Investigation of the effect of B. spergulifolius extracts on insulin-resistant 3T3-L1 adipocytes
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Tarih
2021
Yazarlar
Dergi Başlığı
Dergi ISSN
Cilt Başlığı
Yayıncı
Adana Alparslan Türkeş Bilim ve Teknoloji Üniversitesi
Erişim Hakkı
info:eu-repo/semantics/openAccess
Özet
Diabetes mellitus (DM), yetersiz insülin sekresyonu ve / veya insülin etkisi kusuru nedeniyle metabolik sonuçlar, kronik hiperglisemi ile karakterize metabolik bir hastalıktır. DM sıklıkla kardiyovasküler hastalıklara, göz, böbrek ve sinir sisteminde çeşitli komplikasyonlara neden olur. Tip 2 DM (T2DM), toplam DM'nin yaklaşık % 90'ını oluşturur. Bu nedenle, T2DM için yeni terapötik yaklaşımların ve potansiyel sinyal yolaklarındaki molekülleri hedefleyen alternatif, doğal ajanların araştırılması çok önemlidir. Tıbbi bitkiler, alternatif yeni ilaç etken maddelerinin araştırılmasında çok önemli kaynaklardır. Bolanthus spergulifolius (B. spergulifolius), Caryophyllaceae ailesinin cinslerinden biridir. Bu çalışmada B. spergulifolius ekstraktlarının 3T3-L1 adipositler üzerindeki potansiyel anti-diyabetik etkilerinin in-vitro olarak araştırılması amaçlanmıştır. B. spergulifolius, metanol (MeOH), etilasetat (EA) ve su ile ekstrakte edildi ve toplam fenolik içerikleri (TPC) Folin-Ciocalteu yöntemi ile değerlendirildi. Yüksek TPC (sulu< MeOH< EA) gösteren B.spergulifolius ekstraktlarının olgun 3T3-L1 adipositlerideki sitotoksik etkisi MTT asay ile değerlendirildi ve hücre canlılığında %50 azalmaya neden olan ekstrakt konsantrasyonları (IC50) belirlendi. MeOH, EA ve Sulu ekstraktlarının IC50 konsantrasyonları sırasıyla 305.7 ± 5.583 µg/mL, 567.4 ± 3.008 µg/mL ve 418.3 ± 4.390 µg/mL olarak gözlendi ve ileri deneylerde kullanılmak üzere seçildi için. Ölü/canlı hücre asay ile MeOH, EA ve Sulu ekstrelerinin sitotoksik etkisi doğrulanmıştır (sıırasıyla, %69.75 ± 1.70, %61.75 ± 1.70, %70 ± 4.24 ve hepsi için P< 0.05). Ayrıca, ekstrelerin olgun 3T3-L1 adipositlerinde lipid birikimi üzerindeki etkileri Oil-Red-O boyama testi ile değerlendirildi. Tedavi edilmemiş olgun adipositlere kıyasla ekstraktlar ile tedavi edilmiş olgun adipositlerde lipid birikimi etkili bir şekilde azalmıştır (tümü için P< 0.05). Ayrıca ekstraktların Bax ve Bcl-2 tarafından düzenlenen apoptoz üzerindeki etkisi qRT-PCR ile araştırılmıştır. Tedavi edilmemiş adipositlere kıyasla ekstraktlar, pro-apoptotik Bax ekspresyonunu artırarak, anti-apoptotik Bcl-2 gen ekspresyonunu azaltarak apoptozu önemli ölçüde indükledi (hepsi için P< 0.05). İnsülin direnci ile bağlantılı Glut-4 ekspresyonu, qRT-PCR, Western-blot ve Immunofluorescence boyama ile değerlendirildi. Buna paralel olarak, tedavi edilmemiş olgun adipositlere kıyasla ekstraktlarla tedavi edilen adipositlerde Glut-4 ekspresyonu, önemli ölçüde daha yüksekti (hepsi için P< 0.05). Ekstraktlar, çizik yara asay uygulamasından 30 saat sonra hücre göçünü önemli ölçüde baskıladı (hepsi için P< 0.05). Hücre morfolojisi ve hücre-çapı faz-kontrast mikroskobu, taramalı elektron mikroskobu (SEM), İmmünofloresans (anti-F-aktin ile) ve Giemsa boyaması ile değerlendirildi. Ekstraktlarla tedavi edilen adipositler kısmen küresel morfolojiyi kaybetti ve tedavi edilmemiş adipositlere kıyasla daha küçük hücre çapı gösterdi (hepsi için P< 0.05). Sonuç olarak, çalışmamız B. spergulifolius ekstraktlarının apoptozu indüklediğini, lipid birikimini azalttığını, yarayı iyileşmesini geciktirdiğini, Glut-4 seviyesini arttırdığını ve bu yolla DM'de insülin direncinin azalmasına katkıda bulunabileceğini göstermektedir. Anahtar Kelimeler: Bolanthus spergulifolius, diyabet, insulin dinci, 3T3-L1 adipositler, glikoz taşıyıcı tip 4, anti-diyabetik
Diabetes mellitus (DM) is a metabolic disease with chronic hyperglycemia characterized by metabolic outcomes due to insufficient insulin secretion and/or insulin effect defect. DM often causes cardiovascular diseases and various complications on the eye, kidney and nervous system. Type 2 DM (T2DM) accounts for approximately 90% of the total DM. Therefore, it is very important to investigate new therapeutic approaches for T2DM and alternative, natural agents that target molecules in potential signal pathways. Medicinal plants are very significant resources in the research of alternative new drug active ingredients. Bolanthus spergulifolius (B. spergulifolius) is one of the genera of the family Caryophyllaceae. In the present study, it was aimed to investigate the potential anti-diabetic effects in-vitro of B. spergulifolius extracts on 3T3-L1 adipocytes. B. spergulifolius were extracted with methanol (MeOH), ethylacetate (EA) and Aqueous and evaluated for their total phenolic content (TPC) by Folin-Ciocalteu method. B. spergulifolius extracts showing highly TPC (Aqueous< MeOH< EA) and their different concentrations were caried out on preadipocytes differentiated in to mature 3T3-L1 adipocytes to investigate their half-maximal (50%) inhibitory concentration (IC50) by using MTT assay. The IC50 concentrations of MeOH, EA and Aqueous extracts were observed as 305.7 ± 5.583 µg/mL, 567.4 ± 3.008 µg/mL and 418.3 ± 4.390 µg/mL and were used for further experiments. A live/dead assay further confirmed the cytotoxic effect of MeOH, EA and Aqueous extracts (respectively, 69.75 ± 1.70%, 61.75 ± 1.70%, 70 ± 4.24% and for all P< 0.05). Also, effects of extracts on lipid accumulation in mature 3T3-L1 adipocytes were evaluated by Oil-Red O staining assay. The extracts effectively decreased lipid-accumulation compared to untreated adipocytes (for all P< 0.05). Morever, effect of extracts on apoptosis regulated by the Bax and Bcl-2 was investegated by qRT-PCR. The extracts significantly induced apoptosis by up-regulating pro-apoptotic Bax expression but down-regulating anti-apoptotic Bcl-2 gene expression compared to untreated adipocytes (for all P< 0.05). The Glut-4 expression linked with insulin resistance was determined by qRT-PCR, Western-blot and Immunofluorescence staining. In parallel, expression of Glut-4 in adipocytes treated with extracts was significantly lower compared to untreated adipocytes (for all P< 0.05). Extracts significantly supressed cell migration after 30 h of wounding in a scratch-assay (for all P< 0.05). Cell morphology and diameter were further evaluated by phase-contrast microscopy, Scanning electron microscope (SEM), Immunofluorescence (with F-Actin) and Giemsa staining. The adipocytes treated with extracts partially lost spherical morphology and showed smaller cell-diameter compared to untreated adipocytes (for all P< 0.05). In conclusion, these results suggest that extracts of B. spergulifolius causes to an induce apoptosis, a decrease lipid-accumulation, wound healing, Glut-4 level and migth contribute to the reducing of insulin-resistance in DM. Key Words: Bolanthus spergulifolius, diabetes mellitus, insulin-resistant, 3T3-L1 adipocytes, Glucose transporter-4, anti-diabetic
Diabetes mellitus (DM) is a metabolic disease with chronic hyperglycemia characterized by metabolic outcomes due to insufficient insulin secretion and/or insulin effect defect. DM often causes cardiovascular diseases and various complications on the eye, kidney and nervous system. Type 2 DM (T2DM) accounts for approximately 90% of the total DM. Therefore, it is very important to investigate new therapeutic approaches for T2DM and alternative, natural agents that target molecules in potential signal pathways. Medicinal plants are very significant resources in the research of alternative new drug active ingredients. Bolanthus spergulifolius (B. spergulifolius) is one of the genera of the family Caryophyllaceae. In the present study, it was aimed to investigate the potential anti-diabetic effects in-vitro of B. spergulifolius extracts on 3T3-L1 adipocytes. B. spergulifolius were extracted with methanol (MeOH), ethylacetate (EA) and Aqueous and evaluated for their total phenolic content (TPC) by Folin-Ciocalteu method. B. spergulifolius extracts showing highly TPC (Aqueous< MeOH< EA) and their different concentrations were caried out on preadipocytes differentiated in to mature 3T3-L1 adipocytes to investigate their half-maximal (50%) inhibitory concentration (IC50) by using MTT assay. The IC50 concentrations of MeOH, EA and Aqueous extracts were observed as 305.7 ± 5.583 µg/mL, 567.4 ± 3.008 µg/mL and 418.3 ± 4.390 µg/mL and were used for further experiments. A live/dead assay further confirmed the cytotoxic effect of MeOH, EA and Aqueous extracts (respectively, 69.75 ± 1.70%, 61.75 ± 1.70%, 70 ± 4.24% and for all P< 0.05). Also, effects of extracts on lipid accumulation in mature 3T3-L1 adipocytes were evaluated by Oil-Red O staining assay. The extracts effectively decreased lipid-accumulation compared to untreated adipocytes (for all P< 0.05). Morever, effect of extracts on apoptosis regulated by the Bax and Bcl-2 was investegated by qRT-PCR. The extracts significantly induced apoptosis by up-regulating pro-apoptotic Bax expression but down-regulating anti-apoptotic Bcl-2 gene expression compared to untreated adipocytes (for all P< 0.05). The Glut-4 expression linked with insulin resistance was determined by qRT-PCR, Western-blot and Immunofluorescence staining. In parallel, expression of Glut-4 in adipocytes treated with extracts was significantly lower compared to untreated adipocytes (for all P< 0.05). Extracts significantly supressed cell migration after 30 h of wounding in a scratch-assay (for all P< 0.05). Cell morphology and diameter were further evaluated by phase-contrast microscopy, Scanning electron microscope (SEM), Immunofluorescence (with F-Actin) and Giemsa staining. The adipocytes treated with extracts partially lost spherical morphology and showed smaller cell-diameter compared to untreated adipocytes (for all P< 0.05). In conclusion, these results suggest that extracts of B. spergulifolius causes to an induce apoptosis, a decrease lipid-accumulation, wound healing, Glut-4 level and migth contribute to the reducing of insulin-resistance in DM. Key Words: Bolanthus spergulifolius, diabetes mellitus, insulin-resistant, 3T3-L1 adipocytes, Glucose transporter-4, anti-diabetic
Açıklama
Fen Bilimleri Enstitüsü, Biyomühendislik Ana Bilim Dalı
Anahtar Kelimeler
Biyomühendislik, Bioengineering ; Moleküler Tıp
