Polyinosine-polycytidylic Acid Promotes HMGB1 Acetylation Amplified Kidney Endothelial Cells Damage In Trichloroethylene Hypersensitivity Syndrome Mice

Background and objective Trichloroethylene(TCE)is a chlorinated solvent that is used to remove metal parts or lens grease.Workers who are exposed to TCE in the work environment for a long time develop an allergic syndrome called occupational drug rash-like dermatitis of trichloroethylene(ODMT).OMDT is mainly characterized by skin lesions such as extensive skin and mucous membrane sensitization with fever,jaundice and lymphomegaly,in addition to abnormal kidney function in most patients and even lifethreatening in severe cases.Several studies have found that viral infection or reactivation is the key to the development of OMDT.Previous studies by our group have shown that poly(I:C),a viral mimic,can enhance the damage caused by TCE sensitization.However,whether Poly(I:C)plays a role in TCE-induced kidney injury remains to be elucidated.Renal vascular endothelial cells,as highly differentiated functional cells in the kidney,are key drivers and targets for exacerbating the renal inflammatory response in several renal diseases,with functions such as regulating blood flow to the local tissue bed and maintaining the glomerular filtration barrier and tubular reabsorption.A previous study found increased expression of adhesion molecules in TCE-sensitized mice,suggesting that renal vascular endothelial cells may be involved in the pathogenic process of TCE and that renal endothelial cell injury may be an important cause of kidney damage.To this end,we established a trichloroethylene sensitized mouse model and evaluated the kidney injury in mice,especially renal endothelial cell dysfunction.The activation of high mobility group protein 1(HMGB1)was further examined to investigate the role of Poly(I:C)in trichloroethylene sensitized kidney injury.MethodsTo establish a TCE sensitized mouse model,63 female BALB/c mice at 6 weeks of age were selected and randomly divided into a blank control group(6),a solvent control group(6),a TCE treated group(25)and a combined TCE+Poly(I:C)treated group(26),where the combined TCE+Poly(I:C)treated group was injected intraperitoneally before terminal excitation with Poly(I:C).Mice were scored 24 h after final excitation according to their dorsal skin manifestations and divided into sensitized and nonsensitized groups.The kidney and eye venous blood were collected under aseptic conditions,and serum Cre and BUN levels were measured.Immunohistochemistry was used to detect the expression of renal VCAM-1,ICAM-1 and E-selectin.q RT-PCR was used to detect the gene transcription levels of HMGB1 and TLR3.Results1.Skin sensitization rate: The control and non-sensitized mice had normal skin colour,while the TCE-treated and Poly(I:C)-treated groups showed erythema on the back of mice,with sensitization rates of 32% and 34.61% respectively.2.Kidney damage in TCE-sensitized mice: BUN and Cre levels were increased in the TCE-sensitized group,and Poly(I:C)significantly up-regulated BUN and Cre levels in the mice;pathological tissue sections revealed glomerular lesions in the mice in the TCE-sensitized group,including proliferation of thylakoid cells and disorganized cell arrangement.3.Nucleoplasmic translocation of HMGB1 in TCE-sensitized mice: Elisa results showed that the level of HMGB1 in the peripheral blood serum of the TCE-sensitized mice was significantly higher than that of the other groups,while the difference between the other groups was not statistically significant;further immunohistochemical examination of the kidney tissues of the mice showed that there was extracellular translocation of the nucleoplasm of HMGB1 in the kidneys of the TCE-sensitized mice.4.Activation of endothelial cells in the kidneys of TCE-sensitized mice:Immunohistochemical results showed that the expression of VCAM-1,ICAM-1 and Eselection was increased in the kidneys of TCE-sensitized mice,and more significantly in the TCE+Poly(I:C)sensitized group.Western Blot showed that the expression of Syndecan 1 and Glypican 1 was significantly lower in the TCE-sensitized group compared to the TCE-unsensitized group(P<0.05).The expression of Syndecan 1 and Glypican 1 in the sensitized group was significantly lower than that in the TCEpositive group(P<0.05).5.Renal TLR3 overexpression in TCE-sensitized mice after Poly(I:C)pretreatment:Immunofluorescence experiments showed that TLR3 expression was elevated in the kidneys of mice in the TCE-sensitized group compared to the control group,while the renal TLR3 expression in the TCE+Poly(I:C)-sensitized group was higher than that in the TCE-sensitized group;Western Blot results likewise showed that,compared to the TCE-sensitized group The western blot results also showed that the expression level of TLR3 protein in the kidney of the TCE+Poly(I:C)-sensitized group was significantly higher than that of the TCE-sensitized group,and the difference was statistically significant(P<0.05).q RT-PCR data further confirmed the higher expression level of TLR3 m RNA in the TCE+Poly(I:C)-sensitized group.6.Poly(I:C)pretreatment promoted nucleoplasmic translocation of HMGB1 in the kidneys of TCE-sensitized mice: Labeled endothelial cell marker CD31 was colocalized with HMGB1 immunofluorescence and HMGB1 extra nuclearization was found to be more pronounced in the endothelial cells of the kidneys of TCE-sensitized mice after Poly(I:C)treatment;the level of HMGB1 protein expression was assessed by Western Blot in the kidneys of mice.The total HMGB1 expression levels in the solvent control group,TCE-sensitized group and TCE+Poly(I:C)-sensitized group were not significantly different(P>0.05).In contrast,cytoplasmic HMGB1 levels were elevated in the TCE-sensitized group compared to the solvent group.cytoplasmic HMGB1 levels were significantly higher in the TCE+Poly(I:C)sensitized group than in the TCE-sensitized group.In contrast,nuclear HMGB1 levels were lower in the TCE-sensitized group than in the solvent group and were further reduced in the TCE+Poly(I:C)-sensitized group.7.Poly(I:C)pretreatment enhanced renal HMGB1 acetylation in TCE-sensitized mice:Immunofluorescence revealed that renal endothelial cells were one of the major reservoirs of acetylated HMGB1;Western Bolt results showed that ac-HMGB1 expression was upregulated in the TCE-sensitized group compared to the TCEunsensitized group,while ac-HMGB1 expression was upregulated in the TCE+Poly(I:C)-sensitized group showed significantly higher ac-HMGB1 levels than the TCE-sensitized group(P<0.05).Conclusions1.Renal structure and function damage in TCE sensitized mice were induced by activation and injury of renal endothelial cells.2.Poly(I:C)preconditioning binding with its ligand TLR3 is amplified in kidney injury induced by TCE in mice.3.Poly(I:C)promoted the acetylation of HMGB1 and induced cytoplasmic migration to participate in the injury of renal endothelial cells in TCE sensitized mice.