日粮添加不同水平芦丁对热应激小鼠睾丸组织的影响

本试验旨在探讨日粮添加不同水平芦丁对缓解热应激小鼠睾丸组织损伤的影响。将30只5周龄体重相近（20～22 g）的ICR系雄性小鼠随机分为5组，各组小鼠分别饲喂基础日粮添加0（CON组）、0（HS组）、250（HS+R250）、500（HS+R500）和1 000（HS+R1000） mg·kg^-1芦丁的日粮，饲养10 d后，除对照组（CON）外，所有小鼠在每天10：00至14：00之间置于42℃恒温箱中连续处理8 d后屠宰取样分析。结果显示：1）与CON组相比，HS组小鼠睾丸指数无显著变化（P>0.05），而日粮添加250 mg·kg^-1芦丁显著提高热应激小鼠睾丸指数（P<0.05）；小鼠睾丸组织切片结果显示，与CON组相比，HS组小鼠生精小管直径和横截面积显著降低（P<0.05），生精细胞脱落比率显著增加（P<0.05）；与HS组相比，HS+R250组小鼠生精小管横截面积显著增加（P<0.05），生精细胞脱落比率显著降低（P<0.05），HS+R500组生精细胞脱落比率也显著降低（P<0.05），HS+R1000组小鼠生精小管直径显著增加（P<0.05）；小鼠睾丸指数、生精小管直径及生精小管脱落比率在芦丁处理组与CON组之间无显著差异（P>0.05），但HS+R250组小鼠睾丸生精小管横截面积显著高于CON组（P<0.05）。2）与CON组相比，HS组小鼠睾丸组织丙二醛（MDA）含量显著升高（P<0.05），总抗氧化能力（T-AOC）与还原型谷胱甘肽（GSH）含量以及血红素酶-1（HO-1） mRNA的表达量均显著降低（P<0.05）；日粮添加250 mg·kg^-1芦丁显著降低热应激小鼠睾丸组织中MDA含量（P<0.05），提高T-AOC与GSH含量（P<0.05），并增强核因子E2相关因子2（Nrf2）、HO-1和谷胱甘肽过氧化物酶（GSH-Px） mRNA的表达量（P<0.05），而添加500和1 000 mg·kg^-1芦丁也显著提高了GSH含量（P<0.05）；与CON组相比，除HS+R250组Nrf2 mRNA表达量显著高于CON组（P<0.05）外，芦丁组热应激小鼠睾丸组织抗氧化相应基因与酶活指标均无显著性变化（P>0.05）。3）热应激小鼠睾丸中核因子-κB （NF-κB）、TOLL样受体4（TLR-4）和Bax的mRNA表达量显著增加（P<0.05），而Bcl-2表达量显著降低（P<0.05）；日粮添加250 mg·kg^-1芦丁显著降低NF-κB、TLR-4、髓样分化因子88（MyD88）、白细胞介素-Iβ（IL-1β）和Bax mRNA表达量（P<0.05），增强Bcl-2的表达水平（P<0.05）；添加500 mg·kg^-1芦丁显著降低NF-κB和TLR-4表达量（P<0.05），而1 000 mg·kg^-1芦丁能够显著增加Bcl-2的表达（P<0.05）；小鼠睾丸组织中免疫和增值凋亡相关基因的表达量在添加芦丁组及CON组之间无显著性差异（P>0.05）。结果提示，日粮添加适宜剂量芦丁具有改善热应激小鼠睾丸组织形态及功能的效果，其机制可能与芦丁通过Nrf2信号通路缓解氧化应激，通过TLR-4/NF-κB信号通路抑制炎症反应及调控Bax/Bcl-2 mRNA的表达密切相关。本试验条件下日粮添加250 mg·kg^-1芦丁的效果较好。

Effects of Dietary Supplementation of Different Levels of Rutin on Testicular Tissue in Mice under Heat Stress

The purpose of this experiment was to explore the effects of dietary supplementation of different levels of rutin on alleviating testicular tissue damage in heat-stressed mice. Thirty 5-week-old male mice of ICR strain with similar body weight (20-22 g) were randomly divided into 5 groups. Mice in each group were fed basal diet supplemented with 0 (CON group), 0 (HS group), 250 (HS+R250), 500 (HS+R500) and 1 000 (HS+R1000) mg·kg^-1 rutin for 10 days, respectively. Except for control group (CON), all mice were treated in an incubator at 42℃ between 10:00 and 14:00 every day for 8 consecutive days, and then slaughtered for sampling and analysis. The results showed as follows:1) Compared with CON group, the testis index of mice in HS group had no significant change (P>0.05), but dietary 250 mg·kg^-1 rutin significantly increased the testis index of mice under heat stress (P<0.05). The results of mouse testicular tissue sections showed that compared with the CON group, the cross-sectional area and diameter of the seminiferous tubules in the HS group were significantly reduced (P<0.05), and the rate of spermatogenic cell shedding was significantly increased (P<0.05). Compared with the HS group, the cross-sectional area of the seminiferous tubules in the HS+R250 group was significantly increased (P<0.05), the rate of spermatogenic cells shedding was significantly reduced (P<0.05), while the rate of spermatogenic cell shedding in the HS+R500 group was significantly reduced (P<0.05), and the diameter of the seminiferous tubules in the HS+R1000 group was significantly increased (P<0.05); There was no significant difference in testis index, seminiferous tubule diameter and seminiferous tubule shedding ratio between the rutin-treated groups and the CON group (P>0.05), however, the cross-sectional area of the seminiferous tubules in the HS+R250 group was significantly higher than that in the CON group (P<0.05). 2) Compared with CON group, malondialdehyde (MDA) content in testis tissue of HS group increased significantly (P<0.05), while total antioxidant capacity (T-AOC), glutathione (GSH) content and the mRNA expression of hemolytic enzyme 1 (HO-1) in testis tissue of mice were significantly decreased (P<0.05). The supplementation of 250 mg·kg^-1 rutin significantly decreased the MDA content (P<0.05), increased the T-AOC and GSH content in testis of heat-stressed mice (P<0.05), and enhanced nuclear factor E2 related factors 2 (Nrf2), HO-1 and glutathione peroxidase (GSH-Px) mRNA expression (P<0.05), while the supplementation of 500 and 1 000 mg·kg^-1 rutin also significantly increased the GSH content (P<0.05); Compared with the CON group, except that the Nrf2 mRNA expression in the HS+R250 group was significantly higher than that in the CON group (P<0.05), there were no significant changes in the antioxidant genes and enzyme activity indexes in the testis tissue of the heat-stressed mice in the rutin groups. (P>0.05). 3) The mRNA expression levels of nuclear factor -κB (NF-κB), Toll-like receptor 4 (TLR-4) and Bax in testis of heat-stressed mice were significantly increased (P<0.05), while the expression level of Bcl-2 was significantly decreased (P<0.05). Dietary supplementation of 250 mg·kg^-1 rutin significantly reduced the mRNA expression of NF-κB, TLR-4, myeloid differentiation factor 88 (MyD88), interleukin-Iβ (IL-1β) and Bax (P<0.05), enhanced the expression level of Bcl-2 (P<0.05); Adding 500 mg·kg^-1 rutin to the diet significantly reduced the expression of NF-κB and TLR-4 (P<0.05), while adding 1 000 mg·kg^-1 rutin significantly increased the expression of Bcl-2 (P<0.05). There was no significant difference in the expression of immune and proliferation apoptosis-related genes in mouse testis between the rutin groups and the CON group (P>0.05). These results suggest that dietary supplementation with appropriate dose of rutin can improve the morphology and function of testis in heat-stress mice, and the mechanism may be closely related to the relief of oxidative stress through Nrf2 signaling pathway, the inhibition of inflammatory response through TLR-4/NF-κB signaling pathway and the regulation of Bax/Bcl-2 mRNA expression. The effect of dietary 250 mg·kg^-1 rutin was better under the experimental conditions.