| 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. |
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