基于Cx43研究产前应激对子代大鼠睾丸发育的影响及左归丸干预作用

Zuogui Pills improve testicular development of offspring rats exposed to prenatal stress via Cx43

基于缝隙连接蛋白43(connexin 43, Cx43)研究产前应激大鼠影响子代睾丸发育的机制及左归丸的防治作用。制备SD孕鼠40只,随机分为空白对照组、模型组、左归丸高剂量组(18.9 g·kg^(-1))、左归丸低剂量组(9.45 g·kg^(-1))、维生素E组(1.44 mg·kg^(-1))。除空白对照组以外,其余各组通过慢性不可预测轻度应激法复制产前应激(prenatal stress, PS)孕鼠模型。采用糖水偏好实验、旷场实验、酶联免疫吸附法(enzyme linked immunosorbent assay, ELISA)检测糖皮质激素(glucocorticoids, GC)含量评估PS孕鼠模型;ELISA法检测血清甲状腺素4(thyroxine 4, T4)、睾酮(testosterone, T)、促卵泡生长激素(follicle-stimulating hormone, FSH)评估肾虚状态及性激素水平;苏木素-伊红(hematoxylin-eosin, HE)染色评估睾丸生殖细胞状况;全自动精子分析仪检测精子质量;免疫荧光双染检测子代大鼠睾丸Cx43蛋白、促卵泡激素受体(follicle-stimulating hormone receptor, FSHR)表达;实时荧光定量聚合酶链式反应法(real-time fluorescent quantitative PCR, RT-PCR)检测Cx43、促卵泡生长激素受体(FSHR)、磷脂酰肌醇-3-激酶(phosphatidylinositol 3 kinase, PI3K)、蛋白激酶B(protein kinase B, Akt)基因mRNA表达;蛋白免疫印迹(Western blot, WB)法检测子代大鼠睾丸Cx43、PI3K、FSHR、Akt蛋白表达。PS诱发子代大鼠睾丸发育障碍,HE染色结果显示,出生当天子代雄鼠睾丸曲精小管数量减少,血清FSH升高,睾丸组织Cx43水平降低,60日龄子代雄鼠睾丸生精功能降低,精子质量下降,血清FSH升高,T降低,睾丸组织Cx43、FSHR、PI3K、Akt蛋白及mRNA水平均降低;而给予左归丸防治后对子代大鼠上述睾丸发育异常及功能障碍均有改善。综上,左归丸可能通过激活PI3K/Akt通路调节睾丸Cx43表达改善PS对子代大鼠睾丸发育及生精功能的影响。

This study aims to reveal the mechanism of prenatal stress in affecting the testicular development of offspring rats and the intervention effects of Zuogui Pills via connexin 43(Cx43). Forty pregnant SD rats were randomized into a blank control group, a mo-del group, a high-dose(18.9 g·kg^(-1)) Zuogui Pills group, a low-dose(9.45 g·kg^(-1)) Zuogui Pills group, and a vitamin E(1.44 mg·kg^(-1)) group. The other groups except the blank control group was subjected to chronic unpredictable mild stress for the modeling of prenatal stress. The model was evaluated by sucrose preference test, open field test, and enzyme-linked immunosorbent assay(ELISA) of the glucocorticoid level. ELISA was employed to measure the thyroxine 4(T4), testosterone(T), and follicle-stimulating hormone(FSH) levels to assess kidney deficiency. Hematoxylin-eosin(HE) staining was employed to evaluate the status of testicular germ cells. An automatic sperm analyzer was used to measure the sperm quality. Immunofluorescence double staining was employed to detect the expression of Cx43 and follicle-stimulating hormone receptor(FSHR) in the testes of offspring rats. The mRNA and protein levels of Cx43, FSHR, phosphatidylinositol 3-kinase(PI3K), and protein kinase B(Akt) were determined by real-time fluorescence quantitative polymerase chain reaction and Western blot, respectively. Prenatal stress induced testicular development disorders in offspring rats. The HE staining results showed that on the day of birth, the model group had reduced seminiferous tubules in the testes, elevated FSH level in the serum, and lowered Cx43 level in the testicular tissue. Male offspring rats of 60 days old had reduced testicular spermatogenic function, decreased sperm quality, elevated FSH level and lowered T level in the serum, and down-regulated protein and mRNA levels of Cx43, FSHR, PI3K, and Akt in the testicular tissue. Zuogui Pills alleviated the abnormal development and dysfunction of testicles in the offspring rats caused by prenatal stress. In summary, Zuogui Pills may weaken the effects of prenatal stress on testicular development and spermatogenic function of offspring rats by activating the PI3K/Akt pathway to regulate Cx43 expression in the testicular tissue.