| With the improvement of people’s living standards, the place of milk in the national economy is increasing. In order to increase milk production, a large number of highly concentrated feed were often added in dairy cattle diets. Although it improved the milk production to some extent, but it very easily caused subacute rumen acidosis(SARA). As a common metabolic disease, the incidence of sub-acute ruminal acidosis is greater than 20% in the primary and middle lactation of dairy cows, which causes significant economic damage to dairy cattle cultivation. When SARA occurs, the bacterial lipopolysaccharide(LPS) concentrations are significantly increased. LPS could stimulate the body to release a large amount of inflammatory cytokines, And these proinflammatory cytokines in dairy cows can lead to chronic mastitis and changes in metabolism, which can ultimately influence the milk composition and milk yield. Therefo re, how to inhibit the inflammatory cytokine production induced by LPS, thereby improving the cow’s milk quality have become an urgent problem.The 14-3-3 proteins family are highly conserved dimeric proteins and exist widely in almost all organisms cell. It have seven subunits(β, γ, ε, δ, ε, ζ and η) in mammals and plays an important role in inhibiting cells apoptosis and mediated signal transduction. Many studies showed that 14-3-3 gamma protein took part in the development and occurrence of many diseases, and its expression was increased in some pathological processes. These results implayed us that 14-3-3 gamma may play a very important role in improving the body’s response to injury and anti-injury repair and so on. Our prior research has shown that 14-3-3 gamma overexpression can promote the activity and proliferation of dairy cow mammary epithelial cells(DCMECs) and increase cell viability and promote the secretion of milk protein and milk fat and improve DCMEC lactation ability. So, whether 14-3-3 gamma can inhibit signal transduction pathway of inflammation induced and improve lactationby to improve milk production and milk quality of SARA cows become the focus of our study.In this study, culture method of tissue block was used to primary culture DC MECs as the cell model in vitro; The best concentration of LPS was screened by testing toxic effects of different concentrations of LPS on DCMECs to build cellular inflammation model; We detected the cell viability and apoptosis by CASY-TT Analyser System, determination of LDH activity, acridine orange fluorescence staining and flow cytometry. 14-3-3 gamma overexpression or gene silencing in DCMECs using liposomal transfection method. Lactation related genes with quantitative real time polymerase chain reaction(q RT-PCR) and western blotting technology. The protein expression changes of inflammation-related pathway were detected by western blotting technology; The m RNA expression and secretion of proinflammatory cytokines were detected by q RT-PCR and enzyme-linked immunosorbent assay. Meanwhile, we detected the content of lactose, triglyceride and ?-casein secretion respectively using ?-casein assay kit, lactose/galactose(rapid) kit, TG detection kit and western blotting technology.The changes of fatty acid synthase activity were detected by Enzyme Activity Assay Kit.Experiment results showed that:1. LPS had a certain toxic effect on DCMECs The results of CASY analysis, determination of LDH activity, acridine orange fluorescence staining and flow cytometry showed that LPS could reduce viability and proliferation of DCMECs and induce DCMECs apoptosis. And its toxic effect had the relationship of dose-effect and time-effect.2. LPS influenced lactating function and expression of lactation-related genes in DCMECs 1The results of testing triglyceride, lactose and beta-casein secretion of DCMECs showed that LPS could reduce lactation function of DCMECs. When LPS concentration greater than 100ng/m L, the content of triglyceride significantly decreased compared to the control group(P<0.05); When LPS concentration greater than 1 000ng/m L, the content of lactose significantly decreased compared to the control group(P<0.05); When LPS concentration greater than 100ng/m L, the expession of β-casein decreased compared to the control group. 2 The results of q RT-PCR showed that LPS down-regulated the m RNA expression of lactation related genes in DCMECs. The expression of m TOR, STAT5, S6K1, AKT1, 4EBP1, PPARγ, SREBP1, FABP3, ACC, FAS and GLUT1 were significantly decreased compared to the control group(P<0.05 or P<0.01), after 1 000ng/m L LPS adding in DCMECs. 3The results of western blotting technology showed that LPS down-regulated the protein expression of lactation related genes in DCMECs. 1 000ng/m L LPS decreased the protein expression of m TOR, p-m TOR, S6K1, p-S6K1, AKT1, p-AKT1, SREBP1, PPARγ and GLUT1 compared to the control group(P<0.05).3. LPS increased the expression of 14-3-3 gamma in DCMECs, it implied us that 14-3-3 gamma may be involved inflammatory process.4. 14-3-3 gamma overexpression inhibited secretion of LPS-induced inflammatory cytokines and down-regulated pro-inflammatory signaling pathway in DCMECs 1The results of q RT-PCR and ELISA showed that 14-3-3 gamma overexpression significantly inhibited the m RNA expression of TNF-α, IL-6, IL-1β and i NOS in LPS-induced DCMECs, and significantly decreased the level of TNF-α and IL-6 in the supernatan of LPS-induced DCMECs compared to the LPS control group(P<0.05). 2The results of western blotting technology showed that 14-3-3 gamma overexpression inhibited the protein expression of TLR4 and down-regulated the signaling pathway of NF-κB and MAPK. 14-3-3 gamma overexpression inhibited the phosphorylation of IκB-α, ERK1/2, p38 and NF-κB nuclear translocation compared to the LPS control group(P<0.05).5. The effect of 14-3-3 gamma on the cells activity and lactation of LPS-induced DCMECs 1 CASY and flow cytometry test results showed that 14-3-3 gamma overexpression promoted the viability and proliferation of LPS-induced DCMECs and inhibited cells apoptosis compared to the LPS control group; 14-3-3 gamma gene silencing inhibited the viability and proliferatio of LPS-induced DCMECs and promoted cells apoptosis compared to the LPS control group. It hinted that 14-3-3 gamma had a protective effect on DCMECs injury induced by LPS. 2The detection results of triglyceride, lactose and β-casein in LPS-induced DCMECs showed that 14-3-3 gamma overexpression could promote the synthesis of triglyceride, lactose and β-casein, 14-3-3 gamma gene silencing inhibited the synthesis of triglyceride, lactose and β-casein compared to the LPS control group. 3 The results of q RT-PCR and western blotting showed that 14-3-3 gamma overexpression increased the m RNA and protein expression of m TOR, S6K1, AKT1, PPARγ, SREBP1 and GLUT1 in LPS-induced DCMECs compared to the LPS control group; 14-3-3 gamma gene silencing decreased the m RNA and protein expression of m TOR, S6K1, AKT1, PPARγ, SREBP1 and GLUT1 in LPS-induced DCMECs compared to the LPS control group.In summary, our findings suggest that 14-3-3 gamma may have a beneficial effect in preventing LPS-induced inflammatory response in DCMECs. The mechanisms by which 14-3-3 gamma overexpression can exert its anti-inflammatory effects correlate with the inhibition of the expression of TNF-α, IL-6, IL-1β and i NOS via the inactivation of the NF-κB and MAPK signaling pathways, meanwhile, 14-3-3 gamma overexpression can promote the expression of lactation-related genes. And then 14-3-3 gamma could promote cells activity and synthesis of milk composition in LPS-induced DCMECs. This study provided new theory references for controling lactation capacity reduced by LPS and improving milk quality researches. |
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