The Role Of PP2A In Microcystin-LR-Induced Premature Senescence In Hepatocytes

Objectives:Microcystins are potent hepatotoxins,produced by several cyanobacteria including Microcystis,Anabaena,and Oscillatoria,those often form blooms in waters where the nutrients are enriched.So far,more than 90 structural analogues of microcystins have been identified.Microcystin-LR(MC-LR)is the most commonly encountered microcystin with strong hepatotoxicity.MC-LR can induce oxidative damage in hepatocytes by its active transport uptake system.It inhibits eukaryote serine/threonine protein phosphatases(PP)1 and 2A,leading to increases in protein phosphorylation.Cellular senescence refers to the cell transition into a stable state of irreversible cell cycle arrest,and resulting in immune clearance.Cellular senescence can be divided into replicative senescence due to the attrition of telomere and premature senescence.Cellular premature senescence is caused by DNA damage,hypoxia or metabolic stress,or oncogene activation resulting in cell cycle arrest and secreting senescence associated cytokines,causing immune clearance.Cellular premature senescence is an important mechanism against DNA damage at the cellular level,it' plays an important role in aging,degenerative diseases and tumorigenesis.Several reports have described the DNA damage and apoptosis induced by MC-LR,but whether MC-LR can induce premature senescence was scarcely reported.In our study,we used human embryonic L-02 hepatocytes to explore whether MC-LR can induce premature senescence and its molecular mechanisms.Object:To establish the model of premature senescence induced by microcystin-LR in L-02 hepatocytes,and to explore the role and mechanism of PP2A in microcystin-LR-induced premature senescence in hepatocytes.Methods:L-02 hepatocytes were cultured in RPMI-1640 medium with 10%fetal bovine serum,10OU/ml Penicillin,1000?g/ml streptomycin,maintained at 37? in 5%CO2.When the cells had entered exponential growth phase they were divided into four groups,the Control group(with 0.2%DMSO);10 nmol/L DES group,in this group cells were treated with lOnmol/L DES for 12h;20?mol/L MC-LR group;DES Antagonistic group,this group of cells were incubated with 20?mol/L MC-LR after treatment with l0nmol/L DES for 12h.After three weeks of exposure,the cell viabilities were measured with Cell Counting Kit-8(CCK-8),senescence associated ?-galactosidase activity were measured by(3-gal stained;cell cycle distribution were measured by Propidium Iodide based counting using flow cytometer.Senescence-associated heterochromatic foci(SAHF)forming rate was observed by Diamidino Phenylindole stained using Confocal Laser Scanning Microscope.Total cellular RNA was extracted and hydrolysed,reverse transcription of cDNA was conducted;and the mRNA expression of cell cycle regulation proteins p21,p16 and Gadd45a were then measured by Real-time PCR.Results:(1)After 3 weeks' exposure,the MC-LR group have a significant decrease on the cell viability(P<0.05).(2)Compared with the control group,SA-?-gal staining positive cells of MC-LR group increased,the difference was statistically significant(P<0.05).SA-?-gal staining positive cells in DES antagonistic groups were significantly lower than the MC-LR exposure group,the difference was statistically significant(P<0.05).(3)Compared with the control group,cells in the proportion of G0/G1 phase of MC-LR group increased,the proportion of cells in S phase decreased,correspondingly,the differences were statistically significant(P<0.05).The G0/G1 phase cells in DES antagonistic group were lower than the MC-LR exposure group(P<0.05).(4)After 3 weeks' exposure,SAHF was scarcely found in any of the four groups.(5)After 3 weeks' exposure,PP2A activity of MC-LR group was lower than the control group,which was 55.07%of the control cells;PP2A activity of DES antagonistic group increased for 1.43 times the MC-LR exposure group,the difference was statistically significant(P<0.05).(6)The mRNA expression levels of p21,p16,and Gadd45 for the MC-LR group were 2.12,1.57,and 4.16 times as high as those of the control group,these differences were statistically significant(P<0.05).The p21,p16 mRNA expression levels of DES antagonistic groups were lower than MC-LR exposure group,the differences were statistically significant(P<0.05).Conclusions:(1)MC-LR induced premature senescence in L-02 hepatocytes,showing characteristic of senescence such as cell proliferation inhibition,cell cycle G1 arrest and an increase in the(3-galactosidase activity.(2)MC-LR inhibited PP2A activity,and up-regulated the expressions of p21,p16 and Gadd45a mRNA in hepatocytes.In addition,DES suppressed MC-LR-induced premature senescence and cell cycle arrest.Thus,PP2A,p21 and p16 might be involved in microcystin-LR-induced premature senescence.