| Background & Objectivecardiovascular disease is the leading reason of deaths. Myocardial fibrosis is the most important pathological change in ardiovascular disease progression, it would cause myocardial remodeling, myocardial stiffness, ventricular diastolic dysfunction, decrease coronary arteries reserves, and sudden death.Therefore, prevention and treatment of myocardial fibrosis is important.Myocardial fibrosis is refer to the myocardial tissue excess collagen deposition, a significant increase in the concentration of collagen and collagen volume fraction, the proportion of each type of collagen disorders and disordered.The basal cell biology of myocardial fibrosis are the abnormal proliferation of myocardiac fibroblasts and extracellular matrix increased.myocardial fibrosis have closely relation with a variety of clinical cardiovascular disease. Effectively inhibit the proliferation of myocardiac fibroblasts and collagen synthesis may delay or block the development of myocardial fibrosis.A lot of factors are included in pathagenesis of myocardial fibrosis, these factors are the renin-angiotensin-aldosteronesystem (RAAS), endothelin (ET), bradykinin (BK), catecholamines (CA), transforming growth factor-β (TGF-β), connective tissue growth factor (CTGF), matrix metalloproteinase (MMPs) and intracellular calcium. As one of the most important regulatory factors, and a trigger co-mediator of myocardial fibrosis, TGF-β1 can stimulate cardiac fibroblast proliferation, induce cardiac fibroblast phenotype transformation, promote extracellular matrix deposition and suppression the collagenase degradation. TGF-β1 is the most important target of treatment of fibrosis.The effect of TGF-β1 on the cardiovascular system are mainly manifested in:(1) effcet the endothelial cells function,TGF-β1 can induce endothelial cells to synthesize nitric oxide and endothelin-1, it can directly affect endothelial barrier function in vivo. (2) induce cell proliferation and collagen synthesis:TGF-β1 can promote the cardiac fibroblasts proliferation and induce cardiac fibroblasts converted into myofibroblasts,,which is characterized by the expression of a-smooth muscle actin (a-SMA) in myofibroblasts,enhance the ability of collagen synthesis. (3) regulate extracellular matrix degradation:TGF-β1 is not only inhibit matrix metalloproteinase activity, but also induce the synthesis protease inhibitors to reduce the degradation of extracellular matrix, comprehensive regulation of extracellular matrix deposition. (4)Participate in adrenaline-mediated cardiac remodeling.TGF-β1 is a must trigger co-mediator of myocardial fibrosis caused by many factors in the final, it has now been recognized as the target in the treatment of fibrosis.In the Myocardial fibrosis, the associate Smads protein are mainly Smad2, Smad3 and Smad7 with TGF-β1. While Smad7 can inhibition the other two Smads to mediate the effects of TGF-β1 to play negative immunomodulatory effects.Various types of Smads protein molecules precisely coordination between each other to complete the physiological and pathological condition under the the biological effects of TGF-β1The associate Erk protein with TGF-β1 is most close associated with cardiac growth of fibroblasts, proliferation and differentiation. Phosphorylated Erkl/2 can promote the phosphorylation Smad2/3 accumulation in the nucleus, with the inner nuclear regulatory factors to regulate the expression of collagen gene transcription and together promote the deposition of collagen matrix. Therefore, depth research of the Smads protein and Erk protein associate with TGF-β1 not only can provide more theoretical basis for the pathogenesis of myocardial fibrosis, but also can provide more effective way to prevention and treatment of myocardial fibrosis.In chinese traditional medicine theory, blood stasis and phlegm are related to myocardial fibrosis. So herbal that have some effects of Yiqihuoxue and Huatan in traditional Chinese medicine would be used to reverse cardiac fibrosis. Currently, researches of many chinese medicine compound and formula against cardiac fibroblast have achieved some progress in treatment of myocardial fibrosis. Tan ⅡA have significant anti-myocardial fibrosis effect in vivo and in vitro, and its mechanism may be related to TGF-β1/Smads signaling pathway; tetramethylpyrazine reverse cardiac fibrosis may be related to endothelin and nitric oxide nitrogen; Matrine can reverse cardiac fibrosis, and it has numerous of mechanisms; Chinese medicine formula like tongxinluo, Baoxinjiangyakang,Wendantang anti-myocardial fibrosisare relate with decrease myocardial tissue collagen content in the spontaneously hypertensive rats.Our previous research have shown that Xue-Fu-Zhu-Yu dectoctin can reduce the content of hydroxyproline, inhibit of myocardial tissue fibrosis, reduce the expression of TGF-β1 protein in spontaneously hypertensive rat model. It suggests that Xue-Fu-Zhu-Yu dectoctin have some effects of anti-myocardial fibrosis, but the mechanismes are not clear. Therefore, we will investigate the effects of Xue-Fu-Zhu-Yu dectoctin anti-cardiac fibrosis and its possible mechanism in vitro.Methods & Content1. To establish chromatographic fingerprints of Xue-Fu-Zhu-Yu decoction: Formula extracted ultrasonically with 250 mL of 50% methanol for 45 min,the extraction was filtrated through 0.45 um membrane filter into an HPLC sample vial for HPLC analysis.The results were analyse by Similarity Evaluation System for Chromatographic Fingerprint of Traditional Chinese Medicine (Version 2004).10 batches of samples were import to chromatographic fingerprint similarity evaluation system at least, select the representative common peaks as Mark peak to establish Xue-Fu-Zhu-Yu decoction fingerprint pattern.2. Myocardial fibrosis model was established in vitro:cardiac fibroblast were incubated with different concentrations of TGF-β1 for 24h or 48h. The protein expression of Col Ⅰ-. ColⅢ and α-Smooth muscle actin in cardiac fibroblast were detected using Western blot.3. To investigate the effect of Xue-Fu-Zhu-Yu dectoctin on TGF-β1 induced myocardial fibrosis model:cardiac fibroblast in the Xue-Fu-Zhu-Yu dectoctin pretreatment group were incubated for 2 h, and then, the cells were treated as in the TGF-β1 group. The proliferation of cardiac fibroblast was detected using CCK-8. The mRNA expression of collangen Ⅰ, collangenⅢ, α-Smooth musucle ation was detected using real time-quantitative polymerase chain reaction.The protein expression of collangen Ⅰ, collangenⅢ, a-smooth musucle ation using western blot.4. Explore the possible mechanism of Xue-Fu-Zhu-Yu dectoctin on TGF-β1 induced myocardial fibrosis:cardiac fibroblast in the Xue-Fu-Zhu-Yu dectoctin pretreatment group were incubated for 2 h, and then, the cells were treated as in the TGF-β1 group.The mRNA expression of Smad2、Smad3、Smad7、Erk was detected using real time-quantitative polymerase chain reaction.The protein expression of Smad2、P-Smad2、Smad3、P-Smad3、Smad7、Erk1/2、P-Erkl/2 using western blot.Results(1) Xue-Fu-Zhu-Yu decoction chromatographic fingerprint results shown: When HPLC fingerprints of 10 batches of samples after six common characteristic peaks alignment. the similarity values was above 0.901, the relative standard deviation (RSD) was 1.61%.(2) Myocardial fibrosis model results shown:there was a significant increase in the protein expression of collangen I, collangenIII,a-smooth muscle ation as compared to control group(P<0.05). for 24 and 48 h.. The relative protein expression of total collagen were significant in 24h.(3) Effect of Xue-Fu-Zhu-Yu dectoctin on TGF-β1 induced myocardial fibrosis model:① CCK-8 results shown:Compared to control group,there was a significant increase in cell proliferation in TGF-β1 group (P<0.01),the rate was 126.28±0.92%. Compared with TGF-β1 group,Xue-Fu-Zhu-Yu decoction(1:40,1:60,1:80) remarkablely reduced the proliferative response of cardiac fibroblasts,the rates were 100.44±1.31%,109.03±5.34%,113.46±3.81%(P<0.05).③real time-quantitative polymerase chain reaction results shown:Compared with control group.the mRNA expression of collangen Ⅰ,α-smooth muscle ation increased significantly in TGF-β1 group(P<0.05).Compared with TGF-β1 group,Xue-Fu-Zhu-Yu dectoctin (1:40,1:60,1:80) can reduce the mRNA expression of collangen I, α-smooth muscle ation(P<0.01);④ western blot results shown:Compared with control group,the protein expression of collangen Ⅰ,collangenⅢ, α-smooth muscle ation increased significantly in TGF-β1 group (P<0.01). Compared with TGF-β1 group, Xue-Fu-Zhu-Yu dectoctin (1:40,1:60,1:80) can reduce the protein expression of collangen Ⅰ, collangenⅢ,α-smooth muscle ation (P<0.05).(4) The mechanisms of Xue-Fu-Zhu-Yu dectoctin on TGF-β1 induced myocardial fibrosis:real time-quantitative polymerase chain reaction results shown: compared with control group,the mRNA expression of Smad2 increased significantly in TGF-β1 group(P<0.05). Compared with TGF-β1 group, Xue-Fu-Zhu-Yu dectoctin (1:40,1:60,1:80) can reduce the mRNA expression of Smad2(P<0.05) and increase the mRNA expression of Smad7(P<0.05).②western blot results shown:compared with control group,the protein expression of P-Smad2、 P-Smad3 increased significantly in TGF-β1 group (P<0.01). Compared with TGF-β1 group, Xue-Fu-Zhu-Yu decoction(1:40,1:60,1:80) can reduce the expression of P-Smad2、 P-Smad3 (P<0.05) and increase the expression of Smad7 protein (P<0.05). No concentration of Xue-Fu-Zhu-Yu decoction influenced the expression of Smad3、 Erk1/2、P-Erk1/2 (P>0.05).Conclusion1、The similarity values of the 10 batches of Xue-Fu-Zhu-Yu decoction chromatographic fingerprintsin was 0.901 above, the quality of Xue-Fu-Zhu-Yu decoction was stable and controllable and can be as our latter experiments.2、TGF-β1 can induce cardiac fibroblasts collagen matrix deposit and promote transformed phenotype, It shown that we constructed a model of myocardial fibrosis successfully in vitro.3、Xue-Fu-Zhu-Yu decoction can inhibit cardiac fibroblasts proliferation、 collagen matrix deposit and cardiac fibroblasts transformed phenotype induce by TGF-β1,it can anti-cardiac fibrosis in vitro.4、The effect of Xue-Fu-Zhu-Yu decoction anti-myocardial fibrosis induced by TGF-β1 may be related to up regulation the expression of P-Smad2、 Smad2、P-Smad3 protein and down regulation the expression of Smad7 protein. |
PDF Full Text Request