PC12 cell is a tumor cell line isolated from rat adrenal pheochromocytoma. The differentiated PC12 cell has typical neuronal characteristics in morphology and function, and is widely used as a cell model to study apoptosis and differentiation of nerve cells. Hydrogen peroxide (H2O2) is thought to be the major precursor of ROS and is widely used to induce oxidative stress injury. Exogenous H2O2 can easily immerse into cells through cell membrane, producing a large number of ROS, thus is commonly is used to simulate cell peroxidation damage in vitro. There are many studies on the apoptosis of PC12 cells induced by H2O2, and the conclusions are consistent[10, 11], which can reflect the pathological changes of nerve cells. However, the effective concentration and action time are different. In general, low and moderate concentrations (50–500 micromol/L) of H2O2 can induce oxidative stress, while high concentrations can rapidly cause cell necrosis. In this study, we found that PC12 cells treated with H2O2 for a certain period of time, the cells did not stretch well, the process disappeared, the cell body retracted, a large number of particles accumulated in rough cytoplasm, and some cells did not adhere to the wall,which suggesting that H2O2 may induce cell apoptosis by interfering with cell metabolism and changing cell adhesion. The apoptosis of PC12 cells was analyzed by morphological observation and MTT. The results showed that the apoptosis of PC12 cells could be induced by 100um mol/L H2O2 for 24 hours.
The pathway of H2O2 induced apoptosis is not uniform. Dumont et al found that H2O2 induced apoptosis of T cells mainly depended on mitochondrial ROS and NF-kB activation. H2O2 promotes apoptosis through activating Caspase-3 of HL-60 cells. Kitamura et al found that Bcl-2 and Bax did not increase significantly after treated with H2O2, but mediated apoptosis by increasing P53 expression. H2O2 induced apoptosis of hepatoblastoma cells, not only up-regulated the expression of P53, but also decreased the levels of Bcl-2 protein and Bax protein. In this study, we found that H2O2 can up-regulate the expression of Bax, Caspase-3 and down-regulate the expression of Bcl-2. It is suggested that the mechanism of apoptosis of PC12 cells induced by H2O2 may involve increasing pro-apoptotic protein and decreasing anti-apoptotic protein, thus changing PC12 cell apoptotic environment. This is consistent with recent research that H2O2 can increase Bax and cleaved caspase-3 expression in PC12 cells.
The process of neuronal apoptosis is similar to that of other cells. After death signals, apoptosis-promoting proteins such as Bax and Bid are translocated to the extracellular membrane of mitochondria and interact with anti-apoptotic proteins such as Bcl-2, which deprive the anti-apoptotic protein of its inhibitory effect on apoptosis, increase the permeability of mitochondrial membrane, release cytc into cytoplasm and activate Caspase-3, which eventually leads to apoptosis. Bax/Bcl-2 ratio plays an decisive role in determining whether cells enter the apoptotic state, so the expression ratio of two genes is often used to study the degree of apoptosis. The results of Realtime Q-PCR and Western blot showed that the expression levels of pro-apoptotic factors Bax and Caspase-3, anti-apoptotic factor Bcl-2 and Bax/Bcl-2 were significantly increased after treated with 100 mmol/L H2O2 for 24 hours. TUNEL staining also confirmed this view. PC12 cells treated with 100 micromol/L H2O2 could obviously induce apoptosis of PC12 cells, the apoptosis rate was 26.8%.
PPARγ is a ligand-activated nuclear transcription factor. After being activated by its ligand, PPARγ can combine with specific DNA response elements to regulate the transcription and expression of genes. Our previous studies demonstrated that PPARγ agonist rosiglitazone can upregulate PPARγ mRNA and protein expression in aged SHRs, which was accompanied by markedly decreased expression of oxidative stress markers (iNOS and gp47phox) and pro-apoptotic markers (Bax and caspase-3). Karen et al. demonstrated that rosiglitazone up-regulates the Bcl-2 protein in neurons and induces mitochondrial stabilization and protection against oxidative stress and apoptosis. These results indicated that the PPARγ agonist, rosiglitazone, may exert neuroprotective effects through antioxidative and anti-apoptotic mechanisms. To confirm the protective effect of PPARγ agonist on PC12 cells injured by H2O2, PC12 cells were incubated with different concentrations of pioglitazone before exposure to H2O2 for one hour. MTT results showed that pioglitazone concentration-dependently increased the survival rate of PC12 cells and played a neuroprotective role. The results of flow cytometry and TUNEL staining also confirmed the conclusion of MTT experiment. The early and late apoptotic cells were significantly less in the pioglitazone-protected group than in the H2O2-injured group, and the apoptotic rate was significantly lower. Realtime Q-PCR and Western blot assays confirmed that pioglitazone significantly increased PPARγ expression in PC12 cells, which was 4.4 times higher than that in the control group. This confirms previous reports that neuroprotective doses of pioglitazone can induce a five-fold increase in PPARγ expression, thereby maintaining the responsiveness to cortical neurons by increasing the expression of its receptors.
In addition, we also observed that hydrogen peroxide could also induce the increased expression of PPARγ, which may be a compensatory protective mechanism for the cells damaged. Western blot was used to detect the expression of Bax, Bcl-2 and Caspase-3 proteins. The results showed that 100 micromol/L H2O2 could increase the expression of Bax and Caspase-3 proteins, decrease the expression of Bcl-2 protein and increase the ratio of Bax to Bcl-2. Pioglitazone could down-regulate the expression of Bax and Caspase-3 protein and up-regulate Bcl-2 protein expression, thus reducing the ratio of Bax to Bcl-2. These results suggest that pioglitazone can attenuate the pro-apoptotic environment of PC12 cells induced by H2O2. To further explore whether rosiglitazone can play a role in the activation of PPARγ. We used PPARγ antagonist GW9662 and PPARγ siRNA to block the expression of PPARγ in PC12 cells. The results demonstrated that pretreatment with GW9662 significantly decreased the increased Bax/Bcl-2 ratio and Caspase-3 expression in PC12 cells injured by H2O2. PPARγ siRNA had the same effect. So GW9662 and PPARγ siRNA can offset the protective effect of pioglitazone on PC12 cells injured by H2O2.
In conclusion, the neuronal apoptosis model induced by oxidative stress was established in vitro, and the neuroprotective effect of pioglitazone was studied. The results showed that pioglitazone could increase the activity of PC12 cells damaged by H2O2, increase the expression of Bcl-2, decrease the expression of Bax and Caspase-3, thus decreased the pro-apoptotic environment and the apoptotic rate of PC12 cells. PPARγ antagonist or PPARγ siRNA blocked the expression of endogenous PPARγ, inhibited the protective effect of pioglitazone on PC12 cells injured by H2O2, suggesting that pioglitazone could protect PC12 cells from oxidative stress by reducing the expression ratio of Bax/Bcl-2 and Caspase-3 through PPARγ activation pathway. In conclusion, pioglitazone can exert anti-apoptotic effect and promote the survival of PC12 cells in the presence of oxidative stress injury. This effect is through PPARγ activation pathway. This study thus suggests that PPARγ activation might have potential for intervention in neurodegenerative disorders.