拟除虫菊酯生殖和神经毒性及补肾健脾中药干预作用的研究
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摘要
目的
1.以溴氰菊酯染毒妊娠大鼠模型为实验对象,观察、分析拟除虫菊酯对母体妊娠及子代生长发育和生殖能力的影响,评估拟除虫菊酯所致妊娠缺失、胚胎缺陷情况。并初步揭示其机理。
2.通过研究补肾健脾复方助孕3号对溴氰菊酯所致妊娠缺失、胚胎缺陷的干预作用,评估中药对拟除虫菊酯生殖毒性的防治作用,并初步阐明作用机理。
3.从另一角度验证中药复方的母胎保护作用,为中药制剂在母胎保护方面的推广应用提供理论依据,促进中医药在妊娠期母胎保护和优生优育等方面的推广应用。
方法
1.溴氰菊酯对母鼠妊娠的影响及中药干预作用研究:以72只成年健康妊娠的SD大鼠为研究对象。分6组,其中模型组、模型+中药高剂量组、模型+中药中剂量组、模型+中药低剂量组、模型+牛磺酸组共5组自妊娠第1天至第15天经口灌服1/20LD_(50)(即6.93mg/kg)溴氰菊酯,每日1次,染毒4—6小时后分别灌服生理盐水、相当于成人等效剂量或其2倍、4倍的中药药液及牛磺酸溶液(0.2克/kg)。另一组为空白对照组。妊娠中期(第15天)杀死半数母鼠,余下一半继续妊娠至分娩。观察母鼠的一般情况、母鼠体重增长情况、妊娠情况、分娩情况,以及脾、卵巢、子宫等脏器系数、胎鼠数量及发育情况,并在孕中期及分娩后采血检测β—内啡肽、内分泌激素E2、P和细胞因子IFN—β、IL-2、IL-4、IL-10等指标。
2.溴氰菊酯对胎鼠发育的影响及中药干预作用:以妊娠第15天处死之母鼠子宫内的胎鼠为研究对象。观察胎仔外形有无畸形;胎鼠总数、吸收胎数、活胎数,计算活胎率;测定胎鼠体重、身长、尾长,并取部分胎鼠用荧光定量PCR检测胚胎总Pax3和Cx43mRNA,用免疫组化法测胎鼠脑Cx43蛋白水平。
3.溴氰菊酯对仔鼠生长发育的影响及中药干预作用:以实验各组母鼠分娩的仔鼠为研究对象。观察并比较仔鼠出生时及出生后同一时间点发育情况、仔鼠性别比例、出生存活率和哺育存活率;观察并记录出毛、张耳、睁眼、睾丸下降、
Objective1. The study employed deltamethrin (DM) to jeopardize pregnant rats modeling as the experimental object. It was observed and analyzed about how pyrethroid (Py) affected the pregnant SD rat itself, the filial generation and the reproductive capability. In prospect to those outcomes, we could evaluate pyrethroid based on the outcome of pregnancy and embryonic defect. In addition, the study could explore the mechanism of action towards reproductive system.2. The study used Formula Zhuyun III decoction as the treatment to benefit kidney and spleen. It applied deltamethrin to interfere abortion and embryonic defect in order to theorize the precaution and protection of Chinese medicine and its mechanism of action.3. On the other hand, the study could assess Chinese herbal combined decoction to act on mother-infant protection. It also provided a theoretical evidence emphasizing Chinese medicine approaching reproductive science.Method1. The experimental study of Chinese herbal medicine affecting pregnant rats being poisoned by deltamethrin (DM) with toxic response: 72 SD rats were divided into six groups including (1) model group, (2) model
plus high dose treatment, (3) model plus middle dose treatment, (4) model plus low dose treatment, (5) model plus taurine , (6) Blank control group. Group 1-5 were orally fed l/20LD_50 (6. 93mg/kg) DM daily from Day 1 to 15 when proved pregrancy. Then, physiological saline was given after being poisoned after 4 to 6 hours. The quantities were 4 times, 2 times, 1 time of adult dosage of Formula Zhuyun III and taurine (0. 2g/kg) . Group 6 was blank control. Half pregnant rats were killed on the fifteenth day. The rest was kept until labour. The experiments were to observe their general condition, weight, pregnancy symptoms, spleen, ovary and uterus, number of embryo and its development. Blood tests were carried out in the mid stage of pregnancy and delivery respectively to generate data of β — endorphone, estradiol, prostaglandin and IFN— γ, IL-2 IL-4 IL-10.2. The experimental study of Chinese herbal medicine to affecting embryonic development whilst maternal rats being poisoned by deltamethrin (DM): The embryos were taken from the fifteenth day pregnant rats. The study observed the embryonic shape and defect; weight, length, tail, and RNA isolation and PCR test of Pax3 and Cx43mRNA. Embryonic brains were examined by Cx43 protein test under immunofluorescence test.3. The experimental study of Chinese herbal medicine affecting infant' s growth whilst maternal rats being poisoned by deltamethrin (DM): The infant rats were observed with their growing conditions, sex proportion, life and death rate. The growing conditions included fur growth, ears and eyes opening, testis and vaginal opening for assessment. Forearm reaction, turn-around reaction, up and down reaction were also being assessed. The observation also tested learning skill and memory ability.
4. The experimental study of deltamethrin (DM) affecting the level of brain NO concentration and NOS of the infant rats: On the fourth day and tenth day afer the infant rats were given birth, 12 infant rats were randomly drawn from each group, devitalized. Then, the whole brain was taken, the blood was bleached and the meninx was peeled, physiological saline was applied to bend and grind 10% of brain tissue, estimated NO level and NOS activation was estimated.5. The experimental study of Chinese herbal medicine affecting genital system and the level of endocrine: On the sixtieth day afer the infant rats were given birth, (1) six male rats were taken randomly and six female rats in anestrus were taken by using vaginal test. Sera FSh\ LH T of male were examined. E_2 P FSH LH of female were examined; (2) genital organ was examined and weighed including testis, ovary and uterus; (3) applied Blazak method to obtain sperm count and sperm growth rate (4) immunofluorescence method was applied to assess ovarian protein expression.Results1. Pregnant rat could not find any sign and toxic symptom duringearly/middle pregnancy after being poisoned by l/20LDM deltamethrin (DM).2. With respect to abortion rate, model group showed statistical significance when compared with other groups. Model group also showed progesterone level was low, Thl/Th2 came closely to Thl type. Formula Zhuyun DI is a formula to enhance kidney and spleen. With the treatment Formula Zhuyun HI, it had desirable alleviating effect in abortion rate, higher level of progesterone, and desirable effect to regulate DM preventing from deviation of Thl and TH2. Taurine Group maintained high rate of abortion, but progesterone could not remain high level. The result demonstrated that DM had toxicity on reproduction, it could lead
to abortion which was related to endocrine and immune function for pregnant rats. Formula Zhuyun III could regulate the above condition and relief DM toxicity. Taurine could not influence DM to prevent abortion.3. The labour time of all groups was within the normal range. The study showed no significant difference in DM factor.4. Growing condition after being poisoned by DM: Growing rate dropped, length, weight and tail significantly fell. It implied that DM toxicity might transfer to embryo through placenta to induce the side effect. Regarding the rate of life, middle and high dosages were high, high dosage found significant difference in their health condition. However, there was no difference in low dosage. It showed that the positive correlation existed.5. Brain tissue of Cx43 protein expression was stronger when being poisoned by DM. The result indicated that there was positive correlation according to different concentration of Formula Zhuyun III. The higher the concentration, the higher efficiency. Cx43 protein expression fell in taurine and blank control group. The level of Pax3mRNA and Cx43mRNA of embryonic could not show the same trend as the Cx43 protein expression of brain tissue.6. Health condition: all groups were dysfunctioned by DM except blank control group. It implied that DM could affect infant rats' development.7. The early development of infant rats reached to normal growth. But the fur, open ears, male testis drop of blank control group was better than other group.8. Compared to the blank control group, the sex proportion, life rate of all group poisoned by DM had no significant difference.9. DM could affect NOS activation and NO level or infant rats. The brain
NOS activation and NO level of the fourth day infant rats in all group poisoned by DM were much higher than blank control group. However, there was no neural toxic symptoms and signs.10. Pregnant rats contacted DM would affect the filial generation which was reflected through endocrine. There showed that male infant rats were affected more obvious relatively. To male, the level of estosterone and production of sperm fell. To female, uterus deficiency was discovered. There implied that endocrine might be affected by DM. Formula Zhuyun III could improve the drawback through the regulation of endocrine.Conclusion1. DM is a typical medicine which proved its reproductive toxicity leadingto pregnant defect and abortion. It could affect the embryo and its development. The endocrine of pregnant rats was affected. However, no neurotoxicity of DM could be found regarding the filial generation.2. From the view point of Traditional Chinese Medicine, DM , one of the factors in the environmental pollution, was treated as a kind of toxin. It could affect the embryo and pregnant mother among dysfunction of organ, imbalance of Yin-yan and damage of reproductive system which could lead to abortion. The infant might lead to deficiency of genetic factor and slow development.3. Formula Zhuyun HI strengthened kidney and spleen to resist DM toxicity. Thus, the abortion rate fell obviously. It could also accelerate the infant development and the reproductive performance of filial generation. It might be caused by the regulation of endocrine and immune system. According to the results, we believe that the medicine with strengthening function of kidney and spleen had a great potential to interfere ECDs reproductive toxicity and to avoid pregnant abortion and embryonic defect.
1.以溴氰菊酯染毒妊娠大鼠模型为实验对象,观察、分析拟除虫菊酯对母体妊娠及子代生长发育和生殖能力的影响,评估拟除虫菊酯所致妊娠缺失、胚胎缺陷情况。并初步揭示其机理。
2.通过研究补肾健脾复方助孕3号对溴氰菊酯所致妊娠缺失、胚胎缺陷的干预作用,评估中药对拟除虫菊酯生殖毒性的防治作用,并初步阐明作用机理。
3.从另一角度验证中药复方的母胎保护作用,为中药制剂在母胎保护方面的推广应用提供理论依据,促进中医药在妊娠期母胎保护和优生优育等方面的推广应用。
方法
1.溴氰菊酯对母鼠妊娠的影响及中药干预作用研究:以72只成年健康妊娠的SD大鼠为研究对象。分6组,其中模型组、模型+中药高剂量组、模型+中药中剂量组、模型+中药低剂量组、模型+牛磺酸组共5组自妊娠第1天至第15天经口灌服1/20LD_(50)(即6.93mg/kg)溴氰菊酯,每日1次,染毒4—6小时后分别灌服生理盐水、相当于成人等效剂量或其2倍、4倍的中药药液及牛磺酸溶液(0.2克/kg)。另一组为空白对照组。妊娠中期(第15天)杀死半数母鼠,余下一半继续妊娠至分娩。观察母鼠的一般情况、母鼠体重增长情况、妊娠情况、分娩情况,以及脾、卵巢、子宫等脏器系数、胎鼠数量及发育情况,并在孕中期及分娩后采血检测β—内啡肽、内分泌激素E2、P和细胞因子IFN—β、IL-2、IL-4、IL-10等指标。
2.溴氰菊酯对胎鼠发育的影响及中药干预作用:以妊娠第15天处死之母鼠子宫内的胎鼠为研究对象。观察胎仔外形有无畸形;胎鼠总数、吸收胎数、活胎数,计算活胎率;测定胎鼠体重、身长、尾长,并取部分胎鼠用荧光定量PCR检测胚胎总Pax3和Cx43mRNA,用免疫组化法测胎鼠脑Cx43蛋白水平。
3.溴氰菊酯对仔鼠生长发育的影响及中药干预作用:以实验各组母鼠分娩的仔鼠为研究对象。观察并比较仔鼠出生时及出生后同一时间点发育情况、仔鼠性别比例、出生存活率和哺育存活率;观察并记录出毛、张耳、睁眼、睾丸下降、
Objective1. The study employed deltamethrin (DM) to jeopardize pregnant rats modeling as the experimental object. It was observed and analyzed about how pyrethroid (Py) affected the pregnant SD rat itself, the filial generation and the reproductive capability. In prospect to those outcomes, we could evaluate pyrethroid based on the outcome of pregnancy and embryonic defect. In addition, the study could explore the mechanism of action towards reproductive system.2. The study used Formula Zhuyun III decoction as the treatment to benefit kidney and spleen. It applied deltamethrin to interfere abortion and embryonic defect in order to theorize the precaution and protection of Chinese medicine and its mechanism of action.3. On the other hand, the study could assess Chinese herbal combined decoction to act on mother-infant protection. It also provided a theoretical evidence emphasizing Chinese medicine approaching reproductive science.Method1. The experimental study of Chinese herbal medicine affecting pregnant rats being poisoned by deltamethrin (DM) with toxic response: 72 SD rats were divided into six groups including (1) model group, (2) model
plus high dose treatment, (3) model plus middle dose treatment, (4) model plus low dose treatment, (5) model plus taurine , (6) Blank control group. Group 1-5 were orally fed l/20LD_50 (6. 93mg/kg) DM daily from Day 1 to 15 when proved pregrancy. Then, physiological saline was given after being poisoned after 4 to 6 hours. The quantities were 4 times, 2 times, 1 time of adult dosage of Formula Zhuyun III and taurine (0. 2g/kg) . Group 6 was blank control. Half pregnant rats were killed on the fifteenth day. The rest was kept until labour. The experiments were to observe their general condition, weight, pregnancy symptoms, spleen, ovary and uterus, number of embryo and its development. Blood tests were carried out in the mid stage of pregnancy and delivery respectively to generate data of β — endorphone, estradiol, prostaglandin and IFN— γ, IL-2 IL-4 IL-10.2. The experimental study of Chinese herbal medicine to affecting embryonic development whilst maternal rats being poisoned by deltamethrin (DM): The embryos were taken from the fifteenth day pregnant rats. The study observed the embryonic shape and defect; weight, length, tail, and RNA isolation and PCR test of Pax3 and Cx43mRNA. Embryonic brains were examined by Cx43 protein test under immunofluorescence test.3. The experimental study of Chinese herbal medicine affecting infant' s growth whilst maternal rats being poisoned by deltamethrin (DM): The infant rats were observed with their growing conditions, sex proportion, life and death rate. The growing conditions included fur growth, ears and eyes opening, testis and vaginal opening for assessment. Forearm reaction, turn-around reaction, up and down reaction were also being assessed. The observation also tested learning skill and memory ability.
4. The experimental study of deltamethrin (DM) affecting the level of brain NO concentration and NOS of the infant rats: On the fourth day and tenth day afer the infant rats were given birth, 12 infant rats were randomly drawn from each group, devitalized. Then, the whole brain was taken, the blood was bleached and the meninx was peeled, physiological saline was applied to bend and grind 10% of brain tissue, estimated NO level and NOS activation was estimated.5. The experimental study of Chinese herbal medicine affecting genital system and the level of endocrine: On the sixtieth day afer the infant rats were given birth, (1) six male rats were taken randomly and six female rats in anestrus were taken by using vaginal test. Sera FSh\ LH T of male were examined. E_2 P FSH LH of female were examined; (2) genital organ was examined and weighed including testis, ovary and uterus; (3) applied Blazak method to obtain sperm count and sperm growth rate (4) immunofluorescence method was applied to assess ovarian protein expression.Results1. Pregnant rat could not find any sign and toxic symptom duringearly/middle pregnancy after being poisoned by l/20LDM deltamethrin (DM).2. With respect to abortion rate, model group showed statistical significance when compared with other groups. Model group also showed progesterone level was low, Thl/Th2 came closely to Thl type. Formula Zhuyun DI is a formula to enhance kidney and spleen. With the treatment Formula Zhuyun HI, it had desirable alleviating effect in abortion rate, higher level of progesterone, and desirable effect to regulate DM preventing from deviation of Thl and TH2. Taurine Group maintained high rate of abortion, but progesterone could not remain high level. The result demonstrated that DM had toxicity on reproduction, it could lead
to abortion which was related to endocrine and immune function for pregnant rats. Formula Zhuyun III could regulate the above condition and relief DM toxicity. Taurine could not influence DM to prevent abortion.3. The labour time of all groups was within the normal range. The study showed no significant difference in DM factor.4. Growing condition after being poisoned by DM: Growing rate dropped, length, weight and tail significantly fell. It implied that DM toxicity might transfer to embryo through placenta to induce the side effect. Regarding the rate of life, middle and high dosages were high, high dosage found significant difference in their health condition. However, there was no difference in low dosage. It showed that the positive correlation existed.5. Brain tissue of Cx43 protein expression was stronger when being poisoned by DM. The result indicated that there was positive correlation according to different concentration of Formula Zhuyun III. The higher the concentration, the higher efficiency. Cx43 protein expression fell in taurine and blank control group. The level of Pax3mRNA and Cx43mRNA of embryonic could not show the same trend as the Cx43 protein expression of brain tissue.6. Health condition: all groups were dysfunctioned by DM except blank control group. It implied that DM could affect infant rats' development.7. The early development of infant rats reached to normal growth. But the fur, open ears, male testis drop of blank control group was better than other group.8. Compared to the blank control group, the sex proportion, life rate of all group poisoned by DM had no significant difference.9. DM could affect NOS activation and NO level or infant rats. The brain
NOS activation and NO level of the fourth day infant rats in all group poisoned by DM were much higher than blank control group. However, there was no neural toxic symptoms and signs.10. Pregnant rats contacted DM would affect the filial generation which was reflected through endocrine. There showed that male infant rats were affected more obvious relatively. To male, the level of estosterone and production of sperm fell. To female, uterus deficiency was discovered. There implied that endocrine might be affected by DM. Formula Zhuyun III could improve the drawback through the regulation of endocrine.Conclusion1. DM is a typical medicine which proved its reproductive toxicity leadingto pregnant defect and abortion. It could affect the embryo and its development. The endocrine of pregnant rats was affected. However, no neurotoxicity of DM could be found regarding the filial generation.2. From the view point of Traditional Chinese Medicine, DM , one of the factors in the environmental pollution, was treated as a kind of toxin. It could affect the embryo and pregnant mother among dysfunction of organ, imbalance of Yin-yan and damage of reproductive system which could lead to abortion. The infant might lead to deficiency of genetic factor and slow development.3. Formula Zhuyun HI strengthened kidney and spleen to resist DM toxicity. Thus, the abortion rate fell obviously. It could also accelerate the infant development and the reproductive performance of filial generation. It might be caused by the regulation of endocrine and immune system. According to the results, we believe that the medicine with strengthening function of kidney and spleen had a great potential to interfere ECDs reproductive toxicity and to avoid pregnant abortion and embryonic defect.
引文
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