摘要
目的:探讨疏肝补肾毓麟汤能通过调控葡萄糖转运蛋白4(GLUT4)/胰岛素受体底物-1(IRS-1)/磷脂酰基醇3-激酶/蛋白激酶B(PI3K/Akt)信号通路调节下丘脑葡萄糖摄取从而明显改善肝气郁结型大鼠少弱精子症的作用机制。方法:将雄性SD大鼠随机分成空白组,肝郁模型组,疏肝补肾毓麟汤高、低剂量组及氟西汀组,每组8只,用慢性束缚结合夹尾激怒法制备肝气郁结大鼠模型,持续14 d。造模完成后,疏肝补肾毓麟汤低(9.75 g·kg^(-1))、高(39 g·kg^(-1))剂量组分别给予疏肝补肾毓麟汤灌胃给药,氟西汀组(0.021 g·kg^(-1))给予氟西汀溶液灌胃,连续14 d给药;采用矿场实验、糖水偏好实验评估情绪压力;依据中医理论通过舌色观察肝郁大鼠的造模情况,于光线良好处测量舌体及舌边颜色;精密天平称取大鼠体质量,取睾丸和附睾并称质量,根据公式分别计算睾丸和附睾脏器指数;检测下丘脑-垂体-肾上腺(HPA)轴变化:酶联免疫吸附测定法(ELISA)检测大鼠血液中内啡肽(β-EP)、促肾上腺皮质激素(ACTH)、皮质酮(CORT)、下丘脑促肾上腺皮质激素释放激素(CRH)含量;苏木素-伊红(HE)染色观察睾丸形态评估生精功能;附睾取精子,全自动精子分析仪检测精子密度和活动率,苯胺蓝染色法观察精子成熟度;蛋白免疫印迹法(Western blot)检测大鼠下丘脑组织GLUT4、IRS-1、磷脂酰肌醇3-激酶(PI3K)、磷酸化(p)-PI3K、蛋白激酶B(Akt)、p-Akt、睾丸组织促卵泡激素受体(FSHR)、μ-阿片受体(MOR)。结果:与空白组比较,模型组大鼠在矿场行为学实验中移动总距离和总时长明显降低、休息时间明显增加(P<0.05,P<0.01);糖水实验中模型组大鼠对糖水的摄入显著减少(P<0.01),显示大鼠情绪受到刺激快感缺失;观测舌体时,模型组舌体颜色偏暗红或紫红色;睾丸切片显示模型组大鼠睾丸形态出现萎缩,睾丸生精细胞减少,细胞排列不规则;睾丸和附睾脏器指数显著降低(P<0.01);精子密度、活动率均显著降低(P<0.01),精子苯胺蓝染色显示成熟度降低;血液中ACTH、CORT、CRH、β-EP的水平显著升高(P<0.01);GLUT4、IRS-1、PI3K、p-PI3K、Akt、p-Akt、FSHR蛋白表达显著降低(P<0.01)。与模型组比较,给药治疗组大鼠舌体颜色由深红色变浅;睾丸和附睾脏器指数明显上升(P<0.05,P<0.01);大鼠睾丸形态改善,生精功能改善;精子成熟度、密度、活动率显著上升(P<0.01);血液中ACTH、CORT、CRH、β-EP的水平明显升高(P<0.05,P<0.01);且GLUT4、IRS-1、PI3K、Akt、FSHR蛋白表达明显升高(P<0.05,P<0.01),MOR蛋白表达降低(P<0.05,P<0.01)。结论:负性情绪诱发机体应激反应会引起下丘脑葡萄糖代谢异常导致HPA轴紊乱最终影响精子质量,疏肝补肾毓麟汤可通过调控GLUT4/IRS-1/PI3K/Akt信号通路增加下丘脑葡萄糖摄取来调节HPA轴从而明显改善肝气郁结型大鼠少弱精子症。
Objective:To investigate the mechanism by which Shugan Bushen Yulin decoction improves oligoasthenozoospermia in rats with liver qi stagnation by regulating the glucose transporter 4(GLUT4)/insulin receptor substrate-1(IRS-1)/phosphatidylinositol 3-kinase/protein kinase B(PI3K/Akt)signaling pathway and modulating glucose uptake in the hypothalamus.Methods:Male Sprague-Dawley rats were randomly divided into a blank control group,a liver Qi stagnation model group,high-and low-dose Shugan Bushen Yulin decoction groups,and a fluoxetine group,with eight rats in each group.A liver Qi stagnation model was established by chronic restraint combined with tail-pinching for 14 days.After modeling,the low-(9.75 g·kg^(-1))and high-dose(39 g·kg^(-1))decoction groups received the decoction by gavage,while the fluoxetine group(0.021 g·kg^(-1))received fluoxetine solution by gavage for 14 consecutive days.Emotional stress was evaluated using the open-field test and the sucrose preference test.According to the theory of traditional Chinese medicine(TCM),the modeling of liver Qi stagnation was assessed by observing tongue color and tongue edge color under good lighting.Body weight was measured using a precision scale.The testes and epididymis were removed and weighed to calculate organ indices.Changes in the hypothalamicpituitary-adrenal(HPA)axis were determined by enzyme-linked immunosorbent assay(ELISA)to measure the levels ofβ-endorphin(β-EP),adrenocorticotropic hormone(ACTH),corticosterone(CORT),and corticotropin-releasing hormone(CRH)in the blood.Testicular morphology and spermatogenic function were assessed by hematoxylin-eosin(HE)staining.Sperm from the epididymis was analyzed for density and motility using a computer-assisted sperm analysis system,and sperm maturity was evaluated by aniline blue staining.Western blot was used to detect the expression of GLUT4,IRS-1,PI3K,phosphorylated PI3K(p-PI3K),Akt,phosphorylated Akt(p-Akt)in hypothalamic tissue,and follicle-stimulating hormone receptor(FSHR)andμ-opioid receptor(MOR)in testicular tissue.Results:Compared with the blank group,rats in the model group showed significantly reduced total movement distance and duration,and increased resting time in the open-field test(P<0.05,P<0.01).Sucrose consumption significantly decreased(P<0.01),indicating anhedonia induced by emotional stress.The tongues of model rats appeared dark red or purplish.Testicular sections showed atrophy,reduced spermatogenic cell number,and disordered cell arrangement.The testicular and epididymal indices,sperm density,and motility were significantly reduced(P<0.01),and aniline blue staining revealed decreased sperm maturity.Serum levels of ACTH,CORT,CRH,andβ-EP were significantly increased(P<0.01),while the expression of GLUT4,IRS-1,PI3K,p-PI3K,Akt,p-Akt,and FSHR proteins was significantly decreased(P<0.01).Compared with the model group,rats in the treatment groups showed lighter tongue color,significantly increased testicular and epididymal indices(P<0.05,P<0.01),improved testicular morphology and spermatogenic function,and significantly increased sperm maturity,density,and motility(P<0.01).Serum ACTH,CORT,CRH,andβ-EP levels were significantly increased(P<0.05,P<0.01),while the expression of GLUT4,IRS-1,PI3K,Akt,and FSHR proteins was significantly upregulated(P<0.05,P<0.01),and MOR protein expression was significantly downregulated(P<0.05,P<0.01).Conclusion:Negative emotions induce a stress response that causes abnormal glucose metabolism in the hypothalamus,leading to HPA axis dysfunction and impaired sperm quality.Shugan Bushen Yulin decoction can improve oligoasthenozoospermia in rats with liver Qi stagnation by regulating the GLUT4/IRS-1/PI3K/Akt signaling pathway to enhance hypothalamic glucose uptake and modulate the HPA axis.
作者
张振斌
萧闵
赵纯纯
石遵环
刘阳
何欢
王朝阳
ZHANG Zhenbin;XIAO Min;ZHAO Chunchun;SHI Zunhuan;LIU Yang;HE Huan;WANG Chaoyang(School of Basic Medical Sciences,Hubei University of Chinese Medicine,Wuhan 430065,China;Hubei Shi Zhen Laboratory,Wuhan 430065,China;Laboratory Animal Center,Hubei University of Chinese Medicine,Wuhan 430065,China;College of Physical Education and Health,Hubei University of Chinese Medicine,Wuhan 430065,China)
出处
《中国实验方剂学杂志》
北大核心
2025年第23期75-84,共10页
Chinese Journal of Experimental Traditional Medical Formulae
基金
湖北省自然科学基金-中医药创新发展联合基金项目(2024AFD233)。
