水凝胶递送碱性成纤维细胞生长因子对氧糖剥夺后NIH3T3细胞功能的影响

doi:10.13481/j.1671-587X.20230611

摘要/Abstract

摘要：

目的探讨水凝胶递送碱性成纤维细胞生长因子（bFGF）对氧糖剥夺（OGD）后小鼠成纤维NIH3T3细胞功能的影响，并阐明负载bFGF的水凝胶对OGD条件下成纤维细胞氧化应激反应的改善作用。方法制备bFGF明胶水凝胶，将对数生长期NIH3T3细胞分为空白组、20%明胶组和20%明胶+戊二醛组，采用CCK-8法检测6、12和24 h水凝胶浸出液共培养的NIH3T3细胞活性。将NIH3T3细胞分为对照组、OGD组和OGD+不同质量 bFGF负载组（OGD+1 ng bFGF组、OGD+10 ng bFGF组和OGD+100 ng bFGF组）。采用Western blotting法检测各组细胞中α-平滑肌肌动蛋白（α-SMA）、Ⅰ型胶原蛋白和Ⅲ型胶原蛋白表达水平。检测各组细胞中丙二醛（MDA）水平和乳酸脱氢酶（LDH）及超氧化物歧化酶（SOD）活性。采用酶联免疫吸附试验（ELISA）法检测bFGF体外释放率。结果 CCK-8法检测，与空白组比较，不同浸出时间20%明胶组和20%明胶+戊二醛组NIH3T3细胞活性差异无统计学意义（P>0.05）。Western blotting法检测，与对照组比较，OGD组NIH3T3细胞中α-SMA和Ⅰ型胶原蛋白表达水平明显升高（P<0.05或P<0.01）；与OGD组比较，OGD+1 ng bFGF组、OGD+10 ng bFGF组和OGD+100 ng bFGF组NIH3T3细胞中α-SMA蛋白表达水平均明显降低（P<0.01），Ⅰ型胶原蛋白表达水平均明显降低（P<0.05或P<0.01），并呈剂量依赖性。与对照组比较，OGD组NIH3T3细胞中MDA水平和LDH活性升高（P<0.05），SOD活性降低（P<0.05）。与OGD组比较，OGD+1 ng bFGF组NIH3T3细胞中MDA水平和LDH活性降低（P<0.05），SOD活性升高（P<0.05）。ELISA法检测，在4 h内约10% bFGF由水凝胶中释放，在12 h内约15% bFGF由水凝胶中释放，在24 h内约21% bFGF由水凝胶中释放。结论负载bFGF的水凝胶可以调控OGD条件下成纤维细胞的转化，抑制成纤维细胞Ⅰ型胶原蛋白表达，并改善细胞氧化应激反应，且该系统具有一定的持续释药能力。

关键词: 水凝胶, 碱性成纤维细胞生长因子, NIH3T3细胞, 胶原蛋白, 氧化应激

Abstract:

Objective To discuss the effect of hydrogel-based delivery of basic fibroblast growth factor （bFGF） on the function of the NIH3T3 fibroblast cells after oxygen-glucose deprivation （OGD），and to clarify the improvement effect of bFGF-loaded hydrogel on the oxidative stress responses in the fibroblasts under the OGD condition. Methods The bFGF-containing agarose hydrogel was prepared， and the NIH3T3 cells at logarithmic growth phase were divided into blank group， 20% agarose group， and 20% agarose + glutaraldehyde group. The viabilities of the NIH3T3 cells after co-cultured with the hydrogel-eluted fluid for 6， 12， and 24 h were detected by CCK-8 assay. The NIH3T3 cells were divided into control group， OGD group， and OGD + different masses of bFGF-loaded groups （OGD + 1 ng bFGF group， OGD+10 ng bFGF group， and OGD+100 ng bFGF group）. The expression levels of α-smooth muscle actin （α-SMA）， type Ⅰ collagen， and type Ⅲ collagen proteins in the NIH3T3 cells in various groups were detected by Western blotting method； the content of malondialdehyde （MDA） and activities of lactate dehydrogenase （LDH） and superoxide dismutase （SOD） in the NIH3T3 cells were detected by assay kits； the in vitro release rate of bFGF in the NIH3T3 cells in various groups was detected by enzyme-linked immunosorbent assay （ELISA） method. Results The CCK-8 assay results showed that compared with blank group， the viability of the NIH3T3 cells between blank group， 20% agarose group， and 20% agarose+ glutaraldehyde group at different elution times had no significant difference（P>0.05）. The Western blotting results showed that the expression levels of α-SMA and type Ⅰ collagen proteins in the NIH3T3 cells in OGD group were significantly higher than that in control group （P<0.05 or P<0.01）.Compared with OGD group，the expression levels of α-SMA and type Ⅰ collagen proteins in the NIH3T3 cells in OGD+1 ng bFGF， OGD+10 ng bFGF， and OGD+100 ng bFGF groups were significantly decreased （P<0.05 or P<0.01） in a dose-dependent manner.Compared with control group，the level of MDA and activity of LDH in the NIH3T3 cells in OGD group were increased（P<0.05）， while the activity of SOD was decreased （P<0.05）. Compared with OGD group， the level of MDA and the activity of LDH in the NIH3T3 cells in OGD + 1 ng bFGF group were decreased （P<0.05）， while the activity of SOD was increased （P<0.05）.The ELISA results showed that about 10% bFGF was released from the hydrogel within 4 h， about 15% bFGF was relleased from the bydrogel with in 12 h， and about 21% bFGF was released from the hydrogel within 24 h. Conclusion The bFGF-loaded hydrogel can modulate the transformation of the fibroblasts under the OGD condition，inhibit the expression of typeⅠ collagen protein，and improve the oxidative stress responses in the fibroblasts. Moreover，this system exhibits a sustained drug-release ability.

Key words: Hydrogel, Basic fibroblast growth factor, NIH3T3 cells, Collagen, Oxidative stress

中图分类号:

R966

惠文婷,宋潼潼,黄敏,陈霞. 水凝胶递送碱性成纤维细胞生长因子对氧糖剥夺后NIH3T3细胞功能的影响[J]. 吉林大学学报(医学版), 2023, 49(6): 1484-1490.

Wenting HUI,Tongtong SONG,Min HUANG,Xia CHEN. Effect of hydrogel-based delivery of bFGF on function of NIH3T3 cells after oxygen-glucose deprivation[J]. Journal of Jilin University(Medicine Edition), 2023, 49(6): 1484-1490.

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参考文献 26

1	SHINDE A V， FRANGOGIANNIS N G. Fibroblasts in myocardial infarction： a role in inflammation and repair［J］. J Mol Cell Cardiol， 2014， 70： 74-82.
2	DOBACZEWSKI M， DE HAAN J J， FRANGOGIANNIS N G. The extracellular matrix modulates fibroblast phenotype and function in the infarcted myocardium［J］. J Cardiovasc Transl Res， 2012， 5（6）： 837-847.
3	FRANGOGIANNIS N G. The inflammatory response in myocardial injury， repair， and remodelling［J］. Nat Rev Cardiol， 2014， 11（5）： 255-265.
4	KONG P， CHRISTIA P， FRANGOGIANNIS N G. The pathogenesis of cardiac fibrosis［J］. Cell Mol Life Sci， 2014， 71（4）： 549-574.
5	ORNITZ D M， ITOH N. The fibroblast growth factor signaling pathway［J］. Wiley Interdiscip Rev Dev Biol， 2015， 4（3）： 215-266.
6	ORNITZ D M， MARIE P J. Fibroblast growth factor signaling in skeletal development and disease［J］. Genes Dev， 2015， 29（14）： 1463-1486.
7	WANG Y G， WANG D， WU C， et al. MMP 9-instructed assembly of bFGF nanofibers in ischemic myocardium to promote heart repair［J］. Theranostics， 2022， 12（17）： 7237-7249.
8	FORMIGA F R， TAMAYO E， SIMÓN-YARZA T， et al. Angiogenic therapy for cardiac repair based on protein delivery systems［J］. Heart Fail Rev， 2012， 17（3）： 449-473.
9	AWADA H K， JOHNSON N R， WANG Y D. Sequential delivery of angiogenic growth factors improves revascularization and heart function after myocardial infarction［J］. J Control Release， 2015， 207： 7-17.
10	RAO Z H， SHEN D P， CHEN J H， et al. Basic fibroblast growth factor attenuates injury in myocardial infarction by enhancing hypoxia-inducible factor-1 alpha accumulation［J］. Front Pharmacol， 2020， 11： 1193.
11	LI P， HU J J， WANG J， et al. The role of hydrogel in cardiac repair and regeneration for myocardial infarction： recent advances and future perspectives［J］. Bioengineering， 2023， 10（2）： 165.
12	AHMED E M. Hydrogel： preparation， characterization， and applications： a review［J］. J Adv Res， 2015， 6（2）： 105-121.
13	MAMIDI N， VELASCO DELGADILLO R M， BARRERA E V. Covalently functionalized carbon nano-onions integrated gelatin methacryloyl nanocomposite hydrogel containing γ-cyclodextrin as drug carrier for high-performance pH-triggered drug release［J］. Pharmaceuticals， 2021， 14（4）： 291.
14	宋潼潼，惠文婷，顾逸雯，等. 水凝胶负载bFGF对氧糖剥夺条件下树突状细胞的影响［J/OL］. 中国免疫学杂志， 2023： 1-8. （2023-05-15）. .
15	LI P， FENG Z P， YU Z Y， et al. Preparation of chitosan-Cu²⁺/NH₃ physical hydrogel and its properties［J］. Int J Biol Macromol， 2019， 133： 67-75.
16	WANG X， SONG T T， SUN Y P， et al. Proteomic analysis reveals the effect of trichostatin A and bone marrow-derived dendritic cells on the fatty acid metabolism of NIH3T3 cells under oxygen-glucose deprivation conditions［J］. J Proteome Res，2021，20（1）： 960-971.
17	ZHOU X H， HE X L， SHI K， et al. Injectable thermosensitive hydrogel containing erlotinib-loaded hollow mesoporous silica nanoparticles as a localized drug delivery system for NSCLC therapy［J］. Adv Sci， 2020， 7（23）： 2001442.
18	FAN C X， SHI J J， ZHUANG Y， et al. Myocardial-infarction-responsive smart hydrogels targeting matrix metalloproteinase for on-demand growth factor delivery［J］. Adv Mater， 2019， 31（40）： e1902900.
19	VAN DEN BORNE S W M， DIEZ J， BLANKESTEIJN W M， et al. Myocardial remodeling after infarction： the role of myofibroblasts［J］. Nat Rev Cardiol， 2010， 7（1）： 30-37.
20	ROG-ZIELINSKA E A， NORRIS R A， KOHL P，et al.The living scar： cardiac fibroblasts and the injured heart［J］. Trends Mol Med， 2016， 22（2）： 99-114.
21	LIU J， ZHANG M M， QIN C S， et al. Resveratrol attenuate myocardial injury by inhibiting ferroptosis via inducing KAT5/GPX4 in myocardial infarction［J］. Front Pharmacol， 2022， 13： 906073.
22	GONZÁLEZ-SANTAMARÍA J， VILLALBA M， BUSNADIEGO O， et al. Matrix cross-linking lysyl oxidases are induced in response to myocardial infarction and promote cardiac dysfunction［J］. Cardiovasc Res， 2016， 109（1）： 67-78.
23	SINGH D， RAI V， AGRAWAL D K. Regulation of collagen Ⅰ and collagen Ⅲ in tissue injury and regeneration［J］.Cardiol Cardiovasc Med，2023，7（1）：5-16.
24	GUO Z， FAN D， LIU F Y， et al. NEU1 regulates mitochondrial energy metabolism and oxidative stress post-myocardial infarction in mice via the SIRT1/PGC-1 alpha axis［J］. Front Cardiovasc Med， 2022， 9： 821317.
25	LIN Y J， WANG Y， LI P F. Mutual regulation of lactate dehydrogenase and redox robustness［J］. Front Physiol， 2022， 13： 1038421.
26	ZHANG Z， ZHAO X R， GAO M， et al. Dioscin alleviates myocardial infarction injury via regulating BMP4/NOX1-mediated oxidative stress and inflammation［J］. Phytomedicine， 2022， 103： 154222.

Group	Activity of NIH3T3 cells
Group	(t/h) 6	12	24
Blank	0.950±0.049	1.063±0.055	1.005±0.054
20% gelatin	0.956±0.047	0.998±0.041	1.061±0.073
20% gelatin+ glutaraldehyde	0.976±0.058	1.115±0.064	1.069±0.072

Group	α-SMA	Type Ⅰ collagen	Type Ⅲ collagen
Control	0.807±0.077	0.388±0.046	0.564±0.062
OGD	0.984±0.077^*	0.669±0.043^**	0.522±0.068
OGD+1 ng bFGF	0.653±0.034^△△	0.507±0.043^△	0.566±0.038
OGD+10 ng bFGF	0.506±0.072^△△	0.467±0.035^△△	0.518±0.038
OGD+100 ng bFGF	0.628±0.053^△△	0.410±0.053^△△	0.521±0.065

Group	MDA [c_B/(mmol·L^－1)]	LDH [c_B/(U·mL^－1)]	SOD(U/10⁴ cells)
Control	0.071±0.007	0.381±0.008	0.022±0.001
OGD	0.122±0.016^*	0.419±0.005^*	0.014±0.001^*
OGD+1 ng bFGF	0.100±0.014^△	0.383±0.009^△	0.018±0.002^△

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