基于骨骼肌脂联素信号通路探讨参芪复方从脾论治改善衰老糖尿病大鼠糖脂代谢异常及骨骼肌损伤的作用

Based on the adiponectin signaling pathway of skeletal muscle discussing the effect of Shenqi compound on improving the abnormal glucose and lipid metabolism and skeletal muscle injury in aging diabetes rats by treating from the perspective of spleen

关键词:  参芪复方  糖尿病  衰老  骨骼肌  脂联素

Key words:  Shenqi compound  diabetes  senility  skeletal muscle  adiponectin

摘要:

[目的]探讨参芪复方从脾论治，对衰老糖尿病大鼠糖脂代谢异常、骨骼肌损伤病理进程，以及骨骼肌脂联素信号通路的影响。[方法] 4月龄Wistar雄性大鼠10只为空白对照（C）组，17月龄Wistar雄性大鼠10只为衰老模型（SMo）组，另17月龄雄性Wistar大鼠30只予高脂高糖饲料喂养+链脲佐菌素（STZ）注射建立衰老糖尿病模型，成模后随机分为衰老糖尿病模型（STMo）组、中药（SQFF）组和西药（X）组，与前面C组、SMo组合为5组大鼠。STMo组、SQFF组、X组，予以高脂饲料喂养；C组及SMo组大鼠，予以普通饲料喂养。SQFF组予以参芪复方冻干粉灌胃，X组予以西格列汀混悬液灌胃，C组、SMo组及STMo组予氯化钠溶液灌胃，连续干预8周。检测指标为各组大鼠血浆脂联素（APN）、空腹血糖（FBG）、胰岛素（INS）、三酰甘油（TG）、总胆固醇（T-CHO）、游离脂肪酸（NEFA）；计算胰岛素抵抗指数（HOMA-IR）；光镜及电镜下观察腓肠肌病理形态学；实时荧光定量逆转录聚合酶链反应（qRT-PCR）检测脂联素受体1（AdipoR1）、脂联素受体2（AdipoR2）、腺苷单磷酸活化蛋白激酶（AMPK）、过氧化物酶体增殖物激活受体α（PPARα）的mRNA表达；蛋白免疫印迹（Western blot）法检测腓肠肌组织AdipoR1、AdipoR2、AMPK、磷酸化腺苷单磷酸活化蛋白激酶（pAMPK）、PPARα蛋白的相对表达量。[结果]与C组相比，STMo组血浆FBG、TG、T-CHO、NEFA、HOMA-IR均显著升高（P<0.05）；血浆APN，腓肠肌AdipoR1、AdipoR2、AMPK、PPARα的mRNA表达，以及AdipoR1、AdipoR2、AMPKα、pAMPKα、PPARα蛋白相对表达量均显著降低（P<0.05）；与STMo组相比，SQFF组及X组血浆FBG、TG、T-CHO、NEFA均显著降低（P<0.05），血浆APN，腓肠肌AMPK、PPARα的mRNA表达，以及腓肠肌AdipoR1、AdipoR2、AMPKα、pAMPKα、PPARα蛋白相对表达量均显著升高（P<0.05）。此外与STMo组相比，X组HOMA-IR显著降低（P<0.05），SQFF组仅表现出下降趋势；与STMo相比，SQFF组AdipoR2的mRNA表达显著增加（P<0.05），X组仅表现出上升趋势。光镜下观察腓肠肌损伤程度显示：参芪复方可改善骨骼肌炎症损伤，肌纤维变性、坏死、断裂、溶解，以及纤维组织的增生。电镜下观察显示：参芪复方可改善骨骼肌纤维结构破坏，不同程度的线粒体损伤等。[结论]参芪复方健脾助运，通过恢复脾主运化、主肌肉的功能，改善衰老糖尿病大鼠的糖脂代谢紊乱及腓肠肌损伤，其可能与脂联素信号通路AdipoR1/AMPK及AdipoR2/PPARα均相关。

Abstract:

[Objective] To investigate the effects of Shenqi compound on abnormal glucose and lipid metabolism，pathological process of skeletal muscle injury，and adiponectin signaling pathway in skeletal muscle of aging diabetes rats. [Methods] Ten 4-monthold male Wistar rats were included as control（C）group，ten 17-month-old male Wistar rats were included as aging model（SMo）group，and 30 17-month-old male Wistar rats were fed with high-fat and high-sugar diet and injected with STZ included as aging diabetes model. After modeling，they were randomly divided into aging diabetes model（STMo）group，traditional Chinese medicine（SQFF）group and Western medicine（X）group. They were combined with the former blank control（C）group and aging model（SMo）group to form five groups of rats. STMo group，SQFF group，and X group were fed with high-fat diet. Rats in group C and SMo were fed with regular feed. The SQFF group was given Shenqi compound freeze-dried powder by gavage，the X group was given sitagliptin suspension by gavage， and the C group，SMo group，and STMo group were given sodium chloride solution by gavage，with continuous intervention for 8 weeks. Detection indicators：Plasma APN，FBG，INS，TG，T-CHO，NEFA detection in each group of rats；HOMA-IR calculation；pathological morphology observation of gastrocnemius muscle under light and electron microscopy；qRT-PCR was used to detect the mRNA expression of AdipoR1，AdipoR2，AMPK，and PPAR-α；Western blot was used to detect the relative expression levels of AdipoR1， AdipoR2，AMPK，pAMPK，and PPARα proteins in gastrocnemius muscle tissue. [Results] Compared with group C，plasma FBG，TG，TCHO，NEFA，and HOMA-IR were significantly increased in the STMo group（P<0.05）；the mRNA expression of plasma APN， gastrocnemius muscle AdipoR1，AdipoR2，AMPK，PPARα，as well as the relative expression levels of AdipoR1，AdipoR2，AMPKα， pAMPKα，and PPARα proteins were significantly reduced（P<0.05）；compared with the STMo group，the plasma FBG，TG，T-CHO， and NEFA in the SQFF group and X group were significantly reduced（P<0.05），while the mRNA expression of plasma APN， gastrocnemius muscle AMPK，PPARα，and the relative expression levels of gastrocnemius muscle AdipoR1，AdipoR2，AMPKα， pAMPKα，and PPARα proteins were significantly increased（P<0.05）. In addition，compared with the STMo group，the HOMA-IR of X group was significantly reduced（P<0.05），while the SQFF group only showed a downward trend；compared with STMo，the mRNA expression of AdipoR2 in the SQFF group significantly increased（P<0.05），while the X group only showed an upward trend. The injury degree of gastrocnemius muscle under light microscope showed that Shenqi compound could improve the inflammation injury of skeletal muscle，muscle fibrosis，necrosis，fracture，dissolution，and proliferation of fiber tissue. Observation under electron microscope showed that Shenqi compound could improve the damage of skeletal muscle fiber structure and different degree of mitochondrial damage. [Conclusion] The Shenqi compound strengthens the spleen and promotes transportation. By restoring the spleen’s functions of transportation and transformation as well as muscle management，it improves the disorders of glucose and lipid metabolism and gastrocnemius muscle injury in aging diabetic rats. This may be related to the adiponectin signaling pathways AdipoR1/AMPK and AdipoR2/ PPARα.