DEHP Causes Liver Tissue Disorders And The Effects In T Cell Subsets In Rats

Plasticizers,are compounds of a class of polymeric materials that are closely related to human life.In the processing of plastic products,adding such substances can increase the flexibility and flexibility of hard polymers such as polyvinyl chloride.Because the chemical structure of the plasticizer is similar to the chemical structure of hormones secreted in vivo,it is also called an “environmental endocrine disruptor.” In vitro and in vivo studies have shown that DEHP and MEHP have anti-inflammatory effects as PPAR-alpha ligands,but it has also been reported that PPAR-alpha ligands have pro-inflammatory effects.Examining the literature finds that in vivo and in vitro studies,interpretation of DEHP-induced hepatotoxicity in immunology has rarely been reported.According to reports,DEHP can directly or indirectly regulate the immune system,and DEHP induces the pathogenesis of liver diseases,which usually involves the participation of the compound itself and its metabolites.They directly affect the differentiation of cells and cause immune responses.In the body,the liver plays a key role in various metabolic functions,and it acts as a central "immunological" organ,and is often in an immune surveillance state compared to other organs.Reading the literature found that DEHP can act as a regulator of the immune system,which interferes with the immune system by regulating lymphocyte differentiation and cytokine synthesis.And the Th1/Th2,Th17/Treg imbalance may play an important role in the promotion of liver inflammation and its damage,and the role of Th1/Th2 and Th17/Treg imbalance in DEHP-mediated liver injury is rarely reported.Our group assumes that DEHP may exert its hepatic impairment through interfering with Th1/Th2 and Th17/Treg ratios.Objective: Explain the effect of DEHP on the disorder of liver function and the change of immune system T cell subsetsMethods: 1.In vivo test: Male SD rats were selected and were 4 weeks old and weighed(140±20)g.After being cultured for one week in the animal experiment center,they were randomly divided into four groups,normal group(unmodeled),Low-dose DEHP group(0.05mg/kg),middle-dose DEHP group(5mg/kg),high-dose DEHP group(500mg/kg),8 in each group.DEHP group was punctually fed from 10 am to 10 am every Monday to Saturday.Once on the stomach,the body weight of the rats was weighed at 8 am Tuesday,and the gavage dose was adjusted for a total of 16 weeks.After the end of the model,the body weight of the rats was weighed,and then the femoral artery was bled and the peripheral blood was collected.Cervical dislocation was performed and the liver and spleen were separated and weighed.The liver index and spleen index of each group of rats were calculated.The indicators of liver injury ALT,AST and oxidative stress indexes SOD,MDA,and GSH were performed with a microplate reader.HE staining was used to observe the pathological changes of liver tissue and spleen in each group.Immunohistochemistry was used to detect the expression of α-SMA protein in liver tissue.Flow cytometry was used to detect T cells(CD3+)and T Levels of cells(CD3+CD4+),Th1,Th17,and Treg.2.In vitro experiments: PBMCs from healthy peripheral blood were selected and divided into 4 groups,which were divided into control group and three treatment groups(5μmol/m L,25μmol/m L,100μmol/m L DEHP group).Different concentrations of DEHP were added to culture medium of human PBMC for culture.The effect of DEHP on the apoptosis of PBMCs was detected by cell probe/PI double staining cell apoptosis method.Th1 and Th2 in peripheral blood PBMCs were detected by flow cytometry.Levels of Th17,Treg and Q-PCR were used to detect changes in T-bet,GATA3,ROR-ΥT,and Foxp3.Result: 1 Evaluation of toxicity of DEHP in SD rats Compared with the normal group,the rats in the low-dose group had lower body weight and hepatic index increased,but there was no significant difference.The body weight in the high-dose group was significantly lower,and the hepatic index was significantly increased in the middle-dose group and the high-dose group,with significant differences.It was demonstrated that DEHP had an effect on body weight and liver index in rats.Compared with the normal group,the spleen index decreased in the high administration group,indicating that DEHP has an effect on the spleen of rat.The ALT and AST levels were measured using a microplate reader.Compared with the normal group,the serum ALT and AST levels in middle-dose group,and high-dose group increased.Microplate readers were used to detect MDA and SOD levels.Compared with the normal group,the levels of MDA in the liver homogenates were increased in high dose groups,and the SOD levels in the liver homogenates were decreased in the middle and high dose groups.HE staining was used to detect the long-term exposure of DEHP to liver damage.Observed under a microscope,the normal hepatic lobule of the rats showed normal,the hepatic cords arranged neatly around the central vein,and there were no obvious lesions in the hepatocytes.Compared with the normal group,the hepatocytes of the low-dose group,the middle-dose group,and the high-dose group exhibited lesions with different degrees,and the liver cords were arranged disorderly.In the gavage group,as the dose of DEHP increased the hepatocellular injury,different degrees of edema and hyaline degeneration occurred,and inflammation and edema increased.In the high-dose group,the hepatocellular injury was the most serious,the cytoplasm was loose,the cells were arranged in disorder,and the necrosis of the lesion was obvious.Immunohistochemistry was used to detect the expression of α-SMA protein.In normal liver tissue,there was mild expression.Compared with normal group,low dose group was used.The expression of α-SMA protein in the middle and high dose groups increased.HE staining was used to detect the effect of prolonged exposure to DEHP on spleen injury.Observed under the microscope,compared with the normal group,the medium density of the spleen tissue around the arterial lymphatic sheath increased the cell density,red pulp congestion and lymph node hyperplasia phenomenon,and high dose group,the density of the peripheral arterial lymphatic sheath cells Increased,white pulp diffuse hyperplasia,red pulp congestion,lymph node hyperplasia phenomenon.Flow cytometry was used to detect the effects of long-term exposure to DEHP on T cells and helper T cells.Compared with the normal group,the levels of T cells and helper T cells in the spleen ofthe middle dose group,and the high dose group increased.Flow cytometry was used to detect changes in Th1 cell subsets.Compared with the normal group,the Th1 levels in the high-dose group decreased,with significant differences.Flow cytometry was used to examine the effect of Th17 cell subsets.Compared with the normal group,the levels of Th17 in the middle and high dose groups increased,with significant differences.The effect of DEHP on Treg cell subsets was detected by flow cytometry.Compared with the normal group,the Treg levels in the high-dose group decreased,with significant differences.2 DEHP evaluation of peripheral blood PBMC in healthy people The effect of DEHP on the apoptosis of PBMC was detected by cell probe/PI double staining apoptosis assay.Compared with the control group,DEHP(25μmol/ml,100μmol/ml)decreased the late apoptosis rate of peripheral blood PBMCs.There are significant differences.Flow cytometry was used to detect changes in Th1 levels.Compared with the control group,DEHP(100μmol/ml)significantly decreased Th1 levels.Flow cytometry was used to detect changes in Th2 levels.Compared with the control group,25μmol/ml,and 100μmol/ml significantly increased Th2 levels.Flow cytometry was used to detect changes in Th17 levels.Compared with controls,25μmol/ml and 100μmol/ml significantly increased Th17 levels.Flow cytometry was used to detect changes in Treg levels.Compared with the control group,DEHP at 25μmol/ml and 100μmol/ml significantly decreased the level of Treg.The transcription factors T-bet,GATA3,ROR-ΥT,and Foxp3 were detected by Q-PCR.Compared with the control group,DEHP at 25μmol/ml and 100μmol/ml significantly decreased the expression levels of T-bet and Foxp3 transcription factors,while DEHP at 25μmol/ml and 100μmol/ml significantly increased GATA3 and ROR-γT transcription factors.conclusion: DEHP promotes the occurrence and development of hepatic injury,and it acts as an immunogenic substance that has pro-inflammatory effects,induces oxidative stress disorder,and thus affects the differentiation of T cell subsets,and the increase of pro-inflammatory Th2 and Th17 Causes hepatic inflammation to worsen.