基于AMPK调节能量平衡探讨丹参对奶牛子宫内膜炎的治疗机制

A variety of Traditional Chinese Medicine preparations containing Salvia miltiorrhiza are effective in treating clinical dairy cow endometritis, and have good antibacterial activity to a few pathogenic bacteria. However, the extensive bacteriostatic effect of these drugs is still unproven, which seriously hinders their application. Previous research demonstrated that AMPK modified energy negative balance of the cow can strengthen immunity, fundamentally solve the uterus pathogenic bacteria infection and inflammation, thus cure dairy cow endometritis, and several main compositions in the Tonify Deficiency Medicine Salvia miltiorrhiza can activate AMPK. The present project incorporates “weakness” in Traditional Chinese Veterinary Medicine theory and “body negative energy balance” in Modern pharmacological theory, mimic cell negative energy balance condition by low-glucose medium, utilize immunofluorescence, laser confocal microscopy, Western Blot, PCR technologies, verify the effect of Salvia miltiorrhiza main compositions (Tanshinone IIA, cryptotanshinone, Dihydrotanshinone I, salvianolic acid A, salvianolic acid B) on dairy cow endometrial epithelial cells AMPK activity, energy balance, inflammation signaling (TLR4—MYD88—NF-κB/MAPK), clarify the treatment mechanism of Salvia miltiorrhiza in dairy cow endometrial epithelial cell inflammation, provide theoretical support for Traditional Chinese Veterinary Medicine replace antibiotics, popularization and application, promote positive role on dairy cow breeding industry and food safety.

多种含有丹参的中药制剂对治疗奶牛子宫内膜炎临床疗效显著，且对少数致病菌有较好的抑菌活性。但该类药物的广泛抑菌作用仍未经证实，严重阻碍其推广应用。研究表明，通过AMPK改善奶牛机体能量负平衡可增强机体免疫力，从根本上解决子宫内致病菌感染和炎症发生，达到治疗奶牛子宫内膜炎的目的,而补虚药丹参中多个主要成分均能激活AMPK。本项目将中兽医药理论的“体虚”与现代药理学的“机体能量负平衡”相结合，借助低糖培养液模拟细胞能量负平衡，运用免疫荧光、激光共聚焦、Western Blot、PCR等技术，验证丹参主要成分丹参酮IIA、隐丹参酮、二氢丹参酮I、丹酚酸A、丹酚酸B对奶牛子宫内膜上皮细胞AMPK活性、能量平衡、炎症信号（TLR4—MYD88—NF-κB/MAPK）的影响，阐明丹参对奶牛子宫内膜上皮细胞炎症的治疗机制，为相关中药替代抗生素的推广应用提供理论支持，对奶牛养殖和食品安全起到积极的推动作用。

中兽医药理论认为，奶牛子宫内膜炎由体虚引起。丹参在多个治疗奶牛子宫内膜炎的方剂中均被使用，且临床疗效显著。目前，关于中药方剂对奶牛子宫内膜炎治疗机制的研究多集中在中药的抗菌消炎活性方面。.本项目研究表明，脂多糖刺激能引起奶牛子宫内膜上皮细胞ATP含量降低，阻碍细胞生长，并刺激细胞炎性因子（IL-1，IL-6，IL-8）分泌。利用低糖培养液可成功模拟细胞能量负平衡状态。在能量不足环境下，奶牛子宫内膜上皮细胞能量显著低于健康细胞，且细胞的炎性因子分泌量升高。细胞处于正常能量状态下，丹参主要活性成分丹参酮ⅡA、隐丹参酮、丹酚酸A和丹酚酸B均能抑制细胞炎性因子分泌并提高细胞能量水平。当细胞处于能量负平衡状态下，丹参酮ⅡA、丹酚酸A和丹酚酸B可抑制细胞炎性因子分泌并提高细胞能量水平。丹参酮ⅡA、隐丹参酮、丹酚酸A和丹酚酸B分别通过AKT、ERK、P38、JNK及其下游蛋白NF-Kb、AP-1阻断炎性因子IL-1、IL-6、 IL-8的分泌，从而抑制炎症发生。同时，丹参酮ⅡA、隐丹参酮、丹酚酸A和丹酚酸B可通过影响生热反应和AMPK信号通路提高细胞能量。一方面，丹参酮ⅡA、隐丹参酮、丹酚酸A和丹酚酸B可通过抑制PPARɤ、P38、mTORC1及其下游信号，阻断线粒体解偶联蛋白UCP1活化，通过影响生热反应提高细胞能量。另一方面，丹参酮ⅡA、隐丹参酮、丹酚酸A和丹酚酸B均能激活AMPK，进而通过调节糖代谢、脂肪酸合成及细胞周期，进而抑制细胞炎性反应。.本项目以调节能量平衡为切入点，通过增强免疫力，从根本上解决致病菌感染和炎症发生。将中兽医药理论中的“体虚”与现代药理学理论中的“机体能量负平衡”相结合，利用中药丹参的补虚功效调节能量平衡，代替抗生素类药物治疗奶牛子宫内膜炎，可更加安全、高效地解决奶牛养殖业的难题，进一步保障食品安全，为治疗奶牛子宫内膜炎中药制剂的广泛应用提供理论依据。