黑茸补肾颗粒对白消安诱导的...障碍小鼠模型治疗作用的研究_杨子朝.pdf

NJA中华男科学杂志National Journal of AndrologyZhonghua Nan Ke Xue Za Zhi2022，28(11):1031 1037http:/www androl cn论著Traditional Chinese Medicine(中医中药)黑茸补肾颗粒对白消安诱导的精子发生障碍小鼠模型治疗作用的研究杨子朝1，王小龙2，张迪3，马帅3，郑连文3(1 长春中医药大学中医学院，吉林 长春 130041;2 吉林省妇幼保健院中医科，吉林 长春 130041;3 吉林大学第二医院生殖医学中心，吉林 长春 130041)【摘要】目的:探索黑茸补肾颗粒对白消安诱导的小鼠精子发生障碍的治疗作用，并揭示其调控睾丸生精功能的机制。方法:将 48 只雄性小鼠随机分为 6 组，分别为空白对照(BC)组、阴性对照组(NC)、HKTG-1 组、HK-TG-2 组、HKTG-3 组、HKTG-4 组。除 BC 组外，其余组通过腹腔注射 40 mg/kg 体重的白消安建立精子发生障碍小鼠模型，BC 组、NC 组给予生理盐水灌胃，HKTG-1 组给予黑茸补肾颗粒药剂 1 中添加鹿茸 400 mg/kg 体重、HKTG-2组给予黑茸补肾颗粒药剂 1 中添加鹿茸 800 mg/kg 体重、HKTG-3 组给予黑茸补肾颗粒药剂 2 中添加黑蚁400 mg/kg体重、HKTG-4 组给予黑茸补肾颗粒药剂 2 中添加黑蚁 800 mg/kg 体重，药剂研磨后以生理盐水为溶剂灌胃给小鼠，1 次/d，连续给药 5 周，记录各组小鼠每日平均体重，造模结束后记录各组小鼠睾丸重量，HE 染色检测小鼠睾丸组织显微结构，实时荧光定量 PC 检测标记基因表达水平。结果:与 BC 组相比，NC 组和黑茸补肾颗粒治疗组小鼠的体重及日增重无显著差异;与 NC 组相比，HKTG-1 组睾丸重量显著增加(P 0 05);睾丸组织 HE染色结果显示，与 NC 组相比，HKTG-1 和 HKTG-2 组的生精小管中有大量生殖细胞，包括管腔中已变形的精子细胞，HKTG-3 和 HKTG-4 组的生精小管中也出现了一定数量的生殖细胞(精原精母细胞)，但管腔中几乎没有变形的精子细胞。睾丸组织的实时荧光定量 PC 结果显示，与 NC 组相比，HKTG-1 组的总生殖细胞标记基因(Ddx4)、精原细胞标记基因(Dazl)、精母细胞标记基因(Sycp3)相对表达量显著上调，精子细胞标记基因(Tnp1)则显著上调(P0 05)。HKTG-1 组的支持细胞数量显著降低(P 0 05)，支持细胞标记基因(Sox9)下调，这与其生殖细胞数量多相对应。HKTG-3、HKTG-4 组的 Ddx4、Dazl、Sycp3 和 Tnp1 基因相对表达量无显著变化，支持细胞数量也没有显著变化。与 NC 组相比，HKTG-1 组显著改善小鼠睾丸组织的精子发生，表现为上调减数分裂相关基因(Meioc、Stra8、Spo11)的表达。结论:黑茸补肾颗粒可能是通过促进减数分裂的发生，改善小鼠睾丸组织内生精细胞的功能，进而增加精子的生成。【关键词】黑茸补肾颗粒;非梗阻性无精子症;白消安;小鼠中图分类号:698+2文献标志码:Adoi:10 13263/j cnki nja 2022 11 011Therapeutic effect of Heirong Kidney-Tonifying Granule on busulfan-induceddyszoospermia in model miceYANG Zi-zhao1，WANG Xiao-long2，ZHANG Di3，MA Shuai3，ZHENG Lian-wen31 College of Traditional Chinese Medicine，Changchun University of Chinese Medicine，Changchun，Jilin 130041，China;2 Department of Traditional Chinese Medicine，Jilin Maternal and Child Health Hospital，Changchun，Jilin130041，China;3 Center of eproductive Medicine，The Second Hospital of Jilin University，Changchun，Jilin1301基金项目:吉林省科技发展计划医药健康专项(20200708065YY)作者简介:杨子朝(1996-)，男，河北保定市人，硕士研究生，从事中医男科学研究。Email:864309058 qq com通讯作者:郑连文，Email:362683485 qq com130041，China【Abstract】Objective:To explore the therapeutic effect of Heirong Kidney-Tonifying Granule(HKTG)on busulfan-induceddyszoospermia in mice，and its mechanism in regulating testicular spermatogenesisMethods:Forty-eight male mice were randomlydivided into six groups of an equal number:blank control(BC)，negative control(NC)，HKTG-1，HKTG-2，HKTG-3 and HKTG-4The model of dyszoospermia was established in the latter five groups by intraperitoneal injection of busulfan at 40 mg/kg and，30 daysafter modeling，the mice in the BC and NC groups were given gavage of normal saline，and those in the latter four groups treated withHKTG+pilose antler at 400 mg/kg/d，HKTG+pilose antler at 800 mg/kg/d，HKTG+black ants at 400 mg/kg/d and HKTG+black ants at 800 mg/kg/d，respectively，all for 5 consecutive weeks The mean body weight of the mice was recorded daily，and theirtestes weighed after treatment The microstructure of the testis tissue was detected by HE staining，and the localization and expressionof spermatogenesis markers in the testis were determined by immunofluorescence stainingesults:The mice in the BC and NCgroups showed no statistically significant difference from those in the HKTG groups in the body weight and daily body weight gain(P 0 05)Compared with the NC mice，the animals in the HKTG-1 group exhibited significantly increased testis weight(P 0 05)，and those in the HKTG-1 and HKTG-1 groups presented a large number of germ cells in the seminiferous tubules，including deformedsperm cells in the lumen，and some seminomatogonia in the seminogenic tubules，but almost no deformed sperm cells The expressionsof the total germ cell marker gene Ddx4，spermatogonial cell marker gene Dazl，spermatic cell marker gene Sycp3 and sperm cell mark-er gene Tnp1 were significantly upregulated(P 0 05)while that of the Sertoli cell marker gene Sox9 downregulated(P 0 05)inthe HKTG-1 group The number of Sertoli cells in the HKTG-1 group was remarkably reduced(P 0 05)，corresponding to the in-creased number of germ cells in the HKTG-1 group There were no significant changes in the relative expressions of the DDX4，Dazl，Sycp3 and Tnp1 genes，nor in the number of Sertoli cells in the HKTG-3 and HKTG-4 groups The expressions of meiosis-related genesMeioc，Stra8 and Spo11were markedly upreguated in the HKTG-1 group，indicating significantly improved spermatogenesis in the testistissue of the miceConclusion:HKTG improves the function of spermatogenic cells and increases sperm production in the testis tissue ofmice by promoting meiosis【Key words】Heirong kidney-tonifying granule;non-obstructive azoospermia;busulfan;mouseSupported by a grant from Sci-Tech Development Program of Jilin Province Specialized Project of Medicine and Health(20200708065YY)Correspondence to:ZHENG Lian-wen，email:362683485 qq comeceived:May 17，2022;accepted:October 9，2022全球范围内大约有 15%的夫妇受到不孕不育问题的困扰，而这些病例中有 20%70%是由男性因素造成的1。其中，在男性不育患者中无精子症所占比高达 11 2%2。现阶段，关于无精子症，特别是非梗阻性无精子症(non-obstruction azoosper-mia，NOA)的病因尚不明确，其治疗方法仍处于