| BackgroundGraphene oxide(GO),as two-dimensional carbon nanomaterials with large surface area,is generated from graphene.Recent years,GO has been applied widely in electronic,machine,optics,food and clothing product fields,especially in biomedicine including gene and drug delivery,biosensor and image monitor for disease diagnosis and therapy.Human can be exposed to GO by inhalation in occupational environment,by biomagnification in water and soil,by oral and intravenous routes in biomedicine.Such wide applications and complex exposure routes of GO cause increasing healthy risks to human and environment.In addition,embryo-fetus development during pregnant period,sensitive to endogenous and exogenous exposures,is demonstrated to be associated with postnatal health,even with lifelong diseases.A large number of epidemiological and animal studies have confirmed that PM2.5,cadmium and tobacco can cause a series of adverse pregnancy outcomes such as miscarriage,low birth weight,increased malformation rate and stillbirths.However,existing studies about reproductive and development toxicity of GO are only limited in zebrafish and Caenorhabditis elegans models,indicating that GO exposure can increase mortality of embryo,larval development delay and organ malformation.It is regrettable there is no study about embryo toxicology of GO exposure during pregnant period in mammals.Gut microbiome is a large microbial community that colonizes in intestinal tract,in which the number of bacteria accounts for>99%.Different bacteria interact with each other and keep in a balanced proportion,promoting host energy absorption,nutrition metabolism and immune regulation.With a deep understanding of the associations between gut microbiome with physiological and pathological conditions,the role of maternal gut microbiome in mother health and fetus development attracts more and more interests recently.It has been confirmed that maternal gut microbiome in the late trimester differs from other pregnant stages,showing an increasing community diversity to satisfy energy and metabolic demands.Conversely,gut microbiome dysbiosis is indicated to link with intrauterine growth restriction of embryo,premature birth,stillbirths and preeclampsia.Even exogenous exposures such as PM2.5 and Ti02 nanomaterials,can modulate maternal gut microbiome and induce embryo-fetus development toxicology.Therefore,it can be concluded that maternal gut microbiome can affect pregnant outcomes.GO can cause cell rupture even death by mechanical injury or adhesion to cell membrane owing to its two-dimensional structure.And,it is indicated that oral exposure of GO can impair gut microbiome of non-pregnant mice and zebrafish.Above findings suggest GO exposure during pregnancy has the potential of modulating maternal gut microbiome,which is unknown to corelate with GO-associated pregnant outcomes.Additionally,fecal microbiota transplantation(FMT)has been applied in clinical aspects to treat inflammatory bowel disease,diarrhea and other intestinal diseases with cure rate up to 90%.Even,FMT can alleviate the symptoms of hepatic encephalopathy and Parkinson’s disease caused by toxic.FMT is an effective strategy to intervene pathological damages and furtherly identify the role of gut microbiome.Herein,we aimed to explore the effects of GO exposure on maternal microbiome and to clarify the causality between GO-mediated gut microbiome and pregnancy outcomes by FMT intervention.ObjectiveWe firstly aimed to established pregnant mice model by oral exposure of GO during pregnancy,to indicating the embryo toxicology of GO.We furtherly detected placenta changes to understand the mechanism of embryo toxicology caused by GO.Then,establish FMT model to verify the effects of oral exposure of GO on gut microbiome and its role in embryo toxicology.Finally,we selected intravenous exposure route,indirectly contacting to intestinal tract,to explore whether the role of GO-mediated gut microbiome in embryo toxicology linked with exposure routs.The results could provide reference and intervention strategy for safety assessments and mechanism study of GO or other exogenous exposures during pregnancy.Methods(一)Oral exposure of GO during pregnancy caused embryo toxicology and placenta damages1.Characteristics of GO①Transmission Electron Microscope(TEM)was used to observe the morphology of GO.②Atomic Force Microscopy(AFM)was used to observe the surface morphology and thickness of GO in water and simulated intestinal fluid.③Dynamic Light Scattering(DLS)was used to detect size distributions and Zeta potentials of GO in water and artificial intestinal fluid.2.Establishment of pregnant mice model by oral exposure of GOICR pregnant mice on gestational day 1(GD1)were randomly divided into the control group,low(2mg/kg),medium(10mg/kg)and high(40mg/kg)dose groups(n=10),in which pregnant mice were orally exposed to GO solutions by 0.2mL/10g(v/w)daily on GD7-GD16.Fresh samples were collected on GD19 for further detections.3.Maternal systemic toxicology assessment①Maternal body weight and important organ weight were recorded on GD19 to calculate net body weight,net body weight gain and organ coefficients.②Serum biochemical indexes were detected by AU480 automatic analyzer.4.Embryo toxicology assessment①The number of abortion,absorbed fetus,stillbirth and live fetus were recorded on GD19.②Average birth weight,tail length and head-hip length of fetus were recorded.③ Alizarin red staining was performed to observe fetus skeletal development.5.Detections of placenta structure and factor expression levels① H&E staining was used to observe placenta structure.②The ratio of glutathione(GSH)/oxidized glutathione(GSSH)and the activity of superoxide dismutase(SOD)were determined by kits.③mRNA levels of Claudin1,Occludin,vascular endothelial grown factor A(VEGFA)and interleukin-6(IL-6)were detected by RT-qPCR.④ELISA was used to detect VEGFA and IL-6 protein levels.⑤Western blot was used to detect Claudinl and Occludin protein levels.⑥Immunofluorescence(IF)was used to qualitatively observe the distributions of Claudinl and Occludin.6.Statistical analysisThe data were recorded in Excel,then SPSS 20.0 software was used for statistical analysis with two-tail test(P<0.05).For homogeneity variables,ANOVA was used to analyze the differences among groups and Dunnett’s T test was used to analyze inter-group differences.The differences of categorical variables were analyzed by x2 test.(二)The role of gut microbiome associated with oral exposure of GO in embryo toxicology1.Intestinal damage examinations①H&E staining was used to observe colon structure.②Fluorescence intensity of 4kDa FITC-Dextran in serum was detected to indicate intestinal permeability changes.③mRNA levels of Claudinl and IL-6 in colon were detected by qRCR.2.Gut microbiome was detected by 16s RNA sequencing①Fresh feces of pregnant mice were collected on GD19.②Genomic DNA extraction.③PCR amplification and purification.④Library establishment and sequencing.⑤Bioinformatics analysis including species annotation,diversity analysis,community components and correlation analysis,were carried out based on the sequencing results.3.Establishment of FMT model①We designed control,GO group by oral exposure and FMT group.②Pregnant mice in GO group and FMT group exposed to GO by gavage daily from GD7-GD16 at the dose of 40mg/kg.③Fresh feces from pregnant mice in control group were dissolved into sterile PBS,and the bacterial suspensions were prepared after being mixed,centrifugated and washed.④Pregnant mice in FMT group were given 100μl fecal bacteria suspension by clyster daily from GD14 to GD18,and control pregnant mice were treated by PBS.⑤Fresh tissues and feces were collected on GD19.(三)To verify the role of GO-mediated gut microbiome in embryo toxicology under intravenous exposure route1.Establishment of pregnant mice model by intravenous exposure of GO①Exposure mode:intravenous injection of 5μL/g saline or GO solutions daily on GD12 and GD13.②Calculate LD50 by fitting the curve between GO exposure dose and mortality rate of pregnant mice.③We designed the control group,low(0.25mg/kg),medium(1.25mg/kg)and high dose(2.5mg/kg)groups(n=10).④Record variables of pregnancy outcomes and collect fresh tissues on GD19.2.Establishment of FMT model①We designed control,GO group by intravenous exposure and FMT group.②Pregnant mice in GO group and FMT group exposed to GO by intravenous injection daily on GD12 and GD13 at the dose of 2.5mg/kg.③The method of FMT was stated as described above.Results(一)Oral exposure of GO during pregnancy caused embryo toxicology and placenta damages1.Characteristics of GOThe TEM images revealed the wrinkle on surface,meaning a good flexibility of GO.The AFM images showed that the thickness of GO was about 4nm and the size diameters were 0.1-0.5μm and 0.2-1μm,respectively in water and simulated intestinal fluid.DLS data showed the average hydrodynamic diameters and Zeta potentials of GO were 236±5nmand-17±3 mV in water,766±179nm and-22±7 mV in simulated intestinal fluid,respectively.These results suggested well dispersion and good stability of GO,judging from a higher Zeta potential in simulated intestinal fluid.2.No obvious systemic toxicology caused by GOWith comparison to the control,maternal body weights on GD19 in medium and high dose groups significantly decreased by 5.67g(P<0.05)and 8.38g(P<0.01),and total body weight gains decreased 5.6g(P<0.01)and 7.58g(P<0.01),no obvious changes were seen in low dose group.Exclusion of gravid uterus weight,there were no differences seen in net maternal body weight on GD19 and net body weight gain among groups.Additionally,no differences were seen in serum biochemical indexes among groups.3.Oral exposure of GO caused embryo toxicologyComparison to the control,oral exposure of GO caused dose-dependently increasing number of abortions,resorbed fetus and stillbirth,meanwhile,average number of live fetus decreased gradually.Then,average fetus birth weight in medium and high dose groups decreased by 60mg(P<0.05)and 100mg(P<0.01),contributing to a decreasing weight of gravid uterus and single uterus(P<0.01).There were no morphology and visceral deformity in GO groups.While,head-hip length in medium and high dose groups shortened by 2mm(P<0.01)and 3mm(P<0.01).Skeletal staining results showed that GO induced occipital,rib and four limb bone dysplasia.Especially,ossification points of posterior phalanges and carpus in high dose group reduced significantly(P<0.05).4.Oral exposure of GO caused placenta damages(1)Placenta structure:H&E staining results showed no obvious placenta structure abnormality in low dose group.When the exposure dose was over 10mg/kg,the trophoblast cells arranged disorderly and spiral artery narrowed.(2)In generally,factors levels in placenta changed dose-dependently.Compared with the control,mRNA and protein levels of VEGFA in medium and high dose groups decreased by about 1/3(P<0.05)and 3/4(P<0.01).mRNA levels of Claudin1 and Occludin in the medium and high dose groups only accounted for 30%-50%of that in the control group(P<0.05),while protein levels accounted for 40%-60%(P<0.05).SOD activity and GSH/GSSG ratio in medium and high dose groups obviously decreased by 25%and 35%(P<0.05).IL-6 mRNA levels in medium and high dose groups increased by 100%(P<0.05)and 170%(P<0.01),and protein levels increased by 20%(P<0.05)and 43%(P<0.01).(二)The role of gut microbiome associated with oral exposure of GO in embryo toxicology1.Oral exposure of high dose of GO induced intestinal damagesH&E straining results showed high dose of GO exposure induced incomplete intestinal epithelium,epithelial cell disarrangement,and obvious vacuolization.Serum fluorescence intensity of FITC-Dextran in exposure group doubled to that of the control group(P<0.01).Comparison with the control,mRNA levels of Claudinl decreased by 87%(P<0.05),while IL-6 mRNA levels increased significantly(P<0.05).2.Oral exposure of high dose of GO induced gut microbiome dysbiosis(1)Species richness and α diversity:The number of OTUs noted in GO group(1132)was significantly lower than that the control(1540),consisted with the rank and rare curves.Additionally,α diversity indexes including observed species,ACE and Chao1 in GO group significantly decreased compared to the control(P<0.05).(2)Community components:The results of principal component analysis showed that gut microbial community in GO group significantly differed from the control group.β diversity in GO group was significantly lower than the control group(P<0.05).The relative abundances of the top ten species at the phylum level changed obviously in GO group,with increased Firmicutes/Bacteroide ratio.(3)Spearman correlation analysis:Decreased a diversity indexes and species richness negatively correlated with IL-6 level in placenta(P<0.05),and positively correlated with pregnancy outcome variables and other factors levels in placenta.3.FMT improved GO-mediated gut microbiome dysbiosis(1)Species richness and a diversity:Compared with GO group,the number of OTUs being shared in FMT group and the control group increased.Care curve showed the decreased species richness could partly reversed by FMT.In addition,FMT could obviously restore the decreased a diversity indexes caused by GO exposure.(2)Bacterial community components:Different bacteria in GO group could be reversed in a certain extent by FMT.Increased Firmicutes/Bacteroide ratio in GO group was significantly restored by FMT.T-test results showed that the relative abundance of Lactococcus in GO group decreased to 0.74%(P<0.05)and increased to 4.4%in the FMT group,a little lower than the control(5.5%).4.FMT partly alleviate embryo toxicology caused by oral exposure of GOCompared to the GO group(55.08±3.88g),maternal body weight on GD19 and total body weight gain in FMT group increased 2.32g,while lower than that of the control group(63.44±3.814g)(P<0.01).Exclusion of gravid uterus weight,there were no differences in net body weight on GD19,net body weight gain and important organ coefficients among groups.Then,differences analysis results of pregnancy outcome showed FMT had no effects on the number of resorbed fetus,stillbirths and average live fetus in GO group.Average fetus birth weight in GO group was 1.37g,a decline of 100mg compared to the control(P<0.01),while FMT improved fetus birth weight to 1.40g,that had no difference to the control.Gravid uterus weight and single weight in FMT group increased significantly compared to that in GO group.(三)To verify the role of GO-mediated gut microbiome in embryo toxicology under intravenous exposure route1.No obvious maternal system toxicology caused by intravenous exposure of GOThere were no differences of net body weight on GD19,net body weight and important organ coefficients between GO group and the control.H&E straining results showed GO nanomaterials deposited in lung in high dose group.While,no differences were seen in serum biochemical indexes among groups.2.Intravenous exposure of GO caused dose-dependent embryo toxicologyCompared to the control,the number of abortions,resorbed fetus and stillbirth in GO groups increased dose-dependently.Average number of live fetus in medium and high dose groups decreased by 28.15%(P<0.05)and 43.64%(P<0.01),and birth weight of live fetus decreased by 70mg and 130mg(P<0.01).Meanwhile,gravid uterus and single uterus weight reduced obviously(P<0.01).There were no differences of the above variables between low dose group and the control.3.FMT improved the modulated gut microbiome caused by intravenous exposure of GO(1)Intestinal permeability:Serum fluorescence intensity of FITC-dextran on GO group and FMT group increased significantly compared to the control(P<0.05).(2)Species richness and a diversity:The number of microbial species and specific OTUs in FMT group slightly increased compared to GO group,but still less than that in control group(P<0.05).FMT could partly reverse the significant decline of ACE and Chaol in GO group.(3)Community components:β diversity in both FMT group and control group were higher than that in GO group(P<0.01).FMT could reverse the increased abundances of Firmicutes and Staphylococccus in GO groups,which were opposite to the changing trends of Bacteroide,Acidobacteria and Lactococcus.Abnormal Firmicutes/Bacteroide ratio and hydrocarbon degradation ability in GO group could be corrected by FMT.4.FMT improved low birth weight of fetus caused by intravenous exposure of GOMaternal body weight on GD19 and total body weight gain in FMT group,though lower than the control(P<0.01),increased significantly(P<0.05)with comparison to that in GO group.While,no obvious differences in net body weight on GD19,net body weight gain and organ coefficients among groups.In addition,FMT had no functions on the increased number of resorbed fetus and stillbirths,also the decreased average number of live fetus in GO groups.Importantly,average birth weight of fetus in FMT group(1.40±0.02g)was lower than that in control group(1.47±0.04g)(P<0.01),but higher than that in GO group(1.34±0.04g)(P<0.01).5.No effects of FMT on placenta damages caused by intravenous exposure of GOH&E straining results indicated abnormal placenta structure in both GO and FMT group with disarranged trophoblast cells and narrowed spiral artery.FMT had no effects on the decreased levels of VEGFA mRNA and protein,also increased levels of IL-6 mRNA and protein levels.Conclusion1.Both oral and intravenous exposure of GO during gestation induced dose-dependent embryo toxicology,showing decreased maternal body weight and gravid uterus weight,increased number of resorbed fetus and stillbirths,decreased number and birth weight of live fetus,also skeletal development delays.Under similarly decreased decline of fetus birth weight,intravenous exposure of GO induced a larger number of resorbed fetus and stillbirths,lower gravid uterus weight and fetus birth weight.2.Intravenous exposure of GO induced more obviously decreased gut microbial species richness and components than oral routes.In addition,β diversity and Lactococcus decreased,oppositely to Firmicutes/Bacteroide ratio,in GO-exposed pregnant mice by both exposure routes.3.FMT reversed the decreased species richness and a diversity caused by oral exposure of GO rather than intravenous route;FMT had functions on the decreased βdiversity,Lactococcus and Firmicutes/Bacteroide ratio.4.FMT could improve maternal body weight and fetus birth weight,but didn’t affect placenta structure damage and inflammatory response.The results indicated that GO-mediated gut microbiome dysbiosis aggravated embryo toxicology.5.GO exposure induced placenta damages including structure abnormality,decreased levels of placenta barrier factors,increased oxidative stress and inflammatory response.But,FMT had no effects on GO-induced placenta changes. |
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