The necroptosis-mediated imbalance of mitochondrial dynamics is involved in DEHP-induced toxicity to immature testes via the PGAM5-DRP1 interaction.

Di-(2-ethylhexyl) phthalate (DEHP) is a widely used plasticizer that has been shown to impair male reproduction, but the potential mechanism underlying testicular injury caused by DEHP remains unclear. In vivo, rats were gavaged consecutively from postnatal day (PND) 21 to PND 31 with 0, 250, or 500 mg/kg DEHP for 10 days, and impaired mitochondria and increased necroptosis were observed in immature testes. In vitro, the GC-1 and GC-2 cell lines were exposed to monoethylhexyl phthalate (MEHP) at 100, 200 and 400 µM for 24 h, and this exposure induced oxidative stress damage, necroptosis and mitochondrial injury. Necroptosis and mitochondrial fission were inhibited by the reactive oxygen species (ROS) inhibitor acetylcysteine, and the imbalanced mitochondrial dynamics were rescued by the RIPK1 inhibitor necrostatin-1. Colocalization and co-IP analyses confirmed an interaction between dynamin-related protein 1 (DRP1) and phosphoglycerate mutase 5 (PGAM5), indicating that PGAM5 dephosphorylates DRP1 at serine 637 to induce mitochondrial fragmentation and thereby induces germ cell damage. Drug prediction with Connectivity Map (cMap) identified sulforaphane as a therapeutic drug. In summary, our findings indicate that DEHP triggers necroptosis and mitochondrial injury via a ROS storm in immature testes and that the PGAM5-DRP1 interaction is involved in this process.

Inhibition of the NF-κB Signaling Pathway Alleviates Pyroptosis in Bladder Epithelial Cells and Neurogenic Bladder Fibrosis.

Most children with a neurogenic bladder (NB) have bladder fibrosis, which causes irreversible bladder dysfunction and damage to the upper urinary tract. However, the mechanism of bladder fibrosis remains unclear. This study aimed to investigate the underlying causes of bladder fibrosis. Here, the lumbar 6 (L6) and sacral 1 (S1) spinal nerves of Sprague Dawley rats were severed bilaterally to establish NB models. Using RNA-seq, we discovered that the NF-κB signaling pathway and inflammation were upregulated in spinal cord injury (SCI)-induced bladder fibrosis. Subsequent Western blotting, enzyme-linked immunosorbent assays, immunohistochemical staining, and immunofluorescence staining verified the RNA-seq findings. To further clarify whether the NF-κB signaling pathway and pyroptosis were involved in bladder fibrosis, a TGF-ß1-treated urinary epithelial cell line (SV-HUC-1 cells) was used as an in vitro model. Based on the results of RNA-seq, we consistently found that the NF-κB signaling pathway and pyroptosis might play important roles in TGF-ß1-treated cells. Further experiments also confirmed the RNA-seq findings in vitro. Moreover, using the NLRP3 inhibitor MCC950 rescued TGF-ß1-induced fibrosis, and the NF-κB signaling pathway inhibitor BAY 11-7082 effectively rescued TGF-ß1-induced pyroptosis and the deposition of extracellular matrix by SV-HUC-1 cells. In summary, our research demonstrated for the first time that the NF-κB signaling pathway inhibition rescued bladder epithelial cells pyroptosis and fibrosis in neurogenic bladders.

Epigallocatechin gallate alleviates mono-2-ethylhexyl phthalate-induced male germ cell pyroptosis by inhibiting the ROS/mTOR/NLRP3 pathway.

Mono-2-ethylhexyl phthalate (MEHP) exposure is known to induce severe testicular injury via reactive oxygen species (ROS). However, few effective treatments are available for the precise treatment of MEHP-induced germ cell damage. Epigallocatechin gallate (EGCG), one of the major polyphenols in green tea, has potential antioxidant activity and can alleviate many diseases induced by oxidative stress. This study explored whether EGCG protects germ cells from MEHP-induced oxidative stress damage. Cells were treated with 400 µM MEHP and 60 µM EGCG for 24 h. EGCG reduced MEHP-induced ROS overgeneration in the spermatogonial cell line GC-1 and spermatocyte cell line GC-2. Western blotting and immunofluorescence showed that the MEHP+EGCG group exhibited lower nuclear factor (erythroid-derived 2)-like 2 (NRF2), heme oxygenase (decycling) 1 (HO-1), and superoxide dismutase (SOD) expression than the MEHP group. Moreover, activation of the mammalian target of rapamycin (mTOR) pathway was decreased. The expression of key factors of pyroptosis was downregulated, and interleukin-10 (IL-10) expression was reduced. Additionally, apoptosis was inhibited by EGCG. The findings indicate that EGCG protects against MEHP-induced germ cell pyroptosis by scavenging ROS, suppressing the mTOR pathway, and inhibiting pyroptosis. EGCG may thus be a potential treatment for MEHP-related spermatogenic dysfunction.

Wnt10a downregulation contributes to MEHP-induced disruption of self-renewal and differentiation balance and proliferation inhibition in GC-1 cells: Insights from multiple transcriptomic profiling.

Di (2-ethylhexyl) phthalate (DEHP), one of phthalic acid esters, has been widely used in daily products. Its main metabolite, mono (2-ethylhexyl) phthalate (MEHP) was reported to possess higher testicular toxicity than DEHP. To explore the precise mechanism in MEHP-induced testis damage, multiple transcriptomic sequencing was employed in spermatogonia cell line GC-1 cells treated with MEHP (0, 100, and 200 µM) for 24 h. Integrative omics analysis and empirical validation revealed that Wnt signaling pathway was downregulated and wnt10a, one of hub genes, may be the key player in this process. Similar results were observed in DEHP-exposed rats. MEHP-induced disturbance of self-renewal and differentiation was dose-dependent. Moreover, self-renewal proteins were downregulated; the differentiation level was stimulated. Meanwhile, GC-1 proliferation was decreased. Stable transformation strain of wnt10a overexpression GC-1 cell line constructed from lentivirus was utilized in this study. The upregulation of Wnt10a significantly reversed the dysfunction of self-renewal and differentiation and promoted the cell proliferation. Finally, retinol, predicted to be useful in CONNECTIVITY MAP (cMAP), failed to rescue the damage caused by MEHP. Cumulatively, our findings revealed that the downregulation of Wnt10a induced the imbalance of self-renew and differentiation, and inhibition of cell proliferation in GC-1 cells after MEHP exposure.

Hypoxia-Induced HIF-1α Expression Promotes Neurogenic Bladder Fibrosis via EMT and Pyroptosis.

BACKGROUND: Neurogenic bladder (NB) patients exhibit varying degrees of bladder fibrosis, and the thickening and hardening of the bladder wall induced by fibrosis will further affect bladder function and cause renal failure. Our study aimed to investigate the mechanism of bladder fibrosis caused by a spinal cord injury (SCI). METHODS: NB rat models were created by cutting the bilateral lumbar 6 (L6) and sacral 1 (S1) spinal nerves. RNA-seq, Western blotting, immunofluorescence, cell viability and ELISA were performed to assess the inflammation and fibrosis levels. RESULTS: The rats showed bladder dysfunction, upper urinary tract damage and bladder fibrosis after SCI. RNA-seq results indicated that hypoxia, EMT and pyroptosis might be involved in bladder fibrosis induced by SCI. Subsequent Western blot, ELISA and cell viability assays and immunofluorescence of bladder tissue confirmed the RNA-seq findings. Hypoxic exposure increased the expression of HIF-1α and induced EMT and pyroptosis in bladder epithelial cells. Furthermore, HIF-1α knockdown rescued hypoxia-induced pyroptosis, EMT and fibrosis. CONCLUSION: EMT and pyroptosis were involved in the development of SCI-induced bladder fibrosis via the HIF-1α pathway. Inhibition of the HIF-1α pathway may serve as a potential target to alleviate bladder fibrosis caused by SCI.

Leveraging sequence-based faecal microbial community survey data to identify alterations in gut microbiota among patients with Parkinson's disease.

Parkinson's disease is a common degenerative disease of the elderly. Although the majority of studies have focused on the central nervous system (CNS) features of Parkinson's disease, recent findings suggest there is a functional link between the gut microbiome and the hallmarks of the disease. PubMed, Web of Science, EMBASE and other Chinese and English databases were searched for relevant literature. Studies on changes to intestinal microbiota in Parkinson's patients were retrieved and systematically reviewed. Quality filtering, clustering and species annotation were performed on 16s sequencing raw data from retrieved studies to achieve comparability across studies. Alpha-diversity indices and a random effect model were used to analyse significantly altered microbiota. A total of nine studies were included in this retrospective analysis, four of which contained raw data. Alpha diversity was significantly different between control and Parkinson's disease patients in two of the four studies. Using the raw data from four individual studies, we observed differences in the phlya Bacteroidetes and Actinobacteria. Additionally, differences were observed between control and Parkinson's disease patients at the level of family (Prevotellacaea and Lactobacillaceae) and genus (Bifidobacterium and Clostridium). This study confirmed that changes in the microbiome are a consistent feature of Parkinson's disease patients and, therefore, may contribute to the onset of disease.