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Recombinant Spidroin Films Attenuate Individual Markers of Glucose Induced Aging in NIH 3T3 Fibroblasts

A. M. Moysenovich1#, M. M. Moisenovich1#,a, A. K. Sudina1, V. V. Tatarskiy2,3,4, A. I. Khamidullina3, M. A. Yastrebova3, L. I. Davydova5,6, V. G. Bogush5,6, V. G. Debabov5,6, A. Yu. Arkhipova1,7, K. V. Shaitan1,8, A. A. Shtil2,3, and I. A. Demina1,9

1Faculty of Biology, Lomonosov Moscow State University, 119234 Moscow, Russia

2Blokhin National Medical Research Center of Oncology, 115478 Moscow, Russia

3Institute of Gene Biology, Russian Academy of Sciences, 119334 Moscow, Russia

4National University of Science and Technology “MISiS”, 119049 Moscow, Russia

5NRC “Kurchatov Institute” – GOSNIIGENETIKA, 117519 Moscow, Russia

6NRC “Kurchatov Institute”, 123182 Moscow, Russia

7Moscow Regional Research and Clinical Institute (MONIKI), 129110 Moscow, Russia

8Semenov Institute of Chemical Physics, Russian Academy of Sciences, 119991 Moscow, Russia

9Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology and Immunology, 117198 Moscow, Russia

# These authors contributed equally to this work

* To whom correspondence should be addressed.

Received March 28, 2020; Revised May 15, 2020; Accepted May 22, 2020
The effect of bioresorbable materials on aging in cultured mouse NIH 3T3 fibroblasts treated with elevated glucose concentration was investigated. The cells were grown on films produced from the silkworm fibroin and rS1/9, a recombinant analog of Nephila clavipes spidroin 1. Exposure to 50 mM glucose of the cells grown on uncoated glass support resulted in the cell growth retardation. The average areas of the cells and nuclei and the percentage of apoptotic cells increased, whereas the amount of soluble collagen decreased. In contrast, on the fibroin and spidroin films, the cell density and the percentage of 5-bromo-2′-deoxyuridine (BrdU)-positive cells were higher vs. the cells grown on the glass support. The films protected NIH 3T3 fibroblasts from the glucose-induced death. The most prominent effects on the cell density, BrdU incorporation, and apoptosis prevention were observed in the cells cultured on spidroin films. Unlike the cells grown on glass support (decrease in the soluble collagen production) or fibroin (no effect), production of soluble collagen by the cells grown on spidroin films increased after cell exposure to 50 mM glucose. Molecular analysis demonstrated that 50 mM glucose upregulated phosphorylation of the NFκB heterodimer p65 subunit in the cells grown on the glass support. The treatment of cells grown on fibroin films with 5.5 mM or 50 mM glucose had no effect on p65 phosphorylation. The same treatment decreased p65 phosphorylation in the cells on the spidroin films. These results demonstrate the anti-aging efficacy of biomaterials derived from the silk proteins and suggest that spidroin is more advantageous for tissue engineering and therapy than fibroin.
KEY WORDS: cell aging, recombinant spidroin, regeneration, artificial skin

DOI: 10.1134/S0006297920070093