Activation Of CGAS-STING Pathway Is Involved In Renal Tubular Injury In Trichloroethylene-sensitized Mice

Background and Objective Trichoroethylene(TCE)is an organic solvent,which is widely used in metal surface cleaning,organic synthesis,etc.It is one of the common environmental pollutants,and reach the body through a variety of ways,such as skin and respiratory tract.Occupational environmental TCE exposure can cause occupational medicamentosa-like dermatitis due to trichloroethylene(OMDT).Numerous studies have shown that,OMDT patients have severe liver damage and skin damage.In addition,kidney damage is also one of key reasons for the increase of disease load of OMDT.Previous studies of our group have found that TCE-sensitized mice have mitochondrial swelling and vacuolar degeneration,and mitochondrial damage is a common cause of acute and chronic kidney diseases.However,the exact mechanism by which mitochondrial damage causes immune kidney injury in TCE-sensitized mice is still unclear.This study aims to explore the mechanism of TCE-induced immune kidney injury in mice by TCE-sensitized mouse model combined with in vitro cell experiments.Methods TCE sensitization model was established,and the mice were randomly divided into blank control group,solvent control group,TCE treatment group and TCE+C176treatment group.The kidney,blood and urine samples were collected for tests.HE was used to observe the structure of the kidney,and ELASA was used to detect the levels ofα1-MG and β2-MG in serum and urine.The changes of mitochondrial structure were observed by electron microscopy,and the expressions of COX-Ⅳ and TFAM were detected by WB and immunohistochemistry to reflect the damage of TCE sensitization on mitochondria of renal tubular epithelial cells.The intracellular mtDNA level was detected by RT-qPCR,and the protein expressions of cGAS and STING were detected by WB to reflect the release of mtDNA to the cytoplasm and the activation of cGAS-STING pathway.Western blotting and histochemistry were used to measure the expression and distribution of NF-κB and P-IRF3 in cytoplasm and nucleus.RT-qPCR was used to measure the mRNA levels of typeⅠ interferons(IFNα,IFNβ)and inflammatory cytokines(IL-6,IL-1β,TNFα).To reflect the role of cGAS-STING pathway activation in TCEinduced nuclear translocation of P-IRF3 and NF-κB and renal inflammation.For cell experiments,HK2 cells were treated with 0,10,100,200,500ng/ml mtDNA for24 h to find the best time dose effect,and then the following experiments were carried out after the best time dose treatment.Western blot was used to detect the expression of cGAS and STING proteins.Immunofluorescence was used to detect the nuclear expression of NF-κB and P-IRF3.The mRNA levels of type Ⅰ interferons(IFNα,IFNβ)and inflammatory cytokines(IL-6,IL-1β,TNFα)were detected by RT-qPCR.The role of mtDNA in cGAS-STING pathway activation and cell inflammation was explored in vitro.Results 1.The skin score of each group showed that there was no significant change in the skin of the control group,while 11 mice in the TCE treatment group showed skin edema and erythema(11/35),with a sensitization rate of 40%,and 16 mice in the TCE+C176 treatment group showed skin edema and erythema(16/35),with a sensitization rate of 45.7%.There was no significant difference in sensitization rate between the two groups(P>0.05).2.Mitochondrial swelling and vacuolar degeneration were observed in renal tubular epithelial cells of TCE sensitized mice under electron microscope.WB and immunohistochemistry results showed that the level of COX-Ⅳ in TCE sensitization positive group decreased,and RT-qPCR showed that the mRNA expression of COX-Ⅳalso decreased significantly.That is,TCE sensitization can cause mitochondrial damage.3.The results of WB and immunohistochemistry showed that the expression level of TFAM was decreased in the TCE sensitization positive group,and RT-qPCR showed that the expression level of TFAM mRNA was also significantly decreased.RT-qPCR showed that the expression levels of mtDNA-related genes(mt-co1,mt-ND6,mt-RNR2,and mtCYTB)in the cytoplasm were increased.It can be seen that TCE sensitization can cause TFAM deletion and increased cytoplasmic mtDNA levels.4.WB results showed that the expression levels of cGAS and STING proteins in TCE sensitization positive group were increased,and the expression levels in C176+TCE treatment positive group were lower than those in TCE sensitization positive group.When HK2 cells were treated with different concentrations of mtDNA at a dose of 100ng/ml for24 h,the protein expression levels of cGAS and STING were significantly increased.RTqPCR results also showed that the expression levels of cGAS and STING genes were increased after treatment with 100ng/ml for 24 h.That is,cytosolic mtDNA can activate the cGAS-STING signaling pathway.5.The expression of P-IRF3 and NF-κB in cytoplasm and nucleus were detected by WB,and both indicated that the express levels of P-IRF3 and NF-κB were elevated in the TCEsensitized positive group,and the expression was decreased in the C176+TCE-sensitized positive group compared with the TCE-sensitized positive group.In cell experiments,Western blotting results showed that the expression levels of P-IRF3 and NF-κB in the nucleus were increased in the mtDNA treatment group,and the immunofluorescence results also showed that the expression levels of P-IRF3 and NF-κB in the nucleus were significantly increased in the mtDNA treatment group.It was concluded that TCE sensitization could activate P-IRF3 and NF-κB and their pathways,while STING inhibitor(C176)could inhibit this effect.6.In animal and cell experiments,the expression levels of type Ⅰ interferon(IFNα,IFNβ)and inflammatory cytokines(IL-6,IL-1β,TNFα)were detected by RT-qPCR in TCE sensitization positive group and mtDNA treatment group,respectively.The expression of C176+TCE group was lower than that of TCE group.WB analysis of IFNβ and IL-6showed the same conclusion.That is,nuclear translocation of P-IRF3 and NF-κB can increase the levels of IFN and cytokines in the kidneys of TCE-sensitized mice,and STING inhibition can alleviate the abnormal increase.7.The HE results of the mice kidney showed that compared with the TCE sensitization positive group,the TCE+C176 sensitization positive group had significantly reduced kidney injury.The results of ELASA in serum and urine of mice showed that the expression levels of α1-MG and β2-MG in C176+TCE sensitization positive group were significantly lower than those in TCE sensitization positive group.These results indicate that STING inhibition can alleviate renal injury induced by TCE sensitization in mice.Conclusion 1.TCE sensitization causes mitochondrial damage in renal tubular epithelial cells,which leads to the down-regulation of TFAM,and eventually leads to the release of mtDNA to the cytoplasm.2.The increase of mtDNA in the cytoplasm activates the cGAS-STING pathway,causes nuclear translocation of NF-κB and P-IRF3,promotes the synthesis and secretion of typeⅠ interferon and inflammatory cytokines,and eventually leads to kidney inflammatory injury.