| Objective: 1,2-Dichloroethane(1,2-DCE)is a halogenated hydrocarbon compound,mainly used in the synthesis of vinyl chloride monomer.It can also be used as an organic solvent,such as binder,degreaser and cleaning.1,2-DCE is a highly toxic substance with fat-soluble,and is mainly inhaled by the respiratory minner in the production environment and rapidly distributed in various organ tissues of the whole body,wherein the brain tissue is the main target organ for accumulation and damage.In recent years,the incidence of this type of poisoning accident has been increased.However,the research data was really little on the mechanism of toxic cerebral edema in 1,2-DCE so far,and further research was urgently needed.Abnormal expression of matrix metalloproteinases(MMPs)plays a key role in the formation and development of vasogenic cerebral edema.Among them,MMP-9 is the most important matrix metalloproteinase in brain tissue,which can degrade the extracellular matrix(ECM)and the proteolytic enzyme of collagen in the vascular basement membrane.Our previous study showed that up-regulation of MMP-9 expression in brain tissue was an early event in the formation of subacute 1,2-DCE toxic brain edema.Since the vascular basement membrane and ECM are important structural basis for maintaining the integrity of the blood-brain barrier(BBB),which determines the permeability of BBB,up-regulation of MMP-9 can destroy the integrity of BBB and lead permeability of BBB increased,which in turn promotes the foemation of vasogenic cerebral edema.In addition,studies have been considered that the increased expression of MMP-9 could also degrade tight junction proteins(TJs)to further destroying integrity of BBB.The p38 Mitogen-activated portein kinase(MAPK)signaling pathway plays an important role in promoting the expression of MMP-9,and the p38 MAPK signaling pathway can activate nuclear factor-?B(NF-?B).Furthermore,the promoter of MMP-9 has a NF-?B binding site and the activation of NF-?B can positively regulate the expression of MMP-9.On the other hand,the study found that the inflammatory response was closely related to the formation and development of cerebral edema.p38 MAPK and NF-?B were considered to be important signaling pathways in the development of inflammation.In addition to up-regulating MMP-9 protein expression,the activated p38/NF-?B signaling pathway could also increase inflammatory factors,such as cell vascular cell adhesion molecule-1(VCAM-1),intercellular adhesion molecule-1(ICAM-1)and inducible nitric oxide synthase(i NOS).The cell adhesion molecules VCAM-1 and ICAM-1 could also adhere to peripheral circulating monocytes and transfer them to brain tissue through BBB,further aggravating BBB destruction and expanding inflammatory reactions.In addition,studies have shown that astrocytes were involved in major physiological processes in the central nervous system,such as the function of the blood-brain barrier and intercellular communication;proinflammatory activation of microglia can reduce brain edema by reducing inflammation response.At the same time,p38 MAPK signaling pathway could also promote the expression of cytokine IL-1?,which could be induced by inflammatory response or cellular stress,further activating the p38 MAPK signaling pathway and expand the inflammatory response.Based on the established experimental animal model of subacute 1,2-DCE toxic brain edema,this study investigated the effect of p38 MAPK/NF-?B on MMP-9 during the formation in 1,2-dichloroethane toxic brain edema and its role in the development of inflammatory response,and further exploring the effect of MMP-9 on TJs in brain tissue of toxic cerebral edema caused by 1,2-DCE as well as the accompanying inflammatory response in the formation of cerebral edema,which would provide new research directions and experimental reference data for revealing the mechanism of toxic brain edema induced by 1,2-DCE.Methods: 1.To establish a subacute 1,2-DCE toxic brain edema experimental animal model by static inhalation method.Healthy SPF Kunming female mice were selected and weighed 20-22 g.After one week of adaptive feeding,mice were was randomly divided into groups acrroding to per experimental scheme.The mice were placed in a 100 L static poisoning cabinet(5 /cabinet)and inhaled for 3.5 h with 1.2 mg/L 1,2-DCE daily.The mice in each group were sacrificed the day after the end of the exposure.2.The first part: mice were randomly divided into control group,1,2-DCE one-,two-and three-day exposure groups.Mice in the control group were placed in the chamber for 3.5 h/day without 1,2-DCE exposure and other treatment methods were same as the exposure group.The symptoms of poisoning and changes of weight in mice were observed;the water content of brain tissue was determined by dry-wet weight method;p38,phosphorylated p38(p-p38),NF-?B(p65 and p50),phosphorylated NF-?B(p-p65),I?B,phosphorylated I?B(p-I?B),MMP-9 and i NOS as well as tight junction proteins occludin,ZO-1,claudin 5,JAM-1 were detected by Western Blot;Real-time RT-PCR for detection of p38,p65,p50,I?B and MMP-9 m RNA expression level;HE staining for pathological observation of brain tissue and sodium fluorescein assay for BBB permeability;ELISA method for detection of IL-1? content in brain tissue.3.The second part:(I)Intervention model of p38 inhibitor:The mice were randomly divided into blank control group,solvent control group,p38 MAPK inhibitor control group,1,2-DCE intoxicated group,low,medium and high dose of p38 MAPK inhibitor groups.Except for the control groups,mice in other groups were exposed to 1,2-DCE for 3 days.The mice in low,medium and high doses of p38 MAPK inhibitor group were intraperitoneally injected with 3.75 mg/kg,7.5 mg/kg and 15 mg/kg p38 MAPK inhibitor(SB202190)200 ?L before exposure;the mice in p38 MAPK inhibitor control were intraperitoneally injected with same volume of p38 MAPK(15 mg/kg)inhibitor;the mice in solvent control group and the 1,2-DCE intoxicated group were intraperitoneally injected with same volume of DMSO;the mice in blank control group was injected intraperitoneally with the same volume saline.Measurement indicators of brain tissue were as follows: brain water content;protein expression levels of p38,p-p38,p65,p50,p-p65,I?B,p-I?B,MMP-9 and i NOS,as well as TJs;m RNA expression level of p38,p65,p50,I?B and MMP-9 m RNA expression level;pathological observation;EMSA assay for nuclear transcription factor NF-?B DNA binding activity;IL-1? content.The corresponding method is same as described above,which was also same for mentioned later.(II)Intervention model of NF-?B inhibitors: mice were randomly divided into control group,1,2-DCE intoxicated group,low and high dose NF-?B inhibitor groups.Except for the control group,mice in other groups were exposed to 1,2-DCE for 3 days.The mice in low and high doses of NF-?B inhibitor group were intraperitoneally injected with 10 mg/kg and 100 mg/kg NF-?B inhibitor PDTC 200 ?L before the 1,2-DCE exposure;the mice in the control group and 1,2-DCE intoxicated group were intraperitoneally injected with same volume of saline.Measurement indicators of brain tissue were as follows: brain water content;protein expression levels of MMP-9,TJs,i NOS and IL-1?;m RNA expression level of MMP-9;pathological observation;DNA binding activity of NF-?B.(III)Intervention model of MMP-9 inhibitors: mice were randomly divided into control group,1,2-DCE intoxicated group,low and high dose MMP-9 inhibitor group.Except for the control group,mice in other groups were exposed to 1,2-DCE for 3 days.The mice in low and high doses of MMP-9 inhibitor group were intraperitoneally injected with 12.5 mg/kg and 25 mg/kg MMP-9 inhibitor(SB-3CT)200 ?L before the 1,2-DCE exposure;the mice in the control group and 1,2-DCE intoxicated group were intraperitoneally injected with same volume of solvent.Changes in brain water content and protein expression levels of TJs were examined.4.The third part:(I)NF-?B inhibitor and p38 inhibitor intervention model: grouping and intervention methods were same as before.The p38 inhibitor intervention model was used to detect the expression levels of p-p65,GFAP,Iba-1,ICAM-1 and VCAM-1 in brain tissue.NF-?B inhibitor intervention model was used to detect the protein expression levels of GFAP,Iba-1,ICAM-1 and VCAM-1 in brain tissue;the m RNA levels of ICAM-1 and VCAM-1.The expression of Iba-1 and VCAM-1 in brain tissue were detected by immunohistochemical staining;observed ICAM-1 single staining as well as VCAM-1 and GFAP double staining by immunofluorescence.(II)Intervention model of IL-1? receptor inhibitors: mice were randomly divided into control group,inhibitor control group,1,2-DCE intoxicated group,low and high dose IL-1? receptor inhibitor group,five in each group.Except for the control group,mice in other groups were exposed to 1,2-DCE for 3 days.Low and high dose IL-1? receptor inhibitor group and inhibitor control group were intracerebroventricular injection with 2 ?g and 4 ?g in 4 ?L of IL-1? receptor inhibitor IL-1ra;the mice in the control group and 1,2-DCE intoxicated group were intracerebroventricular injection with same volume of saline.The protein levels of ICAM-1,VCAM-1,GFAP,Iba-1,i NOS,IL-1?,p-p38,p-p65,p-I?B and the m RNA levels of IL-1? were detected.Results: 1.In the first part,mice in three-day exposure group showed typical behavioral symptoms of brain poisoning injury along with lighten of weight.The brain water content and BBB permeability of mice were significantly increased from the second day of exposure(P?0.05).HE staining showed pathological changes of typical cerebral edema: that the nerve cell body in cerebral cortex was swollen and degenerated,as well as the edge was blurred,the cytoplasm was lightly stained,the perinuclear space was widened,and some of the perivascular interstitial were loose.In two-and three-day exposure group mice,the expressions of MMP-9 and IL-1? protein and gene levels in brain tissue were significantly higher than those in the control group(P?0.05),while the ratios of p-p38/p38 were significantly higher start with the one-day exposure group as compared with the control group.The expression levels of p65,p-p65 and p-I?B in brain tissue of mice in two-and three-day exposure group were markedly higher than those in the control group.In contrast,the protein expression levels of I?B in two-and three-day exposure group were significantly lower than those in the control group;m RNA expression levels of the p65 in two-and three-day exposure group,and I?B in three-day exposure group were significantly higher than those in the control group(P?0.05).There was no significant difference in the expression levels of p38 and p50 protein and m RNA as compared with the control group.The protein expression of tight junction proteins occludin,ZO-1,and claudin 5 decreased along with the exposure days,while the expression of JAM-1 increased significantly in the three-day exposure group(P?0.05).The expression levels of i NOS protein decreased along with the exposure days,starting with seconed day of 1,2-DCE exposure(P?0.05).2.In the second part,pretreatment with p38 MAPK inhibitor significantly improved body weight loss and brain tissue water content in 1,2-DCE poisoning mice.Compared with the intoxicated group,the p-p38/p38 protein ratios were significantly decreased among p38 inhibitor group,and the MMP-9 m RNA expression levels in the medium and high dose p38 inhibitor groups,and the MMP-9 protein expression levels in the high-dose p38 inhibitor group were also decreased significantly(P?0.05).Compared with the exposed group,levels of p-p65 protein expression and the levels of p65 and I?B m RNA expression down-regulated markedly,the levels of p-I?B protein expression down-regulated in a dose-dependent manner,and the I?B protein expression levels were significantly increased in low and high dose p38 inhibitors in the brain tissue of mice(P ? 0.05).In addition,the intervention of NF-?B inhibitors could effectively improve the water content and prevent decrease of weight in 1,2-DCE exposed mice.The levels of MMP-9 protein in NF-?B inhibitors intervention group were lower than those in the intoxicated group,and TJs protein levels were higher than those in the intoxicated group(P ? 0.05).After intervention with MMP-9 inhibitor SB-3CT,TJs protein levels and brain water content were increased as compared with the intoxicated group(P?0.05).Both NF-?B inhibitor and p38 inhibitor significantly could reduce overexpressed i NOS and IL-1? in brain tissue of 1,2-DCE exposured mice.3.In the third part,compared with the control group,the activation markers Iba-1 and GFAP of microglia and astrocytes,as well as inflammatory factors VCAM-1 and ICAM-1 were significantly increased in the brain tissue of the mice in intoxicated group of each model(P < 0.05).The p38 inhibitor intervention not only significantly inhibited up-regulation of Iba-1 and GFAP,but also down-regulated the expression of over-expressed VCAM-1 and ICAM-1 proteins(P < 0.05).In addition,NF-?B inhibitor intervention also inhibited Iba-1 and GFAP significantly,and down-regulated VCAM-1 and ICAM-1 protein expression in the brain tissue of mice in intoxicated group(P < 0.05).The results of immunostaining of GFAP and Iba-1 were consistent with the results of protein detection.NF-?B inhibitor intervention significantly reduced the activation of astrocytes and microglia.The results of immunostaining of VCAM-1 and ICAM-1 were also consistent with their protein expression results,and ICAM-1 immunofluorescence staining showed an increase in the number of ICAM-1 positive blood vessels in 1,2-DCE poisoning mice.Compared to the control group and high-dose IL-1ra receptor inhibitor intervention group,protein expression levels of p-p38/p-38,p-p65,p-I?B and VCAM-1,ICAM-1 as well as Iba-1,and protein and m RNA expression levels of IL-1? were down-regulated markedly(P < 0.05).The protein expression levels of i NOS and GFAP were not affected by the intervention of IL-1ra receptor inhibitors.Conclusion: 1.Subacute 1,2-DCE exposure could cause brain edema in mice,p38 MAPK and NF-?B were activated in brain tissue of mice,and up-regulated p-p38 levels is an early event in the process of brain edema formation,meanwhile,along with the up-regulation of MMP-9 and the decreased levels of tight junction proteins expression as well as the destruction of BBB permeability.2.p38 MAPK increased the expression of MMP-9 at the transcriptionaly via NF-?B signaling pathway,and then degrading TJs and destroying the integrity of BBB.3.Subacute 1,2-DCE-induced toxic cerebral edema was accompanied with inflammatory response,which was mainly regulated by p38 MAPK and NF-?B signaling pathways.In the formation of cerebral edema in mice with ,2-DCE poisoning,IL-1? is not the main initiating factor of inflammation,but it has partial inflammatory amplification due to the protein change of p-p38 earlier than IL-1?. |
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