健脾消痰方对高脂血症大鼠AQP3、AQP4表达的影响

发布时间：2018-05-25 04:01

本文选题：高脂血症 + 健脾消痰方　；参考：《河北医科大学》2017年硕士论文

【摘要】：目的:观察健脾消痰方对高脂血症大鼠血清中甘油三酯(TG)、胆固醇(TC)、高密度脂蛋白胆固醇(HDL-C)、低密度脂蛋白胆固醇(LDL-C)的含量及结肠组织中水通道蛋白3(AQP3)、水通道蛋白4(AQP4)表达的影响,探讨其对高脂血症的治疗作用及其可能的作用机制。方法:选用60只健康的SD大鼠,体重在160g~180g,雄性,适应性喂养一周后,按照体重分层的原则,随机分为6组,分别是:正常对照组(简称正常组)、模型对照组(简称模型组)、辛伐他汀对照组(简称对照组)、健脾消痰方低剂量组(简称低剂量组)、健脾消痰方中剂量组(简称中剂量组)、健脾消痰方高剂量组(简称高剂量组)。采用喂饲高脂饲料的方法复制高脂血症模型,除正常组喂饲普通饲料外,其余各组均喂饲高脂饲料。与此同时,正常组与模型组灌服适量蒸馏水,其余各组灌服治疗药或对照药,用药量均按照1ml/100g计算。第5周末,于最后一次给药后,将大鼠禁食不禁水24小时,麻醉状态下,股动脉取血,将血样离心,分离血清,检测血清中TG、TC、HDL-C、LDL-C的含量,并迅速剖取肝脏,将肝脏最大叶部分肝组织放入4%的多聚甲醛溶液中,用于病理观察。同时剖取结肠,取位置相对固定的3块结肠组织,剪开冲洗干净后,将其中1块放入4%多聚甲醛溶液中,运用免疫组织化学法观察AQP3及AQP4的表达,其余2块放入冻存管内,运用RT-PCR及Western Blot法观察AQP3mRNA、AQP4mRNA、AQP3及AQP4的表达。结果:1各组大鼠血清中TG、TC、HDL-C、LDL-C含量的变化与正常组相比,模型组大鼠血清中TC、TG、LDL-C的含量均明显升高(P0.01),HDL-C含量明显降低(P0.01或P0.05),提示大鼠造模成功。药物干预后,各用药组TG、TC、LDL-C的含量均较模型组降低,HDL-C含量均升高;与对照组相比,高剂量组血脂无明显变化(P0.05),中、低剂量组TG、TC、LDL-C的含量升高,HDL-C含量降低(P0.01或P0.05);健脾消痰方各剂量组之间比较,高剂量组TG、TC、LDL-C的含量低于中、低剂量组,hdl-c的含量高于中、低剂量组(p0.01或p0.05);与低剂量组相比,中剂量组tc含量降低(p0.05),但tg、hdl-c、ldl-c的含量无显著差异(p0.05)。2各组大鼠的肝组织形态学的变化正常组大鼠肝组织肝细胞排列整齐,形状为规则六边形,围绕中央静脉呈放射状分布,肝血窦结构清晰,无脂肪滴及炎性细胞浸润。模型组大鼠肝组织中肝细胞排列混乱,且出现大量脂肪滴,细胞核位置被挤压偏离,肝血窦充血水肿。对照组肝细胞结构清晰,脂肪滴明显减少,肝血窦充血水肿减轻,但存在细胞坏死及炎性细胞浸润。健脾消痰方低中高剂量组较模型组结构排列清晰,脂肪滴也不同程度减少,肝血窦形态趋于正常,尤以高剂量组效果显著。3各组大鼠结肠组织中aqp3表达的变化免疫组织化学法:正常组大鼠阳性细胞表达相对较少,着色较浅,模型组大鼠结肠上皮细胞的细胞核、细胞浆及肠腺上皮细胞阳性表达相对较多,着色较深,结缔组织有少量阳性表达,提示模型组大鼠结肠组织aqp3的表达较正常组明显升高(p0.01)。对照组、低剂量组、中剂量组、高剂量组阳性细胞的表达及着色程度依次降低;药物干预后,各用药组aqp3的表达均低于模型组(p0.01);其中,高、中、低剂量组aqp3的表达明显低于对照组(p0.01);三个剂量组中,高剂量组aqp3明显低于中、低剂量组(p0.01);中剂量组aqp3的表达又低于低剂量组(p0.05)。rt-pcr法:模型组大鼠结肠组织aqp3mrna的表达较正常组明显升高(p0.01);药物干预后,各用药组aqp3mrna的表达均低于模型组(p0.01);其中,高、中剂量组aqp3mrna的表达低于对照组(p0.01),而低剂量组与对照组相比无差异(p0.05);三个剂量组中,高剂量组aqp3mrna低于中、低剂量组(p0.01),中剂量组aqp3mrna的表达有低于低剂量组(p0.01)。westernblot法:研究结果显示,模型组大鼠结肠组织aqp3的表达较正常组明显升高(p0.01);药物干预后,各用药组aqp3的表达均低于模型组(p0.01);其中,高、中剂量组aqp3的表达明显低于对照组(p0.01),而低剂量组与对照组相比无差异(p0.05);三个剂量组中,高剂量组aqp3的表达明显低于中、低剂量组(p0.01),中剂量组aqp3的表达又低于低剂量组(p0.01)。4各组大鼠结肠组织中aqp4表达的变化免疫组织化学法:正常组大鼠阳性细胞表达相对较少,着色较浅,模型组大鼠结肠上皮细胞的细胞核、细胞浆及肠腺上皮细胞阳性表达相对较多,着色较深,结缔组织有少量阳性表达,提示模型组大鼠结肠组织中aqp4的表达较正常组明显升高(p0.01)。对照组、低剂量组、中剂量组、高剂量组阳性细胞的表达及着色程度依次降低;药物干预后,各用药组aqp4的表达均低于模型组(p0.01);其中,高、中、低剂量aqp4的表达低于对照组(p0.01或p0.05);三个剂量组中,高剂量组aqp4的表达低于中、低剂量组(p0.01),中剂量组aqp4的表达与低剂量组无显著差异(p0.05)。rt-pcr法:模型组大鼠结肠中aqp4mrna的表达较正常组明显升高,(p0.01);药物干预后,各用药组aqp4mrna的表达均低于模型组(p0.01);其中,高、中剂量aqp4mrna的表达明显低于对照组(p0.01),而低剂量组与对照组相比无显著性差异(p0.05)。三个剂量组中,高剂量组aqp4mrna的表达低于中、低剂量组(p0.01),中剂量组aqp4mrna的表达又低于低剂量组(p0.05)。westernblot法:模型组大鼠结肠中aqp4的表达较正常组明显升高(p0.01);药物干预后,各用药组aqp4的表达均低于模型组(p0.05);其中,对照组aqp4的表达明显高于低、中、高剂量(p0.01或p0.05)。三个剂量组中,高剂量组aqp4的表达低于中、低剂量组(p0.05或p0.01),中剂量组aqp4的表达又低于低剂量组(p0.05)。结论:1健脾消痰方能够降低血清中tc、tg、ldl-c水平,升高hdl-c水平,减轻肝细胞的脂变程度,具有良好地调节血脂作用。2健脾消痰方通过调控结肠组织中aqp3、aqp4、aqp3mrna、aqp4mrna的表达,可有效地改善结肠因过度重吸收水液导致的水液停聚,减少痰湿的生成,消除高脂血症的病因,从而达到防治高脂血症的目的。
[Abstract]:Objective: To observe the effect of Jianpi Xiao Fu Fang on the content of triglyceride (TG), cholesterol (TC), high density lipoprotein cholesterol (HDL-C), low density lipoprotein cholesterol (LDL-C) and the expression of water channel protein 3 (AQP3) and water channel protein 4 (AQP4) in the colon tissue of hyperlipidemia rats, and to explore the therapeutic effect and possibility of the hyperlipidemia on hyperlipidemia. Methods: 60 healthy SD rats were selected and the body weight was 160g~180g, male, after adaptive feeding for one week, according to the principle of weight stratification, they were randomly divided into 6 groups: normal control group (normal group), model control group (abbreviated as model group), simvastatin control group (abbreviated as control group), low dose group of Jianpi eliminating phlegm prescription (abbreviated for short) The low dose group), the middle dose group of Jianpi eliminating phlegm prescription (medium dose group), the high dose group of Jianpi eliminating phlegm prescription (high dose group). The hyperlipidemia model was replicated by feeding high fat diet. Besides the normal diet, the other groups were fed with high fat diet. At the same time, the normal group and the model group were given proper amount of distilled water. Each group was given the treatment or control, and the dosage was calculated according to 1ml/100g. At the end of the fifth week, after the last administration, the rats were fasted for 24 hours. Under the anesthetic state, the femoral artery was taken blood, the blood samples were centrifuged, the serum was separated and the content of TG, TC, HDL-C, LDL-C in the serum was detected, and the liver was quickly dissected and the liver tissue of the largest lobe of the liver was dissected. In 4% of the 4% polycondensation Formaldehyde Solution, it was used for pathological observation. At the same time, the colon was dissected and 3 colonic tissues with relative fixed position were taken. After sciscent and rinse, 1 of them were put into 4% polycondensation of Formaldehyde Solution. The expression of AQP3 and AQP4 was observed by immunohistochemistry. The remaining 2 pieces were placed in the cryopreservation tube, and AQP3mR was used to observe AQP3mR by RT-PCR and Western Blot. The expression of NA, AQP4mRNA, AQP3 and AQP4. Results: 1 the content of TG, TC, HDL-C, LDL-C in serum of rats in each group was significantly higher than that in the normal group. The content of TC, TG, LDL-C in the serum of the model rats was significantly increased (P0.01), which suggested that the rat model was successful. Compared with the control group, the level of HDL-C in the high dose group had no obvious change (P0.05), and the content of TG, TC, LDL-C in the low dose group increased and the content of HDL-C decreased (P0.01 or P0.05) in the low dose group; the content of TG, TC, LDL-C in the high dose group was lower than in the high dose group, and the low dose group was higher than in the low dose group, and the low dose was higher than the low dose. Compared with low dose group (P0.01 or P0.05), the content of TC in middle dose group decreased (P0.05), but the content of TG, HDL-C, LDL-C had no significant difference (P0.05) the change of liver histomorphology in each group of rats (P0.05).2 group, the liver cells in the normal group were arranged orderly, the shape was regular hexagon, the central vein was radially distributed, and the structure of hepatic sinusoid was clear. In the model group, the liver cells in the model group were arranged in confusion and a large number of fat drops, the nucleus position was squeezed off and the hepatic sinusoid congestion and edema. The control group had clear liver cell structure, the fat drops obviously decreased, the hepatic sinusoid congestion and edema lightened, but there was cell necrosis and inflammatory cell infiltration. Invigorating the spleen and eliminating phlegm. In the middle and high dose group, the structure of the model group was clearer than the model group, the fat droplets were reduced in different degrees and the morphology of the hepatic sinusoids tended to be normal. The effect of the high dose group on the expression of AQP3 in the colon tissue of the.3 rats was significant. The expression of the positive cells in the normal group was less, the coloring was shallow, and the colon epithelium of the model group rats. The positive expression of the cell nucleus, cytoplasm and intestinal gland epithelial cells was relatively more, the coloring was deeper, and the connective tissue had a small number of positive expressions, suggesting that the expression of AQP3 in the colon tissue of the model group was significantly higher than that of the normal group (P0.01). The expression and coloring degree of the positive cells in the low dose group, middle dose group and high dose group decreased in turn; The expression of AQP3 in each group was lower than that of the model group (P0.01), and the expression of AQP3 in the high, middle and low dose groups was significantly lower than that of the control group (P0.01); in the three dose group, the AQP3 in the high dose group was significantly lower than the low dose group (P0.01); the expression of AQP3 in the middle dose group was lower than the low dose group (P0.05).Rt-pcr method: the colon tissue of the model group was AQP3. The expression of mRNA was significantly higher than that in the normal group (P0.01), and the expression of aqp3mrna in each drug group was lower than that in the model group (P0.01), and the expression of aqp3mrna in the middle dose group was lower than that of the control group (P0.01), while the low dose group had no difference compared with the control group (P0.05); in the three dose group, the high dose group aqp3mrna was lower than the low dose group (P0.01), The expression of aqp3mrna in the medium dose group was lower than the low dose group (P0.01).Westernblot method. The results of the study showed that the expression of AQP3 in the colon tissue of the model group was significantly higher than that of the normal group (P0.01). The expression of AQP3 in each drug group was lower than that of the model group (P0.01), and the expression of AQP3 in the middle dose group was significantly lower than that of the control group (P0.01), but the expression of the medium dose group was significantly lower than that of the control group (P0.01). There was no difference between the low dose group and the control group (P0.05); in the three dose group, the expression of AQP3 in the high dose group was significantly lower than that in the middle, low dose group (P0.01), and the expression of AQP3 in the middle dose group was lower than that in the colon tissue of the low dose group (P0.01).4. The expression of AQP4 in the colon tissues of the rats of each group was less than that in the normal group. In the model group, the nucleus of the colonic epithelial cells in the model group, the cytoplasm and the intestinal gland epithelial cells were more positive, the coloring was more deep, and the connective tissue had a small amount of positive expression. It suggested that the expression of AQP4 in the colon tissue of the model group was significantly higher than that of the normal group (P0.01). The control group, the low dose group, the middle dose group, the high dose group positive cells. The expression and coloring degree decreased in turn, and the expression of AQP4 in each drug group was lower than that in the model group (P0.01), and the expression of high, medium and low dose AQP4 was lower than that of the control group (P0.01 or P0.05); in the three dose group, the expression of AQP4 in the high dose group was lower than that in the low dose group (P0.01), and the expression of AQP4 in the middle dose group was not significantly worse than that in the low dose group. P0.05.Rt-pcr method: the expression of AQP4mRNA in the colon of the model rats was significantly higher than that in the normal group, (P0.01), and the expression of AQP4mRNA in each drug group was lower than that of the model group (P0.01). The expression of AQP4mRNA in the middle dose group was significantly lower than that of the control group (P0.01), but there was no significant difference between the low dose group and the control group (P0.05). Three In the dose group, the expression of AQP4mRNA in the high dose group was lower than that in the low dose group (P0.01), and the expression of AQP4mRNA in the middle dose group was lower than that of the low dose group (P0.05).Westernblot method. The expression of AQP4 in the colon of the model group was significantly higher than that in the normal group (P0.01). The expression of AQP4 in each drug group was lower than that of the model group (P0.05), and the control group was AQ (P0.05); and the control group AQ was aq. The expression of P4 was significantly higher than low, middle and high dose (P0.01 or P0.05). The expression of AQP4 in the high dose group was lower than that of the middle, low dose group (P0.05 or P0.01), and the expression of AQP4 in the middle dose group was lower than that of the low dose group (P0.05). Conclusion: 1 the prescription of strengthening spleen and eliminating phlegm can reduce TC, TG, LDL-C level, increase HDL-C level, and reduce the lipid change of liver cells. By regulating the expression of AQP3, AQP4, aqp3mrna and AQP4mRNA in colon tissue, the expression of.2 Jianpi eliminating phlegm can effectively improve the accumulation of water caused by excessive heavy absorption of water in colon, reduce the formation of phlegm dampness, eliminate the cause of hyperlipidemia, and thus achieve the purpose of preventing hyperlipidemia.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R285.5

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