Di（2-ethylhexyl） Phthalate （DEHP） Promotes Oxidative Stress And Activates The NF-κB/NLRP3 Pathway To Exacerbate IgA Nephropathy In Mice

Di（2-ethylhexyl）phthalate（DEHP）is an endocrine disruptor commonly used in a variety of plastic products.Epidemiological studies have shown that long-term exposure to DEHP may cause impairment of renal function.When the body is in a state of chronic kidney injury,exposure to DEHP may cause the severity of kidney disease to become more serious.IgA nephropathy is the most common primary glomerulonephritis worldwide.However,the effect of DEHP on IgA nephropathy has not been reported.In the course of this study,we used bovine serum albumin（BSA）combined with carbon tetrachloride（CCl₄）and lipopolysaccharide（LPS）to establish a mouse model of IgA nephropathy,to study the effect of DEHP on mouse IgA nephropathy,and to investigate its potential toxicological mechanisms were excavated.At the same time,in the mouse glomerular mesangial cell（MCs）experiment,we obtained results consistent with the in vivo experiment.In summary,DEHP may aggravate IgA nephropathy in mice by aggravating oxidative stress and activating the NF-κB/NLRP3 pathway.OBJECTIVE:The IgA nephropathy model of BALB/c mice was induced by BSA combined with CCl₄and LPS,and the effect of DEHP on the pathological changes of IgA nephropathy mice was investigated.Meanwhile,MCs cells were used for in vitro experiments to further study the role of oxidative stress and NF-κB/NLRP3pathway in IgA nephropathy.METHODS:In in vivo experiments,we constructed a mouse IgA nephropathy model by gavage of BSA,subcutaneous injection of CCl₄and tail vein injection of LPS.During the modeling period,DEHP（0.1,10,1000 mg/kg）was administered by gavage,and only DEHP 1000 mg/kg was given as a control.After modeling,the mice were sacrificed according to the methods prescribed by animal ethics.Through HE staining and PAS staining of kidney tissue,the pathological damage of DEHP to IgA nephropathy mice induced by BSA combined with CCl₄and LPS was observed.IgA deposition in the glomerular mesangial area of mice was detected by immunofluorescence method.The corresponding kidney index was obtained by dividing the weight of the mouse’s unilateral kidney by the body weight.The24-hour urine protein content of mice was detected by ELISA.The level of interleukin 1-β（IL-1-β）in kidney tissue homogenate was detected by ELISA.Various biochemical indicators in mouse serum and kidney tissue homogenate were detected by biochemical kits,including creatinine（Cr）,urea nitrogen（BUN）,superoxide dismutase（SOD）,glutathione（GSH）,malondialdehyde（MDA）.The expression of p-p65,NLRP3 and other proteins in kidney tissue was detected by Western blot.In the in vitro experiment,the following six groups were set up:control group,LPS group（2μg/m L）,LPS+DEHP（12.5,50,200μmol/L）group,and DEHP 200μmol/L group.After 48 hours,the effect of DEHP on the proliferation of MCs cells was detected by high-content imaging;the effect of DEHP on the production of ROS in MCs cells was detected by fluorescent probes;the protein expression of p-p65 and NLRP3 in MCs cells was detected by Western blot.RESULTS:1.The effect of DEHP exposure on renal function indexes and 24-hour urine protein in mice with IgA nephropathyAfter 2 months of DEHP exposure,the kidney index of the mice increased obviously;the results of serum biochemical indexes showed that DEHP obviously increased the levels of Cr and BUN.The 24-hour urine protein content of mice detected by ELISA kit showed that DEHP1000 mg/kg group obviously increased the24-hour urine protein content of mice.It shows that the intake of DEHP makes the kidney function of IgA nephropathy mice worse.2.The effect of DEHP exposure on histopathological changes in mice with IgA nephropathyThe results of HE staining and PAS staining showed that DEHP exposure significantly promoted the proliferation of glomerular mesangial cells and mesangial matrix,renal tubular atrophy,degeneration of basement membrane vacuoles,and infiltration of renal interstitial inflammatory cells in IgA nephropathy mice.IgA immunofluorescence results of mouse kidney tissue sections showed that IgA was significantly deposited in the glomerular mesangial area of IgA nephropathy mice,which was significantly increased after DEHP exposure.It shows that DEHP aggravates the pathological damage of IgA nephropathy mice.3.The effect of DEHP exposure on the oxidative and antioxidant systems in the kidney tissue of mice with IgA nephropathyThe results of biochemical kits showed that DEHP can obviously increase the MDA content in the kidney tissue of mice with IgA nephropathy,and significantly reduce the activities of SOD and GSH.It indicated that the intake of DEHP further disrupted the oxidative balance in the kidney tissue of IgA nephropathy mice.It indicated that the intake of DEHP further disrupted the oxidative balance in the kidney tissue of IgA nephropathy mice.4.Laser confocal was used to detect the effect of DEHP exposure on the expression of NLRP3 and IL-1βprotein in the kidney tissue of mice with IgA nephropathyAccording to the laser confocal results,the protein expressions of NLRP3 and IL-1βin the kidney tissue of IgA nephropathy mice were obviously increased after DEHP exposure.5.Western blot was used to detect the effect of DEHP exposure on the expression of p-p65,NLRP3,and caspase-1 protein in the kidney tissue of mice with IgA nephropathyCompared with the normal group,the protein expressions of p-p65,NLRP3 and caspase-1 in the kidney tissue of IgA nephropathy mice were obviously increased;compared with the IgA nephropathy group,DEHP exposure could significantly promote the IgA nephropathy mice induced by BSA combined with CCl₄and LPS.p-p65,NLRP3,caspase-1 protein expression in kidney tissue.It indicates that DEHP aggravates IgA nephropathy in mice,and its mechanism may be related to the NF-κB/NLRP3 pathway.6.The effect of DEHP exposure on the proliferation of mouse glomerular mesangial cells（MCs）Compared with the normal group,the LPS group obviously promoted the proliferation of MCs cells.After DEHP intervention,DEHP（6.25,12.5μmol/L）promoted the proliferation of MCs cells,while DEHP（25,50,100,200μmol/）restricted the proliferation of MCs cells.7.The effect of DEHP on ROS production in MCs cells stimulated by LPSROS in MCs cells were labeled with fluorescent probes.The results showed that the ROS content in MCs cells in the DEHP 12.5μmol/L group did not change obviously,while the ROS content in MCs cells in the DEHP 50 and 200μmol/L groups increased obviously.8.Laser confocal was used to detect the effect of DEHP on the expression of IL-1βin MCs cells stimulated by LPSThe expression of IL-1βprotein in MCs cells was detected by laser confocal method,and the results showed that DEHP obviously increased the expression of IL-1βprotein in MCs cells.9.The effect of DEHP on the expression of p-p65,NLRP3,and caspase-1 protein in MCs cells stimulated by LPSIt was found that the overall trend of cell experiments was consistent with the results of in vivo experiments.Compared with the LPS group,DEHP could significantly promote the expression of p-p65,NLRP3,and caspase-1 in MCs cells.CONCLUSIONS1.Exposure to DEHP aggravates IgA nephropathy in mice.After DEHP intervention,the renal index of IgA nephropathy mice increased,the levels of renal function indexes Cr and BUN increased,and the proliferation of mesangial cells and the infiltration of renal interstitial inflammatory cells were aggravated.2.DEHP promotes oxidative stress.DEHP increased the content of MDA in the kidney tissue homogenate of IgA nephropathy mice and decreased the activities of SOD and GSH.Meanwhile,DEHP promoted the production of ROS in MCs cells.3.DEHP activates the NF-κB/NLRP3 pathway.DEHP up-regulated the expression of p-p65,NLRP3 and caspase-1 protein in kidney tissue of IgA nephropathy mice.A consistent trend was also observed in MCs cells.