连翘对岭南湿热模型小鼠的作用及其肠道菌群变化的研究
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摘要
目的探究连翘对岭南湿热模型小鼠的作用及对模型小鼠肠道菌群的影响。方法将21只SPF级雄性BALB/c小鼠随机分为正常组、模型组、连翘组,每组7只。将模型组和连翘组放入人工气候箱[T:(30±0.15)℃,HR:80%～85%]每天12 h,隔日灌服白酒0.3 mL+跑步30 min,每日以高蛋白饲料喂养的"外湿+内湿+脾虚"的复合因素造模。造模49 d后,连翘组小鼠连续7 d进行连翘水溶液灌胃,模型组小鼠连续7 d超纯水灌胃。实验结束后用酶联免疫吸附法(ELISA)对各组小鼠血清、肿瘤坏死因子α(TNF-α)、干扰素γ(INF-γ)及内毒素(LPS)进行检测,对各组小鼠舌、小肠组织进行病理学检测,对粪便进行16s rRNA检测。结果与正常组比较,模型组小鼠血清TNF-α、IFN-γ、LPS含量增加(P <0.01,P <0.001);舌组织黏膜层角化增加并且有明显的过角质化突起,固有层结缔组织破坏明显;小肠组织黏膜层上皮细胞被破坏,固有层腺体损伤;肠道菌群结构发生紊乱,拟杆菌门(Bacteroidetes)、变形菌门(Proteobacteria)所占肠道菌群的比例均有所增加(P <0.05),厚壁菌门(Firmicutes)所占比例减少(P <0.01);肠道有益菌普氏菌属(Prevotella)和颤螺菌属(Oscillospira)含量减少(P <0.05,P <0.01),致病菌大肠杆菌属(Escherichia)含量增加(P <0.05)。与模型组比较,连翘组小鼠血清TNF-α的含量有减少趋势,IFN-γ的含量减少明显(P <0.01),但血清LPS含量没有显著变化;舌组织黏膜层过角化突起没有改善,舌乳头排列较均匀,固有层形态规整;小肠组织黏膜层上皮细胞结构清晰,排列规则,固有层腺体结构清晰;肠道菌群结构改善,拟杆菌门所占比例降低(P <0.05),厚壁菌门所占比例升高(P <0.01);肠道有益菌普氏菌属和颤螺菌属含量增加(P <0.05),致病菌螺杆菌属(Helicobacter)含量降低(P <0.05)。结论连翘对中医湿热证有积极的治疗作用,能够治疗炎症,改善舌和小肠组织病变,降低肠道致病菌的含量并且对肠道菌群结构有调节作用。
Objective To explore the effects of Forsythiae fructus on Lingnan damp-heat model mice and their intestinal florae. Methods Twenty-one SPF male BALB/c mice were randomly divided into normal group, model group and Forsythiae fructus group with 7 mice in each group. The model group and Forsythiae fructus group were fed with liquor 0.3 mL + running for 30 minutes every other day during the stay for 12 hours per day in an artificial climate box [T:(30±0.15)℃, RH: 80%-85%]. To establish the models of compound"external dampness +internal dampness + spleen deficiency",mice were fed with high protein diet. The mice in Forsythiae fructus group were intragastrically administered Forsythiae fructus solution for 7 consecutive days after 49 days of modeling;at the same time,the model group mice were given ultrapure water for 7 consecutive days. At the end of the experiment,the tongue and small intestine tissues of mice in each group were taken for pathological examination. The serum inflammatory factor interferon-gamma(INF-γ),tumor necrosis factor-alpha(TNF-α)and endotoxin(LPS)of mice in each group were detected by enzyme-linked immunosorbent assay(ELISA),and the feces were used for 16 S rRNA detection. Results Compared with the normal group,the serum levels of TNF-α,IFN-γ and LPS in the model group increased(P < 0.01,P < 0.001);the keratinization of tongue mucosa increased and there were obvious hyperkeratinization processes,and the destruction of lamina propria connective tissue was obvious;the epithelial cells of intestinal mucosa and lamina propria glands were damaged;the structure of intestinal flora was disordered,the proportion of Bacteroidetes and Proteobacteria increased(P < 0.05),Firmicutes decreased(P < 0.01);the proportion of Prevotella and Oscillospira decreased(P < 0.05,P < 0.01),and that of Escherichia increased(P < 0.05). Compared with the model group,the content of TNF-α in serum of mice in Forsythiae fructus group tended to decrease and the serum IFN-γ content decreased significantly(P < 0.01),but the serum LPS content did not change significantly;hyperkeratosis of tongue mucosa was not alleviated, papillae were arranged uniformly and lamina propria was regular;the structure of intestinal mucosa epithelial cells was clear and regular,lamina propria gland structure was clear; the structure of intestinal flora was improved, the proportion of Bacteroides decreased(P < 0.05), the proportion of Firmicutes increased(P < 0.01);the contents of Prevotella and Oscillospira increased(P < 0.05),and that of Helicobacter decreased(P < 0.05). Conclusion Forsythiae fructus has a positive therapeutic effect on dampheat syndrome of traditional Chinese medicine. It can treat inflammation,improve the pathological changes of tongue and small intestine,reduce the content of intestinal pathogenic bacteria and regulate the structure of intestinal flora.
Objective To explore the effects of Forsythiae fructus on Lingnan damp-heat model mice and their intestinal florae. Methods Twenty-one SPF male BALB/c mice were randomly divided into normal group, model group and Forsythiae fructus group with 7 mice in each group. The model group and Forsythiae fructus group were fed with liquor 0.3 mL + running for 30 minutes every other day during the stay for 12 hours per day in an artificial climate box [T:(30±0.15)℃, RH: 80%-85%]. To establish the models of compound"external dampness +internal dampness + spleen deficiency",mice were fed with high protein diet. The mice in Forsythiae fructus group were intragastrically administered Forsythiae fructus solution for 7 consecutive days after 49 days of modeling;at the same time,the model group mice were given ultrapure water for 7 consecutive days. At the end of the experiment,the tongue and small intestine tissues of mice in each group were taken for pathological examination. The serum inflammatory factor interferon-gamma(INF-γ),tumor necrosis factor-alpha(TNF-α)and endotoxin(LPS)of mice in each group were detected by enzyme-linked immunosorbent assay(ELISA),and the feces were used for 16 S rRNA detection. Results Compared with the normal group,the serum levels of TNF-α,IFN-γ and LPS in the model group increased(P < 0.01,P < 0.001);the keratinization of tongue mucosa increased and there were obvious hyperkeratinization processes,and the destruction of lamina propria connective tissue was obvious;the epithelial cells of intestinal mucosa and lamina propria glands were damaged;the structure of intestinal flora was disordered,the proportion of Bacteroidetes and Proteobacteria increased(P < 0.05),Firmicutes decreased(P < 0.01);the proportion of Prevotella and Oscillospira decreased(P < 0.05,P < 0.01),and that of Escherichia increased(P < 0.05). Compared with the model group,the content of TNF-α in serum of mice in Forsythiae fructus group tended to decrease and the serum IFN-γ content decreased significantly(P < 0.01),but the serum LPS content did not change significantly;hyperkeratosis of tongue mucosa was not alleviated, papillae were arranged uniformly and lamina propria was regular;the structure of intestinal mucosa epithelial cells was clear and regular,lamina propria gland structure was clear; the structure of intestinal flora was improved, the proportion of Bacteroides decreased(P < 0.05), the proportion of Firmicutes increased(P < 0.01);the contents of Prevotella and Oscillospira increased(P < 0.05),and that of Helicobacter decreased(P < 0.05). Conclusion Forsythiae fructus has a positive therapeutic effect on dampheat syndrome of traditional Chinese medicine. It can treat inflammation,improve the pathological changes of tongue and small intestine,reduce the content of intestinal pathogenic bacteria and regulate the structure of intestinal flora.
引文
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[20]SATOKARI R M,VAUGHAN E E,AKKERMANS A D,et al.Bifidobacterial diversity in human feces detected by genus-specific PCR and denaturing gradient gel electrophoresis[J].Appl Environ Microbiol,2001,67(2):504-513.
[21]KOVATCHEVA-DATCHARY P,NILSSON A,AKRAMI R,et al.Dietary fiber-induced improvement in glucose metabolism is associated with increased abundance of prevotella[J].Cell Metab,2015,22(6):971-982.
[22]ANA I L,FILIPA F V,MóNICA O,et al.Helicobacter pylori infection-recent developments in diagnosis[J].World J Gastroenterol,2014,20(28):9299-9313.
[23]RAJILI-STOJANOVI M,HEILIG H G,TIMS S,et al.Long-term monitoring of the human intestinal microbiota composition[J].Environ Microbiol,2013,15:1146-1159.
[24]MACFARLANE G T,ALLISON C,GIBSON S A,ET AL.Contribution of the microflora to proteolysis in the human large intestine[J].J Appl Bacteriol,1988,64(1):37-46.
[25]ALBERT M J,ALAM K,ISLAM M,et al.Hafnia alvei,a probable cause of diarrhea in humans[J].Infect Immun,1991:59(4):1507-1513.
[26]KONIKOFF T,GOPHNA U.Oscillospira:a central,enigmatic component of the human gut microbiota[J].Trends Microbiol,2016,24(7):523-524.
[2]国家药典委员会.中华人民共和国药典[M].北京:中国医药科技出版社,2015:170-171.
[3]WANG H,ZHANG W,ZUO L,et al.Intestinal dysbacteriosis contributes to decreased intestinal mucosal barrier function and increased bacterial translocation[J].Lett Appl Microbiol,2014,58(4):384-392.
[4]吕冠华,劳绍贤.脾胃湿热证动物模型的建立与评价[J].广州中医药大学学报,2005,22(3):231-235.
[5]谷莉.不同鼠种的肠道菌群在不同饮食结构干预中的组成改变[D].长沙:中南大学,2014.
[6]黄继汉,黄晓晖,陈志扬,等.药理试验中动物间和动物与人体间的等效剂量换算[J].中国临床药理学与治疗学,2004,(9):1069-1072.
[7]王瑾,陈宜鸿,赵志玲.中医温病湿热证动物模型实验的研究[J].解放军药学学报,2002,18(4):209-211.
[8]阎翔,郭明阳.温病湿热证湿重于热动物模型舌的病理观察[J].安徽中医学院学报,2005,24(1):19-21.
[9]李学,魏连波,罗炳德,等.中医大肠湿热证模型SD大鼠肠道的病理变化[J].河北中医药学报,2004,19(1):4-6.
[10]MA T Y,IWAMOTO G K,HOA N T,et al.TNF-α-induced increase in intestinal epithelial tight junction permeability requires NF-κB activation[J].Am J Physiol Gastrointest Liver Physiol,2004,286:G367-G376.
[11]ADAMS R B,PLANCHON S M,ROCHE J K.IFN-gamma modulation of epithelial barrier function.Time course,reversibility,and site of cytokine binding[J].J Immunol,1993(150):2356-2363.
[12]YUE C,MA B,ZHAO Y,et al.Lipopolysaccharide-induced bacterial translocation is intestine site-specific and associates with intestinal mucosal inflammation[J].Inflammation,2012,35(6):1880-1888.
[13]BISCHOFF SC,BARBARA G,BUURMAN W,et al.Intestinal permeability--a new target for disease prevention and therapy[J].BMC Gastroenterol,2014(14):189.
[14]LEE J J,KIM K H,KIM E J,et al.Anti-inflammatory activity of the decoction of Forsythia suspensa(Thunb.)Vahl is related to Nrf2and A20[J].J Ethnopharmacol,2018,227:97-104.
[15]EMILY B,KEVIN R,RUTH A L.Structure and function of the healthy pre-adolescent pediatric gut microbiome[J].Microbiome,2015,3:36.
[16]BACKHED F,LEY RE,SONNENBURG JL,et al.Host-bacterial mutualism in the human intestine[J].Science,2005,307(5717):1915-1920.
[17]KELLY D,CONWAY S,AMINOV R.Commensal gut bacteria:mechanisms of immune modulation[J].Trends Immunol,2005,26(6):326-333.
[18]RASHID M U,WEINTRAUB A,Nord C E.Effect of new antimicrobial agents on the ecological balance of human microflora[J].Anaerobe,2012:249-253.
[19]MIRJANA R S,WILLEM M V.The first 1000 cultured species of the human gastrointestinal microbiota[J].FEMS Microbiol Rev,2014,38(5):996-1047.
[20]SATOKARI R M,VAUGHAN E E,AKKERMANS A D,et al.Bifidobacterial diversity in human feces detected by genus-specific PCR and denaturing gradient gel electrophoresis[J].Appl Environ Microbiol,2001,67(2):504-513.
[21]KOVATCHEVA-DATCHARY P,NILSSON A,AKRAMI R,et al.Dietary fiber-induced improvement in glucose metabolism is associated with increased abundance of prevotella[J].Cell Metab,2015,22(6):971-982.
[22]ANA I L,FILIPA F V,MóNICA O,et al.Helicobacter pylori infection-recent developments in diagnosis[J].World J Gastroenterol,2014,20(28):9299-9313.
[23]RAJILI-STOJANOVI M,HEILIG H G,TIMS S,et al.Long-term monitoring of the human intestinal microbiota composition[J].Environ Microbiol,2013,15:1146-1159.
[24]MACFARLANE G T,ALLISON C,GIBSON S A,ET AL.Contribution of the microflora to proteolysis in the human large intestine[J].J Appl Bacteriol,1988,64(1):37-46.
[25]ALBERT M J,ALAM K,ISLAM M,et al.Hafnia alvei,a probable cause of diarrhea in humans[J].Infect Immun,1991:59(4):1507-1513.
[26]KONIKOFF T,GOPHNA U.Oscillospira:a central,enigmatic component of the human gut microbiota[J].Trends Microbiol,2016,24(7):523-524.