| Background Trichloroethylene(TCE)is an excellent organic solvent commonly used for metal degreasing,dry cleaning,and extraction of paraffin.The health damage caused by TCE mainly includes occupational dermatitis medicamentosa-like of trichloroethylene(OMLDT),nervous system damage and liver and kidney damage.Acute renal failure can occur in OMLDT patients,and threatens their lives.Our previous study found that TCE-sensitized mice had significant tubular damage and C5 a expression,and C5b-9 expressed in liver cells.C5b-9 is the final product of complement system activation,which activates the intracellular signaling pathway when it exerts the sublytic effect,and promotes the assembly of NLRP3 inflammasome,releases inflammatory factors,and participated in the body's inflammatory response.Objective This study aims to investigate the level of C5b-9 and whether NLRP3 inflammasome activated in renal tubular epithelial cells of TCE-sensitized mouse.Then injecting recombinant rat s C D59-Cys into mice and detecting the deposition of C5b-9 in renal tubular epithelial cells and the renal injury of each group mice to determine whether NLRP3 inflammsome was activated by the sublytic levels of C5b-9.Finally exploring the mechanism of TCE-sensitized mice renal immune injury.Methods The BALB/c mouse were used for building TC E-sensitized mouse model.Part of the mouse were used for renal tubular epithelial cells immunohistochemistry and immunofluorescence detection and lactate dehydrogenase level determination.A total of 52 mouse were randomly divided into blank control group(n=5),solvent control group(n=5),TCE treatment group(n=18),TCE+s C D59-Cys treatment group(n=24).Then established a TCE sensitization mouse model.On the experiment 19 th day,mouse in TC E + s CD59-Cys treated group were intraperitoneally injected with 10 ?l of phosphate buffer containing 25 ?g of s CD59-Cys before the last challenge treatment.All mouse were divided into TCE sensitized group and TCE non-sensitized group 24 hours after the last challenge and the blood in eyeball and kidney were taken form sacrificed mouse at 72 h after the last challenge.Mouse serum were used for renal tubular neutrophil gelatinase-associated apolipoprotein(NGAL)levels measurement.The kidney were used for the pathological injury detection,the renal inflammation index NLRP3,IL-1? and IL-18 detection by immunohistochemical method,the protein level of N LRP3,ASC,Caspase-1 and IL-1? detection by Western Blot,and the gene level of NLRP3,ASC,Caspase-1,IL-1?,IL-18 detection.Results 1 The sensitization rate of the blank control group and the solvent control group was 0.The sensitization rate of TCE group and TC E+s CD59-Cys group was 38.9% 41.6% respectively.2 Immunohistochemistry and immunofluorescence identification of renal tubular epithelial cells showed that the extracted cells were cobblestone-like and had basically covered the entire field of view.The CK18 protein is abundantly expressed in almost all cel s.3 According to the formula for cytotoxicity or mortality,the cell lactate dehydrogenase(LDH)release rate is less than 5%,the C5b-9 is at sublytic level of tubular epithelial cel s.4 The renal pathological results of the mice showed that some epithelial cells vacuolated,a small amount of epithelial cells cytoplasmic dissolved and disappeared,and the inflammatory cells infiltrated in TCE sensitized positive group.TCE+s C D59-Cys sensitization-positive group also occasionally showed vacuolated tubular epithelial cells,kidney injury was significantly reduced compared with TCE-sensitized group.The results of immunohistochemistry of mouse kidney injury molecule-1(KIM-1)showed that in TCE-sensitized group,a large amount of KIM-1 was expressed in renal tubules,and the expression level of KIM-1 in the TCE+s C D59-Cys-sensitized group was significantly reduced.Serum NGAL levels were significantly elevated in the TC E-sensitized group,and the NGAL level in the TCE-sensitized group was higher than TCE+s CD59-Cys-sensitized group.5 Immunohistochemistry was used to detect the expression of renal C5b-9.Results from TC E sensitized positive group showed a large number of C5b-9 mainly expressed in renal tubular epithelial cells.TCE+s CD59-Cys sensitized positive group also expressed C5b-9 in renal tubular epithelial cells,but the expression level was significantly decreased compared with the TCE sensitized positive group.6 The expression level of N LRP3,IL-1? and IL-18 representing for kidney inflammation index were detected by immunohistochemistry.TC E sensitized positive group showed a large number of NLRP3,IL-1? and IL-18 mainly expressed in re nal tubular epithelial cells.The protein of NLRP3,IL-1? and IL-18 were also observed in TCE+s C D59-Cys sensitized positive group,but the expression level was significantly decreased compared with the TCE sensitized positive group.7 Results from Western Blot showed that compared to solvent control group,N LRP3,ASC,Caspase-1 and IL-1? significantly increased in TC E sensitized positive group and TCE+s C D59-Cys sensitized positive group.But they expressed in TCE+s C D59-Cys sensitized positive group were significantly lower than TCE sensitized positive group.8 The results of renal PCR showed that compared to TCE+s C D59-Cys-sensitized group,the gene levels of NLRP3,ASC,Caspase-1,IL-1?,IL-18 in TC E-sensitized positive group were significantly increased.Compared to solvent control group,the IL-1? expression level of TCE+s CD59-Cys sensitized positive group did not increased significantly.Compared with TC E+s CD59-Cys sensitized negative group,the expression levels of N LRP3 and IL-1? in TCE+s CD59-Cys sensitized positive mice did not increase significantly.Conclusions 1 This study found that sub-lytic C5b-9 activates NLRP3 inflammasome in renal tubular epithelial cells of TC E-sensitized mice,IL-1?and IL-18 up-regulated,and renal tubular epithelial cell was damaged in TCE-sensitized mice.2 After injection of s CD59-Cys,the damage of renal tubular epithelial cells were alleviated.Results suggest that the decrease or lack of CD59 a expression in renal tubular epithelial cells of TCE-sensitized mice leads to its weakening of C5b-9 inhibition.The massive deposition of C5b-9 exerts its sub-lytic effect to activate NLRP3 inflammasome and up-regulate IL-1?,IL-18 and other inflammations.3 After injection of CD59 protein,the damage of renal tubular epithelial ce lls can not be completely controlled,suggesting that there are other mechanisms of action on renal tubular epithelial cell damage,which needs further study. |
PDF Full Text Request