12种中草药水煎剂体外抑制弓形虫感染效果的real-time PCR评价
|
摘要
为探究苍术、川乌等12种中草药的体外抗弓形虫感染效果,经制备水煎剂、测取吸光值和干重,评价其对Vero细胞的毒害作用;合成引物,优化real-time PCR条件,用9种回归模型拟合不同感染孔内弓形虫数目,求得24孔板单孔适合感染量;用适合数目RH速殖子与适当浓度水煎剂作用6 h、12 h、1 d、2 d和3 d,real-time PCR法定量水煎剂的体外抗弓形虫效果。结果显示,所得水煎剂的吸光值大小与干重间无线性关系,高浓度水煎剂不利于Vero细胞生长。抗感染结果显示,槟榔、白薇、青蒿、蒲公英和柴胡作用3 d时弓形虫速殖子数均较空白组显著降低(P<0.05),其他水煎剂和时间点则作用不明显(P>0.05),表明槟榔、白薇、青蒿、蒲公英和柴胡可抑制弓形虫体外增殖,能用作研发抗弓形虫感染特效药的重要研究对象。
To evaluate in vitro anti-Toxoplasma gondii efficacy of twelve kinds of Chinese herbal medicines,their optical densities,dry weights and toxicities for Vero cells were examined by preparing the boil preparations.PCR amplifications were performed to identify the synthesized primers and the real-time PCR conditions.Based on nine kinds of frequent regression models,the curves were matched according to the number of T.gondii parasites in wells,and their parameters were further used to calculate the suitable infection doses of this parasite for 24-well plate.Anti-T.gondii efficacy of boilpreparations was evaluated by the created real-time PCR at 6 h,12 h,1 d,2 d, and 3 d post infection.The data showed that the same dry weight of boil preparations own significantly different optical densities,and the host cell growth was severely affect by higher doses.The results of real-time PCR indicated that the numbers of T.gondii were statistically reduced at 3 days post preparation addition in Semen Arecae catechu-,Radix Cynanchi atrati-,Herba Artemisiae apiaceae-,Hr Teraxaci mongolicicum radiceor Radix Bupleuri-contained wells compared with the controls(P<0.05),and no difference was detected at other time points and the last seven kinds of boil preparations(P>0.05),suggesting that in vitro proliferation of T.gondii was significantly inhibited by Semen Arecae catechu,Radix Cynanchi atrati,Herba Artemisiae apiaceae,Hr Teraxaci mongolicicum radice and Radix Bupleuri,which could be further used to exploit the miracle drugs for the clinical treatments of toxoplasmosis.
To evaluate in vitro anti-Toxoplasma gondii efficacy of twelve kinds of Chinese herbal medicines,their optical densities,dry weights and toxicities for Vero cells were examined by preparing the boil preparations.PCR amplifications were performed to identify the synthesized primers and the real-time PCR conditions.Based on nine kinds of frequent regression models,the curves were matched according to the number of T.gondii parasites in wells,and their parameters were further used to calculate the suitable infection doses of this parasite for 24-well plate.Anti-T.gondii efficacy of boilpreparations was evaluated by the created real-time PCR at 6 h,12 h,1 d,2 d, and 3 d post infection.The data showed that the same dry weight of boil preparations own significantly different optical densities,and the host cell growth was severely affect by higher doses.The results of real-time PCR indicated that the numbers of T.gondii were statistically reduced at 3 days post preparation addition in Semen Arecae catechu-,Radix Cynanchi atrati-,Herba Artemisiae apiaceae-,Hr Teraxaci mongolicicum radiceor Radix Bupleuri-contained wells compared with the controls(P<0.05),and no difference was detected at other time points and the last seven kinds of boil preparations(P>0.05),suggesting that in vitro proliferation of T.gondii was significantly inhibited by Semen Arecae catechu,Radix Cynanchi atrati,Herba Artemisiae apiaceae,Hr Teraxaci mongolicicum radice and Radix Bupleuri,which could be further used to exploit the miracle drugs for the clinical treatments of toxoplasmosis.
引文
[1]MONTOYA J G,LIESENFELD O.Toxoplasmosis[J].Lancet,2004,363(9425):1965-1976.
[2]GARCíA-GARCíA C,CASTILLO-ALVAREZ F,AZCONAGUT-IERREZ J M,et al.Spinal cord toxoplasmosis in human immunodeficiency virus infection/acquired immunodeficiency syndrome[J].Infectious Diseases(Lond),2015,47(5):277-282.
[3]NISSAPATORN V.Toxoplasmosis in HIV/AIDS:a living legacy[J].Southeast Asian Journal of Tropical Medicine and Public Health,2009,40(6):1158-1178.
[4]MCAULEY J B.Congenital toxoplasmosis[J].Journal of the Pediatric Infectious Diseases Society,2014,3(Suppl1):S30-S35.
[5]VARGAS-VILLAVICENCIO J A,BESNE-MERIDA A,CORREAD.Vertical transmission and fetal damage in animal models of congenital toxoplasmosis:a systematic review[J].Veterinary Parasitology,2016,223:195-204.
[6]SARAF P,SHWAB E K,DUBEY J P,et al.On the determination of Toxoplasma gondii virulence in mice[J].Experimental Parasitology,2017,174:25-30.
[7]JAURIGUE J A,SEEBERGER P H.Parasite carbohydrate vaccines[J].Frontiers in Cellular and Infection Microbiology,2017,7:248.
[8]ZHANG N Z,WANG M,XU Y,et al.Recent advances in developing vaccines against Toxoplasma gondii:an update[J].Expert Review of Vaccines,2015,14(12):1609-1621.
[9]ALDAY P H,DOGGETT J S.Drugs in development for toxoplasmosis:advances,challenges,and current status[J].Drug Design Development and Therapy,2017,11:273-293.
[10]戚畅,刘炘.中药方剂联合阿奇霉素抗支原体感染对小儿支原体肺炎的临床疗效分析[J].中国中医基础医学杂志,2017,23(11):1590-1592.QI Chang,LIU Xin.Clinical analysis of the efficacy of Chinese herbs combined with azithromycin anti-mycoplasma infection of Mycoplasma pneumonia in children[J].Journal of Basic Chinese Medicine,2017,23(11):1590-1592.(in Chinese)
[11]高学敏.中药学[M].北京:中国中医药出版社,2002:82-541.GAO Xue-min.Science of Chinese Traditional Medicine[M].Beijing:China Press of Traditional Chinese Medicine,2002:82-541.(in Chinese)
[12]LIU C,CHANG L,JIA T,et al.Real-time PCR assays for hepatitis B virus DNA quantification may require two different targets[J].Virology Journal,2017,14(1):94.
[13]WANG P,ZHAI G,BAI Y.Values of miR-34a and miR-218expression in the diagnosis of cervical cancer and the prediction of prognosis[J].Oncology Letters,2018,15(3):3580-3585.
[14]叶定江,张世臣,吴皓.中药炮制学(第2版)[M].北京:人民卫生出版社,2011:1-795.YE Ding-jiang,ZHANG Shi-chen,WU Hao.Science of Chinese materia medica processing(second edition)[M].Beijing:People's Medical Publishing House,2011:1-795.(in Chinese)
[15]SOLAV D,CAMOMILLA V,CEREATTI A,et al.Bone orientation and position estimation errors using Cosserat point elements and least squares methods:application to gait[J].Journal of Biomechanics,2017,62:110-116.
[16]郭海婷,胡超,严普,等.多聚甲醛处理的昆明小鼠T细胞及其亚群存放不同时间分析[J].细胞与分子免疫学杂志,2017,33(4):460-464.GUO Hai-ting,HU Chao,YAN Pu,et al.Analysis of the storage time in paraformaldehyde-fixed Tcells and their subsets in Kunming mice[J].Chinese Journal of Cellular and Molecular Immunology,2017,33(4):460-464.(in Chinese)
[17]WHITE N J,PUKRITTAYAKAMEE S,HIEN T T,et al.Malaria[J].Lancet,2014,383(9918):723-735.
[18]南旭梅,张敬,金小雪,等.青蒿浸膏对昆明小鼠和Wistar大鼠的急性毒性试验[J].中国兽医科学,2018,48(2):247-255.NAN Xu-mei,ZHANG Jing,JIN Xiao-xue,et al.Acute toxicity tests of the extract from Artemisia annua in Kunming mice and Wistar rat[J].Chinese Veterinary Science,2018,48(2):247-255.(in Chinese)
[19]王赛君,伍振峰,杨明,等.中药提取新技术及其在国内的转化应用研究[J].中国中药杂志,2014,39(8):1360-1367.WANG Sai-jun,WU Zhen-feng,YANG Ming,et al.Research status and translational application of new extraction techniques of traditional Chinese medicine[J].China Journal of Chinese Materia Medica,2014,39(8):1360-1367.(in Chinese)
[20]GOMEZ PEREZ M,FOURCADE L,MATEESCU M A,et al.Neutral red versus MTT assay of cell viability in the presence of copper compounds[J].Analytical Biochemistry,2017,535:43-46.
[21]YANG Z,CHEN Y,HU B,et al.Identification and validation of reference genes for quantification of target gene expression with quantitative realtime PCR for tall fescue under four abiotic stresses[J].PLoS One,2015,10(3):e0119569.
[22]BAMIAS G,GOUKOS D,LAOUDI E,et al.Comparative study of candidate housekeeping genes for quantification of target gene messenger RNA expression by real-time PCR in patients with inflammatory bowel disease[J].Inflammatory Bowel Diseases,2013,19(13):2840-2847.
[2]GARCíA-GARCíA C,CASTILLO-ALVAREZ F,AZCONAGUT-IERREZ J M,et al.Spinal cord toxoplasmosis in human immunodeficiency virus infection/acquired immunodeficiency syndrome[J].Infectious Diseases(Lond),2015,47(5):277-282.
[3]NISSAPATORN V.Toxoplasmosis in HIV/AIDS:a living legacy[J].Southeast Asian Journal of Tropical Medicine and Public Health,2009,40(6):1158-1178.
[4]MCAULEY J B.Congenital toxoplasmosis[J].Journal of the Pediatric Infectious Diseases Society,2014,3(Suppl1):S30-S35.
[5]VARGAS-VILLAVICENCIO J A,BESNE-MERIDA A,CORREAD.Vertical transmission and fetal damage in animal models of congenital toxoplasmosis:a systematic review[J].Veterinary Parasitology,2016,223:195-204.
[6]SARAF P,SHWAB E K,DUBEY J P,et al.On the determination of Toxoplasma gondii virulence in mice[J].Experimental Parasitology,2017,174:25-30.
[7]JAURIGUE J A,SEEBERGER P H.Parasite carbohydrate vaccines[J].Frontiers in Cellular and Infection Microbiology,2017,7:248.
[8]ZHANG N Z,WANG M,XU Y,et al.Recent advances in developing vaccines against Toxoplasma gondii:an update[J].Expert Review of Vaccines,2015,14(12):1609-1621.
[9]ALDAY P H,DOGGETT J S.Drugs in development for toxoplasmosis:advances,challenges,and current status[J].Drug Design Development and Therapy,2017,11:273-293.
[10]戚畅,刘炘.中药方剂联合阿奇霉素抗支原体感染对小儿支原体肺炎的临床疗效分析[J].中国中医基础医学杂志,2017,23(11):1590-1592.QI Chang,LIU Xin.Clinical analysis of the efficacy of Chinese herbs combined with azithromycin anti-mycoplasma infection of Mycoplasma pneumonia in children[J].Journal of Basic Chinese Medicine,2017,23(11):1590-1592.(in Chinese)
[11]高学敏.中药学[M].北京:中国中医药出版社,2002:82-541.GAO Xue-min.Science of Chinese Traditional Medicine[M].Beijing:China Press of Traditional Chinese Medicine,2002:82-541.(in Chinese)
[12]LIU C,CHANG L,JIA T,et al.Real-time PCR assays for hepatitis B virus DNA quantification may require two different targets[J].Virology Journal,2017,14(1):94.
[13]WANG P,ZHAI G,BAI Y.Values of miR-34a and miR-218expression in the diagnosis of cervical cancer and the prediction of prognosis[J].Oncology Letters,2018,15(3):3580-3585.
[14]叶定江,张世臣,吴皓.中药炮制学(第2版)[M].北京:人民卫生出版社,2011:1-795.YE Ding-jiang,ZHANG Shi-chen,WU Hao.Science of Chinese materia medica processing(second edition)[M].Beijing:People's Medical Publishing House,2011:1-795.(in Chinese)
[15]SOLAV D,CAMOMILLA V,CEREATTI A,et al.Bone orientation and position estimation errors using Cosserat point elements and least squares methods:application to gait[J].Journal of Biomechanics,2017,62:110-116.
[16]郭海婷,胡超,严普,等.多聚甲醛处理的昆明小鼠T细胞及其亚群存放不同时间分析[J].细胞与分子免疫学杂志,2017,33(4):460-464.GUO Hai-ting,HU Chao,YAN Pu,et al.Analysis of the storage time in paraformaldehyde-fixed Tcells and their subsets in Kunming mice[J].Chinese Journal of Cellular and Molecular Immunology,2017,33(4):460-464.(in Chinese)
[17]WHITE N J,PUKRITTAYAKAMEE S,HIEN T T,et al.Malaria[J].Lancet,2014,383(9918):723-735.
[18]南旭梅,张敬,金小雪,等.青蒿浸膏对昆明小鼠和Wistar大鼠的急性毒性试验[J].中国兽医科学,2018,48(2):247-255.NAN Xu-mei,ZHANG Jing,JIN Xiao-xue,et al.Acute toxicity tests of the extract from Artemisia annua in Kunming mice and Wistar rat[J].Chinese Veterinary Science,2018,48(2):247-255.(in Chinese)
[19]王赛君,伍振峰,杨明,等.中药提取新技术及其在国内的转化应用研究[J].中国中药杂志,2014,39(8):1360-1367.WANG Sai-jun,WU Zhen-feng,YANG Ming,et al.Research status and translational application of new extraction techniques of traditional Chinese medicine[J].China Journal of Chinese Materia Medica,2014,39(8):1360-1367.(in Chinese)
[20]GOMEZ PEREZ M,FOURCADE L,MATEESCU M A,et al.Neutral red versus MTT assay of cell viability in the presence of copper compounds[J].Analytical Biochemistry,2017,535:43-46.
[21]YANG Z,CHEN Y,HU B,et al.Identification and validation of reference genes for quantification of target gene expression with quantitative realtime PCR for tall fescue under four abiotic stresses[J].PLoS One,2015,10(3):e0119569.
[22]BAMIAS G,GOUKOS D,LAOUDI E,et al.Comparative study of candidate housekeeping genes for quantification of target gene messenger RNA expression by real-time PCR in patients with inflammatory bowel disease[J].Inflammatory Bowel Diseases,2013,19(13):2840-2847.