猫爪草多糖对小鼠腹腔巨噬细胞活力的调节作用
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摘要
此次研究旨在探讨猫爪草多糖对体外培养的正常状态下的原代小鼠腹腔巨噬细胞活性的调节作用,以及小鼠腹腔巨噬细胞在体外培养条件下的活力变化情况。以原代培养的小鼠腹腔巨噬细胞为研究对象,设对照组(加入100μL DMEM培养基)和实验组(分别加入25μg/mL, 50μg/mL, 100μg/mL, 200μg/mL,400μg/mL的猫爪草多糖),分别采用噻唑蓝(MTT)比色法、CCK-8法、乳酸脱氢酶释放法和中性红吞噬实验检测不同浓度的猫爪草多糖对体外培养的小鼠腹腔巨噬细胞活力的调节作用;同时设置24 h、36 h、48 h、60 h和72 h的不同培养时间,观察在体外培养条件下,小鼠腹腔巨噬细胞活力的变化情况。结果表明:与对照组相比,不同浓度的猫爪草多糖均能增强小鼠腹腔巨噬细胞的活力,且猫爪草多糖浓度在100～400μg/mL的细胞活力极显著增强(p<0.01)。此外,各处理组的巨噬细胞在体外培养24～72 h不更换培养液的条件下,48 h处活性最佳。体外培养条件下,一定浓度的猫爪草多糖可以激活小鼠腹腔巨噬细胞,通过猫爪草多糖激活巨噬细胞,可能是猫爪草发挥提升机体免疫力的作用机制之一。此外,体外培养的巨噬细胞虽能存活长达一个月,但仍有一个最佳活力时间。
The aim of this research was to explore the effect of polysaccharide from Radix Ranunculi Ternati(PRT) on the cell viability of the normal murine peritoneal macrophages in vitro, as well as the the change of cell viability of the murine peritoneal macrophages cultured in vitro. This study took mouse peritoneal macrophages of primary culture as research object, designed a control group(only added with DMEM) and experimental groups(added with different concentration of PRT such as 25 μg/m L, 50 μg/m L, 100 μg/mL, 200 μg/mL, 400 μg/mL). And MTT assay, CCK-8 assay, the secretion of lactate dehydrogenase(LDH) as well as the phagocytic activity of neutral red were used to test the regulation of PRT on the activity of macrophages and the change of activity of the research object at different culture time in vitro(24 h, 36 h, 48 h, 60 h, 72 h). The results showed that compared with control group, PRT with different concentration could promote vitality of MPM, and that the concentration groups between 100 μg/mL and 400 μg/mL significantly elevated cellular viability. In addition, the macrophages cultured for 48 h in vitro showed the best activity. These results collectively indicated that the function of enhancing the body immunity of PRT might be correlated to its activation of macrophages. Despite the macrophages cultured in vitro might survive for weeks, it still possessed a optimizing period of time for cell vitality.
The aim of this research was to explore the effect of polysaccharide from Radix Ranunculi Ternati(PRT) on the cell viability of the normal murine peritoneal macrophages in vitro, as well as the the change of cell viability of the murine peritoneal macrophages cultured in vitro. This study took mouse peritoneal macrophages of primary culture as research object, designed a control group(only added with DMEM) and experimental groups(added with different concentration of PRT such as 25 μg/m L, 50 μg/m L, 100 μg/mL, 200 μg/mL, 400 μg/mL). And MTT assay, CCK-8 assay, the secretion of lactate dehydrogenase(LDH) as well as the phagocytic activity of neutral red were used to test the regulation of PRT on the activity of macrophages and the change of activity of the research object at different culture time in vitro(24 h, 36 h, 48 h, 60 h, 72 h). The results showed that compared with control group, PRT with different concentration could promote vitality of MPM, and that the concentration groups between 100 μg/mL and 400 μg/mL significantly elevated cellular viability. In addition, the macrophages cultured for 48 h in vitro showed the best activity. These results collectively indicated that the function of enhancing the body immunity of PRT might be correlated to its activation of macrophages. Despite the macrophages cultured in vitro might survive for weeks, it still possessed a optimizing period of time for cell vitality.
引文
Bai S.B.,Chen H.X.,Zhong J.J.,Feng S.M.,Li T.,and Luo X.G.2011,RAW264.7 cell viability via MTT assay and possible factor analysis,Zhongguo Xiandai Yixue Zazhi(China Journal of Modern Medicine),21(23):2831-2833(白生宾,陈红香,钟近洁,冯树梅,李甜,罗学港,2011,MTT法检测RAW264.7细胞活力及可能因素分析,中国现代医学杂志,21(23):2831-2833)
Chen W.,Lin X.H.,Chen J.,Luo H.B.,and Huang L.Y.,2005,Activation effects of polysaccharide from Aloe barbadensis on mouse peritoneal macrophages in vitro,Zhongguo Yaoxue Zazhi(Chinese Pharmaceutical Journal),40(1):34-37(陈伟,林新华,陈俊,罗红斌,黄丽英,2005,库拉索芦荟多糖对小鼠腹腔巨噬细胞的体外激活作用,中国药学杂志,40(1):34-37)
Dai Y.,Xu M.S.,Shangguan X.C.,Jiang Y.,Zheng G.D.,and Wang W.J.,2017,Immunoregulation effects of polysaccharides from pine needles on mouse peritoneal macropages,Dongwu Yingyang Xuebao(Chinese Journal of Animal Nutrition),29(2):670-677(戴艺,徐明生,上官新晨,蒋艳,郑国栋,王文君,2017,松针多糖对小鼠腹腔巨噬细胞免疫调节作用的研究,动物营养学报,29(2):670-677)
Hu Z.K.,Miao M.S.,and Liu H.L.,2010,Study on immunocompetence of the active constituent extracted from Radix Ranunculi Ternati,Liaoning Zhongyiyao Daxue Xuebao(Journal of Liaoning University of Traditional Chinese Medicine),12(3):24-26(胡泽开,苗明三,刘会丽,2010,中药猫爪草免疫活性部位的筛选,辽宁中医药大学学报,12(3):24-26)
Ji W.,Tang N.,Zhao D.,and Zhang B.J.,2016,The pharmacological effects on Hericium erinaceus and its cultivation and application,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),35(5):1252-1257(纪伟,唐宁,赵端,张边江,2016,猴头菇的药理作用及栽培与应用,基因组学与应用生物学,35(5):1252-1257)
Jia H.Q.,2006,The study on polysaccharides from Ranunculus ternatus Thunb.,Thesis for M.S.,Shaanxi Normal University,Supervisor:Zhang Z.Q.,pp.1-48(贾慧青,2006,猫爪草多糖的研究,硕士学位论文,陕西师范大学,导师:张志琪,pp.1-48)
Ke M.Y.,Wang H.,Zhang M.,Tian Y.W.,Wang Y.Z.,Li B.,Yu J.,Dou J.,Xi T.,and Zhou C.L.,2014,The anti-lung cancer activity of SEP is mediated by the activation and cytotoxicity of NK cells via TLR2/4 in vivo,Biochem.Pharmacol.,89(1):119-130
Koizumi K.,Hojo S.,Akashi T.,Yasumoto K.,and Saiki I.,2007,Chemokine receptors in cancer metastasis and cancer cell-derived chemokines in host immune response,Cancer Sci.,98(11):1652-1658
Liu J.,Wang J.M.,Ji B.,Zhang H.W.,and Zhao Q.T.,2012,Reseach on extraction process of crude polysaccharide from Radix Ranunculus Ternati,Zhongyi Xuebao(China Journal of Chinese Medicine),27(12):1622-1623(刘菊,王君明,纪彬,张红伟,赵群涛,2012,猫爪草粗多糖提取工艺研究,中医学报,27(12):1622-1623)
Lv X.H.,Wang H.M.,Han H.X.,Lv S.J.,and Qin D.Y.,2010,Effects of polysaccharide of Radix Ranunculi Ternati on immunomodulation and anti-oxidation,Zhongguo Zhongyao Zazhi(China Journal of Chinese Materia Medica),35(14):1862-1685(吕小华,王会敏,韩红霞,吕世静,覃冬云,2010猫爪草多糖免疫调节及抗氧化活性研究,中国中药杂志35(14):1862-1865)
Schepetkin I.A.,and Quinn M.T.,2006,Botanical polysaccharides:macrophage immunomodulation and therapeutic potential,Int.Immunopharmacol.,6(3):317-333
Shinchi H.,Crain B.,Yao S.Y.,Chan M.,Zhang S.S.,Ahmadiiveli A.,Suda Y.,Hayashi T.,Cottam H.B.,and Carson DA.,2015,Enhancement of the immunostimulatory activity of a TLR7 ligand by conjugation to polysaccharides,Bioconjug.Chem.,26(8):1713-1723
Sun D.L.,Xie H.B.and Xia Y.Z.,2013,A study on the inhibitory effect of polysaccharides from Radix Ranunculus Ternati on human breast cancer MCF-7 cell lines,Afr.J.Tradit.Complement.Altern.Med.,10(6):439-443
Sun L.Q.,Chu J.L.,Sun Z.L.,and Chen L.H.,2016,Physicochemical properties,immunomodulation and antitumor activities of polysaccharide from Pavlova viridis,Life Sci.144:156-161
Tuvaanjav S.,Han S.Q.,Komata M.,Ma C.J.,Kanamoto T.Nakashima H.,and Yoshida T.,2016,Isolation and antivira activity of water-soluble Cynomorium songaricum Rupr Polysaccharides,J.Asian Nat.Prod.Res.,18(2):159-171
Wan W.T.,Li N.,Liu J.,and Jin L.H.,2010,Comparative study on the optimal experiment conditions between CCK-8 and MTT in cell proliferation assay of PC3 cell,Shizhen Guoy Guoyao(Lishizhen Medicine and Materia Medica Research)21(12):3046-3048(万文婷,李宁,刘静,金林红,2010,CCK-8法与MTT法检测人前列腺癌PC3细胞活性的比较研究时珍国医国药,21(12):3046-3048)
Wang X.L.,Liao C.S.,Cheng Y.B.,Zhen Y.C.,and Cheng X.C.2014,Effects of glycyrrhizin,glycyrrhiza polysaccharide and glabridin on the toxicity and immunologic function of murine peritoneal macrophages,Zhongguo Shouyi Kexue(Chinese Veterinary Science),44(3):320-325(王晓利,廖成水,程源斌,甄永臣,程相朝,2014,甘草甜素甘草多糖和光甘草定对小鼠巨噬细胞的毒性与免疫功能的调节中国兽医科学,44(3):320-325)
Wang A.W.,Wang M.,Yuan J.R.,Tian J.K.,Wu L.M.,and Geng H.,2004,The study on antitumor effects in vitro of different extract in Radix Ranunculi Ternati,Tianran Chanwu Yanjiu Yu Kaifa(Natural Product research and Development),16(6):529-531(王爱武,王梅,袁久荣,田景奎,吴丽敏,耿晖,2004,猫爪草提取物体外抗肿瘤作用的研究,天然产物研究与开发,16(6):529-531)
Wang Z.Y.,Zhou F.,and Quan Y.,2014,Antioxidant and immunological activity in vitro of polysaccharides from Phellinus nigricans mycelia,International Journal of Biological Macromolecules,64(2):139-143
Yang D.P.,Yang X.X.,and Yu Y.,2017,Comparative study on the optimal experiment conditions between MTT assay and CCK-8 assay in rabbit retinal pigment epithelial cell,Zhongguo Minzu Minjian Yiyao(Chinese Journal of Ethnomedicine and Ethnopharmacy),26(6):31-33,45(杨冬萍,杨晓旭,俞洋,2017,MTT法与CCK-8法检测兔视网膜色素上皮细胞活性的比较研究,中国民族民间医药,26(6):31-33,45)
Zhang Z.L.,Wang L.,Wu X.J.,and Zhang H.X.,2007,The study about immunocompetence of polysaccharide of Radix Ranunculi Ternati,Shizhen Guoyi Guoyao(Lishizhen Medicine and Materia Medica Research),18(3):537-539(张振凌,王磊,吴筱菁,张惠霞,2007,中药猫爪草多糖的免疫活性研究,时珍国医国药,18(3):537-539)
Zhou L.J.,Wang Z.X.,Long T.T.,Zhou X.,and Bao Y.X.,2017,Roles of Astragalus polysaccharides in coculture system of breast cancer cells and macrophages in vitro,Mianyixue Zazhi(Immunological Journal),33(6):469-476(周丽菁,汪志学,龙婷婷,周星,鲍依稀,2017,黄芪多糖在体外巨噬细胞-乳腺癌细胞共培养体系中的作用,免疫学杂志,33(6):469-476)
Zhu Z.Y.,Liu N.,Si C.L.,Liu Y.,Ding L.N.,Jing C.,Liu A.J.,and Zhang Y.M.,2012,Structure and anti-tumor activity of a high-molecular-weight polysaccharide from cultured mycelium of Cordyceps gunnii,Carbohydrate Polymers,88(3):1072-1076
Zong A.Z.,Cao H.Z.,and Wang F.S.,2012,Anticancer polysaccharides from natural resources:A review of recent research,Carbohydr.Polym.,90(4):1395-1410
Chen W.,Lin X.H.,Chen J.,Luo H.B.,and Huang L.Y.,2005,Activation effects of polysaccharide from Aloe barbadensis on mouse peritoneal macrophages in vitro,Zhongguo Yaoxue Zazhi(Chinese Pharmaceutical Journal),40(1):34-37(陈伟,林新华,陈俊,罗红斌,黄丽英,2005,库拉索芦荟多糖对小鼠腹腔巨噬细胞的体外激活作用,中国药学杂志,40(1):34-37)
Dai Y.,Xu M.S.,Shangguan X.C.,Jiang Y.,Zheng G.D.,and Wang W.J.,2017,Immunoregulation effects of polysaccharides from pine needles on mouse peritoneal macropages,Dongwu Yingyang Xuebao(Chinese Journal of Animal Nutrition),29(2):670-677(戴艺,徐明生,上官新晨,蒋艳,郑国栋,王文君,2017,松针多糖对小鼠腹腔巨噬细胞免疫调节作用的研究,动物营养学报,29(2):670-677)
Hu Z.K.,Miao M.S.,and Liu H.L.,2010,Study on immunocompetence of the active constituent extracted from Radix Ranunculi Ternati,Liaoning Zhongyiyao Daxue Xuebao(Journal of Liaoning University of Traditional Chinese Medicine),12(3):24-26(胡泽开,苗明三,刘会丽,2010,中药猫爪草免疫活性部位的筛选,辽宁中医药大学学报,12(3):24-26)
Ji W.,Tang N.,Zhao D.,and Zhang B.J.,2016,The pharmacological effects on Hericium erinaceus and its cultivation and application,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),35(5):1252-1257(纪伟,唐宁,赵端,张边江,2016,猴头菇的药理作用及栽培与应用,基因组学与应用生物学,35(5):1252-1257)
Jia H.Q.,2006,The study on polysaccharides from Ranunculus ternatus Thunb.,Thesis for M.S.,Shaanxi Normal University,Supervisor:Zhang Z.Q.,pp.1-48(贾慧青,2006,猫爪草多糖的研究,硕士学位论文,陕西师范大学,导师:张志琪,pp.1-48)
Ke M.Y.,Wang H.,Zhang M.,Tian Y.W.,Wang Y.Z.,Li B.,Yu J.,Dou J.,Xi T.,and Zhou C.L.,2014,The anti-lung cancer activity of SEP is mediated by the activation and cytotoxicity of NK cells via TLR2/4 in vivo,Biochem.Pharmacol.,89(1):119-130
Koizumi K.,Hojo S.,Akashi T.,Yasumoto K.,and Saiki I.,2007,Chemokine receptors in cancer metastasis and cancer cell-derived chemokines in host immune response,Cancer Sci.,98(11):1652-1658
Liu J.,Wang J.M.,Ji B.,Zhang H.W.,and Zhao Q.T.,2012,Reseach on extraction process of crude polysaccharide from Radix Ranunculus Ternati,Zhongyi Xuebao(China Journal of Chinese Medicine),27(12):1622-1623(刘菊,王君明,纪彬,张红伟,赵群涛,2012,猫爪草粗多糖提取工艺研究,中医学报,27(12):1622-1623)
Lv X.H.,Wang H.M.,Han H.X.,Lv S.J.,and Qin D.Y.,2010,Effects of polysaccharide of Radix Ranunculi Ternati on immunomodulation and anti-oxidation,Zhongguo Zhongyao Zazhi(China Journal of Chinese Materia Medica),35(14):1862-1685(吕小华,王会敏,韩红霞,吕世静,覃冬云,2010猫爪草多糖免疫调节及抗氧化活性研究,中国中药杂志35(14):1862-1865)
Schepetkin I.A.,and Quinn M.T.,2006,Botanical polysaccharides:macrophage immunomodulation and therapeutic potential,Int.Immunopharmacol.,6(3):317-333
Shinchi H.,Crain B.,Yao S.Y.,Chan M.,Zhang S.S.,Ahmadiiveli A.,Suda Y.,Hayashi T.,Cottam H.B.,and Carson DA.,2015,Enhancement of the immunostimulatory activity of a TLR7 ligand by conjugation to polysaccharides,Bioconjug.Chem.,26(8):1713-1723
Sun D.L.,Xie H.B.and Xia Y.Z.,2013,A study on the inhibitory effect of polysaccharides from Radix Ranunculus Ternati on human breast cancer MCF-7 cell lines,Afr.J.Tradit.Complement.Altern.Med.,10(6):439-443
Sun L.Q.,Chu J.L.,Sun Z.L.,and Chen L.H.,2016,Physicochemical properties,immunomodulation and antitumor activities of polysaccharide from Pavlova viridis,Life Sci.144:156-161
Tuvaanjav S.,Han S.Q.,Komata M.,Ma C.J.,Kanamoto T.Nakashima H.,and Yoshida T.,2016,Isolation and antivira activity of water-soluble Cynomorium songaricum Rupr Polysaccharides,J.Asian Nat.Prod.Res.,18(2):159-171
Wan W.T.,Li N.,Liu J.,and Jin L.H.,2010,Comparative study on the optimal experiment conditions between CCK-8 and MTT in cell proliferation assay of PC3 cell,Shizhen Guoy Guoyao(Lishizhen Medicine and Materia Medica Research)21(12):3046-3048(万文婷,李宁,刘静,金林红,2010,CCK-8法与MTT法检测人前列腺癌PC3细胞活性的比较研究时珍国医国药,21(12):3046-3048)
Wang X.L.,Liao C.S.,Cheng Y.B.,Zhen Y.C.,and Cheng X.C.2014,Effects of glycyrrhizin,glycyrrhiza polysaccharide and glabridin on the toxicity and immunologic function of murine peritoneal macrophages,Zhongguo Shouyi Kexue(Chinese Veterinary Science),44(3):320-325(王晓利,廖成水,程源斌,甄永臣,程相朝,2014,甘草甜素甘草多糖和光甘草定对小鼠巨噬细胞的毒性与免疫功能的调节中国兽医科学,44(3):320-325)
Wang A.W.,Wang M.,Yuan J.R.,Tian J.K.,Wu L.M.,and Geng H.,2004,The study on antitumor effects in vitro of different extract in Radix Ranunculi Ternati,Tianran Chanwu Yanjiu Yu Kaifa(Natural Product research and Development),16(6):529-531(王爱武,王梅,袁久荣,田景奎,吴丽敏,耿晖,2004,猫爪草提取物体外抗肿瘤作用的研究,天然产物研究与开发,16(6):529-531)
Wang Z.Y.,Zhou F.,and Quan Y.,2014,Antioxidant and immunological activity in vitro of polysaccharides from Phellinus nigricans mycelia,International Journal of Biological Macromolecules,64(2):139-143
Yang D.P.,Yang X.X.,and Yu Y.,2017,Comparative study on the optimal experiment conditions between MTT assay and CCK-8 assay in rabbit retinal pigment epithelial cell,Zhongguo Minzu Minjian Yiyao(Chinese Journal of Ethnomedicine and Ethnopharmacy),26(6):31-33,45(杨冬萍,杨晓旭,俞洋,2017,MTT法与CCK-8法检测兔视网膜色素上皮细胞活性的比较研究,中国民族民间医药,26(6):31-33,45)
Zhang Z.L.,Wang L.,Wu X.J.,and Zhang H.X.,2007,The study about immunocompetence of polysaccharide of Radix Ranunculi Ternati,Shizhen Guoyi Guoyao(Lishizhen Medicine and Materia Medica Research),18(3):537-539(张振凌,王磊,吴筱菁,张惠霞,2007,中药猫爪草多糖的免疫活性研究,时珍国医国药,18(3):537-539)
Zhou L.J.,Wang Z.X.,Long T.T.,Zhou X.,and Bao Y.X.,2017,Roles of Astragalus polysaccharides in coculture system of breast cancer cells and macrophages in vitro,Mianyixue Zazhi(Immunological Journal),33(6):469-476(周丽菁,汪志学,龙婷婷,周星,鲍依稀,2017,黄芪多糖在体外巨噬细胞-乳腺癌细胞共培养体系中的作用,免疫学杂志,33(6):469-476)
Zhu Z.Y.,Liu N.,Si C.L.,Liu Y.,Ding L.N.,Jing C.,Liu A.J.,and Zhang Y.M.,2012,Structure and anti-tumor activity of a high-molecular-weight polysaccharide from cultured mycelium of Cordyceps gunnii,Carbohydrate Polymers,88(3):1072-1076
Zong A.Z.,Cao H.Z.,and Wang F.S.,2012,Anticancer polysaccharides from natural resources:A review of recent research,Carbohydr.Polym.,90(4):1395-1410