The Function Of PGC-1α Under Cold Stress In Zebrafish Cells

PGC-1α is called peroxisome proliferator activated receptor γ(PPARγ)and is a transcriptional coactivator that regulates genes involved in energy metabolism.It is also a major regulator of mitochondrial biogenesis.PGC-1α is mainly distributed in a large number of functional tissues,such as brown adipose tissue(BAT),liver,brain,muscle,and heart,and has tissue-specific regulation,not only participating in mitochondrial biosynthesis,but also Involved in glucose metabolism,lipid metabolism,skeletal muscle fiber type conversion,and thermogenesis under cold stress.PGC-1α interacts with nuclear receptor PPAR-γ,cAMP response element binding protein(CREB)and nuclear respiratory factor(NRF)and can regulate its activity.The structure of PGC-1α includes a C-terminal transcriptional activation domain,an intermediate transcriptional repression domain,a motif structure rich in serine and arginine residues,and an N-terminal RNA binding domain.PGC-1α can bind to multiple transcriptional coactivators which determine its functional diversity.As a key regulatory molecule involved in various physiological and biochemical processes,PGC-1α is considered to be the main receptor for signals from the body or cells.It can be activated by many factors: 1.Reactive oxygen species(ROS)and reactive nitrogen species(RNS).Both of these are by-products of metabolism that are endogenously formed by the cell,but are upregulated when the cell is under stress.2.PGC-1α is strongly induced by cold exposure and can adapt to the effects of low temperature environment by promoting thermogenesis in the body.3.Exercise and prolonged endurance training can all increase the expression of PGC-1α.There are other multiple signaling pathways that activate PGC-1α transcription and expression.PGC-1α was originally discovered as a cold-induced regulator of increased adaptive heat production in brown fat under cold exposure.As one of the important environmental factors,temperature affects all aspects of biology.As a temperature-changing animal,fish has its physiological activities such as growth,reproduction and metabolism controlled by temperature.Mammals are exposed to cold environments.They are homothermal animals,primarily through skeletal muscle tremors,increased aerobic respiration to produce ATP and calories to maintain normal physiological activity at low temperatures;plants are exposed to cold conditions,protein and nucleic acid conformation changes,mobility of cell membranes decreased,mainly by increasing the expression of coldinducible proteins,and causes downstream signaling pathways to adapt to low temperatures.The role of PGC-1α in cold stress is mainly concentrated in mammals and is rarely reported in fish.Therefore,in this study,ZF4 cells of zebrafish embryonic fibroblasts were used as a research model.PGC-1α in ZF4 cells was knocked down through lentivirus interfering,and stable cell lines were selected by puromycin.After that the cells were under cold treatment at 18°C for 7 days or 10°C for 7 days,and stained with CM-H2 DCFDA probe to detect the ROS content in cells by flow cytometry.In addition,the cells were stained with JC-1 probe to detecte the mitochondrial membrane potential changes after cold treatment to explore the function of PGC-1α under cold stress process in ZF4.After treatment with wild-type zebrafish ZF4 cells at 18°C for 1~7 d,the mRNA expression of PGC-1α increased significantly compared with that at 28°C.The above confirmed that the expression of PGC-1α in ZF4 cells was increased by low temperature stimulation.To investigate the role of PGC-1α in ZF4 cells under cold stimulation,four shRNAs of PGC-1α were designed based on the coding sequence of the PGC-1α gene on the NCBI website: start from the 165,457,1486,and 2487 base site,21 bp in length.After transfection of the lentiviral plasmid,the expression of PGC-1α in ZF4 cells was detected.The knockdown efficiency of the four PGC-1α sites were 43%,38%,25% and 50%,respectively,so the ZF4 cell transfected with PGC-1α-shRNA4 was selected as a subsequent experimental group.The study was divided into three groups: blank control group(pLKO.DEST.EGFP),negative control group(pLKO.1-shNC)and experimental group(pLKO.1-shRNA).The above three groups of cells were treated at 18°C and 10°C for 1d,4d and 7d respectively.After staining with CM-H2 DCFDA probe,three groups of control group,PGC-1α-shNC and PGC-1α-sh4 were detected by flow cytometry.Intracellular ROS levels revealed that knockdown of PGC-1α did not affect intracellular ROS levels at 28°C,whereas PGC-1α knockdown group(PGC-1α-sh4)was induced after ZF4 cells were stimulated at 18°C for different times at different temperatures.)The intracellular ROS content was higher in the blank control group and the negative control group: 1 day at 18°C(0.93±0.03 vs 0.79±0.03,p<0.001)and 4 days at 18°C(2.10±0.15 vs 1.24±0.06,p<0.01).)and 18°C for 7 days(4.00±0.16 vs 2.87±0.15,p<0.01).After treated with ZF4 cells at 10 °C for 1 d,4 d and 7 d,the expression of ROS increased and with the prolonged cold treatment time,ROS accumulated in cells.This also from the side reaction in the low temperature stimulation,PGC-1α can make the ROS content in the body to maintain a relatively stable level range,thereby slowing down the damage caused by ROS produced by cells at low temperatures.Mitochondrial aerobic respiration is accompanied by the production of ROS.If ROS are accumulated in large amounts in the cells,it will first harm the mitochondria where ROS are generated.Therefore,we also used JC-1 staining.This probe is a fluorescence indicator that can be combined with the membrane structure of mitochondria.ZF4 cells after knockdown of PGC-1α were detected by BD C6 flow cytometry after cell staining.After cold treatment,its mitochondrial membrane potential(MMP)significantly decreased.Under normal circumstances,the normal membrane potential of cells accounted for more than 99% of all cells,and after low temperature treatment,the membrane potential of the cells will be reduced to some extent,PGC-1α knocked out after the cell membrane potential increased.At the same time,the knockdown of PGC-1α cells resulted in the apoptosis of cells after MMP decline,and the proportion of early apoptotic cells was significantly higher than that of cells not knocked out by PGC-1α.On the other hand,the ratio of both bax(a protein that promotes apoptosis of cells)and bcl-2(a protein that resists apoptosis)was significantly reduced in PGC-1α knockdown cells,and the protein level of bax was significantly decreased.Increased sexuality,in contrast to a significant decrease in the protein level of bcl-2,indicating that PGC-1α knocked down the ZF4 cells after low-temperature stimulation followed by apoptosis.In summary,the knockdown of PGC-1α during cold stress causes an increase in intracellular ROS,a decrease in mitochondrial membrane potential,an increase in the expression of the pro-apoptotic protein bax and a decrease in the expression of the antiapoptotic protein bcl-2.Initiate mitochondrial pathway-mediated apoptosis.This also shows from the side that PGC-1α can play a role in protecting cells during the cold stress of cells and can adapt to the effects of cold stress by maintaining intracellular ROS levels and mitochondrial membrane potential.