Histone demethylase KDM4A mediating macrophage polarization: A potential mechanism of trichloroethylene induced liver injury.

Trichloroethylene (TCE) is a commonly used organic solvent in industry. Our previous studies have found that TCE can cause liver injury accompanied by macrophage polarization, but the specific mechanism is unclear. The epigenetic regulation of macrophage polarization is mainly focused on histone modification. Histone lysine demethylase 4A (KDM4A) is involved in the activation of macrophages. In this study, we used a mouse model we investigated the role of KDM4A in the livers of TCE-drinking mice and found that the expression of KDM4A, M1-type polarization indicators, and related inflammatory factors in the livers of TCE-drinking mice. In the study, BALB/c mice were randomly divided into four groups: 2.5 mg/mL TCE dose group and 5.0 mg/mL TCE dose group, the vehicle control group, and the blank control group. We found that TCE triggered M1 polarization of mouse macrophages, characterized by the expression of CD11c and robust production of inflammatory cytokines. Notably, exposure to TCE resulted in markedly increased expression of KDM4A in macrophages. Functionally, the increased expression of KDM4A significantly impaired the expression of H3K9me3 and H3K9me2 and increased the expression of H3K9me1. In addition, KDM4A potentially represents a novel epigenetic modulator, with its upregulation connected to ß-catenin activation, a signal critical for the pro-inflammatory activation of macrophages. Furthermore, KDM4A inhibitor JIB-04 treatment resulted in a decrease in ß-catenin expression and prevented TCE-induced M1 polarization and the expression of the pro-inflammatory cytokines TNF-α and IL-1ß. These results suggest that the association of KDM4A and Wnt/ß-catenin cooperatively establishes the activation and polarization of macrophages and global changes in H3K9me3/me2/me1. Our findings identify KDM4A as an essential regulator of the polarization of macrophages and the expression of inflammatory cytokines, which might serve as a potential target for preventing and treating liver injury caused by TCE.

The role of PANoptosis in renal vascular endothelial cells: Implications for trichloroethylene-induced kidney injury.

Trichloroethylene (TCE), a widely distributed environmental chemical contaminant, is extensively dispersed throughout the environment. Individuals who are exposed to TCE may manifest occupational medicamentose-like dermatitis due to trichloroethylene (OMDT). Renal impairment typically manifests in the initial phase of OMDT and is intricately linked to the disease progression and patient outcomes. Although recombinant human tumor necrosis factor-α receptor II fusion protein (rh TNFR:Fc) has been employed in the clinical management of OMDT, there was no substantial improvement in renal function observed in patients following one week of treatment. This study primarily examined the mechanism of TNFα- and IFNγ-induced endothelial cells (ECs) PANoptosis in TCE-induced kidney injury and hypothesized that the synergistic effect of TNFα and IFNγ could be the key factor affecting the efficacy of rh TNFR:Fc therapy in OMDT patients. A TCE-sensitized mouse model was utilized in this study to investigate the effects of TNFα and IFNγ neutralizing antibodies on renal vascular endothelial cell PANoptosis. The gene of interferon regulatory factor 1 (IRF1) in human umbilical vein endothelial cells (HUVEC) was silenced by using small interfering RNA (siRNA), and the cells were then treated with TNFα and IFNγ recombinant protein to investigate the mechanism of TNFα combined with IFNγ-induced PANoptosis in HUVEC. The findings indicated that mice sensitized to TCE exhibited increased levels of PANoptosis-related markers in renal endothelial cells, and treatment with TNFα and IFNγ neutralizing antibodies resulted in a significant reduction in PANoptosis and improvement in renal function. In vitro experiments demonstrated that silencing IRF1 could reverse TNFα and IFNγ-induced PANoptosis in endothelial cells. These results suggest that the efficacy of rh TNFR:Fc may be influenced by TNFα and IFNγ-mediated PANoptosis in kidney vascular endothelial cells. The joint application of TNFα and IFNγ neutralizing antibody represented a solid alternative to existing therapeutics.

Hepatocyte mitochondrial DNA mediates macrophage immune response in liver injury induced by trichloroethylene.

We have previously shown that excessive activation of macrophage proinflammatory activity plays a key role in TCE-induced immune liver injury, but the mechanism of polarization is unclear. Recent studies have shown that TLR9 activation plays an important regulatory role in macrophage polarization. In the present study, we demonstrated that elevated levels of oxidative stress in hepatocytes mediate the release of mtDNA into the bloodstream, leading to the activation of TLR9 in macrophages to regulate macrophage polarization. In vivo experiments revealed that pretreatment with SS-31, a mitochondria-targeting antioxidant peptide, reduced the level of oxidative stress in hepatocytes, leading to a decrease in mtDNA release. Importantly, SS-31 pretreatment inhibited TLR9 activation in macrophages, suggesting that hepatocyte mtDNA may activate TLR9 in macrophages. Further studies revealed that pharmacological inhibition of TLR9 by ODN2088 partially blocked macrophage activation, suggesting that the level of macrophage activation is dependent on TLR9 activation. In vitro experiments involving the extraction of mtDNA from TCE-sensitized mice treated with RAW264.7 cells further confirmed that hepatocyte mtDNA can activate TLR9 in mouse peritoneal macrophages, leading to macrophage polarization. In summary, our study comprehensively confirmed that TLR9 activation in macrophages is dependent on mtDNA released by elevated levels of oxidative stress in hepatocytes and that TLR9 activation in macrophages plays a key role in regulating macrophage polarization. These findings reveal the mechanism of macrophage activation in TCE-induced immune liver injury and provide new perspectives and therapeutic targets for the treatment of OMDT-induced immune liver injury.

C5b-9 promotes ferritinophagy leading to ferroptosis in renal tubular epithelial cells of trichloroethylene-sensitized mice.

Trichloroethylene (TCE) is a common environmental contaminant that can cause a severe allergic reaction called TCE hypersensitivity syndrome, which often implicates the patient's kidneys. Our previous study revealed that C5b-9-induced tubular ferroptosis is involved in TCE-caused kidney damage. However, the study did not explain how tubule-specific C5b-9 causes free iron overload, a key event in ferroptosis. Here, we aimed to explore the role of NCOA4-mediated ferritinophagy in C5b-9-induced iron overload and ferroptosis in TCE-sensitized mice. Our results showed that TCE sensitization does not affect iron import or export, but does affect iron storage, causing ferritin degradation and free iron overload. In addition, mitochondrial ROS was upregulated, and these changes were blocked by C5b-9 inhibition. Interestingly, TCE-induced ferritin degradation and ferroptosis were significantly antagonized by the application of the mitochondrial ROS inhibitor, Mito-TEMPO. Moreover, all of these modes of action were further verified in C5b-9-attack signalling HK-2 cells. Further investigation demonstrated that C5b-9-upregulated mitochondrial ROS induced a marked increase in nuclear receptor coactivator 4 (NCOA4), a master regulator of ferritinophagy. In addition, the application of NCOA4 small interfering RNA not only significantly reversed ferritinophagy caused by C5b-9 but also reduced C5b-9-induced ferroptosis in HK-2 cells. Taken together, these results suggest that tubule-specific C5b-9 deposition activates NCOA4 through the upregulation of mitochondrial ROS, causing ferritin degradation and elevated free iron, which ultimately leads to tubular epithelial cell ferroptosis and kidney injury in TCE-sensitized mice.

C5a/C5aR1 axis as a key driver promotes epithelial-to-mesenchymal transition in airway epithelial cells in silica nanoparticles-induced pulmonary fibrosis.

Previous studies have shown that silica nanoparticles (SiNPs) exposure can affect the respiratory, cardiovascular, reproductive and other systems, with the lung being the primary target organ for the direct effect, causing damage with a central feature of pulmonary inflammation and fibrosis. However, the underlying mechanisms of pulmonary fibrosis due to SiNPs are not fully understood. The aim of the study was to investigate the role of complement anaphylatoxin C5a in SiNPs-induced pulmonary fibrosis. A mouse model of SiNPs-induced pulmonary fibrosis was established, and pulmonary fibrosis-related indicators, epithelial-to-mesenchymal transition (EMT), C5a/C5aR1 and high mobility group protein B1 (HMGB1) proteins were measured. An in vitro study using the human lung epithelial cell line BEAS-2B investigated whether C5a leads to epithelial-to-mesenchymal trans-differentiation. In vivo studies revealed that SiNPs-induced pulmonary fibrosis mainly manifested as EMT trans-differentiation in airway epithelial cells, which subsequently led to excessive deposition of extracellular matrix (ECM). Furthermore, we found that C5a and C5aR1 proteins were also increased in SiNPs-induced pulmonary fibrosis tissue. In vitro studies also showed that C5a directly activated HMGB1/RAGE signaling and induced EMT in BEAS-2B cells. Finally, treatment of SiNPs-exposed mice with the C5aR1 inhibitor PMX205 effectively reduced C5aR1 levels and inhibited the activation of HMGB1/RAGE signaling and the expression of EMT-related proteins, culminating in a significant alleviation of pulmonary fibrosis. Taken together, our results suggest that C5a/C5aR1 is the main signaling pathway for SiNPs-induced pulmonary fibrosis, which induces EMT in airway epithelial cells via the HMGB1/RAGE axis.

The role of Kupffer cell activation in immune liver damage induced by trichloroethylene associated with the IFN-γ/STAT1 signaling pathway.

Trichloroethylene (TCE) is a metal detergent commonly used in industry that can enter the human body through the respiratory tract and skin, causing occupational medicamentosa-like dermatitis due to TCE (OMDT) and multiple organ damage, including liver failure. However, the pathogenesis of liver injury remains unclear. Kupffer cells (KCs) are important tissue macrophages in the body because the polarization of KCs plays a crucial role in immune-mediated liver injury. However, the mechanism of KCs polarization in TCE-induced immune liver injury has not been thoroughly elucidated. In this study, we investigated the effect of TCE-induced KCs polarization on liver function and signal transduction pathways using the TCE sensitization model developed by our group. BALB/c mouse skin was exposed to TCE for sensitization, and an increase in the expression of M1 macrophage-specific markers (CD16/CD32, iNOS), M1 macrophage-specific cytokines IL-1ß, and IFN-γ, P-JAK-1 and P-STAT1 levels were also found to be dramatically increased. When using low doses of gadolinium trichloride (GdCl3), the expression of these proteins and mRNA was significantly reduced. This phenomenon indicates that GdCl3 blocks TCE-induced polarization of KCs and suggests that the IFN-γ/STAT1 signaling pathway may be involved in the polarization process of KCs. These findings clarify the relationship between the polarization of KCs and immune liver injury and highlight the importance of further study of immune-mediated liver injury in TCE-sensitized mice.

Macrophage autophagy contributes to immune liver injury in trichloroethylene sensitized mice: Critical role of TNF-α mediating mTOR pathway.

Trichloroethylene (TCE) induces occupational medicamentosa-like dermatitis due to TCE (OMDT) with immune liver injury, and TNF-α plays an important role in macrophage polarization and liver injury. However, TNF-α regulating macrophage polarization in liver injury induced by TCE is still unknown. Thus, on the basis of our previous research, we established the TCE-sensitized BALB/c mouse model with R7050, a specific inhibitor of TNFR1. Then, we observed significant decreases in autophagy related protein and gene levels in M1 macrophage in TCE positive group, and R7050 can relieve M1 macrophage autophagy. We also found the phosphorylated form of mammalian target of Rapamycin (mTOR) was activated and the expression of p-mTOR protein increased induce by TCE. In vitro, we found TNFR1 and CD11c were increased in RAW264.7 cell line with TNF-α. And then we use Zafirlukast (Zaf), an TNFR1 antagonist, CD11c and TNFR1 reduced significantly, we also found p-mTOR expression increased after TNF-α treatment, but decreased in TNF-α + Zaf group. Further, we used Rapamycin (RAP), a mTOR-specific inhibitor, to establish a TCE-sensitized mice model and found the expression levels of p62 and p-mTOR proteins increased and LC3B decreased in the TCE positive group, while RAP treatment reversed the trends of all of these proteins. Rapamycin prevented the TNF-α-induced p-mTOR increase and dramatically downregulated IL-1ß expression in the RAW264.7 cell line with TNF-α treatment. The results uncover a novel role for TNF-α/TNFR1, which promotes M1 polarization of macrophage and suppresses macrophage autophagy via the mTOR pathway.

IP3R-dependent mitochondrial dysfunction mediates C5b-9-induced ferroptosis in trichloroethylene-caused immune kidney injury.

Patients with occupational medicamentose-like dermatitis due to trichloroethylene often suffer from immune kidney injury. Our previous study reveals that C5b-9-dependent cytosolic Ca2+ overload-induced ferroptosis is involved in trichloroethylene sensitized kidney injury. However, how C5b-9 causes cytosolic Ca2+ rise and the specific mechanism whereby overloaded Ca2+ induces ferroptosis remain unknown. The purpose of our study was to explore the role of IP3R-dependent mitochondrial dysfunction in C5b-9 mediated ferroptosis in trichloroethylene sensitized kidney. Our results showed that IP3R was activated, and mitochondrial membrane potential was decreased in the renal epithelial cells of trichloroethylene-sensitized mice, and these changes were antagonized by CD59, a C5b-9 inhibitory protein. Moreover, this phenomenon was reproduced in a C5b-9-attacked HK-2 cell model. Further investigation showed that RNA interference with IP3R not only alleviated C5b-9-induced cytosolic Ca2+ overload and mitochondrial membrane potential loss but also attenuated C5b-9-induced ferroptosis in HK-2 cells. Mechanistically, IP3R-dependent cytosolic Ca2+ overload activated the mitochondrial permeability transition pore, resulting in the loss of mitochondrial membrane potential and ferroptosis of HK-2 cells. Finally, cyclosporin A, a mitochondrial permeability transition pore inhibitor, not only ameliorated IP3R-dependent mitochondrial dysfunction but also blocked C5b-9-induced ferroptosis. Taken together, these results suggest that IP3R-dependent mitochondrial dysfunction plays an important role in trichloroethylene sensitized renal tubular ferroptosis.

HMGB 1 acetylation mediates trichloroethylene-induced immune kidney injury by facilitating endothelial cell-podocyte communication.

More and more clinical evidence shows that occupational medicamentose-like dermatitis due to trichloroethylene (OMDT) patients often present immune kidney damage. However, the exact mechanisms of cell-to-cell transmission in TCE-induced immune kidney damage remain poorly understood. The present study aimed to explore the role of high mobility group box-1 (HMGB 1) in glomerular endothelial cell-podocyte transmission. 17 OMDT patients and 34 controls were enrolled in this study. We observed that OMDT patients had renal function injury, endothelial cell activation and podocyte injury, and these indicators were associated with serum HMGB 1. To gain mechanistic insight, a TCE-sensitized BALB/c mouse model was established under the interventions of sirtuin 1 (SIRT 1) activator SRT 1720 (0.1 ml, 5 mg/kg) and receptor for advanced glycation end products (RAGE) inhibitor FPS-ZM 1 (0.1 ml, 1.5 mg/kg). We identified HMGB 1 acetylation and its endothelial cytoplasmic translocation following TCE sensitization, but SRT 1720 abolished the process. RAGE was located on podocytes and co-precipitated with extracellular acetylated HMGB 1, promoting podocyte injury, while SRT 1720 and FPS-ZM 1 both alleviated podocyte injury. The results demonstrate that interventions to upstream and downstream pathways of HMGB 1 may weaken glomerular endothelial cell-podocyte transmission, thereby alleviating TCE-induced immune renal injury.

Impaired autophagy-accelerated senescence of alveolar type II epithelial cells drives pulmonary fibrosis induced by single-walled carbon nanotubes.

BACKGROUND: The rapid increase in production and application of carbon nanotubes (CNTs) has led to wide public concerns in their potential risks to human health. Single-walled CNTs (SWCNTs), as an extensively applied type of CNTs, have shown strong capacity to induce pulmonary fibrosis in animal models, however, the intrinsic mechanisms remain uncertain. RESULTS: In vivo experiments, we showed that accelerated senescence of alveolar type II epithelial cells (AECIIs) was associated with pulmonary fibrosis in SWCNTs-exposed mice, as well as SWCNTs-induced fibrotic lungs exhibited impaired autophagic flux in AECIIs in a time dependent manner. In vitro, SWCNTs exposure resulted in profound dysfunctions of MLE-12 cells, characterized by impaired autophagic flux and accelerated cellular senescence. Furthermore, the conditioned medium from SWCNTs-exposed MLE-12 cells promoted fibroblast-myofibroblast transdifferentiation (FMT). Additionally, restoration of autophagy flux with rapamycin significantly alleviated SWCNTs-triggered senescence and subsequent FMT whereas inhibiting autophagy using 3-MA aggravated SWCNTs-triggered senescence in MLE-12 cells and FMT. CONCLUSION: SWCNTs trigger senescence of AECIIs by impairing autophagic flux mediated pulmonary fibrosis. The findings raise the possibility of senescence-related cytokines as potential biomarkers for the hazard of CNTs exposure and regulating autophagy as an appealing target to halt CNTs-induced development of pulmonary fibrosis.

Endoplasmic reticulum stress mediates nickel chloride-induced epithelial­mesenchymal transition and migration of human lung cancer A549 cells through Smad2/3 and p38 MAPK activation.

BACKGROUND: The endoplasmic reticulum (ER) is a cellular membrane-bound organelle whereby proteins are synthesized, folded and glycosylated. Due to intrinsic (e.g., genetic) and extrinsic (e.g., environmental stressors) perturbations, ER proteostasis can be deregulated within cells which triggers unfolded protein response (UPR) as an adaptive stress response that may impact the migration and invasion properties of cancer cells. However, the mechanisms underlying the nickel compounds on lung cancer cell migration and invasion remain uncertain. OBJECTIVE: We aimed to study whether Nickel chloride (NiCl2) induces ER stress in lung cancer cells, and whether ER stress is involved in modulating epithelial-mesenchymal transition (EMT) and migration by Smads and MAPKs pathways activation following NiCl2 treatment. METHODS: A549 cells were treated with NiCl2 to determine the cell viability using MTT assay. The wound healing assay was used to evaluate cell migration ability. ER ultrastructure was observed by transmission electron microscopy. Western blotting assay was performed to evaluate the protein levels of BIP, PERK, IRE-1α, XBP-1 s, and ATF6 for ER stress and UPR, E-cadherin and Vimentin for EMT, p-Smad2/3, p-ERK, p-JNK, and p-P38 for activation of Smads and MAPKs signaling pathways. RESULTS: The expression levels of BIP, PERK, IRE-1α, XBP-1 s, and ATF6 were significantly increased following treatment with NiCl2 in time- and dose-effect relationship. The ER stress inhibitor 4-PBA downregulated the expression levels of the above five proteins, and reversed the decrease in E-cadherin protein level and the increase in vimentin protein expression and cell migration abilities caused by NiCl2. Furthermore, 4-PBA significantly reduced nickel chloride-induced Smad2/3 and p38 MAPK pathway activation, while not affected ERK and JNK MAPK pathways. CONCLUSION: NiCl2 triggers ER stress and UPR in A549 cells. Moreover, 4-PBA alleviates NiCl2-induced EMT and migration ability of A549 cells possibly through the Smad2/3 and p38 MAPK pathways activation, rather than ERK and JNK MAPK pathways.

Renal tubular in TCE-sensitization-induced immune kidney injury: Role of mitochondrial DNA in activating the cGAS-STING signaling pathway.

Occupational medicamentose-like dermatitis due to trichloroethylene (OMDT) is a systemic allergic disease similar to drug eruption-like dermatitis that occurs in workers after exposure to trichloroethylene. In addition to skin and mucosa damage, OMDT patients often accompanied by severe multiorgan damage, including kidney injury. However, the mechanism remains unclear. The aim of our research was to explore the role of increased cytosolic mitochondrial DNA in the activation of cGAS-STING signaling and in the kidney injury of trichloroethylene sensitization mice using a mouse model and an in vitro model. By analyzing the kidneys of TCE-sensitized mice, we found obvious tubular mitochondrial damage, decreased expression of COX-IV and TFAM proteins and increased cytosolic mitochondrial DNA in TCE-sensitized-positive mice. Further study found that cytosolic mitochondrial DNA activated cGAS-STING signaling, resulting in the nuclear translocation of P-IRF3 and NF-κB P65 and the transcription and synthesis of type Ⅰ interferons and cytokines, which ultimately led to immune kidney injury in trichloroethylene-sensitized mice. Interestingly, pretreatment with C-176, a STING inhibitor, not only blocked the nuclear translocation of P-IRF3 and NF-κB P65, but also alleviated the kidney injury induced by TCE sensitization. Consistently, in vitro studies also found that mitochondrial DNA pretreatment can activate the cGAS-STING pathway, causing the nuclear translocation of P-IRF3 and NF-κB P65 and the transcription of type Ⅰ interferons and cytokines in HK-2 cells. Overall, our results suggested that cytosolic mitochondrial DNA plays an important role in the activation of the cGAS-STING pathway and TCE sensitization-induced immune kidney injury.

ROS-mediated inflammatory response in liver damage via regulating the Nrf2/HO-1/NLRP3 pathway in mice with trichloroethylene hypersensitivity syndrome.

Trichloroethylene hypersensitivity syndrome (THS), mainly caused by occupational exposure to trichloroethylene (TCE), can give rise to serious and fatal hepatic damage. To date, the precise mechanisms of hepatic damage in THS remain unclear. Recent studies showed that reactive oxygen species (ROS) play a core role in cell death and inflammatory response. Therefore, the present study sought to explore whether ROS-mediated inflammatory responses contribute to the hepatic damage in TCE sensitization. To this end, a mouse model of TCE sensitization was established; in some cases, hosts were pretreated with tempol, an ROS scavenger. The results showed that TCE sensitization caused hepatic pathological/functional changes, ROS generation, and oxidative stress, alterations of the anti-oxidant defense Nrf2/HO-1/NLRP3 pathway, and pro-inflammatory cytokine formation in the liver. ROS scavenging via pretreatment with tempol was found not only to inhibit the hepatic oxidative stress, but also to regulate Nrf2/HO-1/NLRP3 pathway activity. In all cases, tempol was able to mitigate the pathologic changes induced by TCE sensitization. In summary, the results here demonstrated a novel molecular mechanism wherein ROS-mediated inflammatory responses play a central role in TCE-induced liver damage. Therapies targeting ROS scavenging could help to protect against hepatic damage by regulating Nrf2/HO-1/NLRP3 pathway activities in TCE-sensitized hosts.

Trichloroethylene induces immune renal tubular injury through SIRT 1/HSP 70/TLR 4 pathway in BALBc mice.

Trichloroethylene (TCE) is a volatile chlorinated solvent widely used for cleaning and degreasing industrial metal parts. Due to the widespread use and improper disposal of TCE, exposure to TCE causes a variety of adverse effects on human and animal health. However, the underlying mechanism of the damage remains unclear. The purpose of this study is to investigate the role of Sirtuin-1 (SIRT 1) in TCE-induced immune renal tubular injury. 6-8-week-old female BALB/c mice were used to construct a TCE sensitized mouse model. SIRT 1 activator, SRT 1720 (0.1 ml, 5 mg/kg) and toll like receptor 4 (TLR 4) inhibitor, TAK-242 (0.1 ml, 3 mg/kg) were used for treatment. Results show that SIRT 1 and heat shock protein 70 (HSP 70) levels are significantly down-regulated in renal tubules, serum and urine HSP 70 levels are significantly increased, and inflammatory cytokines levels are significantly increased in renal tubules in TCE-sensitized positive mice. After SRT 1720 treatment, intracellular HSP 70 level is significantly increased and extracellular HSP 70 level is decreased, and inflammatory cytokines levels get alleviated. In addition, HSP 70 and Toll-like Receptor 4 (TLR 4) proteins exist an interaction that can be significantly attenuated by SIRT 1. Subsequently, inflammation of the renal tubules mediated by SIRT 1 downregulation is attenuated after TAK-242 treatment. In conclusion, SIRT 1 alleviates renal tubular epithelial cells immune injury by inhibiting the release of HSP 70 and thereby weakening interaction with HSP 70 and TLR 4.

C5b-9 mediates ferroptosis of tubular epithelial cells in trichloroethylene-sensitization mice.

Occupational medicamentose-like dermatitis due to trichloroethylene (OMDT) is a key but unresolved question. OMDT patients often present multiple organ damage, including kidney damage. However, the underlying mechanism remains unknown. The purpose of our study was to explore the effect of tubule-specific C5b-9 deposition induced by TCE sensitization on renal tubular ferroptosis and its mechanism. By analyzing pathological changes of TCE-sensitization-mice kidney, we observed a significant renal tubular ferroptosis, which was alleviated by CD59, a C5b-9 inhibitory protein. Moreover, this phenomenon was also replicated in a C5b-9-attacked HK-2 cell model. Further experiments identified that C5b-9 induced cytosolic Ca2+ overload in renal tubular epithelia cells from TCE-sensitization-mice and HK-2 cells. Furthermore, in vitro experiments showed that BAPTA-AM, an intracellular Ca2+ chelator, could rescued ferroptosis induced by C5b-9 in HK-2 cells. Taken together, TCE sensitization induced renal tubular ferroptosis is mediated by C5b-9 and cytosolic Ca2+ overload may play a key role.

Effects of mitochondrial reactive oxygen species-induced NLRP3 inflammasome activation on trichloroethylene-mediated kidney immune injury.

This study aimed to investigate the activating mechanism of the NLRP3 inflammasome in trichloroethylene-sensitized mice. In total, 88 BALB/c female mice were used to establish the trichloroethylene (TCE)-sensitized mouse model. Some of the mice received MitoTEMPO, MCC 950 or soluble recombinant CD59-Cys to inhibit mitochondrial reactive oxygen species (mtROS) production, NLRP3 assembly, or C5b-9 formation. Mouse tubular epithelial cell expression levels of NLRP3, ASC, Caspase 1, IL-1ß, IL-18 and mitochondrial antiviral signaling protein (MAVS) were detected by western blot. Mitochondrial numbers, membrane potential (ΔΨm) and mtROS were detected by using MitoScene Green II, JC-1 dye and MitoSOX Red indicator, respectively. Tubular epithelial cell calcium levels were detected by a Fluo-8 no wash calcium assay kit. Human kidney-2 (HK-2) cells were cultured and stimulated by C5b6 and normal human serum (NHS) to verify the role of C5b-9-induced mitochondrial ROS in activating NLRP3 inflammasome. Urine α1-MG, ß2-MG, and mtROS production and calcium levels were increased, while mitochondrial numbers were decreased in TCE-sensitized positive mice. After treatment with MitoTEMPO, renal tubular injury was alleviated, JC-1 fluorescence and mitochondrial numbers were significantly increased, and mitochondrial ROS were inhibited. The NLRP3 inflammasome was activated in TCE-sensitized positive mice, while Mito TEMPO inhibited MAVS expression and NLRP3 inflammasome activation. The in vitro studies proved that C5b-9 can induce mtROS release and activate the assembly of NLRP3 inflammasome in HK-2 cells. In conclusion, in TCE-sensitized positive mouse renal tubular epithelial cells, C5b-9 caused calcium influx and thus induced mitochondrial injury and mtROS overexpression, finally inducing MAVS expression and NLRP3 inflammasome activation and kidney injury.

Wnt 5a mediated inflammatory injury of renal tubular epithelial cells dependent on calcium signaling pathway in Trichloroethylene sensitized mice.

Patients with trichloroethene-induced Trichloroethylene hypersensitivity syndrome (THS) often present kidney injury. However, the role of Wnt 5a/Ca2+ pathway in renal tubular injury in Trichloroethylene (TCE) sensitized mice remains unclear. This study aimed to investigate how Wnt 5a/Ca2+ pathway induced renal tubular epithelial cell injury in TCE sensitized mice. A total of 84 female BALB/c Specific Pathogen Free mice aged 6-8 weeks were used to establish TCE sensitized mouse models. Renal histology and serum levels of α1-MG and ß2-MG were used to assess the renal injury. The renal protein levels of Wnt 5a, ROR2, FZD5, PLC, p-CaMKII, IκB α, p-IκB α, NF-κB(p65), TNF α, IL 6 and IL 1ß were measured. The levels of serum α1-MG and ß2-MG and TNF α, IL 6 and IL 1ß levels in the kidney tissue were significantly increased in TCE sensitized positive group. However, Box5 pretreatment inhibited the expression of PLC, p-CaMKII, p65 and attenuated the injury of renal tubular epithelial cells and suppressed the upregulated expression of the above cytokines. In addition, KN93 also reduced nuclear translocation of p65 and renal injury as well as the elevated cytokines by inhibiting CaMKII. These data identify Wnt 5a binding to ROR2 and FZD5, p65 nuclear translocation, and inflammatory cytokine release as a novel mechanism for renal tubular epithelial cells injury by sensitization with TCE. Box5 or KN93 pretreatment can block the expression of inflammatory cytokines and reduce the injury of renal tubular epithelial cells.

Membrane Attack Complex C5b-9 Promotes Renal Tubular Epithelial Cell Pyroptosis in Trichloroethylene-Sensitized Mice.

Trichloroethylene (TCE), a commonly used organic solvent, is known to cause trichloroethylene hypersensitivity syndrome (THS), also called occupational medicamentosa-like dermatitis due to TCE (OMDT) in China. OMDT patients presented with severe inflammatory kidney damage, and we have previously shown that the renal damage is related to the terminal complement complex C5b-9. Here, we sought to determine whether C5b-9 participated in TCE-induced immune kidney injury by promoting pyroptosis, a new form of programed cell death linked to inflammatory response, with underlying molecular mechanisms involving the NLRP3 inflammasome. A BALB/c mouse-based model of OMDT was established by dermal TCE sensitization in the presence or absence of C5b-9 inhibitor (sCD59-Cys, 25µg/mouse) and NLRP3 antagonist (MCC950, 10 mg/kg). Kidney histopathology, renal function, expression of inflammatory mediators and the pyroptosis executive protein gasdermin D (GSDMD), and the activation of pyroptosis canonical NLRP3/caspase-1 pathway were examined in the mouse model. Renal tubular damage was observed in TCE-sensitized mice. GSDMD was mainly expressed on renal tubular epithelial cells (RTECs). The caspase-1-dependent canonical pathway of pyroptosis was activated in TCE-induced renal damage. Pharmacological inhibition of C5b-9 could restrain the caspase-1-dependent canonical pathway and rescued the renal tubular damage. Taken together, our results demonstrated that complement C5b-9 plays a central role in TCE-induced immune kidney damage, and the underlying mechanisms involve NLRP3-mediated pyroptosis.

Endothelin-1 down-regulated vascular endothelial growth factor A is involved in trichloroethene-induced kidney injury.

The mechanism of kidney injury in occupational medicamentosa-like dermatitis due to trichloroethylene exposure is not well understood. This study aimed to investigate the role of endothelin-1 (ET-1)/vascular endothelial-derived growth factor-A (VEGF-A) in trichloroethylene (TCE)-induced renal injury. Forty BALB/c female mice were used in this study to build the TCE-sensitization mouse model. Transmission electron microscopic observation, histological examination, periodic acid-Schiff staining, serum urea nitrogen, creatinine, and urinary total protein levels were used to reflect renal injury. Glypican1, syndecan1, ET-1 and VEGF-A protein levels were measured by western blot. Serum ET-1 level was also measured. Tumor necrosis factor alpha (TNF-α) and vascular cell adhesion molecule-1 (VCAM-1) were detected by immunohistochemistry. The results showed that TCE-sensitized mouse kidneys were damaged and accompanied by increased serum ET-1. After treatment with CGS 35066, the inhibitor of endothelin converting enzyme-1 (ECE-1), kidney ET-1, TNF-α and VCAM-1 levels decreased, and renal function improved in TCE+CGS 35066-sensitized positive mice. In addition, kidney VEGF-A, glomerular endothelial cell glypican1 and syndecan1 levels increased, and endothelial cell damage was alleviated after treatment with CGS 35066. The results suggest that inhibiting ECE-1 could alleviate glomerular endothelial cell injury by inhibiting ET-1 expression, thus promoting endothelial cell repair by upregulating VEGF-A.

TNF-α/TNFR1 regulates the polarization of Kupffer cells to mediate trichloroethylene-induced liver injury.

We have previously shown trichloroethylene (TCE) induced immune liver injury, and TNF-α/TNFR1 pathway as a probably mechanism underlying the immune damage, but the pathogenic mechanism is still unclear. The study aims to investigate whether TNF-α and its receptors regulate Kupffer cell polarization and downstream inflammation signaling pathways during TCE sensitization, to clarify the mechanism of TCE-mediated immune liver injury. 6-8 weeks old SPF BALB/c female mice were used to establish a TCE sensitization model. We found that in the TCE sensitization positive group, liver injury was aggravated, Kupffer cells activated and polarized to M1 type. The expression of M1 Kupffer cell marker proteins CD11c and CD16/32 increased in the TCE positive group, so did TNF-α and TNFR1 in liver. The expression of P-IKK protein, PP65 protein and P-STAT3 protein increased in the TCE sensitization positive group, and the downstream inflammatory factors IL-1ß and IL-6 also increased in the TCE sensitization positive group. After using the TNFR1 inhibitor R7050, we found that M1 Kupffer cell polarization, TNF-α expression, signal pathway expression and inflammatory factors IL-1ß and IL-6 expression declined, and the liver damage relieved. Briefly, the use of R7050 to inhibit TNF-α/TNFR1 changing the polarization of liver M1 Kupffer cell, thereby inhibiting the activation of related downstream signaling pathways and reducing the secretion of inflammatory factors. TNF-α/TNFR1 regulates the polarization of M1 Kupffer cells inflammatory play an important role in liver immune damage.