芪参健脾方对自发性高血压大鼠肾间质纤维化的影响及机制研究
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摘要
目的：本实验以芪参健脾方为干预因素,采用逆转录聚合酶链式反应(RT-PCR)、蛋白印迹(Western-Blot)、酶联免疫吸附测定法(ELISA)及免疫组化等多种方法,观察芪参健脾方对自发性高血压大鼠(SHR)血压、肾功能、肾组织形态学和肾Ang-Ⅱ含量、肾Ⅰ、Ⅲ、Ⅳ型胶原含量、肾MMP-1、2、9、TGF-β1、Smad3、5、6基因表达以及BMP-7蛋白表达的影响,从整体水平、组织形态学、基因和蛋白等各方面,观察芪参健脾方对SHR的降压及肾脏保护作用,探讨其作用机制和作用靶点,为该方应用于高血压病的临床治疗提供实验依据,并为中药对高血压病的治疗和靶器官保护作用的研究和开发提供思路和理论依据。
材料与方法：
1.实验动物：24周龄雄性自发性高血压大鼠(SHR)60只,体质量(360±20)g,同周龄雄性Wister-Kyoto(WKY)大鼠10只,体质量(350±20)g；大鼠于实验前常规观察1周,然后进行实验。
2.实验动物分组：SHR大鼠60只随机分为6组：SHR组(模型组)、培哚普利组(西药组)、中药加西药组(培哚普利联合中药中剂量组)、中药高、中、低剂量组,每组各10只；WKY大鼠10只作为WKY组(正常对照组)。
3.选用药物：SHR组给予蒸馏水；根据《人和动物按体表面积折算的等效剂量比值表》,培哚普利组用药量为0.4mg*kg-1*d-1,用药时将药溶于蒸馏水中；中药治疗组采用标准水煎工艺,浓缩生药含量4g/mL,高、中、低剂量组用药量按照大鼠体重折算分别为4g/100g,2g/100g和1g/100g;中药加西药组用药为同剂量培哚普利溶解于中药中剂量方中。给药体积均为每100g体重给予药液1mL。大鼠每日一次灌胃,连续治疗6周。
4.收缩压测量：采用大鼠尾动脉间接测压法,在室温22±2℃条件下,将安静、清醒状态大鼠于40℃下预热(用温控器调节温度恒定)约15min,用BP-98A型大鼠智能无创血压计,测量大鼠尾动脉收缩压,连续测量3次,取其均值。分别于实验开始前与药物干预后4小时、2周、4周和6周末进行测量。
5.采用ELISA方法测定尿微量白蛋白(MALb)、尿β2-微球蛋白(β2-MG)和肾组织Ang-Ⅱ的含量。
6.全自动生物化学分析仪检测各组大鼠血清肌酐(SCr)和尿素氮(BUN)含量。
7.常规HE染色,镜下观察各组大鼠肾脏的形态学变化。
8.免疫组化方法检测各组大鼠肾脏组织中Ⅰ型胶原、Ⅲ型胶原和Ⅳ型胶原积分光密度值的变化。
9.采用RT-PCR法检测各组大鼠肾脏组织中MMP-1、MMP-2、MMP-9、TGF-β、Smad3、 Smad5、Smad6的基因表达水平。
10.采用Western Blot法检测各组大鼠肾脏组织中BMP-7的蛋白表达水平。
结果：
1.实验开始前与用药后4小时、2周、4周和6周分别应用智能无创血压计监测各组大鼠尾动脉收缩压的变化,观察到用药前SHR各组大鼠血压均高于WKY组,WKY组与其他各组比较有显著统计学意义(P<0.01),其余各组间比较无统计学意义(P>0.05)：给药后4小时：各组大鼠收缩压无明显变化,WKY组与其他各组比较有显著统计学意义(P<0.01)；给药后2周：中药加西药组收缩压明显降低,与SHR组相比有统计学意义(P<0.05),而培哚普利组、中药高、中、低剂量组大鼠收缩压也有一定程度的下降,但与SHR组比较无统计学意义(P>0.05)；给药后4周：大鼠收缩压持续下降,培哚普利组、中药加西药组与SHR组相比具有显著统计学差异(P<0.01),中药高、中、低剂量组与SHR组比较无统计学意义(P>0.05),培哚普利组与中药加西药组比较无统计学意义(P>0.05)；给药后6周：培哚普利组、中药加西药组与SHR组相比具有显著统计学差异(P<0.01),中药高、中、低剂量组与SHR组比较无统计学意义(P>0.05),培哚普利组与中药加西药组比较无统计学意义(P>0.05)。
2.SHR组大鼠尿微量白蛋白含量明显增加,与WKY组比较有显著性差异(P<0.01)：经药物干预后各治疗组尿微量白蛋白含量明显下降,与SHR组相比,均有显著性差异(P<0.01),其中,中药加西药组下降最明显,是SHR组的0.60倍(P<0.01),中药高剂量组是SHR组的0.62倍(P<0.01),培哚普利组是SHR组的0.64倍(P<0.01),中药中剂量组是SHR组的0.66倍(P<0.01),中药低剂量组是SHR组的0.70倍(P<0.01)；其他各组间比较无统计学意义(P>0.05)。
3.与WKY组相比,SHR组尿β2-微球蛋白含量明显增加,具有显著性差异(P<0.01)：经药物干预后各治疗组尿β2-微球蛋白含量明显下降,与SHR组相比,均有显著性差异(P<0.01),其中,以中药加西药组下降最明显,是SHR组的0.29倍(P<0.01)；培哚普利组是SHR组的0.38倍(P<0.01),中药高剂量组是SHR组的0.32倍(P<0.01),中药中剂量组是SHR组的0.47倍(P<0.01),中药低剂量组是SHR组的0.50倍(P<0.01)；中药加西药组、中药中、低剂量组与培哚普利组比较有统计学意义(P<0.05或P<0.01)。
4.SHR组大鼠血肌酐含量明显增加,与WKY组比较有显著性差异(P<0.01)；经药物干预后各治疗组血肌酐含量明显下降,与SHR组相比,均有显著性差异(P<0.01),其中,中药加西药组下降最明显,是SHR组的0.50倍(P<0.01),中药高剂量组是SHR组的0.53倍(P<0.01),培哚普利组是SHR组的0.59倍(P<0.01),中药中剂量组是SHR组的0.70倍(P<0.01),中药低剂量组是SHR组的0.78倍(P<0.01)；中药加西药组、中药高、中、低剂量组与培哚普利组相比有统计学意义(P<0.01)。
5.SHR组大鼠血尿素氮含量明显增加,与WKY组比较有显著性差异(P<0.01)；经药物干预后各治疗组血尿素氮含量明显下降,与SHR组相比,均有显著性差异(P<0.01)其中中药加西药组下降最明显,是SHR组的0.50倍(P<0.01),中药高剂量组是SHR组的0.60倍(P<0.01),培哚普利组是SHR组的0.61倍(P<0.01),中药中剂量组是SHR组的0.63倍(P<0.01),中药低剂量组是SHR组的0.70倍(P<0.01)；中药加西药组、中药中、低剂量组与培哚普利组比较有统计学意义(P<0.01或P<0.05)。
6.HE染色在光镜下观察结果显示：SHR组肾小球出现轻度萎缩,管腔形态不规则,近、远端小管管腔结构不清晰,可见慢性炎细胞浸润产生的大量粘液性物质,上皮细胞可见颗粒变性、增生、排列不规则,肾小球血管管壁增厚,肾间质出现纤维组织增生。各治疗组肾小管、肾小球的形态、炎性粘液浸润以及间质纤维化均有所改善,其中中药加西药组改善最为明显。
7.SHR组大鼠肾组织中Ang-Ⅱ浓度明显升高,与WKY组比较有显著性差异(P<0.01)；经药物干预后各治疗组Ang-Ⅱ含量明显下降,与SHR组相比,均有显著性差异(P<0.01),其中,中药加西药组下降最明显,是SHR组的0.40倍(P<0.01),培哚普利组是SHR组的0.41倍(P<0.01),中药高剂量组是SHR组的0.48倍(P<0.01),中药中剂量组是SHR组的0.52倍(P<0.01),中药低剂量组是SHR组的0.58倍(P<0.01)；中药高、中、低剂量组与培哚普利组比较有统计学意义(P<0.01)；中药加西药组与培哚普利组比较无统计学意义(P>0.05)；中药高剂量组与中药中、低剂量组比较有统计学意义(P<0.01)。
8.SHR组大鼠肾组织中Ⅰ型胶原的积分光密度值明显升高,与WKY组比较有显著性差异(P<0.01)；经药物干预后的结果显示,各治疗组大鼠Ⅰ型胶原的积分光密度值均出现降低,与SHR组比较(P<0.01或P<0.05),其中以中药加西药组降低最明显,其次是培哚普利组、中药高、中、低剂量组。与培哚普利组相比,中药加西药组和中药高剂量组无统计学意义(P>0.05),而中药中、低剂量组与培哚普利组比较有统计学意义(P<0.05)；中药高剂量组与中药中、低剂量组比较有统计学意义(P<0.05)。
9.SHR组大鼠肾组织中Ⅲ型胶原的积分光密度值明显升高,与WKY组比较有显著性差异(P<0.01)；经药物干预后的结果显示,各治疗组大鼠Ⅲ型胶原积分光密度值均有所降低,与SHR组比较(P<0.01或P<0.05),其中以中药加西药组降低最明显,其次是中药高剂量组、培哚普利组、中药中剂量组和中药低剂量组。与培哚普利组相比,中药加西药组、中药高、中剂量组无统计学意义(P>0.05),而中药低剂量组与培哚普利组比较有统计学意义(P<0.05)；中药高剂量组与中药中、低剂量组比较有统计学意义(P<0.05)。
10.SHR组大鼠肾组织中Ⅳ型胶原的积分光密度值明显升高,与WKY组比较有显著性差异(P<0.01)；经药物干预后的结果显示,各治疗组大鼠Ⅳ型胶原积分光密度值均明显降低,与SHR组比较(P<0.01或P<0.05),其中以培哚普利组下降最明显,其次是中药高剂量组、中药加西药组、中药中、低剂量组。与培哚普利组相比,中药加西药组和中药高剂量组无统计学意义(P>0.05),而中药中、低剂量组与培哚普利组比较有统计学意义(P<0.05)。
11.SHR组大鼠肾组织中MMP-1基因表达明显降低,与WKY组比较有显著统计学意义(P<0.01)；经药物干预后的结果显示,与SHR组比较,各治疗组MMP-1基因表达均升高(P<0.01或P<0.05),其中以培哚普利组升高最明显；培哚普利组与中药加西药组、中药高、中剂量组比较无统计学意义(P>0.05),培哚普利组与中药低剂量组比较有统计学意义(P<0.05)。
12.SHR组大鼠肾组织中MMP-2基因表达明显升高,与WKY组比较有显著统计学意义(P<0.01)；经药物干预后的结果显示,与SHR组比较,各治疗组MMP-2基因表达均有所下降(P<0.01或P<0.05),其中以培哚普利组下降最明显；培哚普利组与中药加西药组、中药高剂量组比较无统计学意义(P>0.05)；与中药中、低剂量组比较有统计学意义(P<0.05)。
13.SHR组大鼠肾组织中MMP-9基因表达明显升高,与WKY组比较有显著统计学意义(P<0.01)；经药物干预后的结果显示,与SHR组比较,各治疗组MMP-9基因表达均出现降低(P<0.01或P<0.05),其中以培哚普利组降低最明显；培哚普利组与中药加西药组、中药高剂量组比较无统计学意义(P>0.05)；与中药中、低剂量组比较有统计学意义(P<0.05)。
14.SHR组大鼠肾组织中TGF-β1基因表达明显高于WKY组(P<0.01)；经药物干预后的结果显示,与SHR组比较,培哚普利组、中药加西药组、中药高、中剂量组TGF-β,基因表达均降低(P<0.01或P<0.05),其中以中药加西药组降低最明显；而中药低剂量组与SHR组比较无统计学意义(P>0.05)；培哚普利组与中药低剂量组比较有统计学意义(P<0.05),培哚普利组与中药加西药组、中药高、中剂量组比较无统计学意义(P>0.05)。
15.SHR组大鼠肾组织中Smad3基因表达明显高于WKY组(P<0.01)；经药物干预后的结果显示,与SHR组比较,培哚普利组、中药加西药组、中药高、中剂量组Smad3基因表达均明显降低(P<0.01或P<0.05),其中以中药加西药组下降最为明显,而中药低剂量组与SHR组比较无统计学意义(P>0.05)；与培哚普利组比较,中药加西药组、中药高、中剂量组无明显差异(P>0.05),中药低剂量组与培哚普利组比较有统计学意义(P<0.05)。
16.SHR组大鼠肾组织中Smad5基因表达明显低于WKY组(P<0.01)；经药物干预后的结果显示,与SHR组比较,培哚普利组和中药加西药组Smad5基因表达均明显升高(P<0.01),中药加西药组升高最为明显；而中药高、中剂量组也有显著升高(P<0.05),中药低剂量组升高不明显(P>0.05)；中药加西药组、中药高、中剂量组与培哚普利组比较无统计学意义(P>0.05),中药低剂量组与培哚普利组比较有统计学意义(P<0.05)。
17.SHR组大鼠肾组织中Smad6基因表达明显高于WKY组(P<0.01)；经药物干预后的结果显示,与SHR组比较,中药加西药组和培哚普利组Smad6基因表达均明显降低(P<0.01),以中药加西药组最为明显；而中药高、中剂量组也有明显降低(P<0.05),中药低剂量组下降不明显(P>0.05)；中药加西药组、中药高、中剂量组与培哚普利组比较无统计学意义(P>0.05),培哚普利组与中药低剂量组比较有统计学意义(P<0.05)。
18.SHR组大鼠肾组织中BMP-7蛋白表达明显低于WKY组(P<0.01)；经药物干预后的结果显示,与SHR组比较,各治疗组BMP-7蛋白表达均明显升高(P<0.01),中药加西药组升高最明显,其次是培哚普利组、中药高、中、低剂量组。培哚普利组与中药中、低剂量组比较有统计学意义(P<0.05),培哚普利组与中药加西药组和中药高剂量组比较无统计学意义(P>0.05)。
结论：
1.芪参健脾方可以降低SHR的血压,但其降压效果不及血管紧张素转换酶抑制剂(ACEI)培哚普利,与培哚普利联合用药可以达到最佳的降压效果。
2.芪参健脾方可以降低SHR尿β2-微球蛋白(β2-MG)和微量白蛋白(MALb)(?)的含量,降低血尿素氮(BUN)和肌酐(SCr)的含量,改善肾间质纤维化的病理改变,具有保护肾功能的作用。
3.芪参健脾方对肾脏保护作用的机制可能是通过降低SHR肾组织中Ang-Ⅱ的水平,上调MMP-1、下调MMP-2、MMP-9的基因表达,从而降低肾组织中Ⅰ型、Ⅲ型、Ⅳ型胶原含量,减少ECM的异常聚集,增加ECM的降解,减轻肾小管和间质的纤维化。
4.芪参健脾方肾脏保护的作用靶点,可能是通过下调Smad6的基因表达,逆转其对BMP-7/Smad5的表达的抑制,从而负向调节TGF-β1/Smad3信号传导通路,抑制病理条件下的肾小管上皮细胞向间充质细胞的转分化(EMT),减少胶原的产生,促进ECM的降解,逆转肾间质纤维化的进程,促进肾功能的恢复。
Purposes:
This experiment was to observe the effect of qishen spleen-invigorating prescription on the pressure of spontaneous hypertensive rat (SHR), renal function and the Ang-Ⅱ content of renal、the collagen Ⅰ、Ⅲ、Ⅳ type of renal and on the gene expression of MMP-1、MMP-2、MMP-9、TGF-β、Smad3、Smad5、Smad6and the protein expression of BMP-7, which have selected qishen spleen-invigorating prescription to be the intervention factor and adopted reverse transcription polymerase chain reaction (PT-PCR)、western-blot、ELISA and immunohistochemistry methods. And observe the effect of qishen spleen-invigorating prescription on decompression of SHR and the protection of kidney from the aspect of The overall level、histomorphology、the gene、protein and so on, investigating its mechanisms and targets. Thus, we can provide experimental basis for its application to the hypertensive clinical treatment and offer a basic thinking and theory supports of the treatment of Chinese medicine on hypertension and the protection of Chinese medicine on target organs.
Materials and methods:
1. Experimental animals:60twenty four-week-old male spontaneously hypertensive rats, body mass (360±20) g;10age-matched male Wistar-Kyoto rats, body mass (350±20) g; until one week routine observation can we experimentize.
2. Experimental animal grouping:60SHE rats were randomly divided into6groups: SHR group (model group), perindopril group (western medicine group), perindopril combined with the medium dosage of traditional Chinese group, the high dosage group, the medium dosage group and the low dosage group of the traditional Chinese medicine, all the groups have10rats individually; Ten Wistar-Kyoto rats were selected as WKY group(normal control group).
3. Selecting drugs:The SHR group were given distilled water; according to the body surface area ratio table in human and animals, perindopril dosage was0.4mg*kg-1*d-1, and the drug should be dissolved in distilled water; Traditional Chinese group adopted standard decoction process; the content dosage of concentrated crude drug was4g/mL; According to the rats' weight, the drugs dose of high, medium and low dosage group were4g/100g,2g/100g and1g/100g respectively; And about the perindopril combined with traditional Chinese group, the same dose of western medicine dissolved in the medium dosage of Chinese medicine. The administration volume is per100g of body weight givenlml liquid. The rats were given gastric perfusion per day with6weeks continuous treatment.
4. Systolic blood pressure measurement:under the22±2℃room temperature, we use the rats'caudal arterial pressure indirect manometric method, preheating the waking rats to40℃about40min (using the thermostat regulates the constant temperature); We introduced the BP-98A rat intelligent non-invasive blood pressure monitor to measure rat tail pressure in the quiet state and the waking state three times, and got the average value. Measure rat tail pressure before the start of the experiment and after4hours medication,2weeks medication,4weeks medication and6weeks medication.
5. Using the ELISA method to determine the content of the urinary micro albumin, urinary β2-microglobulin and renal tissue Ang-Ⅱ.
6. Serum creatinine and urea nitrogen were detected by automatic biochemical analyzer.
7. Renal morphological changes were observed under microscopy by normal HE dyeing.
8. The Integrated Optical Density (IOD)) changes of collagen Ⅰ、collagenⅢ and collagen IV in each tissues of rats were detected by immunohistochemical method.
9. The gene expression levels of MMP-1, MMP-2, MMP-9, TGF-β1, Smad3、Smad5and Smad6in each rats' kidney tissue were detected by RT-PCR method.
10. The protein levels of BMP-7in each set of rats' kidney tissue were detected by Western-blot method.
Results:
1. We used the intelligent non-invasive blood pressure monitor to monitor rats' caudal arterial systolic pressure before the experiment began and after4hours medication,2weeks medication,4weeks medication and6weeks medication, and we noticed that, before the experiment started, SHR rats pressure in each group were higher than in WKYs'. WKY group had statistical significance compared with other groups (P<0.01), and there was no statistical significance between the comparison of the rest groups; After the administration of4hours, each group of systolic pressure in rats did not change significantly, WKY group has statistical significance compared with other groups(P<0.01); After the administration of2weeks, the perindopril combined with traditional Chinese group's systolic blood pressure reduced significantly, and had statistical significance compared with SHR group (P<0.05), while the rats'systolic blood pressure in perindopril group, the high dosage、the medium dosage and the lower dosage of the Chinese medicine group also had a certain degree of decline, and had no statistical significance compared with SHR group (P>0.05); After the administration of4weeks, the rats'systolic blood pressure decreased continuously. Compared with SHR group, the perindopril group and the perindopril combined with traditional Chinese group had great statistical significance (P <0.01), while the high dosage、the medium dosage and the lower dosage of the Chinese medicine group had no statistical significance compared with SHR group (P>0.05), and compared with perindopril combined with traditional Chinese group, the perindopril group had no statistical significance (P>0.05)。After the administration of6weeks, the perindopril group, the perindopril combined with traditional Chinese group, the medium dosage and the lower dosage of the Chinese medicine group all had statistical significant differences (P<0.01), the high dosage of the Chinese medicine group had statistical significance compared with SHR group (P<0.05), the perindopril group had no statistical significance compared with the perindopril combined with traditional Chinese group (P>0.05)。
2. In SHR group, rats' urinary micro globulin increased significantly, and had evident differences compared with WKY group (P<0.01); After intervention with drug, micro globulin contents of each treatment group decreased significantly and all had significant difference (P<0.01) compared with SHR group. Among them the Chinese medicine combined with western medicine group declined evidently, and was0.66times that of SHR group(P<0.01), the lower dosage of Chinese medicine was0.70times that of SHR group (P<0.01); Comparison between other groups was not statistical significance (P>0.05)
3. In SHR group, rats' urinary β2-microglobulin increased significantly and had remarkable statistics meaning compared with WKY group (P<0.01); After intervention with drug, urinary (32-microglobulin contents of each treatment group decreased significantly, and had remarkable statistics meaning compared with SHR group (P<0.01), among them the perindopril combined with traditional Chinese group declined evidently and was0.29times that of the SHR group(P <0.01), the perindopril group was0.32times that of the SHR group (P<0.01), the medium dosage of the Chinese medicine group was0.47times that of the SHR group(P<0.01), the lower dosage of the Chinese medicine group was0.50times that of the SHR group(P<0.01);Meanwhile, compared with the perindopril group, the medium dosage, the lower dosage of the Chinese medicine group and the perindopril combined with traditional Chinese group had statistical significance (P<0.05or P<0.01)
4. In SHR group, rats' serum creatinine increased significantly, and had evident differences compared with WKY group (P<0.01); After intervention with drug, serum creatinine contents of each treatment group declined evidently and all had significant difference (P<0.01) compared with SHR group. Especially Chinese medicine combined with western medicine group declined evidently, and was0.50times that of SHR group (P<0.01); The high dosage of Chinese medicine was0.53times that of SHR group (P<0.01); The perindopril group was0.59times that of SHR group (P<0.01); The medium dosage of Chinese medicine was0.70times that of SHR group (P<0.01); The lower dosage of Chinese medicine was0.78times that of SHR group (P<0.01); The difference between perindopril combined with traditional Chinese group、the high dosage、the medium dosage and the lower dosage of the Chinese medicine group compared with the perindopril group had statistical significance (P<0.01)
5. In SHR group, rats' urea nitrogen increased significantly, and had evident differences compared with WKY group (P<0.01); After intervention with drug, urea nitrogen contents of each treatment group declined evidently and all had significant difference (P<0.01) compared with SHR group. Especially Chinese medicine combined with western medicine group declined evidently, and was0.50times that of SHR group (P<0.01); The high dosage of Chinese medicine was0.60times that of SHR group (P<0.01); The perindopril group was0.61times that of SHR group (P<0.01); The medium dosage of Chinese medicine was0.63times that of SHR group (P<0.01); The lower dosage of Chinese medicine was0.70times that of SHR group (P<0.01); Compared with the perindopril group, the Chinese medicine combined with western medicine group,the medium dosage and the lower dosage of Chinese medicine, had statistical significance (P<0.01or P<0.05)
6. Under the light microscope observation, HE staining showed that glomerular atrophic slightly in SHR group, and lumen shape is Irregular; Lumen structure of Proximal and distal tubule is not clear, we can saw a lot of mucinous substances from the invasion of chronic inflammatory cell. we can also saw granular degeneration hyperplasia、Irregular arrangement from renal tubular epithelial cells. vascular wall of glomerular were thicken, renal interstitial were hyperplasia. The morphology of renal tubular and glomerular in each treatment group have improved, especially, the Chinese medicine combined with western medicine group improved obviously.
7. In SHR group, Ang-Ⅱ concentrations in rat renal tissues increased significantly, and had evident differences compared with WKY group (P<0.01) After intervention with drug, serum creatinine contents of each treatment group declined evidently and all had significant difference (P<0.01) compared with SHR group. Especially Chinese medicine combined with western medicine group declined evidently, and was0.40times that of SHR group (P<0.01); the perindopril group was0.41times that of SHR group (P<0.01); The high dosage of Chinese medicine was0.48times that of SHR group (P<0.01); The medium dosage of Chinese medicine was0.52times that of SHR group (P<0.01); The lower dosage of Chinese medicine was0.58times that of SHR group (P<0.01); The high dosage、 the medium dosage and the lower dosage of the Chinese medicine group had statistical significance (P<0.01) compared with the perindopril group; The Chinese medicine combined with western medicine group had no statistical significance compared with the perindopril group (P>0.05); The high dosage of the Chinese medicine group have statistical significance compared with the medium dosage、the lower dosage of the Chinese medicine group (P<0.01)
8. In SHR group, collagen Ⅰ concentrations in rat renal tissues increased significantly, and had evident differences compared with WKY group (P<0.01) After intervention with drug, the results showed that compared with the model group, collagen Ⅰ in each treatment group all have decreased (P<0.01or P<0.05), the increased Chinese medicine combined with western medicine group was the most obvious, next came perindopril group, the high dosage of the Chinese medicine group, the medium dosage of the Chinese medicine group, the lower dosage of the Chinese medicine group. Compared with the perindopril group, the Chinese medicine combined with western medicine group and the high dosage of the Chinese medicine group had no statistical significance(P>0.05), the medium dosage and the lower dosage of the Chinese medicine group had statistical significance compared with the perindopril group (P<0.05); the high dosage of the Chinese medicine group had statistical significance compared with the medium dosage and the lower dosage of the Chinese medicine group(P<0.05);
9. In SHR group, collagen Ⅲ concentrations in rat renal tissues increased significantly, and had evident differences compared with WKY group (P<0.01) After intervention with drug, the results showed that compared with the SHR group, collagen Ⅲ in each treatment group all have decreased (P<0.01or P<0.05), the increased Chinese medicine combined with western medicine group was the most obvious, next came the high dosage of the Chinese medicine group, perindopril group, the medium dosage of the Chinese medicine group, the lower dosage of the Chinese medicine group. Compared with the perindopril group, the Chinese medicine combined with western medicine group and the high dosage and the medium dosage of the Chinese medicine group had no statistical significance(P>0.05), the lower dosage of the Chinese medicine group had statistical significance compared with the perindopril group(P<0.05); the high dosage of the Chinese medicine group had statistical significance compared with the medium dosage and the lower dosage of the Chinese medicine group (P<0.05);
10. In SHR group, collagen Ⅳ concentrations in rat renal tissues increased significantly, and had evident differences compared with WKY group (P<0.01) After intervention with drug, the results showed that compared with the model group, collagen Ⅳ in each treatment group all have decreased (P<0.01or P<0.05), the increased perindopril group was the most obvious, next came the high dosage of the Chinese medicine group, Chinese medicine combined with western medicine group, the medium dosage of the Chinese medicine group, the lower dosage of the Chinese medicine group. Compared with the perindopril group, the Chinese medicine combined with western medicine group and the high dosage of the Chinese medicine group had no statistical significance(P>0.05), the medium dosage and the lower dosage of the Chinese medicine group had statistical significance compared with the perindopril group(P<0.05);
11. In SHR group, MMP-1gene expression in rats reduced significantly, and had evident statistical significance compared with WKY group (P<0.01); After intervention with drug, the results showed that MMP-1gene expression of each treatment group had risen (P<0.01or P<0.05), especially, the increased perindopril group was the most obvious; Comparison between the perindopril group、the Chinese medicine combined with western medicine group and the high dosage、the medium dosage of the Chinese medicine group had no statistical significance(P>0.05), the perindopril group had statistical significance compared with the lower dosage of the Chinese medicine group(P<0.05)
12. In SHR group, MMP-2gene expression in rats increased significantly, and had evident statistical significance compared with WKY group (P<0.01); After intervention with drug, the results showed that MMP-2gene expression of each treatment group had decreased (P<0.01or P<0.05), especially, the reduced perindopril group was the most obvious; perindopril group had no statistical significance compared with Chinese medicine combined with western medicine group、the high dosage of the Chinese medicine group(P>0.05), perindopril group had statistical significance compared with the medium dosage、the lower dosage of the Chinese medicine group(P<0.05).
13. In SHR group, MMP-9gene expression in rats increased significantly, and had evident statistical significance compared with WKY group (P<0.01); After intervention with drug, the results showed that MMP-9gene expression of each treatment group had decreased (P<0.01or P<0.05), especially, the reduced perindopril group was the most obvious; perindopril group had no statistical significance compared with Chinese medicine combined with western medicine groups the high dosage of the Chinese medicine group(P>0.05), perindopril group had statistical significance compared with the medium dosage、the lower dosage of the Chinese medicine group.(P<0.05).
14. The gene expression of rats TGF-β1in SHR group was higher than that of WKY group(P<0.01); After intervention with drug, by comparing to SHR group, the gene expression of TGF-β1in perindopril group, Chinese medicine combined with western medicine group, the high dosage and the medium dosage of the Chinese medicine group all was decreased (P<0.01or P<0.05), the decreased Chinese medicine combined with western medicine group was the most obvious; while the lower dosage of the Chinese medicine group had no statistical significance compared with SHR group (P>0.05); perindopril group had statistical significance compared with the lower dosage of the Chinese medicine group.(P <0.05), perindopril group had no statistical significance compared with Chinese medicine combined with western medicine group、the high dosage、the medium dosage of the Chinese medicine group(P>0.05)
15. The gene expression of rats Smad3in SHR group was higher than that of WKY group(P<0.01); After intervention with drug, by comparing to SHR group, the gene expression of Smad3in perindopril group, Chinese medicine combined with western medicine group, the high dosage and the medium dosage of the Chinese medicine group all was decreased (P<0.01or P<0.05), the decreased Chinese medicine combined with western medicine group was the most obvious; while the lower dosage of the Chinese medicine group had no statistical significance compared with SHR group (P>0.05); perindopril group had no statistical significance compared with Chinese medicine combined with western medicine group、the high dosage、the medium dosage of the Chinese medicine group(P>0.05), perindopril group had statistical significance compared with the lower dosage of the Chinese medicine group.(P<0.05).
16. The gene expression of rats Smad5in SHR group was lower than that of WKY group(P<0.01); After intervention with drug, by comparing to SHR group, the gene expression of Smad5in perindopril group and Chinese medicine combined with western medicine group all have increased (P<0.01), the increased Chinese medicine combined with western medicine group was the most obvious; while the high dosage and the medium dosage of the Chinese medicine group were also markedly elevated(P<0.05), the lower dosage of the Chinese medicine group were not markedly elevated(P>0.05); perindopril group had no statistical significance compared with Chinese medicine combined with western medicine group、the high dosage and the medium dosage of the Chinese medicine group (P>0.05), perindopril group had statistical significance compared with the lower dosage of the Chinese medicine group.(P<0.05).
17. The gene expression of rats Smad6in SHR group was higher than that of WKY group (P<0.01); After intervention with drug, by comparing to SHR group, the gene expression of Smad6in perindopril group and Chinese medicine combined with western medicine group all have decreased (P<0.01), the decreased Chinese medicine combined with western medicine group was the most obvious; while the high dosage and the medium dosage of the Chinese medicine group were also markedly elevated(P<0.05), the lower dosage of the Chinese medicine group were not markedly elevated(P>0.05); perindopril group had no statistical significance compared with Chinese medicine combined with western medicine group、the high dosage and the medium dosage of the Chinese medicine group (P>0.05), perindopril group had statistical significance compared with the lower dosage of the Chinese medicine group.(P<0.05).
18. SHR rats BMP-7protein expression of model group was lower than that of WKY group (P<0.01); After intervention with drug, by comparing to SHR group, BMP-7protein expression in each treatment group increased evidently (P<0.01), the increased Chinese medicine combined with western medicine group was the most obvious, next came perindopril group, the high dosage, the medium dosage the lower dosage of the Chinese medicine group. perindopril group had statistical significance compared withthe medium dosage、the lower dosage of the Chinese medicine group (P<0.05), perindopril group had no statistical significance compared with Chinese medicine combined with western medicine group and the high dosage of the Chinese medicine group (P>0.05)
Conclusions:
1. Qishen spleen-invigorating prescription can reduce the systolic pressure of SHR rats', but antihypertensive effect is less than angiotensin converting enzyme inhibitors (ACEI), Qishen spleen-invigorating prescription combined with perindopril achieve the best antihypertensive effect.
2. Qishen spleen-invigorating prescription can reduce the content of SHR rats' urinal β2-microbulin and micro albumin MALb and the content of blood urea nitrogen and serum creatinine in the SHR rat kidney tissue, Improve the pathological changes of renal interstitial fibrosis and protect the kidney.
3. In order to reduce the abnormal accumulation of the ECM and increase the ECM degradation and reduce renal interstitial fibrosis, Qishen spleen-invigorating prescription to mechanism of protecting the kidney were by reducing the secretion of Ang-Ⅱ content and the type Ⅰ、typeⅢ and type Ⅳ collagen contents. in the SHR rat kidney tissue, raising the gene expression levels of MMP-1ang reducing the gene expression levels of MMP-2、MMP-9.
4. In order to suppression renal tubular epithelial cells to mesenchymal transdifferentiation of interstitial cells in pathological conditions, reduce collagen generation, increase the ECM degradation, reversal renal interstitial fibrosis and promote the recovery of renal function. Role of target Qishen spleen-invigorating prescription protecting the kidney were by reducing the gene expression levels of Smad6, reversalling its inhibition on the expression of BMP-7/Smad5, adjusting negatively TGF-β1/Smad3signal pathway.
材料与方法：
1.实验动物：24周龄雄性自发性高血压大鼠(SHR)60只,体质量(360±20)g,同周龄雄性Wister-Kyoto(WKY)大鼠10只,体质量(350±20)g；大鼠于实验前常规观察1周,然后进行实验。
2.实验动物分组：SHR大鼠60只随机分为6组：SHR组(模型组)、培哚普利组(西药组)、中药加西药组(培哚普利联合中药中剂量组)、中药高、中、低剂量组,每组各10只；WKY大鼠10只作为WKY组(正常对照组)。
3.选用药物：SHR组给予蒸馏水；根据《人和动物按体表面积折算的等效剂量比值表》,培哚普利组用药量为0.4mg*kg-1*d-1,用药时将药溶于蒸馏水中；中药治疗组采用标准水煎工艺,浓缩生药含量4g/mL,高、中、低剂量组用药量按照大鼠体重折算分别为4g/100g,2g/100g和1g/100g;中药加西药组用药为同剂量培哚普利溶解于中药中剂量方中。给药体积均为每100g体重给予药液1mL。大鼠每日一次灌胃,连续治疗6周。
4.收缩压测量：采用大鼠尾动脉间接测压法,在室温22±2℃条件下,将安静、清醒状态大鼠于40℃下预热(用温控器调节温度恒定)约15min,用BP-98A型大鼠智能无创血压计,测量大鼠尾动脉收缩压,连续测量3次,取其均值。分别于实验开始前与药物干预后4小时、2周、4周和6周末进行测量。
5.采用ELISA方法测定尿微量白蛋白(MALb)、尿β2-微球蛋白(β2-MG)和肾组织Ang-Ⅱ的含量。
6.全自动生物化学分析仪检测各组大鼠血清肌酐(SCr)和尿素氮(BUN)含量。
7.常规HE染色,镜下观察各组大鼠肾脏的形态学变化。
8.免疫组化方法检测各组大鼠肾脏组织中Ⅰ型胶原、Ⅲ型胶原和Ⅳ型胶原积分光密度值的变化。
9.采用RT-PCR法检测各组大鼠肾脏组织中MMP-1、MMP-2、MMP-9、TGF-β、Smad3、 Smad5、Smad6的基因表达水平。
10.采用Western Blot法检测各组大鼠肾脏组织中BMP-7的蛋白表达水平。
结果：
1.实验开始前与用药后4小时、2周、4周和6周分别应用智能无创血压计监测各组大鼠尾动脉收缩压的变化,观察到用药前SHR各组大鼠血压均高于WKY组,WKY组与其他各组比较有显著统计学意义(P<0.01),其余各组间比较无统计学意义(P>0.05)：给药后4小时：各组大鼠收缩压无明显变化,WKY组与其他各组比较有显著统计学意义(P<0.01)；给药后2周：中药加西药组收缩压明显降低,与SHR组相比有统计学意义(P<0.05),而培哚普利组、中药高、中、低剂量组大鼠收缩压也有一定程度的下降,但与SHR组比较无统计学意义(P>0.05)；给药后4周：大鼠收缩压持续下降,培哚普利组、中药加西药组与SHR组相比具有显著统计学差异(P<0.01),中药高、中、低剂量组与SHR组比较无统计学意义(P>0.05),培哚普利组与中药加西药组比较无统计学意义(P>0.05)；给药后6周：培哚普利组、中药加西药组与SHR组相比具有显著统计学差异(P<0.01),中药高、中、低剂量组与SHR组比较无统计学意义(P>0.05),培哚普利组与中药加西药组比较无统计学意义(P>0.05)。
2.SHR组大鼠尿微量白蛋白含量明显增加,与WKY组比较有显著性差异(P<0.01)：经药物干预后各治疗组尿微量白蛋白含量明显下降,与SHR组相比,均有显著性差异(P<0.01),其中,中药加西药组下降最明显,是SHR组的0.60倍(P<0.01),中药高剂量组是SHR组的0.62倍(P<0.01),培哚普利组是SHR组的0.64倍(P<0.01),中药中剂量组是SHR组的0.66倍(P<0.01),中药低剂量组是SHR组的0.70倍(P<0.01)；其他各组间比较无统计学意义(P>0.05)。
3.与WKY组相比,SHR组尿β2-微球蛋白含量明显增加,具有显著性差异(P<0.01)：经药物干预后各治疗组尿β2-微球蛋白含量明显下降,与SHR组相比,均有显著性差异(P<0.01),其中,以中药加西药组下降最明显,是SHR组的0.29倍(P<0.01)；培哚普利组是SHR组的0.38倍(P<0.01),中药高剂量组是SHR组的0.32倍(P<0.01),中药中剂量组是SHR组的0.47倍(P<0.01),中药低剂量组是SHR组的0.50倍(P<0.01)；中药加西药组、中药中、低剂量组与培哚普利组比较有统计学意义(P<0.05或P<0.01)。
4.SHR组大鼠血肌酐含量明显增加,与WKY组比较有显著性差异(P<0.01)；经药物干预后各治疗组血肌酐含量明显下降,与SHR组相比,均有显著性差异(P<0.01),其中,中药加西药组下降最明显,是SHR组的0.50倍(P<0.01),中药高剂量组是SHR组的0.53倍(P<0.01),培哚普利组是SHR组的0.59倍(P<0.01),中药中剂量组是SHR组的0.70倍(P<0.01),中药低剂量组是SHR组的0.78倍(P<0.01)；中药加西药组、中药高、中、低剂量组与培哚普利组相比有统计学意义(P<0.01)。
5.SHR组大鼠血尿素氮含量明显增加,与WKY组比较有显著性差异(P<0.01)；经药物干预后各治疗组血尿素氮含量明显下降,与SHR组相比,均有显著性差异(P<0.01)其中中药加西药组下降最明显,是SHR组的0.50倍(P<0.01),中药高剂量组是SHR组的0.60倍(P<0.01),培哚普利组是SHR组的0.61倍(P<0.01),中药中剂量组是SHR组的0.63倍(P<0.01),中药低剂量组是SHR组的0.70倍(P<0.01)；中药加西药组、中药中、低剂量组与培哚普利组比较有统计学意义(P<0.01或P<0.05)。
6.HE染色在光镜下观察结果显示：SHR组肾小球出现轻度萎缩,管腔形态不规则,近、远端小管管腔结构不清晰,可见慢性炎细胞浸润产生的大量粘液性物质,上皮细胞可见颗粒变性、增生、排列不规则,肾小球血管管壁增厚,肾间质出现纤维组织增生。各治疗组肾小管、肾小球的形态、炎性粘液浸润以及间质纤维化均有所改善,其中中药加西药组改善最为明显。
7.SHR组大鼠肾组织中Ang-Ⅱ浓度明显升高,与WKY组比较有显著性差异(P<0.01)；经药物干预后各治疗组Ang-Ⅱ含量明显下降,与SHR组相比,均有显著性差异(P<0.01),其中,中药加西药组下降最明显,是SHR组的0.40倍(P<0.01),培哚普利组是SHR组的0.41倍(P<0.01),中药高剂量组是SHR组的0.48倍(P<0.01),中药中剂量组是SHR组的0.52倍(P<0.01),中药低剂量组是SHR组的0.58倍(P<0.01)；中药高、中、低剂量组与培哚普利组比较有统计学意义(P<0.01)；中药加西药组与培哚普利组比较无统计学意义(P>0.05)；中药高剂量组与中药中、低剂量组比较有统计学意义(P<0.01)。
8.SHR组大鼠肾组织中Ⅰ型胶原的积分光密度值明显升高,与WKY组比较有显著性差异(P<0.01)；经药物干预后的结果显示,各治疗组大鼠Ⅰ型胶原的积分光密度值均出现降低,与SHR组比较(P<0.01或P<0.05),其中以中药加西药组降低最明显,其次是培哚普利组、中药高、中、低剂量组。与培哚普利组相比,中药加西药组和中药高剂量组无统计学意义(P>0.05),而中药中、低剂量组与培哚普利组比较有统计学意义(P<0.05)；中药高剂量组与中药中、低剂量组比较有统计学意义(P<0.05)。
9.SHR组大鼠肾组织中Ⅲ型胶原的积分光密度值明显升高,与WKY组比较有显著性差异(P<0.01)；经药物干预后的结果显示,各治疗组大鼠Ⅲ型胶原积分光密度值均有所降低,与SHR组比较(P<0.01或P<0.05),其中以中药加西药组降低最明显,其次是中药高剂量组、培哚普利组、中药中剂量组和中药低剂量组。与培哚普利组相比,中药加西药组、中药高、中剂量组无统计学意义(P>0.05),而中药低剂量组与培哚普利组比较有统计学意义(P<0.05)；中药高剂量组与中药中、低剂量组比较有统计学意义(P<0.05)。
10.SHR组大鼠肾组织中Ⅳ型胶原的积分光密度值明显升高,与WKY组比较有显著性差异(P<0.01)；经药物干预后的结果显示,各治疗组大鼠Ⅳ型胶原积分光密度值均明显降低,与SHR组比较(P<0.01或P<0.05),其中以培哚普利组下降最明显,其次是中药高剂量组、中药加西药组、中药中、低剂量组。与培哚普利组相比,中药加西药组和中药高剂量组无统计学意义(P>0.05),而中药中、低剂量组与培哚普利组比较有统计学意义(P<0.05)。
11.SHR组大鼠肾组织中MMP-1基因表达明显降低,与WKY组比较有显著统计学意义(P<0.01)；经药物干预后的结果显示,与SHR组比较,各治疗组MMP-1基因表达均升高(P<0.01或P<0.05),其中以培哚普利组升高最明显；培哚普利组与中药加西药组、中药高、中剂量组比较无统计学意义(P>0.05),培哚普利组与中药低剂量组比较有统计学意义(P<0.05)。
12.SHR组大鼠肾组织中MMP-2基因表达明显升高,与WKY组比较有显著统计学意义(P<0.01)；经药物干预后的结果显示,与SHR组比较,各治疗组MMP-2基因表达均有所下降(P<0.01或P<0.05),其中以培哚普利组下降最明显；培哚普利组与中药加西药组、中药高剂量组比较无统计学意义(P>0.05)；与中药中、低剂量组比较有统计学意义(P<0.05)。
13.SHR组大鼠肾组织中MMP-9基因表达明显升高,与WKY组比较有显著统计学意义(P<0.01)；经药物干预后的结果显示,与SHR组比较,各治疗组MMP-9基因表达均出现降低(P<0.01或P<0.05),其中以培哚普利组降低最明显；培哚普利组与中药加西药组、中药高剂量组比较无统计学意义(P>0.05)；与中药中、低剂量组比较有统计学意义(P<0.05)。
14.SHR组大鼠肾组织中TGF-β1基因表达明显高于WKY组(P<0.01)；经药物干预后的结果显示,与SHR组比较,培哚普利组、中药加西药组、中药高、中剂量组TGF-β,基因表达均降低(P<0.01或P<0.05),其中以中药加西药组降低最明显；而中药低剂量组与SHR组比较无统计学意义(P>0.05)；培哚普利组与中药低剂量组比较有统计学意义(P<0.05),培哚普利组与中药加西药组、中药高、中剂量组比较无统计学意义(P>0.05)。
15.SHR组大鼠肾组织中Smad3基因表达明显高于WKY组(P<0.01)；经药物干预后的结果显示,与SHR组比较,培哚普利组、中药加西药组、中药高、中剂量组Smad3基因表达均明显降低(P<0.01或P<0.05),其中以中药加西药组下降最为明显,而中药低剂量组与SHR组比较无统计学意义(P>0.05)；与培哚普利组比较,中药加西药组、中药高、中剂量组无明显差异(P>0.05),中药低剂量组与培哚普利组比较有统计学意义(P<0.05)。
16.SHR组大鼠肾组织中Smad5基因表达明显低于WKY组(P<0.01)；经药物干预后的结果显示,与SHR组比较,培哚普利组和中药加西药组Smad5基因表达均明显升高(P<0.01),中药加西药组升高最为明显；而中药高、中剂量组也有显著升高(P<0.05),中药低剂量组升高不明显(P>0.05)；中药加西药组、中药高、中剂量组与培哚普利组比较无统计学意义(P>0.05),中药低剂量组与培哚普利组比较有统计学意义(P<0.05)。
17.SHR组大鼠肾组织中Smad6基因表达明显高于WKY组(P<0.01)；经药物干预后的结果显示,与SHR组比较,中药加西药组和培哚普利组Smad6基因表达均明显降低(P<0.01),以中药加西药组最为明显；而中药高、中剂量组也有明显降低(P<0.05),中药低剂量组下降不明显(P>0.05)；中药加西药组、中药高、中剂量组与培哚普利组比较无统计学意义(P>0.05),培哚普利组与中药低剂量组比较有统计学意义(P<0.05)。
18.SHR组大鼠肾组织中BMP-7蛋白表达明显低于WKY组(P<0.01)；经药物干预后的结果显示,与SHR组比较,各治疗组BMP-7蛋白表达均明显升高(P<0.01),中药加西药组升高最明显,其次是培哚普利组、中药高、中、低剂量组。培哚普利组与中药中、低剂量组比较有统计学意义(P<0.05),培哚普利组与中药加西药组和中药高剂量组比较无统计学意义(P>0.05)。
结论：
1.芪参健脾方可以降低SHR的血压,但其降压效果不及血管紧张素转换酶抑制剂(ACEI)培哚普利,与培哚普利联合用药可以达到最佳的降压效果。
2.芪参健脾方可以降低SHR尿β2-微球蛋白(β2-MG)和微量白蛋白(MALb)(?)的含量,降低血尿素氮(BUN)和肌酐(SCr)的含量,改善肾间质纤维化的病理改变,具有保护肾功能的作用。
3.芪参健脾方对肾脏保护作用的机制可能是通过降低SHR肾组织中Ang-Ⅱ的水平,上调MMP-1、下调MMP-2、MMP-9的基因表达,从而降低肾组织中Ⅰ型、Ⅲ型、Ⅳ型胶原含量,减少ECM的异常聚集,增加ECM的降解,减轻肾小管和间质的纤维化。
4.芪参健脾方肾脏保护的作用靶点,可能是通过下调Smad6的基因表达,逆转其对BMP-7/Smad5的表达的抑制,从而负向调节TGF-β1/Smad3信号传导通路,抑制病理条件下的肾小管上皮细胞向间充质细胞的转分化(EMT),减少胶原的产生,促进ECM的降解,逆转肾间质纤维化的进程,促进肾功能的恢复。
Purposes:
This experiment was to observe the effect of qishen spleen-invigorating prescription on the pressure of spontaneous hypertensive rat (SHR), renal function and the Ang-Ⅱ content of renal、the collagen Ⅰ、Ⅲ、Ⅳ type of renal and on the gene expression of MMP-1、MMP-2、MMP-9、TGF-β、Smad3、Smad5、Smad6and the protein expression of BMP-7, which have selected qishen spleen-invigorating prescription to be the intervention factor and adopted reverse transcription polymerase chain reaction (PT-PCR)、western-blot、ELISA and immunohistochemistry methods. And observe the effect of qishen spleen-invigorating prescription on decompression of SHR and the protection of kidney from the aspect of The overall level、histomorphology、the gene、protein and so on, investigating its mechanisms and targets. Thus, we can provide experimental basis for its application to the hypertensive clinical treatment and offer a basic thinking and theory supports of the treatment of Chinese medicine on hypertension and the protection of Chinese medicine on target organs.
Materials and methods:
1. Experimental animals:60twenty four-week-old male spontaneously hypertensive rats, body mass (360±20) g;10age-matched male Wistar-Kyoto rats, body mass (350±20) g; until one week routine observation can we experimentize.
2. Experimental animal grouping:60SHE rats were randomly divided into6groups: SHR group (model group), perindopril group (western medicine group), perindopril combined with the medium dosage of traditional Chinese group, the high dosage group, the medium dosage group and the low dosage group of the traditional Chinese medicine, all the groups have10rats individually; Ten Wistar-Kyoto rats were selected as WKY group(normal control group).
3. Selecting drugs:The SHR group were given distilled water; according to the body surface area ratio table in human and animals, perindopril dosage was0.4mg*kg-1*d-1, and the drug should be dissolved in distilled water; Traditional Chinese group adopted standard decoction process; the content dosage of concentrated crude drug was4g/mL; According to the rats' weight, the drugs dose of high, medium and low dosage group were4g/100g,2g/100g and1g/100g respectively; And about the perindopril combined with traditional Chinese group, the same dose of western medicine dissolved in the medium dosage of Chinese medicine. The administration volume is per100g of body weight givenlml liquid. The rats were given gastric perfusion per day with6weeks continuous treatment.
4. Systolic blood pressure measurement:under the22±2℃room temperature, we use the rats'caudal arterial pressure indirect manometric method, preheating the waking rats to40℃about40min (using the thermostat regulates the constant temperature); We introduced the BP-98A rat intelligent non-invasive blood pressure monitor to measure rat tail pressure in the quiet state and the waking state three times, and got the average value. Measure rat tail pressure before the start of the experiment and after4hours medication,2weeks medication,4weeks medication and6weeks medication.
5. Using the ELISA method to determine the content of the urinary micro albumin, urinary β2-microglobulin and renal tissue Ang-Ⅱ.
6. Serum creatinine and urea nitrogen were detected by automatic biochemical analyzer.
7. Renal morphological changes were observed under microscopy by normal HE dyeing.
8. The Integrated Optical Density (IOD)) changes of collagen Ⅰ、collagenⅢ and collagen IV in each tissues of rats were detected by immunohistochemical method.
9. The gene expression levels of MMP-1, MMP-2, MMP-9, TGF-β1, Smad3、Smad5and Smad6in each rats' kidney tissue were detected by RT-PCR method.
10. The protein levels of BMP-7in each set of rats' kidney tissue were detected by Western-blot method.
Results:
1. We used the intelligent non-invasive blood pressure monitor to monitor rats' caudal arterial systolic pressure before the experiment began and after4hours medication,2weeks medication,4weeks medication and6weeks medication, and we noticed that, before the experiment started, SHR rats pressure in each group were higher than in WKYs'. WKY group had statistical significance compared with other groups (P<0.01), and there was no statistical significance between the comparison of the rest groups; After the administration of4hours, each group of systolic pressure in rats did not change significantly, WKY group has statistical significance compared with other groups(P<0.01); After the administration of2weeks, the perindopril combined with traditional Chinese group's systolic blood pressure reduced significantly, and had statistical significance compared with SHR group (P<0.05), while the rats'systolic blood pressure in perindopril group, the high dosage、the medium dosage and the lower dosage of the Chinese medicine group also had a certain degree of decline, and had no statistical significance compared with SHR group (P>0.05); After the administration of4weeks, the rats'systolic blood pressure decreased continuously. Compared with SHR group, the perindopril group and the perindopril combined with traditional Chinese group had great statistical significance (P <0.01), while the high dosage、the medium dosage and the lower dosage of the Chinese medicine group had no statistical significance compared with SHR group (P>0.05), and compared with perindopril combined with traditional Chinese group, the perindopril group had no statistical significance (P>0.05)。After the administration of6weeks, the perindopril group, the perindopril combined with traditional Chinese group, the medium dosage and the lower dosage of the Chinese medicine group all had statistical significant differences (P<0.01), the high dosage of the Chinese medicine group had statistical significance compared with SHR group (P<0.05), the perindopril group had no statistical significance compared with the perindopril combined with traditional Chinese group (P>0.05)。
2. In SHR group, rats' urinary micro globulin increased significantly, and had evident differences compared with WKY group (P<0.01); After intervention with drug, micro globulin contents of each treatment group decreased significantly and all had significant difference (P<0.01) compared with SHR group. Among them the Chinese medicine combined with western medicine group declined evidently, and was0.66times that of SHR group(P<0.01), the lower dosage of Chinese medicine was0.70times that of SHR group (P<0.01); Comparison between other groups was not statistical significance (P>0.05)
3. In SHR group, rats' urinary β2-microglobulin increased significantly and had remarkable statistics meaning compared with WKY group (P<0.01); After intervention with drug, urinary (32-microglobulin contents of each treatment group decreased significantly, and had remarkable statistics meaning compared with SHR group (P<0.01), among them the perindopril combined with traditional Chinese group declined evidently and was0.29times that of the SHR group(P <0.01), the perindopril group was0.32times that of the SHR group (P<0.01), the medium dosage of the Chinese medicine group was0.47times that of the SHR group(P<0.01), the lower dosage of the Chinese medicine group was0.50times that of the SHR group(P<0.01);Meanwhile, compared with the perindopril group, the medium dosage, the lower dosage of the Chinese medicine group and the perindopril combined with traditional Chinese group had statistical significance (P<0.05or P<0.01)
4. In SHR group, rats' serum creatinine increased significantly, and had evident differences compared with WKY group (P<0.01); After intervention with drug, serum creatinine contents of each treatment group declined evidently and all had significant difference (P<0.01) compared with SHR group. Especially Chinese medicine combined with western medicine group declined evidently, and was0.50times that of SHR group (P<0.01); The high dosage of Chinese medicine was0.53times that of SHR group (P<0.01); The perindopril group was0.59times that of SHR group (P<0.01); The medium dosage of Chinese medicine was0.70times that of SHR group (P<0.01); The lower dosage of Chinese medicine was0.78times that of SHR group (P<0.01); The difference between perindopril combined with traditional Chinese group、the high dosage、the medium dosage and the lower dosage of the Chinese medicine group compared with the perindopril group had statistical significance (P<0.01)
5. In SHR group, rats' urea nitrogen increased significantly, and had evident differences compared with WKY group (P<0.01); After intervention with drug, urea nitrogen contents of each treatment group declined evidently and all had significant difference (P<0.01) compared with SHR group. Especially Chinese medicine combined with western medicine group declined evidently, and was0.50times that of SHR group (P<0.01); The high dosage of Chinese medicine was0.60times that of SHR group (P<0.01); The perindopril group was0.61times that of SHR group (P<0.01); The medium dosage of Chinese medicine was0.63times that of SHR group (P<0.01); The lower dosage of Chinese medicine was0.70times that of SHR group (P<0.01); Compared with the perindopril group, the Chinese medicine combined with western medicine group,the medium dosage and the lower dosage of Chinese medicine, had statistical significance (P<0.01or P<0.05)
6. Under the light microscope observation, HE staining showed that glomerular atrophic slightly in SHR group, and lumen shape is Irregular; Lumen structure of Proximal and distal tubule is not clear, we can saw a lot of mucinous substances from the invasion of chronic inflammatory cell. we can also saw granular degeneration hyperplasia、Irregular arrangement from renal tubular epithelial cells. vascular wall of glomerular were thicken, renal interstitial were hyperplasia. The morphology of renal tubular and glomerular in each treatment group have improved, especially, the Chinese medicine combined with western medicine group improved obviously.
7. In SHR group, Ang-Ⅱ concentrations in rat renal tissues increased significantly, and had evident differences compared with WKY group (P<0.01) After intervention with drug, serum creatinine contents of each treatment group declined evidently and all had significant difference (P<0.01) compared with SHR group. Especially Chinese medicine combined with western medicine group declined evidently, and was0.40times that of SHR group (P<0.01); the perindopril group was0.41times that of SHR group (P<0.01); The high dosage of Chinese medicine was0.48times that of SHR group (P<0.01); The medium dosage of Chinese medicine was0.52times that of SHR group (P<0.01); The lower dosage of Chinese medicine was0.58times that of SHR group (P<0.01); The high dosage、 the medium dosage and the lower dosage of the Chinese medicine group had statistical significance (P<0.01) compared with the perindopril group; The Chinese medicine combined with western medicine group had no statistical significance compared with the perindopril group (P>0.05); The high dosage of the Chinese medicine group have statistical significance compared with the medium dosage、the lower dosage of the Chinese medicine group (P<0.01)
8. In SHR group, collagen Ⅰ concentrations in rat renal tissues increased significantly, and had evident differences compared with WKY group (P<0.01) After intervention with drug, the results showed that compared with the model group, collagen Ⅰ in each treatment group all have decreased (P<0.01or P<0.05), the increased Chinese medicine combined with western medicine group was the most obvious, next came perindopril group, the high dosage of the Chinese medicine group, the medium dosage of the Chinese medicine group, the lower dosage of the Chinese medicine group. Compared with the perindopril group, the Chinese medicine combined with western medicine group and the high dosage of the Chinese medicine group had no statistical significance(P>0.05), the medium dosage and the lower dosage of the Chinese medicine group had statistical significance compared with the perindopril group (P<0.05); the high dosage of the Chinese medicine group had statistical significance compared with the medium dosage and the lower dosage of the Chinese medicine group(P<0.05);
9. In SHR group, collagen Ⅲ concentrations in rat renal tissues increased significantly, and had evident differences compared with WKY group (P<0.01) After intervention with drug, the results showed that compared with the SHR group, collagen Ⅲ in each treatment group all have decreased (P<0.01or P<0.05), the increased Chinese medicine combined with western medicine group was the most obvious, next came the high dosage of the Chinese medicine group, perindopril group, the medium dosage of the Chinese medicine group, the lower dosage of the Chinese medicine group. Compared with the perindopril group, the Chinese medicine combined with western medicine group and the high dosage and the medium dosage of the Chinese medicine group had no statistical significance(P>0.05), the lower dosage of the Chinese medicine group had statistical significance compared with the perindopril group(P<0.05); the high dosage of the Chinese medicine group had statistical significance compared with the medium dosage and the lower dosage of the Chinese medicine group (P<0.05);
10. In SHR group, collagen Ⅳ concentrations in rat renal tissues increased significantly, and had evident differences compared with WKY group (P<0.01) After intervention with drug, the results showed that compared with the model group, collagen Ⅳ in each treatment group all have decreased (P<0.01or P<0.05), the increased perindopril group was the most obvious, next came the high dosage of the Chinese medicine group, Chinese medicine combined with western medicine group, the medium dosage of the Chinese medicine group, the lower dosage of the Chinese medicine group. Compared with the perindopril group, the Chinese medicine combined with western medicine group and the high dosage of the Chinese medicine group had no statistical significance(P>0.05), the medium dosage and the lower dosage of the Chinese medicine group had statistical significance compared with the perindopril group(P<0.05);
11. In SHR group, MMP-1gene expression in rats reduced significantly, and had evident statistical significance compared with WKY group (P<0.01); After intervention with drug, the results showed that MMP-1gene expression of each treatment group had risen (P<0.01or P<0.05), especially, the increased perindopril group was the most obvious; Comparison between the perindopril group、the Chinese medicine combined with western medicine group and the high dosage、the medium dosage of the Chinese medicine group had no statistical significance(P>0.05), the perindopril group had statistical significance compared with the lower dosage of the Chinese medicine group(P<0.05)
12. In SHR group, MMP-2gene expression in rats increased significantly, and had evident statistical significance compared with WKY group (P<0.01); After intervention with drug, the results showed that MMP-2gene expression of each treatment group had decreased (P<0.01or P<0.05), especially, the reduced perindopril group was the most obvious; perindopril group had no statistical significance compared with Chinese medicine combined with western medicine group、the high dosage of the Chinese medicine group(P>0.05), perindopril group had statistical significance compared with the medium dosage、the lower dosage of the Chinese medicine group(P<0.05).
13. In SHR group, MMP-9gene expression in rats increased significantly, and had evident statistical significance compared with WKY group (P<0.01); After intervention with drug, the results showed that MMP-9gene expression of each treatment group had decreased (P<0.01or P<0.05), especially, the reduced perindopril group was the most obvious; perindopril group had no statistical significance compared with Chinese medicine combined with western medicine groups the high dosage of the Chinese medicine group(P>0.05), perindopril group had statistical significance compared with the medium dosage、the lower dosage of the Chinese medicine group.(P<0.05).
14. The gene expression of rats TGF-β1in SHR group was higher than that of WKY group(P<0.01); After intervention with drug, by comparing to SHR group, the gene expression of TGF-β1in perindopril group, Chinese medicine combined with western medicine group, the high dosage and the medium dosage of the Chinese medicine group all was decreased (P<0.01or P<0.05), the decreased Chinese medicine combined with western medicine group was the most obvious; while the lower dosage of the Chinese medicine group had no statistical significance compared with SHR group (P>0.05); perindopril group had statistical significance compared with the lower dosage of the Chinese medicine group.(P <0.05), perindopril group had no statistical significance compared with Chinese medicine combined with western medicine group、the high dosage、the medium dosage of the Chinese medicine group(P>0.05)
15. The gene expression of rats Smad3in SHR group was higher than that of WKY group(P<0.01); After intervention with drug, by comparing to SHR group, the gene expression of Smad3in perindopril group, Chinese medicine combined with western medicine group, the high dosage and the medium dosage of the Chinese medicine group all was decreased (P<0.01or P<0.05), the decreased Chinese medicine combined with western medicine group was the most obvious; while the lower dosage of the Chinese medicine group had no statistical significance compared with SHR group (P>0.05); perindopril group had no statistical significance compared with Chinese medicine combined with western medicine group、the high dosage、the medium dosage of the Chinese medicine group(P>0.05), perindopril group had statistical significance compared with the lower dosage of the Chinese medicine group.(P<0.05).
16. The gene expression of rats Smad5in SHR group was lower than that of WKY group(P<0.01); After intervention with drug, by comparing to SHR group, the gene expression of Smad5in perindopril group and Chinese medicine combined with western medicine group all have increased (P<0.01), the increased Chinese medicine combined with western medicine group was the most obvious; while the high dosage and the medium dosage of the Chinese medicine group were also markedly elevated(P<0.05), the lower dosage of the Chinese medicine group were not markedly elevated(P>0.05); perindopril group had no statistical significance compared with Chinese medicine combined with western medicine group、the high dosage and the medium dosage of the Chinese medicine group (P>0.05), perindopril group had statistical significance compared with the lower dosage of the Chinese medicine group.(P<0.05).
17. The gene expression of rats Smad6in SHR group was higher than that of WKY group (P<0.01); After intervention with drug, by comparing to SHR group, the gene expression of Smad6in perindopril group and Chinese medicine combined with western medicine group all have decreased (P<0.01), the decreased Chinese medicine combined with western medicine group was the most obvious; while the high dosage and the medium dosage of the Chinese medicine group were also markedly elevated(P<0.05), the lower dosage of the Chinese medicine group were not markedly elevated(P>0.05); perindopril group had no statistical significance compared with Chinese medicine combined with western medicine group、the high dosage and the medium dosage of the Chinese medicine group (P>0.05), perindopril group had statistical significance compared with the lower dosage of the Chinese medicine group.(P<0.05).
18. SHR rats BMP-7protein expression of model group was lower than that of WKY group (P<0.01); After intervention with drug, by comparing to SHR group, BMP-7protein expression in each treatment group increased evidently (P<0.01), the increased Chinese medicine combined with western medicine group was the most obvious, next came perindopril group, the high dosage, the medium dosage the lower dosage of the Chinese medicine group. perindopril group had statistical significance compared withthe medium dosage、the lower dosage of the Chinese medicine group (P<0.05), perindopril group had no statistical significance compared with Chinese medicine combined with western medicine group and the high dosage of the Chinese medicine group (P>0.05)
Conclusions:
1. Qishen spleen-invigorating prescription can reduce the systolic pressure of SHR rats', but antihypertensive effect is less than angiotensin converting enzyme inhibitors (ACEI), Qishen spleen-invigorating prescription combined with perindopril achieve the best antihypertensive effect.
2. Qishen spleen-invigorating prescription can reduce the content of SHR rats' urinal β2-microbulin and micro albumin MALb and the content of blood urea nitrogen and serum creatinine in the SHR rat kidney tissue, Improve the pathological changes of renal interstitial fibrosis and protect the kidney.
3. In order to reduce the abnormal accumulation of the ECM and increase the ECM degradation and reduce renal interstitial fibrosis, Qishen spleen-invigorating prescription to mechanism of protecting the kidney were by reducing the secretion of Ang-Ⅱ content and the type Ⅰ、typeⅢ and type Ⅳ collagen contents. in the SHR rat kidney tissue, raising the gene expression levels of MMP-1ang reducing the gene expression levels of MMP-2、MMP-9.
4. In order to suppression renal tubular epithelial cells to mesenchymal transdifferentiation of interstitial cells in pathological conditions, reduce collagen generation, increase the ECM degradation, reversal renal interstitial fibrosis and promote the recovery of renal function. Role of target Qishen spleen-invigorating prescription protecting the kidney were by reducing the gene expression levels of Smad6, reversalling its inhibition on the expression of BMP-7/Smad5, adjusting negatively TGF-β1/Smad3signal pathway.
引文
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