| BackgroundHypertension is one of the most common diseases afflicting humans worldwide. It is a major risk factor for stroke, myocardial infarction, vascular disease and chronic kidney disease. Hypertension is the primary cause of myocardial fibrosis, and contributes to the pathophysiology of cardiac damage. Hypertension causes a sustained increase in cardiac pressure overload, triggering excessive proliferation and the accumulation of interstitial and perivascular collagen fibers that leads to myocardial fibrosis. Myocardial fibrosis is an integral component of chronic heart disease, and it is increasingly thought to have a key role in disease progression as well. Therefore, myocardial fibrosis prevention and treatment is a subject of great clinical value and social significance. Now that myocardial fibrosis is an inevitable process of many common heart diseases to the development of end-stage heart disease development and a critical period from decompensated heart function decompensated to change.Myocardial fibrosis isa key factor of cardiovascular disease prognosis. The formation mechanism is very complex, involving a variety of cytokines. One of the most studied is transforming growth factor-β, it has been recognized therapeutic target organ fibrosis.TGF-P is a cytokine with many members of a large family. TGF-βs is the important one among them which have more research. In mammals only found three forms:TGF-β1, TGF-β2 and TGF-β3. TGF-β1 is mainly produced by fibroblasts in the occurrence and development of myocardial fibrosis, the transformation of muscular fibroblasts, the expression of collagen gene and the synthesis of extracellular matrix are promoted by TGF-β1. TGF-β1 is the most important cytokine promoting myocardial fibrosis. Various studies have shown that the increasing of the pathological TGF-β1 have played a key role in the proliferation and differentiation of fibroblasts., anti-TGF-β1 treatment strategies will undoubtedly become an important place in myocardial fibrosis. Recent pharmacological studies targeting TGF-β1 have reported progress in the development of myocardial fibrosis treatments, but the clinical efficacy of these treatments has yet to be established.Smads is a key transduction molecule in TGF-βsignaling pathways. TGF-β can be transmitted directly from cell membrane receptor into cell membrane by Smads. After binding with its receptors, TGF-β activated Smads protein into the nucleus as a ligand, regulate the transcription of target genes jointly. Smads protein has at least eight kinds in mammalian, which are divided into three subtypes according to the different structures and functions:(1) Receptor-activated Smads (R-Smads), including Smad1,2,3,5 and 8. (2) Common-mediator Smads (Co-Smads), for Smad4 in mammals. (3) Inhibitory Smads (I-Smads), including Smad6 and 7. Due to the precise coordination between various types of Smads molecules in TGF-β/Smads signal transduction pathway, the biological effects of TGF-β have completed jointly in both physiological and pathological conditions.Traditional Chinese medicine (TCM) employs "Fufang" to treat patients. It is currently thought that the therapeutic effects that have been demonstrated for many specific TCM therapies are mediated by multiple ingredients. Pharmacologically active ingredients have been demonstrated to be present in several of these formulas, and some of these formulas have been reported to be therapeutically effective against multiple diseases, including tumors, inflammatory conditions and cardiac diseases.Hypertensive myocardial fibrosis belongs to the Chinese medicine category of "palpitations", "Asthma", "edema". The formulas of Xue-Fu-Zhu-Yu decoction (XFZYD) are improved from Tao-Hong-Si-Wu Decoction, composed of eleven kinds of traditional Chinese medicine. In recent years Xue-Fu-Zhu-Yu decoction are widely used in the treatment of hypertensive heart disease in clinical. Numerous studies published in the Chinese medical literature have reported the successful treatment of symptoms related to high blood pressure, such as headaches and dizziness, using the herbal formula Xue-Fu-Zhu-Yu decoction. However, the underlying mechanisms of action and therapeutic effects of treatment with these herbal formulas remain unclear.ObjectiveThis paper is intended to use spontaneously hypertensive rats as experimental subjects, intragastric administration with different doses of Xue-Fu-Zhu-Yu decoction, observing the effects on myocardial fibrosis in spontaneously hypertensive rats. Detect the expression of TGF-β1 and Smad2,3,7 proteins in myocardial tissue to preliminary inquiry the mechanism of reversal myocardial fibrosis by Xue-Fu-Zhu-Yu decoction. Provide experimental basis for the clinical use of Xue-Fu-Zhu-Yu decoction to treat myocardial fibrosis.Methods1.25SHRs were randomly divided into five experimental groups, each group have five rats.In addition five WKY rats were set for negative control group,a total of six group:(1) WKY control group; (2) SHR control group; (3) SHR+6g·kg-1 Xue-Fu-Zhu-Yu decoction group; (4) SHR+12g-kg-1 Xue-Fu-Zhu-Yu decoction group; (5) SHR+18g·kg-1Xue-Fu-Zhu-Yu decoction group; (6) SHR+60mg·kg-1 captopril group。The rats were administered orally, the WKY and SHR control group were given the same amount of distilled water. Timing administered once daily at 10:00 am for consecutive 16 weeks.2. Rat tail arterial pressure measuring instrument were used to detect the tail artery systolic blood pressure before intragastric administration and withdrawal the drug to be put to death, to explore whether the drug affects the change of blood pressure in rats.3. The rats were sacrificed to collect the myocardial tissue and make the appropriate treatment, experimental samples were provided for subsequent experiments.4. To detect hydroxyproline content in myocardial tissue samples by alkali hydrolysis method, for estimating myocardial collagen content.5. Make the specimen of for paraffin section for subsequent MASSON trichromatic collagen staining and immunohistochemical experiment.6. Use the method of Masson trichrome collagen staining for myocardial histopathology testing to calculate the volume fraction of collagen.7. Observing the distribution and expression of TGF-β1 protein in cardiac tissue by immunohistochemistry.8. Detecting the mRNA expression of TGF-β1, Smad2, Smad3, Smad7 in myocardial tissue by Real-time PCR.9. Detecting the protein expression of TGF-β1, Smad2, Smad3, Smad7 in myocardial tissue by Western blot.Results1. The impact of drugs on blood pressure Blood pressure in SHR control group were significantly higher than in the WKY control group, and there is significant difference (P<0.01); Captopril can significantly reduce blood pressure before and after oral administration, there are significant differences (P<0.05), but other groups did not change significantly.2. Hydroxyproline content measured in myocardial tissueThe oxidation products of Hydroxyproline generated by oxidizing agent present purple under the reaction with dimethylaminobenzaldehyde, the Hydroxyproline content can be calculated according to the depth of the color. Hydroxyproline content of myocardial tissue in SHR control group were significantly higher than WKY control group with a significant difference (P<0.001); relative to the SHR control group, the content in high dose Xue-Fu-Zhu-Yu decoction group and Captopril group was significantly lower (P<0.001); moderate dose Xue-Fu-Zhu-Yu decoction group can also significantly reduced hydroxyproline content (P<0.05); while the effect on low dose Xue-Fu-Zhu-Yu decoction group is not obvious, there is no statistical difference (P>0.05).3. MASSON trichromatic collagen staining to detect the degree of myocardial tissue fibrosisAfter MASSON staining, myocardial collagen fibers appear blue, myocardial fibers appear red, cytoplasm was dark blue. Use the Image-Pro Plus 6.0 image analysis system to measure and analysis the collagen area, then calculate the collagen volume fraction (CVF). SHR control group had a significantly higher CVF value than that of WKY control group with a significant difference (P<0.05); the CVF value in Captopril group decreased significantly relative to the SHR control group (P<0.01); high dose Xue-Fu-Zhu-Yu decoction group values can also reduce CVF value significantly (P<0.05); while the CVF value in low and moderate dose Xue-Fu-Zhu-Yu decoction group is not change obvious compared with SHR control group, there is no statistical difference (P>0.05).4. Immunohistochemistry to detect the expression of TGF-β1 protein in myocardial tissueThe appearance of intracytoplasmic brown granules are seen as positive performance. Using Image-Pro Plus 6.0 image analysis system to measure the cumulative value of optical density (IOD) and distribution area (Area) in positive reaction cells, then calculated the average optical density value (Density) in which reflecting the relative amounts of TGF-β1 protein. The content of TGF-β1 protein in SHR control group was significantly higher than WKY control group with a significant difference (P<0.05); high dose Xue-Fu-Zhu-Yu decoction group and Captopril group significantly lower TGF-β1 protein content relative to the SHR control group (P<0.05); while the TGF-β1 protein content is no significant difference in the low and moderate dose Xue-Fu-Zhu-Yu decoction group compared with SHR control group (P>0.05).5. Real-time PCR to detect the mRNA expression of TGF-β1, Smad2, Smad3, Smad7 in myocardial tissueConfirm amplification curve, melting curve and Ct value after Real Time PCR reaction, use 2-△△ct method to calculate the relative starting template solubility of target genes. The expression of TGF-β1, Smad2, Smad3 mRNA increased and Smad7 mRNA levels decreased significantly in SHR control group compared with WKY group (P<0.05); Contrast in SHR control group, TGF-β1, Smad2, Smad3 mRNA reduced and Smad7 mRNA expression levels increased significantly in captopril group with significant difference (P<0.05); in high dose Xue-Fu-Zhu-Yu decoction group, there is lower TGF-β1, Smad2 and higher Smad7 mRNA expression compared with SHR control group (P<0.05), while the expression of Smad3 mRNA does not change significantly (P>0.05); TGF-β1, Smad2, Smad3, Smad7 mRNA expression have no significant difference in low and moderate dose Xue-Fu-Zhu-Yu decoction group relative to the SHR control group (P>0.05).6. Western blot to detect the protein expression of TGF-β1, Smad2, Smad3, Smad7 in myocardial tissueAfter scan the film, analyze the optical density value of the target band by using Image-pro Plus 6.0 image analysis software, to observe the expression of the respective protein. The expression of TGF-β1, Smad2, Smad3 protein increased and Smad7 protein levels decreased significantly in SHR control group compared with WKY group (P<0.05); Contrast in SHR control group, TGF-β1 Smad2, Smad3 protein reduced and Smad7 protein expression levels increased significantly in captopril group with significant difference (P<0.05); in high dose Xue-Fu-Zhu-Yu decoction group, there is lower TGF-β1, Smad2 and higher Smad7 protein expression compared with SHR control group (P<0.05), while the expression of Smad3 protein does not change significantly (P>0.05); TGF-β1, Smad2, Smad3, Smad7 protein expression have no significant difference in low and moderate dose Xue-Fu-Zhu-Yu decoction group relative to the SHR control group (P>0.05).Conclusion1. In line with relevant literature report, compared to WKY rats, the systolic pressure elevated, myocardial collagen content increased and fibrosis can be significantly observed in myocardial tissue in SHRs.2. High dose Xue-Fu-Zhu-Yu decoction group have significant effect on reversing myocardial fibrosis, while the antihypertensive effect is not obvious, suggesting the effect of reversing myocardial fibrosis may not be realized by reducing cardiac pressure overload.3. Based on detecting the mRNA and protein expression of related genes on TGF-β/Smads signal transduction pathways in myocardial tissue, we found thathigh dose Xue-Fu-Zhu-Yu decoction group significantly reduced TGF-β1, Smad2 expression and improved Smad7 expression in mRNA and protein levels. Initially confirmed that the effect of reversing myocardial fibrosis may be regulated through TGF-β/Smads pathway. |
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