Characteristics of Retinal Vascular Degeneration and the Expression of Vessel-Related Claudin Proteins in Retinal Degeneration Mouse.

INTRODUCTION: This study aimed to investigate the characteristics of retinal vascular degeneration and the expression of vessel-related claudin (CLD) proteins in retinal degeneration mouse (Pde6ßrd1/rd1 rd1 mouse). METHODS: Retinas from wild-type (WT) mice and rd1 mice at postnatal day 3 (P3), P5, P8, P11, P13, P15, P18, and P21 were collected. Immunofluorescence staining was used to assess the retinal vascular plexus, cell proliferation, CLD expression, and retinal ganglion cells (RGCs). The distribution of retinal superficial and deep vessels was determined by isolectin B4 fluorescence staining of retinal flat mounts and frozen sections. Hematoxylin and eosin staining and terminal deoxynucleotidyl transferase-mediated dNTP nick-end labeling were used to investigate retinal histological degeneration and apoptosis in rd1 mice, respectively. Quantitative real-time PCR and Western blot were used to measure the expression of vessel-related CLD-1, -2, -3, and -5, vascular endothelial growth factor A (VEGFA), and vascular endothelial growth factor receptor 2 (VEGFR2) in the retinas. RESULTS: Compared to the WT mice, the rd1 mice displayed delayed but completed progressive development in the retinal superficial vascular plexuses (SVPs) and deep vascular plexuses (DVPs). In the rd1 mice, the thickness of retinal layers gradually decreased and the retinas underwent progressive atrophy and degeneration. The deterioration got worse at the late developmental stage. The declined vessel density of SVP and DVP correlated with the decreased thickness of the full and inner parts of the retina and the reduced number of RGCs. DVP degeneration and the thinning of the outer nuclear layer exhibited an obvious reduction at P15. The expression levels of CLD-1, CLD-2, CLD-3, CLD-5, VEGFA, and VEGFR2 decreased and were consistently lower in the rd1 mice than in WT mice since P15. CONCLUSION: Rd1 mice exhibited progressive vascular degeneration of retinal SVP and DVP, the thinning and atrophy of retinal ONL and RGC, and the downregulation of vessel-related CLD proteins during the late developmental period. Thus, the rd1 mouse is a useful model of not only retinal neuro-degeneration but also retinal vascular degeneration.

Radiation-Induced DNA Damage in Uveal Melanoma Is Influenced by Dose Delivery and Chromosome 3 Status.

Purpose: The purpose of this study was to analyze the extent of DNA breaks in primary uveal melanoma (UM) with regard to radiotherapy dose delivery (single-dose versus fractionated) and monosomy 3 status. Methods: A total of 54 patients with UM were included. Stereotactic radiotherapy (SRT) was performed in 23 patients, with 8 undergoing single-dose SRT (sdSRT) treatment and 15 receiving fractionated SRT (fSRT). DNA breaks in the enucleated or endoresected tumors were visualized by a TUNEL assay and quantified by measuring the TUNEL-positive area. Protein expression was analyzed by immunohistochemistry. Co-detection of chromosome 3 with proteins was performed by immuno-fluorescent in situ hybridization. Results: The amount of DNA breaks in the total irradiated group was increased by 2.7-fold (P < 0.001) compared to non-irradiated tissue. Tumors treated with fSRT were affected more severely, showing 2.1-fold more DNA damage (P = 0.007) compared to the cases after single (high) dose irradiation (sdSRT). Monosomy 3 tumors showed less DNA breaks compared to disomy 3 samples (P = 0.004). The presence of metastases after radiotherapy correlated with monosomy 3 and less DNA breaks compared to patients with non-metastatic cancer in the combined group with fSRT and sdSRT (P < 0.05). Conclusions: Fractionated irradiation led to more DNA damage than single-dose treatment in primary UM. As tumors with monosomy 3 showed less DNA breaks than those with disomy 3, this may indicate that they are less radiosensitive, which may influence the efficacy of irradiation.

Association of CUL4B with the pathogenesis of age-related cataract.

PURPOSE: Age-related cataract (ARC) is the most common cause of visual impairment and blindness in older adults. However, the role of CUL4B in the ARC remains unclear. Therefore, we investigated CUL4B expression and its effects on apoptosis. MATERIALS AND METHODS: CUL4B expression levels were detected by a quantitative real-time polymerase chain reaction from the anterior lens capsules of patients with ARC and HLE-B3 cells treated with different concentrations of H2O2. CUL4B expression was silenced by siRNA transfection to evaluate apoptosis. CUL4B and apoptotic proteins B cell lymphoma 2 (Bcl-2), myeloid cell leukemia 1 (Mcl-1), caspase-3, cleaved caspase-3, Bax, Bak, and Bid were assessed using western blot analysis. Apoptosis was monitored using the TUNEL assay. RESULTS: CUL4B expression was downregulated in the anterior lens capsules (P < 0.0001) and H2O2-treated HLE-B3 cells (P = 0.0405). CUL4B protein levels were significantly lower in 100 µmol/L (P = 0.0012) and 200 µmol/L (P = 0.0041) H2O2-treated HLE-B3 cells than in the untreated cells. CUL4B expression was significantly knocked down at the mRNA (P = 0.0043) and protein levels (P = 0.0002) in HLE-B3 cells. Bcl-2 (P = 0.0199), Mcl-1 (P = 0.0042), and caspase-3 (P = 0.0142) were significantly downregulated, whereas cleaved caspase-3 (P = 0.0089) and Bak (P = 0.009) were significantly upregulated in the knockdown group. The TUNEL assay showed a greater induction of apoptosis. CONCLUSIONS: CUL4B downregulation promotes the apoptosis of lens epithelial cells. Our study may help in understanding the role of CUL4B in ARC pathogenesis.

HDAC1/2 and HDAC3 play distinct roles in controlling adult Meibomian gland homeostasis.

PURPOSE: To investigate the roles of HDAC1/2 and HDAC3 in adult Meibomian gland (MG) homeostasis. METHODS: HDAC1/2 or HDAC3 were inducibly deleted in MG epithelial cells of adult mice. The morphology of MG was examined. Proliferation, apoptosis, and expression of MG acinus and duct marker genes, meibocyte differentiation genes, and HDAC target genes, were analyzed via immunofluorescence, TUNEL assay, and RNA in situ hybridization. RESULTS: Co-deletion of HDAC1/2 in MG epithelium caused gradual loss of acini and formation of cyst-like structures in the central duct. These phenotypes required homozygous deletion of both HDAC1 and HDAC2, indicating that they function redundantly in the adult MG. Short-term deletion of HDAC1/2 in MG epithelium had little effect on meibocyte maturation but caused decreased proliferation of acinar basal cells, excessive DNA damage, ectopic apoptosis, and increased p53 acetylation and p16 expression in the MG. By contrast, HDAC3 deletion in MG epithelium caused dilation of central duct, atrophy of acini, defective meibocyte maturation, increased acinar basal cell proliferation, and ectopic apoptosis and DNA damage. Levels of p53 acetylation and p21 expression were elevated in HDAC3-deficient MGs, while the expression of the differentiation regulator PPARγ and the differentiation markers PLIN2 and FASN was downregulated. CONCLUSIONS: HDAC1 and HDAC2 function redundantly in adult Meibomian gland epithelial progenitor cells and are essential for their proliferation and survival, but not for acinar differentiation, while HDAC3 is required to limit acinar progenitor cell proliferation and permit differentiation. HDAC1/2 and HDAC3 have partially overlapping roles in maintaining survival of MG cells.

BMSC Alleviates Dry Eye by Inhibiting the ROS-NLRP3-IL-1ß Signaling Axis by Reducing Inflammation Levels.

PURPOSE: Bone marrow mesenchymal stem cells (BMSC) have multiple biological functions and are widely involved in regulating inflammatory diseases, tissue repair and regeneration. However, the mechanism of their action in dry eye disease (DED) is currently unclear. The purpose of this study was to investigate the effect of BMSCs in the treatment of dry eye mice and to explore its specific therapeutic mechanism. METHODS: Mouse corneal epithelial cells (MCECs) were treated with 500 mOsM sodium chloride hypertonic solution to induce a DED cell model. The dry eye animal model was constructed by adding 5 µL 0.2% benzalkonium chloride solution to mouse eyes. Western blotting was used to detect the expression of related proteins, and flow cytometry, enzyme-linked immunosorbent assay (ELISA), terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining, hematoxylin-eosin (HE) staining, and periodic acid schiff (PAS) staining were used to detect cell and eye tissue damage. RESULTS: The experimental results showed that BMSCs can reduce the levels of reactive oxygen species (ROS) and inflammatory factors in MCECs, promote cell proliferation, inhibit cell apoptosis, improve the integrity of the corneal epithelial layer in vivo, promote an increase in the number of goblet cells, and alleviate DED. Further exploration of the molecular mechanism of BMSCs treatment revealed that BMSCs alleviate the progression of DED by inhibiting the ROS-NLRP3-IL-1ß signaling pathway. CONCLUSION: BMSCs inhibit ROS-NLRP3-IL-1ß signaling axis, reducing inflammation levels and alleviating dry eye symptoms. These findings provide new ideas and a basis for the treatment of DED and provide an experimental basis for further research on the application value of BMSCs in alleviating DED.

In vivo Gonadoprotective Effects of Myricetin on Cisplatin- Induced Testicular Damage via Suppression of TLR4/NF-kB Inflammation Pathway and Heat-Shock Response / Efectos Gonadoprotectores in vivo de la Miricetina sobre el Daño Testicular Inducido por Cisplatino Mediante la Supresión de la Vía de Inflamación TLR4/NF-kB y la Respuesta al Choque Térmico

SUMMARY: The aim of this study is to reveal the gonadoprotective effects of myricetin (MYC), which has many biological properties, on cisplatin (CP)-induced testicular damage in rats. For this purpose, 40 male Wistar albino rats were divided into 4 groups as Control (group given no treatment), MYC (group given 5 mg/kg/i.p myricetin for 7 days), CP (group given 7 mg/kg/i.p cisplatin at 7th day) and MYC + CP (group given 5 mg/kg/i.p myricetin for 7 days before 7 mg/kg/i.p cisplatin injection). After administrations, testicular tissues of animals were extracted and processed according to tissue processing protocol. Hematoxylin & Eosin staining were performed to evaluate the histopathological changes and Johnsen'sTesticular Biopsy Score (JTBS) was applied and mean seminiferous tubule diameters (MSTD) were measured to compare experimental groups in terms of histopathological changes. Moreover, TLR4, NF-kB, HSP70 and HSP90 expression levels were detected by immunohistochemical staining and the density of immunoreactivity were measured to determine the difference in the expression levels of these factors among groups. Additionally, testicular apoptosis was detected via TUNEL assay. JTBS and MSTD data were significantly lower in CP group compared to other groups and MYC administrations significantly protects testicular tissue against CP-induced damage. Moreover, TLR4, NF-kB, HSP70 and HSP90 expressions and apoptotic cells significantly increased in the CP group (p<0.05). However, MYC administrations exerted a strong gonadoprotective effect on testicular tissue in terms of these parameters in MYC+CP group (p<0.05). According to our results, we suggested that MYC can be considered as a protective agent against cisplatin-induced testicular damage.

El objetivo de este estudio es revelar los efectos gonadoprotectores de la miricetina (MYC), que tiene muchas propiedades biológicas, sobre el daño testicular inducido por cisplatino (CP) en ratas. Para este propósito, se dividieron 40 ratas albinas Wistar macho en 4 grupos: Control (grupo que no recibió tratamiento), MYC (grupo que recibió 5 mg/kg/i.p de miricetina durante 7 días), CP (grupo que recibió 7 mg/kg/i.p de cisplatino al séptimo día) y MYC + CP (grupo que recibió 5 mg/ kg/i.p de miricetina durante 7 días antes de la inyección de 7 mg/ kg/i.p de cisplatino). Después de las administraciones, se extrajeron y procesaron tejidos testiculares de animales según el protocolo de procesamiento de tejidos. Se realizó tinción con hematoxilina y eosina para evaluar los cambios histopatológicos y se aplicó la puntuación de biopsia testicular de Johnsen (JTBS) y se midieron los diámetros medios de los túbulos seminíferos (MSTD) para comparar los grupos experimentales en términos de cambios histopatológicos. Además, los niveles de expresión de TLR4, NF-kB, HSP70 y HSP90 se detectaron mediante tinción inmunohistoquímica y se midió la densidad de inmunorreactividad para determinar la diferencia en los niveles de expresión de estos factores entre los grupos. Además, se detectó apoptosis testicular mediante el ensayo TUNEL. Los datos de JTBS y MSTD fueron significativamente más bajos en el grupo CP en comparación con otros grupos y las administraciones de MYC protegen significativamente el tejido testicular contra el daño inducido por CP. Además, las expresiones de TLR4, NF-kB, HSP70 y HSP90 y las células apoptóticas aumentaron significativamente en el grupo CP (p<0,05). Sin embargo, las administraciones de MYC ejercieron un fuerte efecto gonadoprotector sobre el tejido testicular en términos de estos parámetros en el grupo MYC+CP (p<0,05). Según nuestros resultados, sugerimos que MYC puede considerarse como un agente protector contra el daño testicular inducido por cisplatino.

Aripiprazole sensitizes head and neck cancer cells to ionizing radiation by enhancing the production of reactive oxygen species.

Drug repositioning is an alternative process for drug development in cancer. Specifically, it is a strategy for the discovery of new antitumor drugs by screening previously approved clinical drugs. On the basis of this strategy, aripiprazole, an antipsychotic drug, was found to have anticancer activity. In this study, we investigated the radiosensitizing effects of aripiprazole on head and neck cancer cells at sublethal doses of ionizing radiation (IR) in vitro and in vivo. Treatment with aripiprazole suppressed the growth of head and neck cancer cells in a concentration-dependent manner, as evidenced by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Intriguingly, aripiprazole significantly enhanced the sensitivity of these cells to the IC50 dose of IR. The combination of aripiprazole with IR synergistically increased annexin and propidium iodide double-positive and terminal deoxynucleotidyl transferase dUTP nick end labeling-positive cell populations, and induced cleaved poly(ADP-ribose) polymerase and caspase-3 expression, indicating the induction of apoptosis in these cells. Aripiprazole and IR-induced apoptosis were accompanied by an increase in reactive oxygen species and was almost completely suppressed by the addition of the antioxidant, N-acetylcysteine. Finally, aripiprazole greatly sensitized xenograft tumors to IR at doses that did not affect tumor growth. Taken together, these results suggest that aripiprazole could be considered a potent radiosensitizer for head and neck cancer.

Toxicity of Amphotericin B in Rabbit Corneal Epithelial Cells Stored in Optisol™-GS: Corneal Epithelial Cell Morphology and Migration.

PURPOSE: To evaluate the toxicity of Amphotericin B (AmB) in Optisol™-GS Corneal Storage Media (Bausch & Lomb) on corneal epithelial cell (CEC) morphology and migration ability. METHODS: Sclerocorneal strips were removed from male Japanese white rabbits, and then stored at 4 °C in Optisol™-GS containing 0 µg/ml of AmB (control group) and 2.5, 5, 25, and 50 µg/ml of AmB (AmB groups; four eyes per group). After 7 days of storage, CEC morphology was evaluated by hematoxylin-eosin staining, immunohistochemical staining (ZO-1), and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Moreover, to evaluate CEC migration ability, three corneal blocks (6-8 × 3 mm each) from one preserved cornea were cultured for 24 h, and the area of CEC migration (2 mm at the central region) onto the stromal surface was then measured. RESULTS: At 5, 25, and 50 µg/ml of AmB, deformation and vacuolation of CECs were observed in all preserved corneas. ZO-1 expression was significantly reduced in corneas preserved at AmB concentrations of 25 and 50 µg/ml. TUNEL Labeling Index was significantly increased at AmB concentrations of ≥5 µg/ml. CEC migration was inhibited in a dose-dependent manner at AmB concentrations of 25 and 50 µg/ml compared to the control group. CONCLUSIONS: The addition of AmB to Optisol™-GS can be toxic to CECs and inhibit their migration at a concentration of ≥5 µg/ml. AmB at a concentration of 2.5 µg/ml can be considered safe for the preservation of donor corneal tissue used in corneal epithelial transplantation surgery.

Radioprotective effects and mechanism of HL-003 on radiation-induced salivary gland damage in mice.

Ionizing radiation (IR) can cause damage to the structure and function of salivary glands. Our research group independently synthesized the ROS scavenger, HL-003. The aim of this study was to explore the protective effects and underlying mechanisms of HL-003 on radiation-induced salivary gland injury. Salivary flow rate measurement, H&E staining, immunohistochemistry, FRAP, TUNEL, and western blotting were used to evaluate the radioprotective effect on salivary glands. The results showed that HL-003 protected the salivary secretion function by protecting the AQP-5 protein, on the salivary epithelial cell membrane, from IR damage. HL-003 reduced oxidative stress in the salivary gland by regulating the expression of ROS-related proteins NOX4, SOD2, and 8-OHdG. Furthermore, HL-003 downregulated the expression of p-p53, Bax, caspase 3, and caspase 9, and upregulated the expression of Bcl-2, suggesting that it could inhibit the activation of p53 to reduce cell apoptosis. In conclusion, HL-003 is an effective radioprotector that prevents damage of the radiation-induced salivary gland.

Anticancer Effects of Urtica Dioica in Breast Cancer.

OBJECTIVE: The goal of this study is to look into the antiproliferative capabilities of Urtica Dioica (UD) on breast cancer. METHODS: The cytotoxicity of UD extracts against breast cancer cell lines was investigated. Flow cytometry analyses were used to investigate in vitro apoptosis of breast cancer cells using Annexin V labeling. In vivo tests also performed. RESULTS: UD showed cytotoxicity to three cancer cell lines. The number of Annexin-positive cells was higher in UD-treated cell lines than in untreated control cells. When compared to the untreated control group, the rats treated with UD had greater expressions of caspase 3, p53 protein, and TUNEL positive cells. When compared to the control group, Ki-67 expression was reduced in the treatment groups. In vivo tests revealed that, when compared to untreated rats, the mean tumor volume inhibition ratio in the UD group was 38 percent. CONCLUSION: These findings suggest that Urtica Dioica may have antitumoral properties in the treatment of breast cancer.

Protocol to study cell death using TUNEL assay in Drosophila imaginal discs.

Cell death maintains tissue homeostasis by eliminating dispensable cells. Misregulation of cell death is seen in diseases like cancer, neurodegeneration, etc. Therefore, cell death assays like TUNEL have become reliable tools, where fragmented DNA of dying cells gets fluorescently labeled and can be detected under microscope. We used TUNEL assay in Drosophila melanogaster third-instar larval eye-antennal imaginal discs to label and quantify cell death. This assay is sensitive to detect DNA fragmentation, an important event, during apoptosis in retinal neurons. For complete details on the use and execution of this profile, please refer to Wang et al. (1999), Tare et al. (2011), and Mehta et al. (2021).

Kruppel-like factor 2 acts as a tumor suppressor in human retinoblastoma.

Krüppel-like factor 2 (KLF2) belongs to the KLF family of zinc-finger transcription factors and mediates the occurrence and progression of various cancers. However, little is known about its expression pattern and biological role in retinoblastoma (RB). In the present study, we showed that KLF2 was markedly downregulated in human RB tissue compared with retina. KLF2 overexpression significantly inhibited RB cell proliferation and decreased proliferating cell nuclear antigen (PCNA) expression. Subsequently, we confirmed that KLF2 arrested cells at the G1-S phase transition, accompanied by the upregulation of p21 and downregulation of CyclinD1, as well as the activation of mitochondria-mediated apoptosis in RB cells. In addition, KLF2 overexpression contributed to suppressing RB cell migration and invasion by downregulating matrix metallopeptidase 9 (MMP9). On the contrary, KLF2 downregulation promoted RB cells proliferation, migration and invasion. Notably, the KLF2 expression pattern was opposite to that of C-X-C chemokine receptor 4 (CXCR4) in the two RB cell lines, KLF2 overexpression significantly decreased CXCR4 expression, silencing KLF2 had the opposite effect. Furthermore, dual-luciferase reporter and chromatin immunoprecipitation (ChIP) assays confirmed that KLF2 directly bound to the CXCR4 promoter and negatively regulated its expression in RB cells. Collectively, our results suggested that KLF2 function as a tumor suppressor in RB and may represent a potential therapeutic target for RB.

Iron contributes to photoreceptor degeneration and Müller glia proliferation in the zebrafish light-treated retina.

Zebrafish possess the ability to completely regenerate the retina following injury, however little is understood about the damage signals that contribute to inducing Müller glia reprogramming and proliferation to regenerate lost neurons. Multiple studies demonstrated that iron contributes to various retinal injuries, however no link has been shown between iron and zebrafish retinal regeneration. Here we demonstrate that Müller glia exhibit transcriptional changes following injury to regulate iron levels within the retina, allowing for increased iron uptake and decreased export. The response of the zebrafish retina to intravitreal iron injection was then characterized, showing that ferrous, and not ferric, iron induces retinal cell death. Additionally, iron chelation resulted in decreased numbers of TUNEL-positive photoreceptors and fewer proliferating Müller glia. Despite the contribution of iron to retinal cell death, inhibition of ferroptosis did not significantly reduce cell death following light treatment. Finally, we demonstrate that both the anti-ferroptotic protein Glutathione peroxidase 4b and the Transferrin receptor 1b are required for Müller glia proliferation following light damage. Together these findings show that iron contributes to cell death in the light-damaged retina and is essential for inducing the Müller glia regeneration response.

MircoRNA-126-5p inhibits apoptosis of endothelial cell in vascular arterial walls via NF-κB/PI3K/AKT/mTOR signaling pathway in atherosclerosis.

Atherosclerosis is considered as a chronic inflammatory disease. MircoRNA-126-5p (miR-126-5p) may be pathophysiological relevant with the apoptotic processes in the endothelial cells in the arterial wall. Here, this study determined the role of circulating atherosclerosis-regulatory miR-126-5p in atherosclerotic mice and explored the possible mechanism in human aortic endothelial cells (HAECs). Atherosclerotic mice model was established, oxidative stress-induced apoptosis of HAECs was analyzed, and nuclear factor kappa B (NF-κB)/PI3K/AKT/mTOR signaling pathway was investigated both in vitro and in vivo. This study showed that miR-126-5p mice had less coronary atherosclerotic plaque and lower blood lipid than control mice after being induced by high cholesterol diet. Apoptosis of endothelial cells was inhibited and NF-κB/PI3K/AKT/mTOR signal pathway was downregulated in miR-126-5p mice compared to control. MiR-126-5p increased proliferation and inhibited apoptosis of HAECs induced by oxidative stress. In vitro assay showed that miR-126-5p regulated apoptosis of HAECs via downregulation of NF-κB-mediated PI3K/AKT/mTOR signaling pathway. In conclusion, these data indicated that transfection of miR-126-5p rescued apoptosis of HAECs and limited atherosclerosis, introducing a potential therapeutic approach for atherosclerosis.

The W9 peptide inhibits osteoclastogenesis and osteoclast activity by downregulating osteoclast autophagy and promoting osteoclast apoptosis.

The W9 peptide has been shown to act as a receptor activator for nuclear factor-κB ligand (RANKL) antagonist and tumor necrosis factor (TNF)-α antagonist, which can promote bone formation and inhibit bone resorption. Studies on the W9 peptide at the cellular level have mainly focused on osteoblasts, and little research on the mechanism by which the W9 peptide regulates osteoclasts has been reported, which was the aim of this work. In this study, a rat mandibular defect model was established in vivo and implanted with hydrogel containing the W9 peptide for 2 weeks and 4 weeks, and histochemical staining was used to evaluate the formation of new bone and the changes in osteoclasts. RAW264.7 cells were cultured in vitro for osteoclast induction, and different concentrations of W9 peptide were added. Tartrate resistant acid phosphatase staining, monodansylcadaverine staining, TdT-mediated dUTP Nick-End Labeling assay, real-time PCR and Western blot were used to detect osteoclast differentiation, autophagy and apoptosis. Our results showed that the W9 peptide could reduce osteoclastogenesis and osteoclast activity induced by RANKL, and these effects were partly due to the inhibition of osteoclast autophagy. On the other hand, the W9 peptide could promote mature osteoclast apoptosis, in which autophagy might play an antagonistic role. Taken together, these results suggest that the W9 peptide inhibits osteoclastogenesis and osteoclast activity by downregulating osteoclast autophagy and promoting osteoclast apoptosis. Our results will benefit the development and application of new small molecule peptides for the treatment of bone resorption diseases.

Evaluation by different mechanisms of the protective effects of vitamin B12 on methotrexate nephrotoxicity.

Methotrexate is used for cure of many cancer types. It has many side effects. For this reason, obtaining a nephroprotective agent is obligatory. In the study, our aim is to determine probable effects of Vitamin B12 on MTX caused kidney damages in rats. Rats were randomly divided into 4 groups, including 8 animals in each group. Control group, VitB12 group (3 µg-kg-ip B12 throughout 15 days), MTX group (at the 8th day of experiment, a single dose of 20 mg-kg-ip MTX), Vit B12 + MTX group (3 µg-kg-ip B12 throughout 15 days and at the 8th day of experiment, a single dose of 20 mg-kg-ip MTX) Animals were anesthetized and kidney tissues were removed to evaluate biochemically, immunohistochemically and histopathologycally. There were histopathological deteriorations, rises of apoptotic cells, expressions of heat shock proteins, endoplasmic reticulum stress and inflammation markers in the MTX group. In the MTX group, Superoxide Dismutase (SOD), Total Antioxidant Status (TAS) and Catalase (CAT) levels decreased, but Total Oxidant Status TOS, Malondialdehyde (MDA) and interleukin-6 (IL6) levels increased. In addition, there was amelioration in kidney tissue in Vit B12 + MTX group compared to the MTX group. We suggest that Vit B12 can be used to reduce the toxic effects of MTX.

Suppression of MALAT1 alleviates neurocyte apoptosis and reactive oxygen species production through the miR-499-5p/SOX6 axis in subarachnoid hemorrhage.

Subarachnoid hemorrhage (SAH), a common devastating cerebrovascular accident, is a great threat to human health and life. Exploration of the potential therapeutic target of SAH is urgently needed. Previous studies showed that long noncoding RNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) promotes cell apoptosis in various diseases, while its role in SAH remains unclear. In our study, we established a mouse model of SAH and used the oxyhemoglobin (OxyHb) to induce neuronal injury in vitro. Interestingly, MALAT1 was found upregulated in brain tissues of SAH mice and OxyHb-stimulated neurons. In addition, knockdown of MALAT1 attenuated apoptosis and decreased reactive oxygen species (ROS) production in OxyHb-stimulated neurons. Mechanistically, we demonstrated that MALAT1 bound with miR-499-5p. Furthermore, our findings indicated that miR-499-5p bound to SOX6 3' untranslated region (UTR) and negatively regulated SOX6 mRNA and protein levels. Rescue assays suggested that SOX6 overexpression counteracted the effects of MALAT1 knockdown on neurocyte apoptosis, and ROS production in OxyHb-stimulated neurons. The in vivo assays indicated that knockdown of MALAT1 improved brain injury of SAH mice. Our study demonstrates that silencing of MALAT1 alleviates neurocyte apoptosis and reduces ROS production through the miR-499-5p/SOX6 axis after SAH injury.

The role of amifostine in preventing radiotherapy induced testicular tissue damage in rats.

The germinal epithelium of the adult testis is susceptible to radiation induced damage. Amifostine is a drug used to prevent the side effects of radiotherapy (RT) and chemotherapy. We investigated the protective role of amifostine against RT induced damage to rat testis using the TUNEL assay. We used adult male rats divided equally into four groups: untreated control group; amifostine group, 200 mg/kg amifostine/day for 3 days; RT-saline group, 2 Gy/day local irradiation of testes for 3 days; RT-amifostine group, 2 Gy/day local irradiation of testes for 3 days plus 200 mg/kg amifostine 30 min before each irradiation. Four weeks after treatment, rats were sacrificed for histological examination and apoptosis was assessed using the TUNEL method. The TUNEL staining density was obtained by evaluating separate seminiferous tubules selected randomly from each section using the stereological fractionator method. Apoptosis in the seminiferous tubules in the control group and amifostine groups were evaluated as spontaneous. Frequent apoptosis was observed in the RT-saline group; a statistically significant difference was observed between the RT treated and untreated groups. Administration of amifostine 30 min before RT protected the testicular germ cells against apoptosis.

Protective effect of berberine chloride against cisplatin-induced ototoxicity.

BACKGROUND: Cisplatin (CP) is an effective anticancer drug broadly used for various types of cancers, but it has shown ototoxicity that results from oxidative stress. Berberine has been reported for its anti-oxidative stress suggesting its therapeutic potential for many diseases such as colitis, diabetes, and vascular dementia. OBJECTIVE: Organ of Corti of postnatal day 3 mouse cochlear explants were used to compare hair cells after the treatment with cisplatin alone or with berberine chloride (BC) followed by CP. METHODS: We investigated the potential of the anti-oxidative effect of BC against the cisplatin-induced ototoxicity. We observed a reduced aberrant bundle of stereocilia in hair cells in CP with BC pre-treated group. Caspase-3 immunofluorescence and TUNEL assay supported the hypothesis that BC attenuates the apoptotic signals induced by CP. Reactive oxygen species level in the mitochondria were investigated by MitoSOX Red staining and the mitochondrial membrane potentials were compared by JC-1 assay. RESULTS: BC decreased ROS generation with preserved mitochondrial membrane potentials in mitochondria as well as reduced DNA fragmentation in hair cells. In summary, our data indicate that BC might act as antioxidant against CP by reducing the stress in mitochondria resulting in cell survival. CONCLUSION: Our result suggests the therapeutic potential of BC for prevention of the detrimental effect of CP-induced ototoxicity.

Maternal N-Acetyl-Cysteine Prevents Neonatal Hypoxia-Induced Brain Injury in a Rat Model.

Perinatal hypoxia is a major cause of infant brain damage, lifelong neurological disability, and infant mortality. N-Acetyl-Cysteine (NAC) is a powerful antioxidant that acts directly as a scavenger of free radicals. We hypothesized that maternal-antenatal and offspring-postnatal NAC can protect offspring brains from hypoxic brain damage.Sixty six newborn rats were randomized into four study groups. Group 1: Control (CON) received no hypoxic intervention. Group 2: Hypoxia (HYP)-received hypoxia protocol. Group 3: Hypoxia-NAC (HYP-NAC). received hypoxia protocol and treated with NAC following each hypoxia episode. Group 4: NAC Hypoxia (NAC-HYP) treated with NAC during pregnancy, pups subject to hypoxia protocol. Each group was evaluated for: neurological function (Righting reflex), serum proinflammatory IL-6 protein levels (ELISA), brain protein levels: NF-κB p65, neuronal nitric oxide synthase (nNOS), TNF-α, and IL-6 (Western blot) and neuronal apoptosis (histology evaluation with TUNEL stain). Hypoxia significantly increased pups brain protein levels compared to controls. NAC administration to dams or offspring demonstrated lower brain NF-κB p65, nNOS, TNF-α and IL-6 protein levels compared to hypoxia alone. Hypoxia significantly increased brain apoptosis as evidenced by higher grade of brain TUNEL reaction. NAC administration to dams or offspring significantly reduce this effect. Hypoxia induced acute sensorimotor dysfunction. NAC treatment to dams significantly attenuated hypoxia-induced acute sensorimotor dysfunction. Prophylactic NAC treatment of dams during pregnancy confers long-term protection to offspring with hypoxia associated brain injury, measured by several pathways of injury and correlated markers with pathology and behavior. This implies we may consider prophylactic NAC treatment for patients at risk for hypoxia during labor.