印楝素对猪睾丸ST细胞的生长和基因表达的影响
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摘要
印楝素(azadirachtin)是从印楝(Azadirachta indica)种子提取的植物杀虫剂,广泛应用于农田害虫防治。为了明确印楝素是否影响公猪的繁殖力,本研究采用猪(Sus scrofa)睾丸ST细胞(swine testis cells),通过MTT(3-(4, 5-dimethyl thiazol-2-yl)-2, 5-diphenyl tetrazolium bromide)方法和流式细胞术,研究印楝素对猪睾丸细胞生长的影响,以实时荧光定量PCR方法检测印楝素对繁殖相关基因的影响。随着印楝素处理浓度的增加,ST细胞变圆脱落聚集;MTT实验证明印楝素以剂量依赖方式抑制ST细胞的生长,表现出细胞毒作用,24 h的半抑制浓度(half maximal inhibitory concentration, IC50)为7.646 mg/L;流式细胞术检测到印楝素诱导ST细胞凋亡,8 mg/L印楝素引起的早期凋亡率明显高于5 mg/L处理组(P<0.05);基质金属蛋白酶15 (matrix metallopeptidase 15, MMP15)、蛋白激酶Cα相互作用蛋白(protein interacting with Cαkinase, PICK1)、骨形态发生蛋白6 (bone morphogenetic protein 6, BMP6)基因的表达量随印楝素处理浓度的增加而下降;突变同源蛋白4 (MutS homolog 4, MSH4)基因的表达量随印楝素处理的浓度而增加。综上所述,印楝素可抑制猪睾丸细胞的生长,诱导细胞凋亡,并影响繁殖相关基因的表达,提示印楝素可影响公猪的繁殖力。本研究为饲料中印楝素残留控制提供了理论依据。
As a natural insecticidal tetranortriterpenoid compound extracted from the seeds of the neem tree(Azadirachta indica), azadirachtin is currently widely used in agricultural products and vegetables for pest control. A large fraction of azadirachtin is lost and likely affects the environment and domestic animals by straw feeds exploitation. It has been reported that the intra-vas administration of neem oil resulted in the block of spermatogenesis in male rats. It is still unclear whether there is a role of azadirachtin on the function of pig testis. In order to gain insight into the importance of azadirachtin in male pig reproduction, this study was undertaken to investigate the effect of azadirachtin on testis in vitro by using pig testicular ST cell line. The status of ST cell growth was tracked under optical microscope and the proliferation or viability of cells were measured by 3-(4, 5-dimethyl thiazol-2-yl)-2, 5-diphenyl tetrazolium bromide(MTT) assay. Flow cytometry with annexin V FITC/PI double staining was used to detect apoptosis of ST cell before and after azadirachtin treatment. The expression levels of four genes were determined by using quantitative real-time polymerase chain reaction(qRT-PCR) method, in which included MMP15(matrix metallopeptidase 15), PICK1(protein interacting with Cα kinase), BMP6(bone morphogenetic protein 6), and MSH4(MutS homolog 4). Using different dose of azadirachtin from 1 to 9 mg/L, the number of adherent cells was reduced, with cells rounded and suspended and appeared to be clustered under microscope. Based on determination of MTT assay,azadirachtin exhibited cytotoxic effects on the viability of ST cells in a dose and time-dependent manner with IC50 value of 7.646 mg/L at 24 h. Azadirachtin induced the apoptosis of ST cells in a dose-and time-dependent manner with a higher rate of early apoptotic cells at concentrations of 8 mg/L than that of 5 mg/L for 24 h of incubation(P<0.05). As the treatment concentration of azadirachtin changed from 5 mg/L to 8 mg/L, the expression of all genes except MSH4 were rapidly decreased after dealting with azadirachtin at a dosedependent way. The above findings demonstrated that azadirachtin had a cytotoxic impact on pig testis cell,and the induction of apoptosis might be one of the causes. It made a reasonable speculation that the function of pig testis was influenced by azadirachtin because the several gene expressions related with reproduction ability were disturbed intensely. It suggested that azadirachtin might inhibit the fertility of boar and need to have a mention on the detection and amount of azadirachtin residue in plant feedstuff in pig industry. The results could provide a basis for the residue control of azadirachtin in plant feedstuff.
As a natural insecticidal tetranortriterpenoid compound extracted from the seeds of the neem tree(Azadirachta indica), azadirachtin is currently widely used in agricultural products and vegetables for pest control. A large fraction of azadirachtin is lost and likely affects the environment and domestic animals by straw feeds exploitation. It has been reported that the intra-vas administration of neem oil resulted in the block of spermatogenesis in male rats. It is still unclear whether there is a role of azadirachtin on the function of pig testis. In order to gain insight into the importance of azadirachtin in male pig reproduction, this study was undertaken to investigate the effect of azadirachtin on testis in vitro by using pig testicular ST cell line. The status of ST cell growth was tracked under optical microscope and the proliferation or viability of cells were measured by 3-(4, 5-dimethyl thiazol-2-yl)-2, 5-diphenyl tetrazolium bromide(MTT) assay. Flow cytometry with annexin V FITC/PI double staining was used to detect apoptosis of ST cell before and after azadirachtin treatment. The expression levels of four genes were determined by using quantitative real-time polymerase chain reaction(qRT-PCR) method, in which included MMP15(matrix metallopeptidase 15), PICK1(protein interacting with Cα kinase), BMP6(bone morphogenetic protein 6), and MSH4(MutS homolog 4). Using different dose of azadirachtin from 1 to 9 mg/L, the number of adherent cells was reduced, with cells rounded and suspended and appeared to be clustered under microscope. Based on determination of MTT assay,azadirachtin exhibited cytotoxic effects on the viability of ST cells in a dose and time-dependent manner with IC50 value of 7.646 mg/L at 24 h. Azadirachtin induced the apoptosis of ST cells in a dose-and time-dependent manner with a higher rate of early apoptotic cells at concentrations of 8 mg/L than that of 5 mg/L for 24 h of incubation(P<0.05). As the treatment concentration of azadirachtin changed from 5 mg/L to 8 mg/L, the expression of all genes except MSH4 were rapidly decreased after dealting with azadirachtin at a dosedependent way. The above findings demonstrated that azadirachtin had a cytotoxic impact on pig testis cell,and the induction of apoptosis might be one of the causes. It made a reasonable speculation that the function of pig testis was influenced by azadirachtin because the several gene expressions related with reproduction ability were disturbed intensely. It suggested that azadirachtin might inhibit the fertility of boar and need to have a mention on the detection and amount of azadirachtin residue in plant feedstuff in pig industry. The results could provide a basis for the residue control of azadirachtin in plant feedstuff.
引文
崔羽,杨少成,刘娣,等.2014.猪BMP6基因在不同组织中的表达谱分析[J].黑龙江畜牧兽医,(5):9-11.(Cui Y,Yang S C,Liu T,et al.2014.Expression profile of pig BMP6 gene in different tissues was analyzed[J].Heilongjiang Animal Husbandry Veterinarian,(5):9-11.)
李蕊蕊.2013.PICK1、ErbB2在精子中的表达及其与体外受精关系的初步研究[D].硕士学位论文,郑州大学,导师:郭艺红,pp.1-2.(Li R R.2013.Preliminary study on expression of PICK1 and ErbB2 in sperm and its relationship with in vitro fertilization[D].Thesis for M.S.,Zhengzhou University,Supervisor:Guo Y H,pp.1-2.)
李晓东,赵善欢.1996.印楝素对昆虫的毒理作用机制[J].华南农业大学学报,(1):118-122.(Li X D,Zhao S H.1996.Toxicity mechanism of azadirachtin on insects[J].Journal of South China Agricultural University,(1):118-122.)
唐中林,邓宏,李勇,等.2008.应用荧光定量PCR方法研究RPL29基因在通城猪和长白猪不同时期胚胎骨骼肌中的表达[J].畜牧兽医学报,39(8):1007-1012.(Tang ZL,Deng H,Li Y,et al.Fluorescence quantitative PCRwas used to study the expression of RPL29 gene in the skeletal muscle of embryos of tongcheng and changbai pigs at different stages[J].Journal of Animal Husbandry and Veterinary,39(8):1007-1012.)
文吉辉,侯茂林,卢伟,等.2007.印楝素的杀虫活性及其对烟粉虱的驱避作用[J].应用昆虫学报,44(4):491-496.(Wen J H,Hou M L,Lu W,et al.2007.The insecticidal activity of azadirachtin and its repellent effect on bemisia tabaci[J].Journal of Applied Insect,44(4):491-496.)
吴伟,朱小惠.2003.印楝素对鱼的毒性及在鱼类寄生虫病防治上的应用[J].农药学学报,5(2):85-89.(Wu W,Zhu X H.2003.Toxicity of azadirachtin to fish and its application in control of fish parasitic diseases[J].Journal of Agrochemicals,5(2):85-89.)
肖杰文.2008.山羊高繁殖力候选基因GnRHR、BMP6和BMP15的多态性及与繁殖性状的相关性研究[D].硕士学位论文,四川农业大学,导师:李学伟,pp.9-11.(Xiao J W.2008.Polymorphisms of the candidate genes GnRHR,BMP6 and BMP15 and their correlation with reproductive traits in goats with high fertility[D].Thesis for M.S.,Sichuan Agricultural University,Supervisor:Li X W,pp.9-11.)
赵善欢,张业光,蔡德智,等.1989.印楝引种试验初报[J].华南农业大学学报,(2):34-39.(Zhao S H,Zhang Y G,Cai D Z,et al.1989.Preliminary report of neem introduction experiment[J].Journal of South China Agricultural University,(2):34-39.)
Awasthy K S.2001.Genotoxicity of a crude leaf extract of neem in male germ cells of mice[J].Cytobios,106(2):151-164.
Ciller I M,Palanisamy S K,Ciller U A,et al.2016.Postnatal expression of bone morphogenetic proteins and their receptors in the mouse testis[J].Physiological Research,65(4):673-682.
Gashaw I,Grümmer R,Klein-Hitpass L,et al.2005.Gene signatures of testicular seminoma with emphasis on expression of ets variant gene 4[J].Cellular and Molecular Life Sciences,62(19-20):2359-2368.
Gai M N,álvarez C,Venegas R,et al.2011.An HPLC method for determination of azadirachtin residues in bovine muscle[J].Journal of Chromatographic Science,49(4):327-331.
Jarvis A P,Morgan E D,Edwards C.1999.Rapid separation of triterpenoids from neem seed extracts[J].Phytochemical Analysis,10(1):39-43.
Lehmann E,Fargues M,Nfon DibiéJ J,et al.2018.Assessment of water resource contamination by pesticides in vegetable-producing areas in Burkina Faso[J].Environmental Science and Pollution Research,25(4):3681-3694.
Lampert K,Machein U,Machein M R,et al.1998.Experssion of matrix metalloproteinases and their tissue inhibitors in human brain tumors[J].The American Journal of Pathology,153(2):429-437.
Li X,Mao Z,Wu M,et al.2013.Rescuing infertility of Pick1knockout mice by generating testis-specific transgenic mice via testicular infection[J].Scientific Reports,3:2842.
Liu C,Ran X,Wang J F,et al.2018.Detection of genomic structural variations in Guizhou indigenous pigs and the comparison with other breeds[J].PloS One,13(3):e0194282.
Price J F,Schuster D J.1991.Effects of natural and synthetic insecticides on sweetpotato whitefly Bemisia tabaci(Homoptera:Aleyrodidae)and its hymenopterous parasitoids[J].Florida Entomologist,74(1):60-68.
Ross-Macdonalp P,Roeder G S.1994.Mutation of a meiosis specific MutS homolog decreases crossing over but not mismatch correction[J].Cell,79(6):1069-1680.
Schmutterer H.1990.Properties and potential of natural pesticides from the neem tree,Azadirachta indica[J].Annual Review of Entomology,35(35):271-279.
Sengupta P,Raman S,Chowdhury R,et al.2017.Evaluation of apoptosis and autophagy inducing potential of Berber?is aristata,Azadirachta indica,and their synergistic combinations in parental and resistant human osteosarcoma cells[J].Frontiers in Oncology,7:296.
Upadhyay S N,Dhawan S,Talwar G P.1993.Antifertility effects of neem(Azadirachta indica)oil in male rats by single intra-vas Administration:An alternate approach to vasectomy[J].Journal of Andrology,14(4):275-281.
Upadhyay S,Dhawan S,Sharma M G,et al.1994.Long-term contraceptive effects of intrauterine neem treatment(IUNT)in bonnet monkeys:An alternate to intrauterine contraceptive devices(IUCD)[J].Contraception,49(2):161-169.
Visse R,Nagase H.2003.Matrix metalloproteinases and tissue inhibitors of metalloproteinases:Structure,function,and biochemistry[J].Circulation Research,92(8):827-839.
Wen L P,Yuan Q Q,Sun M,et al.2017.Generation and characteristics of human sertoli cell line immortalized by overexpression of human telomerase[J].Oncotarget,8(10):16553-16570.
Xie Y S,Fields P G,Isman M B.1995.Repellency and toxicity of azadirachtin and neem concentrates to three storedproduct beetles[J].Journal of Economic Entomology,88(4):1024-1031.
Xu L,Li S,Ran X,et al.2016.Apoptotic activity and gene responses in Drosophila melanogaster S2 cells,induced by azadirachtin A[J].Pest Management Science,72(9):1710-1717.
李蕊蕊.2013.PICK1、ErbB2在精子中的表达及其与体外受精关系的初步研究[D].硕士学位论文,郑州大学,导师:郭艺红,pp.1-2.(Li R R.2013.Preliminary study on expression of PICK1 and ErbB2 in sperm and its relationship with in vitro fertilization[D].Thesis for M.S.,Zhengzhou University,Supervisor:Guo Y H,pp.1-2.)
李晓东,赵善欢.1996.印楝素对昆虫的毒理作用机制[J].华南农业大学学报,(1):118-122.(Li X D,Zhao S H.1996.Toxicity mechanism of azadirachtin on insects[J].Journal of South China Agricultural University,(1):118-122.)
唐中林,邓宏,李勇,等.2008.应用荧光定量PCR方法研究RPL29基因在通城猪和长白猪不同时期胚胎骨骼肌中的表达[J].畜牧兽医学报,39(8):1007-1012.(Tang ZL,Deng H,Li Y,et al.Fluorescence quantitative PCRwas used to study the expression of RPL29 gene in the skeletal muscle of embryos of tongcheng and changbai pigs at different stages[J].Journal of Animal Husbandry and Veterinary,39(8):1007-1012.)
文吉辉,侯茂林,卢伟,等.2007.印楝素的杀虫活性及其对烟粉虱的驱避作用[J].应用昆虫学报,44(4):491-496.(Wen J H,Hou M L,Lu W,et al.2007.The insecticidal activity of azadirachtin and its repellent effect on bemisia tabaci[J].Journal of Applied Insect,44(4):491-496.)
吴伟,朱小惠.2003.印楝素对鱼的毒性及在鱼类寄生虫病防治上的应用[J].农药学学报,5(2):85-89.(Wu W,Zhu X H.2003.Toxicity of azadirachtin to fish and its application in control of fish parasitic diseases[J].Journal of Agrochemicals,5(2):85-89.)
肖杰文.2008.山羊高繁殖力候选基因GnRHR、BMP6和BMP15的多态性及与繁殖性状的相关性研究[D].硕士学位论文,四川农业大学,导师:李学伟,pp.9-11.(Xiao J W.2008.Polymorphisms of the candidate genes GnRHR,BMP6 and BMP15 and their correlation with reproductive traits in goats with high fertility[D].Thesis for M.S.,Sichuan Agricultural University,Supervisor:Li X W,pp.9-11.)
赵善欢,张业光,蔡德智,等.1989.印楝引种试验初报[J].华南农业大学学报,(2):34-39.(Zhao S H,Zhang Y G,Cai D Z,et al.1989.Preliminary report of neem introduction experiment[J].Journal of South China Agricultural University,(2):34-39.)
Awasthy K S.2001.Genotoxicity of a crude leaf extract of neem in male germ cells of mice[J].Cytobios,106(2):151-164.
Ciller I M,Palanisamy S K,Ciller U A,et al.2016.Postnatal expression of bone morphogenetic proteins and their receptors in the mouse testis[J].Physiological Research,65(4):673-682.
Gashaw I,Grümmer R,Klein-Hitpass L,et al.2005.Gene signatures of testicular seminoma with emphasis on expression of ets variant gene 4[J].Cellular and Molecular Life Sciences,62(19-20):2359-2368.
Gai M N,álvarez C,Venegas R,et al.2011.An HPLC method for determination of azadirachtin residues in bovine muscle[J].Journal of Chromatographic Science,49(4):327-331.
Jarvis A P,Morgan E D,Edwards C.1999.Rapid separation of triterpenoids from neem seed extracts[J].Phytochemical Analysis,10(1):39-43.
Lehmann E,Fargues M,Nfon DibiéJ J,et al.2018.Assessment of water resource contamination by pesticides in vegetable-producing areas in Burkina Faso[J].Environmental Science and Pollution Research,25(4):3681-3694.
Lampert K,Machein U,Machein M R,et al.1998.Experssion of matrix metalloproteinases and their tissue inhibitors in human brain tumors[J].The American Journal of Pathology,153(2):429-437.
Li X,Mao Z,Wu M,et al.2013.Rescuing infertility of Pick1knockout mice by generating testis-specific transgenic mice via testicular infection[J].Scientific Reports,3:2842.
Liu C,Ran X,Wang J F,et al.2018.Detection of genomic structural variations in Guizhou indigenous pigs and the comparison with other breeds[J].PloS One,13(3):e0194282.
Price J F,Schuster D J.1991.Effects of natural and synthetic insecticides on sweetpotato whitefly Bemisia tabaci(Homoptera:Aleyrodidae)and its hymenopterous parasitoids[J].Florida Entomologist,74(1):60-68.
Ross-Macdonalp P,Roeder G S.1994.Mutation of a meiosis specific MutS homolog decreases crossing over but not mismatch correction[J].Cell,79(6):1069-1680.
Schmutterer H.1990.Properties and potential of natural pesticides from the neem tree,Azadirachta indica[J].Annual Review of Entomology,35(35):271-279.
Sengupta P,Raman S,Chowdhury R,et al.2017.Evaluation of apoptosis and autophagy inducing potential of Berber?is aristata,Azadirachta indica,and their synergistic combinations in parental and resistant human osteosarcoma cells[J].Frontiers in Oncology,7:296.
Upadhyay S N,Dhawan S,Talwar G P.1993.Antifertility effects of neem(Azadirachta indica)oil in male rats by single intra-vas Administration:An alternate approach to vasectomy[J].Journal of Andrology,14(4):275-281.
Upadhyay S,Dhawan S,Sharma M G,et al.1994.Long-term contraceptive effects of intrauterine neem treatment(IUNT)in bonnet monkeys:An alternate to intrauterine contraceptive devices(IUCD)[J].Contraception,49(2):161-169.
Visse R,Nagase H.2003.Matrix metalloproteinases and tissue inhibitors of metalloproteinases:Structure,function,and biochemistry[J].Circulation Research,92(8):827-839.
Wen L P,Yuan Q Q,Sun M,et al.2017.Generation and characteristics of human sertoli cell line immortalized by overexpression of human telomerase[J].Oncotarget,8(10):16553-16570.
Xie Y S,Fields P G,Isman M B.1995.Repellency and toxicity of azadirachtin and neem concentrates to three storedproduct beetles[J].Journal of Economic Entomology,88(4):1024-1031.
Xu L,Li S,Ran X,et al.2016.Apoptotic activity and gene responses in Drosophila melanogaster S2 cells,induced by azadirachtin A[J].Pest Management Science,72(9):1710-1717.