丹皮酚纳米乳在肠细胞模型中吸收机制研究

doi:10.5246/jcps.2019.03.017

摘要/Abstract

摘要：

纳米乳剂的使用改善了丹皮酚的生物利用度, 它主要与我们先前研究中的P-糖蛋白(P-gp)介导的外排有关。但其他涉及丹皮酚纳米乳肠道吸收的机制尚不清楚, 因此本研究的目的是研究丹皮酚纳米乳的吸收机制。通过建立Caco-2细胞模型和卵泡相关上皮(FAE)模型, 在不同的外排抑制剂存在下进行摄取研究和丹皮酚纳米乳的双向转运以观察摄入和转运。通过荧光染色Occludin研究细胞旁通路, Western blotting检测外排蛋白和紧密连接蛋白的表达。在这项研究中, 我们发现纳米乳通过增加Caco-2细胞和FAE的摄取并减少外排蛋白的表达来改善丹皮酚的吸收。此外, 丹皮酚纳米乳对紧密连接处的开放没有影响。结果表明, 丹皮酚纳米乳的吸收是Caco-2细胞模型的被动扩散和FAE模型的内吞作用, 这与多药耐药相关蛋白2(MRP2)和乳腺癌耐药蛋白(BCRP)流出有关。丹皮酚纳米乳显著增加药物在肠细胞中的吸收, 可能与两种肠细胞摄取的增加, 外排蛋白表达的下降和药物外排的减少有关。

关键词: 丹皮酚纳米乳, 吸收机制, 摄取, 转运

Abstract:

The use of nanoemulsion has improved the bioavailability of paeonol, and that is mainly because of the P-glycoprotein(P-gp)-mediated efflux described in our previous study. However, other mechanisms involved in the intestinal absorption of paeonol nanoemulsion are still unknown. Therefore, we aimed to investigate additional paeonol nanoemulsion absorption mechanisms. By establishing the Caco-2 cells model and the follicle-associated epithelium (FAE) model, uptake studies and bidirectional transport of paeonol nanoemulsion in the presence of different efflux inhibitors were conducted to observe its intake and transport. The paracellular pathway was evaluated by fluorescent staining of Occludin, and the expressions of efflux proteins and tight junction proteins were detected by Western blotting analysis. In this study, we found that nanoemulsion improved the absorption of paeonol by increasing its uptake across the Caco-2 and FAE cells and by reducing the expressions of efflux proteins. In addition, paeonol nanoemulsion had no effect on the opening of tight junctions. The results showed that the absorption of paeonol nanoemulsion occurred by passive diffusion in the Caco-2 cell model and by endocytosis in the FAE model, which was related to multidrug resistance-associated protein 2 (MRP2) and breast cancer resistance protein (BCRP) efflux. Paeonol nanoemulsion significantly increased the absorption of the drug in intestinal cells, which was possibly related to the increase in intestinal cell uptake, inhibited expressions of efflux protein and reduction of drug efflux.

Key words: Paeonol nanoemulsion, Absorption mechanism, Uptake, Transport

中图分类号:

R962

Supporting:

欧阳怡, 张敬, 吴鸿飞, 戴敏. 丹皮酚纳米乳在肠细胞模型中吸收机制研究[J]. 中国药学(英文版), 2019, 28(3): 174-185.

Yi Ouyang, Jing Zhang, Hongfei Wu, Min Dai. Absorption mechanism of paeonol nanoemulsion using in vitro intestinal cell models[J]. Journal of Chinese Pharmaceutical Sciences, 2019, 28(3): 174-185.

参考文献

[1] Li, H.K.; Dai, M.; Jia, W. Paeonol attenuates high-fat-diet-induced atherosclerosis in rabbits by anti-inflammatory activity. Planta. Med. 2009, 75, 7-11.

[2] Song, A.W.; Wu, H.F.; Dai, M. Paeonol attenuates progression of atherosclerotic lesion formation through lipid regulation, anti-inflammatory and antioxidant activities. J. Chin. Pharm. Sci. 2018, 27, 565-575.

[3] Lee, B.; Shin, Y.W.; Bae, E.A.; Han, S.J.; Kim, J.S.; Kang, S.S.; Kim, D.H. Antiallergic effect of the root of Paeonia lactiflora and its constituents paeoniflorin and paeonol. Arch. Pharm. Res. 2008, 31, 445-450.

[4] Himaya, S.W.; Ryu, B.; Qian, Z.J.; Kim, S.K. Paeonol from Hippocampus kuda Bleeler suppressed the neuro-inflammatory responses in vitro via NF-κB and MAPK signaling pathways. Toxicol. In. Vitro. 2012, 26, 878-887.

[5] Zong, S.Y.; Pu, Y.Q.; Xu, B.L.; Zhang, T.; Wang, B. Study on the physicochemical properties and anti-inflammatory effects of paeonol in rats with TNBS-induced ulcerative colitis. Int. Immunopharmacol. 2017, 42, 32-38.

[6] Lau, C.H.; Chan, C.M.; Chan, Y.W.; Lau, K.M.; Lau, T.W.; Lam, F.C.; Law, W.T.; Che, C.T.; Leung, P.C.; Fung, K.P.; Ho, Y.Y.; Lau, C.B. Pharmacological investigations of the anti-diabetic effect of Cortex Moutan and its active component paeonol. Phytomedicine. 2007, 14, 778-784.

[7] Yao, J.J.; Zhang, Y.X.; Hu, Q.M.; Zeng, D.C.; Hua, F.; Meng, W.; Wang, W.Y.; Bao, G.H. Optimization of paeonol-loaded poly (butyl-2-cyanoacrylate) nanocapsules by central composite design with response surface methodology together with the antibacterial properties. Eur. J. Pharm. Sci. 2017, 101, 189-199.

[8] Chen, Z.X.; Li, B.; Liu, T.; Wang, X.; Zhu, Y.; Wang, L.; Wang, X.H.; Niu, X.; Xiao, Y.; Sun, Q. Evaluation of paeonol-loaded transethosomes as transdermal delivery carriers. Eur. J. Pharm. Sci. 2017, 99, 240-245.

[9] Chen, C.; Jia, F.; Hou, Z.B.; Ruan, S.; Lu, Q.B. Delivery of paeonol by nanoparticles enhances its in vitro and in vivo antitumor effects. Int. J. Nanomedicine. 2017, 12, 6605-6616.

[10] Jiao, Y.; Zheng, X.Q.; Chang, Y.; Li, D.J.; Sun, X.H.; Liu, X.L. Zein-derived peptides as nanocarriers to increase the water solubility and stability of lutein. Food Funct. 2018, 9, 117-123.

[11] Freag, M.S.; Saleh, W.M.; Abdallah, O.Y. Self-assembled phospholipid-based phytosomal nanocarriers as promising platforms for improving oral bioavailability of the anticancer celastrol. Int. J. Pharm. 2018, 535, 18-26.

[12] Fares, A.R.; Elmeshad, A.N.; Kassem, M.A.A. Enhancement of dissolution and oral bioavailability of lacidipine via pluronic P123/ F127 mixed polymeric micelles: formulation, optimization using central composite design and in vivo bioavailability study. Drug Deliv. 2018, 25, 132-142.

[13] Zhang, Q.H.; Polyakov, N.E.; Chistyachenko, Y.S.; Khvostov, M.V.; Frolova, T.S.; Tolstikova, T.G.; Dushkin, A.V.; Su, W.K. Preparation of curcumin self-micelle solid dispersion with enhanced bioavailability and cytotoxic activity by mechanochemistry. Drug. Deliv. 2018, 25, 198-209.

[14] Lu, Z.; Bu, C.P.; Hu, W.C.; Zhang, H.; Liu, M.R.; Lu, M.Q.; Zhai, G.X. Preparation and in vitro and in vivo evaluation of quercetin-loaded mixed micelles for oral delivery. Biosci. Biotechnol. Biochem. 2018, 82, 238-246.

[15] Hao, YL.; Zhong, T.; Du, R.; Zhang, H.; Liu, BL.; Zhang, X. The cellular uptake and anti-tumor activity of conjugated linoleic acid-paclitaxel loaded iRGD-modified lysolipid-containing thermosensitive liposomes. J. Chin. Pharm. Sci. 2019, 28, 121-133.

[16] Bu, Y.Z.; Mu, L.M.; Liu, L.; Lu, W.L. Construction of folate-conjugated epirubicin liposomes for enhancing the cellular uptake and the co-localization with nuclei of invasive breast cancer cells. J. Chin. Pharm. Sci. 2018, 27, 229-240

[17] Wu, L.; Bi, Y.J.; Wu, H.F. Formulation optimization and the absorption mechanisms of nanoemulsion in improving baicalin oral exposure. Drug. Dev. Ind. Pharm. 2018, 44, 266-275.

[18] Gundogdu, E.; Karasulu, H.Y.; Koksal, C.; Karasulu, E. The novel oral imatinib microemulsions: physical properties, cytotoxicity activities and improved Caco-2 cell permeability. J. Microencapsul. 2013, 30, 132-142.

[19] Li, Y.J.; Hu, X.B.; Lu, X.L.; Liao, D.H.; Tang, T.T.; Wu, J.Y.; Xiang, D.X. Nanoemulsion-based delivery system for enhanced oral bioavailability and Caco-2 cell monolayers permeability of berberine hydrochloride. Drug Deliv. 2017, 24, 1868-1873.

[20] Hong, L.; Zhou, C.L.; Chen, F.P.; Han, D.; Wang, C.Y.; Li, J.X.; Chi, Z.; Liu, C.G. Development of a carboxymethyl chitosan functionalized nanoemulsion formulation for increasing aqueous solubility, stability and skin permeability of astaxanthin using low-energy method. J. Microencapsul. 2017, 34, 707-721.

[21] Piazzini, V.; Monteforte, E.; Luceri, C.; Bigagli, E.; Bilia, A.R.; Bergonzi, M.C. Nanoemulsion for improving solubility and permeability of Vitex agnus-castus extract: formulation and in vitro evaluation using PAMPA and Caco-2 approaches. Drug Deliv. 2017, 24, 380-390.

[22] Chen, S.F; Zhang, J.; Wu, L.; Wu, H.F; Dai, M. Paeonol nanoemulsion for enhanced oral bioavailability: Optimization and mechanism. Nanomedicine(Lond). 2018, 13, 269-282.

[23] Hussain, N. Regulatory aspects in the pharmaceutical development of nanoparticle drug delivery systems designed to cross the intestinal epithelium and M-cells. Int. J. Pharm. 2016, 514, 15-23.

[24] Kyd, J.M.; Cripps, A.W. Functional differences between M cells and enterocytes in sampling luminal antigens. Vaccine. 2008, 26, 6221-6224.

[25] Brück, S.; Strohmeier, J.; Busch, D.; Drozdzik, M.; Oswald, S. Caco-2 cells-expression, regulation and function of drug transporters compared with human jejunal tissue. Biopharm. Drug. Dispos. 2017, 38, 115-126.

[26] Min, H.P.; Niu, M.M.; Zhang, W.L.; Yan, J.; Li, J.C.; Tan, X.Y.; Li, B.; Su, M.X.; Di, B.; Yan, F. Emodin reverses leukemia multidrug resistance by competitive inhibition and downregulation of P-glycoprotein. PLoS One. 2017, 12, e0187971.

[27] Lapierre, L.A. The molecular structure of the tight junction. Adv. Drug. Deliv. Rev. 2000, 41, 255-264.

[28] Ferraretto, A.; Bottani, M.; De Luca,.P.; Cornaghi, L.; Arnaboldi, F.; Maggioni, M.; Fiorilli, A.; Donetti, E. Morphofunctional properties of a differentiated Caco2/HT-29 co-culture as an in vitro model of human intestinal epithelium. Biosci. Rep. 2018, 38, BSR20171497.

[29] Noda, S.; Yamada, A.; Nakaoka, K.; Goseki-Sone, M. 1-alpha,25-Dihydroxyvitamin D3 up-regulates the expression of 2 types of human intestinal alkaline phosphatase alternative splicing variants in Caco-2 cells and may be an important regulator of their expression in gut homeostasis. Nutr. Res. 2017, 46, 59-67.

[30] Gullberg, E.; Leonard, M.; Karlsson, J.;Hopkins, A.M.; Bravden, D.; Baird, A.W.; Artursson, P. Expression of specific markers and particle transport in a new human intestinal M-cell model. Biochem. Biophys. Res. Commun. 2000, 279, 808-813.

[31] Brocks, D.R.; Davies, N.M. Lymphatic Drug Absorption via the Enterocytes: Pharmacokinetic Simulation, Modeling, and Considerations for Optimal Drug Development. J. Pharm. Pharm. Sci. 2018, 21, 254s-270s.

[32] des Rieux, A.; Fievez, V.; Théate, I.; Mast, J.; Préat, V.; Schneider, Y.J. An improved in vitro model of human intestinal follicle-associated epithelium to study nanoparticle transport by M cells. Eur. J. Pharm. Sci. 2007, 30, 380-391.

[33] Artursson, P.; Karlsson, J. Correlation between oral drug absorption in humans and apparent drug permeability coefficients in human intestinal epithelial (Caco-2) cells. Biochem. Biophys. Res. Commun. 1991, 175, 880-885.

[34] Liu, Y.X.; Wang, Y.F.; Peng, Y.M.; Liu, B.J.; Ma, F.; Jiang, J.H.; Wang, Q.D.; Chang, J.B. Effects of the antiretroviral drug 2'-deoxy-2'-β-fluoro-4'-azidocytidine (FNC) on P-gp, MRP2 and BCRP expressions and functions. Pharmazie. 2018, 73, 503-507.

[35] Roger, E.; Lagarce, F.; Garcion, E.; Benoit, J.P. Lipid nanocarriers improve paclitaxel transport throughout human intestinal epithelial cells by using vesicle-mediated transcytosis. J. Control. Release. 2009, 140, 174-181.