The Functional Effects Of Cold Stress On ROS Production、MAPK Pathway And Selenoprotein P

Temperature is one of the important factors that limits the distribution of organisms and directly determines the physiological features and behavior of organisms. Fish is the most widespread vertebrates. How the fish adapted to the temperatures changes is an important and meaningful question in the environmental biology. In our previous study, we compared the transcriptome and genome between Antarctic fish and temperate fish and found that genes involved in reactive oxygen species(ROS) scavenge and Mitogen-activated protein kinase(MAPK) pathway were associated with the cold adaptation in fish. Moveover, Selenoprotein P, which is an antioxidant, was duplicated as high as 22 times in genome of Antarctic fish compared with temperate fish. To further study the regulatory network and the reactive oxygen species(ROS) homeostasis under cold stress, we examined the level of ROS and the expression of stress-related proteins in zebrafish ZF4 cells and human He La cells exposed to mildly and severely cold temperatures, 18℃ and 10℃ for ZF4 cells and 28℃ and 18℃ for He La cells, at various time points(1 d, 3 d, and 5 d).The main results in this paper as follows:1) The cell proliferation became slow at low temperatures. After 5 days of cold treatment we observed the damaged and detached ZF4 cells and He La cells at 10℃ and 18℃, repectively. Meanwhile cells viability had the same consequences.2) DCFH-DA probe method was used to determine the level of ROS in cells. We found that the level of ROS in cells was induced by cold stress. The changes of ROS level showed different patterns between ZF4 cells and He La cells. In ZF4 cells, 3 days after cold treatment, the level of ROS in cells significantly increased to 1.23 ± 0.04(P < 0.05) and 2.31 ± 0.08(P < 0.05) times, respectively, at 18℃ and 10℃, compared with that in cells at 28℃; In He La cells, ROS level was immediately upregulated and reached a peak at 3 d after cold treatment. The level of ROS in cells significantly increased to 10.51 ± 2.90(P < 0.05) and 15.03 ± 1.33(P < 0.05) times, respectively, at 28℃ and 18℃, compared with that in cells at 37℃. Morever, ROS were suppressed after treated with NAC in these cells.3) Western blot was conducted to examine the expression of MAPKs(p-JNK p54,p-JNK p46, p-p38 and p-p44/42) in cells under cold stress. In ZF4 cells, the expression of p-JNK and p-p38 was induced by cold stress(18℃ and 10℃), and reached the highest at 3 d, but the activity of p-p44/42 reduced under cold stress. In He La cells, the expression of p-JNK and p-p38 was specifically induced at 18℃.The expression of p-JNK was turned on at 1 d while the expression of p-p38 was turned on at 3 d. The phosphorylation of p44/42 were activated under the two cold stress, and the expression increased as times.4) Western blot of γH2A.X showed that the expression of γH2A.X reached peak after 3 days both at ZF4 cells and He La cells.5) In addition, the m RNA level and protein level of Selenoprotein P increased at 5 d of cold treatment(18℃ and 10℃, 28℃ and 18℃) in ZF4 cells and He La cells.Taking together, these findings showed that cold temperature could induce the production of ROS in ZF4 cells and He La cells and enhanced the proteins of MAPK pathways and DNA damage. The level of ROS in cells is dependent on the stress intensity, duration and species. The first aspect, we observed the differences in the changes of ROS level and expression of MAPK and DNA damage proteins between ZF4 cells and Hela cells, which provides a foundation for understanding the different molecular mechanisms of cold responses between endotherm and ectoderm. The second aspect, we observed the markedly induction of ROS in the both cells at the first 3 days of cold treatment, while the expression of p-JNK、p-p38、p-p44/42 and γH2A.X was markedly changed at the 3 d, indicating that 3 d of cold treatment might be a key time point to determine the protein expression in cells. The third aspect, cold stress induced the expression of Selenoprotein P protein in ZF4 cells and He La cells, and this could lay a foundation for the later antioxidant study of Selenoprotien P.