杨梅黄酮对环磷酰胺致雄性小鼠生殖毒性的保护作用

Myricetin reduces the reproductive toxicity of cyclophosphamide in male mice

目的探究杨梅黄酮对环磷酰胺引起的雄性生殖毒性的影响及其作用机制。方法 6周龄雄性ICR小鼠30只,随机数字表法分为5组:空白对照组、环磷酰胺生殖损伤模型组、杨梅黄酮低中高剂量干预组。除空白对照组以外,其余各组每天腹腔注射环磷酰胺50 mg/kg,连续7 d,杨梅黄酮组自造模第2 d起每天分别灌胃100、200和400 mg/kg杨梅黄酮,连续30 d,空白对照组和模型对照组灌胃等体积0.25%羧甲基纤维素钠溶液。每3天称取体重一次,末次给药次日,颈椎脱臼处死小鼠,迅速取附睾、睾丸。睾丸称重,计算睾丸指数,附睾获取精子,精子分析仪分析精子密度及活力。免疫印迹法检测睾丸组织Bax以及Bcl-2表达情况,流式细胞术检测精子线粒体膜电位水平。结果造模9 d起,模型组小鼠体重低于空白对照组,差异有统计学意义(P<0.05),杨梅黄酮组与模型组相比无差异。模型组小鼠的睾丸指数为(3.93±0.91)mg/g,显著低于空白对照组(6.93±0.98)mg/g,差异有统计学意义(P<0.05),杨梅黄酮干预组睾丸指数上升,100、200和400 mg/kg组睾丸指数分别为(3.94±1.21)、(4.33±0.88)和(4.80±0.43) mg/g,其中200和400 mg/kg组与模型对照组相比,差异有统计学意义(P<0.05和P<0.01)。与对照组组相比,模型组小鼠精子密度、向前运动精子率、非向前运动精子率减少,不动精子率提高,杨梅黄酮组与模型组相比,精子密度、向前运动精子率、非向前运动精子率上升,不动精子率下降,200和400 mg/kg组差异均有统计学意义(P<0.05或P<0.01)。模型组小鼠精子线粒体膜电位水平正常率为(54.70±5.45)%, 100、200和400 mg/kg杨梅黄酮组精子线粒体膜电位正常率为(59.10±9.97)%、(62.10±6.07)%和(77.10±8.87)%,其中400 mg/kg组与模型组相比差异有统计学意义(P<0.05)。模型组Bax/Bcl-2比值为5.92±1.45,100、200和400 mg/kg杨梅黄酮组Bax/Bcl-2比值下降,分别为2.52±0.51、1.71±0.52、1.07±0.29,差异均有统计学意义(P<0.05或P<0.01)。结论杨梅黄酮可以通过改善精子线粒体膜电位水平,抑制睾丸细胞凋亡,对环磷酰胺引发的雄性小鼠生殖毒性起到保护作用。

OBJECTIVE To explore the effect of myrica flavone on male reproductive toxicity induced by cyclophosphamide and its mechanism. METHODS Thirty 6-week-old male ICR mice were randomly divided into 5 groups: blank control group, cyclophosphamide reproductive injury model group, myricetin low-medium high-dose intervention group. Except the blank control group, the other groups were intraperitoneally injected with cyclophosphamide 50 mg/kg daily for 7 consecutive days. The myricetin group received intragastric administration of 100, 200, and 400 mg/kg myricetin daily for 30 consecutive days since the second day of modeling. The blank control group and the model control group were given an equal volume of a 0.25% sodium carboxymethyl cellulose solution. The body weight was measured every 3 days, and the day after the last administration, the mice were sacrificed by cervical dislocation, and the epididymis and testes were quickly taken. Testicular weighing, testicular index calculation, epididymis to obtain sperm, sperm analyzer to analyze sperm density and vitality. The expression of Bax and Bcl-2 in testicular tissues was detected by immunoblotting, and the mitochondrial membrane potential of sperm was detected by flow cytometry. RESULTS After 9 days of modeling, the weight of mice in the model group was lower than that of the blank control group, which was statistically different(P<0.05). There was no difference between the myricetin treatment group and the model group. The testis index of the model group was(3.93±0.91)mg/g, which was significantly lower than that of the blank control group(6.93±0.98)mg/g, and the difference was statistically significant(P<0.05). After treatment with bayberry flavonoids, the testis index increased, in the 100 and 200 groups and 400 mg/kg testis index were(3.94±1.21) mg/g,(4.33±0.88) mg/g, and(4.80±0.43) mg/g, respectively. Compared with model control group, The difference was statistically significant(P<0.05 and P<0.01). Compared with the control group, the sperm density, sperm rate of forward movement, sperm rate of non-forward movement, and decreased sperm rate of non-moving sperm increased in the model group. After treatment with bayberry flavonoids, compared with the model group, the sperm density, sperm rate of forward motion, and sperm rate of non-forward motion increased, and the immobility sperm rate decreased. The 200 and 400 mg/kg groups had statistical significance(P<0.05 or P<0.01);the normal rate of sperm mitochondrial membrane potential in the model group was(54.70±5.45)%, and the normal mitochondrial membrane potential rate after treatment with myricetin of 100, 200 and 400 mg/kg(59.10±9.97)%,(62.10±6.07)% and(77.10±8.87)%, of which the 400 mg/kg group was statistically significant(P<0.05);the ratio of Bax/Bcl-2 in the model group was 5.92±1.45, and the ratio of Bax/Bcl-2 decreased after treatment with myricetin of 100, 200 and 400 mg/kg, which were 2.52±0.51,1.71±0.52 and 1.07±0.29. There were statistical differences(P<0.05 or P<0.01). CONCLUSION Myrica flavone can protect sperm mitochondrial membrane potential, inhibit testicular cell apoptosis, and protect the male mice from reproductive toxicity induced by cyclophosphamide.