β-羟丁酸在低糖诱导奶山羊神经细胞炎性损伤中的保护作用及其机制

Pregnancy toxemia of dairy goats is a metabolic disease and characterized by the typical clinical pathological features including hypoglycemia and hyperketonemia represented by β-hydroxybutyric acid. Hypoglycemia could promote the ketogenesis, thus precedes hyperketonemia in the process of pregnancy toxemia. Studies have shown that hypoglycemia induces neuronal inflammatory injury and affected goats present a series of neurological symptoms, however, a certain concentration of β-hydroxybutyric acid has the neuroprotective effects. Therefore, we hypothesize that β-hydroxybutyric acid has a neuroprotective effect on hypoglycemia-induced neuronal inflammatory injury in dairy goats. In order to make clear the hypoglycemia-induced neuronal inflammatory injury of dairy goats, the levels of blood biochemical indicators, and the changes of cerebellum histopathology and inflammation parameters are measured and compared in both healthy dairy goats and dairy goats with pregnancy toxemia. Moreover, primary cultured neuronal cells in the cerebellum of dairy goats and hypoglycemia condition are established as experiment models in vitro. The expression of the key molecules of the signaling pathways of JNK and P38 MAPK, as well as the related inflammatory factors are determined after adding different concentrations of β-hydroxybutyric acid and using HCAR2 stimulator and inhibitor. This is mainly to explore the protective effects and mechanism of β-hydroxybutyric acid on hypoglycemia-induced neuronal inflammatory injury of dairy goats, which will further reveal the pathogenesis of pregnancy toxemia in dairy goats and establish the theoretical basis for searching new approach for prevention of this disease.

低血糖和以β-羟丁酸为代表的高酮血症是罹患妊娠毒血症奶山羊的典型临床病理学特征。低血糖可促使酮体生成，因此，在妊娠毒血症发病过程中低血糖必先于高酮血症而出现。研究表明，低血糖可致神经细胞炎性损伤并引起神经症状，但一定浓度的β-羟丁酸具有神经保护作用。本项目以“β-羟丁酸通过抑制低糖诱导的奶山羊神经细胞炎性损伤发挥其神经保护作用”为理论假设。通过检测并比较健康与妊娠毒血症奶山羊血液生化指标、小脑组织病理学和炎症相关指标的变化，以确证低糖可致奶山羊神经细胞炎性损伤；通过建立奶山羊小脑神经细胞体外培养体系，并在模拟低糖代谢环境的基础上，添加不同浓度的β-羟丁酸或运用其受体HCAR2兴奋剂和阻断剂，检测JNK和P38 MAPK信号通路关键因子以及相关炎性因子的变化，探究β-羟丁酸对低糖致奶山羊神经细胞炎性损伤的保护作用及其机制，为揭示奶山羊妊娠毒血症发病机制并为探索其科学防治的新途径奠定理论基础。

围产期奶山羊采食量下降使其出现能量负平衡，极易引发能量代谢障碍性疾病，严重威胁奶山羊产业的发展。因此，围产期健康对保证奶山羊生产性能极其重要。低血糖和以β-羟丁酸为代表的高酮血症是罹患妊娠毒血症奶山羊的典型临床病理学特征。低血糖可促使酮体生成，因此，在妊娠毒血症发病过程中低血糖必先于高酮血症而出现。研究表明，低血糖可致神经细胞炎性损伤并引起神经症状，但一定浓度的β-羟丁酸具有神经保护作用。项目深入研究比较和分析了围产期健康奶山羊血液生理指标，明确了围产期健康奶山羊血液生理指标变动范围和规律，以及明确了营养代谢状态对健康奶山羊血液生理指标的具体影响。进一步通过比较妊娠毒血症奶山羊与同时期健康奶山羊血液代谢参数和胰岛素抵抗指数的变化，明确了罹患妊娠毒血症奶山羊血糖浓度与胰岛素的相关性。妊娠毒血症奶山羊产后血液中胰岛素浓度显著升高，表明在泌乳初期妊娠毒血症奶山羊通过增加胰岛素分泌量以维持正常的血糖浓度，且在产后1周妊娠毒血症奶山羊胰岛素敏感性最低；健康奶山羊在分娩当天胰岛素敏感性最低，推测可能与该时期泌乳启动导致能量缺乏有关。运用体外实验，通过培养神经细胞，建立低糖损伤的模型并添加不同浓度的β-羟丁酸，结果表明，β-羟丁酸减少低糖诱导细胞内ROS的积累，并通过介导P38 MAPK 信号通路和Caspase-3表达进一步抑制细胞凋亡。此外，β-羟丁酸通过激活ERK磷酸化抑制细胞凋亡，缓解低糖对神经细胞的损伤。明确了β-羟丁酸对葡萄糖缺乏条件下细胞生存和凋亡的影响以及相关的分子机制。以上结果为揭示奶山羊妊娠毒血症发病机制并为探索其科学防治的新途径奠定了理论基础。

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