Journal of Traditional Medical Complementary Therapies

.: ORIGINAL RESEARCH
The Investigation of Effects of Medicinal Leech Saliva Extract on the Breast Fibroblast Cell Line In Vitro: An Experimental Study
Tıbbi Sülük Tükürük Ekstraktının Meme Fibroblast Hücre Hattı Üzerine Etkilerinin İn Vitro Araştırılması: Deneysel Bir Çalışma
Kübranur ÜNALa, Nihan TIRIKb, Mehmet Emre EROLb, Leyla İBRAHİMKHANLIa, Mualla Pınar ELÇİb, Hüseyin AYHANc
aDepartment of Medical Biochemistry, Gazi University Faculty of Medicine, Ankara, Türkiye
bDepartment of Medical Biochemistry, Gazi University Institute of Health Sciences, Ankara, Türkiye
cYıldırım Beyazıt University Vocational School of Health Services, Ankara, Türkiye
J Tradit Complem Med. 2023;6(2):142-51
doi: 10.5336/jtracom.2022-92875
Article Language: EN
Full Text
ABSTRACT
 Objective: Medicinal leeches benefit by delivering various bioactive substances from their saliva during sucking. It was aimed to investigate the effects of leech saliva extract (LSE) on breast fibroblast cell line (Hs 578Bst) and HUVEC (CRL-1730) line in vitro. Material and Methods: Leech saliva was obtained from medicinal leeches of the Hirudo verbana species. The LSE was obtained from the Hirudo verbana. LSE dose amounts (400 µg/mL, 200 µg/mL, 100 µg/mL and 50 µg/mL) were adjusted for protein concentration. In cell culture; cell viability, apoptosis, cell migration and gene expressions responsible for these effects were evaluated. The effect of LSE on the cell viability was evaluated with MTT assay. The Scratching Test was used to evaluate the effects of LSE on the cell migration. The effect of LSE on mRNA expression levels of fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF) and epidermal growth factor (EGF) was determined with polymerase chain reaction. The effect of LSE on apoptosis was investigated with flow cytometry. Results: The doses of LSE (400-50 µg/mL) significantly increased the cell viability compared with the control group for 24 h and 48 h (p<0.05). In the scratching test, it was observed that the 50 µg/mL dose induced the cell migration and covered all areas with cells at 24 h. It was observed that 200 µg/mL LSE up-regulated the mRNA expression of FGF, VEGF and EGF compared with the control and other groups (p<0.001). All doses of LSE shows no apoptotic or necrotic effects in the cell lines. Conclusion: In this study, it was found that the LSE applied at different doses had significant effects on the cell viability, cell migration and gene expression responsible for these effects in the breast fibroblast cell line.

Keywords: Leech saliva extract; cell viability; angiogenesis; apoptosis; proliferation
ÖZET
 Amaç: Tıbbi sülükler kan emme sırasında tükürüklerinden konakçının kan dolaşımına çeşitli biyoaktif maddeler salarak fayda sağlarlar. Çalışmamızda tıbbi sülük salgı ekstraktının [leech saliva extract (LSE)] meme fibroblast hücre hattı (Hs 578Bst) ve HUVEC (CRL-1730) hattı üzerindeki etkilerinin in vitro olarak araştırılması amaçlanmıştır. Gereç ve Yöntemler: Sülük salgısı, Hirudo verbana türü tıbbi sülüklerinden elde edildi. Bu sülük salgısının toplam protein konsantrasyonu belirlendi ve doz miktarları (400 ug/mL, 200 ug/mL, 100 ug/mL ve 50 ug/mL) seri dilüsyon ile ayarlandı. Hücre kültüründe hücre canlılığı, apoptoz, hücre göçü ve bu etkilerden sorumlu gen ekspresyonları değerlendirildi. LSE'nin hücre canlılığı üzerindeki etkisi MTT analiziyle değerlendirildi. LSE'nin hücre göçü üzerindeki etkisini değerlendirmek için Scratch Testi kullanıldı. LSE'nin fibroblast büyüme faktörü [fibroblast growth factor (FGF)], vasküler endotelyal büyüme faktörü [vascular endothelial growth factor (VEGF)] ve epidermal büyüme faktörünün [epidermal growth factor (EGF)] mRNA ekspresyonları üzerine etkisi polimeraz zincir reaksiyonu ile analiz edildi. LSE'nin apoptoz üzerine etkisi flow sitometri ile araştırıldı. Bulgular: LSE'nin tüm dozları (400-50 µg/mL) kontrol grubuyla karşılaştırıldığında hem 24 saat hem de 48 saatte hücre canlılığını artırdığı gözlemlendi (p<0,05). Scratch testinde, 50 µg/mL dozunun hücre göçünü indüklediği ve 24 saatte tüm alanları hücrelerle kapladığı gözlemlendi. LSE'nin 200 ug/mL dozunun kontrol ve diğer gruplarla karşılaştırıldığında FGF, VEGF ve EGF mRNA ekspresyonunu önemli ölçüde artırdığı gözlemlendi (p<0,001). LSE'nin hücre hatlarında nekrotik veya apoptotik etkisi olmadığı tespit edildi. Sonuç: Bu çalışmada, farklı dozlarda uygulanan LSE'nin meme fibroblast hücre hattında hücre canlılığı, hücre göçü ve bu etkilerden olası sorumlu gen ekspresyonları üzerinde önemli etkileri olduğu bulundu.

Anahtar Kelimeler: Sülük salgı ekstraktı; hücre canlılığı; anjiyogenez; apopitoz; proliferasyon
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