As the prevalence of microbial keratitis increases, it creates an environment conducive to genotoxicity response. A potential connection between growth arrest and DNA-damage-inducible 45 gamma (GADD45G) gene expression has not been proven in the corneal epithelial cells. The aim of this study was to determine whether lipopolysaccharide (LPS) enhances genotoxicity, DNA damage, and inflammatory responses in human corneal epithelial cells (HCECs) in vitro. In a set of parameters, cytotoxicity, reactive oxygen species, mitochondrial membrane potential, DNA damage, inflammatory response, and apoptosis were assessed. LPS (1, 5, and 10 μg/mL) treated HCECs were increased reactive oxygen species formation, mitochondrial membrane depolarization, and genotoxicity in a concentration-dependent manner. Similarly, NF-κB, PARP1, and TP53 were also overexpressed in the LPS treated HCECs. 24 hours after LPS induction, micronucleus scoring, and proapoptotic factors were also increased. Among them, the GADD45G, NF-κB, and γH2AX were overexpressed both on the mRNA and protein levels in LPS (10 μg/mL) treated HCECs. In our study, we show that the GADD45G signaling can trigger genotoxic instability in HCECs exposed to LPS. Therefore, understanding the factors contributing to infectious keratitis, such as GADD45G, NF-κB, and γH2AX signaling, may help to develop antigenotoxic and anti-inflammatory therapies for corneal dystrophy and epithelial cell remodeling.
The objective of the present study was to determine the effect of class I histone deacetylase (HDAC) inhibitor, mocetinostat (MGCD0103) on the expression of Npr1 (coding for natriuretic peptide receptor-A; NPRA) and renal pathology in Npr1 gene-disrupted haplotype (50% of normal) mice. Adult male Npr1 haplotype (1-copy; Npr1 +/- ), wild-type (2-copy; Npr1 +/+ ), and gene-duplicated (3-copy; Npr1 ++/+ ) mice were injected intraperitonealy with MGCD0103 (2 mg/kg) at alternate days for 2-weeks. After MGCD0103 treatment, the renal Npr1 mRNA was increased in Npr1 +/- mice by 7.3-fold (p < 0.01), Npr1 +/+ mice by 4.4-fold (p < 0.05) and Npr1 ++/+ mice by 3.6-fold (p < 0.05) compared with vehicle-treated controls. The MGCD0103 also enhanced renal cGMP levels (pmol/mg protein) in Npr1 +/- (57 ± 7 vs. control, 12 ± 1; p < 0.01), Npr1 +/+ (125 ± 9 vs. control, 66 ± 5; p < 0.01), and Npr1 ++/+ (202 ± 7 vs. control, 127 ± 15; p < 0.05) mice. An increased HDAC activity (ng/min/mg protein) was observed in Npr1 +/- mice (24.4 ± 2.8; p < 0.05); however, Npr1 ++/+ mice showed lower HDAC activity (6.4 ± 0.7; p < 0.05); compared with Npr1 +/+ mice (15.9 ± 1.3). Treatment with MGCD0103 significantly attenuated HDAC activity by almost 50% (15.3 ± 1.2; p < 0.01) in Npr1 haplotype mice. A significant decrease in systolic blood pressure was observed in MGCD0103-treated Npr1 +/- mice (106 ± 0.6 vs. control, 131 ± 1.9, p < 0.001). Significantly lower creatinine clearance (μl/min) was observed in Npr1 +/- (51 ± 11; p < 0.05) vs. Npr1 +/+ mice (130 ± 14.0; p < 0.05) and treatment with MGCD0103 increased creatinine clearance in Npr1 +/- mice (105 ± 13; p < 0.05) vs. controls. Higher urinary albumin to creatinine ratio was detected in Npr1 +/- mice (0.7 ± 0.05) vs. Npr1 +/+ animals (0.5 ± 0.05; p < 0.05) and a complete reversal was observed in drug-treated Npr1 +/- mice (0.4 ± 0.04). The present results provide direct evidence that class I HDAC inhibition upregulates NPRA expression in vivo and repairs the renal pathology in Npr1 +/- haplotype mice. The present findings will have important implications in the treatment and prevention of hypertension and renal pathophysiological conditions.
Background Exposure to cigarette smoking (CS) is a major risk factor for airway inflammation. However, little is known about the effects of CS exposure on eosinophilic rhinosinusitis with nasal polyps (ERSwNPs). Histopathological and molecular studies were performed to investigate its effects using a murine model of ERSwNPs. Methods Mice were assigned to one of the following four groups (n = 8 for each group): control group, CS exposure (CS group), ERSwNP (ERS group), and ERSwNPs exposed to CS (ERS + CS group). Histopathological changes were investigated using various stains, including hematoxylin and eosin for inflammation and polyp-like lesions, Sirius red for eosinophils, toluidine blue for mast cells, Alcian blue for goblet cells, and Masson's trichrome stain for collagen fibers. mRNA expression of cytokines from nasal mucosae was measured. Serum I and systemic cytokine levels were measured by enzyme-linked immunosorbent assays. The expression of vascular endothelial growth factor (VEGF) and hypoxia-inducible factor (HIF) 1-alpha was evaluated by immunohistochemical staining. Results The ERS + CS group showed more severe symptoms, increased the number of polyp-like lesions filtration of eosinophils, goblet cell hyperplasia, and subepithelial fibrosis, compared with the ERS group. Additionally, mRNA expressions of IL-4 and IL-17A were up-regulated in ERS + CS group and higher levels of IL-4, IL-6, IL-17A, and interferon gamma from splenocytes were observed significantly in the ERS + CS group compared with the ERS group. In the ERSwNP murine model, exposure to CS enhanced the expression of VEGF and HIF-1-alpha in nasal epithelial cells. Conclusion Chronic exposure to CS aggravated eosinophilic inflammation and promoted airway remodeling and nasal polyp formation in a murine model of ERSwNPs. The underlying mechanism might involve up-regulated expression of VEGF and HIF-1-alpha.
Atrial and brain natriuretic peptides (ANP and BNP) bind to guanylyl‐cyclase/natriuretic peptide receptor‐A (GC‐A/NPRA) and regulate sodium excretion, water balance, vasorelaxation, and steroidogenesis; all directed to combat hypertension. ANP acting through NPRA lowers blood pressure and blood volume and protects the heart and vascular disorders and end‐organ damage. The two vasoactive hormones, angiotensin II (ANG II; vasoconstrictive) and atrial natriuretic peptide (ANP; vasodilatory) antagonize the biological actions of each other. The objective of the present study was to examine whether ANG II plays critical roles in the transcriptional repression of Npr1 (encoding for NPRA) and receptor function. To delineate the mechanisms of ANG II‐mediated repression of Npr1 transcription and function, we performed the promoter deletional analyses, which showed that region −1182/−914 base pairs upstream from the transcription start site of the Npr1 promoter contains cis‐acting elements for ANG II‐regulated repression of Npr1 . ANG II significantly decreased the levels of NPRA mRNA (60%), NPRA protein (52%), and intracellular accumulation cGMP (65%) in cultured mesangial cells and attenuated ANP‐mediated relaxation of aortic rings ex vivo . The results showed that transcription factors, CREB and HSF‐4a facilitated the ANG II‐mediated repressive effects on Npr1 transcription and receptor function. The current findings suggest that ANG II‐mediated repressive mechanisms of Npr1 transcription and receptor function may provide new molecular targets for the treatment and prevention of hypertension and renal and vascular disorders. Support or Funding Information This work was supported by NIH grant HL062147
Guanylyl cyclase/natriuretic peptide receptor‐A (GC‐A/NPRA) signaling activates natriuresis, diuresis, and vasodilatation, and inhibits the cell proliferation and fibrosis. Mice lacking functional Npr1 gene (coding for GC‐A/NPRA) exhibit hypertension and kidney disorders, however, the mechanisms regulating Npr1 expression and function are not well understood. The objective of the present study was to elucidate the mechanisms of Npr1 expression and receptor function involving epigenetic regulatory mechanisms activated by class I histone deacetylase (HDAC) inhibitor, Mocetinostat (MGCD0103). Male and female (16–20 weeks old) Npr1 gene‐disrupted heterozygous (1‐copy; Npr1 +/− ), wild‐type (2‐copy; Npr1 +/+ ) and gene‐duplicated heterozygous (3‐copy; Npr1 ++/+ ) mice were injected intraperitoneally with MGCD0103 (2 mg/kg) at alternate days for 2‐weeks. The Western blot analysis showed that MGCD0103 significantly increased the renal NPRA protein levels in Npr1 +/− , Npr1 +/+ , and Npr1 ++/+ mice groups compared with vehicle‐treated controls. Female mice showed increased levels of plasma cGMP compared with male mice and treatment with MGCD0103 significantly induced cGMP levels in the drug‐treated mice groups. The renal HDAC activity and HDAC1 and 2 protein levels in male mice were higher than the corresponding female mice in all the three genotypes. MGCD0103 treatment attenuated HDAC activity by 30%‐40% and HDAC1/2 protein levels in the treated mice groups. Male and female mice showed differential acetylation levels of H3‐K9 and H3‐K27 in the renal tissues and treatment with MGCD0103 significantly enhanced the acetylation levels in the treated mice groups. Both male and female Npr1 +/− mice exhibited higher systolic blood pressure (SBP) compared with the corresponding wild type Npr1 +/+ mice, whereas female Npr1 +/− mice exhibited significantly lower SBP than male Npr1 +/− mice. A significant decrease in SBP was observed in MGCD0103‐treated male and female Npr1 +/− mice. The present results suggest that MGCD0103 epigenetically regulates NPRA expression in vivo in male and female Npr1 +/− mice, via inhibition of HDAC1 and 2 protein expression and histone modifications, which will have important implications in the hypertension and renal disease states. Support or Funding Information This work was supported by NIH grants (HL057531 and HL062147) and by an Institutional Development Award (IDeA) from the NIGMS. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .
Although several studies have claimed that mesenchymal stem cells (MSC) derived from human tissues can ameliorate allergic airway inflammation, the immunomodulatory mechanism of MSCs remains unclear.We aimed to determine the effects and the underlying mechanism of tonsil-derived MSCs (T-MSC) on allergic inflammation compared with adipose tissue-derived stem cells (ASC) in a mouse model of allergic rhinitis (AR).MSCs were isolated from human palatine tonsil (T-MSC) and the surface markers were analyzed. The effect of T-MSCs was evaluated in 24 BALB/c mice that were randomly divided into four groups (negative control group, positive control group, T-MSC group, and ASC group). MSCs were administered intravenously to ovalbumin (OVA) sensitized mice (T-MSC and ASC groups) on days 18 to 23, and subsequent OVA challenge was conducted daily from days 24 to 28. Several parameters of allergic inflammation were assessed.T-MSC and ASC had similar characteristics in surface markers. Intravenous injection of T-MSC significantly reduced allergic symptoms, eosinophil infiltration, serum total, and OVA-specific immunoglobulin E (IgE), and the nasal and systemic T-helper (Th) 2 cytokine profile. Further analysis revealed that nasal innate cytokines, such as interleukin (IL) 25 and IL-33, and chemokines, such as CCL11, CCL24, induction was suppressed in T-MSCs injected groups, which explained their underlying mechanism. In addition, the T-MSC group had more inhibition of allergic inflammation than did the ASC group, which might be attributed to the more proliferative activity of T-MSC.Administration of T-MSC effectively reduced allergic symptoms and inflammatory parameters in the mouse model of AR. T-MSC treatment reduced Th2 cytokines and OVA-specific IgE secretion from B cells. In addition, innate cytokine (IL-25 and IL-33) expression and eotaxin messenger RNA expression was inhibited in the nasal mucosa, which is suggestive of the mechanism of reduced allergic inflammation. Therefore, T-MSC treatment is potentially an alternative therapeutic modality in AR.
Antibiotic-resistant bacterial colonies mitigate rapid biofilm formation and have complex cell wall fabrications, making it challenging to penetrate drugs across their biofilm barriers. The objective of this study was to investigate the antibacterial susceptibility of antibiotic-resistant bacteria and contact lens barrenness. Nilavembu Choornam–Gold Nanoparticles (NC–GNPs) were synthesized using NC polyherbal extract and characterized by UV-visible spectrophotometer, SEM-EDX, XRD, Zeta sizer, FTIR, and TEM analysis. Contact lenses with overnight cultures of antibiotic-resistant bacteria K. pneumoniae and S. aureus showed significant differences in growth, biofilm formation, and infection pathogenicity. The NC–GNPs were observed in terms of size (average size is 57.6 nm) and surface chemistry. A zone of inhibition was calculated for K. pneumoniae 18.8 ± 1.06, S. aureus 23.6 ± 1.15, P. aeruginosa 24.16 ± 0.87, and E. faecalis 24.5 ± 1.54 mm at 24 h of NC–GNPs alone treatment. In electron microscopy studies, NC–GNP-treated groups showed nuclear shrinkage, nuclear disintegration, degeneration of cell walls, and inhibited chromosomal division. In contrast, normal bacterial colonies had a higher number of cell divisions and routinely migrated toward cell multiplications. NC–GNPs exhibited antibacterial efficacy against antibiotic-resistant bacteria when compared to NC extract alone. We suggest that NC–GNPs are highly valuable to the population of hospitalized patients and other people to reduce the primary complications of contact lens contamination-oriented microbial infection and the therapeutic efficiency of antibiotic-resistant bacterial pathogenicity.
Ultraviolet radiation is an environmental carcinogenic agent that enhances inflammation and immunological reactions in the exposed human skin cells leading to oxidative photoaging of the epidermal and dermal segment. In the present study, we investigated the protective role of ursolic acid (UA) against ultraviolet B (UVB) radiation- induced photoaging an in vitro model of human skin dermal fibroblasts. UA-pretreated human skin dermal fibroblast (HDF) cells were exposed to UVB radiation to evaluated cell viability, reactive oxygen species (ROS), mitochondrial membrane potential, lipid peroxidation, antioxidant status, DNA damage, proinflammatory response, apoptotic induction, and matrix metalloproteinase (MMP) alteration. The UA pretreatment of HDFs mitigated the UVB irradiation-induced cytotoxicity, ROS generation, and mitochondrial membrane potential alteration and lipid peroxidation, depletion of antioxidant status, DNA damage, and apoptotic induction. UA pretreatment of HDFs also attenuated the UVB-induced expression of inflammatory (TNF-α and NF-κB) and apoptotic (p53, Bax, and caspase-3) and MMPs (MMP-2 and MMP-9) and enhanced the Bcl-2 protein levels in 20 μM UA treatment, when compared to concentrations. Hence, these results revealed that UA has the potential to mitigate UVB-induced extracellular damage by interfering with the ROS-mediated apoptotic induction and photoaging senescence and thus is a potential therapeutic agent to protect the skin against UVB-irradiation induced photooxidative damage.
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