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Vol. 25, No. 2, 2012
Issue release date: February 2012
Section title: Original Paper
Skin Pharmacol Physiol 2012;25:57–64
(DOI:10.1159/000330897)

Water Extract of Gromwell (Lithospermum erythrorhizon) Enhances Migration of Human Keratinocytes and Dermal Fibroblasts with Increased Lipid Synthesis in an in vitro Wound Scratch Model

Kim H.a · Kim J.a · Park J.b · Kim S.H.c · Uchida Y.d · Holleran W.M.d · Cho Y.a
Departments of aMedical Nutrition and bEast-West Medical Science, Graduate School of East-West Medical Science, Kyung Hee University, Yongin-si, and cNutrex Co. Ltd., Seoul, Korea; dDermatology Service and Research Unit, Department of Veterans Affairs Medical Center, University of California, San Francisco, Calif., USA
email Corresponding Author

Abstract

Background: Although organic extracts of gromwell (Lithospermum erythrorhizon) have been shown to promote wound healing, the wound healing effects of water extracts of gromwell (WG) that are commonly used in traditional remedies have not been elucidated. Objective: We investigated whether WG promotes the migration and/or proliferation of cultured human keratinocytes (CHK) or dermal fibroblasts in parallel with increases in lipid synthesis during in vitro wound healing. Methods: CHK or fibroblasts were treated with 1–1,000 µg/ml WG for up to 48 h following scratch wound formation. Cell migration was assessed by measuring coverage (in percent) from the wound margin, while cell proliferation and lipid synthesis were determined by [3H]thymidine incorporation into DNA fractions, and [3H]palmitate or [3H]serine incorporation into lipid fractions, respectively. Results: Low-dose WG (1 µg/ml) enhanced the wound coverage for both CHK and fibroblasts at 24 h, while cell proliferation was not altered in either cell types. Synthesis of both total lipids and individual lipid classes, including phospholipids, sphingolipids and neutral lipids, were found to be increased at 24 h in CHK treated with 1 µg/ml WG; in similarly treated fibroblasts, only the syntheses of sphingolipids (such as ceramides and glucosylceramides), but not other lipid species, were significantly increased. In contrast, a higher dose of WG (10–1,000 µg/ml) did not enhance wound coverage, and 100 µg/ml WG neither altered cell proliferation nor lipid synthesis in both CHK and fibroblasts. Conclusion: Low-dose WG (1 µg/ml) enhances the migration of both CHK and fibroblasts with increased lipid synthesis in an in vitro wound scratch model.

© 2011 S. Karger AG, Basel


  

Key Words

  • Gromwell
  • Migration
  • Lipid
  • Wound healing
  • Keratinocytes
  • Fibroblasts

References

  1. Papageorgiou VP, Assimopoulou AN, Ballis AC: Alkannins and shikonins: a new class of wound healing agents. Curr Med Chem 2008;15:3248–3267.
  2. Ozaki Y, Ohno A, Abe KI, Saito Y, Satake M: Comparative study on the accelerative effect of ‘koushikon’ and ‘nanshikon’ and their constituents on proliferation of granuloma tissue in rats. Biol Pharm Bull 1996;16:683–685.
  3. Ozaki Y, Ohno A, Abe KI, Saito Y, Satake M: Accelerative effect of shikonin, alkannin and acetylshikonin on the proliferation of granulation tissue in rats. Biol Pharm Bull 1994;17:1075–1077.
  4. Ozaki Y, Xing L, Satake M: Accelerative effect of ‘nanshikon’ and its constituents on the proliferation of granulation tissue in rats. Biol Pharm Bull 1996;19:233–236.
  5. Sakaguchi I, Tsujimura M, Ikeda N, Minamino M, Kato Y, Watabe K, Yano I, Kaneda K: Granulomatous tissue formation of shikon and shikonin by air pouch method. Biol Pharm Bull 2001;24:650–655.
  6. Fujita N, Sakaguchi I, Kobayashi H, Ikeda N, Kato Y, Minamino M, Ishii M: An extract of the root of Lithospermum erythrorhizon accelerated wound healing in diabetic mice. Biol Pharm Bull 2003;26:329–335.
  7. Hayashi M: Pharmacological studies of Shikon and Tooki. 3. Effect of topical application of the ether extracts and Shiunko on inflammatory reactions (in Japanese). Nihon Yakurigaku Zasshi 1977;73:205–214.
  8. Hayashi M: Pharmacological studies on crude plant drugs, Shikon and Tooki. 1. Pharmacological effects of the water and ether extracts (in Japanese). Nihon Yakurigaku Zasshi 1977;73:177–191.
  9. Werner S, Grose R: Regulation of wound healing by growth factors and cytokines. Physiol Rev 2003;83:835–870.
  10. Nanney LB, Caldwell RL, Pollins AC, Cardwell NL, Opalenik SR, Davison JM: Novel approaches for understanding the mechanisms of wound repair. J Investig Dermatol Symp Proc 2006;11:132–139.
  11. Kozel’tsev VL, Volodina TV, Pankrushina AN, Guseva VV, Kostyuk NV: Changes in wound field lipids in rat skin. Bull Exp Biol Med 2006;142:493–494.
  12. Vicanova J, Ponec M, Weerheim A, Swope V, Westbrook M, Harriger D, Boyce S: Epidermal lipid metabolism of cultured skin substitutes during healing of full-thickness wounds in athymic mice. Wound Repair Regen 1997;5:329–338.
  13. Pittelkow MR, Scott RE: New techniques for the in vitro culture of human skin keratinocytes and perspectives on their use for grafting of patients with extensive burns. Mayo Clin Proc 1986;61:771–777.
  14. Holleran WM, Williams ML, Gao WN, Elias PM: Serine-palmitoyl transferase activity in cultured human keratinocyte. J Lipid Res 1990;31:1655–1661.
  15. Hennings H, Michael D, Cheng C, Steinert P, Holbrook K, Yuspa SH: Calcium regulation of growth and differentiation of mouse epidermal cells in culture. Cell 1980;19:245–254.
  16. Williams ML, Rutherford SL, Ponec M, Hincenbergs M, Placzek DR, Elias PM: Density-dependent variations in the lipid content and metabolism of cultured human keratinocytes. J Invest Dermatol 1988;91:86–91.
  17. Lee B, Vouthounis C, Stojadinovic O, Brem H, Im M, Tomic-Canic M: From an enhanceosome to a repressosome: molecular antagonism between glucocorticoids and EGF leads to inhibition of wound healing. J Mol Biol 2005;345:1083–1097.
  18. Holleran WM, Gao WN, Feingold KR, Elias PM: Localization of epidermal sphingolipid synthesis and serine palmitoyl transferase activity: alterations imposed by permeability barrier requirements. Arch Dermatol Res 1995;287:254–258.
  19. Farrell AM, Uchida Y, Nagiec MM, Harris IR, Dickson RC, Elias PM, Holleran WM: UVB irradiation up-regulates serine palmitoyltransferase in cultured human keratinocytes. J Lipid Res 1998;39:2031–2038.
  20. Bretscher MS: Getting membrane flow and the cytoskeleton to cooperate in moving cells. Cell 1996;87:601–606.
  21. Hopkins CR, Gibson A, Shipman M, Strickland DK, Trowbridge IS: In migrating fibroblasts, recycling receptors are concentrated in narrow tubules in the pericentriolar area, and then routed to the plasma membrane of the leading lamella. J Cell Biol 1994;125:1265–1274.
  22. Sidhu GS, Singh AK, Banaudha KK, Gaddipati JP, Patnaik GK, Maheshwari RK: Arnebin-1 accelerates normal and hydrocortisone-induced impaired wound healing. J Invest Dermatol 1999;113:773–781.
  23. Pei XW, Wang KZ, Dang XQ, Song JH, Shi ZB, Gao DF: Arnebia root oil promotes wound healing and expression of basic fibroblasts growth factor on the wound surface in rabbits (in Chinese). Zhong Xi Yi Jie He Xue Bao 2006;4:52–55.
  24. Chiu SC, Yang NS: Inhibition of tumor necrosis factor-α through selective blockade of pre-mRNA splicing by shikonin. Mol Pharmacol 2007;71:1640–1645.
  25. Wang WJ, Bai JY, Liu DP, Xue LM, Zhu XY: The antiinflammatory activity of shikonin and its inhibitory effect on leukotriene B4 biosynthesis (in Chinese). Yao Xue Xue Bao 1994;29:161–165.
  26. Futagami A, Ishizaki M, Fukuda Y, Kawana S, Yamanaka N: Wound healing involves induction of cyclooxygenase-2 expression in rat skin. Lab Invest 2002;82:1503–1513.
  27. Konno C, Mizuno T, Hikino H: Isolation and hypoglycemic activity of lithospermans A, B and C, glycans of Lithospermum erythrorhizon roots. Planta Med 1985;2:157–158.
  28. Kim J, Kim Y, Seo D, Kim S, Lee S, Cho Y: Oral supplementation of Lithospermum erythrorhizon prevents the development of atopic dermatitis with reducing ceramide degradation in the epidermis of NC/Nga mice. Phytother Res 2009;23:1250–1256.
  29. Omoto T, Murakami Y, Shimomura K, Yoshihira K, Mori K, Nakashima T, Tanaka M, Ishimaru K: Caffeic acid derivatives in Lamiaceae and Boraginaceae plants. Jpn J Food Chem 1997;4:11–15.
  30. Deters AM, Schröder KR, Hensel A: Kiwi fruit (Actinidia chinensis L.) polysaccharides exert stimulating effects on cell proliferation via enhanced growth factor receptors, energy production, and collagen synthesis of human keratinocytes, fibroblasts and skin equivalents. J Cell Physiol 2005;202:717–722.
  31. Yao H, Chen Y, Li S, Huang L, Chen W, Lin X: Promotion proliferation effect of a polysaccharide from Aloe barbadensis Miller on human fibroblasts in vitro. Int J Biol Macromol 2009;45:152–162.
  32. Thuong PT, Kang KW, Kim JK, Seo DB, Lee SJ, Kim SH, Kim SH, Oh WK: Lithospermic acid derivatives from Lithospermum erythrorhizon increased expression of serine palmitoyltransferase in human HaCaT cells. Bioorg Med Chem Lett 2009;19:1815–1817.

  

Author Contacts

Yunhi Cho
Department of Medical Nutrition, Graduate School of East-West Medical Science
Kyung Hee University
Yongin-si, Gyeonggi-do 446-701 (Korea)
Tel. +82 31 201 3817, E-Mail choyunhi@khu.ac.kr

  

Article Information

H.K. and J.K. contributed equally to this study.

Received: November 29, 2010
Accepted after revision: June 14, 2011
Published online: October 4, 2011
Number of Print Pages : 8
Number of Figures : 2, Number of Tables : 3, Number of References : 32

  

Publication Details

Skin Pharmacology and Physiology (Journal of Pharmacological and Biophysical Research)

Vol. 25, No. 2, Year 2012 (Cover Date: February 2012)

Journal Editor: Lademann J. (Berlin)
ISSN: 1660-5527 (Print), eISSN: 1660-5535 (Online)

For additional information: http://www.karger.com/SPP


Copyright / Drug Dosage / Disclaimer

Copyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher or, in the case of photocopying, direct payment of a specified fee to the Copyright Clearance Center.
Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in goverment regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug.
Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.

Abstract

Background: Although organic extracts of gromwell (Lithospermum erythrorhizon) have been shown to promote wound healing, the wound healing effects of water extracts of gromwell (WG) that are commonly used in traditional remedies have not been elucidated. Objective: We investigated whether WG promotes the migration and/or proliferation of cultured human keratinocytes (CHK) or dermal fibroblasts in parallel with increases in lipid synthesis during in vitro wound healing. Methods: CHK or fibroblasts were treated with 1–1,000 µg/ml WG for up to 48 h following scratch wound formation. Cell migration was assessed by measuring coverage (in percent) from the wound margin, while cell proliferation and lipid synthesis were determined by [3H]thymidine incorporation into DNA fractions, and [3H]palmitate or [3H]serine incorporation into lipid fractions, respectively. Results: Low-dose WG (1 µg/ml) enhanced the wound coverage for both CHK and fibroblasts at 24 h, while cell proliferation was not altered in either cell types. Synthesis of both total lipids and individual lipid classes, including phospholipids, sphingolipids and neutral lipids, were found to be increased at 24 h in CHK treated with 1 µg/ml WG; in similarly treated fibroblasts, only the syntheses of sphingolipids (such as ceramides and glucosylceramides), but not other lipid species, were significantly increased. In contrast, a higher dose of WG (10–1,000 µg/ml) did not enhance wound coverage, and 100 µg/ml WG neither altered cell proliferation nor lipid synthesis in both CHK and fibroblasts. Conclusion: Low-dose WG (1 µg/ml) enhances the migration of both CHK and fibroblasts with increased lipid synthesis in an in vitro wound scratch model.

© 2011 S. Karger AG, Basel


  

Author Contacts

Yunhi Cho
Department of Medical Nutrition, Graduate School of East-West Medical Science
Kyung Hee University
Yongin-si, Gyeonggi-do 446-701 (Korea)
Tel. +82 31 201 3817, E-Mail choyunhi@khu.ac.kr

  

Article Information

H.K. and J.K. contributed equally to this study.

Received: November 29, 2010
Accepted after revision: June 14, 2011
Published online: October 4, 2011
Number of Print Pages : 8
Number of Figures : 2, Number of Tables : 3, Number of References : 32

  

Publication Details

Skin Pharmacology and Physiology (Journal of Pharmacological and Biophysical Research)

Vol. 25, No. 2, Year 2012 (Cover Date: February 2012)

Journal Editor: Lademann J. (Berlin)
ISSN: 1660-5527 (Print), eISSN: 1660-5535 (Online)

For additional information: http://www.karger.com/SPP


Article / Publication Details

First-Page Preview
Abstract of Original Paper

Received: 11/29/2010 3:32:34 PM
Accepted: 6/14/2011
Published online: 10/4/2011
Issue release date: February 2012

Number of Print Pages: 8
Number of Figures: 2
Number of Tables: 3

ISSN: 1660-5527 (Print)
eISSN: 1660-5535 (Online)

For additional information: http://www.karger.com/SPP


Copyright / Drug Dosage

Copyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher or, in the case of photocopying, direct payment of a specified fee to the Copyright Clearance Center.
Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in goverment regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug.
Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.

References

  1. Papageorgiou VP, Assimopoulou AN, Ballis AC: Alkannins and shikonins: a new class of wound healing agents. Curr Med Chem 2008;15:3248–3267.
  2. Ozaki Y, Ohno A, Abe KI, Saito Y, Satake M: Comparative study on the accelerative effect of ‘koushikon’ and ‘nanshikon’ and their constituents on proliferation of granuloma tissue in rats. Biol Pharm Bull 1996;16:683–685.
  3. Ozaki Y, Ohno A, Abe KI, Saito Y, Satake M: Accelerative effect of shikonin, alkannin and acetylshikonin on the proliferation of granulation tissue in rats. Biol Pharm Bull 1994;17:1075–1077.
  4. Ozaki Y, Xing L, Satake M: Accelerative effect of ‘nanshikon’ and its constituents on the proliferation of granulation tissue in rats. Biol Pharm Bull 1996;19:233–236.
  5. Sakaguchi I, Tsujimura M, Ikeda N, Minamino M, Kato Y, Watabe K, Yano I, Kaneda K: Granulomatous tissue formation of shikon and shikonin by air pouch method. Biol Pharm Bull 2001;24:650–655.
  6. Fujita N, Sakaguchi I, Kobayashi H, Ikeda N, Kato Y, Minamino M, Ishii M: An extract of the root of Lithospermum erythrorhizon accelerated wound healing in diabetic mice. Biol Pharm Bull 2003;26:329–335.
  7. Hayashi M: Pharmacological studies of Shikon and Tooki. 3. Effect of topical application of the ether extracts and Shiunko on inflammatory reactions (in Japanese). Nihon Yakurigaku Zasshi 1977;73:205–214.
  8. Hayashi M: Pharmacological studies on crude plant drugs, Shikon and Tooki. 1. Pharmacological effects of the water and ether extracts (in Japanese). Nihon Yakurigaku Zasshi 1977;73:177–191.
  9. Werner S, Grose R: Regulation of wound healing by growth factors and cytokines. Physiol Rev 2003;83:835–870.
  10. Nanney LB, Caldwell RL, Pollins AC, Cardwell NL, Opalenik SR, Davison JM: Novel approaches for understanding the mechanisms of wound repair. J Investig Dermatol Symp Proc 2006;11:132–139.
  11. Kozel’tsev VL, Volodina TV, Pankrushina AN, Guseva VV, Kostyuk NV: Changes in wound field lipids in rat skin. Bull Exp Biol Med 2006;142:493–494.
  12. Vicanova J, Ponec M, Weerheim A, Swope V, Westbrook M, Harriger D, Boyce S: Epidermal lipid metabolism of cultured skin substitutes during healing of full-thickness wounds in athymic mice. Wound Repair Regen 1997;5:329–338.
  13. Pittelkow MR, Scott RE: New techniques for the in vitro culture of human skin keratinocytes and perspectives on their use for grafting of patients with extensive burns. Mayo Clin Proc 1986;61:771–777.
  14. Holleran WM, Williams ML, Gao WN, Elias PM: Serine-palmitoyl transferase activity in cultured human keratinocyte. J Lipid Res 1990;31:1655–1661.
  15. Hennings H, Michael D, Cheng C, Steinert P, Holbrook K, Yuspa SH: Calcium regulation of growth and differentiation of mouse epidermal cells in culture. Cell 1980;19:245–254.
  16. Williams ML, Rutherford SL, Ponec M, Hincenbergs M, Placzek DR, Elias PM: Density-dependent variations in the lipid content and metabolism of cultured human keratinocytes. J Invest Dermatol 1988;91:86–91.
  17. Lee B, Vouthounis C, Stojadinovic O, Brem H, Im M, Tomic-Canic M: From an enhanceosome to a repressosome: molecular antagonism between glucocorticoids and EGF leads to inhibition of wound healing. J Mol Biol 2005;345:1083–1097.
  18. Holleran WM, Gao WN, Feingold KR, Elias PM: Localization of epidermal sphingolipid synthesis and serine palmitoyl transferase activity: alterations imposed by permeability barrier requirements. Arch Dermatol Res 1995;287:254–258.
  19. Farrell AM, Uchida Y, Nagiec MM, Harris IR, Dickson RC, Elias PM, Holleran WM: UVB irradiation up-regulates serine palmitoyltransferase in cultured human keratinocytes. J Lipid Res 1998;39:2031–2038.
  20. Bretscher MS: Getting membrane flow and the cytoskeleton to cooperate in moving cells. Cell 1996;87:601–606.
  21. Hopkins CR, Gibson A, Shipman M, Strickland DK, Trowbridge IS: In migrating fibroblasts, recycling receptors are concentrated in narrow tubules in the pericentriolar area, and then routed to the plasma membrane of the leading lamella. J Cell Biol 1994;125:1265–1274.
  22. Sidhu GS, Singh AK, Banaudha KK, Gaddipati JP, Patnaik GK, Maheshwari RK: Arnebin-1 accelerates normal and hydrocortisone-induced impaired wound healing. J Invest Dermatol 1999;113:773–781.
  23. Pei XW, Wang KZ, Dang XQ, Song JH, Shi ZB, Gao DF: Arnebia root oil promotes wound healing and expression of basic fibroblasts growth factor on the wound surface in rabbits (in Chinese). Zhong Xi Yi Jie He Xue Bao 2006;4:52–55.
  24. Chiu SC, Yang NS: Inhibition of tumor necrosis factor-α through selective blockade of pre-mRNA splicing by shikonin. Mol Pharmacol 2007;71:1640–1645.
  25. Wang WJ, Bai JY, Liu DP, Xue LM, Zhu XY: The antiinflammatory activity of shikonin and its inhibitory effect on leukotriene B4 biosynthesis (in Chinese). Yao Xue Xue Bao 1994;29:161–165.
  26. Futagami A, Ishizaki M, Fukuda Y, Kawana S, Yamanaka N: Wound healing involves induction of cyclooxygenase-2 expression in rat skin. Lab Invest 2002;82:1503–1513.
  27. Konno C, Mizuno T, Hikino H: Isolation and hypoglycemic activity of lithospermans A, B and C, glycans of Lithospermum erythrorhizon roots. Planta Med 1985;2:157–158.
  28. Kim J, Kim Y, Seo D, Kim S, Lee S, Cho Y: Oral supplementation of Lithospermum erythrorhizon prevents the development of atopic dermatitis with reducing ceramide degradation in the epidermis of NC/Nga mice. Phytother Res 2009;23:1250–1256.
  29. Omoto T, Murakami Y, Shimomura K, Yoshihira K, Mori K, Nakashima T, Tanaka M, Ishimaru K: Caffeic acid derivatives in Lamiaceae and Boraginaceae plants. Jpn J Food Chem 1997;4:11–15.
  30. Deters AM, Schröder KR, Hensel A: Kiwi fruit (Actinidia chinensis L.) polysaccharides exert stimulating effects on cell proliferation via enhanced growth factor receptors, energy production, and collagen synthesis of human keratinocytes, fibroblasts and skin equivalents. J Cell Physiol 2005;202:717–722.
  31. Yao H, Chen Y, Li S, Huang L, Chen W, Lin X: Promotion proliferation effect of a polysaccharide from Aloe barbadensis Miller on human fibroblasts in vitro. Int J Biol Macromol 2009;45:152–162.
  32. Thuong PT, Kang KW, Kim JK, Seo DB, Lee SJ, Kim SH, Kim SH, Oh WK: Lithospermic acid derivatives from Lithospermum erythrorhizon increased expression of serine palmitoyltransferase in human HaCaT cells. Bioorg Med Chem Lett 2009;19:1815–1817.