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.

Rab25 is involved in hypospadias via the ß1 integrin/EGFR pathway.

BACKGROUND: Hypospadias is a common congenital abnormality of the penile. Abnormal regulation of critical genes involved in urethral development leads to hypospadias. We used the Rab25-/- mice and foreskin fibroblasts transfected with lentivirus in vitro and in vivo to investigate the role of Rab25 in hypospadias. METHODS: The expression levels of various molecules in tissue samples and foreskin fibroblasts were confirmed using molecular biology methods (western blotting, PCR, immunohistochemistry, etc.). A scanning electron microscope (SEM) was used to visualize the external morphology of genital tubercles (GTs) of gestation day (GD) 18.5 male wild-type (WT) and Rab25-/- mice. RESULTS: An expanded distal cleft and V-shaped urethral opening were observed in GD 18.5 Rab25-/- mice. We demonstrated that Rab25 mediated hypospadias through the ß1 integrin/EGFR pathway. In addition, silencing Rab25 inhibited cell proliferation and migration and promoted apoptosis in the foreskin fibroblasts; Ki-67- and TUNEL-positive cells were mainly concentrated near the urethral seam. CONCLUSION: These findings suggest that Rab25 plays an essential role in hypospadias by activation of ß1 integrin/EGFR pathway, and Rab25 is a critical mediator of urethral seam formation in GD18.5 male fetal mice.

IL-13 alleviates acute kidney injury and promotes regeneration via activating the JAK-STAT signaling pathway in a rat kidney transplantation model.

AIMS: To identify whether and how a younger systemic internal milieu alleviates acute kidney injury (AKI) in grafts after kidney transplantation. MATERIALS AND METHODS: We conducted an allogenic heterotopic rat kidney transplantation model with young and adult recipients receiving similar donor kidneys. We evaluated the renal function, histological damage, apoptosis, dedifferentiation, proliferation, hub regulating cytokines, and signaling pathways involved in young and adult recipients based on transcriptomics, proteomics, and experimental validation. We also validated the protective effect and mechanism of interleukin-13 (IL-13) on tubular epithelial cell injury induced by transplantation in vivo and by cisplatin in vitro. KEY FINDINGS: Compared with adult recipients, the young recipients had lower levels of renal histological damage and apoptosis, while had higher levels of dedifferentiation and proliferation. Serum IL-13 levels were higher in young recipients both before and after surgery. Pretreating with IL-13 decreased apoptosis and promoted regeneration in injured rat tubular epithelial cells induced by cisplatin, while this effect can be counteracted by a JAK2 and STAT3 specific inhibitor, AG490. Recipients pretreated with IL-13 also had lower levels of histological damage and improved renal function. SIGNIFICANCE: Higher levels of IL-13 in young recipients ameliorates tubular epithelial cell apoptosis and promotes regeneration via activating the JAK-STAT signaling pathway both in vivo and in vitro. Our results suggest that IL-13 is a promising therapeutic strategy for alleviating AKI. The therapeutic potential of IL-13 in injury repair and immune regulation deserves further evaluation and clinical consideration.

Protective effects of melatonin on DEHP-induced apoptosis and oxidative stress in prepubertal testes via the PI3K/AKT pathway.

Di(2-ethylhexyl) phthalate (DEHP), an environmental endocrine disruptor, is one of the most common plasticizers and is widely used in various plastic products. DEHP induces apoptosis and oxidative stress and has been shown to have androgenic toxicity. However, the methods to combat DEHP-induced testicular damage and the mechanisms involved remain to be elucidated. In the present study, we used melatonin, which has strong antioxidant properties, to intervene in prepubertal mice and mouse Leydig cells (TM3) treated with DEHP or its metabolite mono(2-ethylhexyl) phthalate (MEHP). The results showed that melatonin protected against DEHP-induced testicular damage in prepubertal mice, mainly by protecting against DEHP-induced structural destruction of the germinal tubules and by attenuating the DEHP-induced decrease in testicular organ coefficients and testosterone levels. Transcriptomic analysis found that melatonin may attenuate DEHP-induced oxidative stress and apoptosis in prepubertal testes. In vitro studies further revealed that MEHP induces oxidative stress injury and increases apoptosis in TM3 cells, while melatonin reversed this damage. In vitro studies also found that MEHP exposure inhibited the expression levels of molecules related to the PI3K/AKT signaling pathway, and melatonin reversed this change. In conclusion, these findings suggest that melatonin protects against DEHP-induced prepubertal testicular injury via the PI3K/AKT signaling pathway, and provide a theoretical basis and experimental rationale for combating male reproductive dysfunction.

Vitamin D levels and the risk of overactive bladder: a systematic review and meta-analysis.

CONTEXT: Overactive bladder is treated mainly with behavioral and drug therapy, and symptoms of urinary frequency and incontinence are challenging to eliminate. There is thus a continuous unmet need for new drugs with a substitution effect mechanism. OBJECTIVE: It not known whether vitamin D deficiency can lead to overactive bladder or urinary incontinence or whether vitamin D supplementation alleviates bladder symptoms. This comprehensive systematic review with meta-analysis was conducted to determine whether overactive bladder is associated with vitamin D deficiency. DATA SOURCES: The PubMed and Cochrane Library databases were searched systematically up to July 3, 2022. DATA EXTRACTION: Initially, 706 articles were identified in the literature search, of which 13 were included in the systematic review: 4 randomized controlled trials, 3 cohort studies, 3 cross-sectional studies, and 3 case-control studies. DATA ANALYSIS: An increased risk of overactive bladder and urinary incontinence was observed with vitamin D deficiency (odds ratio [OR] = 4.46; 95%CI, 1.03-19.33; P = 0.046 and OR = 1.30; 95%CI, 1.01-1.66; P = 0.036, respectively). Vitamin D levels were relatively low in patients with overactive bladder or urinary incontinence (SMD = -0.33; 95%CI, -0.61 to -0.06, P = 0.019). On the basis of existing data, the risk of urinary incontinence was reduced by 66% after vitamin D supplementation (OR = 0.34; 95%CI, 0.18-0.66; P = 0.001). Egger test was conducted to assess publication bias, and the results were tested for robustness using a sensitivity analysis. CONCLUSIONS: Vitamin D deficiency increases the risk of overactive bladder and urinary incontinence, and vitamin D supplementation reduces the risk of urinary incontinence. The development of new strategies to prevent or alleviate bladder symptoms is crucial. Vitamin D supplementation may be gaining recognition as an effective strategy for prevention or alleviation of bladder symptoms such as overactive bladder and incontinence. SYSTEMATIC REVIEW REGISTRATION: PROSPERO registration no. CRD42022351443.

Di-(2-ethylhexyl) phthalate induces ferroptosis in prepubertal mouse testes via the lipid metabolism pathway.

Di-(2-ethylhexyl) phthalate (DEHP), a widely used plasticizer, has been shown to cause reproductive toxicity, but the precise mechanism remains unclear. This study aimed to investigate the possible molecular mechanism of DEHP-induced testicular damage. In vivo study, we administered different doses of DEHP (0, 250, and 500 mg/kg/day) to male C57BL/6 mice from 22 and 35 days after birth. We found that DEHP exposure induced histopathological alterations in prepubertal testes, and testicular lipidomics indicated notable alterations in lipid metabolism and significant enrichment of ferroptosis. Further tests showed that ferrous iron (Fe2+ ) and malondialdehyde (MDA) levels significantly increased after DEHP exposure. Western blotting revealed that DEHP exposure reduced glutathione peroxidase 4 (GPX4) expression, and elevated acyl coenzyme A synthetase long-chain member 4 (ACSL4) and lysophosphatidylcholine acyltransferase 3 (LPCAT3) expression. The in vitro results were consistent with the in vivo results. When Leydig cells and Sertoli cells were treated with ferrostatin-1 and monoethylhexyl phthalate (MEHP), MEHP-induced increases in Fe2+ and MDA levels, accumulation of lipid reactive oxygen species, downregulation of GPX4, and upregulation of ACSL4 and LPCAT3 were reversed. Collectively, our findings suggested that aberrant lipid metabolism and ferroptosis may be involved in prepubertal DEHP exposure-induced testicular damage.

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.

Expression of Rab25 is down-regulated in the foreskin of children with hypospadias.

BACKGROUND: Hypospadias, a congenital malformation of the penis, is one of the newborns' most common developmental defects. The incidence of hypospadias is increasing yearly, and its pathogenesis is closely related to genetic susceptibility and environmental exposure to endocrine disruptors. Exploring the hypospadias' key molecular regulatory mechanism is crucial to reducing its incidence. OBJECTIVE: To examine the differential expression of Rab25 in hypospadias and normal penile tissue and to identify whether it is a candidate gene for exploring the mechanism of hypospadias. STUDY DESIGN: This study included 18 children aged 1-6 years undergoing hypospadias repair surgery at the Children's Hospital of Chongqing Medical University, and foreskin samples were collected. Children diagnosed with cryptorchidism, intersex status, or endocrine abnormalities were excluded from this study. Another 18 children aged 3-8 years with phimosis were included in the control group. The specimens were used for immunohistochemistry, western blotting, immunofluorescence, and polymerase chain reaction to assess the expression of Rab25. RESULTS: Rab25 protein expression was lower in the hypospadias group than in the control group [ (2.101 ± 0.1845), (0.7506 ± 0.1779), p = 0.0008 < 0.05). The hypospadias group showed decreased expression of Rab25 protein in the epithelial cell layer. Rab25 mRNA levels were downregulated in the foreskin of children with hypospadias compared with controls [(1.697 ± 0.2005), (0.7687 ± 0.2130), p = 0.0053 < 0.05)]. DISCUSSION: Rab25 mRNA and protein expressions in the hypospadias group were significantly downregulated compared with the control group. This was consistent with the results of single-cell sequencing of fetal mice reproductive nodules at 15.5 days of gestation (Zhang Z, Liu Z, Zhang Q, et al., unpublished observations). Our study represents the first report of abnormal Rab25 expression in the foreskin tissue of patients with hypospadias. More detailed research on the relationship between Rab25 and urethral development could be conducted to reveal the molecular mechanism of hypospadias. CONCLUSION: The expression of Rab25 in foreskin tissue was lower in the hypospadias group than in the control group. Rab25 is involved in the formation of the urethral seam and the occurrence of hypospadias. The potential mechanism by which Rab25 affects the canalization of the urethral plate needs to be further investigated.

Single-cell transcriptomic dissection of the toxic impact of di(2-ethylhexyl) phthalate on immature testicular development at the neonatal stage.

Di(2-ethylhexyl) phthalate (DEHP) early exposure leads to immature testicular injury, and we aimed to utilize single-cell RNA (scRNA) sequencing to comprehensively assess the toxic effect of DEHP on testicular development. Therefore, we gavaged pregnant C57BL/6 mice with 750 mg/kg body weight DEHP from gestational day 13.5 to delivery and performed scRNA sequencing of neonatal testes at postnatal day 5.5. The results revealed the gene expression dynamics in testicular cells. DEHP disrupted the developmental trajectory of germ cells and the balance between the self-renewal and differentiation of spermatogonial stem cells. Additionally, DEHP caused an abnormal developmental trajectory, cytoskeletal damage and cell cycle arrest in Sertoli cells; disrupted the metabolism of testosterone in Leydig cells; and disturbed the developmental trajectory in peritubular myoid cells. Elevated oxidative stress and excessive apoptosis mediated by p53 were observed in almost all testicular cells. The intercellular interactions among four cell types were altered, and biological processes related to glial cell line-derived neurotrophic factor (GDNF), transforming growth factor-ß (TGF-ß), NOTCH, platelet-derived growth factor (PDGF) and WNT signaling pathways were enriched after DEHP treatment. These findings systematically describe the damaging effects of DEHP on the immature testes and provide substantial novel insights into the reproductive toxicity of DEHP.

Difenoconazole Exposure Induces Retinoic Acid Signaling Dysregulation and Testicular Injury in Mice Testes.

Difenoconazole (DFZ) is a broad-spectrum triazole fungicide that is widely utilized in agriculture. Although DFZ has been demonstrated to induce reproductive toxicity in aquatic species, its toxic effects on the mammalian reproductive system have yet to be fully elucidated. In vivo, male mice were administered 0, 20 or 40 mg/kg/d of DFZ via oral gavage for 35 days. Consequently, DFZ significantly decreased testicular organ coefficient, sperm count and testosterone levels, augmented sperm malformation rates, and elicited histopathological alterations in testes. TUNEL assay showed increased apoptosis in testis. Western blotting results suggested abnormally high expression of the sperm meiosis-associated proteins STRA8 and SCP3. The concentrations of retinoic acid (RA), retinaldehyde (RE), and retinol (ROL) were increased in the testicular tissues of DFZ-treated groups. The mRNA expression level of genes implicated in RA synthesis significantly increased while genes involved in RA catabolism significantly decreased. In vitro, DFZ reduced cell viability and increased RA, RE, and ROL levels in GC-2 cells. Transcriptome analysis revealed a significant enrichment of numerous terms associated with the RA pathway and apoptosis. The qPCR experiment verified the transcriptome results. In conclusion, our results indicate that DFZ exposure can disrupt RA signaling pathway homeostasis, and induce testicular injury in mice testes.

Prepubertal exposure to copper oxide nanoparticles induces Leydig cell injury with steroidogenesis disorders in mouse testes.

Copper oxide nanoparticles (CuONPs) are metallic multifunctional nanoparticles with good conductive, catalytic and antibacterial characteristics that have shown to cause reproductive dysfunction. However, the toxic effect and potential mechanisms of prepubertal exposure to CuONPs on male testicular development have not been clarified. In this study, healthy male C57BL/6 mice received 0, 10, and 25 mg/kg/d CuONPs by oral gavage for 2 weeks (postnatal day 22-35). The testicular weight was decreased, testicular histology was disturbed and the number of Leydig cells was reduced in all CuONPs-exposure groups. Transcriptome profiling suggested steroidogenesis was impaired after exposure to CuONPs. The steroidogenesis-related genes mRNA expression level, concentration of serum steroids hormones and the HSD17B3-, STAR- and CYP11A1-positive Leydig cell numbers were dramatically reduced. In vitro, we exposed TM3 Leydig cells to CuONPs. Bioinformatic analysis, flow cytometry analysis and western blotting analysis confirmed that CuONPs can dramatically reduce Leydig cells viability, enhance apoptosis, trigger cell cycle arrest and reduce cell testosterone levels. U0126 (ERK1/2 inhibitor) significantly reversed TM3 Leydig cells injury and testosterone level decrease induced by CuONPs. These outcomes indicate that CuONPs exposure activates the ERK1/2 signaling pathway, which further promotes apoptosis and cell cycle arrest in TM3 Leydig cells, and ultimately leads to Leydig cells injury and steroidogenesis disorders.

HucMSC exosomes attenuate partial bladder outlet obstruction-induced renal injury and cell proliferation via the Wnt/ß-catenin pathway.

Bladder outlet obstruction (BOO) can cause serious complications including kidney damage; nevertheless, there are currently no animal models for studying BOO-induced kidney damage. Mesenchymal stem cells (MSCs) are widely used in therapeutic studies of renal fibrosis. However, MSC-derived exosomes show improved safety profile and more controllable characteristics compared with those of MSCs. Herein, we established a kidney injury mouse model of partial bladder outlet obstruction (PBOO) and evaluated the effects of human umbilical cord MSC-derived exosomes (hucMSC-Exos) on PBOO-induced reflux kidney injury in this model. Exosomes were isolated from a hucMSC-conditioned medium, purified by ultracentrifugation, and examined. Living image was performed to indicate the distribution of hucMSC-Exos. The PBOO-treated mice interacted with PBS (phosphate-buffered saline) or hucMSC-Exos. Morphologic changes and expression of interstitial-fibrosis-related, cell proliferation and Wnt/ß-catenin signaling-pathway indices were evaluated. At 7 days after induction of PBOO, structural destruction of renal tubules was observed. Expression of the interstitial markers and the cellular-proliferation index increased significantly in the PBOO group compared with the control group. The isolated exosomes were 30-150 nm in diameter, showing a round shape and bilayer membrane structure with CD63, TSG101, Alix expressed, enriched in the kidney of the PBOO group. Administering hucMSC-Exos to post-PBOO mice reversed renal injury and suppressed expression of Wnt/ß-catenin signaling pathway-related proteins. hucMSC-Exos inhibited PBOO-induced kidney injury and cellular proliferation and suppressed the Wnt/ß-catenin signaling pathway. Our findings will spur the development of novel hucMSC-Exo-mediated therapies for treating patients with renal fibrosis.

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.

MK-2206 Alleviates Renal Fibrosis by Suppressing the Akt/mTOR Signaling Pathway In Vivo and In Vitro.

Renal fibrosis is a common pathological feature of various kidney diseases, leading to irreversible renal failure and end-stage renal disease. However, there are still no effective treatments to reverse renal fibrosis. This study aimed to explore the potential mechanism of a targeted drug for fibrosis. Here, unilateral ureteral obstruction (UUO)-treated mice and a TGF-ß1-treated human renal tubular epithelial cell line (HK-2 cells) were used as models of renal fibrosis. Based on the changes of mRNA in UUO kidneys detected by transcriptome sequencing, MK-2206, an Akt inhibitor, was predicted as a potential drug to alleviate renal fibrosis through bioinformatics. We dissolved UUO mice with MK-2206 by gastric gavage and cultured TGF-ß-induced HK-2 cells with MK-2206. Histopathological examinations were performed after MK-2206 intervention, and the degree of renal fibrosis, as well as the expression of Akt/mTOR pathway-related proteins, were evaluated by immunohistochemical staining, immunofluorescence staining, and Western blot. The results showed that MK-2206 significantly improved the pathological structure of the kidney. Furthermore, MK-2206 intervention effectively inhibited UUO- and TGF-ß1-induced epithelial-mesenchymal transition, fibroblast activation, and extracellular matrix deposition. Mechanistically, MK-2206 treatment attenuated the activation of the Akt/mTOR signaling pathway. Taken together, our study revealed for the first time that MK-2206 is a promising drug for the improvement of renal fibrosis.

Antitumor effect of Escherichia coli-derived outer membrane vesicles on neuroblastoma in vitro and in vivo.

Bacterial outer membrane vesicles (OMVs) are spherical microbubbles that contain biological content and are produced by gram-negative bacteria. The use of OMVs as adjuvants for cancer immunotherapy or as drug carriers for targeted therapies has attracted the interest of many scholars. However, it is unclear whether OMVs can exert direct antitumor effects and whether OMVs can inhibit pediatric tumors. Here, we explore the potential of Escherichia coli-derived OMVs to directly suppress neuroblastoma. Our results demonstrate the antitumor effects of OMVs in vitro and in vivo, and no serious adverse reactions were observed. OMV uptake into the cytoplasm and nucleus directly decreases cell stemness, DNA damage, apoptosis and cell cycle arrest, which may be the mechanisms by which OMVs suppress tumors. Our results demonstrate the potential of bacterial OMVs to be used as antitumor adjuvant therapies, increasing the number of candidates for the development of cancer therapies in the future. More relevant studies are urgently needed to demonstrate the efficacy and safety of OMVs.

Retinoic acid can improve autophagy through depression of the PI3K-Akt-mTOR signaling pathway via RARα to restore spermatogenesis in cryptorchid infertile rats.

Cryptorchidism-caused adult infertility is a common component of idiopathic reasons for male infertility. Retinoic acid (RA) has a vital effect on the spermatogenesis process. Here, we found that the expression of c-Kit, Stra8, and Sycp3 could be up-regulated via the activation of retinoic acid receptor α (RARα) after RA supplementation in neonatal cryptorchid infertile rats. We also demonstrated that the protein expression of PI3K, p-Akt/pan-Akt, and p-mTOR/mTOR was higher in cryptorchid than in normal testes, and could be suppressed with RA in vivo. After RA treatment in infertile cryptorchid testis in vivo, the levels of the autophagy proteins LC3 and Beclin1 increased and those of P62 decreased. Biotin tracer indicated that the permeability of blood-testis barrier (BTB) in cryptorchid rats decreased after RA administration. Additionally, after blocking the RARα with AR7 (an RARα antagonist) in testicle culture in vitro, we observed that compared with normal testes, the PI3K-Akt-mTOR signaling pathway and the autophagy pathway was increased and decreased, respectively, which were coincident with cryptorchisd testes in vivo. Additionally, the appropriate concentrations of RA treatment could depress the PI3K-Akt-mTOR signaling pathway and improve the autophagy pathway. The results confirmed that RA can rehabilitate BTB function and drive key protein levels in spermatogonial differentiation through depressing the PI3K-Akt-mTOR signaling pathway via RARα.

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

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.