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.

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.

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.

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.

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.

Novel piRNA MW557525 regulates the growth of Piwil2-iCSCs and maintains their stem cell pluripotency.

BACKGROUND: CSCs play an important role in tumor development. Some studies have demonstrated that piRNAs participate in the progression of various cancers. However, the detailed function of piRNAs in CSCs requires further investigation. This study aimed to investigate the significance of novel piRNA MW557525, one of the top five up-regulated piRNAs screened by gene chip and it has been verified by RT-q-PCR that it is indeed the most obvious up-regulated expression in Piwil2-iCSCs. METHODS AND RESULTS: Differentially expressed piRNAs in Piwil2-iCSCs were screened by gene chip. Target genes were predicted by the miRanda algorithm and subjected to GO and KEGG analysis. One of the differential piRNAs, novel piRNA MW557525, was transfected and its target gene NOP56 was silenced in Piwil2-iCSCs, respectively. RT-qPCR, western blot (WB) and dual luciferase reporter assay were used to investigate the interaction of piRNA MW557525 and NOP56. We identified the effect of piRNA MW557525 and NOP56 knockdown on cell proliferation, migration, invasion, and apoptosis via CCK-8, transwell assay, and flow cytometry. The expressions of CD24, CD133, KLF4, and SOX2 were detected via WB. The results showed that piRNA MW557525 was negatively correlated with NOP56, and it promoted the proliferation, migration, invasion, and inhibited apoptosis in Piwil2-iCSCs, and it also promoted the expressions of CD24, CD133, KLF4, and SOX2, while NOP56 showed the opposite effect. CONCLUSIONS: These findings suggested that novel piRNA MW557525 might be a novel therapeutic target in Piwil2-iCSCs.

Repression of Mafb promotes foreskin fibroblast proliferation through upregulation of CDK2, cyclin E and PCNA.

Mafb plays a significant role in the development and differentiation of various organs, tissues and cells. Nevertheless, its role in the control of external genital cell proliferation and function in the mechanism of hypospadias remains unknown. In this study, the expression of Mafb in foreskin fibroblasts was inhibited by siRNA. The Cell Count Kit-8 (CCK-8) assay showed cell proliferation increased after transfection, and the number of cells entered the S phase significantly increased via flow cytometry. Both mRNA and protein levels of cyclin E, cyclin-dependent kinase 2 (CDK2) and proliferating cell nuclear antigen (PCNA) were significantly upregulated in the siRNA group. Meanwhile, twenty-five prepuce tissue samples were collected from hypospadias repair surgery. These samples were divided into two groups: the severe and mild groups. Normal prepuce tissue specimens were obtained during circumcision as the normal control. The upregulated expression of cyclin E, CDK2 and PCNA and downregulated Mafb expression were observed in the hypospadias group. This study reveals for the first time that the reduction in Mafb promotes the foreskin fibroblast proliferation. Thus, downregulated Mafb expression may cause hypospadias by upregulating CDK2, cyclin E and PCNA. These findings can shed new light on the embryonic development of the urethra.

Laparoscopic versus open inguinal hernia repair in children: A systematic review.

PURPOSE: Considerable debates exist regarding the preferable technique to repair a paediatric inguinal hernia (PIH). This systematic review aims to compare the efficacy and safety of laparoscopic herniorrhaphy (LH) and open herniorrhaphy (OH) in PIH. METHODS: The randomised controlled trials (RCTs) that compared the outcomes of LH and OH in PIH without region and language restrictions searched from the following databases: PubMed, Web of Science Database, Cochrane Library, SciELO Citation Index, Russian Science Citation Index, China National Knowledge Infrastructure, WanFang Data and China Science and Technology Journal Database. RESULTS: A total of 13 RCTs that involving 1207 patients included in the review. The LH displayed a shorter operative time for bilateral hernia repair (weighted mean difference = -8.23, 95% confidence interval [CI]: -11.22~-5.23, P < 0.00001), a lower complication rate (odds ratio [OR] = 0.32, 95% CI: 013-0.83, P = 0.02) along with a lower wound infection (OR = 0.14, 95% CI: 0.04-0.55, P = 0.005) and major male-specific post-operative complications (OR = 0.10, 95% CI: 0.04-0.24, P < 0.00001) and a less contralateral metachronous inguinal hernia (CMIH) incidence rate (OR = 0.09, 95% CI: 0.02-0.42, P = 0.002). No significant difference was found for unilateral operative time, time to full recovery, length of hospital stay, recurrence and hydrocele rates between the two techniques. CONCLUSION: The present review reiterates that both the LH and OH techniques for the PIH repair are comparable. However, in some aspects, the LH is superior to the OH in terms of operative time for bilateral hernias, post-operative complications rate and CMIH incidence rate. Rigorously designed RCTs are anticipated to confirm the clinical effects of both LH and OH.

Weighted gene coexpression network analysis reveals ESR1, FLNA and Furin as hub genes for DEHP-induced prepubertal testicular injury.

Di-(2-ethylhexyl) phthalate (DEHP) is an environmental endocrine disruptor that accumulates in organisms in various ways and induces male reproductive system disorders. In this study, we established a testicular injury model by gavage with different concentrations of DEHP. The testes were then collected for RNA sequencing (RNA-seq), and the results were analyzed by bioinformatics and verified by experiments. Our research results show that different concentrations of DEHP interfere with testicular development differently. Weighted gene coexpression network analysis (WGCNA) generated sixteen modules and identified the turquoise module as key. Then, estrogen receptor 1 (ESR1), filamin A (Flna) and Furin were identified as hub genes. qPCR and immunohistochemistry results revealed that all three hub genes were upregulated. We detected the locations of these genes by immunohistochemistry. ESR1 was mainly located in Leydig cells; Flna immunostaining is observed in the Leydig and some germ cells and Furin staining was seen in almost all types of testicular cells. Furthermore, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses showed enrichment mainly in MAPK signaling pathways, p53 signaling pathways, HIF-1 signaling pathways, protein processing in the endoplasmic reticulum, apoptosis, the cell cycle, RNA degradation, etc. This is the first study using WGCNA to investigate the mechanism of DEHP-induced injury in the prepubertal testis, providing new research angles to further understand the mechanism of DEHP-induced injury in the prepubertal testis.

c-Fos is upregulated in the genital tubercle of DEHP-induced hypospadiac rats and the prepuce of patients with hypospadias.

This study aimed to investigate the expression of Fos proto-oncogene, AP-1 transcription factor subunit (c-Fos) in the genital tubercle (GT) of rats with di(2-ethylhexyl) phthalate (DEHP)-induced hypospadias and in the prepuce of patients with hypospadias compared with unaffected controls. Pregnant rats were given 750 mg/kg/day DEHP orally from gestational days 12-19. Western blotting showed that c-Fos expression was increased in DEHP-induced hypospadiac male offspring. In addition, 30 prepuce tissue specimens obtained during hypospadias repair surgery were divided into 2 groups: the mild hypospadias group (n = 15) and the severe hypospadias group (n = 15). Fifteen normal prepuce tissue specimens were harvested during elective circumcision as normal controls. Real-time quantitative polymerase chain reaction, western blotting and immunohistochemistry analyses were used to assess c-Fos expression. c-Fos protein levels were higher in the GT of DEHP-induced rats than in that of control rats. c-Fos mRNA and protein levels were also higher in the hypospadias groups than in the control group (p < 0.05, p < 0.001), and c-Fos protein levels were significantly higher in the severe hypospadias group than in the mild hypospadias group (p < 0.01). The expression of c-Fos was increased in both the GT of DEHP-induced hypospadiac rats and the prepuce of hypospadias patients. Thus, c-Fos overexpression might contribute to hypospadias.Abbreviations: DEHP: di(2-ethylhexyl) phthalate; c-Fos: Fos proto-oncogene, AP-1 transcription factor subunit; Mafb: the masculinization-regulatory gene v-maf musculoaponeurotic fibrosarcoma oncogene family, protein B; GT: genital tubercle; ED: embryonic day; AGD: anogenital distance; AGI: anogenital distance index; ED: embryonic day.

Multiple transcriptomic profiling: p53 signaling pathway is involved in DEHP-induced prepubertal testicular injury via promoting cell apoptosis and inhibiting cell proliferation of Leydig cells.

Di(2-ethylhexyl) phthalate (DEHP) is a widely-used plasticizer and has long been recognized as an endocrine-disrupting chemical with male reproductive toxicities. DEHP exposure at the prepubertal stage may lead to extensive testicular injury. However, the underlying mechanisms remain to be elucidated. In the present study, we gavaged male C57BL/6 mice with different concentrations of DEHP (0, 250, and 500 mg/kg-bw·d) from postnatal day 22-35, and exposed TM3 Leydig cells with 0, 100, 200, 300, and 400 µM of MEHP (bioactive metabolite of DEHP) for 12-48 h. RNA sequencing was performed both in testicular tissue and TM3 cells. The results showed that DEHP disrupts testicular development and reduces serum testosterone levels in male prepubertal mice. Bioinformatic analysis and experimental verification have revealed that DEHP/MEHP induces cell cycle arrest in TM3 cells and increases apoptosis both in vivo and in vitro. Furthermore, the p53 signaling pathway was found to be activated upon DEHP/MEHP treatment. The inhibition of p53 by pifithrin-α significantly reduced MEHP-induced injuries in TM3 cells. Cumulatively, these findings revealed the involvement of the p53 signaling pathway in DEHP-induced prepubertal testicular injury by promoting cell apoptosis and inhibiting cell proliferation of Leydig cells.

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

BACKGROUND: Hypospadias is the second most common congenital malformation in males. Although the aetiology of hypospadias is not clear, it is generally thought to be affected by both genetic and environmental endocrine-disrupting factors that affect the development of the urethra, leading to deformity. OBJECTIVE: To investigate the difference in expression of the transcription factor Mafb in hypospadias and normal penile tissues and to assess whether it is related to the occurrence of hypospadias. STUDY DESIGN: Penile tissue was obtained from children with hypospadias who underwent surgical repair at the Children's Hospital of Chongqing Medical University. Patients diagnosed with undescended testicles, intersex status or endocrine abnormalities were excluded from the study. Twenty-five cases with hypospadias (average 3.5 years old) and 15 cases with circumcisions (as control) (average 5 years old) were included in this study. Real-time quantitative polymerase chain reaction, Immunochemistry and Western blot were used to detect the expression of Mafb. RESULTS: Mafb mRNA expressions in the prepuce of cases with hypospadias was significantly reduced compared with that in the controls [(1.179 ± 0.1275), (0.6652 ± 0.07506), p < 0.05)]. Hypospadias cases also showed decreased Mafb protein expression in the preputial subcutaneous mesenchymal cell layer. Mafb protein levels were significantly decreased in those with hypospadias compared with controls [(1.932 ± 0.1139), (1.006 ± 0.03312), p < 0.05]. However, no such differences were found in Mafb expression between subjects with mild and severe hypospadias. DISCUSSION: Compared to the normal foreskin, expression of the Mafb gene was down-regulated at both mRNA and protein levels, which was consistent with our RNA-seq sequencing results in Diethylhexyl phthalate (DEHP)-induced hypospadias rats. This study is the first to report abnormal expression of Mafb in the preputial tissue of hypospadias cases. An in-depth study of the relationship between Mafb and cell proliferation, apoptosis, and urethra development may reveal the pathogenesis of hypospadias. CONCLUSION: Expression of the Mafb gene and protein in the foreskin of children with hypospadias is lower than that in normal foreskin. We postulate that such abnormal expression of the Mafb gene may be related to the occurrence of hypospadias and that this abnormal expression may affect the development of the urethra during the embryonic period.

Human umbilical cord mesenchymal stem cell attenuates renal fibrosis via TGF-ß/Smad signaling pathways in vivo and in vitro.

Renal fibrosis is a progressive pathological process that eventually leads to end-stage renal failure with limited therapeutic options. The aim of this study was to investigate the nephron-protective effect of human umbilical cord mesenchymal stem cells (ucMSCs) on renal fibrosis. UcMSCs were intravenously injected into renal fibrosis mice induced by aristolochic acid (AA) and co-cultured with HK-2 cells induced by TGF-ß1, respectively. The kidney functions including serum creatinine (Scr) and blood urea nitrogen (BUN) levels, and histopathology were examined after treated with stem cells and normal saline as control. Immunohistochemical staining, immunofluorescent staining, and Western blot analysis were used to assessed the expression of proteins associated with epithelial to mesenchymal transition (EMT) and TGF-ß/Smad signaling pathway. The results showed that ucMSCs effectively improved the kidney function and pathological structure, reduced AA-induced fibrosis and extracellular matrix deposition. Besides, UcMSCs significantly inhibited the EMT process and TGF-ß1/Smad signaling pathway in AA-induced mice and TGF-ß1-induced HK-2 cells compared to the control (p < 0.05). Our data suggested that ucMSCs play as a nephron-protective role in anti-fibrosis through inhibiting the activation of TGF-ß1/Smad signaling pathway.

MiR-155-5p exerts tumor-suppressing functions in Wilms tumor by targeting IGF2 via the PI3K signaling pathway.

BACKGROUND: MicroRNA-155-5p (miR-155-5p) has been reported to play an oncogenic role in different human malignancies; however, its role in Wilms tumor (WT) remains unclear. METHODS: Differentially expressed miRNAs (DE-miRNAs) and mRNAs (DEGs) in WT blood and tissues were identified by using miRNA microarray and RNA-sequencing. Bioinformatics prediction and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were used to predict the potential functions of DE-miRNAs. DE-miRNAs and DEGs in WT obtained from Gene Expression Omnibus (GEO) and Therapeutically Applicable Research to Generate Effective Treatments (TARGET) were identified by using the "edgeR" package. RT-qPCR was used to explore miR-155-5p and IGF2 expression and their clinical significance in WT specimens. A rhabdoid cell line (G401) and Ewing sarcoma cell line (SK-NEP-1) were used. Immunohistochemical staining, western blotting and dual-luciferase reporter assays were performed to study the mechanisms involved. The CCK-8, flow cytometry, wound healing and transwell assays were performed to identify the effects of miR-155-5p and IGF2 knockdown on cell proliferation, apoptosis, migration and invasion, respectively. RESULTS: MiR-155-5p was downregulated in both blood and tissues from WT patients who did not receive chemotherapy before surgery but was upregulated in tissues from WT patients who had received chemotherapy before surgery. IGF2, PI3K, AKT and mTOR were found to be upregulated in WT tissues. Additionally, miR-155-5p and IGF2 were significantly correlated with TNM stage and lymphatic metastasis in WT patients. Molecular mechanism exploration indicated that IGF2 was downregulated by miR-155-5p via direct binding to its 3' untranslated region in cell lines. Furthermore, IGF2, PI3K, AKT and mTOR expression was inversely correlated with miR-155-5p expression, and PI3K, AKT and mTOR expression was positively correlated with IGF2 expression in cell culture. Functional studies demonstrated that miR-155-5p upregulation and IGF2 knockdown suppressed cell proliferation, migration and invasion and induced cell apoptosis. Moreover, the tumor-suppressing effects of miR-155-5p in cells were abrogated by miR-155-5p inhibitor treatment. CONCLUSIONS: Taken together, these findings suggest that miR-155-5p functions as a tumor suppressor in WT through inactivating the PI3K/AKT/mTOR signaling pathway by directly targeting IGF2. Thus, miR-155-5p might be a novel therapeutic target for WT.

Human ucMSCs seeded in a decellularized kidney scaffold attenuate renal fibrosis by reducing epithelial-mesenchymal transition via the TGF-ß/Smad signaling pathway.

BACKGROUND: Renal fibrosis occurs largely through epithelial-mesenchymal transition (EMT). This study explored the beneficial effects of a human umbilical cord mesenchymal stem cell-loaded decellularized kidney scaffold (ucMSC-DKS) on renal fibrosis in a rodent model of post-transplantation renal failure, and the underlying mechanism. METHODS: Rat-derived DKSs were examined after preparation, and then recellularized with human ucMSCs to prepare cell-loaded patches. A rat model of renal failure was established after subtotal nephrectomy (STN). The cell patches were transplanted to remnant kidneys. Changes in renal function, histology, EMT, and proteins related to the transforming growth factor-ß (TGF-ß)/Smad signaling pathway in the remnant kidneys were examined 8 weeks after surgery, compared with non-cell patch controls. RESULTS: The DKSs were acellular and porous, with rich cytokine and major extracellular matrix components. The ucMSCs were distributed uniformly in the DKSs. Renal function was improved, renal fibrosis and EMT were reduced, and the TGF-ß/Smad signaling pathway was inhibited compared with controls at 8 weeks after ucMSC-DKS patch transplantation. CONCLUSIONS: The ucMSC-DKS restores renal function and reduces fibrosis by reducing EMT via the TGF-ß/Smad signaling pathway in rats that have undergone STN. It provides an alternative for renal fibrosis treatment.