The role of miR-222-2p in exosomes secreted by hexavalent chromium-induced premature senescent hepatocytes as a SASP component.

With the development of world industrialization, the environmental pollution of hexavalent chromium [Cr(VI)] is becoming an increasingly serious problem. In particular, the mechanisms by which long-term and low-dose exposure to Cr(VI) leading the development of related cancers are not well understood. As senescent cells gradually lose their ability to proliferate and divide, they will not be malignantly transformed. However, Senescence-associated secretory phenotype (SASP) released by senescent cells into the cellular microenvironment can act on neighboring cells. Since SASP has a bidirectional regulatory role in the malignant transformation of cells. Hence, It is very necessary to identified the composition and function of SASP which secreted by Cr(VI) induced senescent L02 hepatocytes (S-L02). Exosomes, a vesicle-like substances released extracellularly after the fusion of intracellular multivesicular bodies with cell membrane, are important components of SASP and contain a large number of microRNAs (miRNAs). By establishing Cr(VI)-induced S-L02 model, we collected the exosomes from the supernatants of S-L02 and L02 culture medium respectively, and screened out the highly expressed miRNAs in the exosomes of S-L02, namely the new SASP components. Among them, the increase of miR-222-5p was the most significant. It was validated that as SASP, miR-222-5p can inhibit the proliferation of L02 and S-L02 hepatocytes and at the same time accelerate the proliferation and migration ability of HCC cells. Further mechanistic studies revealed that miR-222-5p attenuated the regulatory effect of protein phosphatase 2A subunit B isoform R2-α (PPP2R2A) on Akt via repressing its target gene PPP2R2A, causing reduced expressions of forkhead box O3 (FOXO3a), p27 and p21, and finally increasing the proliferation of HCC cells after diminishing the negative regulation of on cell cycle. This study certainly provides valuable laboratory evidence as well as potential therapeutic targets for the prevention and further personalized treatment of Cr(VI)-associated cancers.

Curcumin attenuates diphenyl phosphate-induced apoptosis in GC-2spd(ts) cells through activated autophagy via the Nrf2/P53 pathway.

Diphenyl phosphate (DPhP) is one of the frequently used derivatives of aryl phosphate esters and is used as a plasticizer in industrial production. Like other plasticizers, DPhP is not chemically bound and can easily escape into the environment, thereby affecting human health. DPhP has been associated with developmental toxicity, reproductive toxicity, neurodevelopmental toxicity, and interference with thyroid homeostasis. However, understanding of the underlying mechanism of DPhP on the reproductive toxicity of GC-2spd(ts) cells remains limited. For the first time, we investigated the effect of DPhP on GC-2spd(ts) cell apoptosis. By decreasing nuclear factor erythroid-derived 2-related factor (Nrf2)/p53 signaling, DPhP inhibited autophagy and promoted apoptosis. DPhP reduced total antioxidant capacity and nuclear Nrf2 and its downstream target gene expression. In addition, we investigated the protective effects of Curcumin (Cur) against DPhP toxicity. Cur attenuated the DPhP-induced rise in p53 expression while increasing Nrf2 expression. Cur inhibited DPhP-induced apoptosis in GC-2spd(ts) cells by activating autophagy via Nrf2/p53 signaling. In conclusion, our study provides new insights into the reproductive toxicity hazards of DPhP and demonstrates that Cur is an important therapeutic agent for alleviating DPhP-induced reproductive toxicity by regulating Nrf2/p53 signaling.

Mechanism of NO2-induced migraine in rats: The exploration of the role of miR-653-3p/IGF1 axis.

Migraine is a severely disabling primary neurological disorder. Although some studies have confirmed that nitrogen dioxide (NO2) pollution increases the risk of migraine, and our previous study demonstrated the role of the channel protein transient receptor potential cation channel subfamily V member 1 (TRPV1) in NO2-induced migraine, the underlying mechanisms have not been fully elucidated. This study aimed to explore the intrinsic toxicity mechanism of NO2-induced migraines using transcriptome sequencing. First, the differentially expressed genes in NO2-induced migraine, insulin-like growth factor 1 (IGF1) and miRNA miR-653-3p were identified using RNA and small RNA sequencing, and a protein interaction network was constructed using STRING to explore the possible mechanisms. Next, the targeting relationship between miR-653-3p and IGF1 was determined. NO2-induced migraine was verified by silencing miR-653-3p and IGF1, independently or in combination to regulate the protein kinase B (AKT)/TRPV1 signalling pathway through the miR-653-3p/IGF1 axis. These results indicate that the key molecular mechanism of NO2-induced migraine may be that the miR-653-3p/IGF1 axis regulates the AKT/TRPV1 signalling pathway to induce migraine. The findings of this study will further elucidate the neurotoxic mechanism of NO2-induced migraines and lay a new experimental foundation for implementing migraine-related preventive and therapeutic control measures.

Ambient NO2 hinders neutrophil extracellular trap formation in rats: Assessment of the role of neutrophil autophagy.

NO2 has been known to impair immunity and exacerbate susceptibility to infectious diseases. However, scant notice has been taken of the effect of NO2 on neutrophils. Neutrophil extracellular traps (NETs) formation is necessary for NETosis development by neutrophils as an immune system against pathogens. By analyzing the morphology and signature components of NETs, we focused for the first time on finding that 10 ppm of NO2 exposure for 15 consecutive days can hinder the formation of NETs. Next, we used NO2 in vivo derivatives to probe the mechanism for NETs formation in vitro. Our findings showed that NO2 suppression of respiratory burst levels and mitogen-activated protein kinase (MAPK)/Phosphoinositide 3-kinase (PI3K)-protein kinase B (AKT) signaling was related to NO2 reduction in NETs formation. Inhibition of phorbol myristate acetate (PMA)-induced NETs formation by NO2 hindered autophagy, as evidenced by increased mTOR protein expression, decreased LC3 protein expression, and reduced autophagic vesicles. By activating mTOR-mediated autophagy, rapamycin (Rapa) reduced the inhibition of PMA-induced NETs by NO2. This study will provide valuable insights into the mechanisms of immunotoxicity of NO2, new insights into the etiology of diseases linked to NETs formation, and a theoretical basis for protection against such illnesses.

Spleen Toxicity of Organophosphorus Flame Retardant TDCPP in Mice and the Related Mechanisms.

Tris(1,3-dichloro-2-propyl) phosphate (TDCPP) is an organophosphorus flame retardant that has been utilized in recent years as a primary replacement for polybrominated diphenyl ethers (PBDEs) in a wide variety of fire-sensitive applications. However, the impact of TDCPP on the immune system has not been fully determined. As the largest secondary immune organ in the body, the spleen is considered to be an important study endpoint for determining immune defects in the body. The aim of this study is to investigate the effect of TDCPP toxicity on the spleen and its possible molecular mechanisms. In this study, for 28 consecutive days, TDCPP was administered intragastrically (i.g), and we assessed the general condition of mice by evaluating their 24 h water and food intake. Pathological changes in spleen tissues were also evaluated at the end of the 28-day exposure. To measure the TDCPP-induced inflammatory response in the spleen and its consequences, the expression of the critical players in the NF-κB pathway and mitochondrial apoptosis were detected. Lastly, RNA-seq was performed to identify the crucial signaling pathways of TDCPP-induced splenic injury. The results showed that TDCPP intragastric exposure triggered an inflammatory response in the spleen, likely through activating the NF-κB/IFN-γ/TNF-α/IL-1ß pathway. TDCPP also led to mitochondrial-related apoptosis in the spleen. Further RNA-seq analysis suggested that the TDCPP-mediated immunosuppressive effect is associated with the inhibition of chemokines and the expression of their receptor genes in the cytokine-cytokine receptor interaction pathway, including four genes of the CC subfamily, four genes of the CXC subfamily, and one gene of the C subfamily. Taken together, the present study identifies the sub-chronic splenic toxicity of TDCPP and provides insights on the potential mechanisms of TDCPP-induced splenic injury and immune suppression.

Long-term exposure to ambient air pollutants and their interaction with physical activity on insomnia: A prospective cohort study.

Exposure to air pollution or lack of physical activity (PA) increases the risk of insomnia. However, evidence on joint exposure to air pollutants is limited, and the interaction of joint air pollutants and PA on insomnia is unknown. This prospective cohort study included 40,315 participants with related data from the UK Biobank, which recruited participants from 2006 to 2010. Insomnia was assessed by self-reported symptoms. The annual average air pollutant concentrations of particulate matter (PM2.5, PM10), nitrogen oxides (NO2, NOX), sulfur dioxide (SO2) and carbon monoxide (CO) were calculated based on participants' addresses. We applied a weighted Cox regression model to evaluate the correlation between air pollutants and insomnia and newly proposed an air pollution score to assess joint air pollutants effect using a weighted concentration summation after obtaining the weights of each pollutant in the Weighted-quantile sum regression. With a median follow-up of 8.7 years, 8511 participants developed insomnia. For each 10 µg/m³ increase in NO2, NOX, PM10, SO2, the average hazard ratios (AHRs) and 95% confidence interval (CI) of insomnia were 1.10 (1.06, 1.14), 1.06 (1.04, 1.08), 1.35 (1.25, 1.45) and 2.58 (2.31, 2.89), respectively; For each 5 µg/m³ increase in PM2.5 and each 1 mg/m³ increase in CO, the corresponding AHRs (95%CI) were 1.27 (1.21, 1.34) and 1.83 (1.10, 3.04), respectively. The AHR (95%CI) for insomnia associated with per interquartile range (IQR) increase in air pollution scores were 1.20 (1.15, 1.23). In addition, potential interactions were examined by setting cross-product terms of air pollution score with PA in the models. We observed an interaction between air pollution scores and PA (P = 0.032). The associations between joint air pollutants and insomnia were attenuated among participants with higher PA. Our study provides evidence on developing strategies for improving healthy sleep by promoting PA and reducing air pollution.

Effect of propiconazole on neutrophil extracellular traps formation: Assessing the role of autophagy.

Propiconazole (Pcz) is a kind of triazole fungicide which has an important impact on the environment. With the extensive use of Pcz in agricultural production activities, the pesticides are left in soil, water, crops and food, and will enter the organisms in the form of residues. Neutrophils play a key role in the body's innate immunity against pathogens, and the formation of neutrophil extracellular traps (NETs) is an important way for neutrophils to exert their immune function. In the present study, we focused on the effect of Pcz on the NETs of Sprague-Dawley (SD) rats for the first time. Our data demonstrated that Pcz could hinder NETs formation via inhibiting the Phosphoinositide 3-kinase (PI3K)/rapidly accelerated fibrosarcoma (Raf)/extracellular signal-regulated kinase (ERK) signaling. In the meanwhile, we assessed the role of autophagy played in this process and revealed that Pcz may inhibit the respiratory burst in neutrophils. This study provided new insights into the immunotoxic hazards of Pcz and additional laboratory evidence for assessing the impact of Pcz on terrestrial organisms.

Ambient NO2 exposure induces migraine in rats: Evidence, mechanisms and interventions.

Migraine is a complex neurological disorder with a high disability rate. Although the precipitating factors of migraine remain unclear, previous studies suggest that when there is excess nitrogen dioxide (NO2) pollution in the atmosphere, the medical demand due to migraine attacks increases sharply. However, the main role of NO2 as a trigger for migraine is not yet well understood. The purpose of this study was to explore the relationship between NO2 exposure and the occurrence of migraine as well as the possible underlying mechanisms. We first investigated whether repeated short-term NO2 exposure could induce behavioural and biological migraine phenotypes in rats. Next, capsazepine (CZP) was used to block transient receptor potential cation channel subfamily V member 1 (TRPV1) in vivo, and CZP and vitamin E (VE) were used to verify the role of reactive oxygen species (ROS)-TRPV1 signalling in NO2-induced migraine in primary trigeminal neurones in vitro. We demonstrated that short-term repeated NO2 exposure can significantly induce migraine in rats, and its key molecular mechanism may be related to ROS burst and its downstream TRPV1 channel activation. The findings of this study will enhance the understanding of the neurotoxic mechanism of NO2, provide new clues for identifying the aetiology of migraine, and lay a new experimental basis for implementing migraine-related preventive and therapeutic control measures.

Ambient NO2 exposure induced cardiotoxicity associated with gut microbiome dysregulation and glycerophospholipid metabolism disruption.

An average daily increase of 10 µg/m3 in NO2 concentrations could lead to an increased mortality in cardiovascular, cerebrovascular of 1.89%, 2.07%, but the mechanism by which NO2 contributes to cardiotoxicity is rarely reported. In order to assess the cardiotoxicity of NO2 inhalation (5 ppm), we firstly investigate the change of gut microbiota, serum metabonomics and cardiac proteome. Non-targeted LC-MS/MS metabonomics showed that NO2 stress could perturb the glycerophospholipid metabolism in the serum, which might destabilize the bilayer configuration of cardiac lipid membranes. Furthermore, we observed that NO2 inhalation caused augmented intercellular gap and inflammatory infiltration in the heart. Although 16 S rRNA gene amplification sequencing demonstrated that NO2 exposure did not influence the intestinal microbial abundance and diversity, but glycerophospholipid metabolism disruption might be finally reflected in gut microbiom dysregulation, such as Sphingomonas, Koribacter, Actinomarina and Bradyrhizobium Turicibacter, Rothia, Globicatella and Aerococcus. Proteome mining revealed that differentially expressed genes (DEGs) in the heart after NO2 stress were involved in necroptosis, mitophagy and ferroptosis. We further revealed that NO2 increased the number of cardiac mitochondria with depletion of cristae by regulating the expression of Mfn2 and Hsp70. This study indicating Mfn2-meidcated imbalanced mitochondrial dynamics as a potential mechanism after NO2-induced heart injury and suggesting microbiome dysregulation/glycerophospholipid metabolism exerts critical roles in cardiotoxicity caused by NO2.

Hexavalent chromium triggers hepatocytes premature senescence via the GATA4/NF-κB signaling pathway mediated by the DNA damage response.

Hexavalent chromium [Cr(VI)] is a proven toxin, carcinogen and environmental pollutant. Oral intake of Cr(VI) has been shown to lead to an increasing incidence of primary hepatic carcinoma in the population. Cellular senescence is thought to be a natural barrier to malignant transformation of cells, but senescence-associated secretory phenotype (SASP) is secreted and regulated by senescent cells links cellular senescence to malignant transformation in a dynamic way. In the present research, we demonstrated novel mechanisms of premature hepatocytes senescence induced by Cr(VI). Continuous Cr(VI) stimulation led to DNA damaged in hepatocytes, and DNA damage response (DDR) signals were transmitted by ataxia telangiectasia-mutated gene (ATM)/ataxia telangiectasia and Rad-3-related protein (ATR), resulting in zinc finger transcription factor GATA4 escaping p62-mediated selective autophagy, thereby regulating nuclear factor kappa-B (NF-κB) to induce premature senescence in hepatocytes. In contrast to the classical senescence pathway p53-p21WAF1 /CIP1 and Rb/p16INK4a, GATA4 can directly regulate the secretion of SASP during premature senescence. The results will provide valuable clues for targeted prevention and further individualized treatment of Cr(VI)-associated cancers.

Endogenous hydrogen sulfide counteracts polystyrene nanoplastics-induced mitochondrial apoptosis and excessive autophagy via regulating Nrf2 and PGC-1α signaling pathway in mouse spermatocyte-derived GC-2spd(ts) cells.

Nanoplastics (NaPs) has reported to accumulate in the testes and cause degeneration in the seminiferous tubules. Additionally, exogenous hydrogen sulfide (H2S) is proposed to enhance tolerance to oxidative stress. The current work aimed to investigate the mechanisms of NaPs-induced reproductive toxicity in vitro and probable reproductive protection by endogenous H2S. We firstly found that 80 nm fluorescent NaPs could enter into GC-2spd(ts) cells by fluorescent inverted microscope. In addition, we demonstrated that NaPs-induced could induce ROS-dependent mitochondrial apoptosis and autophagy in vitro. Our results showed that the H2S donor NaHS ameliorated NaPs-triggered mitochondrial apoptosis and autophagy in GC-2spd(ts) cells. Moreover, NaPs treatment did not change the interaction between nuclear factor erythroid-derived 2-related factor (Nrf2) and Kelch-like ECH associated protein 1 (Keap1), while inhibiting nuclear accumulation of Nrf2 protein was observed. Meanwhile, NaHS weakened this interaction, subsequently improving antioxidant ability via increasing the protein levels of heme oxygenase-1 (HO-1) and NAD(P)H dehydrogenase quinone 1 (NQO1). Further, invitro experiments showed that NaPs-induced reproductive toxicity associated with reducing PGC-1α. Meanwhile, NaPs-induced higher expression PGC-1α was further enhanced by NaHS co-treatment. Together, this study highlight that exogenous H2S should be an essential therapeutic approach to alleviate NaPs-induced reproductive toxicity via regulating Nrf2/PGC-1α signal.

Clusterin protects against Cr(VI)-induced oxidative stress-associated hepatotoxicity by mediating the Akt-Keap1-Nrf2 signaling pathway.

Hexavalent chromium [Cr(VI)] is a serious environmental pollutant that threatens human life. Cr(VI) is widely used in industrial processes such as metallurgy, leather processing, and electroplating, which can enter the human body through the respiratory or digestive tracts, thus causing a number of human disease, including inflammation and cancer. Although it has been confirmed that oxidative stress is one of the primary mechanism of liver injury caused by Cr(VI) exposure, the related toxic target and effective intervention measures have not been found. Clusterin (CLU) is an acute phase response protein with cytoprotective and apoptosis-delaying effects, and its expression has been confirmed to increase significantly after exposure to Cr(VI). In this study, our data clearly indicates that Cr(VI) is capable of causing hepatocytes damage through the production of large amounts of reactive oxygen species (ROS), causing an increase in aspartate aminotransferase (AST) and alanine aminotransferase (ALT). In contrast, over expression of CLU was able to inhibit ROS production and alleviate Cr(VI)-induced liver injury. The specific mechanisms are that CLU acts on the protein kinase B (PKB/Akt)-Kelch-like ECH-associated protein 1 (Keap1)-nuclear factor E2-related factor 2 (Nrf2) signaling pathway to release Nrf2 into the nucleus. This is to initiate the expression of a downstream protein, heme oxygenase 1 (HO-1), thereby attenuating the ubiquitination ability of Keap1 with Nrf2. We also demonstrated that CLU could affect oxidative stress through the Akt/Nrf2 pathway, which reduced the production of ROS induced by Cr(VI) and protected against Cr(VI)-induced oxidative stress-associated hepatotoxicity. This study demonstrates a mechanism of Cr(VI)-induced hepatotoxicity and indicates that CLU as an intervention target of oxidative stress can provide valuable experimental basis for the prevention and treatment of occupational diseases in Cr(VI)-exposed population. Under the state of Cr(VI)-induced oxidative stress, CLU though phosphorylation Akt, leading to Nrf2 dissociation from Keap1. Activated Nrf2 entered the nucleus and formed the next step, thus binding to the structure of the antioxidant response element ARE, which activated HO-1, resulting in the decrease in intracellular ROS.

ERK/p38/ROS burst responses to environmentally relevant concentrations of diphenyl phosphate-evoked neutrophil extracellular traps formation: Assessing the role of autophagy.

Organophosphorus compounds were proposed to impair immune surveillance and increase the total burden of pathogens. However, scarce attention has been paid to the effects of organophosphate flame retardants (OPFRs) on neutrophils. Previous literature outlined that neutrophil extracellular traps (NETs) death (NETosis) is associated with autophagy-related signaling. Here we found that 20 µM diphenyl phosphate (DPHP) could promote NETs formation via assessing markers of NETs and the morphological changes. Concurrently, flow cytometry and western blot analysis revealed that DPHP-triggered NETs formation was associated with reactive oxygen species (ROS) burst and activation of extracellular signal-regulated kinase (ERK) and p38. Additionally, the results revealed that autophagy occurred in DPHP-triggered NETs formation, manifested as enhanced LC3B protein expressions and reduced p62 protein expressions. Mechanism dissection revealed that inhibition of autophagy by 3-methyladenine (3-MA) alleviated the ROS burst and subsequent NETosis caused by DPHP. Conversely, autophagy enhancer Rapamycin (Rapa) augmented the above effects of DPHP, including the generation of ROS and NETosis. Collectively, these data suggested ERK/p38 signaling and ROS burst might be an important cause of DPHP-triggered NETs formation, while suppression of excessive autophagy could rescue these actions. These observations provided a theoretical basis for the treatment and prevention of OPFRs-induced immunotoxicity.

Curcumin hinders PBDE-47-induced neutrophil extracellular traps release via Nrf2-associated ROS inhibition.

Polybrominated diphenyl ethers (PBDE-47), a kind of lipophilic persistent organic pollutants (POPs) brominated flame retardant, has been widely used in various consumer products. However, the toxicity of PBDE-47 on human immune system has not been well elucidated. Neutrophil extracellular traps (NETs) contribute to the innate immune responses, and the release of NETs is recognized as the most important part of the extracellular killing mechanism. The aim of this study was to investigate the effect of PBDE-47 on NETs and its possible molecular mechanism, as well as the intervention effect of curcumin (Cur). In this study, the formation of PBDE-47-induced NETs was observed by fluorescence microscopy and scanning electron microscopy, and was also quantitatively detected by DNA dye SYTOX green. In addition, we used Cur and Nrf2 inhibitor ML385 to explore the role of reactive oxygen species (ROS), extracellular signal regulated kinase (ERK) and p38 signaling pathway in PBDE-47-induced reticular formation. We demonstrated that PBDE-47 could significantly induce the formation of NETs, and its molecular mechanism might be related to ROS burst. Cur reduced ROS and inhibited PBDE-47-induced NETs formation by interfering with Nrf2. In conclusion, this study revealed that Cur hindered PBDE-47-induced NETs via Nrf2-associated ROS inhibition, which enriched the cytotoxicity mechanism of PBDE-47, and provided a new clue for the development of Cur as an antagonist of PBDE-47-related immune injury.

Hexavalent chromium inhibits the formation of neutrophil extracellular traps and promotes the apoptosis of neutrophils via AMPK signaling pathway.

As the most common heavy metal pollutant, hexavalent chromium [Cr(VI)] has caused serious environmental pollution and health damage. Although the toxic effect of Cr(VI) has been widely studied, and oxidative stress has been confirmed to be the main mechanism of its cytotoxicity, the toxicity of Cr(VI) to human immune system remains to be elucidated. Neutrophil extracellular traps (NETs) participate in the innate immune response, and the release of NETs is considered to be the most important part of the extracellular killing mechanism. We demonstrated in this study that Cr(VI) inhibited the formation of NETs in rat peripheral blood and induced neutrophils apoptosis by inhibiting the AMP-activated protein kinase (AMPK) signaling pathway. Cr(VI)-induced inhibition of NETs was accompanied by down-regulated myeloperoxidase (MPO)/Histones-3 (H3) protein expressions and decreased NETs-associated intracellular and extracellular DNA levels in the neutrophils. Metformin (Met), as an AMPK activator, triggered autophagy and thus alleviated the inhibitory effect of Cr(VI) on NETs. At the same time, Met can reduce the intracellular reactive oxygen species (ROS) level by activating the AMPK/nuclear factor erythroid-2 related factor 2 (Nrf2) signaling pathway, thus alleviating Cr(VI)-induced neutrophils apoptosis. In conclusion, this study elucidated the mechanism of Cr(VI)-induced neutrophils toxicity and the role of AMPK as a key regulatory signal, which could provide valuable experimental basis for the prevention and treatment of related diseases in Cr(VI)-exposed populations.

Polystyrene microplastics trigger hepatocyte apoptosis and abnormal glycolytic flux via ROS-driven calcium overload.

Human health could be affected by the spread of microplastics in the food chain. Our previous research has indicated that microplastics accumulated in the liver and subsequently induce oxidative damage. However, the molecular events linking oxidative stress to calcium ion (Ca2+) signaling during microplastics stress remains elusive. The present research demonstrated that up-regulation of Orai 1 and stromal interaction molecule 1 (Stim1) expression participated in the microplastics-triggered Ca2+ overload, accompanied with the down-regulation of arcoplasmic reticulum Ca2+ ATPase (SERCA). However, when the protein expression of Stim1/SERCA is restored, microplastics-induced Ca2+ overload is ameliorated. Further analysis revealed that inhibiting the microplastics-induced Ca2+ overload was integral to prevent hepatocyte apoptosis and S phase arrest in the L02 hepatocyte. Simultaneously, we observed that inhibiting microplastics-evoked reactive oxygen species (ROS) could alleviate Ca2+ overload via reversing expression of store-operated Ca2+ channels (SOCs). These changes were accompanied by restoration of glycolytic flux, likely due to the regulation of AMP-activated protein kinase (AMPK)-PGC-1α signaling. Our findings highlight the role of SOCs at microplastics-evoked ROS in Ca2+ overload, and its a crucial step in triggering hepatocyte death. Collectively, this study reveals a regulatory paradigm that links ROS with AMPK and Ca2+ signaling in microplastics-triggered hepatotoxicity.

Clusterin inhibits Cr(VI)-induced apoptosis via enhancing mitochondrial biogenesis through AKT-associated STAT3 activation in L02 hepatocytes.

Improper treatment of a large amount of industrial waste makes hexavalent chromium [Cr(VI)] seriously pollute the atmosphere, soil and water, and enter the food chain, seriously affecting the health of workers and local residents. We previously proved that Clusterin (CLU) can inhibit the apoptosis of L02 hepatocytes induced by Cr(VI) through mitochondrial pathway, but the associated molecular mechanism has not been further studied. Mitochondrial biogenesis is an important step in mitochondrial damage repair, but the mechanism of mitochondrial biogenesis in Cr(VI)-induced liver toxicity is still unclear. We demonstrated in the present study that Cr(VI) triggered mitochondrial biogenesis dysfunction-associated apoptosis, and CLU delayed Cr(VI)-induced apoptosis by enhancing mitochondrial biogenesis. Signal transducer and activator of transcription 3 (STAT3) was down-regulated in Cr(VI)-induced apoptosis, and CLU may regulate STAT3 via protein kinase B (PKB/AKT) in Cr(VI)-exposed hepatocytes. We used the STAT3 inhibitor C188-9 and the AKT inhibitor Uprosertib to eliminate the anti-apoptotic effect of CLU, and found that CLU inhibited Cr(VI)-induced apoptosis by up-regulating AKT/STAT3 signal. Based on the fact that both AKT and STAT3 are closely related to mitochondrial biogenesis and mitochondrial pathway-associated apoptosis, this study is the first time to link CLU, STAT3, AKT and mitochondrial biogenesis function after Cr(VI) exposure, to further enrich the experimental basis of Cr(VI)-induced hepatotoxicity, clarify the molecular mechanism of CLU helping cells to escape apoptosis, and also suggest that new ways can be sought to prevent and treat Cr(VI)-induced hepatotoxicity by regulating mitochondrial biosynthesis.

The role of PKA/PP2B-mediated Drp1 phosphorylation and the subsequent EGFR inhibition in Cr(VI)-induced premature senescence.

In recent years, frequent hexavalent chromium [Cr(VI)] pollution incidents have severely damaged the ecology and endangered the public health. It is well known that cell senescence could promote the carcinogenesis, thus the related research on the occurrence of premature senescence is of great significance to the elucidation of the carcinogenic mechanism of Cr(VI). We previously confirmed that long-term low-dose Cr(VI) exposure induced premature senescence, but the key molecular events that determine the occurrence of premature senescence are still unclear. In the present study, we found that Cr(VI) induced phosphorylation of dynamin-relatedprotein 1 (Drp1)-S637 site in premature senescent cells, which was accompanied with the decrease of mitochondrial fission. We also demonstrated that the phosphorylation status of Drp1-S637 after Cr(VI) exposure was related to the antagonism of PKA/PP2B, and continuous dephosphorylation of Drp1-S637 attenuated premature senescence caused by Cr(VI). The epidermal growth factor receptor (EGFR) overexpression significantly alleviated the occurrence of premature senescence, and the expressions of EFGR and its downstream molecules were related to the phosphorylation status of Drp1-S637. In brief, we revealed the role of PKA/PP2B-mediated Drp1 phosphorylation and the subsequent EGFR inhibition in Cr(VI)-induced premature senescence. This study is the first time to link the phosphorylation of Drp1 with Cr(VI)-induced premature senescence, in order to find the key molecular events that determine the occurrence of premature senescence and demonstrate the molecular mechanism of abnormal elongated mitochondria formation in the senescence process. The significance of this study is to explore the carcinogenesis of Cr(VI) and provide new ideas and strategies for the targeted treatment of Cr(VI)-related cancers.

Role of Clusterin/NF-κB in the secretion of senescence-associated secretory phenotype in Cr(VI)-induced premature senescent L-02 hepatocytes.

Hexavalent chromium [Cr(VI)] and its compounds have caused serious environmental pollution and health damage. Senescent cells can actively change the surrounding environment by secreting some factors, which are called senescence associated secretory phenotype (SASP). Our previous work has confirmed that premature senescent hepatocytes induced by Cr(VI) expressed high level of Clusterin (CLU) and secrete interleukin-6 (IL-6) and IL-8. CLU is involved in the regulation of tumor development and drug resistance, but whether CLU regulates SASP components and participates in Cr(VI)-induced malignant transformation is unclear. In this study we demonstrated that Cr(VI) induced the secretion of tumor promoting components of SASP such as IL-6, IL-8, and granulocyte-macrophage colony stimulating factor (GM-CSF) in senescent L-02 hepatocytes, while the levels of the anti-tumor components of SASP such as chemokine (c-x-c motif) ligand-1 (CXCL-1) and monocyte chemoattractant protein-1 (MCP-1) were not altered. CLU shRNA interference significantly reduced the levels of IL-6, IL-8, and GM-CSF in the culture medium of senescent cells, suggesting CLU may regulate SASP. The NF-κB inhibitor PDTC significantly alleviated Cr(VI)-induced increase of IL-6, IL-8, and GM-CSF, confirming that NF-κB can regulate the tumor promoting components of SASP. CLU shRNA interference aggravated the inhibitory effect of PDTC on SASP secretion, indicating that CLU regulated the secretion of SASP in Cr(VI)-induced senescent hepatocytes through the NF-κB signaling. We speculated that SASP secreted by Cr(VI)-induced premature senescent hepatocytes was tightly related to the carcinogenic effect of Cr(VI). Therefore, elucidation of upstream regulatory mechanism of SASP is of great significance. In addition to further clarifying the carcinogenic mechanisms associated with Cr(VI), we could also seek out new targets for treatment of Cr(VI)-related cancer.

Clusterin alleviates Cr(VI)-induced mitochondrial apoptosis in L02 hepatocytes via inhibition of Ca2+-ROS-Drp1-mitochondrial fission axis.

Hexavalent chromium [Cr(VI)] is ubiquitous in the environment and is commonly used in various industrial processes. Clusterin (CLU) is an extracellular chaperone protein which exerts the anti-apoptotic function. In this study, we aimed to explore the effect of CLU on Cr(VI)-induced mitochondrial fission and apoptosis. We revealed that the apoptosis rate of L02 hepatocytes treated with Cr (VI) was increased. CLU over-expression could protect the hepatocytes from Cr(VI)-induced mitochondrial apoptosis. Furthermore, Cr(VI) triggered the intracellular calcium overload, resulting in the activation of xanthine oxidase (XO). Cr(VI) induced reactive oxygen species (ROS) overproduction, led to dynamin-related protein 1 (Drp1) translocation to mitochondria and the subsequent mitochondrial fission, contributing to the caspase-3-dependent mitochondrial apoptosis as evidenced by higher mitochondrial permeability transition pore (mPTP) opening rate, lower mitochondrial membrane potential (MMP), and more alanine transaminase (ALT)/aspartate transaminase (AST) leakage into the culture medium. However, CLU over-expression could trigger the AMP-activated protein kinase (AMPK) pathway, which was followed by the increase of sarcoplasmic reticulum Ca2+-ATPase (SERCA2a) expression. CLU-induced AMPK/SERCA2a activation attenuated calcium overload, caspase-3 activation, and ultimate mitochondrial apoptosis. All in all, the present study demonstrated that Cr(VI) induced hepatocytes apoptosis via Ca2+-ROS-Drp1-mitochondrial fission axis and CLU alleviated the mitochondrial apoptosis through activation of the AMPK/SERCA2a pathway.