Guizhi Fuling Decoction inhibiting the PI3K and MAPK pathways in breast cancer cells revealed by HTS2 technology and systems pharmacology.

As one of the classical traditional Chinese medicine (TCM) prescriptions in treating gynecological tumors, Guizhi Fuling Decoction (GFD) has been used to treat breast cancer (BRCA). Nonetheless, the potential molecular mechanism remains unclear so far. Therefore, systems pharmacology was used in combination with high throughput sequencing-based high throughput screening (HTS2) assay and bioinformatic technologies in this study to investigate the molecular mechanisms of GFD in treating BRCA. By computationally analyzing 76 active ingredients in GFD, 38 potential therapeutic targets were predicted and significantly enriched in the "pathways in cancer". Meanwhile, experimental analysis was carried out to examine changes in the expression levels of 308 genes involved in the "pathways in cancer" in BRCA cells treated by five herbs of GFD utilizing HTS2 platform, and 5 key therapeutic targets, including HRAS, EGFR, PTK2, SOS1, and ITGB1, were identified. The binding mode of active compounds to these five targets was analyzed by molecular docking and molecular dynamics simulation. It was found after integrating the computational and experimental data that, GFD possessed the anti-proliferation, pro-apoptosis, and anti-angiogenesis activities mainly through regulating the PI3K and the MAPK signaling pathways to inhibit BRCA. Besides, consistent with the TCM theory about the synergy of Cinnamomi Ramulus (Guizhi) by Cortex Moutan (Mudanpi) in GFD, both of these two herbs acted on the same targets and pathways. Taken together, the combined application of computational systems pharmacology techniques and experimental HTS2 platform provides a practical research strategy to investigate the functional and biological mechanisms of the complicated TCM prescriptions.

PF4 antagonizes retinal neovascularization via inhibiting PRAS40 phosphorylation in a mouse model of oxygen-induced retinopathy.

Retinal neovascularization (RNV) is a common pathology of blinding proliferative retinopathies. The current treatments to RNV, however, are hindered by limited efficacy, side effects, and drug resistance. A naturally-occurring cytokine in retina that is amicable to immune system and possesses robust anti-neovascular function would facilitate to overcome the hurdles. In this study, retinas from a mouse model of oxygen-induced retinopathy (OIR) underwent a protein array to screen the naturally-occurring cytokines that may antagonize RNV. Among the 62 angiogenesis-associated cytokines, platelet factor 4 (Pf4) stood out with the most prominent upregulation and statistical significance. Moreover, an intravitreal injection of mouse Pf4 demonstrated dramatic anti-vaso-obliteration and anti-neovascularization effects dose dependently in the OIR model; whereas human PF4 inhibited the proliferation, migration, and tubulogenesis of monkey retinal vascular endothelial cells treated with VEGF and TNF-α. These previously undescribed angiostatic effects of PF4 in OIR retinas and retinal vascular endothelial cells support translation of this naturally-occurring chemokine into a therapeutic modality to RNV supplementary to the anti-VEGFs. Mechanistically, a phosphorylation array and western blots indicated that downregulation of proline-rich Akt substrate of 40 kDa (Pras40) and its phosphorylation were necessary for Pf4's anti-neovascular effects in the OIR retinas. Indeed, overexpression of the wildtype Pras40 and the mutant version with deficient phosphorylation abolished and mimicked the Pf4's angiostatic effects in the OIR retinas, respectively. The similar effects were also observed in vitro. This study, for the first time, links PF4's anti-RNV function to an intracellular signaling molecule PRAS40 and its phosphorylation.

The efficacy and safety of aflibercept and conbercept in diabetic macular edema.

Diabetic macular edema (DME) has shown an increasing prevalence during the past years and is the leading cause of diabetic retinopathy blindness. Traditional treatment modalities include laser and corticosteroid therapy, which, however, either act through unclear mechanisms or cause cataracts and elevated intraocular pressure. In recent years, as the pathogenic role of VEGF in DME has been well-recognized, the intravitreal injection of anti-VEGF drugs has become the first-line treatment of DME due to their great efficacy in improving visual acuity and mitigating macular edema. Advantages have been shown for aflibercept and conbercept, the two recombinant decoy receptors that can bind VEGF with high specificity and affinity, in DME treatment in clinical trials conducted both worldwide and in People's Republic of China. This review introduces the structural characteristics and molecular mechanisms of action of these two anti-VEGF drugs, and summarizes the clinical trials evaluating their efficacy and safety, with the hope to provide clues for designing optimal and personalized therapeutic regimens for DME patients.

Α-Melanocyte-Stimulating Hormone Protects Early Diabetic Retina from Blood-Retinal Barrier Breakdown and Vascular Leakage via MC4R.

BACKGROUND/AIMS: Blood-retinal barrier (BRB) breakdown and vascular leakage is the leading cause of blindness of diabetic retinopathy (DR). Hyperglycemia-induced oxidative stress and inflammation are primary pathogenic factors of this severe DR complication. An effective interventional modality against the pathogenic factors during early DR is needed to curb BRB breakdown and vascular leakage. This study sought to examine the protective effects of α-Melanocyte-stimulating hormone (α-MSH) on early diabetic retina against vascular hyperpermeability, electrophysiological dysfunction, and morphological deterioration in a rat model of diabetes and probe the mechanisms underlying the α-MSH's anti-hyperpermeability in both rodent retinas and simian retinal vascular endothelial cells (RF6A). METHODS: Sprague Dawley rats were injected through tail vein with streptozotocin to induce diabetes. The rats were intravitreally injected with α-MSH or saline at Week 1 and 3 after hyperglycemia. In another 2 weeks, Evans blue assay, transmission electron microscopy, electroretinogram (ERG), and hematoxylin and eosin (H&E) staining were performed to examine the protective effects of α-MSH in diabetic retinas. The expression of pro-inflammatory factors and tight junction at mRNA and protein levels in retinas was analyzed. Finally, the α-MSH's anti-hyperpermeability was confirmed in a high glucose (HG)-treated RF6A cell monolayer transwell culture by transendothelial electrical resistance (TEER) measurement and a fluorescein isothiocyanate-Dextran assay. Universal or specific melanocortin receptor (MCR) blockers were also employed to elucidate the MCR subtype mediating α-MSH's protection. RESULTS: Evans blue assay showed that BRB breakdown and vascular leakage was detected, and rescued by α-MSH both qualitatively and quantitatively in early diabetic retinas; electron microscopy revealed substantially improved retinal and choroidal vessel ultrastructures in α-MSH-treated diabetic retinas; scotopic ERG suggested partial rescue of functional defects by α-MSH in diabetic retinas; and H&E staining revealed significantly increased thickness of all layers in α-MSH-treated diabetic retinas. Mechanistically, α-MSH corrected aberrant transcript and protein expression of pro-inflammatory factor and tight junction genes in the diseased retinas; moreover, it prevented abnormal changes in TEER and permeability in HG-stimulated RF6A cells, and this anti-hyperpermeability was abolished by a universal MCR blocker or an antagonist specific to MC4R. CONCLUSIONS: This study showed previously undescribed protective effects of α-MSH on inhibiting BRB breakdown and vascular leakage, improving electrophysiological functions and morphology in early diabetic retinas, which may be due to its down-regulating pro-inflammatory factors and augmenting tight junctions. α-MSH acts predominantly on MC4R to antagonize hyperpermeability in retinal microvessel endothelial cells.

Decoding Noncoding RNAs: Role of MicroRNAs and Long Noncoding RNAs in Ocular Neovascularization.

Ocular neovascularization is a pathological sequel of multiple eye diseases. Based on the anatomical site into which the abnormal neovessels grow, ocular neovascularization can be categorized into corneal neovascularization, choroidal neovascularization, and retinal neovascularization. Each category is intractable, and may lead to blindness if not appropriately treated. However, the current therapeutic modalities, including laser photocoagulation, vitrectomy surgery, and anti-VEGF drugs, raise concerns due to limited efficacy, damage on retinal parenchyma and vasculature, and the patients' unresponsiveness to the treatments. Therefore, the in-depth study on pathogenesis of and the search for novel therapeutic targets to the ocular neovascularization are needed. During the last 10 years or so, a large number of literatures have emerged indicating a critical role of noncoding RNAs, particularly microRNAs (miRNAs) and long noncoding RNAs (lncRNAs), in the pathogenesis and regulation of the ocular neovascularization. This review summarizes the current understanding of the biosynthesis and functions of the miRNAs and lncRNAs, the regulation of the miRNAs and lncRNAs in neovascular eye diseases, as well as the roles of these noncoding RNAs in the disease models of ocular neovascularization, in the hope that it could provide clues for the pathogenesis of and molecular targets to the ocular neovascularization.