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Puerarin, an isoflavone compound extracted from Gegen (Radix Puerariae Lobatae), modulates sclera remodeling caused by extremely low frequency electromagnetic fields
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J Tradit Chin Med 2016 October 15; 36(5): 678-682 ISSN 0255-2922 © 2016 JTCM. All rights reserved.
EXPERIMENTAL STUDY TOPIC
Puerarin, an isoflavone compound extracted from Gegen (Radix Puerariae Lobatae), modulates sclera remodeling caused by extremely low frequency electromagnetic fields
Tian Tian, Cai Xiaojing, Zhu Huang aa Tian Tian, Cai Xiaojing, Zhu Huang, Department of Ophthalmology, Xinhua Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200092, China Supported by the General Program of the Bio-medical Division of the Shanghai Science and Technology Commission: Effect of Extremely Low Frequency Electromagnetic Fields on Myopia Based on RPE-sclera Complex (No.10411966200); the Scientific Research Fund of Chinese Medical of Shanghai Health Bureau: Effect of Puerarin on RPE Cells Exposed to ELF-EMF (No. 2014JP015) Correspondence to: Prof. Zhu Huang, Department of Ophthalmology, Xinhua Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200092, China. drzhuhuang@ 163.com Telephone: +86-21-25076874 Accepted: November 15, 2015
chain reaction , Western blot analysis, respectively. RESULTS: Puerarin reduced the inhibition in cell proliferation, MMP-2 activity, mRNA, protein expression of HFSFs exposed to ELF-EMFs and enhanced the COL1A1 mRNA and protein expression. CONCLUSION: Puerarin was found to participate in the matrix remodeling process. It might be a potential agent for the treatment of extracellular matrix degradation of sclera associated with ocular conditions. © 2016 JTCM. All rights reserved. Key words: Puerarin; Pueraria lobata; Sclera Fibroblast; Matrix metalloproteinase 2; Collagen type Ⅰ; Electromagnetic fields
Abstract OBJECTIVE: To evaluate the protective effect of puerarin [an isoflavone compound extracted from Gegen (Radix Puerariae Lobatae)] in scleral remodeling induced by extremely low frequency electromagnetic fields (ELF-EMFs).
INTRODUCTION The sclera is now known to be a dynamic tissue that undergoes constant remodeling throughout life. Results from research over the last 25 years have established that sclera remodeling is regulated by genetic and environmental influences which have profound effects on ocular size and refraction.1 Extremely low frequency electromagnetic fields (ELF-EMFs) has been common in daily life all over the world. Both epidemiological and laboratory studies have assessed the biological effects of ELF electromagnetic field and explored its relationship with many diseases.2-4 We found that ELF-EMFs decreased collagen Ⅰ (COL1A1) expression, increased Matrix metalloproteinase-2 (MMP-2) expression in Human fetal scleral fibroblasts (HFSFs). We suggested that ELF-EMFs might contribute to
METHODS: Human fetal scleral fibroblasts (HFSFs) were divided into 5 groups: (a) untreated controls; (b) cells treated with ELF-EMFs; (c) cells treated with ELF-EMFs and puerarin 0.1 μM; (d) cells treated with ELF-EMFs and puerarin 1 μM; (e) cells treated with ELF-EMFs and puerarin 10 μM. Cell proliferation activity was measured by the cell-counting kit-8 assay. Matrix metalloproteinase-2 (MMP-2) activity was measured by gelatin enzymography. MMP-2 and collagenⅠ(COL1A1) mRNA, protein expression were measured by Real-Time polymerase JTCM | www. journaltcm. com
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scleral remodeling, which underlies some ocular conditions such as myopia.5,6 Puerarin is the major bioactive ingredient derived from the root of the Puerarialobata (Willd.) Ohwi, widely known as Gegen (Radix Puerariae Lobatae) in Traditional Chinese Medicine (TCM).7 Pharmacological researches of puerarin in various diseases have made great progress.8,9 Studies showed that it can regulate matrix metabolism,10,11 but the effects of puerarin on protecting sclera pathological remodeling caused by ELF-EMFs were less reported. In this paper we detected the effect of puerarin on cell viability, the synthesis and secretion of cytokines, and enzymes in HFSFs.
Gelatin zymography Activity of MMP-2 was assessed in the culture supernatants by gelatin zymography (Invitrogen, CA, USA), which was performed according to the manufacturer's protocol. The supernatants were collected and intracellular protein concentration was measured using the bicinchoninic acid (BCA) Protein Assay. The volume of supernatants were corrected by the concentration, culture medium was electrophoresed (110 V for 120 min) in a 10% SDS-PAGE gel containing 1mg/ml gelatin. White bands were observed against a blue background after de-staining, indicating MMP-2's gelatinolytic activity. Pictures were acquired with a Chemi Doc XR system (Bio-Rad Laboratories).
MATERIALS AND METHODS
Quantitative real-time PCR analysis Total RNA was extracted using Trizol reagent (Invitrogen, CA, USA). The primers used were MMP-2 forward: 5' GGA AAA GAT TGA TGC G 3'; MMP-2 reverse: 5' GGT GCT GGC TGA GTA G 3'; COL1A1 forward: 5' GTG TTG TGC GAT GAC G 3'; COL1A1 reverse: 5' TCG GTG GGT GAC TCT G3'; glyceraldehyde-3-phosphatedehydrogenase (GAPDH) forward: 5' CTC CTC CAC CTT TGA CGC 3'; GAPDH reverse: 5' CCA CCA CCC TGT TGC TGT 3'; All primers were synthesized by Shanghai Sangon Biological Engineering Technology and Services (Shanghai, China). PCR reactions were performed in a GeneAmp PCR System 9700 thermocycler (Applied Biosystems, Foster, CA, USA). The RQ values (2-△ △ Ct) of MMP-2, COL1A1 mRNA were calculated for each group.
Exposure system Helmholtz coils (16 cm length, 14 cm width, 8 cm height and 168 turns copper wire each) were designed to generate an effective magnetic field in the range of 0-2 mT, with a sinusoidal wave of 50 Hz. The magnetic flux density at the center of coil was measured by an electromagnetic field radiation tester (EMF-827, Lutron Electronic Enterprise Co., Ltd., Taiwan, China). The exposure system was put in a humidified incubator at 37 ℃ with 5% CO2 . 5 Cell culture and treatment HFSFs were obtained from Beijing Institute of Ophthalmology (Beijing, China). Cells were cultured in Dulbecco's modified Eagle's medium with penicillin and streptomycin and 10% fetal bovine serum (Gibco BRL, Gaithersburg, MD, USA), incubated at 37 ℃ in a humidified incubator containing 5% CO2. After trypsinization, cells were allowed to attach to the substratum for 12 h; the medium was renewed; puerarin, final concentrations of 0, 0.1, 1 and 10 μΜ, were added in the puerarin groups. Cells of radiation and puerarin groups were placed inside the coils with a sinusoidal 50 Hz electromagnetic field at 0.2 mT for 24 h. Control cells (not exposed to ELF-EMFs) were cultured in another incubator without power coils.
Western blot analysis Cells were lysed in RIPA (Radio-Immunoprecipitation Assay) buffer and protein concentration was determined. Thirty microgram total protein was separated on 10% SDS-PAGE and transferred to PVDF (Polyvinylidene Fluoride) membranes. Mouse anti-MMP-2 (1∶1000 dilution; Abcam, Cambridge, MA, USA). Rabbit anti-Collagen Ⅰ (1∶1000 dilution; Abcam, Cambridge, MA, USA) and mouse anti-β-actin (1∶ 2000 dilution, Abmart, Shanghai, China) were used, followed by HRP-conjugated secondary antibodies. Specific protein bands were visualized using ECL detection kit (Beyotime, Shanghai, China) and quantified with Bio-Rad Chemi Doc TM XRS+.
Plant materials Puerarin powder (Identification of product, 110752201313) was purchased from the control of pharmaceutical and biological products Chinese (Nanjing, China).
Statistical analysis Statistical analysis was performed with SPSS 17.0 Statistical Software (SPSS, Inc., Chicago, IL, USA). Data were expressed as the mean ± standard deviation ( xˉ ± s). Analysis of variance (least significant difference test if needed) and T-test were performed for group comparisons. The significant level was at P < 0.05.
Cell proliferation assay The cells were cultured at initiated number of 6000 cells/well. The rate of cell proliferation was estimated using the cell counting kit-8 (CCK-8) assay (Dojindo, Laboratorie Kumamoto, Japan), which was performed according to the manufacturer's protocol. Briefly, cells grown in a 96-well plate were incubated with the CCK-8 solutions for 1 hr at 37 ℃ , following which the absorbance of each well at 450 nm was recorded. JTCM | www. journaltcm. com
RESULTS Cell proliferation assay In order to examine the effect of ELF-EMFs on HFSFs 679
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viability, the number of live cells growing under 50 Hz sinusoidal ELF-EMFs at the intensity of 0.2 mT were measured at 6, 12, 24 and 48 h after the exposure (Figure 1). Inhibition in cell proliferation began obviously after exposed to magnetic field for 12hrs compared by corresponding control group (t = 3.311, P < 0.05). By 48 h, a 22.49% of reduction in total live cell number was observed compared with control group (t = 11.906, P < 0.01). In order to examine the effect of puerarin on HFSFs viability treated by ELF-EMFs, cells with puerarin at final concentrations of 0, 0.1, 1 and 10 μΜ were grown under ELF-EMFs for 24 h (F = 77.589, P < 0.01) Puerarin at the concentration of 1.0 μM and 10 μM enhanced the proliferation activity of HFSFs compared with radiation group with puerarin at 0 μΜ (Figure 2). 2.5
activity in HFSF by 25% compared to the control. The activity of MMP-2 gradually decreased in the presence of puerarin with increasing concentrations (Figures 3, 4). Quantitative real-time PCR analysis of MMP-2, COL1A1 The RQ values of MMP-2 mRNA expression in HFSFs after exposure to ELF-EMFs for 24 h at 0.2 mT flux density treated with 0, 0.1, 1 and 10 μM puerarin were 1.737 ± 0.031, 1.607 ± 0.021, 1.233 ± 0.058 and 0.953 ± 0.072, respectively. MMP-2 mRNA expression with 0μM puerarin increased by 73.7% compared to the control group (P < 0.01). MMP-2 mRNA expression gradually decreased in the presence of puerarin with increasing concentrations (Figure 5).
b
2.0 OD450
1.5 a Control Radiation
0.5 0.0
6
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a
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4 6 8 Groups Figure 2 Effect of puerarin on HFSF cell proliferation treated by ELF-EMFs 1: untreated controls; 2: cells treated with ELF-EMFs; 3: cells treated with ELF-EMFs and puerarin 0.1 μM; 4: cells treated with ELF-EMFs and puerarin 1 μM; 5: cells treated with ELF-EMFs and puerarin 10 μM. HFSF: human fetal scleral fibroblasts; ELF-EMFs: extremely low frequency electromagnetic fields. Compared with group 2, aP < 0.01.
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3 4 5 Groups Figure 5 Densitometric analysis of the MMP-2 mRNA expression 1: untreated controls; 2: cells treated with ELF-EMFs; 3: cells treated with ELF-EMFs and puerarin 0.1 μM; 4: cells treated with ELF-EMFs and puerarin 1 μM; 5: cells treated with ELF-EMFs and puerarin 10 μM. ELF-EMFs: extremely low frequency electromagnetic fields; MMP-2: matrix metalloproteinase-2. Compared with group 2, aP < 0.01 and bP < 0.05.
Gelatin zymography analysis of MMP-2 enzyme activity MMP-2 enzyme activity plays essential role in regulating extracellular matrix (ECM) degradation. Therefore, how puerarin affects the activity of MMP-2 in HFSFs exposed to ELF-EMFs becomes important. MMP-2 enzyme activity was assayed from collected after 24 h. Activated forms of MMP-2 migrate to positions of 62 kDa. Electromagnetic fields increased MMP-2 enzyme JTCM | www. journaltcm. com
1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0
MMP-2 mRNA expression
1
3
3 4 5 Groups Figure 4 Densitometric analysis of the active-MMP-2 band 1: untreated controls; 2: cells treated with ELF-EMFs; 3: cells treated with ELF-EMFs and puerarin 0.1 μM; 4: cells treated with ELF-EMFs and puerarin 1 μM; 5: cells treated with ELF-EMFs and puerarin 10 μM. ELF-EMFs: extremely low frequency electromagnetic fields; MMP-2: matrix metalloproteinase-2. Compared with group 2, aP < 0.01.
OD450
a
Active-MMP-2
12 24 48 Time (h) Figure 1 Effect of ELF-EMFs on HFSF cell proliferation Control: cells were cultured 6, 12, 24 and 48 h without ELF-EMFs. Radiation: cells were cultured 6, 12, 24 and 48 h with ELF-EMFs. ELF-EMFs: extremely low frequency electromagnetic fields; HFSF: human fetal scleral fibroblasts. Compared to control, aP < 0.05, bP < 0.01. 2.0 1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0
0
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55 Figure 3 Micrograph of MMP-2 enzyme activity analysis 1: untreated controls; 2: cells treated with ELF-EMFs; 3: cells treated with ELF-EMFs and puerarin 0.1 μM; 4: cells treated with ELF-EMFs and puerarin 1 μM; 5: cells treated with ELF-EMFs and puerarin 10 μM. ELF-EMFs: extremely low frequency electromagnetic fields; MMP-2: matrix metalloproteinase-2.
b
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kDa 72
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The RQ values of COL1A1 mRNA expression in HFSFs after exposure to ELF-EMFs for 24 h at 0.2 mT flux density treated with 0, 0.1, 1 and 10 μM puerarin 680
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were 0.66 ± 0.066, 0.717 ± 0.031, 0.883 ± 0.057 and 0.997 ± 0.100, respectively. COL1A1 mRNA expression with 0 μM puerarin decreased by 34% compared to the control group (P < 0.01). COL1A1 mRNA expression gradually increased in the presence of puerarin with increasing concentrations (Figure 6). COL1A1 mRNA expression
1.2 1.0 0.8 0.6 0.4 0.2 0.0 0
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kD 43 130
MMP-2
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Figure 7 Micrograph of MMP-2 and COL1A1 western blot analysis 1: untreated controls; 2: cells treated with ELF-EMFs; 3: cells treated with ELF-EMFs and puerarin 0.1 μM; 4: cells treated with ELF-EMFs and puerarin 1 μM; 5: cells treated with ELF-EMFs and puerarin 10 μM. ELF-EMFs: extremely low frequency electromagnetic fields; MMP-2:matrix metalloproteinase-2; COL1A1: collagen typeⅠ.
a
2
3 4 5 Groups Figure 6 Densitometric analysis of the COL1A1 mRNA expression 1: untreated controls; 2: cells treated with ELF-EMFs; 3: cells treated with ELF-EMFs and puerarin 0.1 μM; 4: cells treated with ELF-EMFs and puerarin 1 μM; 5: cells treated with ELF-EMFs and puerarin 10 μM. ELF-EMFs: extremely low frequency electromagnetic fields; COL1A1: collagen type Ⅰ. Compared with group 2, aP < 0.01 and bP < 0.05.
0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0
b a
0
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a
2 3 4 5 Groups Figure 8 Densitometric analysis of the MMP-2 protein band compared β-actin 1: untreated controls; 2: cells treated with ELF-EMFs; 3: cells treated with ELF-EMFs and puerarin 0.1 μM; 4: cells treated with ELF-EMFs and puerarin 1 μM; 5: cells treated with ELF-EMFs and puerarin 10 μM. ELF-EMFs: extremely low frequency electromagnetic fields; MMP-2: matrix metalloproteinase-2. Compared with group 2, aP < 0.01, bP < 0.05.
Western blot analysis of MMP-2, COL1A1 The MMP-2/β-actin gray value ratio of MMP-2 protein expression in 5 groups were different (P < 0.01). Consistent with PCR results, MMP-2 protein level increased 1.06 folds compared with the control group (P < 0.01). There was dose-dependent decrease in MMP-2 protein expression after puerarin treatment (Figures 7, 8). The COL1A1/β-actin gray value ratio of COL1A1 protein expression in 5 groups were different. (P < 0.01) Consistent with PCR results, COL1A1 protein level decreased 90% compared with the control group (P < 0.01). There was dose-dependent increase in COL1A1 protein expression after puerarin treatment (Figures 7, 9).
COL1A1 relative expression
0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0 0
1
a b
a
a
2 3 4 5 Groups Figure 9 Densitometric analysis of the COL1A1 protein band compared β-actin 1: untreated controls; 2: cells treated with ELF-EMFs; 3: cells treated with ELF-EMFs and puerarin 0.1 μM; 4: cells treated with ELF-EMFs and puerarin 1 μM; 5: cells treated with ELF-EMFs and puerarin 10 μM. ELF-EMFs: extremely low frequency electromagnetic fields; COL1A1: collagen type Ⅰ. Compared with group 2, aP < 0.01 and bP < 0.05.
DISCUSSION The sclera is a dense connective tissue that defines ocular size and shape, which biomechanical properties are determined by its ECM. Defects in scleral ECM remodeling lead to some ocular conditions such as myopia, characterized by increment in activity and expression of matrix metalloproteinases as well as the reduction of collagen.1 In previous studies, it can be seen that ELF-EMFs act as a kind of stimulus causing the matrix remodeling in HFSFs.5,6 Puerarin has the role of lowering intraocular pressure, improving microcirculation and antioxidation.8,9 Purerarin injection and other preparations have been extensively used in the clinic in China.12 Puerarin has also been found to affect cell proliferation activity, regulate matrix metabolism, and the synthesis and secretion of cytokines and enzymes.13 Wang et al 10 confirmed that 0.001 μM puerarin could decrease MMP-9 expression in endometriosis tissue. Puerarin increased JTCM | www. journaltcm. com
1
COL1A1
MMP-2 relative expression
1
a
β-actin
1
the rate of proliferation and mRNA levels of type I collagen.11 Puerarin also has some hormone-like activities and has protective effect against oxidative stress response and osteoporosis.14 We have found that puerarin decreased the expression of MMP-2 mRNA and protein in HFSF cells exposed to ELF-EMFs,15 but whether puerarin has effect on HFSFs cell viability, MMP-2 activity and collagen I synthesis has not yet been reported. It can be seen that puerarin has effect in preventing matrix remodeling caused by harmful stimulus. By 48 h, a 22.49% of reduction in total live cell number was observed. Puerarin at the concentration of 1.0 μM and 10 μM enhanced the proliferation activity of HFSFs compared with radiation group with puerarin at 0 μΜ. MMP-2 activity, mRNA and protein expres681
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sion increased by 1.25, 1.737 and 1.06 folds in HFSFs exposed to ELF-EMFs compared with the control group. While COL1A1 mRNA and protein expression decreased 34% and 90% in HFSFs exposed to ELF-EMFs compared with the control group. Puerarin weakened these changes to a certain extent. In sum, our study showed that ELF-EMFs had a direct effect on cell viability, synthesis and secretion of cytokines in HFSFs. These effects can be partially reversed by puerarin. These findings suggested a potential option for the treatment of extracellular matrix degradation of sclera associated with various ocular conditions.
8
9 10
11
REFERENCES 1
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Harper AR, Summers JA. The dynamic sclera: extracellular matrix remodeling in normal ocular growth and myopia development. Exp Eye Res 2015; 133: 100-111. Sun JW, Li XR, Gao HY, et al. Electromagnetic field exposure and male breast cancer risk: a Meta-analysis of 18 studies. Asian Pac J Cancer Prev 2013; 14(1): 523-528. Mairs RJ, Hughes K, Fitzsimmons S, et al. Microsatellite analysis for determination of the mutagenicity of extremely low-frequency electromagnetic fields and ionising radiation in vitro. Mutat Res 2007; 626(s 1-2): 34-41. Huang CY, Chang CW, Chen CR, et al. Extremely low-frequency electromagnetic fields cause G1 phase arrest through the activation of the ATM-Chk2-p21 pathway. PLoS One 2014; 9(8): e104732. Wang J, Cui J, Zhu H. Suppression of type Ⅰ collagen in human scleral fibroblasts treated with extremely low-frequency electromagnetic fields. Mol Vis 2013; 19: 885-893. Zhu H, Wang J, Cui J, Fan X. Effects of extremely low frequency electromagnetic fields on human fetal scleral fibroblasts. Toxicol Ind Health 2016; 32(6): 1042-1051. Chen R, Xue J, Xie M. Puerarin prevents isoprenaline-in-
JTCM | www. journaltcm. com
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duced myocardial fibrosis in mice by reduction of myocardial TGF-beta1 expression. J Nutr Biochem 2012; 23(9): 1080-1085. Wei SY, Chen Y, Xu XY. Progress on the pharmacological research of puerarin: a review. Chin J Nat Med 2014; 12 (6): 407-414. Zhou YX, Zhang H, Peng C. Puerarin: a review of pharmacological effects. Phytother Res 2014; 28(7): 961-975. Wang D, Liu Y, Han J, et al. Puerarin suppresses invasion and vascularization of endometriosis tissue stimulated by 17 beta-estradiol. PLoS One 2011; 6(9): 685-690. Tiyasatkulkovit W, Malaivijitnond S, Charoenphandhu N, Havill LM, Ford AL, VandeBerg JL. Pueraria mirifica extract and puerarin enhance proliferation and expression of alkaline phosphatase and type Ⅰ collagen in primary baboon osteoblasts. Phytomedicine 2014; 21(12): 1498-1503. Wong KH, Li GQ, Li KM, Razmovski-Naumovski V, Chan K. Kudzu root: traditional uses and potential medicinal benefits in diabetes and cardiovascular diseases. J Ethnopharmacol 2011; 134(3): 584-607. Yang X, Zhang H, Wang J, Zhang Z, Li C. Puerarin decreases bone loss and collagen destruction in rats with ligature-induced periodontitis. J Periodontal Res 2015; 50(6): 748-757. Tiyasatkulkovit W, Charoenphandhu N, Wongdee K, Thongbunchoo J, Krishnamra N, Malaivijitnond S. Upregulation of osteoblastic differentiation marker mRNA expression in osteoblast-like UMR106 cells by puerarin and phytoestrogens from Puerariamirifica. Phytomedicine 2012; 19(13): 1147-1155. Zhu H, Cai XJ, Fan XQ. Effect of puerarin on matrix metalloproteinase-2 in human fetal scleral fibroblasts treated with low frequency electromagnetic fields. J Tradit Chin Med 2013; 33(5): 664-668.
October 15, 2016 | Volume 36 | Issue 5 |
J Tradit Chin Med 2016 October 15; 36(5): 678-682 ISSN 0255-2922 © 2016 JTCM. All rights reserved.
EXPERIMENTAL STUDY TOPIC
Puerarin, an isoflavone compound extracted from Gegen (Radix Puerariae Lobatae), modulates sclera remodeling caused by extremely low frequency electromagnetic fields
Tian Tian, Cai Xiaojing, Zhu Huang aa Tian Tian, Cai Xiaojing, Zhu Huang, Department of Ophthalmology, Xinhua Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200092, China Supported by the General Program of the Bio-medical Division of the Shanghai Science and Technology Commission: Effect of Extremely Low Frequency Electromagnetic Fields on Myopia Based on RPE-sclera Complex (No.10411966200); the Scientific Research Fund of Chinese Medical of Shanghai Health Bureau: Effect of Puerarin on RPE Cells Exposed to ELF-EMF (No. 2014JP015) Correspondence to: Prof. Zhu Huang, Department of Ophthalmology, Xinhua Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200092, China. drzhuhuang@ 163.com Telephone: +86-21-25076874 Accepted: November 15, 2015
chain reaction , Western blot analysis, respectively. RESULTS: Puerarin reduced the inhibition in cell proliferation, MMP-2 activity, mRNA, protein expression of HFSFs exposed to ELF-EMFs and enhanced the COL1A1 mRNA and protein expression. CONCLUSION: Puerarin was found to participate in the matrix remodeling process. It might be a potential agent for the treatment of extracellular matrix degradation of sclera associated with ocular conditions. © 2016 JTCM. All rights reserved. Key words: Puerarin; Pueraria lobata; Sclera Fibroblast; Matrix metalloproteinase 2; Collagen type Ⅰ; Electromagnetic fields
Abstract OBJECTIVE: To evaluate the protective effect of puerarin [an isoflavone compound extracted from Gegen (Radix Puerariae Lobatae)] in scleral remodeling induced by extremely low frequency electromagnetic fields (ELF-EMFs).
INTRODUCTION The sclera is now known to be a dynamic tissue that undergoes constant remodeling throughout life. Results from research over the last 25 years have established that sclera remodeling is regulated by genetic and environmental influences which have profound effects on ocular size and refraction.1 Extremely low frequency electromagnetic fields (ELF-EMFs) has been common in daily life all over the world. Both epidemiological and laboratory studies have assessed the biological effects of ELF electromagnetic field and explored its relationship with many diseases.2-4 We found that ELF-EMFs decreased collagen Ⅰ (COL1A1) expression, increased Matrix metalloproteinase-2 (MMP-2) expression in Human fetal scleral fibroblasts (HFSFs). We suggested that ELF-EMFs might contribute to
METHODS: Human fetal scleral fibroblasts (HFSFs) were divided into 5 groups: (a) untreated controls; (b) cells treated with ELF-EMFs; (c) cells treated with ELF-EMFs and puerarin 0.1 μM; (d) cells treated with ELF-EMFs and puerarin 1 μM; (e) cells treated with ELF-EMFs and puerarin 10 μM. Cell proliferation activity was measured by the cell-counting kit-8 assay. Matrix metalloproteinase-2 (MMP-2) activity was measured by gelatin enzymography. MMP-2 and collagenⅠ(COL1A1) mRNA, protein expression were measured by Real-Time polymerase JTCM | www. journaltcm. com
678
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Tian T et al. / Experimental Study
scleral remodeling, which underlies some ocular conditions such as myopia.5,6 Puerarin is the major bioactive ingredient derived from the root of the Puerarialobata (Willd.) Ohwi, widely known as Gegen (Radix Puerariae Lobatae) in Traditional Chinese Medicine (TCM).7 Pharmacological researches of puerarin in various diseases have made great progress.8,9 Studies showed that it can regulate matrix metabolism,10,11 but the effects of puerarin on protecting sclera pathological remodeling caused by ELF-EMFs were less reported. In this paper we detected the effect of puerarin on cell viability, the synthesis and secretion of cytokines, and enzymes in HFSFs.
Gelatin zymography Activity of MMP-2 was assessed in the culture supernatants by gelatin zymography (Invitrogen, CA, USA), which was performed according to the manufacturer's protocol. The supernatants were collected and intracellular protein concentration was measured using the bicinchoninic acid (BCA) Protein Assay. The volume of supernatants were corrected by the concentration, culture medium was electrophoresed (110 V for 120 min) in a 10% SDS-PAGE gel containing 1mg/ml gelatin. White bands were observed against a blue background after de-staining, indicating MMP-2's gelatinolytic activity. Pictures were acquired with a Chemi Doc XR system (Bio-Rad Laboratories).
MATERIALS AND METHODS
Quantitative real-time PCR analysis Total RNA was extracted using Trizol reagent (Invitrogen, CA, USA). The primers used were MMP-2 forward: 5' GGA AAA GAT TGA TGC G 3'; MMP-2 reverse: 5' GGT GCT GGC TGA GTA G 3'; COL1A1 forward: 5' GTG TTG TGC GAT GAC G 3'; COL1A1 reverse: 5' TCG GTG GGT GAC TCT G3'; glyceraldehyde-3-phosphatedehydrogenase (GAPDH) forward: 5' CTC CTC CAC CTT TGA CGC 3'; GAPDH reverse: 5' CCA CCA CCC TGT TGC TGT 3'; All primers were synthesized by Shanghai Sangon Biological Engineering Technology and Services (Shanghai, China). PCR reactions were performed in a GeneAmp PCR System 9700 thermocycler (Applied Biosystems, Foster, CA, USA). The RQ values (2-△ △ Ct) of MMP-2, COL1A1 mRNA were calculated for each group.
Exposure system Helmholtz coils (16 cm length, 14 cm width, 8 cm height and 168 turns copper wire each) were designed to generate an effective magnetic field in the range of 0-2 mT, with a sinusoidal wave of 50 Hz. The magnetic flux density at the center of coil was measured by an electromagnetic field radiation tester (EMF-827, Lutron Electronic Enterprise Co., Ltd., Taiwan, China). The exposure system was put in a humidified incubator at 37 ℃ with 5% CO2 . 5 Cell culture and treatment HFSFs were obtained from Beijing Institute of Ophthalmology (Beijing, China). Cells were cultured in Dulbecco's modified Eagle's medium with penicillin and streptomycin and 10% fetal bovine serum (Gibco BRL, Gaithersburg, MD, USA), incubated at 37 ℃ in a humidified incubator containing 5% CO2. After trypsinization, cells were allowed to attach to the substratum for 12 h; the medium was renewed; puerarin, final concentrations of 0, 0.1, 1 and 10 μΜ, were added in the puerarin groups. Cells of radiation and puerarin groups were placed inside the coils with a sinusoidal 50 Hz electromagnetic field at 0.2 mT for 24 h. Control cells (not exposed to ELF-EMFs) were cultured in another incubator without power coils.
Western blot analysis Cells were lysed in RIPA (Radio-Immunoprecipitation Assay) buffer and protein concentration was determined. Thirty microgram total protein was separated on 10% SDS-PAGE and transferred to PVDF (Polyvinylidene Fluoride) membranes. Mouse anti-MMP-2 (1∶1000 dilution; Abcam, Cambridge, MA, USA). Rabbit anti-Collagen Ⅰ (1∶1000 dilution; Abcam, Cambridge, MA, USA) and mouse anti-β-actin (1∶ 2000 dilution, Abmart, Shanghai, China) were used, followed by HRP-conjugated secondary antibodies. Specific protein bands were visualized using ECL detection kit (Beyotime, Shanghai, China) and quantified with Bio-Rad Chemi Doc TM XRS+.
Plant materials Puerarin powder (Identification of product, 110752201313) was purchased from the control of pharmaceutical and biological products Chinese (Nanjing, China).
Statistical analysis Statistical analysis was performed with SPSS 17.0 Statistical Software (SPSS, Inc., Chicago, IL, USA). Data were expressed as the mean ± standard deviation ( xˉ ± s). Analysis of variance (least significant difference test if needed) and T-test were performed for group comparisons. The significant level was at P < 0.05.
Cell proliferation assay The cells were cultured at initiated number of 6000 cells/well. The rate of cell proliferation was estimated using the cell counting kit-8 (CCK-8) assay (Dojindo, Laboratorie Kumamoto, Japan), which was performed according to the manufacturer's protocol. Briefly, cells grown in a 96-well plate were incubated with the CCK-8 solutions for 1 hr at 37 ℃ , following which the absorbance of each well at 450 nm was recorded. JTCM | www. journaltcm. com
RESULTS Cell proliferation assay In order to examine the effect of ELF-EMFs on HFSFs 679
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viability, the number of live cells growing under 50 Hz sinusoidal ELF-EMFs at the intensity of 0.2 mT were measured at 6, 12, 24 and 48 h after the exposure (Figure 1). Inhibition in cell proliferation began obviously after exposed to magnetic field for 12hrs compared by corresponding control group (t = 3.311, P < 0.05). By 48 h, a 22.49% of reduction in total live cell number was observed compared with control group (t = 11.906, P < 0.01). In order to examine the effect of puerarin on HFSFs viability treated by ELF-EMFs, cells with puerarin at final concentrations of 0, 0.1, 1 and 10 μΜ were grown under ELF-EMFs for 24 h (F = 77.589, P < 0.01) Puerarin at the concentration of 1.0 μM and 10 μM enhanced the proliferation activity of HFSFs compared with radiation group with puerarin at 0 μΜ (Figure 2). 2.5
activity in HFSF by 25% compared to the control. The activity of MMP-2 gradually decreased in the presence of puerarin with increasing concentrations (Figures 3, 4). Quantitative real-time PCR analysis of MMP-2, COL1A1 The RQ values of MMP-2 mRNA expression in HFSFs after exposure to ELF-EMFs for 24 h at 0.2 mT flux density treated with 0, 0.1, 1 and 10 μM puerarin were 1.737 ± 0.031, 1.607 ± 0.021, 1.233 ± 0.058 and 0.953 ± 0.072, respectively. MMP-2 mRNA expression with 0μM puerarin increased by 73.7% compared to the control group (P < 0.01). MMP-2 mRNA expression gradually decreased in the presence of puerarin with increasing concentrations (Figure 5).
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4 6 8 Groups Figure 2 Effect of puerarin on HFSF cell proliferation treated by ELF-EMFs 1: untreated controls; 2: cells treated with ELF-EMFs; 3: cells treated with ELF-EMFs and puerarin 0.1 μM; 4: cells treated with ELF-EMFs and puerarin 1 μM; 5: cells treated with ELF-EMFs and puerarin 10 μM. HFSF: human fetal scleral fibroblasts; ELF-EMFs: extremely low frequency electromagnetic fields. Compared with group 2, aP < 0.01.
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3 4 5 Groups Figure 5 Densitometric analysis of the MMP-2 mRNA expression 1: untreated controls; 2: cells treated with ELF-EMFs; 3: cells treated with ELF-EMFs and puerarin 0.1 μM; 4: cells treated with ELF-EMFs and puerarin 1 μM; 5: cells treated with ELF-EMFs and puerarin 10 μM. ELF-EMFs: extremely low frequency electromagnetic fields; MMP-2: matrix metalloproteinase-2. Compared with group 2, aP < 0.01 and bP < 0.05.
Gelatin zymography analysis of MMP-2 enzyme activity MMP-2 enzyme activity plays essential role in regulating extracellular matrix (ECM) degradation. Therefore, how puerarin affects the activity of MMP-2 in HFSFs exposed to ELF-EMFs becomes important. MMP-2 enzyme activity was assayed from collected after 24 h. Activated forms of MMP-2 migrate to positions of 62 kDa. Electromagnetic fields increased MMP-2 enzyme JTCM | www. journaltcm. com
1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0
MMP-2 mRNA expression
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3 4 5 Groups Figure 4 Densitometric analysis of the active-MMP-2 band 1: untreated controls; 2: cells treated with ELF-EMFs; 3: cells treated with ELF-EMFs and puerarin 0.1 μM; 4: cells treated with ELF-EMFs and puerarin 1 μM; 5: cells treated with ELF-EMFs and puerarin 10 μM. ELF-EMFs: extremely low frequency electromagnetic fields; MMP-2: matrix metalloproteinase-2. Compared with group 2, aP < 0.01.
OD450
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12 24 48 Time (h) Figure 1 Effect of ELF-EMFs on HFSF cell proliferation Control: cells were cultured 6, 12, 24 and 48 h without ELF-EMFs. Radiation: cells were cultured 6, 12, 24 and 48 h with ELF-EMFs. ELF-EMFs: extremely low frequency electromagnetic fields; HFSF: human fetal scleral fibroblasts. Compared to control, aP < 0.05, bP < 0.01. 2.0 1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0
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55 Figure 3 Micrograph of MMP-2 enzyme activity analysis 1: untreated controls; 2: cells treated with ELF-EMFs; 3: cells treated with ELF-EMFs and puerarin 0.1 μM; 4: cells treated with ELF-EMFs and puerarin 1 μM; 5: cells treated with ELF-EMFs and puerarin 10 μM. ELF-EMFs: extremely low frequency electromagnetic fields; MMP-2: matrix metalloproteinase-2.
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The RQ values of COL1A1 mRNA expression in HFSFs after exposure to ELF-EMFs for 24 h at 0.2 mT flux density treated with 0, 0.1, 1 and 10 μM puerarin 680
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were 0.66 ± 0.066, 0.717 ± 0.031, 0.883 ± 0.057 and 0.997 ± 0.100, respectively. COL1A1 mRNA expression with 0 μM puerarin decreased by 34% compared to the control group (P < 0.01). COL1A1 mRNA expression gradually increased in the presence of puerarin with increasing concentrations (Figure 6). COL1A1 mRNA expression
1.2 1.0 0.8 0.6 0.4 0.2 0.0 0
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Figure 7 Micrograph of MMP-2 and COL1A1 western blot analysis 1: untreated controls; 2: cells treated with ELF-EMFs; 3: cells treated with ELF-EMFs and puerarin 0.1 μM; 4: cells treated with ELF-EMFs and puerarin 1 μM; 5: cells treated with ELF-EMFs and puerarin 10 μM. ELF-EMFs: extremely low frequency electromagnetic fields; MMP-2:matrix metalloproteinase-2; COL1A1: collagen typeⅠ.
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3 4 5 Groups Figure 6 Densitometric analysis of the COL1A1 mRNA expression 1: untreated controls; 2: cells treated with ELF-EMFs; 3: cells treated with ELF-EMFs and puerarin 0.1 μM; 4: cells treated with ELF-EMFs and puerarin 1 μM; 5: cells treated with ELF-EMFs and puerarin 10 μM. ELF-EMFs: extremely low frequency electromagnetic fields; COL1A1: collagen type Ⅰ. Compared with group 2, aP < 0.01 and bP < 0.05.
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2 3 4 5 Groups Figure 8 Densitometric analysis of the MMP-2 protein band compared β-actin 1: untreated controls; 2: cells treated with ELF-EMFs; 3: cells treated with ELF-EMFs and puerarin 0.1 μM; 4: cells treated with ELF-EMFs and puerarin 1 μM; 5: cells treated with ELF-EMFs and puerarin 10 μM. ELF-EMFs: extremely low frequency electromagnetic fields; MMP-2: matrix metalloproteinase-2. Compared with group 2, aP < 0.01, bP < 0.05.
Western blot analysis of MMP-2, COL1A1 The MMP-2/β-actin gray value ratio of MMP-2 protein expression in 5 groups were different (P < 0.01). Consistent with PCR results, MMP-2 protein level increased 1.06 folds compared with the control group (P < 0.01). There was dose-dependent decrease in MMP-2 protein expression after puerarin treatment (Figures 7, 8). The COL1A1/β-actin gray value ratio of COL1A1 protein expression in 5 groups were different. (P < 0.01) Consistent with PCR results, COL1A1 protein level decreased 90% compared with the control group (P < 0.01). There was dose-dependent increase in COL1A1 protein expression after puerarin treatment (Figures 7, 9).
COL1A1 relative expression
0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0 0
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2 3 4 5 Groups Figure 9 Densitometric analysis of the COL1A1 protein band compared β-actin 1: untreated controls; 2: cells treated with ELF-EMFs; 3: cells treated with ELF-EMFs and puerarin 0.1 μM; 4: cells treated with ELF-EMFs and puerarin 1 μM; 5: cells treated with ELF-EMFs and puerarin 10 μM. ELF-EMFs: extremely low frequency electromagnetic fields; COL1A1: collagen type Ⅰ. Compared with group 2, aP < 0.01 and bP < 0.05.
DISCUSSION The sclera is a dense connective tissue that defines ocular size and shape, which biomechanical properties are determined by its ECM. Defects in scleral ECM remodeling lead to some ocular conditions such as myopia, characterized by increment in activity and expression of matrix metalloproteinases as well as the reduction of collagen.1 In previous studies, it can be seen that ELF-EMFs act as a kind of stimulus causing the matrix remodeling in HFSFs.5,6 Puerarin has the role of lowering intraocular pressure, improving microcirculation and antioxidation.8,9 Purerarin injection and other preparations have been extensively used in the clinic in China.12 Puerarin has also been found to affect cell proliferation activity, regulate matrix metabolism, and the synthesis and secretion of cytokines and enzymes.13 Wang et al 10 confirmed that 0.001 μM puerarin could decrease MMP-9 expression in endometriosis tissue. Puerarin increased JTCM | www. journaltcm. com
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MMP-2 relative expression
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the rate of proliferation and mRNA levels of type I collagen.11 Puerarin also has some hormone-like activities and has protective effect against oxidative stress response and osteoporosis.14 We have found that puerarin decreased the expression of MMP-2 mRNA and protein in HFSF cells exposed to ELF-EMFs,15 but whether puerarin has effect on HFSFs cell viability, MMP-2 activity and collagen I synthesis has not yet been reported. It can be seen that puerarin has effect in preventing matrix remodeling caused by harmful stimulus. By 48 h, a 22.49% of reduction in total live cell number was observed. Puerarin at the concentration of 1.0 μM and 10 μM enhanced the proliferation activity of HFSFs compared with radiation group with puerarin at 0 μΜ. MMP-2 activity, mRNA and protein expres681
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sion increased by 1.25, 1.737 and 1.06 folds in HFSFs exposed to ELF-EMFs compared with the control group. While COL1A1 mRNA and protein expression decreased 34% and 90% in HFSFs exposed to ELF-EMFs compared with the control group. Puerarin weakened these changes to a certain extent. In sum, our study showed that ELF-EMFs had a direct effect on cell viability, synthesis and secretion of cytokines in HFSFs. These effects can be partially reversed by puerarin. These findings suggested a potential option for the treatment of extracellular matrix degradation of sclera associated with various ocular conditions.
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