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Pharmaceutics 2023, 15, x FOR PEER REVIEW 11 of 16 Pharmaceutics 2023, 15, 993 blocks IkB-a’s proteasomal degradation and eventually results in the inhibition of the nu- 11 of 15 clear translocation of NF-kB [43]. Nanomaterials could induce inflammatory responses in cells and thus activate the Nrf2 and NF-kB pathways [44]. To enhance our toxicity assess- ment of bG and cG, we tested whether different doses of these compounds influence the In NIH/3T3 cells, an increase in Nrf2 levels was observed at both bG- and cG-treated expression of the proteins Nrf2, NF-kB and HO-1. cells, with the relative amount of protein being significantly higher in cG- (about 2.5-fold In NIH/3T3 cells, an increase in Nrf2 levels was observed at both bG- and cG-treated cehllas,nwgiethctohmerpelatrievdeatmoocuonnttorfoplr)otheiannbebinGg-tsrigenaitfiecdan(talybhoiughte1r-ifnoclGd-c(habaonugte2.)5(-fFoilgdure8a).Aslight change compared to control) than bG-treated (about 1-fold change) (Figure 8a). A slight increase was also observed in HO-1’s relative amount in cG-treated cells (Figure 8c). The increase was also observed in HO-1′s relative amount in cG-treated cells (Figure 8c). The expression of the p65 protein was not affected by the two nanomaterials (Figure 8b). expression of the p65 protein was not affected by the two nanomaterials (Figure 8b). Figiguurer8e.8R.elRateivlaetaimveouanmtofuNnrtf2o(faN),prf625(ba),anpd65HO(b-1) a(cn)dinHNIOH-/13T(3c)ceinllsN.*ISHta/tis3tTic3alclyesllisg.ni*fiScatantistically significant difference from control (p < 0.05). difference from control (p < 0.05). In HaCaT cells, treatments with 50 and 100 μg/mL of bG doubled the expression of In HaCaT cells, treatments with 50 and 100 μg/mL of bG doubled the expression of Nrf2 protein (about 1-fold change). The treatment with 50 μg/mL of cG had the same effect Nrf2 protein (about 1-fold change). The treatment with 50 μg/mL of cG had the same (Figure 9a). This increase was not combined with an increase in HO-1′s expression (Figure 9ecf)f.eTchte(rFeilagtuivrea9mao)u. nTthoifspi6n5crreemaasienewd ausncnhoantgceodminbtirneaetdedwceitllhs (aFniguinrecr9eba).se in HO-1’s expression (Figure 9c). The relative amount of p65 remained unchanged in treated cells (Figure 9b). The treatment with both nanomaterials in THP-1 derived macrophages had a different effect compared to other cell lines. Both bG and cG did not activate Nrf2 in any dose tested (Figure 10a) but there was a mild dose-dependent activation of HO-1 in bG-treated cells compared to the control (Figure 10c). Moreover, cG induced a significant increase in the p65 amount which was also dose-dependent (about 1-fold change and 2-fold change, for 20 and 50 μg/mL, respectively) (Figure 10b). Our Western blot analysis correlates with our previous results and confirms that both nano-compounds act differently in each cell line. According to the literature, the activation and interaction of Nrf2 and NF-kB pathways are cell-type specific [45]. In NIH/3T3 cells, cG induced a higher increase in Nrf2 and HO-1 levels compared to bG- treated cells, indicating a possible stronger influence in the immune system which triggers Nrf2’s activation. In HaCaT cells, high doses of bG and cG activated Nrf2, but this increase was not combined with an increase in HO-1. Finally, in THP-1 cells, bG seems to activate HO-1 but through mechanisms that involve transcription factors other than Nrf2 and NF-kB, such as, activator protein-1 (AP-1) and hypoxia inducible factor (HIF) [46]. Moreover, the significant increase of p65 in cG-treated THP-1-derived macrophages indicates a possible toxicity and inflammatory response induced by high doses of cG [47,48].PDF Image | Green Exfoliation of Graphene
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