补肾抗衰汤对氧化应激所致的秀丽隐杆线虫生育缺陷的影响

1、补肾抗衰汤对氧化应激所致的秀丽隐杆线虫生育缺陷的影响                            作者：曹新国, 侯莉莉, 陈军霞, 陆勤【摘要】  目的：探讨补肾抗衰汤对秀丽隐杆线虫因氧化应激诱导所致生育缺陷的作用。方法：将终浓度为0.33 g/mL的补肾抗衰汤按25%、50%、75%和100%梯度稀释后对L4期线虫

2、培养。应激方式采用紫外线照射20 J/(m2·min)、热休克（36 , 2 h）和百草枯(2 mmol/L, 2 h)处理。通过子宫内卵数、卵的大小和传代时间来评价秀丽隐杆线虫的生育能力。结果：野生型N2线虫经过紫外线照射、热休克和百草枯处理后，子宫内卵数、卵的大小均显著减少，传代时间明显增加；过氧化氢酶和过氧化物歧化酶活性被抑制。补肾抗衰汤对野生型线虫没有毒性或其他副作用。高浓度补肾抗衰汤不仅能够减轻由紫外线照射、热休克和百草枯所导致的生育缺陷，而且可以增加过氧化氢酶和超氧化物歧化酶被各种应激抑制的活性。此外，高浓度补肾抗衰汤可以明显恢复mev1突变体线虫受损的生育能力。 结论：

3、氧化应激对生育能力有负面影响，高浓度补肾抗衰汤可明显逆转氧化应激所致的线虫生育缺陷。 【关键词】  补肾抗衰汤; 氧化应激; 生育; 线虫The reproductive successes of animals and humans depend on a series of careful neuroendocrine events, and the reproduction failure will occur if the timing for any one of these events is disrupted1. Under such conditions, anim

4、al reproduction will be at risk due to the effects of stress, and the reproductive process controlled by the neuroendocrine system appears to be the most vulnerable1. For example, when dairy cattle are subjected to heat stress, reproductive efficiency declines. Cows under heat stress have reduced du

5、ration and intensity of estrus, altered follicular development, and impaired embryonic development2. Stress can be defined as an event (physical, environmental, psychological, etc.) which significantly challenges the homeostasis of the animals. In mammals, stress impacts on the reproductive axis at

6、the hypothalamus and the pituitary gland3. Various stressful conditions have adverse effects on reproduction of animals, and the stress conditions include environmental factors (i.e., temperature, humidity, metals and irradiation), physical factors (i.e., transport, shearing, restraint and strenuous

7、 exercise), metabolic factors (i.e., insulininduced hypoglycaemia), immunological factors (i.e., infection and endotoxins administration), and psychological factors (i.e., isolation, social interactions and mental arithmetic tasks)15.    Caenorhabditis elegans, a classical model organ

8、ism, is a free living nematode with a nervous system, specialized muscles, and digestive and reproductive systems6. The reproductive process has been investigated extensively in C.elegans79. C.elegans hermaphrodites are selffertile, and their reproductive process is modulated by diverse environmenta

9、l cues79. Among these environmental cues, environmental stress can affect the reproductive process of nematodes significantly. The data from Coohill et al10 showed the effects of ultraviolet (UV) irradiation on larval development and fertility. The reproductive process can also be altered by heat st

10、ress in nematodes11. Electromagnetic nanopulse exposure will be resulted in the decreased fertility of C. elegans by interfering with the fertilization or development12. In addition, exposure to heavy metals of lead, silver, nickel and zinc will cause severe defects of brood size and generation time

11、 in exposed nematodes1316.    Bushen Kangshuai Tang (BKT), a compound traditional Chinese herbal medicine, has been widely used for clinical treatment of premature ovarian failure (POF). This medicine can alleviate the inhibitory action of excessive androgen on ovarian granulosa cells

12、 and regulate the ovarian function by promoting follicular development, increasing the levels of estrogen and progestogen, and improving the ratio of estrogen/androgen. In present study, we provided evidences for the retrieval effect of this medicine on oxidative stressinduced reproductive defects u

13、sing a C. elegans model. Three parameters had been selected to evaluate the reproductive process, which were eggs in uterus, brood size, and generation time.    1  Materials and methods    1.1  Materials    1.1.1  Preparation of BKT and che

14、micals  BKT consists of nine medicinal herbs: Semen Cuscutae (Tusizi) 15 g, Rhizoma Polygonati Sibirici (Huangjing) 15 g, prepared Radix Rehmanniae (Shudihuang) 15 g, Herba Cynomorii Songarici (Roucongrong) 10 g, Radix Morindae Officinalis (Bajitian) 10 g, Radix Angelicae Sinensis (Danggui) 10

15、g, Rhizoma Chuanxiong (Chuanxiong) 6 g, Fluoritum (Zishiying) 15 g, and Fructus Schisandrae Chinensis (Wuweizi) 6 g, which were purchased from Pharmacy of Nanjing Maternity and Child Health Care Hospital. The herbs were soaked in 2 000 mL distilled water (DW) for 2 h, and then boiled for 60 min for

16、the first decoction; the second and third decoctions were obtained by adding DW to 1 000 mL each and then boiled for 1 h. After being mixed, the decoction was condensed with water bath to crude drug content 0.33 g/mL and stored at 4 until use. All other chemicals were obtained from SigmaAldrich (St.

17、 Louis, MO, USA).    1.1.2  C.elegans model  All experiments were performed on hermaphrodite C. elegans. The strains used in the current study were wildtype N2, and mutant of mev1 (kn1), originally obtained from the Caenorhabditis Genetics Center (Minneapolis, MN, USA). They

18、 were maintained on nematode growth medium (NGM) plates and seeded with Escherichia coli OP50 at 20 as described by Brenner17. Agesynchronous populations of L2 or L4larva stage nematodes were obtained by collection as described18. The L2 or L4larva stage nematodes were washed with doubledistilled wa

19、ter twice, followed by washing with modified K medium once (50 mmol/L NaCl, 30 mmol/L KCl, 10 mmol/L NaOAc, pH 5.5)19. Exposures were performed in sterile culture plates (glass) for 2 h on L4larva stage nematodes with 25%, 50%, 75%, and 100% of examined BKT diluted with M9 buffer. Experiments of all

20、 concentrations were repeated 3 times respectively. Approximately 100 nematodes were transferred in 100 L to each exposure solution in the micropipetter. All exposures were carried out in 20 incubator for 2 h in the absent of food. All the controls are wildtype C. elegans without BKT administration.

21、    1.2  Methods    1.2.1  Brood size assay  The procedure was performed as previously described15. Briefly, brood size was assayed by placing exposed or control nematodes onto individual culture plates. The examined nematodes were transferred four times

22、to a new well every 1.5 days, and the total number of eggs released on the plates was scored. At least 20 replicates were performed for statistical purposes.    1.2.2  Eggs in uterus  To score the number of eggs in the uterus, the treated or control adult nematodes were indi

23、vidually transferred to a drop of solution containing commercial bleach and 1 mol/L NaOH on a glass slide covered with an agar pad. The bleach solution dissolved the body of the adult nematodes, and eggs were scored immediately under digit optics with a Zeiss microscope.    1.2.3 

24、; Generation time assay  The experiment was performed as previously described15. The generation time refers to the time from day of birth (P0) egg to the first filial generation (F1) egg. At least 20 replicates were performed for statistical purposes.    1.2.4  UV irradiatio

25、n experiments  Approximately 30 L2stage larvae were irradiated on NGM plates without food at 20 J/(m2·min) by using a germicidal bulb (254 nm) for 2 hours as described by Murakami et al20. All UV irradiation assays were performed at 20 , and afterwards further maintained at 20 . All assays

26、 were replicated more than three times.    1.2.5  Heatshock experiments  Approximately 30 L2stage larvae grown at 20 were heatstressed for 2 h at 36 and afterwards further maintained at 20 . All assays were replicated more than three times.    1.2.6  Para

27、quat treatment  Approximately 30 L2stage larvae were treated with 2 mmol/L paraquat solution for 2 h at 20 . All assays were replicated more than three times.    1.2.7  Superoxide dismutase and catalase activities  The treated or control adult nematodes were used for th

28、e assay of superoxide dismutase (SOD) and catalase (CAT) activities as previously described21. The CAT activity was estimated following the method of Abei22. The decrease in absorption was measured spectrophotometrically at 240 nm. An extinction coefficient of 43.6/(mol·cm) was used to determin

29、e the enzyme activity. The SOD activity was measured by using the kit from Randox Laboratories following the manufacturers protocol. The data were the summary of three trials.    1.3  Statistical analysis  All data in this article were expressed as x±s. Oneway analysis

30、of variance (ANOVA) followed by a Dunnetts t test was used to determine the significant differences between the groups. P value 0.05 was considered statistical difference, P value 0.01 was considered statistical significant difference.    2  Results    2.1  Ad

31、ministration of BKT did not affect the reproduction of wildtype N2 nematodes  We first investigated the effects of BKT administration on the reproductive process of wildtype N2 nematodes. The egg number in uterus, brood size, and generation time were examined after 2 h administration of 25%, 50

32、%, 75%, and 100% BKT on L4larvae. As shown in Figure 1, no significant increases or decreases of egg number in uterus, brood size, and generation time could be observed in nematodes after exposed to different concentrations of BKT as compared with the control. Usually, egg number in uterus and brood

33、 size reflect the reproductive capacity, whereas a longer generation time indicates a low reproductive speed. Therefore, administration of BKT did not enhance or suppress the reproductive process under wildtype background in C. elegans. That is, our data suggest that administration of BKT did not ca

34、use toxic or altered effects on animals reproduction.    2.2  Effects of administration of BKT on reproductive defects induced by UV irradiation in C. elegans  In C. elegans, UV irradiation, heatshock, and paraquat treatment can cause oxidative stress2326. Considering the po

35、ssibility that the oxidative stress may negatively regulate the reproductive process, we next examined whether the administration of BKT could retrieve the reproductive defects induced by UV irradiation. As shown in Figure 2, after UV irradiation at 20 J/(m2·min), the egg number in uterus and b

36、rood size of exposed nematodes were significantly decreased as compared with the control (P<0.01), while the generation time was significantly increased as compared with the control (P<0.01). Again, administration of 75% and 100% of BKT would markedly increase the egg number in uterus (75%, P&

37、lt;0.05; 100%, P<0.01) and brood size (P<0.01), and reduce the generation time (75%, P<0.05; 100%, P<0.01) as compared with UV irradiation. However, administration of BKT at 25% and 50% could only moderately alter the reproductive process in UVirradiated nematodes. In addition, the defec

38、ts of egg number in uterus, brood size and generation time induced by UV irradiation could be effectively alleviated by administration of 75% of BKT. The defects of egg number in uterus, brood size, and generation time induced by UV irradiation could be effectively alleviated by administration of 10

39、0% of BKT. Therefore, administration of BKT at higher concentrations could largely retrieve the reproductive defects induced by UV irradiation in C. elegans.    2.3  Effects of administration of BKT in reproductive defects induced by heatshock in C. elegans  We further inves

40、tigated whether the administration of BKT could also largely retrieve the reproductive defects induced by heatshock. As shown in Figure 3, heatshock treatment at 36 for 2 h on L2stage larvae decreased the egg number in uterus and brood size, and increased generation time as compared with the control

41、 (P<0.01). Following heatshock treatment, administration of high concentrations (75% and 100%) of BKT to C. elegans resulted in significant decreases of egg number in uterus and brood size, and increase of generation time as compared with heatshock treatment only (75%, P<0.05; 100%, P<0.01)

42、. In contrast to this, administration of 25% and 50% of BKT would not significantly alter the egg number, brood size, and generation time of heatshocktreated nematodes. Therefore, administration of 75% and 100% of BKT could also largely recover the reproductive defects formed in heatshock treated ne

43、matodes.    2.4  Effects of administration of BKT on reproductive defects induced by paraquat treatment in C. elegans  Paraquat treatment causes oxidative stress through a metabolically catalyzed reaction to result in depletion of cellular nicotinamideadenine dinucleotide ph

44、osphate (NADPH) and production of ROS, primarily superoxide anions27. As shown in Figure 4, treatment with 2 mmol/L paraquat solution caused obvious reduction of egg number and brood size and elongation of generation time as compared with the control (P<0.01). Following paraquat treatment, admini

45、stration of 75% and 100% of BKT could significantly alleviate the defects of egg number in uterus (75%, P<0.05; 100%, P<0.01), brood size (P<0.01), and generation time (P<0.01) induced by paraquat exposure, whereas administration of 25% and 50% of BKT would not have obvious effects on th

46、e reproductive process in paraquattreated nematodes. Moreover, the defects of egg number in uterus, brood size, and generation time induced by paraquat treatment could be effectively alleviated by administration of 75% of BKT by approximately 65.2%, 55.4%, and 62.5%, respectively. In addition, the d

47、efects of egg number in uterus, brood size, and generation time induced by paraquat treatment could be effectively alleviated by administration of 100% of BKT by approximately 83.5%, 77.2%, and 81.6%, respectively. Therefore, administration of high concentrations of BKT would largely retrieve the re

48、productive defects formed in paraquattreated nematodes.    2.5  Administration of BKT after stress exposure altered the CAT and SOD activities in C. elegans  CAT, acting in concert with SOD, belongs to the major defense enzymes against superoxide radicals, and activities of

49、CAT and SOD are directly linked to oxidative stress28, 29. To examine whether the retrieval effects of BKT on reproductive defects induced by UV irradiation, heatshock, and paraquat treatment are through alleviating the damage from oxidative stress, we further investigated the activity changes of CA

50、T and SOD in stressexposed and BKTadministrated nematodes. As shown in Figure 5, UV irradiation, heatshock, and paraquat treatment all caused significant decreases of CAT and SOD activities as compared with the control, suggesting the occurrence of severe damage from oxidative stress. Moreover, admi

51、nistration of 50%, 75%, and 100% of BKT could significantly increase the CAT and SOD activities of UVirradiated (CAT: 50%, P<0.05; 75% and 100%, P<0.01. SOD: P<0.01), heatshocktreated (CAT: P<0.01. SOD: 50%, P<0.05; 75% and 100%, P<0.01), and paraquattreated (CAT: P<0.01; SOD: P

52、<0.01) nematodes as compared with those in stressexposed nematodes without BKT administration. Furthermore, the decreases of CAT activity in UVirradiated, heatshocktreated and paraquattreated nematodes could be effectively alleviated by administration of 50% BKT by approximately 27.1%, 41.5%, and

53、 38.2%, respectively, and the decreases of SOD activity in UVirradiated, heatshocktreated, and paraquattreated nematodes could be effectively recovered by administration of 50% BKT by approximately 44.6%, 39.3%, and 42.6%, respectively. The decreases of CAT activity in UVirradiated, heatshock and pa

54、raquattreated nematodes could be effectively alleviated by administration of 75% of BKT by approximately 47.9%, 56.9%, and 58.3%, respectively, and the decreases of SOD activity in UVirradiated, heatshocktreated, and paraquattreated nematodes could be effectively recovered by administration of 75% B

55、KT by approximately 64.6%, 66.4%, and 65.9%, respectively. In addition, the decreases of CAT activity in UVirradiated, heatshocktreated, and paraquattreated nematodes could be effectively alleviated by administration of 100% of BKT by approximately 63.5%, 71.3%, and 79.4%, respectively, and the decr

56、eases of SOD activity in UVirradiated, heatshocktreated, and paraquattreated nematodes could be effectively recovered by administration of 100% of BKT by approximately 83.6%, 84.2%, and 82.2%, respectively. Therefore, administration of high concentrations of BKT could largely recover the reproductiv

57、e defects induced by UV irradiation, heatshock and paraquat treatment by alleviating the damage from the oxidative stress.    2.6  Administration of BKT largely rescued the reproductive defects formed in mev1 mutant nematodes  mev1 encodes a subunit of succinatecoenzyme Q ox

58、idoreductase in complex of the electron transport chain, and mutation of this gene will cause elevated levels of superoxide and short lifespan30. Mutation of mev1 results in several phenotypes consistent with elevated oxidative stress such as sensitivity to the superoxide generator paraquat or high

59、oxygen, which provides a useful tool for testing compounds or medicines for antioxidant properties31, 32. As shown in Figure 6, mutation of mev1 also caused severe reproductive defects with significant reduction of egg number in uterus and brood size, and elongation of generation time as compared wi

60、th wildtype N2 nematodes (P<0.01), indicating the important role of elevated levels of superoxide in inducing reproductive defects. In addition, we observed that administration of 50%, 75%, and 100% of BKT all would noticeably increase the egg number in uterus and brood size or shorten the genera

61、tion time of mev1 mutant nematodes as compared with those in mev1 mutant nematodes without BKT administration (P<0.01), whereas administration of 25% of BKT would not obviously affect the reproductive phenotypes of mev1 mutant nematodes. Moreover, the egg number in uterus of mev1 mutant nematodes could be recovered by 41.2%, 70.1%, and 89.3%, respectively, after administration of 50% BKT, and the brood size of mev1 mutant nematodes could be alleviated by 38.4%, 69.4%, and 90.7%, respectively, after administration of 75% BKT, and the generation time of mev1 mutant nematodes