| With the continuous development of nanotechnology,superparamagnetic iron oxide nanoparticles(SPIO-NPs)are widely used in the biomedical field.Therefore,the potential health risks in the application of SPIO-NPs,such as the hepatotoxicit or liver injury,deserve attention and discussion.Mitochondrial-associated membrane(MAM)is a specialized membrane-associated coupling structure between mitochondria and endoplasmic reticulum(ER)specific sites,with dozens of proteins enriched in it,acting as mitochondrial and ER interactions.The functional platform is closely related to calcium signaling,lipid transport,ER stress,and cell death.And it is a pivot of cellular activity and outcome regulation,affecting the physiological and toxic state of injury induced by xenobiotic.cyclooxygenase 2(COX-2)is an inducible enzyme that is highly induced by proinflammatory cytokines,tumor promoters,mitogens,and growth factors in various cells.COX-2 is involved in inflammatory reactions,cells proliferation and apoptosis.It plays an important role in hepatotoxicity induced by exogenous chemicals.Our group has previously found that xenobiotic can induce the expression of COX-2,its distribution in the mitochondria and endoplasmic reticulum,and cell functional effects.However,the relationship between COX-2 and MAM as well as the role in regulating the hepatotoxicity induced by SPIO-NPs,remains to be studied.Objectives:This study established a model for the toxicological effects of SPIO-NPs on hepatocytes and liver through the SPIO-NPs exposuring.We explored the role of SPIO-NPs-induced COX-2 in the structure and Ca2+ transfer of MAM.The results provide potential targets for the intervention of SPIO-NPs-induced hepatotoxicity and liver injury.Methods:(1)Characterization:Transmission electron microscopy(TEM)and dynamic light scattering(DLS)were used to detect the morphology,particle size and Zeta potential of SPIO-NPs.(2)In vitro:Immortalized human normal hepatic L02 cells were treated with SPIO-NPs.The relevant indicators were detected.?Western blot(WB)was used to detect the levels of MAM-related protein,COX-2 and mitochondria-dependent apoptotic protein;?Proximity ligation assay(PLA)was used to detect the proximity of the MAM protein components,which indicates the status of MAM structure.?TEM for cell MAM structure and spacing to verify the MAM structure changes;?High content analysis(HCA)for cells detection of intracellular MAM Ca2+ transfer reflects the function of MAM;?Immunofluorescence(IF)was used to detect co-localization of cytochrome C(Cyt C)with mitochondria,COX-2 with mitochondria and ER,and Annexin V positive cells.?MAM fractions were purified to detect the distribution and expression of COX-2.?Co-IP was used to detect the interaction between COX-2 and MAM proteins.? Specific siRNAs intervene with GRP75 or COX-2 to verify the role of MAM or COX-2 in the hepatotoxicity induced by SPIO-NPs.?By using Aspirin and Celecoxib,we establish selective COX-2 inhibition model to verify the role of COX-2 for prevention and therapy of the hepatotoxicity induced by SPIO-NPs.(3)In vivo:8 to 10 weeks old male BALB/c mice were selected,SPIO-NPs was administreated(20 mg/kg·bw)via intravenous injection to establish a exposure model;The Celecoxib preconditioning was performed for 24 hours to establish an intervention model.The whole animals,as well as blood and liver tissue samples were performed as follows:? The distribution of SPIO-NPs in mouse liver cells was detected by magnetic resonance imaging(MRI);?The distribution of SPIO-NPs in liver tissues and cells was detected by TEM.? Observing liver pathological changes by HE staining;? Serum AST,ALT were detected for evaluation of liver function;?MAM fraction separation and purification for testing COX-2 content and changes;?TEM observation of MAM structure and spacing in livers;? WB detection for COX-2,MAM and mitochondrial-dependent apoptosis-related protein expression;? IHC for the expression and distribution of COX-2,MAM and mitochondria-dependent apoptosis-related proteins in liver tissue.Results:(1)The particle size of SPIO-NPs was observed to be 21.4±2.3 nm by TEM.The dispersity was good.The particle size was 102.0±56.7 nm in water.Partial agglomeration phenomenon was observed.The Zeta potential was-0.0474±2.28 mV.Neutrality has a certain reunion.(2)In vitro:Compared with L02 cells in control group,?MAM-related proteins(IP3R,GRP75,and VDAC1),COX-2 and apoptosis-related proteins(Cyt C,Bax,and Caspase 3)were increased in SPIO-NPs-treated cells.?PLA results showed that the fluorescence signal of IP3R and VDAC1 protein increased in the SPIO-NPs-treated group;? TEM showed the adjacent structure of MAM in the cells treated with SPIO-NPs(62.5±4.2)was increased(control group,24.2±5.0,P<0.05);? HCA results showed that the average mitochondrial Ca2+ fluorescence level in the cells treated with SPIO-NPs(2997.5±216.7)was increased(2012.6±36.6 in the control group,P<0.05).The average fluorescence level of cytoplasmic Ca2+(425.7±44.6)was increased(256.8±4.7 in control group,P<0.05).And the average fluorescence level of ER Ca2+(3513.3±501.1)was decreased(5158.7±482.8 in control group,P<0.05).It suggests that SPIO-NPs induce Ca2+ transfer of MAM;?Confocal showed that co-localization of COX-2 with ER-Tracker and MitoTracker,mitochondrial Cyt C release and Annexin V positive cell ratio(40.0±1.4)(control group,1.9±1.9,P<0.05)was enhanced in the cells treated with SPIO-NPs.? MAM fractions were seperated and purified to detect COX-2 protein,which showed COX-2 distribution on MAM.The levels of COX-2 in the fractions was increased in SPIO-NPs treated group;?Co-IP results showed that COX-2 was contained in the precipitate of the IP3R-GRP75-VDAC1 complex,suggesting that COX-2 interacts with the MAM proteins;In the SPIO-NPs-treated group of cells,the level of COX-2 in the precipitate of the IP3R-GRP75-VDAC1 complex per unit of VDAC1 was increased;? In the GRP75 knockdown group cells,compared with the SPIO-NPs-treated group,the average mitochondrial Ca2+ fluorescence level(2434.1±506.5)was decreased(3536.4±337.9 in SPIO-NP treatment group,P<0.05).The level of apoptosis-related protein was decreased and the rate of Annexin V positive cells(7.0±1.1)was decreased(31.4±1.0 in SPIO-NP treated group,P<0.05).These results suggested that MAM plays a key role in the SPIO-NPs-induced Ca2+ transfer and hepatotoxicity.In COX-2 knockdown group cells,compared with SPIO-NPs treated group,MAM protein levels,PLA fluorescence of IP3R-VDAC1,MAM adjacent structure(35±5)(63.3±3.3 in the SPIO-NPs treated group,P<0.05),and the average fluorescence level of mitochondrial Ca2+(3011.0±111.1)(3682.4± 116.4 in SPIO-NP treatment group,P<0.05),the level of apoptotic protein and the ratio of Annexin V positive cells(7.7±0.8)(SPIO-NPs treatment group,37.7±1.3,P<0.05)were decreased,suggesting that COX-2 knockdown attenuates MAM structure,Ca2+ transfer and hepatotoxicity induced by SPIO-NPs.? In Aspirin or Celecoxib treatment group cells,compared with SPIO-NPs treatment group,MAM protein levels,IP3R-VDACI PLA fluorescence signal,mitochondrial Ca2+(2818.6 ± 218.9 and 2863.8 ± 100.0,respectively)(3597.3±244.1 and 3417.0±210.0 in SPIO-NPs treatment group,respectively,P<0.05),Apoptosis protein levels and Annexin V positive cells(13.8±0.5 and 13.4±0.9%,respectively)(42.9±7.1 and 42.0±4.1%,respectively,in SPIO-NP treated groups,P<0.05)were decreased,suggesting COX-2 inhibition attenuates the effect of SPIO-NPs on MAM structure,Ca2+tranfer,and hepatocyte toxicity.(3)In vivo:Compared with the control group,? MRI showed that SPIO-NPs accumulated in the liver of mice treated with SPIO-NPs.?TEM showed that SPIO-NPs was present in liver cells of mice treated with SPIO-NPs.NPs particles were included in hepatocytes and intrahepatic Kuffer cells.?HE staining revealed the hepatic cord breaks and vacuolization in the livers of mice treated with SPIO-NPs.Serum ALT and AST levels were elevated,suggesting that the liver function lesion was induced by SPIO-NPs.In the liver of mice treated with Celecoxib,the hepatic cord rupture and tissue vacuolization induced by SPIO-NPs were reduced,suggesting that COX-2 affects SPIO-NPs-induced liver injury.? GOX-2 was detected in MAM of liver of mice,indicating the distribution of COX-2 on MAM;MAM COX-2 was increased in mice treated with SPIO-NPs.In the MAM in liver of mice treated with Celecoxib,the level of COX-2 was decreased.?The MAM interval(45±5)in the hepatocytes of mice treated with SPIO-NPs is increased(19.9±1.6 in the control group,P<0.05).In the Celecoxib treated groups,MAM adjacent structure(22.5±2.5)was decreased(83.5±7.24,P<0.05 for SPIO-NPs treatment group),suggesting that COX-2 inhibition affects the effect of SPIO-NPs on the structure of MAM.? WB results show IP3R,COX-2 and Caspase 3 protein levels were increased in livers of mice treated with SPIO-NPs,which were decreased in the celecoxib treated group;IHC results and WB results were consistent,verifying the changes of SPIO-NPs induced MAM related proteins and apoptosis.Conclusions:Acute exposuring to SPIO-NPs can induce COX-2 expression,MAM disorders and apoptosis in hepatocytes and mouse livers.The induced COX-2 can be localized on MAM and interact with IP3R-GRP75-VDAC1 complex.COX-2 involved MAM structure and Ca2+ tranfer function and regulated SPIO-NPs-induced mitochondrial-dependent apoptosis and liver injury in hepatocytes.COX-2 targeting intervention can inhibit SPIO-NPs-induced hepatotoxicity via changing the structure and function of MAM.The results of this study can provide clues for the safer and more extensive application of SPIO-NPs in the field of biomedicine and the prevention of hepatotoxicity and hepatic injury induced by xenobiotics. |
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