Expression Of PCNA Research Articles

BPA induces placental trophoblast proliferation inhibition and fetal growth restriction by inhibiting the expression of SRB1

Bisphenol-A (BPA) is a common environmental toxicant that is known to be associated with fetal growth restriction (FGR). However, the mechanisms of how BPA induce FGR is poorly characterized. We conducted proteomics to identify the abnormal expression of SRB1 in female placental tissues with high BPA-induced FGR and further verified its decreased expression in human placenta and BeWo cells. Next, the effect of BPA on fetal development was further confirmed in pregnant C57BL/6 mice. The expression of SRB1 was consistently downregulated in human FGR placentas, BPA-exposed trophoblasts and mouse placentas. In addition, we found that SRB1 interacted with PCNA, and BPA exposure indirectly reduced the expression of PCNA and further inhibited placental proliferation. In vitro studies showed that BPA exposure reduced the expression of CDK1, CDK2, cyclin B and phosphorylated Rb in placental trophoblast cells, indicating cell cycle arrest after exposure to BPA. In addition, the expression of γ-H2AX and phosphorylated ATM was upregulated in BPA-exposed trophoblasts, indicating increased DNA damage. Our results indicate that BPA-induced FGR is achieved by reducing the expression of SRB1, inhibiting placental proliferation and increasing DNA damage. Our findings not only explain the mechanism of BPA-associated developmental toxicity but also shed light upon developing novel therapeutic targets.

The adverse effect of anticancer drug toremifene on vascular smooth muscle cells is an important aspect of its tumor growth inhibition.

Toremifene (TOR) is widely used as an antineoplastic drug and has an inhibitory effect on angiogenesis in mesenteric desmoid tumors and vascular intracranial solitary fibrous tumors. However, no study has investigated the direct effect of TOR on vascular cells. This study aimed at exploring the effect of TOR on the behaviors of vascular smooth muscle cells (VSMCs). Human aortic umbilical vascular smooth muscle cells (HAVSMCs) were treated by TOR. Cell morphology, migration, adhesion, and proliferation assay were investigated. The cell cycle, apoptosis, mitochondrial membrane potential, and reactive oxygen species were assessed using flow cytometry. Caspase-3 and 9 activities were assayed using Caspase-3 and Caspase-9 Activity Assay kits, respectively. Immunofluorescence and Western blot assays were carried out to characterize protein expressions of PCNA, p53, and Rho/ROCK signaling pathway. TOR damaged cytoskeleton, inhibited VSMC proliferation, migration, and adhesion, and induced abnormal cell morphology and apoptosis. The antiproliferative activity of TOR was associated with the induction of G0/G1 phase arrest, blocking the cell cycle. TOR disrupted intracellular reactive oxygen species and mitochondrial membrane potential, and enhanced p53 expression and the activities of caspase-3 and caspase-9. Thus, TOR-induced apoptosis by the mitochondrial signaling pathway. Additionally, TOR induced decreased Rho, ROCK, MLC, and pMLC proteins. Collectively, TOR may affect multiple behaviors of VSMCs by damaging cytoskeleton through the Rho/ROCK pathway. The adverse effect of TOR on VSMCs could be considered as an important aspect of tumor growth inhibition.

Gum Acacia attenuates cisplatin toxic effect spermatogenesis dysfunction and infertility in rats

This study aimed to investigate the potential benefits Gum Arabic/Acacia senegal (GA) in mitigating the harmful effects of cisplatin (CP) on spermatogenesis and testicular health in male adult rats. A total of forty albino rats were used in the study and divided into four groups; control, GA, CP, and Co-treated group, which received both CP and GA concurrently. The results revealed that CP caused a significant increase in oxidative stress and a decrease in antioxidant activities (CAT, SOD, and GSH), which disturbed the testicular machinery. This caused significant histological and ultrastructural damage to the testicular structure, including atrophied seminiferous tubules with severely reduced germinal epithelium. Additionally, CP caused a decrease in reproductive hormones (testosterone and LH), a decline in nucleic proliferation PCNA immunoexpression, and an increase in cytoplasmic apoptotic Caspase-3 protein expression in testicular tissue, when compared to the control and GA groups. Moreover, the CP treatment impaired spermatogenesis and decreased sperm number and motility with abnormal morphology. However, co-administration of GA with CP mitigated the dysfunction in spermatogenesis and reversed testicular damage caused by CP through significantly (P < 0.01) reducing oxidative stress (MDA) and increasing the activities of CAT, SOD, and GSH. Additionally, co-administration of GA elevated the levels of testosterone and luteinizing hormone in blood sera, significantly (P < 0.01) improved the histometric measurements of seminiferous tubules diameter, their epithelial height, Johnsen's score of spermatogenesis, 4-level histological grading scale Cosentino's score, immunohistochemical expression of nucleic PCNA, and cytoplasmic Caspase-3 proteins. Furthermore, TEM examination confirmed the synergistic effect of GA in restoring the germinal epithelial cells ultrastructure, the elongated and transverse sections of spermatozoa in the lumen, and the interstitial tissue. All of these effects resulted in a significant improvement in sperm quality in the Co-treated animals compared with the CP group, as well as, a significant decline in the morphological abnormalities of sperm in Co-treated rats compared to those in the CP group. GA is a valuable agent for ameliorating chemotherapy-related infertility.

A tri-herb formulation protects against ethanol-induced mouse liver injury and downregulates mitogen-activated protein kinase phosphatase 1

BackgroundA tri-herb formulation comprising Ganoderma (the dried fruiting body of Ganoderma lucidum), Puerariae Thomsonii Radix (the dried root of Pueraria thomsonii) and Hoveniae Semen (the dried mature seed of Hovenia acerba) —GPH for short— has been using for treating liver injury; however, the pharmacological basis of this application of GPH is unknown. This study aimed to investigate the liver protective effects and mechanisms of action of an ethanolic extract of GPH (GPHE) in mice. MethodsTo control the quality of GPHE, the contents of ganodermanontriol, puerarin and kaempferol in the extract were quantified by ultra-performance liquid chromatography. An ethanol (6 ml/kg, i.g.)-induced liver injury ICR mouse model was employed to investigate the hepatoprotective effects of GPHE. RNA-sequencing analysis and bioassays were performed to reveal the mechanisms of action of GPHE. ResultsThe contents of ganodermanontriol, puerarin and kaempferol in GPHE were 0.0632%, 3.627% and 0.0149%, respectively. Daily i.g. administration of 0.25, 0.5 or 1 g/kg of GPHE for 15 consecutive days suppressed ethanol (6 ml/kg, i.g., at day 15)-induced upregulation of serum AST and ALT levels and improved histological conditions in mouse livers, indicating that GPHE protects mice from ethanol-induced liver injury. Mechanistically, GPHE downregulated the mRNA level of Dusp1 (encoding MKP1 protein, an inhibitor of the mitogen-activated protein kinases JNK, p38 and ERK), and upregulated expression and phosphorylation of JNK, p38 and ERK, which are involved in cell survival in mouse liver tissues. Also, GPHE increased PCNA (a cell proliferation marker) expression and reduced TUNEL-positive (apoptotic) cells in mouse livers. ConclusionGPHE protects against ethanol-induced liver injury, and this effect of GPHE is associated with regulation of the MKP1/MAPK pathway. This study provides pharmacological justifications for the use of GPH in treating liver injury, and suggests that GPHE has potential to be developed into a modern medication for managing liver injury.

Abstract 5255: Effect of STAT3 inhibitors, TTI-101 and SH5-07, against bladder cancer in preclinical 3D tumor models

Abstract Bladder cancer (BC) is a lethal genitourinary malignancy associated with frequent recurrence and poor survival due to metastatic potential. Identification of key cancer cell signaling networks and developing promising agents is critical for effectively inhibiting tumor growth and progression. In many cancers, including bladder cancer (BC), signal transducer and activator of transcription 3 (STAT3) has emerged as an important molecular pathway due to its role in promoting proliferation, invasion, and chemoresistance. Thus, developing STAT3 targeting, orally bioavailable small molecule inhibitors may be helpful for the prevention of BC progression and improving the survival rate of patients with muscle invasive BC. Monolayer culture has limitations for drug testing. Therefore, spheroid and organoid culture are used extensively as they may mimic in-vivo drug response more accurately. The aim of our study is to examine the preclinical anticancer efficacy of STAT3 inhibitors [TTI-101 (C188-9) and SH5-07] in 3D (spheroid and tumoroid) invitro models of BC. We optimized the spheroid growth using various BC cell lines [human (J82), rat (NBT-II), and mouse (MB49) BC cells]. Similarly, tumoroids from rat (BBN-induced bladder tumors) and transgenic mice (UPII-SV40T) bladder tumors were developed. These spheroids and tumoroids were treated with various concentrations (0 - 50 μM range) of STAT3 inhibitors and evaluated for their viability [Calcein AM (CA) and EtBr staining], ATP production (CellTiter-Glo™ 3D), and ROS production (MitoSOXTM). Effect of drug treatment on biomarkers of cell proliferation, apoptosis, stemness, STAT3 signaling, and immune modulation was determined using western blotting and immunofluorescence. Treatment with TTI-101 (0 - 50 μM or SH5-07 (0 - 50 μM) for 144 hrs resulted in significant reduction in the spheroids size (39-45% smaller Vs untreated; p&amp;lt;0.0001), along with decreased ATP levels (20%-40%, p&amp;lt;0.05). MitoSOXTM staining suggested that STAT3 inhibitors treatment increased ROS production in BC cells. CA and EtBr staining revealed that TTI-101 and SH5-07 treatment resulted in increased cell death in BC spheroids compared to control. Decreased spheroids and organoids size also correlated with increased apoptotic marker (cleaved caspase-3) along with decreased cyclin D1, PCNA, and pSTAT3 protein expression. Drug treated BC spheroids/tumoroids also showed reduction in CD44 (BC stemness and invasion marker) and induction of cGAS-STING pathway (cGAS, STING, TBK1, and IRF3) in comparison to the control. These findings indicate that STAT3 inhibitors, TTI-101 and SH5-07, could inhibit bladder cancer by suppressing STAT3 pathway activation and therefore warrant further study in vivo. (Supported by P30 CA225520 and Kerley-Cade Endowed Chair) Citation Format: Surya P. Singh, Gopal Pathuri, Adam Asch, Brian Cholewa, Robert Shoemaker, Chinthalapally V. Rao, Venkateshwar Madka. Effect of STAT3 inhibitors, TTI-101 and SH5-07, against bladder cancer in preclinical 3D tumor models. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5255.

Abstract 3073: Action of thyroid hormones in sorafenib treatment of thyroid cancer

Abstract Background: Thyroid carcinoma (TC) is the most common endocrine neoplasia. Its incidence has increased in the last 40 years worldwide. Sorafenib (Sor), a tyrosine kinase inhibitor (TKI), was approved for the treatment of TC, being hypothyroidism the most frequent consequence of Sor-induced endocrine dysfunction. We have described that thyroid hormones (TH) increase cell proliferation in TC cell lines. Thus, hormone replacement therapy to treat Sor-induced hypothyroidism could negatively affect its antitumor action. We have also shown that the selective inhibition of the TH membrane receptor, integrin αVβ3, diminishes proliferation. Objective: To study integrin αVβ3 inhibition to enhance Sor antineoplastic activity in TC and the molecular mechanisms involved. Experimental Procedures: 8505C human anaplastic TC cell viability was evaluated by MTS assay. Sor was the TKI used, and Cilengitide (Cile) was used to inhibit integrin αVβ3. Protein modulation was measured by Western blot. Apoptosis induction was determined by APC-Annexin V Propidium iodide staining, followed by flow cytometry. In silico analysis on several databases were performed using CBioPortal (https://cbioportal.org/; TCGA, Cell 2014, n=504) and R2 (http://r2.amc.nl; GSE126729, n=28). Results: We first performed a bioinformatic analysis by cBioPortal on the PanCancer Atlas data and found that thyroid tumors are those with the highest integrin αVβ3 subunits expression. We then studied the role of integrin αVβ3 in Sor inhibition of TH-induced proliferation in TC cells. Treatment of 8505C cells with integrin αVβ3 antagonist Cile inhibits TH-induced proliferation (p&amp;lt0.0001), confirming the participation of the integrin. As expected, Sor treatment decreases proliferation (p&amp;lt0.05) while Cile addition did not change the inhibition level. Also, we found that Sor and Cile treatment diminished PCNA and Cyclin D1 expression levels. Cells were preincubated with Sor and Cile and then treated or not with TH for 48h to analyze apoptosis. Sor treatment increases the number of apoptotic cells relative to untreated control (p&amp;lt0.0001) and the presence of TH reduces the rate of apoptosis (p&amp;lt0.01). We then studied Sor target genes expression by R2 on an anaplastic TC patients’ database. FGFR is highly expressed among Sor targets, and it is correlated with integrin αVβ3 expression. Thus, FGFR expression modulation was studied by Western blot on cells treated as previously described. Finally, we found that TH treatment increased FGFR expression, Sor diminishes expression and in presence of TH, FGFR expression was rescued. Interestingly, Cile addition resensitized cells to Sor in presence of TH. Conclusion: Our results establish that the effective dual Sor and Cile treatment can significantly drive tumor proliferation inhibition and apoptosis, and it could provide alternatives to the treatments currently used for this disease. Citation Format: Mateo N. Campos Haedo, Maria C. Diaz Flaque, Helena A. Sterle, Johanna A. Diaz Albuja, Maria F. Cayrol, Maria M. Debernardi, Marina Perona, Guillermo J. Juvenal, Graciela A. Cremaschi, Cinthia Rosemblit. Action of thyroid hormones in sorafenib treatment of thyroid cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3073.

Identification of fish spermatogenic cells through high-throughput immunofluorescence against testis with an antibody set.

Image-based identification and quantification of different types of spermatogenic cells is of great importance, not only for reproductive studies but also for genetic breeding. Here, we have developed antibodies against spermatogenesis-related proteins in zebrafish (Danio rerio), including Ddx4, Piwil1, Sycp3, and Pcna, and a high-throughput method for immunofluorescence analysis of zebrafish testicular sections. By immunofluorescence analysis of zebrafish testes, our results demonstrate that the expression of Ddx4 decreases progressively during spermatogenesis, Piwil1 is strongly expressed in type A spermatogonia and moderately expressed in type B spermatogonia, and Sycp3 has distinct expression patterns in different subtypes of spermatocytes. Additionally, we observed polar expression of Sycp3 and Pcna in primary spermatocytes at the leptotene stage. By a triple staining of Ddx4, Sycp3, and Pcna, different types/subtypes of spermatogenic cells were easily characterized. We further demonstrated the practicality of our antibodies in other fish species, including Chinese rare minnow (Gobiocypris rarus), common carp (Cyprinus carpio), blunt snout bream (Megalobrama amblycephala), rice field eel (Monopterus albus) and grass carp (Ctenopharyngodon idella). Finally, we proposed an integrated criterion for identifying different types/subtypes of spermatogenic cells in zebrafish and other fishes using this high-throughput immunofluorescence approach based on these antibodies. Therefore, our study provides a simple, practical, and efficient tool for the study of spermatogenesis in fish species.

Platelet-rich Fibrin Promotes the Proliferation and Osteo-/odontoblastic Differentiation of Human Dental Pulp Stem Cells.

Pulp regeneration is a promising strategy that promotes the continued development of young permanent teeth with immature apical foramen. Platelet-rich fibrin (PRF) was found to stimulate the proliferation and differentiation of osteoblasts, but its effects on osteoblast/odontoblast differentiation of human dental pulp stem cells (hDPSCs) are unknown. The hDPSCs were isolated and identified using known surface markers by flow cytometry. The CCK-8 assay and the expression of Ki67 and PCNA were used to examine hDPSC proliferation. After 7 days of culture in an osteo-/odontoblastic induction medium with various concentrations of liquid PRF (0, 10% and 20%), the early stage of osteogenesis-intracellular alkaline phosphatase (ALP) was checked. After 21 days of culture, matrix mineralization was checked using Alizarin Red S and quantified. The mRNA and protein levels of osteo-/odontoblastic genes, including RUNX2, DSPP, DMP1 and BSP, were measured by qRT-PCR. The notch signal was checked by Western blot to analyze three key proteins (Notch 1, Jagged 1 and Hes 1). PRF-treated groups showed higher expression of Ki-67 and PCNA, higher ALP activity, and the higher dose showed a stronger induction. PRF promoted osteo-/odontoblastic differentiation of hDPSCs indicated by elevated protein levels and mRNA levels of the expression of osteo-/odontoblastic markers. The three key proteins in Notch signaling showed an increase compared with the control group and increased as the PRF concentration increased. PRF can promote the proliferation and osteo-/odontoblastic differentiation of hDPSC, which may be through the Notch signaling pathway.

Toxicity mechanism of peri-implantation pesticide beta-cypermethrin exposure on endometrial remodeling in early pregnant mice

Beta-cypermethrin (β-CYP) is a universally used pyrethroid pesticide with adverse effects on human health. β-CYP may impair endometrial remodeling in mice; however, the mechanism remains largely unknown. Endometrial remodeling plays a vital role in embryonic development and the maintenance of pregnancy. Therefore, we explored the mechanism by which peri-implantation β-CYP administration reduces uterine remodeling in pregnant mice. The C57BL/6 J pregnant mice were administered a dose of 20 mg/kg.bw. d β-CYP via oral gavage once daily from day 1 of gestation (GD1) to GD7. Molecular markers of endometrial remodeling, stromal cell proliferation, cell cycle regulation, and the PI3K/Akt/mTOR signaling pathway were evaluated in the decidual tissue of the uterus on GD7. An in vivo pseudopregnancy mouse model, a pregnant mouse model treated with an mTOR activator and an mTOR inhibitor and an in vitro decidualization model of mouse endometrial stromal cells were used to confirm β-CYP-induced defective endometrial remodeling and the key molecules expression of PI3K/Akt/mTOR signaling pathway. The results showed that β-CYP decreased the expression of the endometrial remodeling markers MMP9 and LIF in the uterine decidua. Peri-implantation β-CYP treatment markedly downregulated the expression of endometrial proliferation markers PCNA and Ki67 and decreased decidua thickness. Correspondingly, peri-implantation β-CYP exposure upregulated the expression of FOXO1, P57 and p-4E-BP1 in the decidua. Further experiments showed β-CYP significantly inhibited key molecules in the PI3K/Akt/mTOR pathway: PI3K, p-Akt/Akt, p-mTOR, and p-P70S6K in the uterine decidua. Additional experiments showed that aberrant endometrial remodeling induced by β-CYP was aggravated by rapamycin (an mTOR inhibitor) and partially reversed by MHY1485 (an mTOR agonist). In summary, our results indicated that a reduction in the PI3K/Akt/mTOR pathway may enhance defective endometrial remodeling by downregulating the proliferation and differentiation of endometrial stromal cells in early pregnant mice exposed to β-CYP. Our study elucidates the mechanism of defective endometrial remodeling induced by peri-implantation β-CYP exposure.

Double Immunohistochemical Analysis of PCNA and FAK Expression in Selected Odontogenic Lesions

Double Immunohistochemical Analysis of PCNA and FAK Expression in Selected Odontogenic Lesions

MP20-06 ATORVASTATIN WITH DEGARELIX MODULATES TUMOR PROLIFERATION AND THE PROSTATE TUMOR IMMUNE ENVIRONMENT IN A Myc-CaP/AS MURINE MODEL

You have accessJournal of UrologyCME1 Apr 2023MP20-06 ATORVASTATIN WITH DEGARELIX MODULATES TUMOR PROLIFERATION AND THE PROSTATE TUMOR IMMUNE ENVIRONMENT IN A Myc-CaP/AS MURINE MODEL Ashanda Esdaille, Bing Yang, Tanaya Purohit, Kayla Bahr, Ali Ghasemzadeh, Dagna Sheerar, Douglas McNeel, and David Jarrard Ashanda EsdailleAshanda Esdaille More articles by this author , Bing YangBing Yang More articles by this author , Tanaya PurohitTanaya Purohit More articles by this author , Kayla BahrKayla Bahr More articles by this author , Ali GhasemzadehAli Ghasemzadeh More articles by this author , Dagna SheerarDagna Sheerar More articles by this author , Douglas McNeelDouglas McNeel More articles by this author , and David JarrardDavid Jarrard More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003245.06AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Androgen deprivation therapy (ADT) exerts immunomodulatory effects and induces a unique phenotypic response that accumulating data suggests may be improved with the addition of standard medications. Prior studies have shown that ADT and metformin or statins improves oncologic outcomes versus ADT alone. We hypothesize that these outcomes may be due to the effects of statins and ADT in immune and prostate tumor modulation. We aim to investigate whether Atorvastatin±ADT alters tumor growth, proliferation, apoptosis and the immune infiltrate within myc-CaP/AS murine models. METHODS: FVB/NJ, immunocompetent mice were injected subcutaneously with 1x106 Myc-CaP/AS cells. Treatment was initiated at an average tumor volume of 400mm3. Groups were randomized into short term (5 days post ADT) and long term (10 days post) for a total of 8 mice per group per time period. Mice were injected i.p. with: 1) 5µg/g Atorvastatin, q2 days x 3 doses OR q2 days x 5 doses AND/OR 2) Degarelix 25µg/g x 1 dose OR 3) PBS i.p. for controls. Immunohistochemical staining, followed by flow cytometry, was performed using single cell suspensions of harvested tumors. Western blots were performed to assess expression of PCNA and CC3. RESULTS: ADT + Atorvastatin treated tumors had the greatest decrease in volume (Figure 1). Tumor volume differences were present between post-treatment Atorvastatin vs control (p=0.029) and post-treatment Degarelix +Atorvastatin vs control (p=0.018). On flow cytometric analysis, Atorvastatin-treated tumors had the greatest CD8 T-cell expansion over time. MDSC expression decreased significantly over time for ADT and ADT + Atorvastatin-treated tumors. Early ADT and ADT + Atorvastatin decreased tumor proliferation and late Atorvastatin increased apoptosis in myc-CaP/AS tumors. CONCLUSIONS: Atorvastatin+ADT combination therapy decreased tumor volume and tumor proliferation when compared to ADT or statin drug alone. Atorvastatin alone increased CD8 T-cell presence, important for cell death, and in combination with ADT, decreased MDSC expression, which impedes cancer cell death. Demonstrating synergy between androgen deprivation and immunomodulatory agents has to potential to shift the treatment paradigm in patients with high-risk prostate cancer. Source of Funding: AUA/UCF Grant# 017-822959 © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e277 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Ashanda Esdaille More articles by this author Bing Yang More articles by this author Tanaya Purohit More articles by this author Kayla Bahr More articles by this author Ali Ghasemzadeh More articles by this author Dagna Sheerar More articles by this author Douglas McNeel More articles by this author David Jarrard More articles by this author Expand All Advertisement PDF downloadLoading ...

D-Aspartate Depletion Perturbs Steroidogenesis and Spermatogenesis in Mice.

High levels of free D-aspartate (D-Asp) are present in vertebrate testis during post-natal development, coinciding with the onset of testosterone production, which suggests that this atypical amino acid might participate in the regulation of hormone biosynthesis. To elucidate the unknown role of D-Asp on testicular function, we investigated steroidogenesis and spermatogenesis in a one-month-old knockin mouse model with the constitutive depletion of D-Asp levels due to the targeted overexpression of D-aspartate oxidase (DDO), which catalyzes the deaminative oxidation of D-Asp to generate the corresponding α-keto acid, oxaloacetate, hydrogen peroxide, and ammonium ions. In the Ddo knockin mice, we found a dramatic reduction in testicular D-Asp levels, accompanied by a significant decrease in the serum testosterone levels and testicular 17β-HSD, the enzyme involved in testosterone biosynthesis. Additionally, in the testes of these Ddo knockin mice, the expression of PCNA and SYCP3 proteins decreased, suggesting alterations in spermatogenesis-related processes, as well as an increase in the cytosolic cytochrome c protein levels and TUNEL-positive cell number, which indicate an increase in apoptosis. To further investigate the histological and morphometric testicular alterations in Ddo knockin mice, we analyzed the expression and localization of prolyl endopeptidase (PREP) and disheveled-associated activator of morphogenesis 1 (DAAM1), two proteins involved in cytoskeletal organization. Our results showed that the testicular levels of DAAM1 and PREP in Ddo knockin mice were different from those in wild-type animals, suggesting that the deficiency of D-Asp is associated with overall cytoskeletal disorganization. Our findings confirmed that physiological D-Asp influences testosterone biosynthesis and plays a crucial role in germ cell proliferation and differentiation, which are required for successful reproduction.

Combination of vitamin D and probiotics inhibits chemically induced colorectal carcinogenesis in Wistar rats

The modulation of inflammatory elements, cell differentiation and proliferation by vitamin D and the role of probiotics in the intestinal microbiota and immunogenic response have sparked interest in the application of both in chemotherapeutics and chemoprevention of colorectal tumors. AimsThe present study aimed to investigate the effects of isolated and/or combined treatment of vitamin D3 and probiotics on colorectal carcinogenesis. Main methodsPre-neoplastic lesions were induced with 1,2-dimethylhydrazine in the colon of Wistar rats, which were treated with probiotics and/or vitamin D in three different approaches (simultaneous, pre-, and post-treatment). We investigated the frequency of aberrant crypt foci (ACF) and aberrant crypt (AC) in the distal colon, fecal microbiome composition, gene and protein expression through immunohistochemical and RT-PCR assays, and general toxicity through water consumption and weight gain monitoring. Key findingsResults confirm the systemic safety of treatments, and show a protective effect of vitamin D and probiotics in all approaches studied, as well as in combined treatments, with predominance of different bacterial phyla compared to controls. Treated groups show different levels of Nrf2, GST, COX2, iNOS, β-catenin and PCNA expression. SignificanceThese experimental conditions explore the combination of vitamin D and probiotics supplementation at low doses over pathways involved in distinct stages of colorectal carcinogenesis, with results supporting its application in prevention and long-term strategies.

Lactational exposure to Deoxynivalenol causes mammary gland injury via inducing inflammatory response and impairing blood-milk barrier integrity in mice

Lactation is a unique physiological process to produce and secrete milk. Deoxynivalenol (DON) exposure during lactation has been demonstrated to affect adversely the growth development of offspring. However, the effects and potential mechanism of DON on maternal mammary glands remain largely unknown. In this study, we found the length and area of mammary glands were significantly reduced after DON exposure on lactation day (LD) 7 and LD 21. RNA-seq analysis results showed that the differentially expressed genes (DEGs) were significantly enriched in acute inflammatory response and HIF-1 signaling pathway, which led to an increase of myeloperoxidase activity and inflammatory cytokines. Furthermore, lactational DON exposure increased blood-milk barrier permeability by reducing the expression of ZO-1 and Occludin, promoted cell apoptosis by upregulating the expression of Bax and cleaved Caspase-3 and downregulating the expression of Bcl-2 and PCNA. Additionally, lactational DON exposure significantly decreased serum concentration of prolactin, estrogen, progesterone. All these alterations eventually resulted in a decrease of β-casein expression on LD 7 and LD 21. In summary, our findings indicated that lactational exposure to DON caused lactation-related hormone disorder and mammary gland injury induced by inflammatory response and blood-milk barrier integrity impairment, ultimately resulting in lower production of β-casein.

Platelet Lysate Therapy Attenuates Hypoxia Induced Apoptosis in Human Uroepithelial SV-HUC-1 Cells through Regulating the Oxidative Stress and Mitochondrial-Mediated Intrinsic Apoptotic Pathway.

(1) Background: Ischemia/hypoxia plays an important role in interstitial cystitis/bladder pain syndrome (IC/BPS). Platelet-rich plasma (PRP) has been shown to relieve symptoms of IC/BPS by regulating new inflammatory processes and promoting tissue repair. However, the mechanism of action of PRP on the IC/BPS bladder remains unclear. We hypothesize that PRP might protect the urothelium during ischemia/hypoxia by decreasing apoptosis. (2) Methods: SV-HUC-1 cells were cultured under hypoxia for 3 h and treated with or without 2% PLTGold® human platelet lysate (PL). Cell viability assays using trypan blue cell counts were examined. Molecules involved in the mitochondrial-mediated intrinsic apoptosis pathway, HIF1α, and PCNA were assessed by Western blot analysis. The detection of apoptotic cells and CM-H2DCFDA, an indicator of reactive oxygen species (ROS) in cells, was analyzed by flow cytometry. (3) Results: After 3 h of hypoxia, the viability of SV-HUC-1 cells and expression of PCNA were significantly decreased, and the expression of ROS, HIF1α, Bax, cytochrome c, caspase 3, and early apoptosis rate were significantly increased, all of which were attenuated by PL treatment. The addition of the antioxidant N-acetyl-L-cysteine (NAC) suppressed the levels of ROS induced by hypoxia, leading to inhibition of late apoptosis. (4) Conclusions: PL treatment could potentially protect the urothelium from apoptosis during ischemia/hypoxia by a mechanism that modulates the expression of HIF1α, the mitochondria-mediated intrinsic apoptotic pathway, and reduces ROS.

Nanosecond pulsed electric field ablates rabbit VX2 liver tumors in a non-thermal manner.

Liver tumor remains an important cause of cancer-related death. Nanosecond pulsed electric fields (nsPEFs) are advantageous in the treatment of melanoma and pancreatic cancer, but their therapeutic application on liver tumors need to be further studied. Hep3B cells were treated with nsPEFs. The biological behaviors of cells were detected by Cell Counting Kit-8, 5-ethynyl-20-deoxyuridine, and transmission electron microscopy (TEM) assays. In vivo, rabbit VX2 liver tumor models were ablated by ultrasound-guided nsPEFs and radiofrequency ablation (RFA). Contrast-enhanced ultrasound (CEUS) was used to evaluate the ablation effect. HE staining and Masson staining were used to evaluate the tissue morphology after ablation. Immunohistochemistry was performed to determine the expression of Ki67, proliferating cell nuclear antigen, and α-smooth muscle actin at different time points after ablation. The cell viability of Hep3B cells was continuously lower than that of the control group within 3 days after pulse treatment. The proliferation of Hep3B cells was significantly affected by nsPEFs. TEM showed that Hep3B cells underwent significant morphological changes after pulse treatment. In vivo, CEUS imaging showed that nsPEFs could completely ablate model rabbit VX2 liver tumors. After nsPEFs ablation, the area of tumor fibrosis and the expression of Ki67, proliferating cell nuclear antigen, and α-smooth muscle actin were decreased. However, after RFA, rabbit VX2 liver tumor tissue showed complete necrosis, but the expression of PCNA and α-smooth muscle actin did not decrease compared to the tumor group. nsPEFs can induce Hep3B cells apoptosis and ablate rabbit VX2 liver tumors in a non-thermal manner versus RFA. The ultrasound contrast agent can monitor immediate effect of nsPEF ablation. This study provides a basis for the clinical study of nsPEFs ablation of liver cancer.

Circ_0000595 knockdown alleviates CoCl2-mediated effects in VSMCs by regulating the miR-582-3p/ADAM10 axis.

Thoracic aortic aneurysm (TAA) is a serious vascular disease causing the death of elder people. Accumulating studies have reported that circular RNAs (circRNAs) are implicated in the regulation of aortic aneurysms. However, the role of circ_0000595 in the progression of TAA is still unclear. Quantitative real-time PCR (qRT-PCR) and western blotting were implemented to assess circ_0000595, microRNA (miR)-582-3p, guanine nucleotide-binding protein alpha subunit (ADAM10), PCNA, Bax, and Bcl-2 expression. The proliferation of vascular smooth muscle cells was determined using cell counting kit 8 (CCK-8) and 5-ethynyl-2-deoxyuridine (EdU). Cell apoptosis was measured using flow cytometry, and caspase-3 activity was analyzed using a commercial kit. After bioinformatics analysis, the interaction between miR-582-3p and circ_0000595 or ADAM10 was validated using a dual-luciferase reporter and RNA immunoprecipitation. As compared with controls, TAA tissues and CoCl2-induced VSMCs displayed high expression of circ_0000595 and ADAM10, and low expression of miR-582-3p. CoCl2 treatment evidently suppressed VSMC proliferation and promoted VSMCs apoptosis, and these impacts were reverted by circ_0000595 knockdown. Circ_0000595 acted as a molecular sponge for miR-582-3p, and circ_0000595 silencing-mediated influences in CoCl2-induced VSMCs were overturned by miR-582-3p inhibitor. ADAM10 was confirmed as a target gene of miR-582-3p, and miR-582-3p overexpression-induced influence was almost restored by overexpressed ADAM10 in CoCl2-induced VSMCs. Besides, circ_0000595 contributed to ADAM10 protein expression by sponging miR-582-3p. Our data verified that circ_0000595 silencing might attenuate CoCl2-mediated impacts in VSMCs by regulating the miR-582-3p/ADAM10 axis, providing new potential roads for treating TAA.

Effect of Red Bean Extract (Phaseolus vulgaris L. Sp) on Interleukin-1, Inhibin Β and PCNA Expression in Systemic Lupus Erythematosus Mice Model

Introduction: Systemic Lupus Erythematosus (SLE) is a complex autoimmune disease characterized by multiple organ involvement. Chronic inflammation due to oxidative stress and autoimmune causes damage to related organs, one of which is the ovary which leads to Premature Ovarian Failure (POF) . Red bean (Phaseolus vulgaris L. Sp.) is an anti-inflammatory and antioxidant agent. This study aims to examine the effect of red beans on IL-1, Inhibin B, and PCNA. Methods: This is a true experimental study with a post-test-only controlled group design in vivo. The sample in this study were female Balb/c mice treated with red bean extract (Phaseolus vulgaris L. sp.) at doses of 50 mg/KgBW (P1), 75 mg/KgBW (P2), and 100 mg/KgBW (P3). IL-1, Inhibin B, and PCNA levels in each group were statistically analyzed using the SPSS 16 for Windows software program. Results: Red bean extract (Phaseolus vulgaris L. Sp.) administration at various doses was statistically proven to significantly reduce IL-1 levels (p&lt;0,05) and increase Inhibin levels in all groups (p&lt;0,05). The highest average Inhibin B levels were found in the 100 mg/KgBW dose group (P3), significant increase in PCNA expression in all groups given various doses of red bean extract (p&lt;0,05). Conclusion: Administration of red bean extract (Phaseoulus vulgaris L. Sp.) can reduce levels of Interleukin-1, increase levels of Inhibin B and PCNA expression in SLE mice.

Melatonin alleviates oxidative damage in mouse spermatogenesis and sperm quality parameters induced by exposure to Bisphenol A

Bisphenol A (BPA) exposure is known to cause damage to male fertility. Conversely, melatonin (MLT) has been shown to offer protection against reproductive damage by acting as an antioxidant. Exploration into the molecular mechanisms behind how MLT alleviates BPA-induced male fertility damage remains unclear. In this study, adult male mice were divided into four groups of control, BPA exposure (intragastrically, 50 mg/kg/day), MLT treatment (intraperitoneal injection, 10 mg/kg/day) and combined BPA+MLT treatment group for comparative analysis after five weeks of treatment. BPA exposure led to male mice subfertility characterized by poor sperm quality of decreased sperm counts and motility, and reduced pregnancy rate of female mice in in vivo and blastocyst formation in vitro experiments. In BPA exposed mice, germ cell proliferation was impaired, showing a decrease in PCNA expression. BPA exposure also induced a decrease in the expression of antioxidant molecules Cat, Prdx6, and Sod1, and up-regulated BAX expression. These expression changes resulted in increased germ cell apoptosis detected by TUNEL experiments. MLT treatment significantly maintained male mice sperm quality and fertility adversely affected by BPA through modulating the abovementioned responses. Sperm proteomics identified 118 BPA-regulated proteins whose expression was changed by MLT treatment, including 37 down-regulated proteins related to proteolysis activity, the up-regulated proteins were mainly involved in lipid metabolism. Conclusively, BPA exposure impaired male fertility by negatively affecting germ cell proliferation and sperm quality. MLT treatment could alleviate the side effects of BPA by regulating antioxidant molecules, inhibiting germ cell apoptosis, and maintaining male fertility. This study will provide more information for further exploration into the molecular mechanisms of BPA and MLT in male reproduction.

The ameliorative effect of CangFu Daotan Decoction on polycystic ovary syndrome of rodent model is associated with m6A methylation and Wnt/β-catenin pathway

Objective: This study investigates the effects of CangFu Daotan Decoction (CDD) on m6A methylation and Wnt/β-catenin pathway in rats with polycystic ovary syndrome (PCOS).Methods: The PCOS rat model was established by letrozole gavage. The rats were fed high-fat chow, and their body weight and blood glucose were recorded. The expressions of follicle-stimulating hormone(FSH), luteinizing hormone(LH), and testosterone(T) were quantified by ELISA. Chemical components in CDD were analyzed using UPLC-Q/TOF-MS. Based on network pharmacology methods, related targets of CDD on PCOS were screened. An enrichment analysis according to Tokyo Encyclopedia of Genes and Genomes (KEGG) was conducted to predict the potential signaling pathway of CDD in PCOS. The expressions of Wnt-1, β-Catenin, GSK-3β, C-MYC, Beclin1, LC3II, Bax, and PCNA were detected by western blotting. The expressions of Mettl3, Mettl14, Fto, Alkbh5, Ythdf1, and Ythdf2 were monitored by RT-PCR. The expressions of Mettl3, Fto, and Ythdf1 were detected by western blotting.Results: Letrozole and a high-fat diet induced ovarian dysfunction in rats, which was attenuated by CDD. CDD decreased blood glucose, LH, and T concentrations and increased FSH expression in PCOS. After removing duplicates, a total of 71 compounds were identified by UHPLC-Q/TOF-MS, among which terpenoids and flavonoids account for the main proportion. The clustering analysis showed that the active site of CDD might be in the Wnt/β-catenin pathway. CDD decreased the expressions of Wnt-1, β-Catenin, GSK-3β, C-MYC, Beclin1, LC3II, and Bax and increased PCNA expression in the ovarian tissue of PCOS rats. CDD decreased the m6A gene expressions of Mettl3, Mettl14, Fto, Alkbh5, Ythdf1, and Ythdf2 in peripheral blood and ovarian tissue of PCOS rats. CDD reduced the m6A proteins expressions of Mettl3, Fto, and Ythdf1 in the ovarian tissue of PCOS rats.Conclusion: CDD can regulate m6A modification and inhibit the Wnt/β-catenin signaling pathway in PCOS rats, thereby reducing body weight, lowering blood glucose levels, improving sex hormone disorders, and decreasing autophagy and apoptosis in ovarian tissue to promote the recovery of ovarian morphology.