Dissection of molecular mechanisms of liver injury induced by microcystin-leucine arginine via single-cell RNA-sequencing.

The occurrence of poisoning incidents caused by cyanobacterial blooms has aroused wide public concern. Microcystin-leucine arginine (MC-LR) is a well-established toxin produced by cyanobacterial blooms, which is widely distributed in eutrophic waters. MC-LR is not only hazardous to the water environment but also exerts multiple toxic effects including liver toxicity in both humans and animals. However, the underlying mechanisms of MC-LR-induced liver toxicity are unclear. Herein, we used advanced single-cell RNA sequencing technology to characterize MC-LR-induced liver injury in mice. We established the first single-cell atlas of mouse livers in response to MC-LR. Our results showed that the differentially expressed genes and pathways in diverse cell types of liver tissues of mice treated with MC-LR are highly heterogeneous. Deep analysis showed that MC-LR induced an increase in a subpopulation of hepatocytes that highly express Gstm3, which potentially contributed to hepatocyte apoptosis in response to MC-LR. Moreover, MC-LR increased the proportion and multiple subtypes of Kupffer cells with M1 phenotypes and highly expressed proinflammatory genes. Furthermore, the MC-LR increased several subtypes of CD8+ T cells with highly expressed multiple cytokines and chemokines. Overall, apart from directly inducing hepatocytes apoptosis, MC-LR activated proinflammatory Kupffer cell and CD8+ T cells, and their interaction may constitute a hostile microenvironment that contributes to liver injury. Our findings not only present novel insight into underlying molecular mechanisms but also provide a valuable resource and foundation for additional discovery of MC-LR-induced liver toxicity.

Tris(2-ethylhexyl) phosphate induces cytotoxicity in TM3 Leydig cells by modulating autophagy and endoplasmic reticulum stress.

Tris (2-ethylhexyl) phosphate (TEHP) is a frequently used organophosphorus flame retardant with significant ecotoxicity and widespread human exposure. Recent research indicates that TEHP has reproductive toxicity. However, the precise cell mechanism is not enough understood. Here, by using testicular mesenchymal stromal TM3 cells as a model, we reveal that TEHP induces apoptosis. Then RNA sequencing analysis, immunofluorescence, and western blotting results show that THEP inhibits autophagy flux and enhances endoplasmic reticulum (ER) stress. Moreover, the activation of the ER stress is critical for TEHP-induced cell injury. Interestingly, TEHP-induced ER stress is contributed to autophagic flux inhibition. Furthermore, pharmacological inhibition of autophagy aggravates, and activation of autophagy attenuates TEHP-induced apoptosis. In summary, these findings indicate that TEHP triggers apoptosis in mouse TM3 cells through ER stress activation and autophagy flux inhibition, offering a new perspective on the mechanisms underlying TEHP-induced interstitial cytotoxicity in the mouse testis.

Polystyrene microplastics induce anxiety via HRAS derived PERK-NF-κB pathway.

Exposure to environmentally hazardous substances is recognized as a significant risk factor for neurological associated disorders. Among these substances, polystyrene microplastics (PS-MPs), widely utilized in various consumer products, have been reported to exhibit neurotoxicity. However, the potential association of PS-MPs with abnormal anxiety behaviors, along with the underlying molecular mechanisms and key proteins involved, remains insufficiently explored. Here, we delineated the potential mechanisms of PS-MPs-induced anxiety through proteomics and molecular investigations. We characterized the PS-MPs, observed their accumulation in the brain, leading to anxiety-like behavior in mice, which is correlated with microglia activation and pro-inflammatory response. Consistent with these findings, our studies on BV2 microglia cells showed that PS-MPs activated NF-κB-mediated inflammation resulting in the upregulation of pro-inflammatory cytokines such as TNFα and IL-1ß. Of particular significance, HRAS was identified as a key factor in the PS-MPs induced pro-inflammatory response through whole proteomics analysis, and knockdown of H-ras effectively inhibited PS-MPs induced PERK-NF-κB activation and associated pro-inflammatory response in microglia cells. Collectively, our findings highlight that PS-MPs induce anxiety of mice via the activation of the HRAS-derived PERK-NF-κB pathway in microlglia. Our results contribute valuable insights into the molecular mechanisms of PS-MPs-induced anxiety, and may offer implications for addressing neurotoxicity and prevention the adverse effects of environmentally hazardous substances, including microplastics.

Perfluorooctanoic acid induces Leydig cell injury via inhibition of autophagosomes formation and activation of endoplasmic reticulum stress.

Perfluorooctanoic acid (PFOA) is a man-made chemical broadly distributed in various ecological environment and human bodies, which poses potential health risks. Its toxicity, especially the male reproduction toxicity has drawn increasing attention due to declining birth rates in recent years. However, how PFOA induces male reproductive toxicity remains unclear. Here, we characterize PFOA-induced cell injury and reveal the underlying mechanism in mouse Leydig cells, which are critical to spermatogenesis in the testes. We show that PFOA induces cell injury as evidenced by reduced cell viability, cell morphology changes and apoptosis induction. RNA-sequencing analysis reveals that PFOA-induced cell injury is correlated with compromised autophagy and activated endoplasmic reticulum (ER) stress, two conserved biological processes required for regulating cellular homeostasis. Mechanistic analysis shows that PFOA inhibits autophagosomes formation, and activation of autophagy rescues PFOA-induced apoptosis. Additionally, PFOA activates ER stress, and pharmacological inhibition of ER stress attenuates PFOA-induced cell injury. Taken together, these results demonstrate that PFOA induces cell injury through inhibition of autophagosomes formation and induction of ER stress in Leydig cells. Thus, our study sheds light on the cellular mechanisms of PFOA-induced Leydig cell injury, which may be suggestive to human male reproductive health risk assessment and prevention from PFOA exposure-induced reproductive toxicity.

Insight into Tetrabromobisphenol A-Associated Liver Transcriptional Landscape via Single Cell RNA Sequencing.

In recent years, there has been growing concern over the rising incidence of liver diseases, with increasing exposure to environmental toxins as a significant contributing factor. However, the mechanisms of liver injury induced by environmental pollutants are largely unclear. Here, using tetrabromobisphenol A (TBBPA), a widely used brominated flame retardant, as an example, environmental toxin-induced liver toxicity in mice is characterized via single-cell sequencing technology. Heterogeneous gene expression profiles after exposure to TBBPA in major cell types of the liver are demonstrated. In hepatocytes, pathway analysis of differentially expressed genes reveals the enhanced interferon response and diminished metabolic processes. The disrupted endothelial functions in TBBPA-treated cells are then shown. Moreover, the activation of M2-polarization in Kupffer cells, as well as activated effector T and B cells are unveiled in TBBPA-treated cells. Finally, ligand-receptor pair analysis shows that TBBPA disrupts cell-cell communication and induces an inflammatory microenvironment. Overall, the results reveal that TBBPA-induced dysfunction of hepatocytes and endothelial cells may then activate and recruit other immune cells such as Kuffer cells, and T/NK cells into the liver, further increasing inflammatory response and liver injury. Thus, the results provide novel insight into undesiring environmental pollutant-induced liver injury.

Single-cell RNA sequencing reveals the effects of capsaicin in the treatment of sepsis-induced liver injury.

Sepsis is a difficult-to-treat systemic condition in which liver dysfunction acts as both regulator and target. However, the dynamic response of diverse intrahepatic cells to sepsis remains poorly characterized. Capsaicin (CAP), a multifunctional chemical derived from chilli peppers, has recently been shown to potentially possess anti-inflammatory effects, which is also one of the main approaches for drug discovery against sepsis. We performed single-cell RNA transcriptome sequencing on 86,830 intrahepatic cells isolated from normal mice, cecal ligation and puncture-induced sepsis model mice and CAP-treated mice. The transcriptional atlas of these cells revealed dynamic changes in hepatocytes, macrophages, neutrophils, and endothelial cells in response to sepsis. Among the extensive crosstalk across these major subtypes, KC_Cxcl10 shared strong potential interaction with other cells when responding to sepsis. CAP mitigated the severity of inflammation by partly reversing these pathophysiologic processes. Specific cell subpopulations in the liver act collectively to escalate inflammation, ultimately causing liver dysfunction. CAP displays its health-promoting function by ameliorating liver dysfunction induced by sepsis. Our study provides valuable insights into the pathophysiology of sepsis and suggestions for future therapeutic gain.

A single-cell landscape of triptolide-associated testicular toxicity in mice.

Triptolide is a key active component of the widely used traditional Chinese herb medicine Tripterygium wilfordii Hook. F. Although triptolide exerts multiple biological activities and shows promising efficacy in treating inflammatory-related diseases, its well-known safety issues, especially reproductive toxicity has aroused concerns. However, a comprehensive dissection of triptolide-associated testicular toxicity at single cell resolution is still lacking. Here, we observed testicular toxicity after 14 days of triptolide exposure, and then constructed a single-cell transcriptome map of 59,127 cells in mouse testes upon triptolide-treatment. We identified triptolide-associated shared and cell-type specific differentially expressed genes, enriched pathways, and ligand-receptor pairs in different cell types of mouse testes. In addition to the loss of germ cells, our results revealed increased macrophages and the inflammatory response in triptolide-treated mouse testes, suggesting a critical role of inflammation in triptolide-induced testicular injury. We also found increased reactive oxygen species (ROS) signaling and downregulated pathways associated with spermatid development in somatic cells, especially Leydig and Sertoli cells, in triptolide-treated mice, indicating that dysregulation of these signaling pathways may contribute to triptolide-induced testicular toxicity. Overall, our high-resolution single-cell landscape offers comprehensive information regarding triptolide-associated gene expression profiles in major cell types of mouse testes at single cell resolution, providing an invaluable resource for understanding the underlying mechanism of triptolide-associated testicular injury and additional discoveries of therapeutic targets of triptolide-induced male reproductive toxicity.

Identification of quiescent FOXC2+ spermatogonial stem cells in adult mammals.

In adult mammals, spermatogenesis embodies the complex developmental process from spermatogonial stem cells (SSCs) to spermatozoa. At the top of this developmental hierarchy lie a series of SSC subpopulations. Their individual identities as well as the relationships with each other, however, remain largely elusive. Using single-cell analysis and lineage tracing, we discovered both in mice and humans the quiescent adult SSC subpopulation marked specifically by forkhead box protein C2 (FOXC2). All spermatogenic progenies can be derived from FOXC2+ SSCs and the ablation of FOXC2+ SSCs led to the depletion of the undifferentiated spermatogonia pool. During germline regeneration, FOXC2+ SSCs were activated and able to completely restore the process. Germ cell-specific Foxc2 knockout resulted in an accelerated exhaustion of SSCs and eventually led to male infertility. Furthermore, FOXC2 prompts the expressions of negative regulators of cell cycle thereby ensures the SSCs reside in quiescence. Thus, this work proposes that the quiescent FOXC2+ SSCs are essential for maintaining the homeostasis and regeneration of spermatogenesis in adult mammals.

Decoding the spermatogonial stem cell niche under physiological and recovery conditions in adult mice and humans.

The intricate interaction between spermatogonial stem cell (SSC) and testicular niche is essential for maintaining SSC homeostasis; however, this interaction remains largely uncharacterized. In this study, to characterize the underlying signaling pathways and related paracrine factors, we delineated the intercellular interactions between SSC and niche cell in both adult mice and humans under physiological conditions and dissected the niche-derived regulation of SSC maintenance under recovery conditions, thus uncovering the essential role of C-C motif chemokine ligand 24 and insulin-like growth factor binding protein 7 in SSC maintenance. We also established the clinical relevance of specific paracrine factors in human fertility. Collectively, our work on decoding the adult SSC niche serves as a valuable reference for future studies on the aetiology, diagnosis, and treatment of male infertility.

RNF138 confers cisplatin resistance in gastric cancer cells via activating Chk1 signaling pathway.

Chemotherapy resistance represents a major issue associated with gastric cancer (GC) treatment, and arises through multiple mechanisms, including modulation of the cell-cycle check point. Several ubiquitin kinases, including RING finger protein 138 (RNF138), have been reported to mediate the G2/M phase arrest. In this study, we investigated the role of RNF138 in the development of cisplatin resistance of two GC cell lines. We show that RNF138 levels are higher in cisplatin-resistant cell lines, compared with cisplatin-sensitive cells, and RNF138 expression was elevated during drug withdrawal following the cisplatin treatment. Using gene overexpression and silencing, we analyzed the impact of altering RNF138 level on GC cell viability, apoptosis, and cell cycle phenotypes in two isogenic cisplatin-sensitive and resistant cell lines. We show that RNF138 overexpression increased GC cell viability, decreased apoptosis and delayed cell cycle progression in the cisplatin-sensitive GC cells. Conversely, RNF138 silencing produced opposite phenotypes in the cisplatin-resistant cells. Moreover, RNF138-dependent phosphorylation of Chk1 was seen in GC cells, indicating a novel connection between cisplatin-induced DNA damage and apoptosis. Collectively, these data suggest that RNF138 modulates the cisplatin resistance in the GC cells, thus serving as a potential drug target to challenge chemotherapy failure. In addition, RNF138 can also be used as a marker to monitor the development of cisplatin resistance in GC treatment.