Proliferating Cell Nuclear Antigen Expression Research Articles

Genetic Disruption of Guanylyl Cyclase/Natriuretic Peptide Receptor-A Triggers Differential Cardiac Fibrosis and Disorders in Male and Female Mutant Mice: Role of TGF-β1/SMAD Signaling Pathway.

The global targeted disruption of the natriuretic peptide receptor-A (NPRA) gene (Npr1) in mice provokes hypertension and cardiovascular dysfunction. The objective of this study was to determine the mechanisms regulating the development of cardiac fibrosis and dysfunction in Npr1 mutant mice. Npr1 knockout (Npr1−/−, 0-copy), heterozygous (Npr1+/−, 1-copy), and wild-type (Npr1+/+, 2-copy) mice were treated with the transforming growth factor (TGF)-β1 receptor (TGF-β1R) antagonist GW788388 (2 µg/g body weight/day; ip) for 28 days. Hearts were isolated and used for real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR), Western blot, and immunohistochemical analyses. The Npr1−/− (0-copy) mice showed a 6-fold induction of cardiac fibrosis and dysfunction with markedly induced expressions of collagen-1α (3.8-fold), monocyte chemoattractant protein (3.7-fold), connective tissue growth factor (CTGF, 5.3-fold), α-smooth muscle actin (α-SMA, 6.1-fold), TGF-βRI (4.3-fold), TGF-βRII (4.7-fold), and phosphorylated small mothers against decapentaplegic (pSMAD) proteins, including pSMAD-2 (3.2-fold) and pSMAD-3 (3.7-fold), compared with wild-type mice. The expressions of phosphorylated extracellular-regulated kinase ERK1/2 (pERK1/2), matrix metalloproteinases-2, -9, (MMP-2, -9), and proliferating cell nuclear antigen (PCNA) were also significantly upregulated in Npr1 0-copy mice. The treatment of mutant mice with GW788388 significantly blocked the expression of fibrotic markers, SMAD proteins, MMPs, and PCNA compared with the vehicle-treated control mice. The treatment with GW788388 significantly prevented cardiac dysfunctions in a sex-dependent manner in Npr1 0-copy and 1-copy mutant mice. The results suggest that the development of cardiac fibrosis and dysfunction in mutant mice is predominantly regulated through the TGF-β1-mediated SMAD-dependent pathway.

Reproductive Development of Male Rats Exposed In Utero to Stress and/or Sertraline.

Despite increased prescription of sertraline during pregnancy, little is known about its action on reproductive development. Therefore, this study aimed to investigate the impact that stress, associated or not with sertraline, causes on the reproductive development of male rats. Pregnant Wistar rats were divided into 4 groups (n = 16/group): CO-received filtered water; SE-received 20 mg/kg sertraline; ST-submitted to restraint stress and received filtered water; SS-submitted to restraint stress and received sertraline. The treatment was carried out from gestational days (GDs) 13-20. The animals were euthanized on GD 20 (n = 8/group), postnatal day (PND) 45 (n = 8/group), and PND 110 (n = 8/group). The testes and epididymis were analyzed histologically, and immunohistochemistry was performed on the testes by proliferating cell nuclear antigen (PCNA) and the Wilms tumor protein (Wt1). Sperm quality was also analyzed on PND 110. The evolution of body weight, anogenital distance (AGD), and puberty installation day were also verified. Statistical analysis: 2-way ANOVA or Kruskal-Wallis test (p ≤ .05). Fetal testes presented a large number of acidophilic cells in the sertraline-exposed groups. The SS group also showed a decrease in the nuclear volume of Leydig cells. This same group showed low expression of PCNA and Wt1, decreased weight of the testes and epididymis, lower AGD, and delayed puberty installation. The adulthood groups exposed to sertraline presented alterations in sperm morphology and motility. The results demonstrated that prenatal exposure to sertraline compromises the development of the rat reproductive system.

E2F1 induced neuroblastoma cell migration and invasion via activation of CENPE/FOXM1 signaling pathway

Background: Neuroblastoma (NB) is a common malignancy occurring in infants and young children. Centrosome-associated protein E (CENPE) is a kinetochore-related motor protein highly expressed in NB, with the mechanism largely unknown. This study is committed to investigating the role and mechanism of CENPE in NB.Method: Short hairpin RNAs targeting CENPE and E2F transcription factor 1 (shCENPE and shE2F1) and CENPE overexpression plasmid were transfected into IMR-32 and SK-N-SH cells. The mRNA expressions of CENPE, N-Cadherin, Vimentin, and proliferating cell nuclear antigen (PCNA) in NB cells were detected by qRT-PCR. The viability, migration, and invasion of cells were tested through cell function experiments. Western blot was applied to detect the protein levels of N-Cadherin, Vimentin, PCNA, CENPE and Forkhead box M1 (FOXM1). The relationship between CENPE and E2F1 was verified by dual-luciferase reporter assay, while the interaction between FOXM1 and CENPE in NB cells was analyzed by rescue experiments.Results: CENPE expression was upregulated in NB cells from metastatic sites. Silencing of CENPE suppressed the NB cell viability, migration, and invasion; and decreased N-Cadherin, Vimentin and PCNA expressions, while overexpressed CENPE did oppositely. E2F1 positively targeted CENPE and CENPE partly reversed the effects of shE2F1 on repressing NB cell viability, migration, invasion and the activation of CENPE/FOXM1 signaling pathway. In addition, silenced FOXM1 partly offset the effects of CENPE on promoting NB cell migration and invasion.Conclusion: E2F1 induces NB cell migration and invasion via activating CENPE/FOXM1 pathway.

Expression of epithelial–mesenchymal transition-associated proteins and proliferating cell nuclear antigen in dihydropyridine-induced gingival overgrowth fibroblasts: A preliminary study

Background/purposeThe clinical features of dihydropyridine-induced gingival overgrowth (DIGO), including extracellular matrix accumulation and cell hyperplasia, are regulated by inflammatory factors (e.g., Interleukin-1β [IL-1β]) in combination with calcium channel blockers (e.g., nifedipine [Nif]). We speculated that IL-1β and Nif (IL-1β/Nif) may be the main factor modulating the proliferative potential and turnover of fibroblasts in DIGO. Materials and methodsWe cultured four DIGO fibroblast strains and analysed the possible effects of IL-1β/Nif treatments on epithelial–mesenchymal transition (EMT)-associated proteins. We developed short hairpin ribonucleic acids (shRNAs) and used them to explore the role of IL-1β/Nif in regulating proliferating cell nuclear antigen (PCNA) levels in DIGO tissues. ResultsOur results revealed that compared with control cells, DIGO cells stimulated with IL-1β/Nif had higher levels of the EMT-associated proteins Snail, Slug, and Twist. Moreover, both drugs enhanced androgen receptor (AR), Slug, and PCNA expression. ConclusionTaken together, our data indicate that proinflammatory cytokines in combination with calcium channel blockers can regulate the expression of EMT-associated proteins and increase the proliferative potential of DIGO fibroblasts.

Effects of probenecid on the migration and proliferation ability of platelet derived growth factor-BB induced pulmonary artery smooth muscle cells in rats

To investigate whether probenecid (PROB) could improve the proliferation and migration ability of rats' pulmonary artery smooth muscle cells induced by platelet-derived growth factor-BB (PDGF-BB). Primary pulmonary artery smooth muscle cells (PASMCs) of SD rats were cultured in vitro, and were randomly divided into control group (CON group), PDGF-BB group (10 ng/ml PDGF-BB treatment for 24 h) and PDGF-BB+PROB group (10 ng/ml PDGF-BB and 200 μmol/L PROB treatment for 24 h, PROB is a specific blocker of pannexin-1). CCK-8 method was used to select the suitable intervention concentrations of PROB and PDGF-BB, and to detect the proliferation of PASMCs in each group. The migration ability of PASMCs was detected by TranswellTM assay and cell scratch test. Immunofluorescence cytochemistry and Western blot were used to detect the protein expressions and distribution of osteopontin (OPN) and proliferating cell nuclear antigen (PCNA) in PASMCs. Compared with CON group, the migration and proliferation ability of PASMCs in PDGF-BB group were enhanced (P＜0.05). After treated with PROB, the migration and proliferation ability of PASMCs in PDGF-BB+PROB group were decreased significantly (P＜0.05). Compared with CON group, the expression and protein levels of OPN and PCNA in PDGF-BB group were increased significantly (P＜0.05), while the expression and protein levels of OPN and PCNA in PDGF-BB+PROB were decreased significantly (P＜0.05). Probenecid inhibits the migration and proliferation of PDGF-BB-induced PASMCs by blocking Pannexin-1.

Eriochloa villosa Alleviates Progression of Benign Prostatic Hyperplasia in vitro and in vivo.

IntroductionBenign prostatic hyperplasia (BPH) is a non-neoplastic proliferative disease of the prostate. Eriochloa villosa (EV) reportedly possesses various pharmacological activities, including anti-lipase activity and modulation of various antioxidative enzymes. In this study, we investigate the therapeutic potential of EV against BPH in a testosterone-induced BPH rat model.MethodsRats were subjected to a daily subcutaneous injection of testosterone (3 mg kg−1) for 4 weeks to induce BPH. Along with testosterone, rats in the treatment group were administered finasteride (10 mg kg−1) or EV (150 mg kg−1) via oral gavage. Prostatic cancer (LNCaP) cell line was used to examine the effect of EV.ResultsFinasteride and EV significantly decrease the relative prostate weight, serum levels of dihydrotestosterone and testosterone, and prostate epithelial thickness. Testosterone injection induced prostatic hyperplasia and proliferating cell nuclear antigen expression; however, EV treatment significantly attenuated these effects. Moreover, finasteride- and EV-treated rats exhibit an increase in the number of TUNEL-positive cells and reduced Bcl-2 expression in the prostate tissues compared with the testosterone-treated animals. Furthermore, EV suppresses inflammatory cytokines, including interleukin (IL)-6 and IL-8, in the prostate tissues. Meanwhile, the expression of inflammatory mediator cyclooxygenase-2 is consistently upregulated in testosterone-treated rats, whereas EV treatment significantly reverses this effect. Notably, EV treatment suppresses malondialdehyde (MDA) levels and upregulates testosterone-induced catalase (CAT) expression. In addition, EV suppresses expression of androgen receptor (AR) and prostate-specific antigen (PSA) induced by testosterone in LNCaP cells.ConclusionThe present study results suggest that EV regulates prostatic proliferation, apoptosis, response to inflammation, and oxidative stress in the BPH rat model, and may, therefore, serve as a useful therapeutic agent for BPH.

Therapeutic Versus Preventative Use of Ginkgo biloba Extract (EGb 761) against Indomethacin-Induced Gastric Ulcer in Mice

The main bioactive constituents in the standardized Ginkgo biloba leaf extract (EGb 761) are the terpene lactones and flavonoid glycosides. EGb 761’s antioxidant and anti-inflammatory properties have previously been demonstrated. Indomethacin-induced gastric ulcers have a multifactorial etiology and represent a major restriction to its therapeutic utility. The underlying ulcerogenic process involves oxidative and inflammatory biomolecular insults. This study was performed to explore the curative and preventative benefits of EGb 761 in experimentally-induced ulcers. To develop gastric ulcers in mice, indomethacin (40 mg/kg) was administered orally. EGb 761 (200 mg/kg) was given by gavage for 7 days before (preventative) and after (therapeutic) indomethacin administration. The histological alterations and macroscopic mucosal lesions were assessed. In gastric tissue homogenates, malondialdehyde (MDA), reduced glutathione (GSH), nitric oxide (NO), and inflammatory cytokines were measured. The expressions of cyclooxygenase-2 (COX-2), cytokines, and proliferating cell nuclear antigen (PCNA) in the stomach mucosa were also investigated. The ulcer index, histological alterations, gastric oxidants, and inflammatory biomarkers were all significantly increased by indomethacin. In stomach specimens, it increased COX-2 and PCNA expression. EGb 761 treatments, both prophylactic and therapeutic, resulted in significant reductions in ulcer lesions, nitrosative and oxidative damage, and inflammatory markers, along with the lowering of COX-2 and PCNA expressions. Furthermore, in the fight against stomach ulcers, EGb 761 treatment was found to be more efficient than prevention.

Effects of Granule Dendrobii on chronic atrophic gastritis induced by N-methyl-N'-nitro-N-nitrosoguanidine in rats.

BACKGROUND Dendrobium officinale is an herb of Traditional Chinese Medicine (TCM) commonly used for treating stomach diseases. One formula of Granule Dendrobii (GD) consists of Dendrobium officinale and American Ginseng (Radix Panacis quinquefolii), and is a potent TCM product in China. Whether treatment with GD can promote gastric acid secretion and alleviate gastric gland atrophy in chronic atrophic gastritis (CAG) requires verification.AIMTo determine the effect of GD treatment on CAG and its potential cellular mechanism.METHODSA CAG model was induced by feeding rats N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) for 12 wk. After oral administration of low, moderate, and high doses of GD in CAG rats for 8 wk, its effects on body weight, gastric mucosa histology, mucosal atrophy, intestinal metaplasia, immunohistochemical staining of proliferating cell nuclear antigen (PCNA) and B-cell lymphoma-2, and hemoglobin and red blood cells were examined.RESULTSThe body weights of MNNG-induced CAG model rats before treatment (143.5 ± 14.26 g) were significantly lower than that of healthy rats (220.2 ± 31.20 g, P < 0.01). At the 8th week of treatment, the body weights of rats in the low-, moderate-, and high-dose groups of GD (220.1 ± 36.62 g) were significantly higher than those in the untreated group (173.3 ± 28.09 g, all P < 0.01). The level of inflammation in gastric tissue of the high-dose group (1.68 ± 0.54) was significantly reduced (P < 0.01) compared with that of the untreated group (3.00 ± 0.00, P < 0.05). The number and thickness of gastric glands in the high-dose group (31.50 ± 6.07/mm, 306.4 ± 49.32 µm) were significantly higher than those in the untreated group (26.86 ± 6.41/mm, 244.3 ± 51.82 µm, respectively, P < 0.01 and P < 0.05), indicating improved atrophy of gastric mucosa. The areas of intestinal metaplasia were significantly lower in the high-dose group (1.74% ± 1.13%), medium-dose group (1.81% ± 0.66%) and low-dose group (2.36% ± 1.08%) than in the untreated group (3.91% ± 0.96%, all P < 0.01). The expression of PCNA in high-dose group was significantly reduced compared with that in untreated group (P < 0.01). Hemoglobin level in the high-dose group (145.3 ± 5.90 g/L), medium-dose group (139.3 ± 5.71 g/L) and low-dose group (137.5 ± 7.56 g/L) was markedly increased compared with the untreated group (132.1 ± 7.76 g/L; P < 0.01 or P < 0.05).CONCLUSIONTreatment with GD for 8 wk demonstrate that GD is effective in the treatment of CAG in the MNNG model by improving the histopathology of gastric mucosa, reversing gastric atrophy and intestinal metaplasia, and alleviating gastric inflammation.

Neratinib inhibits proliferation and promotes apoptosis of acute myeloid leukemia cells by activating autophagy-dependent ferroptosis.

Acute myeloid leukemia (AML) is a hematologic malignancy with increased lethality. We focused on elucidating the role of Neratinib, a tyrosine kinase inhibitor, in the progression of AML and identify the potential mechanisms. Upon the treatment of Neratinib, autophagy suppressor 3-methyladenine (3-MA) and ferroptosis stimulator Erastin, the viability and proliferation of HL-60 cells were evaluated by cell counting kit-8 and 5-Ethynyl-20-Deoxyuridinestaining assays. A flow cytometer was to observe cell cycle and apoptosis. Production of reactive oxygen species (ROS) was tested via 2,7-dichlorodihydrofluorescein diacetate assay. Additionally, malondialdehyde (MDA) content and Fe2+ activity were examined with commercial kits. LC3-II expression was examined by using immunofluoresencestaining. Western blot analysis ascertained the expression of proliferation, apoptosis, ferroptosis and autophagy-associated proteins. It was noted that Neratinib notably mitigated cell viability and proliferation, cut down Ki67 and proliferating cell nuclear antigen expression. Moreover, Neratinib hindered cell cycle at G0/G1 phase whereas exacerbated apoptosis. ROS, MDA and Fe2+ activities were elevated by Neratinib, coupled with the reduced glutathione peroxidase 4, ferritin heavy chain 1 expression and enhanced acyl-CoA synthetase long-chain family member 4expression. Furthermore, Neratinib promoted autophagy of HL-60 cells, evidenced by raised LC3-II, ATG5, Beclin1 expression and lessened p62 expression. Importantly, 3-MA eased the impacts of Neratinib on cell ferroptosis, proliferation and apoptosis, which were offset by further administration of Erastin. To conclude, Neratinib could suppress proliferation and promote apoptosis of HL-60 cells through autophagy-dependent ferroptosis.

Red propolis exhibits chemopreventive effect associated with antiproliferative and anti-inflammatory activities.

Red propolis is synthetized from exudates of Dalbergia ecastophyllum (L) Taub. and Symphonia globulifera L.f., presents isoflavones, guttiferone E, xanthochymol, and oblongifolin B and has anti-inflammatory, antioxidant, and antiproliferative activities. This study aimed to evaluate the antigenotoxic and anticarcinogenic potential of red propolis hydroalcoholic extract (RPHE) in rodents. The influence of RPHE in doxorubicin (DXR)-induced genotoxicity was investigated through the micronucleus test in Swiss mice. Blood samples were also collected to investigate oxidative stress, hepatotoxicity, and nephrotoxicity. Was investigated the influence of RPHE in 1,2-dimethylhydrazine (DMH)-induced aberrant crypt foci, as well as its influence in proliferating cell nuclear antigen (PCNA) and the cyclooxygenase-2 (COX-2) expression in colon of rats, by immunohistochemistry. The results showed that RPHE (48mg/kg) reduced DXR-induced genotoxicity. Animals treated with DXR showed significantly lower GSH serum levels in comparison to the negative control. RPHE treatments did not attenuated significantly the DXR-induced GSH depletion. No difference was observed in cytotoxicity parameters of mice hematopoietic tissues between the treatment groups, as well as the biochemical parameters of hepatotoxicity and nephrotoxicity. RPHE (12mg/kg) reduced the DMH-induced carcinogenicity and toxicity, as well as DMH-induced PCNA and COX-2 expression in colon tissue. Therefore, was observed that the RPHE has chemopreventive effect, associated to antiproliferative and anti-inflammatory activities.

Grain-Sized Moxibustion Heightens the AntiTumor Effect of Cyclophosphamide in Hepa1-6 Bearing Mice.

Objective The side effects of chemotherapy as a treatment of liver cancer cannot be ignored. Grain-sized moxibustion, a characteristic external therapy, has been shown to reduce the toxic and side effects of chemotherapy and regulate the immune function. The purpose of this study was to explore the synergistic antitumor activity of grain-sized moxibustion combined with cyclophosphamide (CTX). Methods A hepatoma 1–6 (Hepa1-6)-bearing mouse model was established by injecting mice with Hepa1-6 cancer cells. CTX and grain-sized moxibustion on Dazhui (DU14), Zusanli (ST36), and Sanyinjiao (SP6) were used for treatment, and mouse survival status, body weight, and tumor growth, weight, and volume were measured. White blood cells (WBCs) and bone marrow nucleated cells (BMNCs) were quantified. The spleens and livers of Hepa1-6-bearing mice were pathologically examined and scored. Serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels were measured with enzyme-linked immunosorbent assay (ELISA) kits, and protein and mRNA expression levels of Ki67 and proliferating cell nuclear antigen (PCNA) in tumor tissues were measured with immunohistochemistry and real-time quantitative polymerase chain reaction (RT-qPCR) techniques. Results Both grain-sized moxibustion and CTX could restrain the growth of Hepa1-6 tumors, reducing both tumor volume and weight; the combined treatment had a greater effect. Grain-sized moxibustion down-regulated the expression of proliferation genes Ki67 and PCNA, weakened the proliferation ability of Hepa1-6 tumor cells, inhibited tumor growth, and enhanced the antitumor effect of CTX. In addition, grain-sized moxibustion significantly improved the signs of CTX-induced toxicity (including weight loss, leukopenia, bone marrow suppression, and hepatotoxicity), down-regulated serum AST and ALT levels, reduced spleen and liver inflammation, and improved liver and spleen indices. Conclusion Grain-sized moxibustion can synergize with CTX to enhance the antitumor effect of CTX and alleviate its toxic and side effects. It may be a promising adjuvant therapy to chemotherapy.

Yohimbine, an α2-Adrenoceptor Antagonist, Suppresses PDGF-BB-Stimulated Vascular Smooth Muscle Cell Proliferation by Downregulating the PLCγ1 Signaling Pathway.

Yohimbine (YOH) has antiproliferative effects against breast cancer and pancreatic cancer; however, its effects on vascular proliferative diseases such as atherosclerosis remain unknown. Accordingly, we investigated the inhibitory mechanisms of YOH in vascular smooth muscle cells (VSMCs) stimulated by platelet-derived growth factor (PDGF)-BB, a major mitogenic factor in vascular diseases. YOH (5–20 μM) suppressed PDGF-BB-stimulated a mouse VSMC line (MOVAS-1 cell) proliferation without inducing cytotoxicity. YOH also exhibited antimigratory effects and downregulated matrix metalloproteinase-2 and -9 expression in PDGF-BB-stimulated MOVAS-1 cells. It also promoted cell cycle arrest in the initial gap/first gap phase by upregulating p27Kip1 and p53 expression and reducing cyclin-dependent kinase 2 and proliferating cell nuclear antigen expression. We noted phospholipase C-γ1 (PLCγ1) but not ERK1/2, AKT, or p38 kinase phosphorylation attenuation in YOH-modulated PDGF-BB-propagated signaling pathways in the MOVAS-1 cells. Furthermore, YOH still inhibited PDGF-BB-induced cell proliferation and PLCγ1 phosphorylation in MOVAS-1 cells with α2B-adrenergic receptor knockdown. YOH (5 and 10 mg/kg) substantially suppressed neointimal hyperplasia in mice subjected to CCA ligation for 21 days. Overall, our results reveal that YOH attenuates PDGF-BB-stimulated VSMC proliferation and migration by downregulating a α2B-adrenergic receptor–independent PLCγ1 pathway and reduces neointimal formation in vivo. Therefore, YOH has potential for repurposing for treating atherosclerosis and other vascular proliferative diseases.

Inhibition of KIR2.1 decreases pulmonary artery smooth muscle cell proliferation and migration

The investigation of effective therapeutic drugs for pulmonary hypertension (PH) is critical. KIR2.1 plays crucial roles in regulating cell proliferation and migration, and vascular remodeling. However, researchers have not yet clearly determined whether KIR2.1 participates in the proliferation and migration of pulmonary artery smooth muscle cells (PASMCs) and its role in pulmonary vascular remodeling (PVR) also remains elusive. The present study aimed to examine whether KIR2.1 alters PASMC proliferation and migration, and participates in PVR, as well as to explore its mechanisms of action. For the in vivo experiment, a PH model was established by intraperitoneally injecting Sprague-Dawley rats monocrotaline (MCT). Hematoxylin and eosin staining revealed evidence of PVR in the rats with PH. Immunofluorescence staining and western blot analysis revealed increased levels of the KIR2.1, osteopontin (OPN) and proliferating cell nuclear antigen (PCNA) proteins in pulmonary blood vessels and lung tissues following exposure to MCT, and the TGF-β1/SMAD2/3 signaling pathway was activated. For the in vitro experiments, the KIR2.1 inhibitor, ML133, or the TGF-β1/SMAD2/3 signaling pathway blocker, SB431542, were used to pre-treat human PASMCs (HPASMCs) for 24 h, and the cells were then treated with platelet-derived growth factor (PDGF)-BB for 24 h. Scratch and Transwell assays revealed that PDGF-BB promoted cell proliferation and migration. Immunofluorescence staining and western blot analysis demonstrated that PDGF-BB upregulated OPN and PCNA expression, and activated the TGF-β1/SMAD2/3 signaling pathway. ML133 reversed the proliferation and migration induced by PDGF-BB, inhibited the expression of OPN and PCNA, inhibited the TGF-β1/SMAD2/3 signaling pathway, and reduced the proliferation and migration of HPASMCs. SB431542 pre-treatment also reduced cell proliferation and migration; however, it did not affect KIR2.1 expression. On the whole, the results of the present study demonstrate that KIR2.1 regulates the TGF-β1/SMAD2/3 signaling pathway and the expression of OPN and PCNA proteins, thereby regulating the proliferation and migration of PASMCs and participating in PVR.

Molecular mechanism behind methamphetamine-induced damages in testicular tissue: Evidences for oxidative stress, autophagy, and apoptosis.

Methamphetamine (METH) is shown to cause massive oxidative stress and apoptosis in testicular tissue. This study attempted to investigate the possible effects of METH chronic administration on the crosstalk between oxidative DNA damage (ODD), the ODD repairing process, autophagy, and apoptosis in testicular tissue. For this purpose, 20 rats were divided into control and METH (2.5mg/kg)-received groups (N= 10 rats/group). Following 7 days, the tubular differentiation (TDI) and spermiogenesis (SPI) indices, histomorphometric alterations, intracytoplasmic carbohydrate and lipid storage in germ and Sertoli cells along with expression levels of proliferating cell nuclear antigen (PCNA), as a key element in regulating base excision repair (BER) enzymes expression/activity were assessed. Moreover, the expression levels of uracil-DNA (UDG) and methylpurine (MPG) DNA glycosylases and microtubule-associated protein light chain 3 (LC3-I/II), and apoptotic cells distribution in testicular tissue were evaluated. Observations revealed that METH significantly suppressed spermatogenesis and spermiogenesis development, altered intracytoplasmic carbohydrate and lipid storage, increased ODD, and suppressed the PCNA expression compared to the control group (p< 0.05). Furthermore, METH-received animals exhibited a remarkable (p< 0.05) reduction in UDG and MPG, increment in LC3-I/II expressions, and apoptotic cells distribution. In conclusion, METH consumption results in a failed intracytoplasmic glucose storage (primary metabolites of Sertoli and germ cells) and oxidative stress (OS) circumstance in the testicular tissue. Further, METH can induce ODD by suppressing the expression levels of PCNA and BER enzymes, UDG and MPG. Finally, we demonstrated that METH-induced massive ODD is capable of initiating autophagy signalling that leads to progressive apoptosis in the testicular tissue.

KAP1 phosphorylation promotes the survival of neural stem cells after ischemia/reperfusion by maintaining the stability of PCNA

AimsTo explore the function of phosphorylation of KAP1 (p-KAP1) at the serine-824 site (S824) in the proliferation and apoptosis of endogenous neural stem cells (NSCs) after cerebral ischemic/reperfusion (I/R).MethodsThe apoptosis and proliferation of C17.2 cells transfected with the p-KAP1-expression plasmids and the expression of proliferation cell nuclear antigen (PCNA) and p-KAP1 were detected by immunofluorescence and Western blotting after the Oxygen Glucose deprivation/reperfusion model (OGD/R). The interaction of p-KAP1 and CUL4A with PCNA was analyzed by immunoprecipitation. In the rats MCAO model, we performed the adeno-associated virus (AAV) 2/9 gene delivery of p-KAP1 mutants to verify the proliferation of endogenous NSCs and the colocalization of PCNA and CUL4A by immunofluorescence.ResultsThe level of p-KAP1 was significantly down-regulated in the stroke model in vivo and in vitro. Simulated p-KAP1(S824) significantly increased the proliferation of C17.2 cells and the expression of PCNA after OGD/R. Simulated p-KAP1(S824) enhanced the binding of p-KAP1 and PCNA and decreased the interaction between PCNA and CUL4A in C17.2 cells subjected to OGD/R. The AAV2/9-mediated p-KAP1(S824) increased endogenous NSCs proliferation, PCNA expression, p-KAP1 binding to PCNA, and improved neurological function in the rat MCAO model.ConclusionsOur findings confirmed that simulated p-KAP1(S824) improved the survival and proliferation of endogenous NSCs. The underlying mechanism is that highly expressed p-KAP1(S824) promotes binding to PCNA, and inhibits the binding of CUL4A to PCNA. This reduced CUL4A-mediated ubiquitination degradation to increase the stability of PCNA and promote the survival and proliferation of NSCs.

Immunohistochemical expression of FAP and PCNA in neoplastic epithelial colonic lesions

Background Colorectal carcinoma is one of the leading causes of morbidity and mortality worldwide. Fibroblast-activation protein is expressed in cancer-associated fibroblasts and its expression is upregulated during the transformation of benign colorectal tissue into cancer. Proliferating-cell nuclear antigen expression was used as a marker of cell proliferation and is considered as a marker able to identify aggressive adenomas that may in turn become malignant. Aim To study the expression of fibroblast-activation protein and proliferating-cell nuclear antigen in neoplastic epithelial colonic lesions and their role in adenoma–carcinoma sequence. Patients and methods This retrospective study aimed to evaluate the expression of fibroblast-activation protein and proliferating-cell nuclear antigen in 80 cases of neoplastic epithelial colonic lesions. Results In malignant colorectal epithelial tumors, high fibroblast-activation protein expression was detected with an increase in histological grade, depth of invasion, vascular invasion, and lymph-node metastasis. No fibroblast-activation protein expression was detected in all benign cases. In included benign cases, there was a significant relation between proliferating-cell nuclear antigen labeling index and both histological type and grade of dysplasia. In the included malignant cases, there was a significant relation between proliferating-cell nuclear antigen labeling index with grade of tumor and the depth of invasion. Conclusions Both fibroblast-activation protein and proliferating-cell nuclear antigen labeling index suggested to have a role in early detection of malignant transformation and had a prognostic role in malignant cases.

Therapeutic effects of sulforaphane in ulcerative colitis: effect on antioxidant activity, mitochondrial biogenesis and DNA polymerization

ABSTRACT Objectives Ulcerative colitis (UC), an inflammatory bowel disease, affects mucosal lining of colon leading to inflammation and ulcers. Sulforaphane is a natural compound obtained from cruciferous vegetables. We aimed to investigate potential therapeutic effects of sulforaphane in experimentally induced UC in rats through affection antioxidant activity, mitochondrial biogenesis and DNA polymerization. Methods UC was induced in rats via an intracolonic single administration of 2 ml of 4% acetic acid. UC rats were treated with 15 mg/kg sulforaphane. Samples of colon were used to investigate gene expression and protein levels of peroxisome proliferator-activated receptor-gamma coactivator (PGC-1), mitochondrial transcription factor A (TFAM), mammalian target of rapamycin (mTOR), cyclin D1, nuclear factor erythroid 2-related factor-2 (Nrf2), heme Oxygenase-1 (HO-1) and proliferating cell nuclear antigen (PCNA). Results UC showed dark distorted Goblet cell nucleus with disarranged mucus granules and no distinct brush border with atypical microvilli. All morphological changes were improved by treating with sulforaphane. Finally, treatment with sulforaphane significantly increased expression of PGC-1, TFAM, Nrf2 and HO-1 associated with reduction in expression of mTOR, cyclin D1 and PCNA. Conclusion Sulforaphane could cure UC in rats. The protective activity can be explained by enhancing antioxidant activity, elevating mitochondrial biogenesis and inhibiting DNA polymerization.

The protective effect of curcumin on testicular tissue in a cryptorchid rat model

Cryptorchidism is the most common abnormality of male sexual development. For the protection of testicular functions, antioxidants have emerged as novel options. This study aimed to investigate the protective effect of curcumin (Cur), a strong antioxidant, on the Flutamide-induced cryptorchidism testicular tissue. Pregnant rats were randomly allocated to 3 groups (n=10, each): a control, a model, and a Cur-treated group (100mg/kg/d). All offspring were delivered by days 21-22 of gestation and the male rats were sacrificed at postnatal birth days (PNDs) PND60. The testicles were separated and weighed, followed by TUNEL staining to detect germ cell apoptosis, an ELISA kit to measure SOD and MDA, and Western blot analysis to evaluate the expression of Bax, Bcl-2, and PCNA. Curcumin administration ameliorated the histological appearance of the testis and greatly reduced the level of apoptosis in cryptorchidism rats' testicular cells. After curcumin treatment, the expression of proliferating cell nuclear antigen (PCNA) was restored in the testis tissues of cryptorchidism rats. Curcumin therapy reduced Bax expression while increasing Bcl-2 expression, according to the molecular study. Curcumin therapy also reduced malondialdehyde (MDA) levels and enhanced superoxide dismutase (SOD) levels in cryptorchidism rats' testis tissue. It can be concluded that curcumin administration significantly reduced the germ cell apoptosis in rats with cryptorchidism, which provides new insight for antioxidant therapy in preserving testicular functions before or after surgery in cryptorchidism.

Therapeutic Potential of Mesenchymal Stem Cells versus Omega n - 3 Polyunsaturated Fatty Acids on Gentamicin-Induced Cardiac Degeneration.

This study compared the cardioprotective action of mesenchymal stem cells (MSCs) and PUFAs in a rat model of gentamicin (GM)-induced cardiac degeneration. Male Wistar albino rats were randomized into four groups of eight rats each: group I (control group), group II (gentamicin-treated rats receiving gentamicin intraperitoneally (IP) at dose of 100 mg/kg/day for 10 consecutive days), group III (gentamicin and PUFA group receiving gentamicin IP at dose of 100 mg/kg/day for 10 consecutive days followed by PUFAs at a dose of 100 mg/kg/day for 4 weeks), and group IV (gentamicin and MSC group receiving gentamicin IP at dose of 100 mg/kg/day followed by a single dose of MSCs (1 × 106)/rat IP). Cardiac histopathology was evaluated via light and electron microscopy. Immunohistochemical detection of proliferating cell nuclear antigen (PCNA), caspase-3 (apoptosis), Bcl2, and Bax expression was performed. Moreover, cardiac malonaldehyde (MDA) content, catalase activity, and oxidative stress parameters were biochemically evaluated. Light and electron microscopy showed that both MSCs and PUFAs had ameliorative effects. Their actions were mediated by upregulating PCNA expression, downregulating caspase-3 expression, mitigating cardiac MDA content, catalase activity, and oxidative stress parameters. MSCs and PUFAs had ameliorative effects against gentamicin-induced cardiac degeneration, with MSCs showing higher efficacy compared to PUFAs.

Hydrogen Sulfide Suppresses Skin Fibroblast Proliferation via Oxidative Stress Alleviation and Necroptosis Inhibition.

Keloid is a common dermatofibrotic disease with excessive skin fibroblast proliferation. Hydrogen sulfide (H2S) as the third gasotransmitter improves fibrosis of various organs and tissues. Our study is aimed at investigating the effects and possible mechanisms of H2S on skin fibroblast proliferation. Scar tissues from six patients with keloid and discarded skin tissue from six normal control patients were collected after surgery, respectively. Plasma H2S content and skin H2S production in patients with keloid were measured. Keloid fibroblasts and transforming growth factor-β1- (TGF-β1, 10 ng/mL) stimulated normal skin fibroblasts were pretreated with H2S donor as NaHS (50 μM) for 4 h. Cell migration after scratch was assessed. The expressions of α-smooth muscle actin (α-SMA), proliferating cell nuclear antigen (PCNA), collagen I, and collagen III were detected by immunofluorescence, real-time PCR, and/or Western blot. Intracellular superoxide anion and mitochondrial superoxide were evaluated by dihydroethidium (DHE) and MitoSOX staining, respectively. Mitochondrial membrane potential was detected by JC-1 staining. Apoptotic cells were detected by TDT-mediated dUTP nick end labeling (TUNEL). The expressions of receptor interacting protein kinase 1 (RIPK1), RIPK3, and mixed lineage kinase domain-like protein (MLKL) were measured by Western blot. We found that H2S production was impaired in both the plasma and skin of patients with keloid. In keloid fibroblasts and TGF-β1-stimulated normal skin fibroblasts, exogenous H2S supplementation suppressed the expressions of α-SMA, PCNA, collagen I, and collagen III, reduced intracellular superoxide anion and mitochondrial superoxide, improved the mitochondrial membrane potential, decreased the positive rate of TUNEL staining, and inhibited RIPK1 and RIPK3 expression as well as MLKL phosphorylation. Overall, H2S suppressed skin fibroblast proliferation via oxidative stress alleviation and necroptosis inhibition.