Inhibition of Wilms’ Tumor Proliferation and Invasion by Blocking TGF-β Receptor I in the TGF-β/Smad Signaling Pathway

Wilms’ tumor (WT) is a common embryonal tumor, and nephrogenic rests play a critical role in WT development. The transforming growth factor β (TGF-β) signaling pathway is fundamental to embryo development and cell growth and proliferation. Moreover, TGF-β contributes to WT development, but the mechanisms of disease pathogenicity are unknown. This study investigated whether the TGF-β signaling pathway was involved in WT and whether blocking TβRI receptor inhibited WT growth, proliferation, and invasion. A total of 60 WT patients with clinical data and surgical specimens were evaluated. Immunohistochemistry (IHC) was used to detect the expression of TGF-β1 and P-smad2/3. In vitro, the proliferation, migration, apoptosis, and epithelial-mesenchymal transition (EMT) protein expression were analyzed using the CCK8 assay, wound healing assay, transwell assay, flow cytometry, and western blot, respectively. In vivo, tumor morphology, tumor size, toxicity, and EMT protein expression were analyzed in tumor-bearing mice treated with a TβRI kinase inhibitor or PBS. High protein levels of TGF-β1 and P-samd2/3 were associated with clinical stage and metastasis or invasion. TβRI inhibition effectively suppressed WT proliferation and migration and promoted apoptosis in the human WT cell line G401, consequently decreasing EMT protein expression. In addition, the TβRI kinase inhibitor significantly impaired the subcutaneous growth of WT. It is worth noting that treatment with the TβRI kinase inhibitor did not cause liver and kidney injury. Our results indicate that the TGF-β/Smad signaling pathway plays a crucial role in WT progression. Blocking the TβRI receptor may be a novel strategy to treat and prevent WT

Human umbilical cord mesenchymal stem cell exosomes alleviate acute kidney injury by inhibiting pyroptosis in rats and NRK-52E cells

AbstractHuman umbilical cord mesenchymal stem cells (hucMSCs) have been shown to improve kidney injury. Exosomes have been indicated to be important mediators of renal protection in MSC therapy. In spite of this, the mechanism remains unclear. Our study investigated how exosomes derived from hucMSCs (hucMSC-Ex) improve acute kidney injury (AKI). Exosomes were extracted by using an ultracentrifugation technique and identified by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and Western blot. Twenty-four male SD rats were randomly divided into four groups: sham group, sham + hucMSC-Ex group, ischemia–reperfusion injury (IRI) group, and IRI + hucMSC-Ex group. In vitro, we treated rat proximal renal tubular epithelial cell line (NRK-52E) with cisplatin to mimic in vivo models of AKI. The NRK-52E cells were treated with or without 160 μg/mL hucMSC-Ex, and 1 µg/mL cisplatin was added after 9 h. Cells were harvested after 24 h. In the IRI group, the levels of serum creatinine (Scr) and blood urea nitrogen (BUN) were increased; renal tubules were dilated, epithelial cells were vacuolated, and collagen fibers were deposited in the renal interstitium. After treatment with cisplatin, the NRK-52E cells displayed pyroptotic morphology characterized by pyroptotic bodies. The protein expression levels of fibronectin, α-smooth muscle actin (α-SMA), vimentin, gasdermin D (GSDMD), caspase-1, interleukin-1 (IL-1β) and NLRP3 in IRI tissues and in cisplatin treatment NRK-52E cells were significantly upregulated. However, after the hucMSC-Ex intervention, kidney injury was effectively improved in vivo and in vitro. The current study shows that pyroptosis is involved in AKI and that hucMSC-Ex improves AKI by inhibiting pyroptosis

Retardation of Preputial Wound Healing in Rats with Hypospadias Induced by Flutamide

Objective: The aim of this study was to identify a theoretical support for the prevention of urethral fistula following hypospadias repair, by comparing the preputial wound healing process in Sprague-Dawley (SD) rats with and without hypospadias induced by flutamide. Methods: Fifteen pregnant SD rats were randomly divided into three groups. These rats in one group received the androgen receptor antagonist flutamide (25 mg/kg/day) from gestation days 11–17, to establish a rat model of hypospadias for further study of the molecular mechanisms of the hypospadias etiology. The pregnant rats in the control groups were not administered flutamide. The pups from the control and experiment groups underwent an incision on the dorsal prepuce on postnatal day 25 and were sacrificed on postoperative days 3, 7, and 14 to collect penis samples. The penis morphology was examined in all groups. Subsequently, transforming growth factor β1 (TGF-β1), α-smooth muscle actin (α-SMactin), and signal transducers and activators of the transcription 3 (STAT3) expression levels in the different groups were measured at the indicated time points postoperatively using qRT-PCR and Western blot. Results: There was less regeneration of the subcutaneous tissue in hypospadias rats than in the sham-operated group (P < 0.05) on postoperative day 3. No differences were found in the regeneration of the subcutaneous tissue between these groups on postoperative days 7 or 14. Additionally, there were no differences in the epithelial cell regeneration between the control and the hypospadias groups at any postoperative timepoint. Moreover, the expression levels of TGF-β1, α-SMactin, and STAT3 were all significantly lower in hypospadias group than that in the sham-operated group (P < 0.05). Conclusion: The results from the present work suggest that preputial wound healing is retarded in rats with hypospadias induced by flutamide and that this retardation might result from multi-gene regulation

Correction to: Human umbilical cord mesenchymal stem cell conditioned medium attenuates renal fibrosis by reducing inflammation and epithelial-to-mesenchymal transition via the TLR4/NF-κB signaling pathway in vivo and in vitro

Abstract The original article [1] contains an error whereby the corresponding authorship is mistakenly designated to the author Fengxia Ding

Exosomes from Human Umbilical Cord Mesenchymal Stem Cells Reduce Damage from Oxidative Stress and the Epithelial-Mesenchymal Transition in Renal Epithelial Cells Exposed to Oxalate and Calcium Oxalate Monohydrate

Objective. To investigate whether exosomes from human umbilical cord mesenchymal stem cells (hUC-MSCs) can protect against the toxic effects of oxalate and calcium oxalate monohydrate (COM) crystals in human proximal tubular epithelial (HK-2) cells. Methods. Exosomes were isolated from hUC-MSCs, purified by ultracentrifugation, and verified by examination of cell morphology using transmission electron microscopy and the presence of specific biomarkers. HK-2 cells received 1 of 4 treatments: control (cells alone), hUC-MSC exosomes, oxalate+COM, or oxalate+COM and hUC-MSC exosomes. Cell viability was determined using the MTT assay. Oxidative stress was determined by measuring LDH activity and the levels of H2O2, malondialdehyde (MDA), and reactive oxygen species (ROS). Expressions of N-cadherin, TGF-β, and ZO-1 were determined by immunofluorescence. Expressions of epithelial markers, mesenchymal markers, and related signaling pathway proteins were determined by western blotting. Results. After 48 h, cells in the oxalate+COM group lost their adhesion, appeared long, spindle-shaped, and scattered, and the number of cells had significantly decreased. The oxalate+COM treatment also upregulated TGF-β and mesenchymal markers, downregulated epithelial markers, increased the levels of LDH, H2O2, MDA, and ROS, decreased cell viability, and increased cell migration. The isolated exosomes had double-layer membranes, had hollow, circular, or elliptical shapes, had diameters mostly between 30 and 100 nm, and expressed CD9, CD63, and Alix. Treatment of HK-2 cells with hUC-MSC exosomes reversed or partly reversed all the effects of oxalate+COM. Conclusions. Exosomes from hUC-MSCs alleviate the oxidative injury and the epithelial-mesenchymal transformation of HK-2 cells that is induced by oxalate+COM

Hypoxia-Induced HIF-1&alpha; 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-1&alpha; and induced EMT and pyroptosis in bladder epithelial cells. Furthermore, HIF-1&alpha; 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-1&alpha; pathway. Inhibition of the HIF-1&alpha; pathway may serve as a potential target to alleviate bladder fibrosis caused by SCI

Comprehensive proteomic analysis of exosome mimetic vesicles and exosomes derived from human umbilical cord mesenchymal stem cells

Abstract Background Exosomes derived from mesenchymal stem cells (MSCs) have shown to have effective application prospects in the medical field, but exosome yield is very low. The production of exosome mimetic vesicles (EMVs) by continuous cell extrusion leads to more EMVs than exosomes, but whether the protein compositions of MSC-derived EMVs (MSC-EMVs) and exosomes (MSC-exosomes) are substantially different remains unknown. The purpose of this study was to conduct a comprehensive proteomic analysis of MSC-EMVs and MSC-exosomes and to simply explore the effects of exosomes and EMVs on wound healing ability. This study provides a theoretical basis for the application of EMVs and exosomes. Methods In this study, EMVs from human umbilical cord MSCs (hUC MSCs) were isolated by continuous extrusion, and exosomes were identified after hUC MSC ultracentrifugation. A proteomic analysis was performed, and 2315 proteins were identified. The effects of EMVs and exosomes on the proliferation, migration and angiogenesis of human umbilical vein endothelial cells (HUVECs) were evaluated by cell counting kit-8, scratch wound, transwell and tubule formation assays. A mouse mode was used to evaluate the effects of EMVs and exosomes on wound healing. Results Bioinformatics analyses revealed that 1669 proteins in both hUC MSC-EMVs and hUC MSC-exosomes play roles in retrograde vesicle-mediated transport and vesicle budding from the membrane. The 382 proteins unique to exosomes participate in extracellular matrix organization and extracellular structural organization, and the 264 proteins unique to EMVs target the cell membrane. EMVs and exosomes can promote wound healing and angiogenesis in mice and promote the proliferation, migration and angiogenesis of HUVECs. Conclusions This study presents a comprehensive proteomic analysis of hUC MSC-derived exosomes and EMVs generated by different methods. The tissue repair function of EMVs and exosomes was herein verified by wound healing experiments, and these results reveal their potential applications in different fields based on analyses of their shared and unique proteins

Stereological analysis and transcriptome profiling of testicular injury induced by di-(2-ethylhexyl) phthalate in prepubertal rats

Di-(2-ethylhexyl) phthalate (DEHP) is the most common phthalate that can affect the male reproductive system. DEHP exposure at the prepubertal stage could lead to the injury of immature testes, but the mechanism has not been fully clarified. In the present study, we elucidated the possible underlying mechanism of DEHP-induced prepubertal testicular injury through stereological analysis and transcriptome profiling. Compared with the control group, the DEHP-treated rats had lower body weight gain and decreased testicular weight and organ coefficient. Moreover, lower serum levels of testosterone and LH were observed in the DEHP group, in contrast to the increased FSH level. Additionally, the serum level of estradiol had no significant difference after DEHP exposure. Stereological analysis showed significant reduction in volumes of most testicular structures, especially in the seminiferous tubule and seminiferous epithelium, along with a vast decrease of spermatogenic cells and obvious structural damages with substantial pathological signs (germ cracks, cytoplasmic vacuolization, sloughing, multinucleated giant cell formation, chromatolysis desquamation and dissolution, pyknosis of nuclei) in the seminiferous tubule upon DEHP exposure at the prepubertal stage. Furthermore, transcriptome profiling identified 5548 differentially expressed genes (DEGs) upon DEHP exposure. Pathway enrichment analysis revealed several crucial signaling pathways related to retinol metabolism, oxidative phosphorylation, steroid hormone biosynthesis, and cell adhesion molecules (CAMs). In addition, seven DEGs selected from RNA-seq data were validated by quantitative real-time polymerase chain reaction (qRT-PCR), and the results showed the same trends as the RNA-seq results. In conclusion, the above findings provide basic morphological data and lay a foundation for systematic research on transcriptome profiling in prepubertal testicular injury induced by DEHP