Effects of Short-term High Glucose on NIH/3T3 Fibroblast Proliferation, Apoptosis, and Collagen Type I Production

Abstract

Objective: To investigate the mechanisms of in vitro high glucose induced model of liver
fibrosis.
Material and methods: The effects of high glucose concentration on fibroblast proliferation
were investigated by BrdU immunostaining. Apoptosis and necrosis levels were analyzed by
flow cytometric assay. The content of collagen type I was measured by Collagen Estimation
Assay through ELISA.
Results: A high glucose medium not only increased NIH/3T3 fibroblast proliferation, but also
increased type I collagen synthesis, showing a similar condition to the fibrosis. Moreover, the
high glucose caused an increased level of cellular apoptosis and necrosis.
Conclusions: High glucose modulates the fibrosis in NIH/3T3 fibroblast cells via inducing the
production of type I collagen while maintains the homeostasis by inducing the apoptosis and
necrosis of cells.

Keywords

• High glucose
• Collagen
• NIH/3T3 fibroblast
• Apoptosis

Kısa Süreli Yüksek Glikoz Uygulamasının NIH/3T3 Fibroblastlarında Proliferasyon, Apoptoz ve Kollajen Tip I Üretimi Üzerine Etkileri

Abstract

Amaç: Yüksek glikoza bağlı karaciğer fibrozis mekanizmalarını in vitro model kullanarak
araştırmak amaçlanmıştır.
Gereç ve yöntem: Yüksek glikoz konsantrasyonunun fibroblast proliferasyonu üzerindeki
etkileri BrdU immünositokimya tekniği ile incelenmiştir. Apoptoz seviyeleri ve nekroz akım
sitometri analizi ile tespit edildi. Kollajen tip I içeriği, ELISA ile ölçüldü.
Bulgular: Yüksek glikoz düzeyi sadece NIH/3T3 fibroblast proliferasyonunu arttırmadı, aynı
zamanda fibrozise benzer bir durumun göstergesi olarak tip I kollajen sentezini de arttırdı.
Ayrıca yüksek glikoz, apoptoz ve nekroz seviyelerinin artmasına da neden olmuştur.
Sonuç: Yüksek glikoz, NIH/3T3 fibroblast hücrelerinde fibrozisi, tip I kollajen üretimini
indükleyerek modüle ederken, hücrelerin apoptoz ve nekrozunu indükleyerek homeostazı korur.

Keywords

• High glucose
• Collagen
• NIH/3T3 fibroblast
• Apoptosis

References

1. 1. Tang M, Zhang W, Lin H, et al. High glucose promotes the production of collagen types I and III by cardiac fibroblasts through a pathway dependent on extracellular-signal-regulated kinase 1/2. Mol Cell Biochem 2007;301:109-14.
2. 2. Friedman SL. The cellular basis of hepatic fibrosis: mechanisms and treatment strategies. New Eng J Med 1993;328:1828-1836.
3. 3. Olaso E, Friedman SL. Molecular regulation of hepatic fibrogenesis. J Hepatol 1998;29:836-847.
4. 4. Hynes RO. The extracellular matrix: not just pretty fibrils. Science 2010;326:1216-19.
5. 5. Bataller R, Brenner DA. Liver fibrosis. J Clin Invest 2005;115:209-218.
6. 6. Friedman SL. Mechanisms of hepatic fibrogenesis. Gastroenterology 2008;134:1655- 1669.
7. 7. Dranof JA, Wells RG. Portal fibroblasts: Underappreciated mediators of biliary fibrosis. Hepatology 2010;51:1438-1444.
8. 8. Liu CH, Hu YY, Wang XL, et al. Effcts of salvianolic acid-A on NIH/3T3 fibroblast proliferation, collagen synthesis and gene expression. World J Gastroenterol 2000;6:361-364.

9. 9. Wynn TA, Ramalingam TR. Mechanisms of fibrosis: therapeutic translation for fibrotic disease. Nat Med 2012;18,1028-1040.
10. 10. Dostal DE, Rothblum KN, Conrad KM, et al. Detection of angiotensin I and II in cultured rat cardiac myocytes and fibroblasts. Am J Physiol Cell Physiol 1992;263:C851-C863.
11. 11. Eghbali M. Cardiac fibroblasts: function, regulation of gene expression, and phenotypic modulation. Basic Res Cardiol 1992;87:183-189.
12. 12. Higuchi C, Tanihata Y, Nishimura H, et al. Effcts of glucose and plasminogen activator inhibitor-1 on collagen metabolism in the peritoneum. Ther Apher Dial 2005;9:173-178.
13. 13. Saed GM, Diamond MP. Effct of glucose on the expression of type I collagen and transforming growth factor-beta1 in cultured human peritoneal fibroblasts. Fertil Steril 2003;79:158-163.
14. 14. Lam S, van der Geest RN, Verhagen NA, et al. Connective tissue growth factor and igf-I are produced by human renal fibroblasts and cooperate in the induction of collagen production by high glucose. Diabetes 2003;52:2975-2983
15. 15. Benazzoug Y, Borchiellini C, Labat-Robert J, et al. Effct of high-glucose concentrations onthe expression of collagens and fibronectin by fibroblasts in culture. Exp Gerontol 1998;33:445- 455.
16. 16. Chen CC, Lau LF. Deadly liaisons: fatal attraction between CCN matricellular proteins and the tumor necrosis factor family of cytokines. Journal of Cell Communication and Signaling 2010;4:63-69.
17. 17. Johnson A, DiPietro LA. Apoptosis and angiogenesis: an evolving mechanism for fibrosis. The FASEB Journal 2013;27:3893-3901.
18. 18. Liu WL, Wang XX, Mei Z, et al. BNIP3L promotes cardiac fibrosis in cardiac fibroblasts through [Ca2+]i TGF-βSmad2/3 pathway. Scientific Reports 2017;7:1906.
19. 19. Chu Q, Yang K, Wang A. Research progress on oxidative stress and apoptosis. Journal of Hygiene Research 2003;323:276-279.
20. 20. Kanigur Sultuybek G, Soydas T, Yenmis G. NF- κB as the mediator of metformin’s effct on ageing and ageing-related diseases. Clin Exp Pharmacol Physiol 2019;46:413-422.
21. 21. Soydas T, Yaprak Sarac E, Cinar S et al. The protective effcts of metformin in an in vitro model of aging 3T3 fibroblast under the high glucose conditions. J Physiol Biochem. 2018;74:273-281.
22. 22. Moiseeva O, Deschênes‐Simard X, St‐Germain E, et al. Metformin inhibits the senescence‐ associated secretory phenotype by interfering with IKK/NF‐kB activation. Aging Cell. 2013;12:489-498.