA3 and siRNA4) was constructed through online design of Amnion company and transfected into human endothelial cell line HUVEC with lipofectamine. The nontransfected cells and cells treated with control siRNA were taken as controls. At 24, 48 and 72 hours post various interventions, cell viability was determined by MTS method as well as LDH and ATP releasing tests. egfl7 expressions at protein and mRNA levels were detected by Western blot and RT-PCR respectively. RESULTS: Cell survival rate, LDH and ATP release were significantly reduced in siRNA treated cells compared to control cells (P < 0.05). Similarly, egfl7 expression at protein and mRNA levels was also significantly reduced in siRNA treated cells (P < 0.01), especially in siRNA1 treated cells. CONCLUSION: siRNA inhibited egfl7 gene expression and cell survival in HUVEC.

[Mechanism of reactive oxygen species in manganese chloride-induced apoptosis in PC12 cells].

OBJECTIVE: To explore the mechanism of reactive oxygen species (ROS) in manganese chloride (MnCl(2))-induced apoptosis in PC12 cells. METHODS: The model that MnCl(2) induced apoptosis in PC12 cells was established. The apoptotic effect of MnCl(2) on PC12 cells was analyzed with the MTT, the flow cytometry and the DNA fragmentation. The production of ROS and ATP in MnCl(2)-induced apoptosis of PC12 cells was examined. The influence of MnCl(2) on the expression of bcl-xl, bax and the activity of Caspase 3 was also analyzed. RESULTS: MnCl(2) triggered PC12 cells apoptosis in a dose-and time-dependent manner (P < 0.01). The rate of apoptosis was significantly increased (P < 0.01) when MnCl(2) of 2 mmol/L induced PC12 cells for 36 hours. The production of ROS was increased (P < 0.001) and the quantity of ATP was decreased (P < 0.01) in PC12 cells with the same inducement of MnCl(2). The expression of bcl-xl was inhibited and the bax was activated in this process (P < 0.01). Caspase 3 was also activated (P < 0.01). CONCLUSION: MnCl(2) induces apoptosis of PC12 cells, which is related to the increase of ROS, the inhibition of the mitochondria and the activation of Caspase 3.