Di-(2-ethylhexyl)phthalate Facilitates The Progression Of Hepatocellular Carcinoma In Mice By Activating The JAK2/STAT3 Signaling Pathway

As an endocrine disruptor,Di（2-ethyl）hexyl phthalate（DEHP）is often used as a plasticizer in various PVC plastic products and medical consumables.In epidemiological studies,it has been shown that long-term high intake of DEHP may impair liver function.Liver-related diseases will be affected by long-term exposure to DEHP.When people are exposed to DEHP for a long time,the degree of liver damage may become serious.However,there are few studies on the effects of DEHP on hepatocellular carcinoma（HCC）.In our study,diethylnitrosamine（DEN）combined with carbon tetrachloride（CCl₄）was used to establish HCC model,to study the effects of DEHP on HCC,and discuss its molecular mechanism.It was found that DEHP exposure significantly promotes tumor immune escape and activates signaling pathways involved in related protein expression of tumor immune escape,including PD-L1,JAK2,and STAT3.In addition,the trends observed in the HepG2 cells assay are consistent with vivo conditions.In summary,DEHP may play a tumor-promoting role in HCC mice and IFN-γstimulated HepG2 cells,which may be related to the JAK2/STAT3 signaling pathway.OBJECTIVE:Intraperitoneal injection of CCl₄and DEN were used to induce HCC model to discuss the effects of DEHP on the pathological process of HCC mice.At the same time,HepG2 cells were used in vitro experiments to further explore the role of PD-L1 and JAK2/STAT3 signaling pathways in HCC.METHODS:DEN and CCl₄ were injected into the abdominal cavity of C57BL/6mice to construct HCC model in vivo experiment.The animal experiment was divided into six groups:normal group,DEN+CCl₄group,DEN+CCl₄+0.1 mg/kg DEHP group,DEN+CCl₄+10 mg/kg DEHP group,DEN+CCl₄+1000 mg/kg DEHP group,1000 mg/kg DEHP group.After eighteen weeks,mice were sacrificed.HE staining to observe the pathological changes of DEHP on the liver of mice with hepatocellular carcinoma induced by CCl₄combined with DEN.Divide mouse liver weight by body weight to get the corresponding liver index.Use biochemical kits to detect serum indicators,including aspartate aminotransferase（AST）,alpha-fetoprotein（AFP）,etc.Use vernier calipers to measure the size of tumors and visually observe and count the number of tumors.Then use ELISA to detect the levels in the liver tissues of tumor necrosis factor-α（TNF-α）and interleukin-6（IL-6）.Detect the expression of P-STAT3,P-JAK2 and PD-L1 in liver tissue by Western blot.Distribution and expression of PD-L1 proteins in liver tissue were detected by laser scanning confocal microscopy（LSCM）.In vitro,six groups were set up:normal group,IFN-γ（30 ng/m L）,IFN-γ+DEHP（12.5,50,200μmol/L）,DEHP 200μmol/L control group.After 48 hours of in vitro culture,the effect of DEHP on HepG2 cell viability was studied,and the kit used was CCK-8kit;the effect of DEHP on the expression of PD-L1 m RNA in HepG2 cells was detected by RT-PCR;in the in vitro experiment,To study the effect of DEHP in the migration process of HepG2 cells,the detection method is Transwell detection;the cell protein extraction operation is performed to detect the expression of P-STAT3,P-JAK2 and other proteins in the cell,and the detection method used is Western blotting.（Western blot）;Laser confocal observation of PD-L1 expression and distribution in HepG2 cells.RESULTS:1.Effects of long-term exposure to DEHP on liver function indexes and liver tumors in miceAfter the mice were treated with DEHP by gavage for 3 months,the results showed that the liver index of the mice had a significant upward trend.In the detection of biochemical indicators in serum,the results showed that the activity levels of AST and ALT had a significant upward trend.The tumor size and number of mice in the DEHP high-dose group increased significantly.In ELISA kit detection,DEHP makes the content of AFP in the liver tissue of mice have a significant upward trend.It is reminded that long-term high intake of DEHP will aggravate liver damage in HCC mice.2.Effects of long-term exposure to DEHP on the levels of interleukin 6（IL-6）and inflammatory factor tumor necrosis factor（TNF-α）in liver tissues of mice with HCCComparing the model group and the control group,it is clear that the former has a higher level of IL-6.Comparing the model group and the DEHP group with different concentrations,it is clear that the IL-6 levels of the DEHP 0.1 and 10 mg/kg groups did not change significantly,and the DEHP 1000 mg/kg group had higher IL-6 levels.Comparing the DEHP 1000 mg/kg control group and the control group,it is clear that the former has a higher IL-6 levels.Comparing the model group and the DEHP group with different concentrations,it is clear that the DEHP 10 and 1000 mg/kg groups have higher TNF-αlevel,while the other DEHP groups have no significant changes.Comparing the DEHP 1000 mg/kg control group and the control group,it is clear that the former has a higher TNF-αlevel.3.Histopathological changes in mice with HCC after DEHP exposureThe liver pathological section of the mice in the normal group showed complete liver lobule structure,clear nucleus,full cytoplasm,liver cords arranged neatly,and clear boundaries.Compared with the normal group,the DEHP 1000 mg/kg control group only had unclear cell boundaries,narrow liver sinusoids and slight inflammatory cell infiltration in the vein area.The morphology of cancer cells in model group,DEHP0.1 mg/kg group and DEHP 10 mg/kg dose group was different from that of normal liver cells.The tumor cells were arranged in thin liver plate with large cells and no obvious inner layer.The ratio of cancer cells to nucleoplasm increased slightly.However,in the dose of 1000 mg/kg DEHP,the morphology of cancer cells was significantly different from that of normal hepatocytes,with cytoplasmic decrease,large and irregular nuclei,and a significant increase in the ratio of nucleus to cytoplasm.4.Western blot detect the effects of long-term exposure to DEHP on the expression of PD-L1,P-JAK2 and P-STAT3 proteins in liver tissuesTo detect the expression of P-STAT3,P-JAK2 and PD-L1 proteins,the detection method used is Western blot.Studies have shown that compared with the control group,the expression levels of the model group has increased regardless of the protein.Compared with the model group,no matter what kind of protein,the expression level of DEHP 0.1 mg/kg group did not change significantly.In the DEHP10 and 1000 mg/kg group,the expression levels of STAT3 protein increased,and the expression levels of the other two proteins did not change significantly.Compared with the control group,regardless of the protein,the expression levels of DEHP 1000mg/kg group increased.5.Laser confocal were used to detect the effects of DEHP exposure on the expression and distribution of PD-L1 in liver tissues of mice with HCCIn order to detect the expression and distribution of PD-L1 protein in liver tissue,we used a laser confocal microscope to photograph it.The results displayed that after long-term exposure to DEHP,the expression of PD-L1 protein in mouse liver tissue has a significant upward trend,but DEHP 1000 mg/kg control group PD-L1 The expression of the protein is not obvious.6.Effects of DEHP on the proliferation and migration of HepG2In the results of the study,IFN-γcan significantly reduce the viability of HepG2 cells.At the same time,our results indicated that a lower concentration of DEHP（12.5μmol/L）can increase cell viability than IFN-γgroup,but a higher concentration of DEHP has no significant effect on cell viability.Compared with the normal control group,after administration of IFN-γ,the migration ability of HepG2 cells was severely inhibited.The migration ability of HepG2 cells in the DEHP 12.5,50,and200μmol/L group was significantly higher than that in the IFN-γgroup.Comparing the DEHP 200μmol/L control group and the control group,it is clear that the former has a higher HepG2 cell migration ability.7.Effects of DEHP on HepG2 PD-L1 m RNA expressionComparing the IFN-γtreatment group and the normal control group,it is clear that the former has a higher level of m RNA expression.Comparing the DEHP and IFN-γgroups with different concentrations,it is clear that PD-L1 m RNA has no significant change in the 12.5μmol/L group,and has a higher expression level in the DEHP 200μmol/L group,and in the DEHP 50μmol/L group There is an upward trend in expression.Comparing the normal control group and the DEHP 200μmol/L control group,it is clear that the PD-L1 m RNA expression of the latter has an upward trend.8.Laser confocal were used to detect the effects of DEHP exposure on the expression and distribution of PD-L1 in HepG2 cellsIt can be seen from the result that the expression of PD-L1 protein increased with the increase of DEHP dose,but the expression of PD-L1 protein in the DEHP 200μmol/L control group was not obvious.9.Effects of DEHP on the expression of PD-L1,P-JAK2 and P-STAT3 proteins in HepG2 cells stimulated by IFN-γComparing the results of the in vitro experiment with the results of the in vivo experiment,we found that the two experiments obtained similar results.DEHP caused a significant increase in the expression of P-JAK2,P-STAT3 and PD-L1protein,especially at high doses of 200μmol/L DEHP.CONCLUSIONS1.Long-term exposure to DEHP impaired liver function,increased the levels of ALT,AST and AFP,and long-term exposure to high doses of DEHP promoted tumor growth.2.Long-term exposure to DEHP increased the expression of IL-6 and TNF-αin liver tissues.3.Long-term exposure to DEHP significantly increased the expression of PD-L1 and inhibited anti-tumor immunity.The mechanism may be mediated by up-regulating the level of JAK2/STAT3.4.IFN-γactivated STAT3 in HepG2 cells,and this stimulation was further enhanced by DEHP,which lead to increased expression of PD-L1 in HepG2 cells,and ultimately reduced the sensitivity of tumor cells to CTL.