| Retained fetal membranes(RFM)is a common postpartum reproductive disorder in dairy cows.The previous research results of our team showed that the occurrence of RFM was related to the oxidative stress state of the body,apoptosis and the degree of extracellular matrix(ECM)degradation.Non-esterified fatty acids(NEFA)are lipid metabolites that increase in blood concentration during the perinatal period when dairy cows are in a negative energy balance(NEB)state.Several domestic and foreign studies have found that perinatal hyper NEFA-emia can increase the incidence of RFM,and NEFA is involved in regulating the remodeling of ECM.Therefore,this experiment intends to stimulate the endometrial epithelial cells of dairy cows with different concentrations of NEFA by simulating hyper NEFA-emia in vitro,and use biochemistry and molecular biology methods to determine the correlation between NEFA and RFM occurrence.To clarify the mechanism of the occurrence of RFM caused by the inhibition of ECM degradation in NEB cows with hyper NEFA-emia.The purpose of this experiment was to provide theoretical basis and experimental support for the prevention and treatment of postpartum RFM by using nutritional metabolism regulation technology to target ECM degradation,and further reveal the internal relationship between NEB and RFM in perinatal dairy cows.The experiment was divided into three groups: control group(0 m M),low concentration group(0.2 m M)and high concentration group(1.2 m M)after screening the optimal concentration and time of NEFA in cell nutrient solution.Three groups of NEFA with different concentrations were used to stimulate the endometrial epithelial cells of dairy cows for 12 h.The contents of GSH,GSH-Px,SOD and MDA in the cells were detected by ELISA,the content of ROS was determined by flow cytometry,and the nuclear entry of Nrf2 was observed by fluorescence microscope to determine the effect of NEFA on the oxidative stress state of endometrial epithelial cells.The cell apoptosis rate was detected by flow cytometry,the expression levels of mitochondrial apoptosis pathway related factors caspase-3,caspase-8,Bax and Bcl-2 were detected by q RT-PCR and western blot,and the change of mitochondrial membrane potential was measured by JC-10 method.To determine whether NEFA induces endometrial epithelial cell apoptosis through mitochondrial pathway.Finally,the expression levels of MMP-2,MMP-9,COL-Ⅳ,IL-1β,IL-6,IL-8 and TNF-α were detected by q RT-PCR and western blot to determine the effects of NEFA on collagen degradation and inflammatory response related factors.Results: compared with the control group and the low concentration group,the contents of GSH,GSH-Px and SOD in the high concentration group were decreased after NEFA stimulation for 12 h.The contents of GSH and GSH-Px were significantly decreased(P < 0.01).The content of SOD was decreased significantly(P < 0.05).While the content of MDA in high concentration group was extremely significantly increased(P < 0.01)compared with the control group and the low concentration group.The intracellular ROS content in high concentration group was significantly higher than that in control group(P<0.01)and the low concentration group(P <0.05).Nrf2 entered the nucleus in all three groups,and with the increase of NEFA concentration,the nuclear accumulation of Nrf2 became more obvious.The apoptosis rate of high concentration group was significantly higher than that of control group and low concentration group after 12 h stimulation(P < 0.01).JC-10 detection of mitochondrial membrane potential showed that the ratio of red/green fluorescence in high concentration group was significantly lower than that in control group and low concentration group(P < 0.01).The results of q RT-PCR and western blot showed that the m RNA expression levels of caspase-3,caspase-8 and Bax were significantly increased in the high concentration group compared with the control group and the low concentration group(P < 0.01).And the m RNA expression level of Bcl-2 decreased significantly in high concentration group compared with the control group and the low concentration group(P < 0.05).The protein expression levels of caspase-3,caspase-8,Bax and Bcl-2 in high concentration group were significantly higher than those in control group and low concentration group(P < 0.01).The protein expression level of Bax/Bcl-2 was only significantly higher in low concentration group than in control group(P < 0.01).And the protein expression level of the high concentration group decreased compared with the low concentration group,while there was no significant change compared with the control group.The m RNA expression levels of MMP-2 and COL-Ⅳ were significantly up-regulated at low and high concentrations(P < 0.01),while the m RNA expression level of MMP-9 in high concentration group was significantly down-regulated compared with control group and low concentration group(P < 0.01).Compared with the control group and the low concentration group,the protein expression level of MMP-2 and COL-Ⅳ were significantly increased at the high concentration(P< 0.01),and the protein expression level of MMP-9 decreased significantly in high concentration groups(P < 0.01).Among the inflammatory factors,the m RNA expression level of IL-1β in the high concentration group was significantly up-regulated compared with the control group(P <0.01)and the low concentration group(P < 0.05).The m RNA expression level of IL-6 was significantly increased in both low concentration group and high concentration group(P < 0.01)compared with control group.The m RNA expression level of IL-8 in high concentration group was significantly up-regulated compared with control group and low concentration group(P <0.01).Compared with the control group,the m RNA expression level of TNF-α was significantly up-regulated in the low concentration group(P < 0.05),and extremely up-regulated in the high concentration group(P < 0.01).According to the above experimental results,high concentration of NEFA can induce oxidative stress in endometrial epithelial cells of dairy cows and induce cell apoptosis through mitochondrial apoptosis pathway.It can also reduce the expression of MMPs by inducing aggravated cellular inflammatory response,which impedes collagen degradation and thus impedes ECM degradation.In conclusion,hyper NEFA-emia can induce excessive apoptosis of endometrial epithelial cells and collagen decomposition ability decreased,leading to ECM degradation disorders.This will eventually affect fetal efflux and induce RFM. |
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