Effect Of Graphene Oxide On Lung Injury Induced By Paraquat Poisoning Preliminary Study On Cytotoxic Mechanism

PART I Preliminary study on the role of graphene oxide in acutelung injury caused by paraquatObjective:To observe the effect of graphene oxide on the model of acute lung injury(ALI)induced by paraquat poisoning in rats.Methods:Healthy adult male Sprague-Dawley rats were randomly divided into 3 groups(about 200 g per rat)by random number table,ie normal control group(NS group),paraquat group(PQ group),paraquat + graphene oxide.Group(PQ+GO group),6 in each group.NS group:prepared by intraperitoneal injection of an equal volume of normal saline(NS).PQ group(paraquat ALI model):prepared by single intraperitoneal injection of 20 mg/kg paraquat solution into rats;PQ+GO group:after successful preparation of paraquat acute lung injury model for 1 h,corresponding graphite oxide The ene solution(concentration 6.25 mg/ml)was obtained by one-time gavage.Rats in three groups were quickly sacrificed within 24 hours after successful model making and were harvested(abdominal aortic blood and lung tissue).The wet/dry(W/D)ratio of lung tissue in each group was compared:hematoxylin-eosin staining(HE)was used to observe the gross lung disease,inflammatory cell infiltration,pulmonary congestion and edema in rats;TUNEL apoptosis was observed.The detection method was used to detect the degree of apoptosis of rat lung tissue cells,and the abdominal aortic blood was collected.The tumor necrosis factor-a(TNF-?)was detected by enzyme-linked immunosorbent assay(ELISA).?),the expression level of serum amyloid A1(SAA1).Results:The ALI rat model was successfully established.Compared with the NS group,the wet/dry(W/D)ratio and the pathological lesions of the lungs in the PQ group were significantly increased,while the PQ+GO group had a W/D ratio and a pathological lesion in the lung compared with the PQ group.The results of TUNEL apoptosis showed that the degree of apoptosis in PQ group increased,but the degree of apoptosis decreased after a certain concentration(6.25 mg/ml)of graphene oxide(GO).The difference was statistically significant(P<0.05);TNF-? and SAA1 were significantly increased in arterial blood supernatant.The concentration of TNF-? and SAA1 in rats was significantly increased after a certain concentration(6.25 mg/ml)of graphene oxide(GO).The paraquat group was reduced,the difference was statistically significant(P<0.05).Conclusion:A certain dose of graphene oxide solution(6.25 mg/ml)can relieve AL1 caused by paraquat poisoning.PART ? Study on the toxicity mechanism of graphene oxide onhuman renal tubular epithelial cellsObjective:To investigate whether GO can produce cytotoxic effects on human renal tubular epithelial cells HK2 through oxidative stress-induced apoptosis mechanism mediated by increased ROS.Methods:HK2 cells were treated with different concentrations of GO(0,25,50,75,100 ?g/ml)for 6 h,and the effect of GO on proliferation of HK2 cells was determined by CCK-8 method.Different concentrations of GO were used(0,25,50,HK2 cells were treated with 75 and 100 ?g/ml for 6 h,respectively,and the oxidative stress inhibitor NAC was used to intervene to compare the amount of ROS produced in HK2 cells.The apoptosis rate of the cells was measured by a flow cytometer.Results:GO inhibits the proliferation of HK2 cells in a concentration-dependent manner and promotes apoptosis.After GO treatment of cells,the expression of reactive oxygen species ROS was significantly increased,and the expression of ROS in cells was significantly decreased after pre-application of oxidative stress inhibitor NAC(Acetylcysteine N-acetyl-L-cysteine).The apoptotic rate also decreased,and NAC attenuated the cytotoxic effect of GO on renal tubular epithelial cells.The difference was statistically significant(P<0.05).Conclusion:GO may produce cytotoxic effects on renal tubular epithelial cells HK2 through oxidative stress pathway and induce apoptosis.PART III Study on the Toxicity Mechanism of Graphene Oxide to Human Nasopharyngeal Carcinoma CellsObjective:To investigate the effect of ERS-induced autophagy pathway on GO-induced apoptosis of human nasopharyngeal carcinoma cell line HONE1 and its cytotoxic mechanism.Methods:HONE1 cells were treated with different concentrations of GO(0,25,50,75,100 ?g/ml)for 6 h.The effect of GO on cell proliferation was determined by CCK-8 method.The expression of GRP78 and LC3B was detected by Werstern blot.RT-The expression of GRP78 and LC3B mRNA was detected by PCR.The expression of LC3B was detected by indirect immunofluorescence.Autophagosomes were observed by TEM.The effect of GO on apoptosis was detected by flow cytometry.Results:GO inhibited the proliferation of HONE1 cells in a concentration-dependent manner and promoted apoptosis.After treatment of cells with GO,GRP78,LC3B protein and mRNA expression were increased.Indirect immunofluorescence can detect the enhancement of intracellular LC3B fluorescence intensity.Autophagosomes were observed in TEM.After pretreatment with the endoplasmic reticulum stress inhibitor 4-PBA,4-PBA attenuated GO-induced apoptosis of HONE1 cells and inhibited the expression of LC3B,P-values of less than 0.05 were considered to indicate statistically significant differences.The difference was statistically significant(P<0.05).Conclusion:GO may produce cytotoxicity to HONE1 through endoplasmic reticulum stress-autophagy pathway and induce apoptosis.