益脾补肾方改善肥胖2型糖尿病小鼠生精功能障碍作用及机制

基金项目:宁夏重点研发计划项目（No.2020BFG02010）；宁夏自然科学基金资助项目（No.2022AAC02033）；国家自然科学基金资助项目（No.82201000）；宁夏科技创新领军人才培养项目（No.2020GKLRLX11）；宁夏医科大学校级科研项目（No.XT2019017）

作者	单位
王强强，于普光，彭庆杰，任帅，王春红，苏亚珊，梁晓霞，孙苗，陈小江，杜小利，李广永，何瑞

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中文摘要:

目的观察益脾补肾方改善肥胖2型糖尿病小鼠生精功能障碍的作用，并探讨其作用机制。方法将30只4周龄健康C57BL/6J雄性小鼠随机分为对照组、模型组、益脾补肾方低剂量组（低剂量组）、益脾补肾方高剂量组（高剂量组）、二甲双胍组，每组6只。对照组给予普通饲料，其余各组给予高脂饲料喂养16周，建立肥胖2型糖尿病小鼠模型。低、高剂量组给予益脾补肾方2、4 g/（kg·d），二甲双胍组给予二甲双胍200 mg/（kg·d）灌胃，模型组和对照组灌胃等量的生理盐水，连续灌胃30天。干预前后进行口服葡萄糖耐量试验（OGTT）和胰岛素耐量试验（ITT），计算曲线下面积（AUC）；观察睾丸形态，测量小鼠重量并计算睾丸脏体比；HE染色观察睾丸形态变化；检测精子参数以及空腹血糖（FBG）；ELISA法检测血清中睾酮（T）水平；比色法检测血清中总超氧化物歧化酶（T-SOD）、丙二醛（MDA）、谷胱甘肽过氧化物酶（GSH-Px）的变化； Western Blot检测脂酰肌醇-3-激酶（PI3K）、蛋白激酶B（Akt）、B淋巴细胞瘤-2（Bcl-2）表达。结果对照组小鼠睾丸组织结构清晰，生精小管内各级生精细胞排列整齐；模型组小鼠睾丸组织结构混乱，各级生精细胞排列混乱，且数量显著减少，腔内出现大面积空洞；干预组出现不同程度改善。与对照组比较，模型组体重、体重AUC、FBG、OGTT-AUC、ITT-AUC、精子畸形率、血清MDA水平增加（P<0.05，P<0.01），睾丸系数、附睾系数、精囊腺系数、精子数量、精子活力、精子活率、血清T、血清T-SOD、GSH-Px水平、睾丸PI3K、p-Akt、Bcl-2蛋白表达降低（P<0.05）。与模型组同期比较，干预组体重AUC、体重、OGTT-AUC、ITT-AUC、精子畸形率、血清MDA水平下降（P<0.05，P<0.01），睾丸系数、附睾系数、精囊腺系数、精子数量、精子活力、血清T-SOD、GSH-Px水平、睾丸Bcl-2蛋白表达增加（P<0.05）；二甲双胍组精子活率、血清T、睾丸PI3K表达增加（P<0.05）；低剂量组睾丸p-Akt蛋白表达升高（P<0.05）；高剂量组精子活率、血清T、睾丸PI3K、p-Akt蛋白增加（P<0.05）。与二甲双胍组同期比较，低剂量组干预后体重AUC、体重增加（P<0.05）；高剂量组干预后体重AUC、精子活力、血清T增加（P<0.05），ITT-AUC降低（P<0.05）。结论益脾补肾方可能通过调节PI3K/Akt/Bcl-2通路调节糖代谢来改善肥胖2型糖尿病小鼠生精功能。

英文摘要:

Objective To observe the effect of Yipi Bushen Formula（YBF） on improving the spermatogenic dysfunction in obese type 2 diabetic mice，and explore its mechanism. Methods Totally 30 healthy 4-week-old C57BL/6J male mice were randomly divided into 5 groups，control group，model group，low-dose YBF group，high-dose YBF group， and metformin group，6 mice in each group. The control group was given normal feed，and the other groups were fed high-fat feed for 16 weeks to establish obese type 2 diabetic mouse model. The low-dose and high-dose YBF group were given YBF 2 and 4 g·kg-1·d-1，respectively，the metformin group was given metformin 200 mg·kg-1·d-1 by gavage， the model and control group were given the same amount of normal saline by gavage for 30 consecutive days. Oral glucose tolerance test （OGTT） and insulin tolerance test （ITT） were performed before and after intervention，and the area under the curve（AUC） was calculated. Testicular morphology was observed，mouse weight and testis visceral ratio were measured. Testicular morphology was observed by HE staining. Sperm parameters and fasting blood glucose （FBG） were detected. ELISA was used to detect serum testosterone （T） level. Colorimetry was used to detect serum total superoxide dismutase（T-SOD），malondialdehyde （MDA），and glutathione peroxidase （GSH-Px） level. Western Blot was used to detect the expression of phosphatidylinositide 3-kinases（PI3K），protein kinase B（Akt），B-cell lymphoma-2（Bcl-2）. Results The testicular tissue structure of the control group mice was clear，with orderly arranged germ cells at all levels in the seminiferous tubules，while the testicular tissue structure of the model group mice was chaotic，with disordered arrangement of germ cells at all levels with significantly reduced in number，and large areas of cavities appearing in the tubules. The intervention group showed varying degrees of improvement. Compared with the control group，body weight，body weight AUC，FBG，OGTT-AUC，ITT-AUC，sperm deformity rate，and serum MDA level increased（P<0.05，P<0.01），while the testicular coefficient，epididymal coefficient，seminal vesicle coefficient，sperm count，sperm motility，sperm viability，serum T，T-SOD，GSH-Px level，and testicular PI3K，p-Akt，and Bcl-2 protein expression decreased in model group（P<0.05）. Compared with model group at the same time，body weight AUC，body weight，OGTT-AUC，ITT-AUC，sperm deformity rate，and serum MDA level decreased（P<0.05，P<0.01），testicular coefficient，epididymal coefficient，seminal vesicle coefficient，sperm count，sperm motility，serum T-SOD，GSH-Px level，and testicular Bcl-2 protein expression increased in intervention groups after intervention（P<0.05）. Sperm viability，serum T，and testicular PI3K expression increased in metformin group（P<0.05）. Testicular p-Akt protein expression increased in low-dose YBF group（P<0.05）. Sperm viability，serum T，testicular PI3K，and p-Akt protein expression increased in high-dose YBF group（P<0.05）. Compared with the metformin group at the same time，body weight AUC and body weight increased in low-dose YBF group after intervention（P<0.05）. Body weight AUC，sperm motility，and serum T increased（P<0.05），ITT-AUC decreased in high-dose YBF group after intervention（P<0.05）. Conclusion YBF may improve the reproductive function of obese type 2 diabetic mice by regulating the PI3K/Akt/Bcl-2 pathway to adjust glucose metabolism.

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