Role Of PUMA In Methamphetamine-induced Neuronal Apoptosis

Background:Methamphetamine (methamphetamine, METH), commonly known as "crystal METH", is a white crystalline powder with colourless and odourless. METH belongs to amphetamines central nervous system stimulant. Methamphetamine has become the second used illegal drug in the world widely. Because it works fast, as well as its lasting exciting time, accessible synthetic materials, simple synthesis technology and a variety of ways to absorb, METH abuse quickly spread throughout the world. In China, METH abuse area presents gradually expanded, abuse rate was growing rapidly and abuser showed a trend of getting younger. METH abuse not only damaged their body and spirit, but caused a large number of criminal cases. It damage to public safety seriously. METH addiction not only caused serious damage to the body, but also to neurons. Sudden death cases caused by METH can be found in forensic work. Therefore, study of toxic injury caused METH has become the hotspot on the worldwide, and it also got some achievements.The mechanism of METH neurotoxicity is still not clear entirely, but the majority of studies have shown that METH have toxic effects for animal and human brain dopaminergic neurons. It mainly involves the following mechanisms:high fever, oxidative stress, glutamate excitatory toxic effect, mitochondrial dysfunction, nerve cells apoptosis, glial cell activation and cytokine production, neuron degeneration and so on. Among them, the oxidative stress is closely connected with other mechanisms, and mitochondrial plays an important role in oxidative stress. We found the 53 upregulated modulator of apoptosis (PUMA) obviously higher than in control group in PC 12 cells.PUMA belongs to the BH3-only protein in the Bcl-2 family. The Bcl-2 families are divided into antiapoptotic proteins, promoting apoptosis protein and BH3-only protein. It is the switch of the mitochondrial apoptosis pathway. In recent years, PUMA became a hotspot of research on mitochondrial apoptotic pathways because of its strong function to promote apoptosis. It is closely related to the function of its BH3 domain. PUMA can combine with the antiapoptotic proteins Bcl-2 which locates on the mitochondrial membrane, so as to remove the Bcl-2 to interact with directly promote apoptosis proteins, such as the BID, BIM, p53. It induced cell apoptosis indirectly. PUMA can interact with proteins Bax, direct activation Bax to promote cell apoptosis directly. However, most of these studies focused on the mechanism of apoptosis on the tumor and its prevention, and research about the role of PUMA in METH neurotoxicity reports did not be reported.We planned to establish METH poisoning PC 12 cells and SH-SY5Y cell model, and then observed the morphological change and detected PUMA protein expression changes to further verify the results of gene chip. We next designed the specific design of siRNA to block of PUMA protein expression, and then explore the relationship between apoptosis and PMUA using flow cytometry technology, TUNEL staining and WB, including caspase 3 and PARP expression. In order to further investigate Role of PUMA in methamphetamine-induced neuronal apoptosis, we examined the Bcl-2 and Bax expression and the changes of cytochrome c (Cyt cytochrome c, c).This project intends to prove the role of PUMA in METH mediated apoptosis, to METH neurotoxic injury mechanism research and genetic intervention treatment provide theoretical basis.Objectives:We aimed to establish METH poisoning PC 12 cells and SH-SY5Y cell model, and then observed the morphological change and detected PUMA protein expression changes to further verify the results of gene chip. We next designed the specific design of siRNA to block of PUMA protein expression, and then explore the relationship between apoptosis and PMUA using flow cytometry technology, TUNEL staining and WB, including caspase 3 and PARP expression. In order to further investigate Role of PUMA in methamphetamine-induced neuronal apoptosis, we examined the Bcl-2 and Bax expression and the changes of cytochrome c (Cyt cytochrome c, c).This project intends to prove the role of PUMA in METH mediated apoptosis, to METH neurotoxic injury mechanism research and genetic intervention treatment provide theoretical basis.Methods:1. Establish the METH poisoning PC12 cells and SH SY5Y cells model(1)10% fetal bovine serum (FBS) in DMEM medium respectively inoculated PC12 cells and SH SY5Y cells in 96-well plates, under the condition of 37℃ and 5% CO2. When cell density reaches 70%, respectively, they were cultured using 0 mmol/L,0.5 mmol/L、1.0 mmol/L、1.5 mmol/L、2.0mmol/L、2.5 mmol/L、3 mmol/L、3.5mmol/L、4mmol/L、4.5mmol/L、5 mmol/L METH with 2% FBS in DMEM medium. After 24 hours, the LC25, LC50 of PC12 cells and SH SY5Y cells in different concentrations of METH were detected by CCK-8.(2) PC12 cells and SH SY5Y cells were inoculated in six well plates respectively. When cell density reaches 70%, PC 12 cells with the concentration of Ommol/L、1.0mmol/L、2.0mmol/L、2.5 mmol/L、3.0 mmol/L和4.0 mmol/L, SH SY5Y cells with concentration of 0 mmol/L、0.5 mmol/L、1.0 mmol/L、1.5 mmol/L、 2.0 mmol/L和2.5 mmol/L METH with 2% FBS in DMEM medium for 24 hours. And then we extracted the total protein. Western Blot detected PUMA protein expression changes, in order to select appropriate concentration.(3) We chose a suitable concentration of METH to establish the METH poisoning PC 12 cells and SH SY5Y cells model, and cells were cultured with the concentration of METHrespectively 0 hours,2 hours,4 hours,8 hours,16 hours,24 hours. After 24 hours, total protein were extracted to Western Blot for detection PUMA protein expression changes, in order to select appropriate action time by METH.2. The role of PUMA in METH-induced neuronal apoptosis(1) We designed PUMA siRNA for PC12 cells and SH respectively, and cell transfection by lipo 2000. We detected cell PUMA protein expression by WB to choose the effective interference fragment.(2) The PC 12 cells and SH SY5Y cells can be divided into blank control group (Ctrl) and control group+small interference(siRNA), the drug group (METH), the drug+small interference group (METH+siRNA). After 24 hours, cell protein were collected to detect the expression of PUMA, Caspase 3, PARP.(3)Flow cytometry and TUNEL staining method to detect cell apoptosis on each group.3. PUMA mediated METH-induced neuronal apoptosis by mitochondrial pathway(1) WB to detect the expression of Bcl-2 and Bax cell on each group, and calculate the ratio of Bax/Bcl-2.(2) Extract cytoplasm and mitochondrial proteins, and detect the expression of Cyt c on cytoplasm and mitochondria.Results:1. Establish the METH poisoning PC12 cells and SH SY5Y cells model(1) The survival rate of PC 12 cells and SH SY5Y cells gradually reduced with increasing concentration of METH. LC25 and LC50 of PC12 cells were 3.42 mmol/L和4.53mmol/L respectively, and LC25 and LC50 of SH SY5Y cells were 2.14 mmol/L和3.42mmol/L respectively.(2) After cultured with Ommol/L、1.0mmol/L、2.0 mmol/L、2.5 mmol/L、3.0 mmol/L和4.0 mmol/L METH respectively, Western Blot results show that the expression of PUMA rise gradually with the increase of concentration of METH in PC12 cells. The expression of PUMA was higher than 4.5 times between control group and 3.0 mmol/L group.(3) After cultured with 0 mmol/L、0.5 mmol/L、1.0mmol/L、1.5 mmol/L、2.0 mmol/L和2.5 mmol/L METH respectively, Western Blot results show that the expression of PUMA rise gradually with the increase of concentration of METH in SH-S Y5 Y cells. The expression of PUMA was higher than 2 times between control group and 2.0 mmol/L group.(4) Western Blot results show that the expression of PUMA rise gradually after cultured with 3.0 mmol/L in 0 hours,2 hours,4 hours,8 hours,16 hours,24 hours respectively in PC 12 cells. The expression of PUMA began to rise in 2 hours, reached peak in 16 hours. The expression of PUMA rise gradually after cultured with 2.0 mmol/L in 0 hours,2 hours,4 hours,8 hours,16 hours,24 hours respectively in SH-SY5Y cells. The expression of PUMA began to rise in 2 hours, reached twice as often as control group in 8-24 hours.2. The role of PUMA in METH-induced neuronal apoptosis(1) Using specific siRNA fragment, after different processing 24 hours, cells morphology were observed under inverted microscope cell. We can see cells does not take place obvious change compared with the control group (Ctrl) and control group+small interference (siRNA).And morphology of PC 12 cells or SH SY5Y cells on drug group (METH) changed obviously. The synaptic shortened, cell body became circular, bright area could be see around the cell body, and connections between cells became loose. Part of the cell floated in the medium. And cells on METH+small interference group (METH+siRNA) improved.(2) Using specific siRNA fragment, and found that compared with the METH group, the expression of PUMA fell about 60% on METH+siRNA#1 group and METH+siRNA#2 group. And for SH SY5Y cells, PUMA expression quantity drop about 40% with siRNA# 3, while siRNA# 4 did not work.(3) There were no significant differences on the rate of apoptosis compared with control group and siRNA group by flow cytometry. The total apoptosis rate of PC 12 cells was 17.5±0.13%, and the total apoptosis rate of SH-SY5Y cells was 22.9 ±4.56%. The rate of apoptosis were significantly lower compared with METH group and siRNA+METH group.(4) There were no significant differences on the number of apoptotic cells compared with control group and siRNA group by TUNEL staining. the total number of apoptotic cells increased about 6 times on PC 12 cells, and it increased about 7 times on SH-SY5Y cells. The number of apoptotic cells were significantly lower compared with METH group and siRNA+METH group.3. PUMA mediated METH-induced neuronal apoptosis by mitochondrial pathway(1) Using specific siRNA fragment, after different processing 24 hours, and Western Blot to detect the Bcl-2 and Bax proteins. We found the expression of Bcl-2 reduced, and the expression of Bax increased, the ratio of Bax/Bcl-2 increased compared with METH group and control group.This effect was reversed with siRNA. The expression of Bcl-2 increased, and the expression of Bax reduced, the ratio of Bax/Bcl-2 reduced compared with METH group and siRNA+METH group.(2) Using specific siRNA fragment, after different processing 24 hours, extract the cytoplasm protein and mitochondrial protein, and Western Blot to detect Cyt c protein. We found the expression of Cyt c in the mitochondria decreased, and the expression of Cyt c in the cytoplasm increased compared with METH group and control group. The expression of Cyt c in the mitochondria increased, and the expression of Cyt c in the cytoplasm reduced compared with METH group and siRNA+METH group.(3) Using specific siRNA fragment, after different processing 24 hours, and Western Blot to detect Caspase3 and PARP proteins. We found the expression of Caspase3 and PARP protein increased compared with METH group and control group. And the expression of Caspase3 and PARP protein decreased compared with METH group and siRNA+METH group.Conclusion:1. METH exposure increased PUMA protein in PC12 and SH-SY5Y cells.2. METH elevated Bax, reduced Bcl-2, activated caspase 3 and PARP.3. METH increased the release of cytochrome c from mitochondria to cytoplasm.4. All these effects were attenuated or reversed after silencing PUMA with siRNA.