Hyperosmotic Stress-Induced TRPM2 Channel Activation Stimulates NLRP3 Inflammasome Activity in Primary Human Corneal Epithelial Cells.

Purpose: The purpose of this study was to determine whether either a hyperosmotic or oxidative stress induces NLRP3 inflammasome activation and increases in bioactive IL-1ß secretion through transient receptor potential melastatin 2 (TRPM2) activation in primary human corneal epithelial cells (PHCECs). Methods: Real-time PCR, Western blots, and immunofluorescent staining were used to evaluate TRPM2 and NLRP3, ASC, caspase-1, and IL-1ß mRNA and protein expression levels, respectively. A CCK-8 assay evaluated cell viability. Hyperosmotic 500 mOsm and oxidative 0.5 mM H2O2 stresses were imposed. TRPM2 expression was inhibited with a TRPM2 inhibitor, 20 µM N-(p-amylcinnamoyl) anthranilic acid (ACA), or TRPM2 siRNA knockdown. Results: In the hypertonic medium, TRPM2, NLRP3, ASC, caspase-1, and IL-1ß gene and protein expression levels rose after 4 hours (P ≤ 0.043), whereas ACA preincubation suppressed these rises (P ≤ 0.044). Similarly, H2O2 upregulated TRPM2 protein expression by 80%, and induced both NLRP3 inflammasome activation and increased bioactive IL-1ß secretion (P ≤ 0.036), whereas ACA pretreatment suppressed these effects (P ≤ 0.029). TRPM2 siRNA transfection reduced TRPM2 gene expression by 70% (P = 0.018) in this hyperosmotic medium and inhibited the increases in NLRP3, caspase-1, and IL-1ß gene (P ≤ 0.028) and protein expression (P ≤ 0.037). Conclusions: TRPM2 activation by either a hyperosmotic or oxidative stress contributes to mediating increases in NLRP3 inflammasome activity and bioactive IL-1ß expression because inhibiting TRPM2 activation or its expression blunted both of these responses in PHCECs. This association points to the possibility that TRPM2 is a viable target to suppress hyperosmotic-induced corneal epithelial inflammation.

Myeloid differentiation protein 2 induced retinal ischemia reperfusion injury via upregulation of ROS through a TLR4-NOX4 pathway.

Retinal ischemia reperfusion (I/R) injury is common in many ophthalmic diseases. Recent studies have shown that toll-like receptor 4 (TLR4) is involved in ischemic retinal injury. Activation of TLRs requires specific accessory proteins such as myeloid differentiation protein 2 (MD2), which facilitate in ligand responsiveness. Therefore, inhibiting MD2 may be a novel approach to modulate TLR4 signaling and deleterious downstream effects in ischemic retinal injury. We used human Müller MIO-M1 cells treated with tert-butyl hydroperoxide (TBHP) to establish an in vitro I/R model of oxidative injury and tested the therapeutic effect of inhibiting MD2. Furthermore, we inhibited MD2 in a mouse model of retinal I/R injury and confirmed the results using MD2 knockout mice. Our studies show that pharmacological inhibition of MD2 prevented TBHP-induced reactive oxygen species (ROS) generation, inflammation and subsequent apoptosis in Müller cells. We also show that retinal I/R injury in mice induced functional deficits, increased ROS levels, inflammation and apoptosis. These pathological changes were not observed in MD2 knockout mice and attenuated when MD2 was inhibited in wildtype mice. In addition, we discovered that the mechanism of these therapeutic effects involved regulation of NADPH oxidase 4 (NOX4)-MD2-TLR4 complex formation. This study provides evidence that MD2 plays a key role in the pathogenesis of retinal I/R damage by participating in TLR4-NOX4 complex formation and elaboration of oxidative and inflammatory damage. Hence, inhibition of MD2 may reduce TLR-dependent damage during retinal I/R injury.

Hyperosmolarity-induced AQP5 upregulation promotes inflammation and cell death via JNK1/2 Activation in human corneal epithelial cells.

Tear film hyperosmolarity and anterior ocular inflammation are two clinical signs that may be indicative of dry eye disease (DED). This condition can cause pathological and functional changes to the anterior ocular surface tissues. A contributing factor may be dysfunctional aquaporin 5 (AQP5) water channels as they are the AQP subtype that expressed in the corneal epithelium and contribute to fluid efflux needed for corneal function. We determined if described hyperosmolarity-induced increases in proinflammatory cytokine expression and cell death are mediated through AQP5 upregulation and JNK1/2 MAPK signaling activation in both primary human corneal epithelial cells (HCECs), and in a HCEC line. Real time RT-PCR identified rises in IL-1ß, IL-6, IL-8, TNF-α, caspase-1, and AQP5 mRNA levels upon step increases in osmolarity up to 550 mOsm. Western blot analysis and the TUNEL assay identified corresponding rises in AQP5 and p-JNK1/2 protein expression and cell death respectively. JNK1/2 inhibition with SP600125, or siRNA AQP5 gene silencing reduced hypertonic-induced rises in proinflammatory cytokine expression and cell death. Taken together, hypertonicity-induced AQP5 upregulation leads to increases in proinflammatory cytokine expression and cell death through JNK1/2 MAPK activation. These results suggest that drug targeting AQP5 upregulation may be a therapeutic option in DED management.

High-intensity corneal collagen crosslinking with riboflavin and UVA in rat cornea.

Corneal collagen cross-linking (CXL) halts human corneal ectasias progression by increasing stromal mechanical stiffness. Although some reports describe that this procedure is effective in dealing with some infectious and immunologic corneal thinning diseases, there is a need for more animal models whose corneal thickness more closely resemble those occurring in these patients. To meet this need, we describe here high-intensity protocols that are safe and effective for obtaining CXL in rat corneas. Initially, a range of potentially effective UVA doses were evaluated based on their effectiveness in increasing tissue enzymatic resistance to dissolution. At UVA doses higher than a threshold level of 0.54 J/cm2, resistance to enzymatic digestion increased relative to that in non-irradiated corneas. Based on the theoretical threshold CXL dose, a CXL regimen was established in which the UVA tissue irradiance was 9 mW/cm2, which was delivered at doses of either 2.16, 2.7 or 3.24 J/cm2. Their dose dependent effects were evaluated on ocular surface morphological integrity, keratocyte apoptotic frequency, tissue thickness and endothelial cell layer density. Doses of 2.16 and 2.7 J/cm2 transiently decreased normal corneal transparency and increased thickness. These effects were fully reversed after 14 days. In contrast, 3.24 J/cm2 had more irreversible side effects. Three days after treatment, apoptotic frequency in the CXL-2.16 group was lower than that at higher doses. Endothelial cell losses remained evident only in the CXL-3.24 group at 42 days posttreatment. Stromal fiber thickening was evident in all the CXL-treated groups. We determined both the threshold UVA dose using the high-intensity CXL procedure and identified an effective dose range that provides optimal CXL with minimal transient side effects in the rat cornea. These results may help to provide insight into how to improve the CXL outcome in patients afflicted with a severe corneal thinning disease.

C-MYC-induced upregulation of lncRNA SNHG12 regulates cell proliferation, apoptosis and migration in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is one of the most aggressive subtypes of breast cancer, with a significantly higher recurrence and mortality rate. There is an urgent need to uncover the mechanism underlying TNBC and establish therapeutic targets. Long non-coding RNAs (lncRNAs) are involved in a series of biological functions and provide novel insights into the molecular mechanism of cancer. Based on their expression specificity and large number, lncRNAs are likely to serve as the basis for clinical applications in oncology. In our previous study, we utilized RNA sequencing (RNA-seq) to explore the lncRNAs expression profiles in TNBC and identified that small nucleolar RNA host gene 12 (SNHG12) was remarkably increased in TNBC. However, the role of SNHG12 in TNBC has not been clarified. Herein, we determine that SNHG12 is upregulated in TNBC, and its high expression is significantly correlated with tumor size and lymph node metastasis. Mechanistic investigations show that SNHG12 is a direct transcriptional target of c-MYC. Silencing SNHG12 expression inhibits TNBC cells proliferation and apoptosis promotion, whereas SNHG12 overexpression has the opposite effect. In addition, we reveal that SNHG12 may promote cells migration by regulating MMP13 expression. To the best of our knowledge, it is the first report indicating that SNHG12 is involved in breast cancer. Taken together, our findings suggest that SNHG12 contributes to the oncogenic potential of TNBC and may be a promising therapeutic target.