三七总皂苷对自然衰老大鼠睾丸炎症反应和生殖细胞凋亡的影响

Effect of Panax Notoginseng Saponins on Testicular Inflammatory Reaction and Apoptosis of Germ Cells in Naturally Aging Rats

目的探讨三七总皂苷(PNS)对自然衰老大鼠睾丸炎症反应和生殖细胞凋亡的影响。方法将SPF级SD大鼠随机分为9月龄组(9M),24月龄组(24M),PNS低、中、高剂量组,每组10只。大鼠从18个月开始,PNS低、中、高剂量组分别灌胃给予PNS 10,30,60 mg·kg^(-1),灌胃至24个月,每周灌胃6 d,周日停1 d,连续灌胃6个月。末次给药并禁食不禁水12 h后,处死大鼠,迅速取出睾丸组织。苏木精-伊红(HE)染色法观察睾丸组织形态学变化,Western Blot检测睾丸组织中P21^(WAF1/CIP1)(P21)、核转录因子(NF-κB)以及下游炎症因子白介素-1β(IL-1β)、肿瘤坏死因子-α(TNF-α)、环氧化酶2(COX2)蛋白的表达和凋亡相关蛋白Bcl-2相关X蛋白(Bax)、B淋巴细胞瘤-2基因(Bcl-2)的表达,脱氧核糖核苷酸末端转移酶介导的缺口末端标记法(TUNEL)检测睾丸生殖细胞凋亡。结果 HE染色观察发现,与9M比较,24M大鼠睾丸生精小管形态结构发生明显变化,生精细胞层数减少,细胞稀疏,各级生精细胞脱落。与24M比较,PNS低、中、高剂量组均能在一定程度上改善睾丸组织生精小管结构形态。与9M比较,24M大鼠睾丸组织P21、NF-κB及其下游炎症因子IL-1β、TNF-α、COX2的表达显著上调(P<0.01),PNS低、中、高剂量组可显著下调其表达(P<0.05,P<0.01)。24M大鼠睾丸组织抗凋亡蛋白Bcl-2的表达显著降低(P<0.01),促凋亡蛋白Bax显著升高(P<0.05),Bcl-2/Bax比值显著降低(P<0.01),PNS可显著升高Bcl-2的表达,降低Bax的表达,升高Bcl-2/Bax比值(P<0.05,P<0.01);与9M比较,24M大鼠睾丸内凋亡阳性细胞数显著增多,PNS低、中、高剂量组可明显减少凋亡的阳性细胞数。结论 PNS能够抑制自然衰老大鼠睾丸炎症反应,显著减少睾丸生殖细胞凋亡。

Objective To investigate the effect of Panax Notoginseng Saponins （PNS） on testicular inflammatory reaction and germ cell apoptosis in naturally aging rat model. Methods SPF SD rats were randomly divided into five groups, namely 9-month-aged group, 24-month-aged group, and low-, middle- and high-dose PNS groups, 10 rats in each group. Rats of PNS groups were given gastric gavage of PNS 10, 30, 60 mg ·kg-1 respectively from the 18th month to the 24th month, 6 days every week, lasting for 6 months. The rats were executed on fasting hour 12 after the last medication, and the testes were immediately removed. The testicular histology was observed by haematoxylin-eosin （HE） staining, the protein expression of the P21, nuelear factor kappa B （NF-KB）, downstream inflammatory factors of interleukin 1 beta （IL- 1β）, tumor necrosis factor alpha （TNF- a ）, cyclooxygenase 2 （COX2）, and apoptosis-related proteins Bax and Bcl-2 in testicular tissue were detected by Western blotting. Testieular germ cell apoptosis was detected using TUNEL （TdT-mediated dUTP nick-end labeling）method. Results Compared with 9-month-aged group, 24-month-aged group had obvious changes in the testicular seminiferous tubule morphology,showing as reduced layers of spermatogenic cells, scattered cells, and shedding of various degrees of sperm cells. Low-, middle- and high-dose PNS could improve the histological changes of testicular seminiferous tubule structure in some degrees. Compared with those in the 9-month-aged group, the expression of P21, NF-KB, and downstream inflammation factors of TNF-a, IL-1β and COX2 were significantly upregulated, anti-apoptosis protein Bcl-2 expression and the ratio of the Bcl-2/Bax were significantly reduced（P 〈 0.01 ） while apoptosis-promoting protein Bax was significantly increased（P〈 0.05） in rat testis tissue of 24-month-aged group（P〈 0.01 ）. And PNS at low, middle and high dose had significant counteraction on the above changes in the protein expression of apoptosis-related genes （P 〈 0.05, P 〈 0.01 ）. Compared with that of the 9-month-aged rats, the number of testicular apoptosis-positive cells of 24-month-aged rats was significantly increased, and PNS at low, middle and high dose obviously reduced the number of apoptosis-positive cells. Conclusion PNS can inhibit the testicular inflammation reaction of naturally aging rats and effectively reduce the apoptosis of germ cells.