Effects Of Nonylphenol Subchronic Exposure On Immune-related Factors And Estrogen Receptor Expression In Rats

Part Ⅰ: Effects of subchronic NP exposure on immune function,transcription factors and estrogen receptors in ratsObjective: To explore whether the subchronic exposure of NP180 d in male rats induces immune injury and changes in inflammatory factors,immune factors and estrogen receptors.Methods: SD rats were randomly divided into 5 groups,the control group(Group C)was given 5 ml/kg corn oil every day,the NP dose group: low(L),medium(M),high(H)was given NP(0.4,4,40 mg/kg)by gavage every day,12 rats in each group.In 180 days of continuous gavage,90 days and 180 days were dissected respectively.Rats were anesthetized,dissected and sampled by intraperitoneal injection of 20% ethyl carbamate(0.5ml/100g).Observe the changes of body weight,spleen and thymus organ coefficient of rats;count and quantify the five main inflammatory cells(lymphocytes,neutrophils,monocytes,eosinophils and basophils)under the microscope after Giemsa staining;detect the changes of peripheral blood parameters by blood biochemical analyzer;enzyme linked immunosorbent assay(ELISA,enzyme linked)The levels of IL-4,ER-α and ER-β in serum were measured by immune assay.The ultrastructural changes of peyers’ lymph nodes(periarterial sheath,marginal area,germinal center of lymphoid follicle,lymphocyte,splenic cord and megaphagocyte)in thymus,spleen and ileum were observed by transmission electron microscope.Pathological sections(hematoxylin eosin staining)were used to observe the immune damage of thymus(cortex area,medullary area),spleen(number of splenic nodules,relative area of white pulp,area of lymph nodes),peyers lymph nodes(Lymphatic follicle length meridian germinal center length meridian)in ileum Quantitative analysis was carried out by plus 6.0 software;the levels of Ig E,Ig G and Ig M in serum were detected by UV spectrophotometer;the changes of peripheral blood lymphocyte subsets(T lymphocyte group,B lymphocyte group and NK cell group)were detected by flow cytometry;the changes of ER-α and ER-β in spleen tissue were detected by Immunohistochemistry(IHC)technique,The changes of AP-1,NF-AT and NF-kB in spleen tissue were detected by immunofluorescence(IF)technique.Results: 0.4,4,40 mg / kg NP group was compared with control group: 1)with the change of time and exposure dose,the body weight gradually increased.There was no significant difference among the components in 160 days before exposure(P > 0.05),and there was statistical significance in 160 days after exposure(P < 0.05).There was no statistical difference between each group and the control group(P > 0.05).2)compared with the control group,the weight of thymus and spleen in NP group decreased significantly,and the thymus and spleen coefficients of each group were statistically significant(P < 0.05).3)The results showed that there were significant differences in the total number of neutrophils(P < 0.05),but no significant differences in leukocytes,lymphocytes,monocytes and erythrocytes(P > 0.05).4)The results of HE staining showed that the area of thymic cortex and medulla decreased gradually with the increase of the dose,which was statistically different from that of the blank group(P < 0.05);the number of splenic nodules and the area of splenic nodules,the relative area of white pulp decreased significantly,which was statistically different from that of the blank group(P <0.05).The length of lymph follicle and germinal center in peyers lymph node decreased gradually,which was significantly different from that in the blank group(P < 0.05).5)The results of transmission electron microscopy showed that with the increase of the dose,a small number of apoptotic bodies were formed in the lymphocytes of spleen and thymus,and some of the lymphocytes showed swelling and hypertrophy of mitochondria,local vacuolation,irregular cristae and dilation of endoplasmic reticulum.6)With the increase of dose,the content of Ig G and Ig M in serum decreased compared with the blank control group,the difference between groups was statistically significant(P < 0.05),the content of Ig E in serum increased compared with the blank control group,the difference between groups was statistically significant(P < 0.05).7)Compared with the blank control group,the expression of ER-α and ER-β protein increased significantly(P < 0.05).8)The results of immunofluorescence showed that the expression of AP-1,NF-AT in spleen tissue was relatively decreased.The expression of NF-k B is relatively increased.Compared with blank group,it has statistical significance(P<0.05).Conclusion:1.Sub chronic exposure of 4,40(mg/kg/day)NP for 180 days can lead to immune dysfunction and produce immune function damage and inflammation.2.The relative expression of ER-α and ER-β protein in spleen tissue increased after subclinical exposure of 4,40(mg/kg/day)NP for 180 days.3.Subclinical exposure of 4,40(mg/kg/day)NP for 180 days resulted in a significant increase in IL-4 secretion,and the activity of AP-1,NF-AT,which regulated IL-4 synthesis and secretion,increased,while the relative expression of NF-kB protein decreased.Part Ⅱ:Using bioinformatics to verify and study the relationship between rat estrogen receptors,differential gene bases,and immune-related factorsObjective: To study the m RNA expression of differentially expressed genes in spleen tissue of NP exposed group and control group,and to screen some differentially expressed genes and significant pathway changes between NP exposed group and rats,and to select immune factors related to immune inflammation for further study.The expression of related genes and proteins was observed and verified after subchronic low-dose NP exposure in rats.To explore the relationship between immune injury and inflammation in rats exposed to NP.Methods: High throughput screening technology was used to screen the m RNA expression profiles of 6 independent samples(3 cases in the control group and 3 cases in the high dose group with NP 40mg/kg).The differentially expressed genes of m RNA were obtained by bioinformatics analysis.Furthermore,go and pathway were used to analyze the differentially expressed genes.To integrate and analyze the regulatory network of m RNA and explore its possible molecular regulatory mechanism in estrogen dependent immune inflammation of rats.High performance liquid chromatography(HPLC)was used to detect the content of NP in the spleen and serum of rats after 90 and 180 days of dissection;real-time fluorescence quantitative PCR was used to detect the ER-α and ER-β,NF-kB,IL4,AP-1,NF-AT in the low,medium and high dose NP groups(L,M,H),i.e.the ER-α and ER-β,NF-kB,IL-4,AP-1,NF-AT,The expression of ER-α,ER-β,NF-kB,IL4,AP-1,NF-AT and the expression of differential immune factors were detected by Western blot.The correlation between the above indexes and the content of NP in spleen was analyzed.Results: The high-throughput screening technology was used to screen the different expression patterns of m RNA in the blank control group and the NP 40 mg/kg gavage high-dose group for bioinformatics analysis,810 differentially expressed genes were obtained,363 of which were up-regulated and 447 of which were down regulated.Some of the differentially expressed genes related to immune inflammatory function were down regulated:TLR4,TLR3,TLR11,CD80,Gata3,IL12,TL9 R,MKK3/6,CD21,TGF-α,IAP,XIAP,Up regulated: IL1 R,TNF-a,IL10,TLR2,INOS,IL1 B,CD18,CXCL4.RNA expression of ER-α,ER-β,NF-k B,IL4,AP-1,NF-AT and related differential immune factors was detected by real-time fluorescence quantitative PCR and Western blotting.There was a significant difference between NP(0.4,4,40 mg/kg)group and control group(P < 0.05),The relative expression of ER-α,ER-β,IL4,AP-1,NF-AT m RNA increased gradually(P < 0.05),TLR4,GATA3,NF-kB m RNA decreased gradually,and the change trend of other immune factors was consistent with that of high-throughput screening(P <0.05).Conclusion: The results of high throughput screening verify the results and conclusions of the first part of animal experiments.NP can lead to estrogen dependent immune damage and differential expression of related genes such as immune inflammatory signal transduction,immune response and inflammatory response factors in rats.The above changes may interfere with the function of the immune system and the expression of estrogen.NP may promote the expression of IL-4 by affecting ER-receptor and activating NF-AT transcription factor by binding with AP-1.It can cause the change of NF-kB and other related immunotoxic inflammatory factors,and then affect the expression of the whole immune inflammatory system.With the increase of NP dose and time,the relative expression of ER-α 、ER-β、IL4、AP-1 protein in the spleen of rats increased gradually,while the relative expression of TLR4、GATA3、NF-kB and IL12 protein decreased.There was a significant correlation between the NP content in the spleen and the expression of AP-1,NF-AT,NF-kB,ER-α,ER-β protein in the spleen(P < 0.05),and the levels of IL-4,ER-α,ER-β detected by ELISA.(P < 0.05).