| β-zearalenol(β-zol)is a common contaminant in improperly stored food and animal feed.β-zol has an estrogen-like effect and is considered to be associated with infertility,reduced milk production and high estrogen levels of bovines.HT-2 is the main metabolite of T-2 in vivo and in vitro.HT-2 can cause acute or chronic toxicity in animals and humans.Studies have shown that mycotoxins exhibit carcinogenicity,mutagenicity,teratogenicity and neurotoxicity even at low concentrations.HT-2 toxin can induce oocyte apoptosis,oxidative stress and autophagy,which can reduce oocyte maturation rate,and then affect female reproduction.Melatonin is an endogenous antioxidant involved in apoptosis and oxidative stress.Recent studies have found that melatonin has a significant role in alleviating mycotoxin-induced injury.The antioxidant effect of melatonin can reduce the damage caused by aflatoxin and deoxynivalenol.Granulosa cells and oocytes are closely related during the whole oogenesis process.Granulosa cells support oocyte development by providing nutrients such as amino acids.Apoptosis of granulosa cells can cause follicular atresia and premature ovarian failure and other reproductive problems.At present,the molecular mechanism of the toxicity of β-zol and HT-2 toxins in bovine ovarian granulosa cells is not clear,as is the role of melatonin.Therefore,in this study,primary cultured dairy bovine ovarian granulosa cells were used as materials to explore the molecular mechanism of cytotoxicity induced by β-zol and HT-2 toxins,and whether melatonin could alleviate the cell damage caused by the mycotoxin.The research contents and main results are as follows:1、Primary dairy bovine ovarian granulosa cell culture:Cells were cultured by puncture follicle extraction method,and follicular fluid was extracted from follicles with a diameter of 2-5 mm on the surface of the ovary of cows.After adherent growth,ovarian granulosa cells grew well.The specific follicle stimulating hormone receptor(FSHR)on granulosa cells was used for cell identification.RESULTS:More than 95%of the cells were FSHR positive,which indicated that the purity of the cells was high and could be further study.2、Effects of melatonin on cytotoxicity of bovine ovarian granules induced by β-zol:Experimental groups:(1)Cell proliferation concentration gradient test:divided into control group,treatment group with(3-zol(5,10,15,25,50,100,200 μM);(2)Concentration gradient of melatonin rescue test:control group,different concentrations of melatonin(0,0.01,0.1,1,10,100,1000 μM)pretreated for 12h+25 μM β-zol;(3)Rescue test of melatonin:the experiment was divided into control group,25 μM β-zol,100 μM melatonin treatment for 12 h+25 μM β-zol treatment for 24 h.Flow cytometry,ROS staining,qrt-pcr and Western blot were used to detect apoptosis,oxidative stress and ROS level after the cells were grouped according to the above.RESULTS:With the increase of β-zol concentration,the cell viability decreased in a concentration-dependent manner.The cell viability was 50%after 24 h of 25 μM β-zol treatment,and the cell proliferation inhibited by β-zol was significantly improved after 12 h of 100 μM melatonin pretreatment(p<0.05).Reactive oxygen species(ROS)levels in cells were measured by ROS staining,and intracellular ROS increased sharply by β-zol(p<0.01),while melatonin pretreatment could significantly eliminate ROS(p<0.01).Flow cytometry showed that compared with the control group,the apoptosis of bovine ovarian granulosa cells induced by β-zol was very significant(p<0.01),and melatonin pretreatment could significantly reduce the early apoptosis of bovine ovarian granulosa cells(p<0.01).QRT-PCR showed that melatonin could reverse the up-regulation of apoptosis-related genes Bax/Bcl-2 and Caspase 3 induced by β-zol(p<0.05).Western blot results were consistent with qRT-PCR.(3-zol could activate the P38 MAPK pathway and up-regulate the expression of phosphorylated P38(p<0.01).Detection of oxidative stress-related gene and protein levels showed that the levels of copper-zinc superoxide dismutase(SOD1)and manganese superoxide dismutase(SOD2)in dairy cow ovarian granulosa cells were significantly increased(p<0.01),while the levels of catalase(CAT)gene and protein were significantly decreased(p<0.01).The activity of superoxide dismutase(SOD),glutathione peroxidase(GSH-px)and malondialdehyde(MDA)was detected.The activity of GSH-px(p<0.05),SOD(p<0.01)and MDA(p<0.01)increased after exposure to β-zol.Melatonin pretreatment could significantly change the activity of SOD and GSH-px induced by β-zol(p<0.05),but had little effect on MDA(p>0.05).These results suggest that melatonin can rescue the inhibition of proliferation,apoptosis and oxidative stress induced by β-zol in dairy cow ovarian granulosa cells.3、Effect of melatonin on cytotoxicity of bovine ovarian granules induced by HT-2toxin:Experimental groups:(1)Cell proliferation concentration gradient test:divided into control group,HT-2 toxin treatment group(12.5,25,50,100,200 nM);(2)concentration gradient of melatonin rescue test:divided into control group,different concentrations of melatonin(0,0.01,0.1,1,10,100,1000 μM)pretreatment of 12 h+50 nM HT-2 toxin;(3)melatonin rescue test:divided into control group,50 nM HT-2 toxin.HT-2 toxin and 100 μM melatonin were treated for 12 h+50 nM HT-2 toxin for 24 h.Cells were treated according to the above grouping.Flow cytometry,ROS staining,qRT-PCR and Western blot were used to detect apoptosis,oxidative stress and ROS level.RESULTS:With the increase of HT-2 toxin concentration,the cell viability decreased in a concentration-dependent manner.The cell survival rate was 50%after 24 hours of 50 nM HT-2 toxin treatment.The cell proliferation inhibited by HT-2 toxin was significantly improved after 12 hours of pretreatment with 100 μM melatonin(p<0.01).Flow cytometry showed that HT-2 toxin induced apoptosis of bovine ovarian granulosa cells(p<0.001),and melatonin pretreatment could significantly reduce the early apoptosis of bovine ovarian granulosa cells(p<0.01).QRT-PCR showed that melatonin could reverse the up-regulation of apoptosis-related genes Bax/Bcl-2 and Caspase 3 induced by HT-2 toxin(p<0.05).Western blot showed that HT-2 toxin activated phosphorylation of P38 MAPK and up-regulated the expression of Bax/Bcl-2 and Caspase 3(p<0.01).After exposure to HT-2 toxin,ROS accumulation is accompanied by increased levels of oxidative stress related genes SOD1 and SOD2.Exposure to HT-2 toxin resulted in a significant decrease in the levels of CAT mRNA and protein in bovine ovarian granulosa cells(p<0.01).SOD activity(p<0.05)and gsh-px activity(p<0.01)were increased after treatment with ht-2 toxin,but had no significant effect on the activity of MDA(p>0.05).Melatonin pretreatment attenuated the effects of HT-2 toxin on SOD(p<0.01)and GSH-px(p<0.01).Compared with HT-2 toxin exposure group,melatonin induced a significant increase in MDA activity(p<0.05).There is a close relationship between apoptosis and autophagy.Intranet stress and autophagy-related test results of bovine ovarian granulosa cells exposed to HT-2 toxin showed that HT-2 toxin can induce concentration-dependent endoplasmic reticulum stress in granulosa cells,and induce the expression of CHOP and GRP78 genes in a concentration-dependent manner.Western blot results were consistent with qRT-PCR.Meanwhile,autophagy-related genes LC3-II and P62 were detected.HT-2 toxin induced autophagy in a concentration-dependent manner.The expression of LC3-Ⅱ was detected by immunofluorescence staining.The level of LC3-Ⅱ increased with the increase of HT-2 exposure.After pretreatment with melatonin,melatonin can significantly reduce autophagy and endoplasmic reticulum stress.Mycotoxin-induced apoptosis,oxidative stress,autophagy and endoplasmic reticulum stress were significantly reduced in bovine ovarian granulosa cells pretreated with melatonin.Our results indicate that melatonin has protective effects on mycotoxin-induced apoptosis and oxidative stress in dairy bovine ovarian granulosa cells. |
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