| Dairy industry is one of the most important part of livestock husbandry. It has become a primary problem in restricting healthy development of Chinese dairy industry with regards to milk quality and safety. Protein and fat is the main nutritional content of milk,which both related not only to safety and health of consumers, but also the determination of the economic value and core competitiveness of milk. Ruminants are always fed high-concentrate diet in order to meet the increasing energy requirement for higher milk yield in nowadays dairy-farming industry, which leads to abnormal fermentation in rumen and then to the subacute ruminal acidosis. Lipopolysaccharide (LPS) in the rumen will be accumulating and translocating into circulatory system, and subsequently reach to different organ and tissues such as mammary gland. This is able to activate NF- ?B signaling pathway and inhibit mTOR signaling pathway, which lead to inflammation and inhibition of milk protein synthesis in the mammary gland. Based on the current reality of poor quality roughage in China, how to improve animal performance while maintaining animal health under the current feeding mode has become a crucial issue, which needs to be addressed in urgent. Sodium butyrate can promote the development of gastrointestinal tract and enhance the barrier function, as well as inhibit LPS-mediated NF- ?B signaling pathway, relieve inflammation, and restore lactation. Exogenous perfusion and in vitro experiments were also introduced to further verify the effect of LPS on the innate immune response and casein synthesis function in mammary gland.1. Lipopolysaccharide derived from the digestive tract activates inflammatory gene expression and inhibits casein synthesis in the mammary glands of lactating dairy cowsTo meet the nutrition requirements of lactation, dairy cows are usually fed a high concentrate diet (HC). However, high-grain feeding causes subacute ruminal acidosis(SARA), a metabolic disorder that causes milk protein depression. This study aimed to investigate the effect of lipopolysaccharide (LPS) released in the rumen on inflammatory gene expression and casein synthesis in mammary glands of lactating dairy cows fed a HC diet. We found that milk protein was significantly decreased in the HC group after 15 weeks of feeding. Overall, LPS concentrations in the rumen fluid, lacteal artery and vein were increased in the HC group. Transcriptome microarray was used to evaluate alterations in the signaling pathway in mammary glands. Signaling pathways involved in inflammatory responses were activated, whereas those involved in protein synthesis were inhibited in the HC group. m RNA expression involved in inflammatory responses, including that of TLR4,NF-?B and pro-inflammatory genes, was increased in the HC group, while ?s1-casein(CSN1S1), ?-casein (CSN2), m TOR and S6K gene expression were decreased. Moreover,protein expression was consistent with the corresponding gene expression. After feeding with an HC diet, LPS derived from the rumen increased inflammatory gene expression and inhibited casein synthesis in the mammary glands of lactating dairy cows fed a HC diet.2. Effect of the mammary gland innate immune response and casein synthesis function of lactating goat feeding high concentrate diet with sodium butyrateEighteen multiparous Chinese Sanen lactating goats (38.86±2.06kg) were randomly allocated to three groups (6/group). One group was fed with a diet composed of 60% forage and 40% concentrate (low-concentrate, LC group) as a control while the other two groups,one received a diet composed of 40% forage and 60% concentrate (high-concentrate, HC group), another was fed with a diet which contained 40% forage and 60% concentrate with sodium butyrate (SB) buffer in it (BHC group) as treatments. The experimental period was 20 weeks. High concentrate diets caused a decline in rumen pH, rumen pH values are certain to rise after adding sodium butyrate; Plasma LPS content of HC group were significantly higher than the LC group and BHC group (p <0.01); the main inflammatory cytokines IL-1?, IL-6, TNF-a content in HC group were significantly higher than the LC group and BHC group (p <0.05); The expression of inflammatory gene were significantly decreased , while the expression of protein synthesis related gene were significantly increased in the mammary gland of BHC group. mRNA and protein expression were consistent. Thus, high concentrate diets supplemented with sodium butyrate can relieve inflammation, restore casein synthesis.3. Effect of mesenteric venous infusion of LPS on the mammary gland innate immune response and casein synthesis function of lactating goatEight delivery in three weeks Saanen lactating goat weighing 42 ± 2.8 kg were fed column. Animals were feeding with same diet which was designed according to secrete milk goat breeding standard. The animals were mounted mesenteric vascular catheters.Experimental animals were randomly divided into two groups, one group was mesenteric inject LPS group (Treatment); another group was mesenteric inject saline as control group(Control). 4h after injecting collected the blood samples, and then animals were killed for mammary gland tissue samples collecting. LPS content of plasma in LPS injected group is much higher than the saline group. The expression of inflammation gene was significantly increased in the dairy goat mammary gland of treatment group, and milk protein synthesis related genes expression were down-regulated. TLR4 promoter region of chromatin in a open state, DNA demethylation, and the expression of TLR4 gene was increased in the mammary gland. This study shows that a certain degree of LPS injected by TLR4 expression of epigenetic mechanism of gene regulation, the impact of mammary gland lactation.4. LPS regulates the expression of inflammatory genes through the NF- ?B signaling pathway in the MAC-T cellsIn this experiment, mammary epithelial cell line (MAC-T) was applied and split into five groups: PBS group; LPS (100ng/mL) group; NF-?B over-expression group; LPS +BAY11-7085 group and LPS + MG132 group, respectively. It was found that the expression of TLR4 was positively correlated with the concentration of LPS at the scale of 0.1-1000 ng/mL, while TLR4 expression changed little at the concentration which was higher than 1000 ng / mL. Accordingly, we properly set 100 ng/mL as the concentration for the further treatment. We observed that only when the concentration of LPS was up to 100 ?g/mL could cause apoptosis, which has less biological significance. MAC-T cells treated with LPS were found an increase in the expression of NF-?B and pro-inflammatory factors.Over-expression of NF-?B could increase the mRNA and protein expression of inflammatory factors. BAY11-7085 and MG132 known as NF-?B inhibitors could inhibit the mRNA and protein expression of NF-?B. BAY11-7085 inhibited the expression of inflammatory genes, particular in IL-1B; MG132 also inhibited the expression of these genes, particular in IL-8. Therefore, we conclude that NF-?B plays an important role in LPS-activated TLR4 inflammatory signaling pathway. |
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