外源性脂多糖对马冈鹅胚胎期胸腿肌发育的影响

Effects of exogenous lipopolysaccharide on breast and leg muscle development during embryonic stages in Magang goose

[目的]胚胎期是脊椎动物肌肉发育的重要时期,在鹅胚发育过程中,易受到种鹅体内脂多糖(LPS)垂直污染而导致死胚和弱苗的产生。本研究通过注射外源性LPS,模拟受LPS污染的马冈鹅胚胎,探究外源性LPS对马冈鹅胚胸腿肌发育的影响。[方法]选取8胚龄(E8)马冈鹅胚,分别注射含0、25、50和100μg LPS的生理盐水100μL于尿囊腔。于15胚龄(E15)、23胚龄(E23)和雏鹅1日龄(1 d)时采集尿囊液(1日龄采集血清)进行LPS含量检测,取胸腿肌组织进行HE染色切片并统计肌束面积和密度,检测胸腿肌中基因和1 d时蛋白表达量。[结果]外源性LPS显著降低鹅胚的孵化率。E23时,100μg LPS组雏鹅尿囊液LPS含量显著高于其他3组(P<0.05)。LPS显著降低E23和1 d胸肌肌束面积,显著降低E15肌束密度,显著提高E23胸肌肌束密度,显著降低E15和1 d腿肌肌束面积(P<0.05)。LPS组E15鹅胚胸肌炎症相关因子基因(IL-6、TNF-α)和成肌增殖相关基因(IGF-1、MyoD)的表达量显著降低(P<0.05);1 d时,IGF-1基因表达量显著升高,成肌分化相关基因(Myogenin,Myh1)表达量显著降低(P<0.05);LPS组E15鹅胚腿肌IL-6、TNF-α、MyoD、Myogenin和Myh1基因表达量显著上调,IGF-1基因表达量显著下调(P<0.05),1 d时,仅100μg组IL-6和Myogenin基因表现出显著上调(P<0.05)。蛋白表达方面,1 d时,外源性LPS组胸肌TNF-α蛋白表达量显著升高,MyoD蛋白表达量显著降低(P<0.05);腿肌IL-6、TNF-α和MyHC蛋白表达量显著升高,MyoD蛋白表达量显著降低(P<0.05)。[结论]外源性LPS会导致雏鹅孵化率降低,胸腿肌肌束横截面积显著降低,炎症因子表达量上升,成肌增殖相关基因表达量降低,不利于马冈鹅胚胎肌纤维生长发育。

[Objectives]The embryonic stage is an important period for muscle development of vertebrates.During the development of goose embryo,it is easy to be polluted by lipopolysaccharide(LPS),which leads to the generation of dead embryos and weak seedlings.The objective of this study was to investigate the effects of exogenous LPS on breast and leg muscle development of Magang goose embryos by injecting exogenous LPS to simulate LPS contaminated Magang goose embryos.[Methods]Magang goose embryos of embryonic age day 8(E8)were selected and injected 100 μL of normal saline containing 0,25,50 and 100 μg LPS into allantoic cavity.Allcystic fluid(blood on 1 day-old age goose,1 d)was collected at embryonic age day 15(E15),day 23(E23)and 1 day after birth(1 d)for LPS content detection.Breast and leg muscle tissue was collected for HE staining section to count muscle bundle area and density,and gene and protein expression levels of pectoral and leg muscle from 1 d were detected.[Results]Exogenous LPS significantly decreased the hatchability of goose embryos.At E23,LPS content in allantoic fluid of goslings in 100 μg LPS group was significantly higher than that in the other three groups(P<0.05).LPS decreased breast muscle bundle area at E23 and 1 d,significantly decreased muscle bundle density at E15,significantly increased breast muscle bundle density at E23,and significantly decreased leg muscle bundle area at E15 and 1 d(P<0.05).The expression levels of inflammatory factor-related genes(IL-6,TNF-α)and myoproliferative genes(IGF-1,MyoD)of goose embryo breast muscle significantly decreased in LPS injection group E15(P<0.05).At 1 d,IGF-1 expression level significantly increased,and Myogenin,Myh1 expression levels significantly decreased(P<0.05).In LPS group,the expression levels of IL-6,TNF-α,MyoD,Myogenin and Myh1 at E15 goose embryo leg muscle were significantly upregulated,while the expression levels of IGF-1 were significantly downregulated(P<0.05).At 1 d,only IL-6 and Myogenin genes in 100 μg group showed significant upregulation(P<0.05).In terms of protein expression,the expression level of TNF-α protein in breast muscle significantly increased and the expression level of MyoD protein significantly decreased(P<0.05)at 1 d after injection of exogenous LPS.The protein expression levels of IL-6,TNF-α and MyHC in leg muscle significantly increased,while the protein expression level of MyoD significantly decreased(P<0.05).[Conclusions]Exogenous LPS could decrease the hatchability of goslings,significantly decrease the cross-sectional area of breast and leg muscle bundle,increase the expression levels of inflammatory factors,and decrease the expression levels of genes related to muscle proliferation,which was not conducive to the growth and development of myofibers of Magang goose embryos.