Regulation of astrocyte activity and immune response on graphene oxide-coated titanium by electrophoretic deposition

Abstract

Introduction: Astrocytes play crucial role in modulating immune response in the damaged central nervous system. Numerous studies have investigated the relationship between immune responses in astrocytes and brain diseases. However, the potential application of nanomaterials for alleviating neuroinflammation induced by astrocytes remains unexplored. Method: In this study, we utilized electrophoretic deposition (EPD) to coat graphene oxide (GO) onto titanium (Ti) to enhance the bioactivity of Ti. Results: We confirmed that GO-Ti could improve cell adhesion and proliferation of astrocytes with upregulated integrins and glial fibrillary acidic protein (GFAP) expression. Moreover, we observed that astrocytes on GO-Ti exhibited a heightened immune response when exposed to lipopolysaccharide (LPS). Although pro-inflammatory cytokines increased, anti-inflammatory cytokines and brain-derived neurotrophic factors involved in neuroprotective effects were also augmented through nuclear localization of the yes-associated protein (YAP) and nuclear factor kappa B (NF-κB). Discussion: Taken together, GO-Ti could enhance the neuroprotective function of astrocytes by upregulating the expression of anti-inflammatory cytokines and neuroprotective factors with improved cell adhesion and viability. Consequently, our findings suggest that GO-Ti has the potential to induce neuroprotective effects by regulating cell activity.

Keywords: anti-inflammatory cytokine; astrocyte; electrophoretic deposition; graphene oxide; titanium.

FIGURE 1

Characterization of Titanium (Ti) and Graphene Oxide coated Titanium (GO-Ti) (A) Optical images of Ti and GO-Ti; (B) The microstructure of Ti and GO-Ti obtained using field emission scanning electron microscopy (FE-SEM); (C) EDS spectrum of Ti after a treatment with GO; (D) Raman spectrum of Ti and GO-Ti; (E) Raman mapping of Ti and GO-Ti; (F) The contact angle of Ti and GO-Ti.

FIGURE 2

Cell morphology and attachment of astrocytes on GO-Ti. (A) Representative images of immunocytochemistry with GFAP (green) in primary astrocytes. (B) Representative images of immunocytochemistry with phalloidin (green) in primary astrocytes (left) and cell area of primary astrocytes by image analysis with region of interest (right). (C–F) mRNA expression of adhesion molecules in primary astrocytes at 2, 4 and 24 h by qRT-PCR. Expression of target mRNA was normalized by Hprt. (C) mRNA expression of Itgb1 in primary astrocytes. (D) mRNA expression of Hepacam in primary astrocytes. (E) mRNA expression of Itgav in primary astrocytes. (F) mRNA expression of Itgb3 in primary astrocytes. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. n = 4 from four independent cell preparations and experiments.

FIGURE 3

Cell survival and activity of astrocytes on GO-Ti. (A) Representative images of immunocytochemistry with BrdU (green) and phalloidin (magenta) in primary astrocytes (left) and percentage of primary astrocytes positive for BrdU (right). (B) Cell viability was assayed with CCK-8 for days 1 and 4. (C) mRNA expression of Gfap in primary astrocytes by qRT-PCR at 4 and 24 h mRNA expression of GFAP was normalized by Hprt. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. N = 3 from three independent cell preparations and experiments.

FIGURE 4

The immune response of astrocytes on GO-Ti. (A) mRNA expression of Gfap in primary astrocytes by qRT-PCR after treatment with LPS. (B) mRNA expression of pro-inflammatory cytokines such as Il6, Tnf and Il1b in primary astrocytes by qRT-PCR without or with LPS. (C) mRNA expression of anti-inflammatory cytokines such as Il10 in primary astrocytes by qRT-PCR without or with LPS. (D) Protein expression of BDNF (37 kDa) in primary astrocytes without LPS. (E) Protein expression of BDNF (37 kDa) in primary astrocytes with LPS. Expression of BDNF protein was normalized by $\beta$ -actin (48 kDa). (F) mRNA expression of Tlr4 in primary astrocytes by qRT-PCR with LPS. Expression of target mRNA was normalized by Hprt. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. n = 3 from three independent cell preparations.

FIGURE 5

Total protein expression levels of YAP and NF𝜅B in astrocytes on GO-Ti. (A) Representative images of western blot (YAP) 202 in primary astrocytes without LPS (left) and protein expression of YAP (65kDa) in primary astrocytes without LPS (right). (B) 203 Representative images of western blot (YAP) in primary astrocytes with LPS (left) and protein expression of YAP (65 kDa) in primary 204 astrocytes with LPS (right). (C) Representative images of western blot (NF𝜅B) in primary astrocytes without LPS (left) and protein 205 expression of NF𝜅B (65 kDa) in primary astrocytes without LPS (right). (D) Representative images of western blot (NF𝜅B) in primary 206 astrocytes with LPS (left) and protein expression of NF𝜅B (65 kDa) in primary astrocytes with LPS (right). Expression of YAP and NF𝜅B 207 protein was normalized by β-actin (48 kDa). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. n = 3 from three independent cell 208 preparations.

FIGURE 6

Ratio of YAP and NF𝜅B located in the nucleus of astrocytes on GO-Ti. (A,B) Representative images of 242 immunocytochemistry with phalloidin (green) and YAP (magenta) in primary astrocytes without LPS (left) or with LPS (right) and 243 percentage of YAP located in the nucleus compared to YAP located in the cytosol without LPS (left) or with (right) LPS by image 244 analysis. (C,D) Representative images of immunocytochemistry with phalloidin (green) and NF𝜅B (magenta) in primary astrocytes 245 without LPS (left) or with LPS (right) and percentage of NF𝜅B located in the nucleus compared to NF𝜅B located in the cytosol without 246 LPS (left) or with LPS (right) by image analysis. *p <0.05, **p <0.01, ***p <0.001, ****p <0.0001. For LPS (-) YAP group, n means 247 individual cell numbers. PDL; n = 35, Ti; n = 44 and GO-Ti; n = 48. For LPS (+) YAP group, PDL; n = 35, Ti; n = 42 and GO-Ti; n = 34. For 248 LPS (-) NF𝜅B group, PDL; n = 42, Ti; n = 44 and GO-Ti; n = 44. For LPS (+) NF𝜅B group, PDL; n = 50, Ti; n = 36 and GO-Ti; n = 40.