【作者】 赵兴兵；

【导师】 蔡光先；

【作者基本信息】 湖南中医药大学 ， 中医内科学， 2014， 博士

【摘要】 目的：研究小鼠脾虚便秘造模对肠道微生物、酶活及血常规的影响,为脾虚便秘的治疗提供基础；观察超微铁皮石斛对脾虚便秘的疗效,探明超微铁皮石斛对脾虚便秘小鼠肠道微生物、酶活性和血常规的影响,及对脾虚便秘肠道细菌和乳酸杆菌多样性的影响,为其疗效机理提供依据。方法：采用灌胃番泻叶水煎液7d,然后控制饮食,饥饱失常喂养8天；正常组灌胃等量的无菌水7d后再正常喂养8天；共15d,制备小鼠脾虚便秘模型。造模成功后,分析小鼠体重、肠道菌群和酶活性；通过眼球采血,用CA-500血液自动分析仪进行血常规检测。制备小鼠脾虚便秘模型,治疗组分别灌胃给药铁皮石斛传统汤剂、铁皮石斛超微全量汤剂、铁皮石斛超微50%量汤剂、铁皮石斛超微25%量汤剂和四磨汤口服液,正常组和模型组灌胃等量无菌水,共治疗7d,然后采集肠道内容物分析肠道菌群及酶活；正常组、模型组、铁皮石斛传统汤剂组和铁皮石斛超微50%量汤剂组均通过眼球采血,用CA-500血液自动分析仪进行血常规检测。制备小鼠脾虚便秘模型,铁皮石斛传统汤剂组和铁皮石斛超微50%量汤剂组治疗组分别灌胃给药7d,正常组和模型组灌胃等量无菌水,采集肠道内容物,提取肠道微生物宏基因组,经细菌通用引物和乳酸杆菌特异引物PCR扩展后进行其多样性(?)RDRA分析。结果：脾虚便秘造模后,模型组小鼠体重、体重变化率均显著小于正常组(P<0.05或P<0.01)；肠道细菌总数显著小于正常组(P<0.01),大肠杆菌、乳酸菌、双歧杆菌数和真菌均显著大于正常组(P<0.05或P<0.01)；肠道淀粉酶和蛋白酶活性显著低于正常组(P<0.05或P<0.01),木聚糖酶和纤维素酶则显著高于正常组(P<0.01)。脾虚便秘造模使得小鼠血小板数量及平均体积均显著小于正常组(P<0.05),白细胞数量显著大于正常组(P<0.05),其余各项指标均与正常组差异无统计学意义(P>0.05)。造模成功的脾虚便秘小鼠经铁皮石斛治疗后,模型组脾虚便秘症状无显著改善,体重变化和体重变化率较正常组低,肠道微生物(细菌总数、大肠杆菌、乳酸菌、双歧杆菌和真菌)、纤维素酶活性、木聚糖酶活性、淀粉酶活性和蛋白酶活性与正常组存在显著差异(P<0.01或P<0.05)；各治疗组的脾虚便秘症状均有不同程度的改善,乳酸菌和双歧杆菌数均高于正常组(P<0.01或P<0.05),大肠杆菌数除铁皮石斛超微全量汤剂组极显著高于正常组外(P<0.01),其余各治疗组均较低至正常组水平(P>0.05)；铁皮石斛超微50%量汤剂组和四磨汤组真菌数均达到正常组水平(P>0.05)；铁皮石斛超微全量汤剂组的纤维素酶活性及淀粉酶活性、超微50%量汤剂组纤维素酶活性、超微25%量汤剂组和四磨汤组蛋白酶活性均达到正常组水平(P>0.05),各治疗组的木聚糖酶活性均极显著低于正常组(P<0.01)。经铁皮石斛治疗后,血小板数量和比容显著高于正常组(P<0.01或P<0.05)；增加红细胞数量和比容；降低红细胞体积分布宽度；血红蛋白含量、白细胞和淋巴细胞数量均降低；红细胞平均血红蛋白含量和浓度均降低,但仍显著高于正常组(P<0.01或P<0.05)。超微50%汤剂组红细胞数量和比容显著高于模型组(P<0.05),达到正常组水平(P>0.05)；白细胞和淋巴细胞数量显著低于模型组和传统汤剂组(P<0.01或P<0.05),与正常组差异不显著(P>0.05)。超微铁皮石斛对脾虚便秘小鼠肠道细菌多样性影响的研究结果显示,正常组和铁皮石斛超微50%量汤剂组、模型组和铁皮石斛传统汤剂组的OTUs、Shanon指数和Brillouin指数分别相同,且前两组均大于后两组；铁皮石斛超微50%量汤剂组与正常组群落结构的相似性系数最大,模型组和传统汤剂组较小；当聚类距离≥15且<25时,各组小鼠肠道肠道菌群被聚为2类,正常组和铁皮石斛超微50%量汤剂组为1类,其余两组为1类；当聚类距离<15时,各组小鼠肠道菌群被聚为4类,每组为一类；经主成分分析,正常组和铁皮石斛超微50%量汤剂治疗组对PC1的贡献率较大,模型组和铁皮石斛传统汤剂治疗组对PC2的贡献率相同。超微铁皮石斛对脾虚便秘小鼠肠道乳酸杆菌多样性影响的研究结果显示,正常组、铁皮石斛传统汤剂组和超微50%量汤剂组的OTUs、Shanon指数和Brillouin指数相同,且均大于模型组；铁皮石斛超微50%量汤剂组与正常组乳酸杆菌群落结构的相似性系数最大,为0.3333,其次是模型组为0.1818,传统汤剂组最小,为0.1667；聚类分析和主成分分析结果显示,小鼠肠道乳酸杆菌多样性受脾虚便秘造模影响,发生改变,铁皮石斛两种汤剂通过不同的途径对其进行调控作用。结论：小鼠脾虚便秘造模影响小鼠的体重,破坏了肠道微生物的平衡,并影响肠道酶活性；显著影响血小板数量及平均体积、白细胞数量,影响血液系统的功能。铁皮石斛传统汤剂、超微全量汤剂、超微50%量汤剂和四磨汤对脾虚便秘均有疗效,通过不同的途径调控肠道微生态平衡,调整肠道酶活性,且铁皮石斛超微50%量汤剂、传统汤剂与四磨汤效果相当。经铁皮石斛治疗后,脾虚便秘小鼠的血小板数量及比容、红细胞数量及体积分布宽度、血红蛋白数量及红细胞平均血红蛋白含量和浓度、白细胞及淋巴细胞均有改善,超微50%量汤剂总体疗效优于传统汤剂。脾虚便秘造模使小鼠肠道细菌和乳酸杆菌的多样性均遭到破坏,经铁皮石斛两种汤剂治疗,具有一定的调整作用,且超微50%量汤剂组小鼠肠道细菌和乳酸杆菌多样性更接近正常组,疗效更优。更多还原

【Abstract】 Objective:To study the effect of spleen-deficiency constipation model made on the intestinal microbiota,enzyme activities and blood routine in mice, provide basis on the treatment of spleen-deficiency constipation.The relationship between efficacy of ultra-micro powder Dendrobium officinale on the intestinal microbiota, enzyme activities, blood routine,molecular diversity of intestinal Bacteria and Lactobacillus in mice with spleen-deficiency constipation was studied to provide the basis on its mechanism of action.Methods:The mouse models of spleen-deficiency constipation were established with senna water decoction7days, then diet was restricted, to make them hunger satiety disordered. The mouse of nomal group were were given equivalent amount of sterile water, then were fed normally. The models were made in total15days. The body weight, intestinal microbiota, enzyme activities and blood routine were snalysed.The treatment groups were respectively given the traditional decoction of Dendrobium officinale,the full amount,50%amount,25%amount of ultra-micro powder Dendrobium officinale,Simo decoction,the normal group and the model group were given equivalent amount of sterile water.The intestinal microbiota enzyme and activities were snalysed after7days. Then treatment groups were respectively given the traditional decoction of Dendrobium officinale,50%amount of ultra-micro powder Dendrobium officinale,the normal group and the model group were given equivalent amount of sterile water. Adopted the eyeball blood collection method, and used the CA-500automated hematology analyzer for determination.The mouse with spleen-deficiency were constipation in treatment groups respectively given the traditional decoction of Dendrobium officinale,50%amount of ultra-micro powder Dendrobium officinale.The metagenome DNA of intestinal flora was extracted from intestinal contents. Then ARDRA analysis was made after PCR by universal primer of Bacteria and specific primer of Lactobacillus.Results:The results showed that amount and rate of the body weight change of mice in the model group were smaller than those in the normal group (P<0.05or P<0.01), compared to normal group the amount of bacteria was reduced (P<0.01), the amount of Escherichia coli, Lactobacillus, Bifidobacterrium, Fungus were increased. The activities of amylase, protease were receded (PO.05or PO.01), but xylase, cellulase were enhanced compared to normal group mice were different from the mouse in normal group (PO.01). The mice with spleen-deficiency constipation whose PLT (Platelet) and MPV (Mean Platelet Volume) were significantly lower than normal group (P<0.05). And WBC (White Blood Cell) was significantly higher than normal group (PO.05). The other indexes had no significant difference with normal group (P>0.05).Spleen-deficiency constipation had no significant improvement in model group, amount and rate of the body weight change of mouse were smaller than those in the normal group,the intestinal microbiota(ba.cteria,Escherichia coli, lactobacillus, bifidobacterrium, fungus) and the cellulase, xylase, amylase, protease were had significant difference with the normal group(PO.01or P<0.05).The spleen-deficiency constipation had significant improvement in all treatment groups,and whose lactobacillus,bifidobacterrium were more than the normal group(PO,01or PO.05). Escherichia coli reached the level of normal group in all treatment groups except the full amount of ultra-micro powder Dendrobium offidnale(P<Q.05). The fungus reached the level of normal group in50%amount of ultra-micro powder Dendrobium officnaler group and Simo decoction group.The activities of cellulase and amylase in full amount of ultra-micro powder Dendrobium officnaler group, cellulase in50%amount of ultra-micro powder Dendrobium officnaler group, protease in25%amount of ultra-micro powder Dendrobium officnaler group and Simo decoction group were reached the level of normal group(P>0.05). The activities of xylase in all treatment groups were lower than the normal group(P<0.01).After treatment with Dendrobium officnale, platelet(PLT) and thrombocytocrit(PCT) were significantly higher than normal group(P<0.01or P<0.05);red blood cell (RBC) and haematocrit(HCT)were increased; Rbc Volume Distribution Width (RDW), hemoglobin(HGB), white blood cell(WBC)and lymphocytotoxicity (LYM) were reduced;Mean Corpuscular Hemoglobin(MCH) and Mean Corpuscular Hemoglobin Concentration(MCHC) were also reduced,but still were significantly higher than normal group(P<0.01or P<0.05).The RBC and HCT of50%amount of ultra-micro powder Dendrobium officinale group were significantly higher than model group(P<0.05),reached the lever of normal group(i^>0.05).The WBC and LYM of50%amount of ultra-micro powder Dendrobium officinale group were significantly lower than model group and the traditional decoction of Dendrobium officinale group(P<0.01orP<0.05),but had no significant difference with normal group(P>0.05).The results showed that OTUs,Shanon index and Brillouin index in the nomal group, the50%volume of ultra-micro powder group were the same and larger than in the model group and the traditional decoction group. The similarity coefficient which compared with Bacteria community structure in normal group was the largest in the50%volume of ultra-micro powder group, then in the model group and the traditional decoction group. The cluster analysis showed that groups were clustered in2classes when the clustering distance>15and<25,the nomal group and the50%volume of ultra-micro powder group were in one class,then the others. All groups were clustered in4classes when the clustering distance<15,one group was in one class.The principal components analysis suggested that the nomal group and the50%volume of ultra-micro powder group were had greater contribution to PC1,and the model group and the traditional decoction group were had same contribution to PC2.The molecular diversity of intestinal Bacteria in mice changed by the affect of spleen-deficiency constipation model,and the two kinds of decoction of Dendrobium officinale regulated it in different ways. Study the effect of ultra-micro powder Dendrobium officinale on the molecular diversity of intestinal Lactobacillus in mice with spleen-deficiency constipation. The results showed that OTUs,Shanon index and Brillouin index in the nomal group,the traditional decoction group and the1/2volume of ultra-micro powder group were the same and larger than in the model group.The Czekanowski coefficient0.3333was the largestest in the1/2volume of ultra-micro powder group,0.1818in the model group,0.1667was the least in the traditional decoction group,compare with Lactobacillus community structure in the normal group.The cluster analysis and principal components analysis suggested that molecular diversity of intestinal Lactobacillus in mice changed by the spleen-deficiency constipation model made,and the two kinds decoction of Dendrobium officinale regulated it in different ways.Conclusion The spleen-deficiency constipation was made had affect the body weight, had destroyed the balance intestinal microbiota and influnce intestinal enzyme activities. The modeling spleen-deficiency constipation had significantly influence on PLT, MPV and WBC, which affected the function of blood system in mice. The traditional decoction of Dendrobium officinale, and the full amount,50%amount of ultra-micro powder Dendrobium officinale and Simo decoction had curative effect on spleen-deficiency constipation,regulated the balance of intestinal microflora and the intestinal enzyme activity through different pathways.The50%amount of ultra-micro powder and traditional decoction of Dendrobium officinale had the equal curative effect with Simo decoction. Dendrobium officinale had effect on the RBC,HCT,RBC,RDW,HGB,MCH, MCHC,WBCand LYM in mice with spleen-deficiency constipation,and the overall curative effect of50%amount of ultra-micro powder Dendrobium officinale was better than the traditional decoction.The molecular diversity of intestinal Bacteria and Lactobacillus in mice changed by the affect of spleen-deficiency constipation model,and the two kinds of decoction of Dendrobium officinale regulated it in different ways. The efficacy of50%volume of ultra-micro powder Dendrobium officinale was the better than the traditional decoction group.更多还原