| With the development of the economy,people have increasingly strict requirements for the quality of food and beverages.Among the daily visible beverages,milk is the most suitable for human nutritional needs and is therefore widely favored.Milk is easily digested and absorbed by the body,and its nutrients can comprehensively cover human needs,especially contain animal derived fatty acids-conjugated linoleic acid(CLA).CLA is a stereoisomer of linoleic acid,mainly synthesized by ruminants in the mammary gland after ingesting trans isooleic acid(TVA)from their diet.It has a good preventive and protective effect on various common diseases and is an essential fatty acid that the human body cannot synthesize on its own,which can effectively improve human health.TVA is an essential substrate for the synthesis of CLA and synthesises in the rumen as a precursor of CLA.Lithium,once lacking,can increase the mortality rate and reduce the reproductive rate of ruminants.At present,due to the considerable complexity of the endogenous synthesis of CLA,there is a lack of relevant research.At present,due to the considerable complexity of the endogenous synthesis of CLA,there is a lack of relevant research.The aim of this study is to fill the gap in this area and explore whether exogenous addition of the nutrient lithium chloride(LiCl)can promote endogenous synthesis in the breast by targeting genes through key signaling pathways,increase CLA content,and its specific mechanism of action.Therefore,this study used cow mammary epithelial(MAC-T)cells as an in vitro model to screen for appropriate concentrations of LiCl,which was combined with TVA for treatment,to detect the effect of LiCl on CLA synthesis in MAC-T cells.The experiment was mainly divided into the following three aspects:1.Firstly,the effect of LiCl on key factors in the endogenous synthesis process of CLA and the content of CLA was tested.First,the effects of 0-20 mM LiCl of stearyl coenzyme A desaturase(SCD1)and proteasome α5 subunit(PSMA5)in MAC-T cells were analyzed.The results showed that compared with the control group,the expression of PSMA5 was significantly increased in SCD1 treated with 5,10,and15 mM LiCl,as well as in PSMA5 treated with 1,5,and 10 mM LiCl(P<0.05).Therefore,LiCl at concentrations of 1,10,and 15 mM was selected and used to differentiate MAC-T cells for 4 days,50 μM TVA treated cells for 4 hours.The results showed that TVA significantly inhibited the mRNA expression of SCD1 and PSMA5(P<0.05).Western blot was used to test and verify the effect of LiCl on SCD1 and PSMA5.Compared with the control group,LiCl group significantly increased the protein expression of SCD1 and PSMA5(P<0.05),while TVA significantly inhibited the expression of these two genes(P<0.05);Compared with TVA group,TVA+LiCl group significantly increased the protein expression of SCD1 and PSMA5 to normal level.After the above studies proving that LiCl can indeed improve the expression of genes related to CLA synthesis,therefore further analyze the effect of LiCl on endogenous CLA synthesis.The content of CLA in MAC-T cells treated with 10 mM LiCl for 4 days was detected by gas chromatography.Compared with TVA group,the content of cis-9 trans-11 CLA(c9,t11-CLA)and CLA index(P<0.01)in TVA+LiCl group were significantly increased(P<0.05),which proved that LiCl could promote the endogenous synthesis of CLA in MAC-T cells.2.Secondly,the effect of LiCl on genes related to fat synthesis during the endogenous synthesis of CLA was tested.The results showed that compared with the control group,the LiCl group significantly increased the mRNA expression of sterol regulatory element binding protein 1(SREBP1),acetyl coenzyme A carboxylase(ACC),fatty acid synthase(FASN),lipoprotein lipase(LPL),and phospholipid 2(PLIN2),while the TVA group significantly decreased the expression of most of the above factors(P<0.05);At the same time,compared with TVA group,TVA+LiCl group significantly increased the mRNA expression of the above factors(P<0.05).Further detection of proliferator-activated receptorsγ(PPARγ)after LiCl treatment at protein level.Compared with the control group,the LiCl group significantly increased PPARγ And SREBP1 protein expression(P<0.05);Compared with TVA group,TVA+LiCl group increased the protein expression of these two genes to normal level.The above results indicate that LiCl can effectively promote milk fat synthesis and significantly alleviate the inhibitory effect of TVA on genes related to milk fat synthesis.3.Finally,the effects of LiCl on the Wnt/β-catenin and HIF-1α signaling pathways in the endogenous synthesis of CLA were examined.About the Wnt/β-catenin pathway related genes,compared with TVA group,TVA+LiCl group promoted glycogen synthase kinase3-β(GSK3-β)、β-catenin mRNA expression of.Further test the changes in protein level.Compared with the control group,LiCl group significantly promoted the expression of p-GSK3β、β-catenin、p-β-catenin(P<0.05);Compared with the TVA group,the TVA+LiCl group has no significant effect on GSK3β,but significantly promotes the protein expression of p-GSK3β、β-catenin、p-β-catenin(P<0.05),indicates that LiCl can activate gene expression in the classical Wnt pathway.At the same time,HIF-1α signal pathway related genes were detected.The results of fluorescence quantitative analysis(q PCR)showed that compared with the control group,the LiCl group significantly increased HIF-1α and its downstream factors erythropoietin(EPO),erythropoietin receptor(EPOR)mRNA expression(P<0.05);Compared with TVA group,TVA+LiCl group significantly increased the expression of the above genes(P<0.05).The results at protein level and mRNA level were consistent.Compared with the control group,the TVA group significantly reduced HIF-1α And EPOR protein expression(P<0.05);After combining 10 mM LiCl with TVA,compared with TVA group,the expression of HIF-1α,EPOR and significantly returned to normal state(P<0.05).It can be seen that after LiCl combined with TVA,HIF-1α and its downstream factors have returned to normal expression,proving that LiCl can promote HIF-1α gene expression of the pathway.To sum up,LiCl may activate HIF-1α and its downstream gene expression,Wnt/β-catenin and SREBP1 pathway to increase the expression of key genes SCD and PSMA5 in CLA synthesis,thereby promoting the transformation of TVA into endogenous synthesis of CLA in mammary.The experimental results prove that LiCl is a potential exogenous nutrient additive,which can increase the content of CLA in milk by activating lactation related signal pathways in mammary,ultimately improving milk quality. |
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