Yang Chih-Chao, Chen Yen-Ta, Chen Chih-Hung, Chiang John Y, Zhen Yen-Yi, Yip Hon-Kan
Division of Nephrology, Department of Internal Medicine, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of MedicineKaohsiung 83301, Taiwan, R.O.C.
Division of Urology, Department of Surgery, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of MedicineKaohsiung 83301, Taiwan, R.O.C.
Am J Transl Res. 2017 Dec 15;9(12):5275-5288. eCollection 2017.
Microtubules, maintaining a non-linear structure, are suitable for direct observation in living mammalian by second-harmonic imaging microscopy (SHIM) (a new kind of confocal microscopies). Testes constituted by vast seminiferous microtubules (SM), serve as good candidates for visualization by SHIM. This study employs the SHIM and Western-blot (WB) to assess the cellular-molecular levels of doxorubicin (Dox)-induced mouse testicular damage. The SHIM examination was able to clearly identify the integrity of normal architecture of the living mouse testis, namely, the anatomical features of SM, smooth muscle wall of SM, manchette microtubules, exoplasmic microtubules in Sertoli cells and interstitial connective tissue, as well as the destructive feature of SM in Dox-treated mice (n = 6 per group). By day 21 after Dox-treatment, the testicular weight and testicular length were significantly progressively decreased as Dox dosage was stepwise increased, i.e., 0/5/10/15/20 mg/kg/body-weight (BW) (all p<0.0001). The cross-section area of SM was significantly lower in Dox-treated (15 mg/kg-BW) mice than that in controls (p<0.001). The protein expression of vimentin was significantly progressively increased whereas the protein expression of β-tubulin/androgen-receptor was significantly progressively decreased in stepwise increased Dox dosage (all p<0.001). The protein expressions of inflammatory (MMP-9/IL-1β/TNF-α/iNOX), oxidative-stress (NOX-1/NOX-2/NOX-4/oxidized protein), apoptotic (mitochondrial-Bax/cleaved-caspase-3/PARP), fibrotic (Smad3/TGF-ß) mitochondrial/DNA-damaged (cytosolic cytochrome-C/γ-H2AX/ATM/KU70), and cell apoptotic/death (PTEN/p53) biomarkers were significantly higher in Dox-treated (15 mg/kg-BW) group than those in controls (all p<0.001). In conlusion, the dose-dependent Dox-caused mouse testicular damage can be not only detected by WB in molecular level but also clearly identified by SHIM in living mice.
微管保持非线性结构，适合通过二次谐波成像显微镜（SHIM，一种新型共聚焦显微镜）在活体哺乳动物中进行直接观察。由大量生精微管（SM）构成的睾丸是通过SHIM进行可视化观察的良好候选对象。本研究采用SHIM和蛋白质免疫印迹法（WB）来评估阿霉素（Dox）诱导的小鼠睾丸损伤的细胞分子水平。SHIM检查能够清晰地识别活体小鼠睾丸正常结构的完整性，即SM的解剖特征、SM的平滑肌壁、袖套微管、支持细胞中的外质微管和间质结缔组织，以及Dox处理小鼠（每组n = 6）中SM的破坏特征。在Dox处理后第21天，随着Dox剂量逐步增加，即0/5/10/15/20毫克/千克体重（BW），睾丸重量和睾丸长度显著逐渐降低（所有p<0.0001）。Dox处理（15毫克/千克体重）小鼠的SM横截面积显著低于对照组（p<0.001）。随着Dox剂量逐步增加，波形蛋白的蛋白表达显著逐渐增加，而β-微管蛋白/雄激素受体的蛋白表达显著逐渐降低（所有p<0.001）。炎症（基质金属蛋白酶-9/白细胞介素-1β/肿瘤坏死因子-α/诱导型一氧化氮合酶）、氧化应激（烟酰胺腺嘌呤二核苷酸磷酸氧化酶-1/烟酰胺腺嘌呤二核苷酸磷酸氧化酶-2/烟酰胺腺嘌呤二核苷酸磷酸氧化酶-4/氧化蛋白）、凋亡（线粒体- Bax/裂解的半胱天冬酶-3/聚（ADP-核糖）聚合酶）、纤维化（Smad3/转化生长因子-β）、线粒体/DNA损伤（细胞质细胞色素C/γ-H2AX/毛细血管扩张性共济失调突变基因/库欣综合征70）以及细胞凋亡/死亡（磷酸酶和张力蛋白同源物/p53）生物标志物的蛋白表达在Dox处理（15毫克/千克体重）组中显著高于对照组（所有p<0.001）。总之，剂量依赖性的Dox引起的小鼠睾丸损伤不仅可以通过WB在分子水平上检测到，还可以通过SHIM在活体小鼠中清晰地识别出来。
