| ObjectiveRegarding Tiao Gan Shen Qu Tan Yu Formula (Formula) as the intervention factor, two hours before the peak of blood pressure was set as the best administration time (19:00 and 00:00) for rats in the experiment. The effects of the Formula on spontaneous hypertensive rats (SHR), including their blood pressure, kidney function, kidney histomorphology, the content of Ang Ⅱ, collagen Ⅰ and collagen Ⅳ, the genetic expression of fibronectin (FN) of transforming growth factor-β1 (TGF-β1), Smad2, Smad3, Smad7, connective tissue growth factor (CTGF) and extra cellular matrix (ECM), as well as the protein expression of TGF-β1、CTGF、FN1, were discussed in this paper, by applying RT-PCR, Western blot, ELISA and immunohistochemical method. Based on the overall level, histomorphology, gene and protein, the anti-hypertensive and kidney-protective effect of the Formula on SHR was observed. Moreover, the mechanisms of those effects were also discussed in this paper, so as to provide not only experimental evidences for the time of treating hypertension, but also ideas and theoretical evidences for the research and exploration on Chinese medicines for both hypertension and target organs.Content and methodsStudy content Consist of six parts1. The effect of the Formula taken before the peak of blood pressure on SHR kidney injury on the early stage;2. The effect of the Formula taken before the peak of blood pressure on the histomorphology of SHR heart and kidney;3. The effect of the Formula taken before the peak of blood pressure on the regulation of signal path TGF-β1-CTGF-FN 1 of SHR;4. The effect of the Formula taken before the peak of blood pressure on the regulation of signal path TGF-β1-Smads of SHR.Experiments1. Chinese medicines:Tiao Gan Shen Qu Tan Yu Formula (for an adult per day)The Formula included 25g Shu Di(rehmannia glutinosa),30g Gui Ban(tortoise plastron) (decocted earlier),15g Dan Shen (salviamiltiorrhiza),12g Gua Lou (Trichosanthes kirilowii Maxim.),5g Tian Qi (panax notoginseng) and 12g Gou Teng (uncaria tomentosa) (decocted later).Meeting the criterion by authentication, all medicinal materials above were produced by Chinese Medicine Yinpian Factory of Guangzhou Medicine Company, marked with a batch of numbers as 13093120. The Formula (Concentration: 200mg·mL-1 crude drugs) was decocted and preserved in refrigerator with 4℃ after cooling down.Losartan (Product name:Ke Ya Su; Batch number:130101; Usage: 100mg/tablet; Manufacturer:Hangzhou Moshadong Pharmaceutical Co., Ltd.)2. Experimental animals and grouping30 SPF class-SHR and 10 SD rats, at the age of 11 weeks, were applied in this experiment, with 220+20g for each. Those two different kinds of rats were bought from Vital River Biotechnology Co. Ltd. and Laboratory Animal Center of Guangzhou University of Chinese Medicine respectively.After one-week routine observation, those rats were grouped for the experiment.A total of 30 male SHR were randomly divided into three groups:Formula group, SHR group and Losartan group, with 10 rats respectively, while 10 SD rats were taken as Control group.Rats were fed by solid pellet feeds with high protein all the time.3. Administration:During 12 weeks, the intragastric administration was given in Formula group twice (19:00 and 00:00) per day, with 4g·kg-1 Formula in accordance to the clinical equivalent dose each time; The intragastric administration was given in Losartan group once in the morning per day with 0.03g · kg-1 Losartan, while equal quantity normal saline (NS) 0.144 g · kg-1 was given for SHR group and Control group respectively.4. Systolic pressure measurement:non-invasive sphygmomanometers were applied to measure the blood pressure in the 2nd,4th,6th,8th,10th and 12th week before or after administration, so as to record the systolic pressure of rats in each group.5. The ELISA was used to determine the level of microalbuminuria (mALb) and β2-urinary microglobulin (β2-MG) of rats.6. The automatic biochemical analyzer was applied to determine the content of serum creatinine (SCr) and blood urea nitrogen (BUN) of rats in each group.7. The light microscope and the electron microscope were applied to observe histomorphological changes of rats’kidneys in each group.8. The immunohistochemical method was used to test the change of original integrated optical density (IOD) of Ang Ⅱ, Collagen Ⅰ, Collagen Ⅳ, Smad2, Smad3 and Smad7 in kidney tissues.9. The RT-PCR was used to detect the mRNA expression of TGF-β1, CTGF, FN, Smad2, Smad3 and Smad7 in kidney tissues.10. The Western blot was used to detected the protein expression of TGF-β1, CTGF and FN in kidney tissues.Results1. The changes of blood pressure before and after administrationIn the experiment, the intelligent non-invasive sphygmomanometer was applied to measure the systolic pressure of rats’ caudal artery for each group in the 2nd,4th,6th,8th,10th and 12th week before or after administration. It was observed that the blood pressure of SHR group was higher than that in Control group before administration, and there was a statistical significance between Control group and other groups (P<0.01), however, there was no statistical significance among rest groups except Control group (P>0.05). The 2nd week after administration:the atrophic pressure decreased significantly in Losartan group, and there was a statistical significance by comparing with SHR group (P<0.01); in addition, the atrophic pressure decreased in a certain degree in Formula group, but there was no statistical significance by comparing to SHR group (P>0.05). The 4th week after administration:the atrophic pressure kept decreasing in Losartan group, and there was a statistical difference by comparing with SHR group (P<0.01); in addition, the anti-hypertension effect showed in Formula group, and there was a statistical significance by comparing with both SHR group and Losartan group (P<0.01, respectively). The 6th-10th week after administration:the blood pressure kept decreasing in both Losartan group and Formula group; in Formula group, there was a statistical difference by comparing with SHR group and Losartan group (P<0.01, respectively). Moreover, there was a statistical significance between Losartan group and SHR group (P<0.01).The 12th week after administration:the anti-hypertension effect was steady in both Formula group and Losartan group; the anti-hypertension effect of Formula group gradually approached to Losartan group, and there was no statistical significance between both groups (P>0.05).2. The content of SCr and BUN of rats in each groupCompared with Control group, the content of SCr significantly elevated (P<0.01) in SHR group, and the content of SCr decreased obviously (P<0.01) in Formula group by comparing with SHR group. With the comparison of Control group, there was a significant elevation of the content of BUN in SHR group, Formula group and Losartan group (P<0.01). There was no statistical difference among SHR group, Formula group and Losartan group (P>0.05)3. The content of mALb and β2-MG of rats in each groupCompared with Control group, the protein expression of mALb significantly elevated (P<0.01) in SHR group. The protein expression of mALb decreased obviously (P<0.05) in Formula group, but there was no regulating effect on Losartan group by comparing with SHR group respectively. With the comparison of Control group, there was no significant changes of the protein expression of β2-MG in both SHR group and Losartan group, and the protein expression of β2-MG decreased significantly in Formula group (P<0.05)4. The histomorphological comparison of kidney tissues via light microscopeFormula group:there was no significant hyperplasia in the glomerular mesangial cell and the mesangial matrix; moreover, there were intact epithelial cells of kidney tubules, without tubiform structures in lumen as well as both fibrous connective tissues and inflammatory cells in mesenchyma. Losartan group:there was a mild hyperplasia in the glomerular mesangial cell and the mesangium, without tubiform structures in lumen but intact epithelial cells of kidney tubules. Control group:there was no significant hyperplasia in the glomerular mesangial cell and the mesangial matrix; moreover, there were intact epithelial cells of kidney tubules, without tubiform structures in lumen as well as both inflammatory cells and fibrous connective tissues in mesenchyma. SHR group:the glomerulus was atrophic and necrotic, and the lumen narrowed down slightly as well.5. The histomorphological comparison of kidney tissues via electron microscopeFormula group:there was no significant hyperplasia in the mesangial matrix of glomerular mesangial cell nucleus, with aligned foot processes of epithelial cells. Losartan group:the foot process of glomerular mesangial cells integrated into flakiness, with a mild thickening, and adjacent basement membranes thickened as well. SHR group:there was a large glomerular mesangial cell nucleus, few cytoplasms, a thickening basement membrane and a obvious collagen fibroplasia. Control group:there was no hyperplasia in glomerular mesangial cells and matrix, with an even thick basement membrane and aligned foot processes of epithelial cells.6. The analysis result of protein expression of Collagen Ⅰ and Collagen Ⅳ of rats in each group via immunohistochemical methodCompared with Control group, the protein expression of Collagen Ⅰ and Collagen Ⅳ significantly elevated significantly (P<0.01) in SHR group; the protein expression of Collagen Ⅰ and Collagen Ⅳ decreased obviously (P<0.01) in both Formula group and Losartan group with the comparison of SHR group. There was no statistical difference between both Formula group and Losartan group in the protein expression of Collagen Ⅰ and Collagen Ⅳ(P>0.05). Moreover, There was also no statistical difference between Formula group and Control group in the protein expression of Collagen Ⅰ (P>0.05).7. The analysis result of protein expression of AngⅡ of rats in each group via immunohistochemical methodCompared with Control group, the protein expression of AngⅡ significantly elevated (P<0.01) in SHR group; the protein expression of AngⅡ decreased obviously(P<0.01) in both Formula group and Losartan group with the comparison of SHR group, and there was no statistical difference between both Formula group and Losartan group in the protein expression of AngⅡ (P>0.05). Moreover, There was no statistical difference among Formula group, Losartan group and Control group in the protein expression of AngⅡ (P>0.05)8. The analysis result of protein expression of Smad2, Smad3 and Smad7 of rats’kidney tissues in each group via immunohistochemical methodCompared with the Control group, the protein expression of Smad2 and Smad3 significantly elevated P<0.01) in SHR group, while the protein expression of Smad7 decreased obviously (P<0.01) in SHR group. The protein expression of Smad2 and Smad3 decreased significantly (PM0.01), while the protein expression of Smad7 increased obviously (P<0.01) in both Formula group and Losartan group, by comparing with SHR group. There was no statistical difference between the Formula group and the Losartan group in the protein expression of Smad2, Smad3 and Smad7 (P>0.05). In addition, there was no statistical significance between the Formula group and Control group in the protein expression of Smad7 (P>0.05).9. The mRNA expression of Smad2, Smad3 and Smad7 in kidney tissuesCompared with Control group, the mRNA expression of Smad2 and Smad3 significantly elevated (P<0.01) in SHR group, while the mRNA expression of Smad7 decreased correspondingly (P<0.01) in SHR group. The mRNA expression of Smad2 and Smad3 decreased obviously P<0.01), but the mRNA expression of Smad7 increased significantly (P<0.05) in both Formula group and Losartan group, by comparing with SHR group. There was no statistical difference between Formula group and Losartan group in the mRNA expression of Smad2, Smad3 and Smad7 (P>0.05).10. The mRNA expression of TGF-β1, CTGF and FN in kidney tissuesCompared with Control group, the mRNA expression of TGF-β1, CTGF and FN significantly elevated (P<0.01) in SHR group. In Formula group, the mRNA expression of TGF-β1 significantly decreased (P<0.01) by comparing with SHR group, which was better than Losartan group (P<0.05). The mRNA expression of CTGF and FN significantly decreased (P<0.01) in Formula group, and there was no statistical difference with Losartan group (P>0.05)11. The protein expression of TGF-β1, CTGF and FN in kidney tissues via Western blotCompared with Control group, the protein expression of CTGF and FN significantly elevated (P<0.01)in SHR group. Moreover, the protein expression of TGF-β1, CTGF and FN significantly decreased (P<0.01) in Formula group by comparing with SHR group; the protein expression of TGF-β1 and FN were lowered obviously (P<0.01 or 0.05) in Losartan group. The protein expression of CTGF and FN decreased significantly in Formula group by the comparison with Losartan group (P<0.05).Conelusion1. The Formula taken before the peak of blood pressure could reduce SHR blood pressure.2. The Formula taken before the peak of blood pressure could reduce the content of SCr, mALb and β-MG, so as to improve pathological changes of SHR kidney injury, which had a protective effect on the kidney.3. The mechanism of kidney-protective effect of the Formula was to lower the content of collagen Ⅰ and collagen Ⅳ in kidney tissues, reduce the abnormal aggregation of ECM to improve its degradation, and alleviate the fibrosis of kidney tubules and mesenchyma via the up-regulation of expression of Smad7and the down-regulation of expression of both Smad2 and Smad3.4. The drug target of kidney-protective effect of the Formula was to regulate pathways including both TGF- β1-CTGF-FN and TGF-β1-Smads, keep epithelial cells of kidney tubules from epithelial mesenchymal transition (EMT) under pathological conditions, and reduce the collagen generation, so as to promote the degradation of ECM to delay the kidney injury.Innovation1. The paper was features as the combination of TCM, clinical practice and experiment, by focusing on both western medicine (administration before the peak of blood pressure and the protection of target organs) and TCM (the Formula, the theory of harmony between Heaven and human as well as the theory of movement of Yin-Yang)2. The method of Tiao Gan Shen Qu Tan Yu (regulating liver and kidney, and eliminating phlegm and blood stasis) was applied in the experiment. In this paper, it was discussed that the efficacy and mechanism of kidney protection, as well as the prevention of hypertension, via the pathway of kidney injuries and the regulation of human body in accordance to circadian rhythms. |
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