加味四君子汤对脾气虚犬血清细胞因子水平、肠道免疫球蛋白水平以及脾脏组织形态及功能的影响
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摘要
【目的】研究加味四君子汤对脾气虚犬免疫机能的影响.【方法】将24只1岁龄健康‘比格犬’随机均分为对照组、脾气虚组、自然恢复组和加味四君子汤组,用番泻叶药液复制犬脾气虚模型.第7 d造模成功,脾气虚组犬处死采样,对照组和自然恢复组犬饲喂基础日粮,加味四君子汤组犬在基础日粮中添加2%加味四君子汤.试验期14 d.第0、7、14天,采集血液,采用酶联免疫吸附法测定血清中干扰素(IFN)-γ、白细胞介素(IL)-2、IL-4、IL-6水平;在第14天,全部试验犬处死采样,计算脾脏指数,测定脾脏组织中促吞噬肽(Tuftsin)水平,观察脾脏显微和超微结构的变化;免疫组织化学(IHC)检测小肠各段分泌型免疫球蛋白A(sIgA)蛋白阳性表达水平,荧光定量PCR检测小肠各段免疫球蛋白A(IgA)、IgG、IgM mRNA相对表达量.【结果】与对照组犬相比,脾气虚犬血清细胞因子IL-4、IL-6水平显著升高(P<0.05),IFN-γ、IL-2水平显著降低(P<0.05);脾脏指数、脾脏Tuftsin水平、小肠sIgA蛋白阳性表达水平以及IgA、IgG、IgM mRNA的相对表达量均显著或极显著降低(P<0.05,P<0.01).经加味四君子汤治疗后,脾气虚犬上述指标和脾脏组织结构恢复正常.【结论】脾气虚犬免疫功能低于对照组犬,饲粮中添加加味四君子汤可以显著改善脾气虚症状,增强机体免疫机能.
【Objective】 To study the effect of supplementary Sijunzi decoction(SJZD) on immune function of spleen qi deficiency canines.【Method】 24 one-year-old healthy beagle canines were randomly divided into control group,spleen qi deficiency group,natural recovery group and supplementary SJZD group.The senna solution was used to replicate spleen qi deficiency model canines.The spleen qi deficiency model was established on 7 d,then the canines in spleen qi deficiency group were killed for collecting samples.The canines in the control group and the natural recovery group were fed a basal diet,the canines in supplementary SJZD group were fed a basal diet with 2% supplementary SJZD.The trial lasted for 14 d.The blood samples were taken at the 0,7 and 14 d,and the serum levels of IFN-γ,IL-2,IL-4,IL-6 were detected by enzyme linked immunosorbent assay(ELISA).On the 14 d,all the canines were killed for collecting samples,and the spleen index was calculated,the spleen Tuftsin level was detected,the microscopic and ultrastructural changes in spleen were observed.In small intestine,the protein positive expression level of sIgA was measured by immunohistochemistry(IHC),and the relative expression values of IgA,IgG and IgM mRNA were measured by fluorescent quantitative PCR.【Result】 Compare with the control group canines,the levels of serum cytokines IL-4 and IL-6 in the spleen qi deficiency canines were significantly increased(P<0.05) and the levels of IFN-γand IL-2 were significantly decreased(P<0.05).The spleen index,spleen Tuftsin level and the protein positive expression level of sIgA and the relative expression values of IgA,IgG and IgM mRNA in small intestine were all significantly or extremely significantly decreased(P<0.05,P<0.01).After treatment with supplementary SJZD,the above indexes and spleen tissue structure of the spleen qi deficiency canines returned to normal.【Conclusion】 The immune function of the spleen qi deficiency canines decreased than the control group canines,the basal diet with supplementary can significantly improve the symptoms of spleen qi deficiency and enhance the body's immune function.
【Objective】 To study the effect of supplementary Sijunzi decoction(SJZD) on immune function of spleen qi deficiency canines.【Method】 24 one-year-old healthy beagle canines were randomly divided into control group,spleen qi deficiency group,natural recovery group and supplementary SJZD group.The senna solution was used to replicate spleen qi deficiency model canines.The spleen qi deficiency model was established on 7 d,then the canines in spleen qi deficiency group were killed for collecting samples.The canines in the control group and the natural recovery group were fed a basal diet,the canines in supplementary SJZD group were fed a basal diet with 2% supplementary SJZD.The trial lasted for 14 d.The blood samples were taken at the 0,7 and 14 d,and the serum levels of IFN-γ,IL-2,IL-4,IL-6 were detected by enzyme linked immunosorbent assay(ELISA).On the 14 d,all the canines were killed for collecting samples,and the spleen index was calculated,the spleen Tuftsin level was detected,the microscopic and ultrastructural changes in spleen were observed.In small intestine,the protein positive expression level of sIgA was measured by immunohistochemistry(IHC),and the relative expression values of IgA,IgG and IgM mRNA were measured by fluorescent quantitative PCR.【Result】 Compare with the control group canines,the levels of serum cytokines IL-4 and IL-6 in the spleen qi deficiency canines were significantly increased(P<0.05) and the levels of IFN-γand IL-2 were significantly decreased(P<0.05).The spleen index,spleen Tuftsin level and the protein positive expression level of sIgA and the relative expression values of IgA,IgG and IgM mRNA in small intestine were all significantly or extremely significantly decreased(P<0.05,P<0.01).After treatment with supplementary SJZD,the above indexes and spleen tissue structure of the spleen qi deficiency canines returned to normal.【Conclusion】 The immune function of the spleen qi deficiency canines decreased than the control group canines,the basal diet with supplementary can significantly improve the symptoms of spleen qi deficiency and enhance the body's immune function.
引文
[1] 曾荣华,周露,欧阳厚淦,等.针刺‘足三里’穴对脾虚证模型大鼠肠系膜淋巴结T淋巴细胞亚群的影响[J].中国组织工程研究,2018,22(4):576-581.
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[8] 沈莹,高啸,覃艳琼,等.加味四君子汤通过mTOR信号通路延缓小鼠脑组织衰老的机制研究[J].华中科技大学学报(医学版),2018,47(4):417-420.
[9] 曹立亭,彭代国,张媛媛,等.黄芪多糖与庆大霉素肌肉注射治疗犬脾虚泄泻证的比较[J].中国兽医学报,2014,34(2):293-296.
[10] 何敏,伍春,明海霞,等.甘肃党参水煎剂对D-半乳糖诱导衰老小鼠免疫功能的影响[J].细胞与分子免疫学杂志,2013,29(8):794-797.
[11] Mebius R E,Kraal G.Structure and function of the spleen[J].Nature Reviews Immunology,2005,5(8):606-616.
[12] Nolte M A,Beliёn J A,Schadee-Eestermans I,et al.A conduit system distributes chemokines and small blood?borne molecules through the splenic white pulp[J].J Exp Med,2003,198(3):505-512.
[13] Gao Y L,Chai Y F,Dong N,et al.Tuftsin-derived T-peptide prevents cellular immunosuppression and improves survival rate in septic mice[J].Scientific Reports,2015,5,16725.
[14] Dzierzbicka K,Wardowska A,Rogalska M,et al.New conjugates of muramyl dipeptide and nor-muramyl dipeptide linked to tuftsin and retro-tuftsin derivatives significantly influence their biological activity[J].Pharmacological Reports,2012,64(1):217-223.
[15] Siebert A,Cholewinski G,Dzierzbicka K,et al.Tuftsin-properties and analogs[J].Current Medicinal Chemistry,2017,24:1-18.
[16] 孙佳鑫,侯明星.促吞噬肽免疫学机制的研究进展[J].世界最新医学信息文摘,2018,18(16):53-54,57.
[17] 叶文斌,樊亮,王昱,等.黄果槲寄生果实多糖对环磷酰胺诱导免疫低下小鼠免疫功能影响[J].甘肃农业大学学报,2018,53(1):1-7,14.
[18] 丁杨,朱萱萱,郁晓维.运脾温阳颗粒及拆方含药血清对正常和脾虚小鼠脾淋巴细胞增殖的影响[J].实用中医内科杂志,2008(5):65-66.
[19] 廖乐乐,罗翱翔,唐干益等.四君子汤对大黄脾虚模型小鼠的影响[J].广东药学院学报,2013,29(2):202-205.
[20] Wang R J,Peng Y,Meng H,et al.Protective effect of polysaccharides fractions from Sijunzi decoction in reserpine-induced spleen deficiency rats[J].RSC Advances,2016,6(65):60657-60665.
[21] 金钟晔,李娜,赵宏波,等.逍遥散对慢性束缚应激肝郁脾虚证焦虑模型大鼠血清IL-1β、IL-6、TNF-α的影响[J].中医学报,2016,31(6):822-826.
[22] 韩晓春,季旭明,王彦芳,等.细胞因子对脾虚水湿不化大鼠模型影响的曲线拟合分析[J].世界科学技术-中医药现代化,2015,17(12):2564-2569.
[23] 杨冬花,李家邦,郑爱华,等.脾气虚证模型大鼠Th1/Th2细胞因子的失衡以及四君子汤的干预作用[J].中国医师杂志,2004,6(2):181-183.
[24] 伍杨,邓明会.长叶胡颓子对荷瘤鼠细胞免疫功能影响[J].中国公共卫生,2010,26(5):576-577.
[25] 戈娜,袁慧.肠道免疫屏障功能损伤的研究进展[J].广东畜牧兽医科技,2008(1):9-11.
[26] Sekirov I,Russell S L,Antunes L C M,et al.Gut microbiota in health and disease[J].Physiological Reviews,2010,90(3):859-904.
[2] 曹森林,范利锋,王楠,等.加味三香汤联合复方消化酶治疗对肝郁脾虚型功能性消化不良患者疗效及焦虑抑郁状态的影响[J].中国中西医结合消化杂志,2018,26(1):83-86.
[3] Kennedy P J,Clarke G,Quigley E M M,et al.Gut memories:towards a cognitive neurobiology of irritable bowel syndrome[J].Neuroscience & Biobehavioral Reviews,2012,36(1):310-340.
[4] 王玉杰,谢鸣,杨柳倩,等.疏肝、健脾、疏肝健脾方对肝郁脾虚证模型大鼠T3、T4、TRH、TSH的影响[J].北京中医药大学学报,2010,33(6):381-384.
[5] 朱慧渊,王江,苗琦.基于“脾主运化”理论仙鹤草调控线粒体能量代谢抗脑缺血损伤的探讨[J].中华中医药学刊,2015,33(12):2929-2931.
[6] 吕林,王凤云,唐旭东,等.脾虚型功能性消化不良大鼠胃组织GRP78/BiP蛋白表达及脾虚1号方干预研究[J].中国中西医结合杂志,2018,38(1):54-59.
[7] 李瑞芝,马浩清,金晓董,等.加味四君子汤对结肠癌模型大鼠AQP-1、VEGF、COX-2表达的影响[J].江苏中医药,2018,50(4):84-87.
[8] 沈莹,高啸,覃艳琼,等.加味四君子汤通过mTOR信号通路延缓小鼠脑组织衰老的机制研究[J].华中科技大学学报(医学版),2018,47(4):417-420.
[9] 曹立亭,彭代国,张媛媛,等.黄芪多糖与庆大霉素肌肉注射治疗犬脾虚泄泻证的比较[J].中国兽医学报,2014,34(2):293-296.
[10] 何敏,伍春,明海霞,等.甘肃党参水煎剂对D-半乳糖诱导衰老小鼠免疫功能的影响[J].细胞与分子免疫学杂志,2013,29(8):794-797.
[11] Mebius R E,Kraal G.Structure and function of the spleen[J].Nature Reviews Immunology,2005,5(8):606-616.
[12] Nolte M A,Beliёn J A,Schadee-Eestermans I,et al.A conduit system distributes chemokines and small blood?borne molecules through the splenic white pulp[J].J Exp Med,2003,198(3):505-512.
[13] Gao Y L,Chai Y F,Dong N,et al.Tuftsin-derived T-peptide prevents cellular immunosuppression and improves survival rate in septic mice[J].Scientific Reports,2015,5,16725.
[14] Dzierzbicka K,Wardowska A,Rogalska M,et al.New conjugates of muramyl dipeptide and nor-muramyl dipeptide linked to tuftsin and retro-tuftsin derivatives significantly influence their biological activity[J].Pharmacological Reports,2012,64(1):217-223.
[15] Siebert A,Cholewinski G,Dzierzbicka K,et al.Tuftsin-properties and analogs[J].Current Medicinal Chemistry,2017,24:1-18.
[16] 孙佳鑫,侯明星.促吞噬肽免疫学机制的研究进展[J].世界最新医学信息文摘,2018,18(16):53-54,57.
[17] 叶文斌,樊亮,王昱,等.黄果槲寄生果实多糖对环磷酰胺诱导免疫低下小鼠免疫功能影响[J].甘肃农业大学学报,2018,53(1):1-7,14.
[18] 丁杨,朱萱萱,郁晓维.运脾温阳颗粒及拆方含药血清对正常和脾虚小鼠脾淋巴细胞增殖的影响[J].实用中医内科杂志,2008(5):65-66.
[19] 廖乐乐,罗翱翔,唐干益等.四君子汤对大黄脾虚模型小鼠的影响[J].广东药学院学报,2013,29(2):202-205.
[20] Wang R J,Peng Y,Meng H,et al.Protective effect of polysaccharides fractions from Sijunzi decoction in reserpine-induced spleen deficiency rats[J].RSC Advances,2016,6(65):60657-60665.
[21] 金钟晔,李娜,赵宏波,等.逍遥散对慢性束缚应激肝郁脾虚证焦虑模型大鼠血清IL-1β、IL-6、TNF-α的影响[J].中医学报,2016,31(6):822-826.
[22] 韩晓春,季旭明,王彦芳,等.细胞因子对脾虚水湿不化大鼠模型影响的曲线拟合分析[J].世界科学技术-中医药现代化,2015,17(12):2564-2569.
[23] 杨冬花,李家邦,郑爱华,等.脾气虚证模型大鼠Th1/Th2细胞因子的失衡以及四君子汤的干预作用[J].中国医师杂志,2004,6(2):181-183.
[24] 伍杨,邓明会.长叶胡颓子对荷瘤鼠细胞免疫功能影响[J].中国公共卫生,2010,26(5):576-577.
[25] 戈娜,袁慧.肠道免疫屏障功能损伤的研究进展[J].广东畜牧兽医科技,2008(1):9-11.
[26] Sekirov I,Russell S L,Antunes L C M,et al.Gut microbiota in health and disease[J].Physiological Reviews,2010,90(3):859-904.