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Effects of puerarin on expression of cardiac Smad3 and Smad7 mRNA in spontaneously hypertensive rat
Journal of Ethnopharmacology 138 (2011) 737–740
Contents lists available at SciVerse ScienceDirect
Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jethpharm
Effects of puerarin on expression of cardiac Smad3 and Smad7 mRNA in spontaneously hypertensive rat Nian-Bao Zhang, Zheng-Gui Huang ∗ , Wei-Dong Cui, Bo-Ping Ding Department of Pharmacology & Pharmacology of TCM Grade Three Laboratory, State Administration of Traditional Chinese Medicine of P.R. China, Wannan Medical College, Wuhu, Anhui 241002, PR China
a r t i c l e
i n f o
Article history: Received 3 August 2011 Received in revised form 8 October 2011 Accepted 10 October 2011 Available online 19 October 2011 Keywords: Puerarin TGF-1 Smad3 Smad7 Spontaneously hypertensive rat
a b s t r a c t Aim of the study: Puerarin is a pure extract from traditional Chinese medicine Kudzu root. It has been used to treat hypertension and angina pectoris. In our previous study, it showed a protective effect against cardiac hypertrophy in rats. This study was to observe effects of puerarin on expression of cardiac transforming growth factor 1 (TGF-1 ), Smad3 and Smad7 mRNA in the spontaneously hypertensive rat (SHR), and to explore its possible mechanism of myocardial protection. Materials and methods: Thirty-five 12-week-old SHRs were randomly allocated into 5 groups of 7 rats as follows: 3 groups which received intraperitoneal (i.p.) puerarin (100 mg kg−1 , 50 mg kg−1 and 25 mg kg−1 ), 1 group which received captopril (30 mg kg−1 , i.g.) and 1 control group of untreated SHRs. In addition, a control group of 7 Wistar-Kyoto (WKY) rats was established. Both control groups received i.p. injections of saline. All rats were treated for six weeks. At the end of this period all rats were sacrificed, and their hearts were quickly removed for semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis. Results: Compared with WKY control group, expression of TGF-1 and Smad3 mRNA was increased (p < 0.01) and Smad7 mRNA expression was decreased in SHR control group (p < 0.01). High- and middledose puerarin decreased the expression of TGF-1 and Smad3 mRNA (p < 0.01) and increased the expression of Smad7 mRNA (p < 0.05). Conclusion: Puerarin reduces expression of TGF-1 and Smad3 mRNA and increases that of Smad7 mRNA in SHR myocardium. These changes in gene expression may be a mechanism by which puerarin provides myocardial protection from hypertension. © 2011 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Hypertension is one of the most common worldwide diseases afflicting humans. Over the past several decades advances in research along with widespread patient education have led to decreased hypertension-related morbidity and mortality. During onset and maintenance of sustained hypertension, an increase in angiotensin II levels and macrophage infiltration into the heart play important roles in hypertension-related heart damage (Caglayan et al., 2008). Angiotensin II is known to induce chemotaxis in inflammatory cells such as macrophages, causing them to aggregate. Macrophages can secrete cytokines like TGF-1 , which act through Smads to promote myocardial collagen synthesis, deposition, and fibrosis (Kohno et al., 2008). There are numerous reports in Chinese medical literature describing the successful use of kudzu (Radix pueraria) in the
∗ Corresponding author. Tel.: +86 553 3932464; fax: +86 553 3932531. E-mail address: [email protected] (Z.-G. Huang). 0378-8741/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2011.10.013
treatment of symptoms of high blood pressure such as headache and dizziness. Recent clinical uses of kudzu include treatments for hypertension and angina pectoris. Puerarin is the 8-C-glucoside of daidzein, a pure extract from Kudzu root. In our previous experiments (Huang et al., 2010; Zhang et al., 2010), we found that puerarin had an antihypertensive effect and a role in protecting the heart in rats. In this study, we observed effects of puerarin on expression of TGF-1 , Smad3 and Smad7 mRNA in the hearts of spontaneously hypertensive rats (SHRs), and demonstrated their role in a possible mechanism of myocardial protection. 2. Materials and methods 2.1. Animals Thirty-five 12-week-old SHRs and 7 WKY rats were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. with certificate number: SCXK (Beijing) 2006-0009. Each cage contained one rat which had free access to laboratory chow and water and with the normal circadian rhythm. Ambient temperatures were
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N.-B. Zhang et al. / Journal of Ethnopharmacology 138 (2011) 737–740
between 22 ◦ C and 24 ◦ C with humidity 60–65%. The study protocol was approved by the Animal Review Board of the University. 2.2. Drugs Puerarin injection was purchased from Shandong Fangming Pharmaceutical Group Co., Ltd. with batch number: H20033292. Captopril was obtained from Shanghai Pukang Pharmaceutical Co., Ltd. with batch number: H31021327. Those drugs are commercially available analytical pure products. 2.3. Kits Trizol kit, RT first strand kit and PCR amplification kit were supplied by Beijing Tiangen Biotechnology Co., Ltd. DEPC kit was supplied by Sigma–Aldrich Co., Ltd. 2.4. Instruments Instruments for current study included homogenate machine (PRO250, PRO Scientific Inc., Monroe, CT), gene amplifier (EDC810, Dongsheng International Trade Co., Ltd.), water exclusion thermostatic incubator (GHX-9270B, Shanghai Fuma laboratory equipment Co., Ltd.), and freezing high-speed centrifuge (22R, Beckman Coulter Co., Ltd.). 2.5. Experimental methods 2.5.1. Animal groups and sample collection Thirty-five 12-week-old male SHRs were trained for one week and then randomly divided into five groups of seven rats each. From the start of the second week, the rats were given drugs at 8 am every morning. The doses and routes are as follows: rats in high-, middle- and low-dose puerarin groups were intraperitoneally given 100 mg kg−1 d−1 , 50 mg kg−1 d−1 and 25 mg kg−1 d−1 of puerarin injection, respectively. Rats in captopril group were orally given captopril at the dose of 30 mg kg−1 d−1 . Rats in SHR and WKY control groups were intraperitoneally injected with normal saline. The drugs were administered for six weeks. After this period, all rats were sacrificed with urethane anesthesia. The heart was removed quickly and washed with cold normal saline, then dried with filter paper. 100 mg of the left ventricle of each rat was taken, kept at −70 ◦ C, and prepared for RT-PCR. 2.5.2. Semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) 2.5.2.1. Total RNA extraction. Ventricular myocardium samples were cut into pieces with eye scissors. Trizol was added (100 mg/1 ml) and samples were homogenized on ice and then centrifuged (4 ◦ C, 12,000 r min−1 , 10 min). Next, the supernatant was removed. 0.2 ml chloroform/1 ml Trizol was added and samples were shaken for 10 s at 4 ◦ C for 30 min. Subsequently, the aqueous later was carefully removed. Then 0.5 ml isopropanol/1 ml Trizol was added. Samples were again shaken for 10 s and centrifuged at 4 ◦ C with 12,000 r min−1 for 10 min. The supernatant was discarded and 75% ethanol prepared from DEPC-treated water was added and kept at 4 ◦ C for 30 min after a 1 min shake. Next, samples were centrifuged at 4 ◦ C with 7500 r min−1 for 5 min and their supernatant was discarded. The samples were air-dried for 5 min. Total RNA was dissolved in 20 l DEPC water, from which 1 l of this total RNA solution was taken and diluted 50-fold for determining OD260/OD280. This ratio, which assessed purity of the RNA, was between 1.8 and 2.0 for all samples. The total RNA concentration was calculated according to the following formula: Total RNA concentration (mg/L) = OD260 × 40× dilution factor.
Fig. 1. Optical density ratios of TGF-1 , Smad3 and Smad7 to -actin in all groups. *p < 0.05, **p < 0.01 compared with the SHR group; p < 0.05, p < 0.01 compared with WKY group.
2.5.2.2. cDNA synthesis. cDNA was produced using 2 g of total RNA which was added to the following reaction system: 10× RT mix 2 l, dNTP mixture 2 l, Oligo-dT15 2 l, Quant Reverse Transcriptase 1 l. Then RNase-free water was added to make up a total volume of 20 l, which was incubated at 37 ◦ C for 60 min. 2.5.2.3. Polymerase chain reaction (PCR). The templates and primers used for PCR are as follows: TGF-1 primers: upstream, 5 GAA GCC ATC CGT GGC CAG AT-3 , downstream, 5 -CCA GTG ACG TCA AAA GAC AG-3 , product: 461 bp. -actin: upstream primer, 5 CCG TAA AGA CCT CTA TGC CAA CA-3 , downstream primer, 5 -CGG ACT CAT CGT ACT CCT GCT-3 , product: 230 bp. Smad3: upstream primer, 5 -AGG GCT TTG AGG CTG TCT ACC-3 , downstream primer, 5 -GTC CAC GCT GGC ATC TTC TG-3 , product: 364 bp. Smad7: upstream primer, 5 -TTT TGA GGT GTG GTG GGT-3 , downstream primer, 5 -GAG GCA GTA AGA CAG GGA TGA-3 , product: 478 bp. The primers above were synthesized by Nanjing GenScript Corporation. PCR systems were as follows: 1 g of template DNA, 1 l of both the upstream and downstream primers, and 12.5 l of 2× Master Mix. ddH2 O was added to make the final volume equal 25 l. Each PCR included the following steps: predenaturation at 94 ◦ C for 5 min; denaturation at 94 ◦ C for 30 s; annealing for 30 s (the annealing temperature for TGF-1 , Smad3, Smad7 and -actin were 57 ◦ C, 52 ◦ C, 47 ◦ C and 46 ◦ C, respectively); extension at 72 ◦ C for 60 s, 30 cycles; extension at 72 ◦ C for 5 min, 1 cycle. 2.5.2.4. Analysis of RT-PCR-5 l. RT-PCR products were placed in a 1.7% agarose gel for electrophoresis. Gels were scanned using a gel imaging analysis system (JD801 gel electrophoresis image analysis software) and the optical density ratios of the PCR products relative to -actin were calculated. 2.6. Statistical analysis All data were expressed as x¯ ± S.D. Differences among groups were assessed using ANOVA, and difference between two groups was analyzed using Student’s t-test. A p value less than 0.05 was considered to be statistically significant. 3. Results As shown in Figs. 1 and 2, the expression level of myocardial TGF-1 mRNA in SHR control group was 4.52 ± 0.46, which was significantly higher than 0.87 ± 0.21 in WKY group (p < 0.01). Treatment with puerarin or captopril markedly reduced the expression level of TGF-1 mRNA (p < 0.05 or p < 0.01). There were no significant differences among captopril, middle- and high-dose puerarin groups (p > 0.05). There was a significant difference between highor middle-dose puerarin group and low-dose puerarin group (p < 0.01 or p < 0.05).
N.-B. Zhang et al. / Journal of Ethnopharmacology 138 (2011) 737–740
Fig. 2. The expression levels of TGF-1 mRNA in all groups.
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is a R-Smad, which is phosphorylated upon TGF-1 activation and conveys a signal to the nucleus, amplifying the TGF-1 signaling pathway (Divakaran et al., 2009; Lin et al., 2009). Smad7 is an I-Smad which inhibits the phosphorylation of R-Smads, playing an essential role in the negative-feedback regulation of TGF-1 signaling (Euler-Taimor and Heger, 2006; Wang et al., 2007). Macrophages secrete TGF-1 , and abnormal accumulation of macrophages in local tissue leads to high level of TGF-1 and enhanced the Smad3-mediated signal transduction. Angiotensin II is the key factor that links the renin–angiotensin system with TGF-1 (Rodríguez-Vita et al., 2005; Siddiquee et al., 2011; Zhong et al., 2011), which enhances collagen synthesis and leads to myocardial fibrosis (Caglayan et al., 2008; Kohno et al., 2008). Angiotensin II levels are higher in SHRs (Sullivan et al., 2010), which could increase expression of Smad3 mRNA and decrease that of Smad7 mRNA. Our experimental data show that the expression of Smad3 mRNA, the activated intracellular transduction factor of TGF-1 , was abnormally increased and that the expression of Smad7 mRNA, the inhibitory intracellular transduction factor of TGF-1 , was significantly reduced. This may limit the effects of angiotensin II-induced TGF-1 signaling and contribute to the improvement of myocardial hypertrophy and fibrosis. 5. Conclusion
Fig. 3. The expression levels of Smad3 mRNA in all groups.
Our results demonstrate for the first time that puerarin reduces expression of cardiac TGF-1 and Smad3 mRNA and increases that of cardiac Smad7 mRNA in the SHR. These changes, which should cause improvement of myocardial hypertrophy and fibrosis, may underlie puerarin’s mechanisms for myocardial protection. Thus, our study provides a theoretical basis for using puerarin in the treatment of hypertension and its associated myocardial hypertrophy and fibrosis. Acknowledgments This work was financially supported by the Natural Science Foundation of Anhui Province (No. 070413124) and the Fresh Start Foundation for Doctorate Holder of Wannan Medical College (No. BK00010804) to ZGH.
Fig. 4. The expression levels of Smad7 mRNA in all groups.
Expression level of myocardial Smad3 mRNA was significantly higher in SHR control group than that in WKY group (p < 0.01). Captopril, middle- and high-dose puerarin significantly reduced the expression of Smad3 mRNA (p < 0.01). Low-dose puerarin, however, did not show significant effect (p > 0.05). We found that the increase in expression level of myocardial Smad3 mRNA was consistent with that of myocardial TGF-1 mRNA (Figs. 1 and 3). Expression level of myocardial Smad7 mRNA in SHR control group was significantly lower than that in WKY group (p < 0.01). Captopril, high- and middle-dose puerarin significantly increased the expression of myocardial Smad7 mRNA (p < 0.01). Low-dose puerarin had no significant effect (p > 0.05). We found that the expression level of myocardial Smad7 mRNA was decreased while that of myocardial TGF-1 mRNA was increased (Figs. 1 and 4). 4. Discussion Smads are intracellular proteins that transduce extracellular signals from the TGF- superfamily (Attisano and Wrana, 2002). Based on function and sequence similarity, Smads are divided into 3 subfamilies: receptor-activated Smads (R-Smads), common partner Smads (Co-Smads) and inhibitory Smads (I-Smads). Smad3
References Attisano, L., Wrana, J.L., 2002. Signal transduction by the TGF-beta superfamily. Science 296, 1646–1647. Caglayan, E., Stauber, B., Collins, A.R., Lyon, C.J., Yin, F., Liu, J., Rosenkranz, S., Erdmann, E., Peterson, L.E., Ross, R.S., Tangirala, R.K., Hsueh, W.A., 2008. Differential roles of cardiomyocyte and macrophage peroxisome proliferator-activated receptor gamma in cardiac fibrosis. Diabetes 57, 2470–2479. Divakaran, V., Adrogue, J., Ishiyama, M., Entman, M.L., Haudek, S., Sivasubramanian, N., Mann, D.L., 2009. Adaptive and maladaptive effects of SMAD3 signaling in the adult heart after hemodynamic pressure overloading. Circulation: Heart Failure 2, 633–642. Euler-Taimor, G., Heger, J., 2006. The complex pattern of SMAD signaling in the cardiovascular system. Cardiovascular Research 69, 15–25. Huang, Z.G., Cheng, H.Z., Zhang, N.B., Ding, B.P., 2010. Effects of puerarin on level of angiotensin II in left cardiac ventricle and its protective effect on left ventricular hypertrophy (in Chinese with English abstract). Practical Pharmacy and Clinical Remedies 13, 161–163. Kohno, T., Anzai, T., Naito, K., Sugano, Y., Maekawa, Y., Takahashi, T., Yoshikawa, T., Ogawa, S., 2008. Angiotensin-receptor blockade reduces border zone myocardial monocyte chemoattractant protein-1 expression and macrophage infiltration in post-infarction ventricular remodeling. Circulation Journal 72, 1685–1692. Lin, H.M., Lee, J.H., Yadav, H., Kamaraju, A.K., Liu, E., Zhigang, D., Vieira, A., Kim, S.J., Collins, H., Matschinsky, F., Harlan, D.M., Roberts, A.B., Rane, S.G., 2009. Transforming growth factor-beta/Smad3 signaling regulates insulin gene transcription and pancreatic islet beta-cell function. Journal of Biological Chemistry 284, 12246–12257. Rodríguez-Vita, J., Sánchez-López, E., Esteban, V., Rupérez, M., Egido, J., Ruiz-Ortega, M., 2005. AngiotensinII activates the smad pathway in vascular smooth muscle cells by a transforming growth factor--independent mechanism. Circulation 111, 2509–2517.
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Siddiquee, K., Hampton, J., Khan, S., Zadory, D., Gleaves, L., Vaughan, D.E., Smith, L.H., 2011. Apelin protects against angiotensin II-induced cardiovascular fibrosis and decreases plasminogen activator inhibitor type-1 production. Journal of Hypertension 29, 724–731. Sullivan, J.C., Bhatia, K., Yamamoto, T., Elmarakby, A.A., 2010. Angiotensin (1–7) receptor antagonism equalizes angiotensin II-induced hypertension in male and female spontaneously hypertensive rats. Hypertension 56, 658–666. Wang, B., Omar, A., Angelovska, T., Drobic, V., Rattan, S.G., Jones, S.C., Dixon, I.M., 2007. Regulation of collagen synthesis by inhibitory Smad7
in cardiac myofibroblasts. American Journal of Physiology 293, H1282– H1290. Zhang, N.B., Chen, H.Z., Cui, W.D., Huang, Z.G., 2010. Antihypertensive effect of puerarin in renal hypertensive rats and its effect on level of angiotensin II in renal tissue (in Chinese with English abstract). Pharmacology and Clinics of Chinese Materia Medica 26, 26–29. Zhong, J., Guo, D., Chen, C.B., Wang, W., Schuster, M., Loibner, H., Penninger, J.M., Scholey, J.W., Kassiri, Z., Oudit, G.Y., 2011. Prevention of angiotensin II-mediated renal oxidative stress, inflammation, and fibrosis by angiotensin-converting enzyme 2. Hypertension 57, 314–322.
Contents lists available at SciVerse ScienceDirect
Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jethpharm
Effects of puerarin on expression of cardiac Smad3 and Smad7 mRNA in spontaneously hypertensive rat Nian-Bao Zhang, Zheng-Gui Huang ∗ , Wei-Dong Cui, Bo-Ping Ding Department of Pharmacology & Pharmacology of TCM Grade Three Laboratory, State Administration of Traditional Chinese Medicine of P.R. China, Wannan Medical College, Wuhu, Anhui 241002, PR China
a r t i c l e
i n f o
Article history: Received 3 August 2011 Received in revised form 8 October 2011 Accepted 10 October 2011 Available online 19 October 2011 Keywords: Puerarin TGF-1 Smad3 Smad7 Spontaneously hypertensive rat
a b s t r a c t Aim of the study: Puerarin is a pure extract from traditional Chinese medicine Kudzu root. It has been used to treat hypertension and angina pectoris. In our previous study, it showed a protective effect against cardiac hypertrophy in rats. This study was to observe effects of puerarin on expression of cardiac transforming growth factor 1 (TGF-1 ), Smad3 and Smad7 mRNA in the spontaneously hypertensive rat (SHR), and to explore its possible mechanism of myocardial protection. Materials and methods: Thirty-five 12-week-old SHRs were randomly allocated into 5 groups of 7 rats as follows: 3 groups which received intraperitoneal (i.p.) puerarin (100 mg kg−1 , 50 mg kg−1 and 25 mg kg−1 ), 1 group which received captopril (30 mg kg−1 , i.g.) and 1 control group of untreated SHRs. In addition, a control group of 7 Wistar-Kyoto (WKY) rats was established. Both control groups received i.p. injections of saline. All rats were treated for six weeks. At the end of this period all rats were sacrificed, and their hearts were quickly removed for semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis. Results: Compared with WKY control group, expression of TGF-1 and Smad3 mRNA was increased (p < 0.01) and Smad7 mRNA expression was decreased in SHR control group (p < 0.01). High- and middledose puerarin decreased the expression of TGF-1 and Smad3 mRNA (p < 0.01) and increased the expression of Smad7 mRNA (p < 0.05). Conclusion: Puerarin reduces expression of TGF-1 and Smad3 mRNA and increases that of Smad7 mRNA in SHR myocardium. These changes in gene expression may be a mechanism by which puerarin provides myocardial protection from hypertension. © 2011 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Hypertension is one of the most common worldwide diseases afflicting humans. Over the past several decades advances in research along with widespread patient education have led to decreased hypertension-related morbidity and mortality. During onset and maintenance of sustained hypertension, an increase in angiotensin II levels and macrophage infiltration into the heart play important roles in hypertension-related heart damage (Caglayan et al., 2008). Angiotensin II is known to induce chemotaxis in inflammatory cells such as macrophages, causing them to aggregate. Macrophages can secrete cytokines like TGF-1 , which act through Smads to promote myocardial collagen synthesis, deposition, and fibrosis (Kohno et al., 2008). There are numerous reports in Chinese medical literature describing the successful use of kudzu (Radix pueraria) in the
∗ Corresponding author. Tel.: +86 553 3932464; fax: +86 553 3932531. E-mail address: [email protected] (Z.-G. Huang). 0378-8741/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2011.10.013
treatment of symptoms of high blood pressure such as headache and dizziness. Recent clinical uses of kudzu include treatments for hypertension and angina pectoris. Puerarin is the 8-C-glucoside of daidzein, a pure extract from Kudzu root. In our previous experiments (Huang et al., 2010; Zhang et al., 2010), we found that puerarin had an antihypertensive effect and a role in protecting the heart in rats. In this study, we observed effects of puerarin on expression of TGF-1 , Smad3 and Smad7 mRNA in the hearts of spontaneously hypertensive rats (SHRs), and demonstrated their role in a possible mechanism of myocardial protection. 2. Materials and methods 2.1. Animals Thirty-five 12-week-old SHRs and 7 WKY rats were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. with certificate number: SCXK (Beijing) 2006-0009. Each cage contained one rat which had free access to laboratory chow and water and with the normal circadian rhythm. Ambient temperatures were
738
N.-B. Zhang et al. / Journal of Ethnopharmacology 138 (2011) 737–740
between 22 ◦ C and 24 ◦ C with humidity 60–65%. The study protocol was approved by the Animal Review Board of the University. 2.2. Drugs Puerarin injection was purchased from Shandong Fangming Pharmaceutical Group Co., Ltd. with batch number: H20033292. Captopril was obtained from Shanghai Pukang Pharmaceutical Co., Ltd. with batch number: H31021327. Those drugs are commercially available analytical pure products. 2.3. Kits Trizol kit, RT first strand kit and PCR amplification kit were supplied by Beijing Tiangen Biotechnology Co., Ltd. DEPC kit was supplied by Sigma–Aldrich Co., Ltd. 2.4. Instruments Instruments for current study included homogenate machine (PRO250, PRO Scientific Inc., Monroe, CT), gene amplifier (EDC810, Dongsheng International Trade Co., Ltd.), water exclusion thermostatic incubator (GHX-9270B, Shanghai Fuma laboratory equipment Co., Ltd.), and freezing high-speed centrifuge (22R, Beckman Coulter Co., Ltd.). 2.5. Experimental methods 2.5.1. Animal groups and sample collection Thirty-five 12-week-old male SHRs were trained for one week and then randomly divided into five groups of seven rats each. From the start of the second week, the rats were given drugs at 8 am every morning. The doses and routes are as follows: rats in high-, middle- and low-dose puerarin groups were intraperitoneally given 100 mg kg−1 d−1 , 50 mg kg−1 d−1 and 25 mg kg−1 d−1 of puerarin injection, respectively. Rats in captopril group were orally given captopril at the dose of 30 mg kg−1 d−1 . Rats in SHR and WKY control groups were intraperitoneally injected with normal saline. The drugs were administered for six weeks. After this period, all rats were sacrificed with urethane anesthesia. The heart was removed quickly and washed with cold normal saline, then dried with filter paper. 100 mg of the left ventricle of each rat was taken, kept at −70 ◦ C, and prepared for RT-PCR. 2.5.2. Semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) 2.5.2.1. Total RNA extraction. Ventricular myocardium samples were cut into pieces with eye scissors. Trizol was added (100 mg/1 ml) and samples were homogenized on ice and then centrifuged (4 ◦ C, 12,000 r min−1 , 10 min). Next, the supernatant was removed. 0.2 ml chloroform/1 ml Trizol was added and samples were shaken for 10 s at 4 ◦ C for 30 min. Subsequently, the aqueous later was carefully removed. Then 0.5 ml isopropanol/1 ml Trizol was added. Samples were again shaken for 10 s and centrifuged at 4 ◦ C with 12,000 r min−1 for 10 min. The supernatant was discarded and 75% ethanol prepared from DEPC-treated water was added and kept at 4 ◦ C for 30 min after a 1 min shake. Next, samples were centrifuged at 4 ◦ C with 7500 r min−1 for 5 min and their supernatant was discarded. The samples were air-dried for 5 min. Total RNA was dissolved in 20 l DEPC water, from which 1 l of this total RNA solution was taken and diluted 50-fold for determining OD260/OD280. This ratio, which assessed purity of the RNA, was between 1.8 and 2.0 for all samples. The total RNA concentration was calculated according to the following formula: Total RNA concentration (mg/L) = OD260 × 40× dilution factor.
Fig. 1. Optical density ratios of TGF-1 , Smad3 and Smad7 to -actin in all groups. *p < 0.05, **p < 0.01 compared with the SHR group; p < 0.05, p < 0.01 compared with WKY group.
2.5.2.2. cDNA synthesis. cDNA was produced using 2 g of total RNA which was added to the following reaction system: 10× RT mix 2 l, dNTP mixture 2 l, Oligo-dT15 2 l, Quant Reverse Transcriptase 1 l. Then RNase-free water was added to make up a total volume of 20 l, which was incubated at 37 ◦ C for 60 min. 2.5.2.3. Polymerase chain reaction (PCR). The templates and primers used for PCR are as follows: TGF-1 primers: upstream, 5 GAA GCC ATC CGT GGC CAG AT-3 , downstream, 5 -CCA GTG ACG TCA AAA GAC AG-3 , product: 461 bp. -actin: upstream primer, 5 CCG TAA AGA CCT CTA TGC CAA CA-3 , downstream primer, 5 -CGG ACT CAT CGT ACT CCT GCT-3 , product: 230 bp. Smad3: upstream primer, 5 -AGG GCT TTG AGG CTG TCT ACC-3 , downstream primer, 5 -GTC CAC GCT GGC ATC TTC TG-3 , product: 364 bp. Smad7: upstream primer, 5 -TTT TGA GGT GTG GTG GGT-3 , downstream primer, 5 -GAG GCA GTA AGA CAG GGA TGA-3 , product: 478 bp. The primers above were synthesized by Nanjing GenScript Corporation. PCR systems were as follows: 1 g of template DNA, 1 l of both the upstream and downstream primers, and 12.5 l of 2× Master Mix. ddH2 O was added to make the final volume equal 25 l. Each PCR included the following steps: predenaturation at 94 ◦ C for 5 min; denaturation at 94 ◦ C for 30 s; annealing for 30 s (the annealing temperature for TGF-1 , Smad3, Smad7 and -actin were 57 ◦ C, 52 ◦ C, 47 ◦ C and 46 ◦ C, respectively); extension at 72 ◦ C for 60 s, 30 cycles; extension at 72 ◦ C for 5 min, 1 cycle. 2.5.2.4. Analysis of RT-PCR-5 l. RT-PCR products were placed in a 1.7% agarose gel for electrophoresis. Gels were scanned using a gel imaging analysis system (JD801 gel electrophoresis image analysis software) and the optical density ratios of the PCR products relative to -actin were calculated. 2.6. Statistical analysis All data were expressed as x¯ ± S.D. Differences among groups were assessed using ANOVA, and difference between two groups was analyzed using Student’s t-test. A p value less than 0.05 was considered to be statistically significant. 3. Results As shown in Figs. 1 and 2, the expression level of myocardial TGF-1 mRNA in SHR control group was 4.52 ± 0.46, which was significantly higher than 0.87 ± 0.21 in WKY group (p < 0.01). Treatment with puerarin or captopril markedly reduced the expression level of TGF-1 mRNA (p < 0.05 or p < 0.01). There were no significant differences among captopril, middle- and high-dose puerarin groups (p > 0.05). There was a significant difference between highor middle-dose puerarin group and low-dose puerarin group (p < 0.01 or p < 0.05).
N.-B. Zhang et al. / Journal of Ethnopharmacology 138 (2011) 737–740
Fig. 2. The expression levels of TGF-1 mRNA in all groups.
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is a R-Smad, which is phosphorylated upon TGF-1 activation and conveys a signal to the nucleus, amplifying the TGF-1 signaling pathway (Divakaran et al., 2009; Lin et al., 2009). Smad7 is an I-Smad which inhibits the phosphorylation of R-Smads, playing an essential role in the negative-feedback regulation of TGF-1 signaling (Euler-Taimor and Heger, 2006; Wang et al., 2007). Macrophages secrete TGF-1 , and abnormal accumulation of macrophages in local tissue leads to high level of TGF-1 and enhanced the Smad3-mediated signal transduction. Angiotensin II is the key factor that links the renin–angiotensin system with TGF-1 (Rodríguez-Vita et al., 2005; Siddiquee et al., 2011; Zhong et al., 2011), which enhances collagen synthesis and leads to myocardial fibrosis (Caglayan et al., 2008; Kohno et al., 2008). Angiotensin II levels are higher in SHRs (Sullivan et al., 2010), which could increase expression of Smad3 mRNA and decrease that of Smad7 mRNA. Our experimental data show that the expression of Smad3 mRNA, the activated intracellular transduction factor of TGF-1 , was abnormally increased and that the expression of Smad7 mRNA, the inhibitory intracellular transduction factor of TGF-1 , was significantly reduced. This may limit the effects of angiotensin II-induced TGF-1 signaling and contribute to the improvement of myocardial hypertrophy and fibrosis. 5. Conclusion
Fig. 3. The expression levels of Smad3 mRNA in all groups.
Our results demonstrate for the first time that puerarin reduces expression of cardiac TGF-1 and Smad3 mRNA and increases that of cardiac Smad7 mRNA in the SHR. These changes, which should cause improvement of myocardial hypertrophy and fibrosis, may underlie puerarin’s mechanisms for myocardial protection. Thus, our study provides a theoretical basis for using puerarin in the treatment of hypertension and its associated myocardial hypertrophy and fibrosis. Acknowledgments This work was financially supported by the Natural Science Foundation of Anhui Province (No. 070413124) and the Fresh Start Foundation for Doctorate Holder of Wannan Medical College (No. BK00010804) to ZGH.
Fig. 4. The expression levels of Smad7 mRNA in all groups.
Expression level of myocardial Smad3 mRNA was significantly higher in SHR control group than that in WKY group (p < 0.01). Captopril, middle- and high-dose puerarin significantly reduced the expression of Smad3 mRNA (p < 0.01). Low-dose puerarin, however, did not show significant effect (p > 0.05). We found that the increase in expression level of myocardial Smad3 mRNA was consistent with that of myocardial TGF-1 mRNA (Figs. 1 and 3). Expression level of myocardial Smad7 mRNA in SHR control group was significantly lower than that in WKY group (p < 0.01). Captopril, high- and middle-dose puerarin significantly increased the expression of myocardial Smad7 mRNA (p < 0.01). Low-dose puerarin had no significant effect (p > 0.05). We found that the expression level of myocardial Smad7 mRNA was decreased while that of myocardial TGF-1 mRNA was increased (Figs. 1 and 4). 4. Discussion Smads are intracellular proteins that transduce extracellular signals from the TGF- superfamily (Attisano and Wrana, 2002). Based on function and sequence similarity, Smads are divided into 3 subfamilies: receptor-activated Smads (R-Smads), common partner Smads (Co-Smads) and inhibitory Smads (I-Smads). Smad3
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