Effect of Agkistroden blomhoffi (mamushi) on the proliferation of human fibroblasts Qing Yang, a,b Guoguang Yang, b,c Jianying Zhang, b Noriyoshi Masuoka, d Yun Z. Riffle, b Zhigang Wang, b Hideo Ebinuma, e and Hiroyuki Kodama b, * a Yale University, New Haven, CT 06520, USA b Department of Chemistry, Kochi University Medical School, Kohasu, Oko-cho, Nankoku, Kochi 783-8505, Japan c Department of Orthopaedic Surgery, Musculoskeletal Research Center, University of Pittsburgh Medical Center, Pittsburgh, PA 15213, USA d Department of Biochemistry, Okayama University Graduate School of Medicine and Dentistry, Okayama 700-8558, Japan e Tohtoshu Seizo Co. LTD., 2-17-1, Nagishi, Taitoku, Tokyo 110-0003, Japan Received 7 July 2003; received in revised form 15 October 2003; accepted 15 October 2003 Abstract Objectives: The purpose of this study is to investigate the effect of Agkistroden blomhoffi (mamushi) aqueous extract on human patellar tendon cells in vitro, to pharmacologically explain the natural medicine’s healing effect on tendon, bone and muscle injuries. Design and methods: Human patellar tendon fibroblasts (HPTF) were incubated in media containing different concentrations of mamushi aqueous extract. Cell proliferation was studied by microscopic observations and total protein, actin, collagen I, and cyclooxygenase-2 (Cox 2) expressions. Results: Mamushi aqueous extract enhanced HPTF proliferation when its concentration was lower than 333 Ag/ml. Cells cultured in manushi-containing medium showed developed intercellular structure and increased protein production. However, mamushi extract higher than 500 Ag/ml oppressed cell growth. At 667 Ag/ml, mamushi induced Cox 2 production, a sign of cytotoxicity. Conclusion: A. blomhoffi aqueous extract was found to directly stimulate the proliferation and protein production, particularly collagen I synthesis, of HPTF in a dose-dependent manner. D 2003 The Canadian Society of Clinical Chemists. All rights reserved. Keywords: Agkistroden blomhoffi (mamushi); Human patellar tendon fibroblasts (HPTF); Cell proliferation; Type I collagen; Actin Introduction Agkistroden blomhoffi (mamushi), a venomous snake, has been well known for its tonic effect and used as a source of stamina in ancient China [1]. Mamushi pit viper has been taken orally in Asia to raise male vigor and strength. Among the earliest recorded use of snakes in Chinese medicine is the application of snake slough for the treatment of superficial diseases, including skin eruptions, eye infections or opacities, sore throat, and hemorrhoids [2]. Ecdysis of snake suggests that they have a regenera- tive quality for treating chronic skin problems. Snake gallbladder and bile are used to improve visual acuity, whooping cough, rheumatic pain, high fever, infantile convulsion, hemiplegia, hemorrhoids, gum bleeding, and skin infections [3,4]. Whole bodies, parts and derivatives like organs, tissues, secretions and feces of snakes are the main items used for kidney functions [5]. They are believed to release the excess fire accumulated in the body, regulate the balance between air and water, espe- cially improving the circulation of fluids [5]. Snakes are also frequently used in treatments involving repair of bone, muscle and tendon tissues. Using agkistro- don-containing formula to treat 118 cases of rheumatoid arthritis in China, 16 showed complete recovery, 58 showed good improvement and 39 cases showed some relief, with a total efficiency of 95.64% [6]. Agkistrodon acutus is found 0009-9120/$ - see front matter D 2003 The Canadian Society of Clinical Chemists. All rights reserved. doi:10.1016/j.clinbiochem.2003.10.012 Abbreviations: HPTF, human patellar fibroblast; PBS, phosphate buffer solution; CLAP, chymostatin, leupeptin, antipain and pepstatin A; FBS, fetal bovine serum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Cox 2, cyclooxygenase-2; Mamushi, Agkistroden blomhoffi (mamushi). * Corresponding author. Fax: +81-888-655937. E-mail address: hkodama@kochi-ms.ac.jp (H. Kodama). Clinical Biochemistry 37 (2004) 138 – 145