mersin univ saglık bilim derg Mersin Üniversitesi Sağlık Bilimleri Dergisi 1308-0830 Mersin University 10.26559/mersinsbd.538541 Health Care Administration Sağlık Kurumları Yönetimi Effect of the bacterial lipopolysaccharide on preadipocyte differentiation: possible contribution of the NO and Rho-kinase Bakteriyel lipopolisakkaridin preadiposit diferensiyasyonu üzerine etkisi: NO’nun ve Rho-kinaz enziminin olası katkısı https://orcid.org/0000-0002-6690-119X Yurtsever Ahmet Sencer MERSİN ŞEHİR HASTANESİ https://orcid.org/0000-0003-4117-6013 Büyükafşar Kansu MERSİN ÜNİVERSİTESİ, TIP FAKÜLTESİ 08 30 2019 12 2 257 270 03 12 2019 04 05 2019 Copyright © 2008, Mersin Üniversitesi Sağlık Bilimleri Dergisi 2008 Mersin Üniversitesi Sağlık Bilimleri Dergisi

Aim: Differentiation of preadipocytes is one of importantsteps for the adipogenesis. Adipogenesis is a metabolic disorder thataccompanied by low grade inflammation and posses a lot of complications. Weaimed to investigate effect of the LPS which one of the inflammatory responsegenerating endotoxins on differentiation of 3T3-L1 cells and contribution of NOand Rho/ROCK pathways to this effect. Method:Fibroblast origin 3T3-L1 cells used for the differentiation ofpreadipocytes to adipocytes. Seeding was performed as 20000 cells on the everywell of 24well plates and standard preadipocyte differentiation protocol wasapplied. In order to inducing differentiation, 0.25 µM dexamethasone, 0.5 mMizobuthylmethylxhantine and 1μM insulin containing 10% FBS/DMEM treated at 0-2thdays. Cells treated with 10% FBS/DMEM containing 1μM insulin on 2-4thdays of the protocol. 10% FBS/DMEM alone applied to wells at 4-8thdays. Incubation was maintained until 8th day. LPS (10, 100 ng/ml)treatment was performed with or without L-NAME (NG-nitro-L-argininemethyl esther, 2 and 5x10-4 M) on certain time points (0-2, 2-4,4-8, 0-8th days) of the differentiation protocol. Differentiationevaluated with Oil Red-O staining method performed at 8th day.Effect of the LPS on iNOS and Rho/Rho-kinase enzyme expressions was evaluatedwith Western Blot analysis. Besides nitrite levels in cell culture media wasmeasured with Griess method on the presence of LPS and L-NAME. Results: LPS treatment on 3T3-L1 cellculture is significantly supressed the differentiationtime points except 0-2th days. In spite of the pretreatment ofthe cells with L-NAME did not any effect on the differentiation suppressionproduced by LPS, L-NAME treatment significantly supressing the differentiationevery time points except 0-2th days. LPS increased both iNOS andROCK-2 expression. ROCK expression increasing effect of the LPS did not changedby the L-NAME treatment. When treated alone, L-NAME increased the ROCK-2expression in a similar vein. Conclusion:LPS which is bacterial endotoxin, supressed the differentiation of 3T3-L1cells. This effect could mediated by inflammatory mediator(s) other than NO.Besides, LPS could supressed the preadipocyte differentiation by a Rho/ROCKdependent mechanism.

Amaç: Preadipositlerin diferensiyasyonuadipogenezis için önemli basamaklardan biridir. Adipogenezis, düşük düzeydeinflamasyonun eşlik ettiği ve pek çok komplikasyonu olan metabolik birhastalıktır. Bu çalışmamızda, inflamatuar yanıt oluşturan bakteriyelendotoksinlerden LPS’nin 3T3-L1 hücrelerinde diferensiyasyon üzerine etkisinive bu etkiye NO ve Rho/ROCK yolağının katkısını araştırmayı amaçladık. Yöntem: Preadipositlerin adipositlerediferensiyasyonu için fibroblast kökenli 3T3-L1 hücreleri kullanıldı. 24kuyucuklu pleytlere 20.000 hücre olacak şekilde ekim yapıldı ve standartpreadiposit diferensiyasyon protokolü uygulandı. Diferensiyasyonun indüklenmesiiçin protokolün 0-2. günü 0.25 µM deksametazon, 0.5mM izobutilmetilksantin ve1μM insülin içeren %10FBS/DMEM uygulandı. Protokolün 2-4. günleri 1μM insüliniçeren %10 FBS/DMEM uygulandı. 4-8. gün ise kuyucuklara sadece %10 FBS/DMEMkonuldu. İnkübasyon 8. güne kadar sürdürüldü. Diferensiyasyon protokolününbelirli zaman noktalarında (0-2, 2-4, 4-8, 0-8. günler) bakteriyel LPS (10-100ng/ml), L-NAME (2-5x10-4 M) varlığında ya da yokluğunda uygulandı.Diferensiyasyon, 8’inci günde Oil Red-O boyaması ile değerlendirildi. LPS’niniNOS ve Rho/Rho-kinaz ekspresyonları üzerine etkileri de Western-blot analiziile değerlendirildi. Ayrıca, kültür ortamında nitrit düzeyleri, LPS ve L-NAMEvarlığında Griess yöntemi ile ölçüldü. Bulgular:LPS uygulaması, 0-2. gün dışındaki zaman aralıklarında diferensiyasyonuanlamlı bir şekilde baskıladı. L-NAME ön uygulaması, bu süpresyonu ortadankaldırmadı ancak tek başına L-NAME, 0-2. gün dışında tüm zaman aralıklarındadiferensiyasyonu süprese etti. LPS hem iNOS hem de ROCK-2 ekspresyonunuarttırdı. LPS’nin ROCK ekspresyonunu arttırıcı etkisi L-NAME tarafındandeğiştirilmedi. L-NAME tek başına uygulandığında LPS’ye benzer şekilde ROCK-2ekspresyonunu arttırdı. Sonuç: Birbakteriyel endotoksin olan LPS, 3T3-L1 hücrelerinde diferensiyasyonubaskılamaktadır. Bu etkiye NO değil ancak onun dışındaki bir inflamatuarmediyatör(ler) aracılık edebilir. Ayrıca LPS, Rho/ROCK bağımlı bir mekanizmaile preadiposit diferensiyasyonunu süprese edebilir.

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