| Background Trichloroethylene(TCE)is a commonly used organic solvent.It has been widely used in industry for more than 100 years.It is not only used as a raw material for polymer compounds,but also widely used in hardware,toys,electronics and other industries.Used as a metal part and electronic component cleaning agent [1-4].The application of TCE is also difficult to find other better alternatives.In addition,the lack of effective protection for workers exposed to poisons has become a prominent problem in the health hazards of workers in China.China has named the occupational disease-like dermatitis due to TCE(OMLDT)in 2005.In recent years,studies have found that the clinical manifestations of OMLDT not only have severe systemic skin damage,but also liver failure is one of the important causes of death.Therefore,the prevention and treatment of liver damage in OMRDT has attracted widespread attention from domestic occupational health physicians and clinical therapists.The pathogenesis of OMLDT and its immunological liver injury is still unclear.Most studies consider antigen-specific T lymphocyte-mediated delayed type IV allergic reaction,and the pathogenesis of immune-induced liver injury induced by TCE It was found that T lymphocyte-mediated type IV allergic reaction could not fully explain its pathogenesis,but related studies also found that complement activation may play a role in OMLDT immunological liver injury,Kallikrein-Kinin System,KKS)has strong biological correlation with the complement system.Some studies have found that the liver of guinea pigs with TCE-sensitized guinea pigs has increased sinusoidal Kupffer cells.Kupffer cell activation and expression of inflammatory factors play a role in TCE-induced liver injury.Important role,however,the mechanism of Kupffer cell inflammatory response in this process is not clear,some studies have found that BK and its receptor B2 R binding can activate mitogen-activated protein kinases(MAPK)and STAT3 signaling pathway,We hypothesized that the activation product BK of the KKS system binds to its receptor during TCE-induced immunological liver injury.Live MAPK pathway,so that the liver Kupffer cells secrete anti-inflammatory cytokines and proinflammatory changes,further amplify cellular immune response involved in autoimmune liver injury.Objective Using the whole animal experiment combined with Kupffer cell isolation technology and detection technology and using KKS system inhibitor PKSI-527 and B2 R specific inhibitor HOE-140 to study the binding of BK to receptor B2 R to activate MAPK pathway and secrete inflammatory factors in liver Kupffer cells.The effects of TCE-sensitized mouse Kupffer cells were determined in vitro,and the pathological changes of Kupffer cells and the changes of inflammatory factors were determined from the positive and negative aspects.The mechanism of TCE-immune liver injury was discussed..The results of this study will provide a theoretical basis for the pathogenesis of TCE-induced immune liver injury,and serve the prevention and treatment of OMLDT.Part one.Study of Kallikrein-kinin system activation in trichloroethylene sensitized miceMethods The TCE percutaneous sensitized BALB/c mouse model was established,and the sensitized mice were pretreated with the selective inhibitor PKSI-527 of kallikrein-kinin system high.The KKS system-related protein indexes in plasma and liver of each group were observed.Express the situation.The expression levels of BK,PK and HMWK in mouse plasma were detected by ELISA.The expression of BK and its receptors in the liver of mice in each group were detected by immunofluorescence.To investigate the activation and expression of KKS system in TCE-sensitized mice and liver.Results1.Skin sensitization of mouse modelIn the modeling process,no animals died in each group of mice.The blank control group and the solvent control group mice were observed,and no erythema and edema appeared in the back skin.TCE sensitized mice showed different degrees of erythema and edema in the back skin.Patients with severe edema had skin bulge.They were touched with 200μl gun head,and the skin edema thickened obviously.TCE was not sensitized to the skin of the mice.No obvious erythema and edema were found.2.Analysis of sensitization rate in each group TCE-treated mice,24 of 60 TCE-treated mice were sensitized,with 11 skin scores of 1,9 with a score of 2,4 with a score of 3,and 36 with no sensitization.The sensitization rate was 40%;13 of the 60 TCE + PKSI-527 treated mice were sensitized,including 7 with a skin score of 1,3 with a score of 3,3 with a score of 3,and 47 none sensitized,the sensitization rate reached 21.67%.3.Changes in body weight during modeling of each group of mice The body weight of each group was monitored during the modeling process.The study found that the body weight of the blank control group and the solvent control group was stable and there was no obvious abnormality.In the modeling stage,the weight gain of mice in the TCE sensitized group was slower.After the first sensitization on the 18 th day,the body weight showed a significant "V" type depression.The weight change of the TCE + PKSI-527 group was similar to that of the TCE treatment group.However,there was no obvious "V" type subsidence.The weight of the 20 th day of each group was subtracted from the initial body weight,and the difference in body weight was calculated during the modeling period.The difference in body weight between the TCE-sensitized group and the control group was statistically significant(P<0.05),TCE + Body weight changes were also significantly reduced in the PKSI-527 group(P<0.05).4.Changes in liver coefficient of mice in each group After the animals were sacrificed,the livers of each group were weighed and weighed to calculate the liver organ coefficient.Statistical analysis showed that there was no significant difference between the blank control group and the solvent control group(P>0.05);The liver coefficient of the TCE-sensitized group was significantly higher than that at 48 h and 72 h,and the difference was statistically significant(P<0.05).At the same time,the organ coefficients of the TCE + PKSI-527 sensitized group were also significantly increased at 48 h and 72 h.5.Plasma PK and HMWK expression levels in each group of miceThe plasma concentrations of PK and HMWK in each group were detected by ELISA.There was no significant difference in plasma PK levels between the blank control group and the solvent control group(P>0.05).Compared with the blank control group,the plasma PK concentration in the TCE-sensitized group was significantly increased at 48 h and 72 h,and the difference was statistically significant(P<0.05).At 24 h,48h,72 h and 7d,PK The concentration reached the peak at 72 h.After treatment with PKSI-527,the plasma PK concentration of each group was significantly decreased.The plasma PK concentration of TCE+PKSI-527 sensitized mice was significantly lower than that of the corresponding TCE sensitization group at 48 h and 72 h.The difference was statistically significant(P<0.05).There was no significant difference in the plasma HMWK levels between the blank control group and the solvent control group(P>0.05).Compared with the blank control group,the plasma HMWK concentration in the 24 h and 48 h TCE sensitized mice was significantly increased,and the difference was statistically significant(P<0.05).After treatment with PKSI-527,the plasma HMWK concentration of each group was significantly decreased.The plasma HMWK concentration in the 48 h TCE+PKSI-527 sensitized group was significantly lower than that in the corresponding TCE sensitized group,and the difference was statistically significant(P<0.05)6.Plasma BK concentration in each group of miceThe plasma BK expression levels of the mice in each group were detected by ELISA.The difference between the blank control group and the solvent control group was not statistically significant(P>0.05).The plasma BK concentration in the TCE sensitized group was observed at 24 h,48h and 72 h.Compared with the blank control group,the difference was statistically significant(P<0.05).At the same time,we found that the BK expression level reached the highest peak at 72 h,and the plasma BK of the sensitized mice at each time point in the TCE+PKSI-527 treatment group.The concentration was lower than that in the TCE sensitization group,and the difference was statistically significant(P<0.05).The plasma BK concentration in the TCE+PKSI-527 sensitized group was significantly decreased in 72 h.7.BK expression levels in liver of different treatment groupsImmunofluorescence was used to detect the expression of B2 R in the liver tissues of the control group,the solvent control group,the 72 h TCE sensitization group,and the 72 h TCE+PKSI-527 sensitized group.The B2 R in the liver of the blank control group and the solvent control group was found.There was a small amount of expression,and the expression of B2 R in the liver of the 72 h TCE sensitized group was significantly increased.After using PKSI-527,the expression level decreased.Conclusions 1.The model of TCE percutaneous sensitized mice was successfully established.After the first stimulation,the body weight of the mice showed a significant "V"-type subsidence trend;2.The KKS system was activated in TCE-sensitized mice,and the plasma PK,HMWK and BK expression levels were significantly increased in tce-sensitized mice;3.The expression of B2R in liver of TCE-sensitized mice was increased.After using PKSI-527,the expression of B2R was decreased.Part two.Bradykinin Receptor B2 R Regulating Kupffer cell Activation Mediated Immune Liver Injury Induced by TrichloroethyleneMethods In the study one,we found that the activation of KKS system and its active products BK and B2 R increased in the liver of trichloroethylene-sensitized mice,but its role in immune liver injury,the specific mechanism is still unclear.In order to reveal the molecular mechanism of KKS activation in immune-induced liver injury in TCE-sensitized mice,we further selected a specific B2 R antagonist HOE140 based on previous animal experiments to detect BK/B2 R signaling pathway and immunize TCE-sensitized mice.The effect of liver injury;detection of Kupffer cell activation in TLB-sensitized BALB/c mice,Kupffer cells were isolated from the liver of experimental animals,and the activation of MAPK and STAT3 signaling pathways and the secretion of related cytokines were observed.Results 1.Analysis of sensitization rate in each group of miceEach group of mice did not die during the modeling process.According to the skin reaction score of Table 2,the mice were divided into a sensitized positive group and a sensitized negative group on the 20 th day.The sensitization rate was calculated by TCE treatment group and TCE + HOE140 treatment group,respectively.The sensitization rate was 39.58%(19/48)in TCE treatment group and 32.69%(17/52)in TCE + HOE140 group.There was no significant difference between the two groups.2.CD68 expression in liver tissue of mice in each groupCD68 is a highly glycosylated membrane protein.When Kupffer cells were activated,CD68 expression increased and acted as a marker of Kupffer cell activation.In this study,IHC detection showed no significant difference in the expression of CD68+ cells in the blank control group and the solvent control group,and the difference was not statistically significant;TCE treatment After percutaneous sensitization,compared with the blank control group and the solvent control group,the number of CD68+ cells in the TCE sensitized group increased significantly at 24 h and 72 h.At the same time,compared with the blank control group and the solvent control group,24 h and 72 h TCE + The number of CD68+ cells in the liver of HOE140 sensitized group also increased significantly.There was no significant difference in the number of CD68+ cells between the TCE-sensitized group and the TCE + HOE140-sensitized group.3.CD16/CD32 expression in liver tissue of mice in each groupIHC method further detected the expression of CD16/CD32 on the surface of M1 Kupffer cells,which was consistent with the expression of CD68.There was no significant difference in the expression of CD16/CD32+ cells between the blank control group and the solvent control group,and the difference was not statistically significant;TCE treatment After percutaneous sensitization,compared with the blank control group and the solvent control group,the number of CD16/CD32+ cells in the liver of the TCE sensitized group increased significantly at 24 h and 72h;meanwhile,compared with the blank control group and the solvent control group,24 h and The number of CD16/CD32+ cells in the liver of 72 h TCE + HOE140 sensitized group also increased significantly.There was no significant difference in the number of CD16/CD32+ cells between the TCE sensitized group and the TCE + HOE140 sensitized group.4.B2 R expression in liver tissue of mice in each groupThe study found that liver injury in the TCE-treated group was the most severe at 72 h.Therefore,we chose to detect the expression of B2 R protein at the 72 h time point.The expression of B2 R in liver tissue of each group was detected by Western-blot method.It was found that there was a certain amount of B2 R expression in the blank control group and the solvent control group,and the expression was less.There was no significant difference between the two groups(P>0.05).The expression of B2 R in liver of 72 h TCE sensitized group was significantly higher than that of the control group,which was 6~8 times higher than that of the control group(P<0.05).The expression of B2 R in the sensitized group was not significantly higher than that in the blank control group.There was no significant difference between the two groups(P>0.05).The expression of B2 R in the TCE-sensitized group was significantly higher than that in the non-sensitized group,and the difference was statistically significant(P<0.05).After using HOE140,the 72 h TCE + HOE140 sensitization group was significantly different from the blank control group,the solvent control group and the 72 h TCE + HOE140 non-sensitized group(P<0.05).At the same time,72 h TCE + HOE140 sensitization Compared with TCE sensitized group,there was no significant change in liver B2 R expression,and the difference was not statistically significant(P>0.05).5.The role of HOE140 in TCE-treated miceHOE140 is a specific antagonist of B2 R.The binding of BK and B2 R was detected by fluorescence double labeling.It was found that BK and B2 R were more expressed in TCE sensitized group,and BK and B2 R were combined(dark yellow).After using HOE140,The combination of BK and B2 R was reduced(red and green),and the results showed that HOE140 exerted an antagonistic effect on B2 R with obvious effect.6.Liver injury in each group of mice6.1 Liver function detection The results of liver function test are shown in the figure.There was no significant difference between the blank control group and the solvent control group(P>0.05).Compared with the blank control group,the AST of the TCE sensitized group was significantly increased at 24 h and 72 h,the difference was statistically significant(P<0.05).Compared with the corresponding unsensitized group,the AST of the TCE sensitized group was significantly increased at 24 h and 72 h.The difference was statistically significant(P<0.05).Compared with the blank control group,the AST of the 24 h TCE + HOE140 sensitized group was significantly increased,the difference was statistically significant(P<0.05),however,compared with the blank control group,72 h The increase of AST in TCE + HOE140 sensitized group was not obvious,and the difference was not statistically significant(P>0.05).The results of ALT examination showed that there was no significant difference between the blank control group and the solvent control group(P>0.05).The ALT level was significantly higher in the 72 h sensitized group than in the blank control group,the difference was statistically significant(P<0.05).The difference was statistically significant(P<0.05)compared with the corresponding unsensitized group.Compared with the blank control group,the AST increase in the TCE + HOE140 sensitized group was not significant at 24 h and 72 h,and the difference was not statistically significant(P>0.05).6.2 Ultrastructural inspection The structure of the blank control group and the blank control group was intact and no significant change was observed by transmission electron microscopy.At 24 h and 72 h, the organelles in the liver cells of TCE-sensitized group disappeared,a large number of vacuoles appeared in the liver cells,mitochondria disappeared,glycogen particles decreased,endoplasmic reticulum ruptured,and the nuclear membrane boundary was unclear.At the same time,the histomorphological changes of TCE + HOE140 positive group were alleviated in 24 h and 72 h,the ribosomal part of hepatocytes was decreased,and the mitochondria were vacuolar degeneration.The majority of mitochondria in the TCE + HOE140 positive group were clear in 72 h.7.MAPK pathway and STAT3 protein expression in liver of each group7.1 Expression of P38 MAPK and its phosphorylated protein in mouse liver Western blot analysis showed that there was no significant difference in the expression of p-P38 between the blank control group and the solvent control group(P>0.05).The expression of p-P38 in the TCE + HOE140 sensitized group was 72 h in the 72 h TCE sensitized group.Compared with the blank control group,the expression was slightly increased,but the difference was not statistically significant(P>0.05),and no significant difference was found between the two groups(P>0.05).7.2 ERK MAPK and its phosphorylated protein expression levels in mouse liver Western blot analysis showed that there was no significant difference in the expression of p-ERK between the blank control group and the solvent control group(P>0.05).The expression of p-ERK in the TCE sensitized group was significantly higher than that in the blank control group.The difference was statistically significant(P<0.05).Compared with the 72 h TCE unsensitized group,the expression was significantly increased(P<0.05).72 h TCE + HOE140 sensitized group and 72 h TCE sensitized group.Compared with p-ERK expression,the difference was statistically significant(P<0.05).7.3 JNK MAPK and its phosphorylated protein expression levels in mouse liver Western blot analysis showed that there was no significant difference in the expression of p-JNK between the blank control group and the solvent control group(P>0.05).The expression of p-JNK in the TCE-sensitized group was significantly higher than that in the blank control group.The difference was statistically significant(P<0.05).Compared with the 72 h TCE unsensitized group,the expression was significantly increased(P<0.05).72 h TCE + HOE140 sensitized group and 72 h TCE sensitized group.Compared with p-JNK expression,the difference was statistically significant(P<0.05).7.4 Expression of STAT3 and its phosphorylated protein in liver of mice Western blot analysis showed that there was no significant difference in the expression of p-STAT3 between the blank control group and the solvent control group(P>0.05).The expression of p-STAT3 in the TCE-sensitized group was significantly higher than that in the blank control group.The difference was statistically significant(P<0.05).Compared with the 72 h TCE unsensitized group,the expression was significantly increased(P<0.05).72 h TCE + HOE140 sensitized group and 72 h TCE sensitized group.Compared with p-STAT3 expression,the difference was statistically significant(P<0.05).8.Expression of MAPK pathway protein in liver Kupffer cells of mice in each group8.1 P38 MAPK and its phosphorylated protein expressed in Kupffer cells After separating Kupffer cells,the protein was extracted and detected by Western blot.The expression of p-P38 in the blank control group and the solvent control group was not significantly different(P>0.05),72 h TCE sensitized group p-P38 The expression was significantly increased compared with the blank control group,the difference was statistically significant(P<0.05),and the expression was enhanced compared with the 72 h TCE unsensitized group,the difference was statistically significant(P<0.05),72 h TCE + HOE140 sensitization Compared with the 72 h TCE sensitized group,the expression of p-P38 was decreased,and the difference was statistically significant(P<0.05).8.2 ERK MAPK and its phosphorylated protein expressed in Kupffer cells After separating Kupffer cells,the protein was extracted and detected by Western blot.The expression of p-ERK in the blank control group and the solvent control group was not significantly different(P>0.05),72 h TCE sensitized group p-ERK The expression was significantly increased compared with the blank control group,the difference was statistically significant(P<0.05),and the expression was enhanced compared with the 72 h TCE unsensitized group,the difference was statistically significant(P<0.05),72 h TCE + HOE140 sensitization Compared with the 72 h TCE sensitized group,the expression of p-ERK was decreased,and the difference was statistically significant(P<0.05).8.3 JNK MAPK and its phosphorylated protein expressed in Kupffer cells After separating Kupffer cells,the protein was extracted and detected by Western blot.The expression of p-JNK in the blank control group and the solvent control group was not significantly different(P>0.05).72 h TCE sensitized group p-JNK The expression was significantly increased compared with the blank control group,the difference was statistically significant(P<0.05),and the expression was enhanced compared with the 72 h TCE unsensitized group,the difference was statistically significant(P<0.05),72 h TCE + HOE140 sensitization Compared with the 72 h TCE sensitized group,the expression of p-JNK was decreased,and the difference was statistically significant(P<0.05).9.Kupffer cell inflammatory factor mRNA expression levels in each group of miceAfter separating Kupffer cells,total RNA was extracted,and the expression levels of Kupffer cell inflammatory factors IL-1β,IL-6 and TNF-α m RNA were detected by real-time quantitative PCR.The results showed that there was no significant difference in the expression of IL-1β,IL-6 and TNF-α m RNA between the blank control group and the solvent control group(P>0.05),and the IL-1β,IL-6 and Kupffer cells in the TCE sensitized positive group at 72 h.The expression of TNF-α m RNA was significantly increased.Compared with the blank control group and the 72 h TCE sensitized negative group,the difference was statistically significant(P<0.05).After treatment with HOE140,the Kupffer cells decreased IL-1β,IL-6.The expression of TNF-α m RNA was significantly different between TCE + HOE140 sensitized group and TCE sensitized group(P<0.05).10.Expression levels of inflammatory factors in liver tissue of mice in each group 10.1 IL-1β expression levels in the liverThe expression levels of IL-1β in the liver tissues of each group of mice are as shown.There was no significant difference between the blank control group and the solvent control group(P>0.05).The scores of the TCE sensitized group were significantly higher than those of the blank control group at 24 h and 72 h,the difference was statistically significant(P<0.05).The sensitization 72 h group had the highest expression,and the 24 h,72h TCE sensitization group had no difference.Significance(P>0.05);however,the expression of IL-1β in the TH + HOE140 sensitized group was significantly lower than that in the blank control group(P>0.05),and the difference was statistically different from the 72 h sensitized group.Academic significance(P<0.05).10.2 IL-6 expression levels in the liver The expression levels of IL-6 in the liver tissues of each group of mice are as shown.There was no significant difference between the blank control group and the solvent control group(P>0.05).The scores of the TCE sensitized group were significantly higher than those of the blank control group at 24 h and 72 h,the difference was statistically significant(P<0.05).The sensitization 72 h group had the highest expression,and the 24 h,72h TCE sensitization group had no difference.Significance(P>0.05);however,the expression of IL-6 in the 24 h TCE + HOE140 sensitized group was significantly lower than that in the blank control group,the difference was not statistically significant(P>0.05),and the difference was statistically different from the 24 h sensitized group.The significance of learning(P<0.05);72h TCE + HOE140 sensitized group IL-6 expression was significantly lower,compared with the blank control group,the difference was not statistically significant(P>0.05),compared with the 72 h sensitized group,the difference was statistically significant.Significance(P<0.05).10.3 TNF-α expression levels in the liver The expression levels of TNF-α in the liver tissues of each group of mice are as shown.There was no significant difference between the blank control group and the solvent control group(P>0.05).The scores of TCE sensitized group were significantly higher than those of the blank control group at 24 h and 72 h,the difference was statistically significant(P<0.05).The 72 h sensitized group had the highest expression,and the difference between TCE sensitized group at 24 h and 72 h was not statistically significant.Significance(P>0.05);however,the expression of TNF-α in the TH + HOE140 sensitized group was significantly lower than that in the control group(P>0.05),and the difference was statistically different from the 72 h sensitized group.Academic significance(P<0.05).Conclusions 1.The expression of BK and B2R in liver tissue of TCE-sensitized mice was significantly increased,accompanied by Kupffer cell activation;2.HOE140 can significantly improve immune liver injury in TCE-sensitized mice;3.MAPK signaling pathway and STAT 3 protein were enhanced in the liver of TCE-sensitized mice,and HOE140 could significantly reduce its expression;Kupffer cell MAPK signaling pathway and its mediated expression of inflammatory factors IL-1β,IL-6 and TNF-α were significantly enhanced,and HOE140 could improve its expression level;4.BK and its receptor B2 R mediate MAPK regulation of Kupffer cell inflammatory factor release plays an important role in immune liver injury in TCE-sensitized mice... |
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