|Year : 2015 | Volume
| Issue : 4 | Page : 605-609
Puerarin exhibits greater distribution and longer retention time in neurons than astrocytes in a co-cultured system
Shu-Yong Wei1, Jie Tong2, Qiang Xue2, Fang-hong Shang2, Yan-jun Li2, Yang Liu2, Bin-bin Feng3, Xiao-yu Xu2
1 Pharmaceutical Sciences College & Chinese Medicine College of Southwest University; Chongqing Engineering Research Center for Pharmacodynamics Evaluation, Chongqing; Rongchang Campus of Southwest University, Chongqing, China
2 Pharmaceutical Sciences College & Chinese Medicine College of Southwest University; Chongqing Engineering Research Center for Pharmacodynamics Evaluation, Chongqing, China
3 Chongqing Three Gorges Medical College, Chongqing, China
|Date of Acceptance||22-Jan-2015|
|Date of Web Publication||30-Apr-2015|
Pharmaceutical Sciences College & Chinese Medicine College of Southwest University; Chongqing Engineering Research Center for Pharmacodynamics Evaluation, Chongqing
Source of Support: This study was supported by the National Natural Science Foundation of China, No. 31402237, 81473549; the Fundamental Research Funds for Central Universities in China, No. XDJK2014C058, XDJK2014D023, XDJK2015D016; a grant from the National Key New Drug Development Project of China, No. 2014ZX09304-306-04; the Fundamental and Front Research Funds of Chongqing of China, No. CSTC2014jcyjA80023; and a grant from the Natural Science Foundation of Chongqing of China, No. CSTC2012jjA10012., Conflict of Interest: None
The phytoestrogen puerarin has been shown to protect neurons and astrocytes in the brain, and is therefore an attractive drug in the treatment of Alzheimer's disease, Parkinson's disease and cerebral ischemia. Whether puerarin exhibits the same biological processes in neurons and astrocytes in vitro has rarely been reported. In this study, cortical neurons and astrocytes of newborn Sprague-Dawley rats were separated, identified and co-cultured in a system based on Transwell membranes. The retention time and distribution of puerarin in each cell type was detected by fluorescence spectrophotometry and fluorescence microscope. The concentration of puerarin in both co-cultured and separately cultured neurons was greater than that of astrocytes. Puerarin concentration reached a maximum 20 minutes after it was added. At 60 minutes after its addition, a scant amount of drug was detected in astrocytes; however in both separately cultured and co-cultured neurons, the concentration of puerarin achieved a stable level of about 12.8 ng/mL. The results indicate that puerarin had a higher concentration and longer retention time in neurons than that observed in astrocytes.
Keywords: nerve regeneration; puerarin; in vitro experiments; co-culture; neurons; astrocytes; Transwell; neonatal rats; neural regeneration
|How to cite this article:|
Wei SY, Tong J, Xue Q, Shang Fh, Li Yj, Liu Y, Feng Bb, Xu Xy. Puerarin exhibits greater distribution and longer retention time in neurons than astrocytes in a co-cultured system. Neural Regen Res 2015;10:605-9
|How to cite this URL:|
Wei SY, Tong J, Xue Q, Shang Fh, Li Yj, Liu Y, Feng Bb, Xu Xy. Puerarin exhibits greater distribution and longer retention time in neurons than astrocytes in a co-cultured system. Neural Regen Res [serial online] 2015 [cited 2019 Sep 20];10:605-9. Available from: http://www.nrronline.org/text.asp?2015/10/4/605/155435
Author contributions: SYW, BBF and XYX designed the study. SYW, JT, QX and FHS performed the experiments. SYW, YJL and YL analyzed the data. SYW and XYX wrote the paper. All authors approved the final version of this paper.
Conflicts of interest: None declared.
| Introduction|| |
As the main active constituent of the plant Pueraria lobata (Willd.) Ohwi (Fabaceae), puerarin (Pur) is considered to be one of the most important phytoestrogens and has been widely used in China in clinical treatment (Wei et al., 2014). The neuroprotective effects of Pur have been established in vivo in Alzheimer's disease (Xu and Zhao, 2002; Xu et al., 2004), Parkinson's disease (Li et al., 2003; Zhu et al., 2010), and cerebral ischemia (Xu et al., 2007; Chang et al., 2009; Wu et al., 2009), and in vitro on primary cultured neurons (Li et al., 2003, 2010; Zou et al., 2013), gliocytes (Zheng et al., 2012), and other cell lines (Lin et al., 2010; Xing et al., 2011; Zhang et al., 2012; Zhu et al., 2012). The potential mechanisms of these neuroprotective effects include alleviating mitochondrial oxidative stress (Xu and Zhao, 2002; Li et al., 2010; Zou et al., 2013), anti-apoptosis (Li et al., 2003; Chang et al., 2009; Wu et al., 2009; Lin et al., 2010; Zhu et al., 2010), inhibiting excitatory toxicity (Xu et al., 2004, 2007), and promoting cell proliferation (Xing et al., 2011; Zhang et al., 2012; Zhu et al., 2012).
Although various studies have indicated neuroprotective effects of Pur, the difficulty in completely separating each kind of neurocyte and subsequently detecting individual Pur concentration in vivo means that the target of Pur in the brain is still unclear. Our team has reported Pur concentration in the cerebrospinal fluid of Sprague-Dawley (SD) rats (Wang et al., 2012), but until now, there has been no report on the different distributions and retention time lengths of this drug in cortical neurons and astrocytes. In the present study, we established a system to co-culture cortical neurons and astrocytes. The different distributions and retention time lengths of Pur in these two kinds of neurocytes were analyzed with the purpose of providing further insight into the mechanism underlying the neuroprotection of Pur.
| Materials and Methods|| |
SD rats aged 24-72 hours were used to culture neurons and astrocytes. Rats were obtained from the Experimental Animal Center, Chongqing Research Institute of Traditional Chinese Medicine (China; production license No. SCXK (Yu) 02007-0006). Temporary rearing was performed at the Experimental Animal Center, College of Pharmaceutical Sciences & College of Chinese Medicine, Southwest University (Chongqing, China; experimental animal use license No. SYXK (Yu) 2013-0002).
Isolation and purification of neurons and astrocytes
The cerebral cortex was isolated in ice-cold D-Hank's solution and separated into 1 mm 3 pieces and digested in Trypsin solution (Amresco, Solon, OH, USA) (37°C, 1.25 mg/mL) for 8 minutes, then medium with 10% fetal bovine serum (FBS; Hyclone, Thermo Scientific, Waltham, MA, USA) was used to suspend the digesting process, and the solution was filtered with 10-mm pore size nylon mesh and centrifuged at 150 × g for 4 minutes. The precipitates were re-suspended in DMEM/F12 medium (Invitrogen, Carlsbad, CA, USA) containing 2% B27 (Invitrogen) for neurons and in DMEM/F12 medium containing 10% FBS for astrocytes to a concentration of 1 × 10 5 cells/mL. Cells were then seeded in plates pre-coated with 0.1 mg/mL poly-L-lysine (molecular weight 70,000-150,000; Hyclone, Thermo Scientific) and cultured at 37°C and 5% CO 2 for 4 hours. Finally, the cells were transferred into new plates and cultured for 24 hours, and the medium was changed every 2 days for 7-10 days. To purify astrocytes, the plate was shaken twice at 220 r/min (37°C, 18 hours).
Immunocytochemical identification of target cells
Briefly, purified cells were fixed with 4% paraformaldehyde at 25°C for 30 minutes, washed with PBS and then incubated in 10% goat serum containing 0.5% Triton X-100 (pH 7.4) for 1 hour. Cells were then probed with different rabbit anti-rat primary antibodies (1:200) overnight at 4°C; microtubule-associated protein-2 (MAP2; Santa Cruz Biotechnology, Santa Cruz, CA, USA) for neurons, and glial fibrillary acidic protein (GFAP; ZSGB-BIO, Beijing, China) for astrocytes. Cells were detected with goat anti-rabbit FITC-IgG (1:800) at 37°C for 1 hour, and the cell nuclei were stained with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI; Santa Cruz Biotechnology). Images were captured by fluorescence microscope (Carl Zeiss, Oberkochen, Germany) and the purity of the cells was analyzed by Image-Pro Plus 6.0 (Media Cybernetics, MD, USA).
Co-culture of neurons and astrocytes
The method used to co-culture the two kinds of cells was carried out as previously described but with some modifications (Xue et al., 2013). As shown in [Figure 1], astrocytes (1.5 × 10 5 cells/cm 2 ) were seeded on the outside of a Transwell membrane (Corning, Corning, NY, USA) coated with poly-L-lysine. After culturing cells for 6 hours and subsequently washing with D-Hank's solution, the Transwell was inserted into a plate well containing neurons that had already had been seeded at a density of 1 × 10 5 cells/cm 2 and subsequently grown for 10 days. DMEM/F12 (1:1) complete medium with 10% FBS and 2% B27 was used to culture cells for 3 days, and the medium was given a daily change before analysis.
Preparation of cell extracts
Pur was added into the medium of co-cultured or cultured neurons and astrocytes at a final concentration of 1,000 ng/mL. At 0, 10, 20, 30, 60, and 90 minutes after the drug was added, the cells were washed with 0.01 M PBS (twice for 30 seconds) and digested with trypsin (1.25 mg/mL) separately and re-suspended in PBS at a concentration of 5 × 10 5 cells/mL into a final volume of 2 mL. Cells were completely broken using ultrasonication, and after centrifugation at 1.5 × 10 4 × g for 20 minutes, the supernatant was collected as cell extracts and detected by a fluorospectrophotometer (Hitachi, Tokyo, Japan).
Neuron and astrocyte cell extracts were obtained at a rate of 1:1. 50 ng/mL Pur was prepared with the cell extract to detect the most intensive excitation and emission wavelengths, and both the excitation and the emission slits were 10 nm (Chauhan et al., 2011). Samples of 6.25, 12.5, 25, 50, and 100 ng/mL of Pur prepared with the cell extract were used to fit the standard curve of fluorescence spectrophotometry, and samples of 6.25, 25, and 100 ng/mL Pur were prepared to detect the precision and the percentage recovery; cell extracts without Pur was used to detect the specificity of the method.
Fluorescence spectrophotometry and fluorescence
At 0, 10, 20, 30, 60, and 90 minutes after 1,000 ng/mL Pur was added into the medium, the concentration of Pur in cells was detected through the use of the spectrofluorometer and determined from the standard curve. For fluorescence microscope detection, 20 and 60 minutes after the addition of Pur, neurons and astrocytes were separately washed in DMEM/F12 medium twice and then immediately detected under blue emission of a fluorescence microscope (Carl Zeiss, Oberkochen, Germany).
The mean, standard deviation, and relative standard deviation of the concentration of Pur in cells were analyzed by SPSS 19.0 (IBM, Armonk, NY, USA). A value of P < 0.05 was considered significantly different.
| Results|| |
Identification of neurons and astrocytes
After detection by anti-MAP2 and anti-GFAP, the purities of neurons and astrocytes were 96.50 ± 0.87% and 97.67 ± 0.10%, respectively (n = 6).
Establishment and validation of the analytical method
As shown in [Figure 2], the λex/em of Pur was 342/461 nm, the slit of ex/em was 10 nm, the standard curve exhibited a good linear relationship between absorbance and Pur concentration in the tested concentrations (y = 4.5107x-0.375, R 2 = 0.9978), and the limit of detection was 5.45 ng/mL. The specificity, precision, and percentage recovery of the method were shown in [Figure 2] and [Table 1].
|Figure 2 Absorption spectrum and standard curves of puerarin (Pur). (A) The excitation spectrum of Pur; (B) the emission spectrum excited at 342 nm; (C) the absorption of cell extraction in detected condition; (D) the standard curve of Pur (R2 = 0.9978).|
Click here to view
Fluorescence spectrophotometry detection for the
concentration of Pur in cells
Different distributions and retention time of Pur in tested cells are shown in [Figure 3]. Briefly, peak concentration of Pur in separately cultured or co-cultured neurons was higher than that observed in astrocytes, in particular the concentration of Pur in both co-cultured and separately cultured neurons were kept at a steady level of approximately 12.80 ng/mL, but a scant amount of Pur was detected in astrocytes when co-cultured with Pur for 60-90 minutes.
|Figure 3 Concentration of puerarin (Pur) in tested cells after co-cultured with Pur for 0– 90 minutes.|
S-neurons and S-astrocytes: Separately cultured neurons and astrocytes; C-neurons and C-astrocytes: co-cultured neurons and astrocytes.
Click here to view
Fluorescence microscope detection for the distribution of Pur in cells
As shown in [Figure 4], when cells were co-cultured with Pur for 20 minutes, blue fluorescence was detected in both neurons ([Figure 4]A-2) and astrocytes ([Figure 4]B-2), and a scant amount of blue fluorescence was detected in the cell medium ([Figure 4]A-2, B-2). When co-cultured with Pur for 60 minutes, blue fluorescence was detected in the medium ([Figure 4]C-2, D-2), weak fluorescence was detected in the neurons ([Figure 4]C-2), but a scant amount of fluorescence was detected in astrocytes ([Figure 4]D-2).
|Figure 4 Distribution of puerarin (Pur) in neurons and astrocytes detected by fluorescence microscope.|
At 20 minutes after Pur was added, the cell morphologies of neurons (A-1) and astrocytes (B-1) were detected under white light. Blue fluorescence was detected in the neurons (A-2) and astrocytes (B-2), and weak blue fluorescence was detected in the medium (A-2, B-2) under ultraviolet light source. At 60 minutes after Pur was added, the cell morphologies of neurons (C-1) and astrocytes (D-1) were detected under common sources, weak blue fluorescence was detected in the neurons (C-2), and no fluorescence was detected in astrocytes (D-2), but blue fluorescence was detected in the medium (C-2, D-2) under ultraviolet light source. Scale bar: 50 ƒÊm.
Click here to view
| Discussion|| |
The injection of Pur has been widely used in clinical therapy of ischemic cerebrovascular disease, coronary heart disease, angina pectoris, myocardial infarction, and diabetes in China, but its beneficial outcomes on human health are still unknown (Wang et al., 2006; Tan et al., 2008; Wei et al., 2014). Correctly assessing cytoprotection of Pur remains a major challenge and target cells in different organs should be confirmed. The plasma pharmacokinetics of Pur in rats were widely researched (Okamura et al., 1999; Li et al., 2013), but were limited by the difficulties in detecting the concentration of drugs in each kind of neurocyte in brain tissue; to the best of our knowledge, there have been no studies addressing the distribution of Pur in different kinds of neurocytes until now. In this study, a system for co-culturing neurons and astrocytes was established and first used to analyze the distribution and retention time of Pur in neurons and astrocytes. This offered a new method for detecting the distribution of drugs in different neurocytes. Results showed that there was a broader distribution and longer retention time of Pur in neurons than in astrocytes, which might provide some information towards understanding the neuroprotective mechanism of this significant natural medicine.
Several methods have been used for the detection of Pur in different sample matrices, such as high-performance liquid chromatography (Okamura et al., 1999), ultra-performance liquid chromatography tandem mass-spectrometry (Li et al., 2013), capillary zone electrophoresis (Liu et al., 2012), and flow-injection chemiluminescence (Yang et al., 2011), but all these methods are time-consuming and costly. Based on the isoflavone structure, Pur showed intense blue fluorescence observed under UV light, so the content of Pur in Pueraria tuberosa could be detected by a spectrofluorometric method (Chauhan et al., 2011). However, there is no report on this method being used to detect Pur in cells, and so a rapid and simple fluorometric assay method for the measurement of Pur concentration in cells (which had been previously confirmed to have acceptable linearity, precision, and recovery), was established in this study and used to detect the distribution of Pur in co-culture and separately cultured neurons and astrocytes. [Table 2] shows Pur distribution in different kinds of samples detected by different methods. Results from this study showed that when co-cultured with Pur for 60-90 minutes, there was a scant amount of Pur in the tested astrocytes, but the concentration of Pur in both separately cultured and co-cultured neurons was maintained at a relatively stable level, and the underlying mechanism should be further investigated.
|Table 2 Comparison of the proposed fluorescence spectrophotometry method with other methods for determination of puerarin|
Click here to view
| References|| |
Chang Y, Hsieh CY, Peng ZA, Yen TL, Hsiao G, Chou DS, Chen CM, Sheu JR (2009) Neuroprotective mechanisms of puerarin in middle cerebral artery occlusion-induced brain infarction in rats. J Biomed Sci 16:9-21.
Chauhan NS, Gupta NK, Sharma V, Dixit VK (2011) Spectrofluorimetric estimation of puerarin in Pueraria tuberosa. Acta Pol Pharm 68:453-456.
Li J, Wang G, Liu J, Zhou L, Dong M, Wang R, Li X, Li X, Lin C, Niu Y (2010) Puerarin attenuates amyloid-beta-induced cognitive impairment through suppression of apoptosis in rat hippocampus in vivo. Eur J Pharmacol 649:195-201.
Li N, Deng Y, Wang D, Qiao Y, Li F (2013) Determination of glibenclamide and puerarin in rat plasma by UPLC-MS/MS: application to their pharmacokinetic interaction study. Talanta 104:109-115.
Li X, Sun S, Tong E (2003) Experimental study on the protective effect of puerarin to Parkinson disease. J Huazhong Univ Sci Technol Med Sci 23:148-150.
Lin CM, Lin RD, Chen ST, Lin YP, Chiu WT, Lin JW, Hsu FL, Lee MH (2010) Neurocytoprotective effects of the bioactive constituents of Puerariathomsonii in 6-hydroxydopamine (6-OHDA)-treated nerve growth factor (NGF)-differentiated PC12 cells. Phytochemistry 71:2147-2156.
Liu L, Feng F, Shuang S, Bai Y, Choi MM (2012) Determination of puerarin in pharmaceutical and biological samples by capillary zone electrophoresis with UV detection. Talanta 91:83-87.
Okamura N, Miki H, Orii H, Masaoka Y, Yamashita S, Kobayashi H, Yagi A (1999) Simultaneous high-performance liquid chromatographic determination of puerarin, daidzin, paeoniflorin, liquiritin, cinnamic acid, cinnamaldehyde and glycyrrhizin in Kampo medicines. J Pharm Biomed Anal 19:603-612.
Tan Y, Liu M, Wu B (2008) Puerarin for acute ischaemic stroke. Cochrane Database Syst Rev 23:CD004955.
Wang Q, Wu T, Chen X, Ni J, Duan X, Zheng J, Qiao J, Zhou L, Wei J (2006) Puerarin injection for unstable angina pectoris. Cochrane Database Syst Rev 3:CD004196.
Wang Q, Xing M, Chen W, Zhang J, Qi H, Xu X (2012) HPLC-APCI-MS/MS method for the determination of catalpol in rat plasma and cerebrospinal fluid: application to an in vivo pharmacokinetic study. J Pharm Biomed Anal 70:337-343.
Wei SY, Chen Y, Xu XY (2014) Progress on the pharmacological research of puerarin: a review. Chin J Nat Med 12:407-414.
Wu HQ, Guo HN, Wang HQ, Chang MZ, Zhang GL, Zhao YX (2009) Protective effects and mechanism of puerarin on learning-memory disorder after global cerebral ischemia-reperfusion injury in rats. Chin J Integr Med 15:54-59.
Xing G, Dong M, Li X, Zou Y, Fan L, Wang X, Cai D, Li C, Zhou L, Liu J, Niu Y (2011) Neuroprotective effects of puerarin against beta-amyloid-induced neurotoxicity in PC12 cells via a PI3K-dependent signaling pathway. Brain Res Bull 85:212-218.
Xu X, Hu Y, Ruan Q (2004) Effects of puerarin on learning - memory and amino acid transmitters of brain in ovariectomized mice. Planta Med 70:627-631.
Xu XH, Zhao TQ (2002) Effects of puerarin on D-galactose-induced memory deficits in mice. Acta Pharmacol Sin 23:587-590.
Xu XH, Zheng XX, Zhou Q, Li H (2007) Inhibition of excitatory amino acid efflux contributes to protective effects of puerarin against cerebral ischemia in rats. Biomed Environ Sci 20:336-342.
Xue Q, Liu Y, Qi H, Ma Q, Xu L, Chen W, Chen G, Xu X (2013) A novel brain neurovascular unit model with neurons, astrocytes and microvascular endothelial cells of rat. Int J Biol Sci 9:174-189.
Yang R, Wang Q, Zeng H, Qin Z, Li J, Qu L (2011) Determination of puerarin in biological samples and its application to a pharmacokinetic study by flow-injection chemiluminescence. Luminescence 26:368-373.
Zhang Q, Huang WD, Lv XY, Yang YM (2012) Puerarin protects differentiated PC12 cells from H2O2-induced apoptosis through the PI3K/Akt signaling pathway. Cell Biol Int 36:419-426.
Zheng GM, Yu C, Yang Z (2012) Puerarin suppresses production of nitric oxide and inducible nitric oxide synthase in lipopolysaccharide-induced N9 microglial cells through regulating MAPK phosphorylation, O-GlcNacylation and NF-êB translocation. Int J Oncol 40:1610-1618.
Zhu G, Wang X, Chen Y, Yang S, Cheng H, Wang N, Li Q (2010) Puerarin protects dopaminergic neurons against 6-hydroxydopamine neurotoxicity via inhibiting apoptosis and upregulating glial cell line-derived neurotrophic factor in a rat model of Parkinson's disease. Planta Med 6:1820-1826.
Zhu G, Wang X, Wu S, Li Q (2012) Involvement of activation of PI3K/Akt pathway in the protective effects of puerarin against death. Neurochem Int 60:400-408.
Zou Y, Hong B, Fan L, Zhou L, Liu Y, Wu Q, Zhang X, Dong M (2013) Protective effect of puerarin against beta-amyloid-induced oxidative stress in neuronal cultures from rat hippocampus: involvement of the GSK-3â/Nrf2 signaling pathway. Free Radic Res 47:55-63.
[Figure 1], [Figure 3], [Figure 2], [Figure 4]
[Table 1], [Table 2]
|This article has been cited by|
||Brain Pharmacokinetics and the Pharmacological Effects on Striatal Neurotransmitter Levels of Pueraria lobata Isoflavonoids in Rat
| ||Bingxin Xiao,Zengxian Sun,Fangrui Cao,Lisha Wang,Yonghong Liao,Xinmin Liu,Ruile Pan,Qi Chang |
| ||Frontiers in Pharmacology. 2017; 8 |
|[Pubmed] | [DOI]|
||Pathways Involved in Oxygen Glucose Deprivation Damage of Astrocytes
| ||Shuyong Wei,Jie Tong,Qiang Xue,Yang Liu,Xiaoyu Xu |
| ||Journal of Molecular Neuroscience. 2016; |
|[Pubmed] | [DOI]|