Graphene Oxide Induce Pyroptosis Through Mitochondrial Oxidative Damage In SH-SY5Y Cell

BackgroundGraphene oxide(GO),as a kind of new carbon-based nanomaterials,has unique physical and chemical characteristics,such as high electrical and thermal conductivity,which make them widely used in the field of neuromedicine.However,studies have reported that GO could cause many kinds of nerve injury,its mechanism of toxicity still remains unclear.It has shown that GO have strong oxidation-promoting effect.When GO uptake by cells,they can induce the generation of reactive oxygen species(ROS),which enhance intracellular redox imbalance,and enhance oxidative stress effect,and ultimately lead to neurotoxicity.However,how does the oxidative stress effect lead to toxicity of nerve cells needs to further study.This study will further explore whether oxidative stress induced by GO mediated mitochondrial oxidative damage and its downstream biological effects.Materials and methodsChapter 1:Characterization of GO and detection of its intracellular uptake and cytotoxicity1.The original particle size and surface morphology of GO were observed by scanning electron microscope(SEM)and atomic force microscope(AFM).2.The chemical bond and element content of GO were detected by X-ray photoelectron spectroscopy(XPS).3.The hydration particle size and agglomeration degree of GO in the suspension were detected by dynamic light scattering(DLS).4.The effect of GO on SH-SY5Y cells survival was examined by CCK-8 cytotoxicity proliferation assay.5.The intracellular uptake of GO was observed by TEM.Chapter 2:GO induced mitochondrial oxidative damage through oxidative stress in SH-SY5Y cells were detected1.The level of cellular ROS after GO treatment were detected by Immunofluorescence(IF).2.The protein levels of mitochondrial antioxidant proteins Trx2 and PRDX3 after GO treatment were detected by Western blotting(WB).3.The level of ROS in mitochondria(mtROS)after GO treatment were detected by IF.Chapter 3:GO induced pyroptosis in SH-SY5Y cells1.The release of intracellular LDH after GO treatment was measured by Lactate dehydrogenase(LDH)kit.2.The expression and distribution of PI were detected by IF.3.The expression of NLRP3,Caspase-1,Cleaved Caspase-1,and GSDMD after treatment of GO were detected by WB.4.The levels of inflammatory cytokines IL-1β and IL-18 in cell medium were detected by enzyme-linked immunosorbent assay(ELISA).Chapter 4:The important role of mtROS in GO-induced pyroptosis1.The level of mtROS after Mitotempo and GO treatment was detected by IF.2.The integrity of cell membrane after Mitotempo and GO treatment was detected by LDH kit and IF.3.The level of pyroptosis related proteins after Mitotempo and GO treatment was measured by WB.4.The levels of IL-1β and IL-18 after Mitotempo and GO treatment was measured by ELISA.Results1.SEM and AFM images showed that GO was lamellar with an average particle size of 300～800 nm,the thickness was 1 nm,the surface was rich in oxygencontaining functional groups;In aqueous solution,the average particle size was 741.50±205.40 nm,and the Zeta potential was-29.40 ± 5.10 mV.2.TEM detected that GO was absorbed into SH-SY5Y cells;With the increase of GO concentration,the survival rate of SH-SY5Y cells decreased significantly.3.GO increased the generation of ROS,decreased mitochondrial antioxidant enzymes PRDX3,Trx2 protein expression,and thus increased the formation of mtROS and mitochondrial oxidative damage.4.GO upregulated NLRP3 inflammasome,Cleaved Caspase-1 and GSDMD expression,and increased the level of IL-1β and IL-18,which induced pyroptosis in SH-SY5Y cells.5.Mitotempo inhibited the production of mtROS,decreased the expression of NLRP3,Cleaved Caspase-1,and GSDMD,inhibited the release of inflammatory cytokines IL-1β and IL-18,and alleviated GO-induced pyroptosis.Conclusion1.GO can cause concentration-dependent cytotoxicity to SH-SY5Y cells.2.GO can promote mtROS accumulation and enhance mitochondrial oxidative damage by increasing intracellular ROS generation and inhibiting the expression of PRDX3 and Trx2.3.GO induce pyroptosis by activating the NLRP3 inflammasome,promoting the Caspase-1 cascade,and enhancing the release of IL-1β and IL-18.4.Mitotempo can inhibit the production of mtROS and alleviate GO-induced pyroptosis.