| Left ventricular pathological remodeling after acute myocardial infarction results in the decrease of cardiac compliance, decreased diastolic systolic function, is ultimately the main cause of the development of heart failure. The remodeling of the left ventricle after myocardial infarction is mainly presented by the compensatory hypertrophy of the myocardial cells, myocardial interstitial fibroblasts hyperplasia, fibrosis, and extracellular matrix collagen network of metabolic abnormalities, especially the increase of the typeⅠcollagen(CollagenⅠ, Col- Ⅰ) and type Ⅲ collagen(Collagen Ⅲ, Col-Ⅲ), and the collagen’s ratio change. Studies have shown that transforming growth factor-β1(TGF-β1)can inhibit the cardiac fibroblasts(CFs) proliferation via activating the pathway of the TGF- β/Smads, regulate the expression of the typeⅠandⅢcollagen, and improve the remodeling of the ventricle [1]. TGF-β is a multifunctional cytokines to inhibit or stimulate a variety of cells, especially by the signal transduction of the Smads protein-mediated, plays a extremely important role in maintaining normal physiological functions of the cells. Numerous studies confirm that the signal transduction pathways of the TGF-β / Smads are involved in the occurrence and development of multiple organs and tissues, such as the fibrosis diseases of the heart, liver, kidney and other organs[2,3,4]. The cardiac fibroblasts will prolifrate by pathological stimulation, and transform into fibroblasts that have extracellular matrix secretion function, which provides significant meaning in the ventricle remodeling [5]. Inhibiting the myocardial fibrosis(MF) is crital to the prevention and treatment of the remodeling of the ventricle after myocardial infarction. Therefore, the research on inhibiting the proliferation of CFs, and identifying the relevant therapeutic objectives and molecular mechanisms of clinical medicine, is proven has obvious significance.As an active ingredient of traditional Chinese medicine, Paeonol(Pae) and of Panax notoginseng saponins(PNS) has a wide range of pharmacological activity, such as protecting myocardiu, defending myocardial ischemia, antiarrhythmic and other effects[6,7]. Research has shown that Pae and PNS have effects on improving MF and reversing the effect of the remodeling of the ventricle [8,9],but it was never reported that if there is any synergy of the compositive application of the two. This research was established on proliferating rats CFs by using angiotensin Ⅱ(Ang Ⅱ), observing the combination effect of the Pae and PNS through the proliferation of the CFs, and discussing the combination of the compositive application of the Pae and PNS to verify whether there is any synergistic effect and molecular mechanisms or not. From both theoretical and experimental perspectives, this research provided valuable basis of the clinical feasibility of using the Pae and PNS compositively to prevent and treat the ventricle remodeling after myocardial infarction.Objective:Investigate the combination effect of the Pae and PNS on the AngⅡ derivational proliferation of the CFs and the signal transduction pathways of the TGF-β / Smads, explore the mechanism of its anti myocardial fibrosis, and provide a theoretical and experimental basis of preventing the remodeling of the ventricle after myocardial infarction.Methods:1 cμltivation and identification of rats CFs.Adopt primary culture by using 0.08% trypsin digestion to cultivate the neonatal rats CFs, and subculture by using 0.125% trypsin digestion. Then, make the immunocytochemistry staining on the two generation of the cells in the experiment to do the identification, where the vimentin staining results in positive are identified as CFs, and purity is more than 95%. And establish the model of proliferation of rats CFs by Ang Ⅱ’s inducing.2 Pae, PNS and the combination dose screening.Set arithmetic progression of the drug intervention dose, that is set the final concentration of Pae as 0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80 mg·L-1, and the final concentration of PNS as 0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 mg·L-1. Use MTT method to screen out the safe dose of the Pae and PNS for CFs when separately applied. Again, use the MTT method to test the cytotoxicity of the different combinations of high and low dose(Pae 45, 30 mg·L-1 + PNS 50, 30 mg·L-1) groups, record the results, and calculate the survive rate of the cells.3 Experiment groups and administration.After identified the safe dose for the compositive application of the Pae and PNS, divide the cμltured CFs into 6 groups,(1)normal control group: no intervention factor;(2) model group: AngⅡ(10-7 mol·L-1);(3)Pae group: Pae(45 mg·L-1) + Ang Ⅱ(10-7 mol·L-1);(4) PNS group: PNS(50 mg·L-1)+ Ang Ⅱ(10-7 mol·L-1);(5) Low-dose combination group: Pae(30 mg·L-1) + PNS(30 mg·L-1) + Ang Ⅱ(10-7 mol·L-1);(6) High-dose combination group: Pae(45 mg·L-1)+ PNS(50 mg·L-1)+ AngⅡ(10-7 mol·L-1)4 Measuring the CFs proliferation via MTT method.5 Detecting the α-SMA protein expression via the immunocytochemistry method.6 Detecting the m RNA expression of TGF-β1, Col-Ⅰ and Col-Ⅲ via RT-PCR method.7 Detecting the protein levels of TGF-β1, p-Smad 2/3, Col-Ⅰand Col-Ⅲ via Western blot method.Result:1 Cultivation and identification of rats CFs.Observe under the reversed microscope, the primary cultured CFs present in the fusiform, angle or flat star shapes, network distribution in low density, and beam shaped or radial distribution in high density. Subculture cells are similar to the primary culture cells in the shape and growth pattern. Use the immunocytochemical staining to identify the CFs, the vimentin staining results in positive are identified as CFs, and purity is more than 95%.2 Pae, PNS and the combination dose screening.The safe dose range screened out by MTT method is 5 ~ 75 mg·L-1 for the Pae, and 5 ~ 85 mg·L-1 for PNS. In the subsequent experiments: The separate intervention dose of the Pae was 45 mg·L-1; The separate intervention dose of the PNS was 50 mg·L-1; The Pae of the low intervention dose in the combination groups was 30 mg·L-1; The Pae of the high intervention dose in the combination groups was 45 mg·L-1; The PNS of the low intervention dose in the combination groups was 30 mg·L-1; The PNS of the high intervention dose in the combination groups was 50 mg·L-1. The MTT test results of dose in the combination groups showed that the survival rates of the low and high dose combination were 69.2% and 56.1% respectively, and the cells in both of them have no cytotoxicity.3 Measuring the CFs proliferation via MTT method.Compared to the normal control group, the proliferation of CFs in model group is significantly higher(P < 0.01). Compared to the model group, the Pae group, the PNS group, the low-dose and high-dose of the combination group showed obvious depressing in the proliferation of CFs by Ang II’s inducing(P < 0.01). Compared to the Pae group and the PNS group, the low-dose and high-dose combination group showed obvious enhancement in the proliferation of the CFs(P < 0.01); Compared to the low-dose combination group, the high-dose combination group showed better effect in the proliferation of the CFs(P < 0.05), and showed a certain degree of the dose dependency.4 Detecting the α-SMA protein expression via the immunocytochemistry method.After the CFs being induced by Ang Ⅱ, compared to the normal control group, the α-SMA protein expression in the model group was significantly increased(P < 0.01). After the drug intervention, compared to the model group, the α-SMA protein expression in the Pae group, the PNS group, the high-dose and low-dose of the combination group decreased significantly(P < 0.01). Compared to the Pae group and the PNS group, the α-SMA protein expression in the high-dose and low-dose of the combination group decreased significantly(P < 0.01). Compared to the low-dose combination group, the α-SMA expression in the high-dose combination group decreased significantly(P < 0.01), and showed a certain degree of the dose dependency.5 Detecting the m RNA expression of TGF-β1, Col-Ⅰ and Col-Ⅲ via RT-PCR method.Compared to the normal control group, the TGF-β1, Col-I and Col-III’s m RNA expression and the value of I/III of the rats CFs in the model group significantly increased(P < 0.01). Compared to the model group, the TGF-β1, Col-I, Col-III’s m RNA expression in the Pae group, the PNS group, and the high-dose and low-dose combination group significantly decreased(P < 0.01), and the value of I/III in high-dose and low-dose compatibility group significantly decreased(P < 0.01). Compared to the Pae group, the PNS group, the TGF-β1, Col-I and Col-III’s m RNA expression in the high-dose and low-dose combination group significantly decreased(P < 0.01, P < 0.05). Compared to the Pae group, the value of I/III in the low-dose combination group significantly decreased(P < 0.05). Compared to the low-dose combination group, the inhibition effect on the above index increased significantly in the high dose combination group(P < 0.05), and showed a certain degree of the dose dependency.6 Detecting the protein levels of TGF-β1, p-Smad2/3, Col-Ⅰ and Col-Ⅲ via Western blot method.Compared to the normal control group, the rats CFs’ TGF- β1, p-Smad2/3, Col- I, Col- III’s protein expression and the value of I/III in the model group significantly increased(P < 0.01). Compared to the model group, the TGF- β1, p-Smad2/3, Col-I, Col-III’s protein expression and the value of I/III in the Pae group, the PNS group and the high-dose and low-dose combination group significantly reduced(P < 0.01). Compared to the Pae group and the PNS group, the inhibition effect of the TGF-β1, p-Smad2/3, Col-I, Col-III’s protein expression and the value of I/III in the high-dose and low-dose combination group significantly decreased(P < 0.01, P < 0.05). Compared to the low-dose combination group, the TGF-β1, p-Smad2/3, Col-I, Col-III’s protein expression in high-dose combination group significantly decreased(P < 0.01), and showed a certain degree of the dose dependency.Conclusion:1 The Pae and PNS can significantly inhibit the proliferation of the CFs and the expression of TGF-β1, reduce the activation of the p-Smad2/3, and then down regulate the expression of the type I and type III collagen.2 Compositive application of the Pae and PNS in inhibiting the proliferation of CFs shows obvious synergistic effect and a degree of the concentration dependency.3 Compositive application of the Pae and PNS in inhibiting the expression of TGF-β1, reducing the activation of the p-Smad2/3, and then down regulating the expression of the type I and type III collagen shows synergistic effect and a degree of the concentration dependency. |
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