益精方对环磷酰胺小鼠精子胞内钙离子浓度的干预作用

The effect of Yijingfang on the concentration of calcium ion in mouse spermatophores with peritoneal injection of cyclophosphamide

目的从精子胞内钙离子的角度阐述益精方治疗弱精子症的机制。方法将40只雄性昆明小鼠随机分为对照组（CG）、模型组（MG）、小剂量中药治疗组（SG）和大剂量中药治疗组（LG），按60mg／kg体质量给CG小鼠腹腔注射生理盐水，60mg／kg体质量给MG、SG、LG小鼠腹腔注射环磷酰胺，qd，连续5d，第6d开始按体重分别给SG和LG小鼠灌服益精方，剂量分别为人类常规用量（以60kg为标准）的1倍和5倍，MG小鼠给予等体积的生理盐水灌胃，qd，连续34d，CG常规饲养，第40天对小鼠进行精液常规分析，对精子行FITC—AV／PI染色后用流式细胞仪检测精子凋亡率，用Ca^2＋荧光探针Fluo-3／AM处理精子后用流式细胞仪检测精子胞内Ca^2＋浓度。结果CG、MG、SG和LG精子胞内Ca^2＋浓度分别为133．14±11．86、80．84±6．92、88．05±3．31、99．08±7．17，MG胞内Ca^2＋浓度显著低于CG（P〈0．05），治疗后LG胞内Ca2＋浓度显著升高，与MG比较差异有统计学意义（P〈0．05）；精子活力分别为（14．49±0．30）％、（6．64±1．88）％、（11．99±1．01）％和（19．40±3．13）％；精子活率分别为（68．39±15．13）％、（39．96±4．89）％、（62．28±4．43）％和（73．61±5．05）％；精子密度分别为（5．20±1．34）×10^6／ml、（1．73±0．03）×10^6／ml、（2．08±0．01）×10^6／m和（3．31±0．56）×10^6／ml，MG活力、活率、密度显著低于CG（P〈0．05），经治疗后LG活力、活率、密度明显增加，与MG比较差异有统计学意义（P〈0．05），而与CG在活力和活率上没有明显区别；精子凋亡率分别为8．38±0．45、14．84±1．22、13．25±1．83、8．35±2．1，MG凋亡率显著高于CG（P〈0．05），治疗后LG凋亡率显著降低，与MG比较差异有统计学意义（P〈0．05）而与CG没有明显区别。结论大剂量益精方可提高因腹腔注射环磷酰胺造成的小鼠精子活力、活率低下状态，其改善精子活力的作用可能与益精方增加精子胞内Ca^2＋浓度有关。

Objective To observe the effect of Yijingfang on the concentration of calcium ion in mouse spermatophores with peritoneal injection of cyclophosphamide. Methods 40 Kunming male mice were randomly divided into the control group（CG）, model group（MG）, small dose group of Yijingfang （SG）, and large dose group of Yijingfang（LG）. During the first 5 days, CG was given NS 60mg/kg weight once a day by peritoneal injection, while MG, SG and LG were given peritoneal injection ofcyclophosphamide. On the 5th or 6th days, according to the weight of mice, Yijingfang was given to SG and LG once a day by intragastric administration for 34 days, the dose for SG was double of human common dose （human weight 60kg）, while 5 folds for LG. and MG MG was given the same volume of NS by gavage once day for 34 days. CG was bred normally. On the 40th day, the sperm count, motility and survival rate were measured, and the apoptosis of sperm dyed with FITC-AV/PI was measured by a flow cytometer. The concentration of calcium ion of sperm dyed with Fluo-3/AM was also measured by a flow cytometer. Results The concentration of calcium ion in the spermtophores of CG, MG, SG and LG was （133.14±11.86）, （80,84±6.92）, （88.05±3.31）and （99.08±7.17） respectively, That in MG was obviously lower than that in CG（P〈0.05）, while that in LG was higher than that in MG（P〈0.05）. The sperm motility ofCG, MG, SG and LG was （14.49±0.30）%, （6.64±1.88）%, （11.99±1.01）% and （19.40±3.13）% respectively. That in MG was obviously lower than that in CG（P〈0.05）, while that in LG was higher than that in MG（P〈0.05）. The sperm survival rate of CG, MG, SG and LG was （68.39±15.13）%, （39.96±4.89）%, （62.28±4.43）% and （73.61±5.05）% respectively, That in MG was obviously lower than that in CG（P〈0.05）,while that in LG was higher than that in MG（P〈0.05）. The sperm count ofCG, MG, SG and LG was （5.20±1.34）, （1.73±0.03）, （2.08±0.01）and （3.31±0.56） （× 10^6/ml） respectively, That of MG was obviously lower than that of CG（P〈0.05）,while that of LG was higher than that of MG（P〈0.05）.The spermatozoa apoptosis of CG, MG, SG and LG was （8.38±0.45）, （14.84±1.22）, （13.25±1.83） and （8.35±2.1） respectively. That of LG was lower than that of MG（P〈0.05） but there was no difference compared with CG（P〉0.05）： Conclusion Administration of large dose of Yijingfang can increase the motility and survival rate of mice when it is decreased by peritoneal injection of cyclophosphamide, and the effect may be related to the increased concentration of calcium ion in spermatophores, by Yijingfang.