| ObjectiveEssential hypertension, the worldwide disease causing the lesion of heart, brain and kidney, seriously damage human's health. The onset tendency of hypertension in China is severe in recent years. According to the epidemiological survey in 2002, the morbidity of hypertension has increased to 18.8% from 11.88% in 1981. Myocardial fibrosis (MF), the pathological result of hypertension , is characteristic of increased fibroblast proliferation and collagen deposition , which also means cardial reorganization. MF is responsible to many cardiovascular events and become one of the main targets for current anti-hypertensive therapy. It is important to develop ways to inhibit, even reverse MF. Although some progress has been made in the studying MF, the accurate mechanism has not been elucidated. Therefore the therapeutic methods and the clinical effect for MF are limited. Traditional Chinese Medicine (TCM) has important role in anti-hypertension treatment, accumulating many experence in this field. For example, the traditional formula Tian Ma Gou Teng Yin (TMGTY) has been proved to be effective and nontoxic. In the treatment of MF, TCM has advantages in many aspects considering the simultaneous treatment of principal and subordinate symptoms, few adverse effects and relatively low cost. In this study, according to the pathophysiologocal analysis to MF by TCM, the clinical effect and therapeuticmechanism of TCM to MF was explored from the aspects of animal study, cell study and gene regulation in order to search the effective ways to control MF by TCM method.Prof. Li Cheng Zhao has been engaging in hypertension study for many years. With comprehensive regonization to the pathophysiology of hypertension, he believes in that the pathophysiology of hypertention in TCM theory can be summarized as asthenia in origin and sthenia in superficiality, exhibiting in five aspects as wind, fire, phlegm, asthenia and blood stasis in the TCM concept. Under his observation and analysis in long clinical period, he found that the essence of hypertension is due to the abnormity of heart, which was thought to in charge of vascular systems according to TCM theory. This function of heart can keep the normal raising and falling of QI and blood transport, and also is the basis of blood stasis and phlegm formation. It is a long period from hypertension to MF. According to TCM theory, long-time disease makes asthenia, blood stasis and phlegm formation. Furthermore, Phlegm and blood stasis interact with each other. Phlegm makes the blood hard to flow, and blood stasis makes the phlegm hard to diminish. Therefore, long-time phlegm is prone to making blood stasis and long period of blood stasis is also prone to making phlegm. Long-time disease induces the weakness of heart and QI, deficiency of heart-YANG and kidney-YANG, deficiency of both QI and YIN. The stagnation of QI and blood make stagnant blood in heart, and the retention of water-damp also makes phlegm block the heart collaterals. From the preceding analysis by TCM theory, it can conclude that phlegm, blood stasis and athenia are more prominent than wind and fire in the pathogenesis of MF. Therefore, the pathophysiological character of hypertension-related MF is organs asthenia, presenting with asthenia YING causing predominant YANG, and the tangle of phlegm and blood stasis. Blood stasis and phlegm run through the whole process of this disease. Generally, aesthetic YING with predominant YANG , blood stasis in the heart and phlegm are the fundamental pathophysiological change in this disease. In our previous studies, 85 formulas for treating hypertension were randomly selected and their therapeutic principles were analyzed and categorized. The result was as follows: twenty-eight formulas adopting the principle of supporting healthy energy to eliminate evils, 19 adopting the principle of calming the liver and subduing YANG, 15 adopting the principle of activating blood circulationto dissipate blood stasis, 8 adopting the principle of tonification, 5 adopting the principle of dissipating phlegm and 10 using other principles. From this data we understand that the principle of calm the liver and subdue YANG, activating blood circulation to dissipate blood stasis, dissipating phlegm and their combination take an important role in hypertension treatment. By combination of the TCM theory and clinical practice, we chose the representative formulas for calming the liver and subduing YANG, activating blood circulation to dissipate blood stasis, dissipating phlegm, which were Tian-Ma-Gou-Teng-Yin (TMGTY), Xue-Fu-Zu-Yu-Tang (XFZYT) and Wen-Dan-Tang (WDT) respectively, to study their therapeutic effect on MF with captopril as the positive control.The pathogenic mechanism of MF is not elucidated yet. What we currently know is that generally it is the result of imbalance of collagen deposition and lysis in heart tissue , regulated by many factors such as the rein-angiotension-aldosterone system (RAAS) , vasoactive substance (endothelin, catecholamine, nitric oxide, prostaglandin, bradykinin et al), cytokine (TGF-p, transfer growth factor |3;IGF-1, insulin-like growth factor-1), intracellular Ca2+, oxidative stress and haemodynamics, which have been proved to be vital in the process of MF.In the clinical part of this study, the hypertension patients randomly received TMGTY, XFZYT , WDT or catopril for 8 weeks and the serum level of angiotensin II (Ang II), aldosterone (ALDO), precollagen III ( PCIII), laminin (LN), hyaluronic acid (HA) were tested in order to explore the mechanism of the 3 therapies designed according to the TCM theory.In the laboratory part of this study, drug-containing sera were collected from rabbits fed with the preceding formulas. Their effects to the proliferation of heart fibroblast of spontaneous hyertensive rat (SHR) were observed. The effects of the 3 formulas to the serum PCIII, LN, HA;gene expression of TGF-p and IGF - 1 in the heart, and AnglK ALDO concentration in both plasma and heart tissue were also examined. With these data we try to explore the mechanisms by which the 3 kinds of therapy affecting the process of MF. This study is beneficial to searching an ideal anti-hypertension remedy, discovering the mechanism of TCM therapy and improving the clinical practice to hypertension.MethodsPart I Clinical Study1.1 SubjectsAccording to the diagnosis criteria of primary hypertension as well as differentiation of symptoms and signs by TCM theory, ninety-two patients were divided into 4 groups, receiving the treatment of captopril (positive control), catopril+TMGTY, catopril+XFZYT and catopril+WDT respectively.1.2 Treatment and indexThe patients took their remedy for 8 weeks. The blood pressure, symptoms and serum level of PCIIL LN> HA, Angll and ALDO were recorded before and after the treatment.Part II Laboratory Study 2. 1 Cellular Experiment2.1.1 Preparation of the drug-containing SeraRabbits were fed with TMGTY, XFZYT, WDT and catopril with the equivalent dose to the human used for 10 days. Then the rabbits were sacrificed and the serum was collected and stored at the -20°C.2. 1.2 Cell CulturePrimary cardial fibroblast was collect by clipping and trypsin digestion from neonate SHR and purified from other cells by their difference in cell adherence.2. 1. 3 Effect of drug-containing sera on the cardial fibroblast proliferationThe cardial fibroblasts were incubated with drug-containing sera with different doses. The proliferation of fibroblasts was determined by CCK-8 kit.2.2 Animal experimentThe group of WKY rats (normal control) and 5 groups of SHR were treated with catopril, catopril+TMGTY, catopril+XFZYT and catopril+WDT respectively for 8 weeks. The systolic blood pressure (SBP), left ventricular mass (LVM), serum PIIINP, LN, HA were detected and the gene expression of IGF - 1 and TGF - P in the heart tissue were analyzed. Also checked were Angll and ALDO both in heart tissue and in plasma.2.2.1 Effects of the 3 therapies on SBP and LVMThe SBPs of the rat-tail arteries were checked every 4 weeks. LVM was weighted by at the 8th week and the ratio of LVM and rat weight was calculated.2.2.2 Effects of the 3 therapies on Angll and ALDOAngll and ALDO concentrations in the plasma and in the heart were detected by radio-immunity method.2.2.3 Effects of the 3 therapies on the gene expression of IGF - 1 and TGF - 3 in the heart tissueThe gene expression of IGF - 1 and TGF - 0 in the heart tissue was determined by RT-PCR.2.2.4 Effects of the 3 therapies on PIIINP, LN and HAThe serum level of PIIINP, LN and HA were detected by radio-immunity method.Result1. All subjects in the 4 groups are comparable in the age, the gender and the course of disease (P>0. 05). Statistic analysis to the clinical data show that ? when TMGTY combined captoril, a trend of synergistic effect showed in decrease blood pressure although it was not statistically significant. Captoril combining XFZYT or WDT didn' t show any difference with captoril alone in treating hypertension. (2) Combination of captopril with TMGTY, XFZYT or WDT is superior than captoril alone in improving the symptoms (P<0. 05), and every combination show similar effects, implying that TMGTY, XFZYT and WDT was advantageous in alleviate symptoms and combination of captopril with TCM formula can provide overall superiority in hypertension treatment. ?Although captopril alone or combining any of the preceding formula all decreased the serum levels of PCIII, LN, HA, Angll, ALDO in hypertensive patients, the combination therapy was superior to captopril alone. Captopril combining XFZYT was superior than combining TMGTY or WDT in decreasing HA, while combining TMGTY was better than combining the other two formulas in decreasing Angll. Captopril combining XFZYT or WDT was more advantageous than combining TMGTY in decreasing serum ALDO.These data suggest that TMGTY, XFZYT and WDT all have significant anti-MF effect. Captopril combining the formula is superior to captopril alone. The synergistic effects of the formula are related to decreasing serum PCIIL LN, HA, Anglland ALDO.2. Cardial fibroblasts were successfully isolated and the phenotype was identified by immunohistochemistry method. The first 3 passages were suitable for the cell experiments.3. Drug-containing sera were collected from rabbits fed with the preceding formulas. Their effects to the proliferation of heart fibroblast were detected by CCK-8 kit. The blank control, culture medium with 5%, 10% and 20% normal rabbit serum showed comparable effect on the fibroblast proliferation. The 5% serum from TMGTY-treated or captopril-treated rabbits significantly inhibited the cardial fibroblasts proliferation compared with 5% serum from the normal rabbit (P<0. 01). The 5% serum from WDT-treated or XFZYT-treated rabbits showed no difference to the cardial fibroblasts proliferation compared with 5% serum from the normal rabbit (P>0. 05), but 10% and 20% serum from captopril and the formulas all showed significant inhibitory effects to cardial fibroblasts proliferation. As to the 10% drug-containing serum, the inhibitory effects of the formulas were inferior to captopril while all the 20% drug-containing serum showed similar inhibitory effects to fibroblasts proliferation. The 20% serum from XFZYT showed a tendency of superiority than captopril, TMGTY and WDT in inhibiting cardial fibroblasts proliferation.4. SBP is much higher in 24-week old SHR than in 16-week old SHR. TMGTY, XFZYT and WDT alleviate the increased SBP, though no statistical significance showed. The left ventricular mass and left ventricular mass index increased in the 24-week old SHR, which were significantly inhibited by captopril and the other 3 formulas and XFZYT had the most powerful effect in the aspect.5. The Angll and ALDO both in circulation and cardial tissue were significantly elevated in 24-week old SHR, implying the activation of RAAS. The formulas showed better effect than captopril in inhibiting ALDO both in circulation and cardial tissue. But captopril showed better effect than the formulas in reducing cardial Angll, although no statistical significant difference exited between it and XFZYT, WDT.6. The cardial Gene expressions of TGF-p and IGF-1 significantly increased in 24-week old SHR and were significantly inhibited by TMGTY, XFZYT, WDT and captopril. TMGTY and WDT have similar effect with captopril (P>0. 05) and XFZYT showed stronger effect than TMGTY and WDT.7. The serum surrogate of fibrosis, PIIINP, LN and HA were all increased in 24-week old SHR, which also could be significantly inhibited by TMGTY, XFZYT, WDT and captopril. There was no difference in the inhibiting PIIINP and LN among captorpil and the formulas, but XFZYT and WDT showed superior effect to TMGTY and captopril in decreasing HA.These data showed that the 24-week old SHR develops significant MF, which could be alleviated by TMGTY, XFZYT, WDT or captopril. XFZYT and WDT were superior to TMGTY, especially that XFZYT had advantage in inhibiting the proliferation of cardial fibroblasts, gene expression of TGF-p and IGF-1 and the serum level HA than the other 2 formulas. The mechanism that the 3 formulas inhibiting MF development maybe relate to their effect of inhibiting the proliferation of cardial fibroblasts, gene expression of TGF-p and IGF-1 and the serum level of ALDO, Angll , PIIINP, LN, and HA.Conclusion and perspectivePrimary hypertension is a chronic, progressive disease. Because the treatment of hypertension is a long and continuous course, the observation of a therapy should comply with this character. In this study, the TCM formulas didn' t show satisfying anti-hypertension effect. It might be attributed to the short observation and the effect of decreasing or stabilizing hypertension might show after a long-term treatment. The present study proved that TMGTY, XFZYT and WDT had similar short effect with captopril except the inferiority in anti-hypertension. The TCM formulas not only showed better effect than western drugs in improving the patients' symptoms , but also showed significant effect in inhibiting MF development in addition to the lighter side effect. Therefore, TCM formulas should take advantage in the long-term observation, which is our next work to do.This study proved that TCM formulas had good treatment effect on hypertension related MF. The mechanism maybe rely on their effect of inhibiting the proliferation of cardial fibroblasts, gene expression of TGF-pand IGF-1 and the serum level of ALDO, Angll, PIIINP, LN, and HA. But the detail mechanism about every formula is still not known, including whether there are interactions in it. Furthermore, because of the complexity of TCM formula, it is hard to determine the accurate key substance in treatment. Therefore, it is still a long way to go in the process of exploring the mechanism of TCM on hypertension. By the way, the relationship between the indexes studied in the research and the pathogenic mechanism in TCM is another interesting field in TCM study.The pathogenic mechanism of hypertension and MF is not clear yet, which is related to multiple organs and systems. This study is based on the well-accepted viewpoints and theories, thus the explanation need to be updated if new theory or viewpoint appear. With the progression in researching hypertension and MF, more reasonable explanation will be given regarding the mechanism of TCM formulas.
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