【作者】 司银梅；

【导师】 向楠；

【作者基本信息】 湖北中医药大学 ， 中医内科学， 2014， 博士

【摘要】 目的探讨温肾健脾化痰方治疗肥胖病的疗效，进一步明确温肾健脾化痰方减肥的作用机制，为临床应用提供依据。方法本研究从理论研究、实验研究和临床研究三个方面进行。理论研究：从肥胖病的易患人群、肥胖病的分类和肥胖病的病因病机方面进行了系统的论述，并从理、法、方、药四个方面对温肾健脾化痰方治疗肥胖病的机制进行了理论探讨。实验研究：将60只体重100～150g的健康雄性SD大鼠随机分为2组，一组为正常对照组，10只，给予普通饲料喂养，自由取食饮水；其余50只给予高脂饲料喂养，自由取食饮水。每周测量体重2次，喂养4周后选取体质量超出正常组平均体质量20%的40只作为造模成功大鼠。将40只造模成功的大鼠随机分为4组，分别为模型组、低剂量组、中剂量组和高剂量组，每组10只。正常组和模型组大鼠给予生理盐水1ml/100g/d灌胃；低、中、高剂量组大鼠分别给予低、中、高剂量温肾健脾化痰方1ml/100g/d灌胃。正常组继续喂以普通饲料，其他4组继续喂以高脂饲料，实验过程中每周测量体重2次，并根据体重调整给药量，连续治疗10周。观察各组大鼠一般情况和体质量的变化，采用全自动生化分析仪检测各组大鼠血脂及肝功能。运用HE染色观察各组大鼠肝脏组织和脂肪组织的病理形态学改变。采用ELISA法检测血清瘦素和脂联素水平，运用免疫组织化学法及RT-PCR法观察温肾健脾化痰方对各组大鼠肝脏组织和脂肪组织FTO基因表达的影响。临床研究：随机将符合纳入标准的60例患者分为治疗组和对照组，每组各30例，对照组采取单纯的饮食运动疗法，治疗组在饮食、运动基础上给予温肾健脾化痰方口服，每日一剂，水煎分两次温服，疗程2个月。比较两组患者治疗前后体质量、体质量指数（BMI）、腰围、臀围、腰臀比（WHR）的变化以及总胆固醇（TC）、甘油三酯（TG）、低密度脂蛋白胆固醇（LDL-C）、高密度脂蛋白胆固醇（HDL-C）的变化。结果实验研究：1）各组大鼠体质量的变化：温肾健脾化痰方低、中、高剂量均能明显控制肥胖大鼠体重的增加，使体重增加的速度明显变缓，与模型组相比具有统计学意义（P<0.01），且中剂量组和高剂量组效果明显优于低剂量组并具有统计学意义（P<0.01），而中、高剂量组之间差异无统计学意义。2）各组大鼠肝脏组织及脂肪组织病理学变化：正常组肝细胞形态排列正常，肝小叶规则，细胞质均匀；模型组均出现程度不等的弥漫性肝细胞脂肪变性，肝细胞体积增大，胞浆内可见数量不等、大小不一的圆形脂滴或脂肪空泡，胞核被推向周边；低剂量组脂变肝细胞与模型组相当；中、高剂量组脂变肝细胞较模型组明显减少，可见少量脂肪空泡。与正常组大鼠相比，模型组大鼠脂肪细胞明显增大，单位视野内脂肪细胞数明显减少；低剂量组大鼠脂肪细胞与模型组相当；中、高剂量组大鼠脂肪细胞较模型组明显减小，单位视野内脂肪细胞数明显增多，且中剂量组的变化更为显著。3）各组大鼠肝功能比较：与正常组相比，模型组与低、中、高剂量组的ALT、AST均显著升高（P＜0.01，P＜0.05）；与模型组相比，温肾健脾化痰方低、中、高剂量均可显著降低ALT、AST水平（P＜0.01），且中、高剂量组效果显著优于低剂量组（P＜0.01），高剂量组在降低AST水平方面又显著优于中剂量组（P＜0.01），但在降低ALT水平方面则无明显区别（P＞0.05）。4）各组大鼠血脂比较:与正常组相比，模型组与低、中、高剂量组的TC、TG、LDL-C均显著性升高（P＜0.01），HDL-C显著性降低（P＜0.01），提示肥胖大鼠模型存在明显的血脂紊乱。与模型组相比，温肾健脾化痰方低、中、高剂量组均可显著性降低TC、TG以及LDL-C（P＜0.01），升高HDL-C（P＜0.01），且三组之间比较也有显著性差异（P＜0.01）。5）各组大鼠瘦素水平的比较：与正常组比较，模型组、低、中、高剂量组瘦素水平均显著升高（P＜0.01）；与模型组比较，药物治疗组瘦素水平均显著下降（P＜0.01）；治疗组低、中、高剂量之间比较也有显著性差异（P＜0.01），高剂量组瘦素水平下降最明显。6）各组大鼠脂联素水平的比较：与正常组比较，模型组、低、中、高剂量组脂联素水平均显著下降（P＜0.01）；与模型组比较，药物治疗组脂联素水平均显著升高（P＜0.01）；治疗组低、中、高剂量之间比较也有显著性差异（P＜0.01），高剂量组脂联素水平升高最明显。7）各组大鼠肝脏组织FTO基因表达：模型组大鼠肝脏组织内FTO基因表达量显著升高（P＜0.01），经温肾健脾化痰方治疗后，与模型组比较，药物治疗组FTO基因表达均显著下降（P＜0.01），其中高剂量组与低、中剂量组相比有显著性差异（P＜0.01），与正常组相比无明显区别（P＞0.05）。8）各组大鼠脂肪组织FTO基因表达：模型组大鼠脂肪组织内FTO基因表达量显著升高（P＜0.05），经温肾健脾化痰方治疗后，与模型组比较，药物治疗组FTO基因表达均显著下降（P＜0.01），其中高、中剂量组相比无明显区别（P＞0.05），但与低剂量组相比有显著性差异（P＜0.01）。9）各组大鼠肝脏组织FTO免疫组化检测结果：光镜下肝细胞的细胞核呈棕黄色或棕褐色的为FTO阳性细胞，阴性细胞核为蓝色或蓝紫色。观察各组大鼠的肝细胞：模型组大鼠肝细胞内FTO阳性细胞明显较正常组增多，经温肾健脾化痰方药物治疗的大鼠肝细胞内FTO阳性细胞较模型组明显减少，以中、高剂量组较明显。IPP分析结果也显示：与正常组相比，模型组、低、中、高剂量组FTO阳性表达率明显升高（P＜0.05），经治疗后，低、中、高剂量组大鼠肝脏FTO阳性表达率较模型组显著下降（P＜0.01）。10）各组大鼠脂肪组织FTO免疫组化检测结果：光镜下脂肪细胞的细胞核呈棕黄色或棕褐色的为FTO阳性细胞，阴性细胞核为蓝紫色。观察各组大鼠的脂肪细胞：模型组大鼠脂肪细胞内FTO阳性细胞明显较正常组增多，经温肾健脾化痰方药物治疗的大鼠脂肪细胞内FTO阳性细胞较模型组明显减少，以中、高剂量组较明显。IPP分析结果也显示：与正常组相比，模型组、低、中、高剂量组FTO阳性表达率明显升高（P＜0.05），经治疗后，低、中、高剂量组大鼠脂肪组织FTO阳性表达率较模型组显著下降（P＜0.05）。临床研究：治疗组患者的症状得到明显改善，体质量、BMI、腰围、WHR、血脂与治疗前相比均有显著性差异（P<0.05），总有效率90%，明显优于对照组（P<0.05）。结论理论研究：肥胖病的病理因素属本虚标实，本虚表现为以脾肾虚衰为主，标实以痰湿、瘀血等为主。温肾健脾化痰方的组方体现了肥胖病治疗的根本大法，为运用其治疗肥胖病提供了理论依据。实验研究：1）温肾健脾化痰方在控制肥胖模型大鼠体重增加，减轻肝功能损害，减少腹内脂肪，降低血清胆固醇及甘油三酯，减轻肝脏脂肪变性方面有良好作用。2）温肾健脾化痰方能控制食欲，使能量消耗增加，抑制脂肪合成，改善糖、脂代谢，减轻肥胖，可能是通过调节肥胖模型大鼠的瘦素水平和脂联素水平来实现的。3）温肾健脾化痰方可降低肥胖模型大鼠肝脏组织和脂肪组织内FTO基因的表达，而FTO基因表达水平的下降不仅可直接影响食欲和饮食，提高能量消耗效率，还能通过调节瘦素、脂联素等内分泌因子，达到减轻肥胖的目的。临床研究：温肾健脾化痰方在治疗肥胖病方面具有较好的疗效，值得临床推广应用。更多还原

【Abstract】 objectiveThis study discussed WSJPHT Decoction effect of the treatmentof obesity,further clarify the mechanism and provide the basis forclinical application.MethodThis study carried out theoretical studies,experimental andclinical research in three aspects.Theoretical studies:Investigated systematically from obesitysusceptible populations,classification and pathogenesis,discussed the mechanism of WSJPHT Decoction from four aspectsprinciple,method,recipe and medicines.Experimental studies:The60healthy male SD rats weighing100～150g were randomly divided into two groups,one for the normalcontrol group,10,given normal diet,free access to food andwater,rest of the50given high fat diet, free access to food andwater too.Measured weight of rats twice a week,selected40ratswhich weight beyond the normal average to20%after feeding4weeksas a successful model.The40successful model rats were randomlydivided into four groups,namely model group,low-dose group,middle dose group and high-dose groups of10. Normal group and model grouprats given saline1ml/kg/d gavage;low,medium and high dose grouprats were given low, medium and high doses WSJPHT decoction1ml/kg/dgavage respectively. Normal group fed with normal diet continues,the other four groups were fed with high fat diet continues,measured weights of rats twice weekly and adjusted dose basing onweight, treatment for10weeks continuous. Observed changes inweight and general conditions of the rats, using automaticbiochemical analyzer to detect the serum lipid and liver functions.Observed pathological changes of rats in liver tissue and adiposetissue HE stained.Tested leptin and adiponectin levels byELISA,observed WSJPHT decoction effect on the FTO gene expressionin liver tissue and adipose tissue by IHCand RT-PCR.Clinical studies:60patients meeting the inclusion criteriawere randomly divided into treatment group and control group,eachgroup had30cases.The control group adopted the diet and exercisetherapy alone,the treatment group was given WSJPHT decoction orallyon the basis of the diet and exercise,a daily dose,decoction orallytwice one day, The course of treatment was2months.Two groups werecompared before and after treatment about weight,BMI,waistcircumference,hip circumference,WHR and the changes ofTC,TG,LDL-C,HDL-C.ResultExperimental studies:1) Changes in weight of rats in each group:The WSJPHT decoctioncan significantly slow the growth rate of obese rats weight,compared with the model group with statistical significance (P<0.01),the middle dose group and high dose group was significantly better than the low-dose group and was statistically significant(P <0.01), but the difference between the medium and high dosegroups was not statistically significant.2)The pathological changes of rat liver tissue and adiposetissue in each group: Normal liver cells are arranged normally,lobular rules, The cytoplasm uniform; model group appeared varyingdegrees of diffuse hepatic steatosis, liver cell volume increases,varying amounts and different sizes round lipid droplets or fatvacuoles were seen in the cytoplasm,the nucleus are pushed to thesurrounding;Low-dose group steatosis hepatocytes with the modelgroup quite Low-dose group steatosis hepatocytes are equal to themodel group; The medium and high-dose group was significantlyreduced hepatic steatosis cells than the model group, showing asmall amount of adipose vacuoles.Compared with the normal grouprats,adipose cells volume in the rats of model group increasedsignificantly, adipose cells number decreased vision unit.Low-dosegroup adipose cells are epual to the model group.The volume ofadipose cells in medium and high dose groups significantly reducedand the number increased significantly vision unit compared withthe model group,the changes in medium dose group was significantlymore.3)Comparison of liver function in rats:compared with the normalgroup,model group and the low,middle and high dose group,ALT andAST were significantly increased(P<0.01,P<0.05); Compared with themodel group, WSJPHT decoction can significantly reduce ALT and ASTlevels (P<0.01); The effect of middle and high dose group wassignificantly better than the low-dose group (P<0.01), High-dosegroup was significantly better than the middle dose group (P<0.01) in reducing AST levels,but in reducing ALT levels had no significantdifference (P>0.05).4) Comparison of serum lipid in rats: compared with the normalgroup, model group and the low,middle and high dose group TC, TG,LDL-C were significantly higher (P<0.01), HDL-C was significantlylower(P<0.01), prompt obesity rat model exists dyslipidemiaobvious. Compared with the model group, WSJPHT decoction cansignificantly reduce TC,TG and LDL-C(P<0.01),increaseHDL-C(P<0.01),there was significant difference among the threegroups(P<0.01).5)Comparison of leptin levels in rats:compared with the normalgroup, model group and the low,middle and high dose group leptinlevels were significantly increased (P<0.01),Compared with themodel group, WSJPHT decoction can significantly decrease leptinlevels(P<0.01),there was significant difference among the threegroups(P<0.01),and the effect of high dose group was most obvious.6)Comparison of adiponectin levels in rats:compared with thenormal group,model group and the low,middle and high dose groupadiponectin levels were significantly decreased (P<0.01), Comparedwith the model group, WSJPHT decoction can significantly increaseadiponectin levels(P<0.01), there was significant difference amongthe three groups(P<0.01),and the effect of high dose group was mostobvious.7)Expression of FTO gene in liver tissue of rats in eachgroup:the expression level of FTO gene in liver of the model groupwas significantly increased(P<0.01). the expression levels of FTOgene in liver of the treatment group by WSJPHT decoction weresignificantly decreased(P<0.01).There was significant difference among the three groups(P<0.01),but no significantdifference(P>0.05) compared with the normal group.8)Expression of FTO gene in adipose tissue of rats in each group:the expression level of FTO gene in adipose tissue of the model groupwas significantly increased(P<0.05). The expression levels of FTOgene in adipose tissue of the treatment group by WSJPHT decoctionwere significantly decreased(P<0.01).High, medium dose groupshowed no significant difference(P>0.05),but there was asignificant difference compared with the low dose group were (P<0.01).9)FTO gene immunohistochemistry results in liver tissue of ratsin each group:Liver cell nuclei in brown yellow or brown areFTO-positive cells under light microscope, negative nuclei in blueor blue purple. Observing liver cells of rats in each group,theFTO-positive cells in liver cells of the model group rats were morethan that of the normal group significantly, The FTO-positive cellsin liver cells of the treatment group by WSJPHT decoction weresignificantly decreased,the effect of middle and high dose groupwere most obvious. The IPP analysis results also show that: comparedwith the normal group,model group and the low,middle and high dosegroup FTO-positive expression was significantlyincreased(P<0.05),after treatment,the expression of FTO-positivein liver was significantly decreased compared with the modelgroup(P<0.01).10)FTO gene immunohistochemistry results in adipose tissue ofrats in each group: adipose cell nuclei in brown yellow or brownare FTO-positive cells under light microscope, negative nuclei inblue purple. Observing adipose cells of rats in each group, the FTO-positive cells in adipose cells of the model group rats weremore than that of the normal group significantly, The FTO-positivecells in adipose cells of the treatment group by WSJPHT decoctionwere significantly decreased,the effect of middle and high dosegroup were most obvious. The IPP analysis results also show that:compared with the normal group,model group and the low,middle andhigh dose group FTO-positive expression was significantlyincreased(P<0.05),after treatment,the expression of FTO-positivein adipose was significantly decreased compared with the modelgroup(P<0.05).Clinical studies: Symptoms of the treated patients wereimproved significantly, there were significant differences aboutweight,BMI,waist circumference,WHR, serum lipids after treatmentcompared with before(P<0.05).The total effective rate was90%,significantly better than the control group (P<0.05).ConclusionTheoretical studies: Pathological factors of obesity isdeficiency ben and excessive biao.The performance of the deficiencyin spleen and kidney deficiency,phlegm and blood stasis insuperficiality mainly.The compositions of WSJPHT decoctionembodies the fundamental law of obesity treatment,provides atheoretical basis for the treatment of obesity.Experimental studies:1)The WSJPHT decoction has a good role in controlling weightgain in obese rat model,reducing the damage of liver function，decreasing intra-abdominal fat and hepatic steatosis,reducingserum cholesterol and triglycerides.2)The WSJPHT decoction can reduce the leptin level in obese rats, increase adiponectin level, thus suppressing appetite, increasingenergy consumption, inhibiting the synthesis of fat,improveglucose and lipid metabolism and reducing weight.3)The WSJPHT decoction could reduce the expression of FTO genein liver tissue and fatty tissues of the obesity model rats,and theexpression level of FTO gene decline not only can directly affectthe appetite and diet, improve the efficiency of energy consumption,but also reduce obesity through the regulation of leptin andadiponectin.Clinical studies: The WSJPHT decoction has a good effect in thetreatment of obesity, worthy of clinical application.更多还原