Amphibian-derived peptide RL-RF10 ameliorates paraquat-induced pulmonary fibrosis injury.

Pulmonary fibrosis is the result of dysfunctional repair after lung tissue injury, characterized by fibroblast proliferation and massive extracellular matrix aggregation. Once fibrotic lesions develop, effective treatment is difficult, with few drugs currently available. Here, we identified a short cyclic decapeptide RL-RF10 derived from frog skin secretions as a potential novel lead molecule for the amelioration of pulmonary fibrosis. In vivo experiments indicated that RL-RF10 treatment ameliorated lung histopathological damage and fibrogenesis after paraquat (PQ) induction in a concentration-dependent manner. On day 7, bronchoalveolar lavage fluid assays performed on mice showed that RL-RF10 exerted anti-inflammatory effects by decreasing the expression of inflammation-related factors, including transforming growth factor-ß1 (TGF-ß1) and tumor necrosis factor-α, in lung tissue. In addition, RL-RF10 down-regulated the levels of collagen I, collagen III, and vimentin, while increasing the expression of E-cadherin to inhibit epithelial-mesenchymal transition. Further research demonstrated that the SMAD2/3 signaling pathway, which is strongly linked to TGF-ß1, played a critical function in enhancing the pulmonary fibrosis relief achieved by RL-RF10. Both in vivo and in vitro assays showed that RL-RF10 treatment led to a significant reduction in the phosphorylation levels of SMAD2 and SMAD3 following PQ induction. Overall, we investigated the protective effects and underlying mechanisms of the RL-RF10 peptide against pulmonary fibrosis and demonstrated its potential as a novel therapeutic drug candidate for the treatment of pulmonary fibrotic diseases.

Peptide OA-VI12 restrains melanogenesis in B16 cells and C57B/6 mouse ear skin via the miR-122-5p/Mitf/Tyr axis.

Excessive melanogenesis leads to hyperpigmentation, which is one of the common skin conditions in humans. Existing whitening cosmetics cannot meet market needs due to their inherent limitations. Thus, the development of novel skin-whitening agents continues to be a challenge. The peptide OA-VI12 from the skin of amphibians at high altitude has attracted attention due to its remarkable anti light damage activity. However, whether OA-VI12 has the skin-whitening effect of inhibiting melanogenesis is still. Mouse melanoma cells (B16) were used to study the effect of OA-VI12 on cell viability and melanin content. The pigmentation model of C57B/6 mouse ear skin was induced by UVB and treated with OA-VI12. Melanin staining was used to observe the degree of pigmentation. MicroRNA sequencing, quantitative real-time PCR (qRT-PCR), immunofluorescence analysis and Western blot were used to detect the change of factor expression. Double luciferase gene report experiment was used to prove the regulatory relationship between miRNA and target genes. OA-VI12 has no effect on the viability of B16 cells in the concentration range of 1-100 µM and significantly inhibits the melanin content of B16 cells. Topical application of OA-VI12, which exerted transdermal potency, prevented UVB-induced pigmentation of ear skin. MicroRNA sequencing and double luciferase reporter analysis results showed that miR-122-5p, which directly regulated microphthalmia-associated transcription factor (Mitf), had significantly different expression before and after treatment with OA-VI12. Mitf is a simple helix loop and leucine zipper transcription factor that regulates tyrosinase (Tyr) expression by binding to the M-box promoter element of Tyr. qRT-PCR, immunofluorescence analysis and Western blot showed that OA-VI12 up-regulated the expression of miR-122-5p and inhibited the expression of Mitf and Tyr. The effects of OA-VI12 on melanogenesis inhibition in vitro and in vivo may involve the miR-122-5p/Mitf/tyr axis. OA-VI12 represents the first report on a natural amphibian-derived peptide with skin-whitening capacity and the first report of miR-122-5p as a target for regulating melanogenesis, thereby demonstrating its potential as a novel skin-whitening agent and highlighting amphibian-derived peptides as an underdeveloped resource.

OL-FS13 Alleviates Cerebral Ischemia-reperfusion Injury by Inhibiting miR-21-3p Expression.

BACKGROUND: OL-FS13, a neuroprotective peptide derived from Odorrana livida, can alleviate cerebral ischemia-reperfusion (CI/R) injury, although the specific underlying mechanism remains to be further explored. OBJECTIVE: The effect of miR-21-3p on the neural-protective effects of OL-FS13 was examined. METHODS: In this study, the multiple genome sequencing analysis, double luciferase experiment, RT-qPCR, and Western blotting were used to explore the mechanism of OL-FS13. RESULTS: Showed that over-expression of miR-21-3p against the protective effects of OL-FS13 on oxygen- glucose deprivation/re-oxygenation (OGD/R)-damaged pheochromocytoma (PC12) cells and in CI/R-injured rats. miR-21-3p was then found to target calcium/calmodulin-dependent protein kinase 2 (CAMKK2), and its overexpression inhibited the expression of CAMKK2 and phosphorylation of its downstream adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK), thereby inhibiting the therapeutic effects of OL-FS13 on OGD/R and CI/R. Inhibition of CAMKK2 also antagonized up-regulated of nuclear factor erythroid 2-related factor 2 (Nrf-2) by OL-FS13, thereby abolishing the antioxidant activity of the peptide. CONCLUSION: Our results showed that OL-FS13 alleviated OGD/R and CI/R by inhibiting miR-21-3p to activate the CAMKK2/AMPK/Nrf-2 axis.

miR-6315 silencing protects against spinal cord injury through the Smo and anti-ferroptosis pathway.

Spinal cord injury (SCI) causes permanent damage and has a high disability rate. Currently, no efficient therapeutic strategy is available for SCI. The present study investigated the mechanisms of microRNAs (miRNAs) in rats with spinal cord injury. Whole transcriptome sequencing (WTS) was used for analyzing miRNA and messenger RNA (mRNA) expression patterns in rat spinal cord tissue at different time points after SCI. Gene Ontology (GO) and KEGG pathways were analyzed to obtain crucial functional pathways. miR-6315 was the most significantly up-regulated and differentially expressed miRNA after 24 h of SCI; the expression of miR-6315 gradually decreased after 3 and 7 days of SCI. Bioinformatics analysis was conducted to predict the targeting relation of miR-6315 with Smo, and qRT-PCR and dual-luciferase reporter assays were conducted for verification. The miR-6315 silencing (miR-6315-si) adenovirus was successfully constructed. miR-6315 knockdown treatment significantly promoted functional behavioral recovery in rats post-SCI through using Basso-Beattie-Bresnahan (BBB) locomotor rating scale and the inclined plane test. The neuronal axon regeneration and neuronal migration were promoted, and cell apoptosis was attenuated in treated SCI rats and Glu-treated neurons after miR-6315 knockdown using immunofluorescence and scratch assays. We discovered that Smo and anti-ferroptosis pathway factors, xCT, GSH, and GPX4, may be involved in miR-6315-regulated SCI repair. The expression of miR-6315 was negatively correlated with Smo, xCT, GSH, and GPX4. In conclusion, miR-6315 may be a potential target in the treatment of SCI.

Peptide RL-QN15 promotes regeneration of epidermal nerve fibers and recovery of sensory function in diabetic skin wounds.

Epidermal nerve fiber regeneration and sensory function are severely impaired in skin wounds of diabetic patients. To date, however, research on post-traumatic nerve regeneration and sensory reconstruction remains scarce, and effective clinical therapeutics are lacking. In the current study, localized treatment with RL-QN15, considered as a drug candidate for intervention in skin wounds in our previous research, accelerated the healing of full-thickness dorsal skin wounds in diabetic mice and footpad skin wounds in diabetic rats. Interestingly, nerve density and axonal plasticity in the skin wounds of diabetic rats and mice, as well as plantar sensitivity in diabetic rats, were markedly enhanced by RL-QN15 treatment. Furthermore, RL-QN15 promoted the proliferation, migration, and axonal length of neuron-like PC12 cells, which was likely associated with activation of the phosphatidylinositol-3 kinase/protein kinase B (PI3K/Akt) signaling pathway. The therapeutic effects of RL-QN15 were partially reduced by blocking the PI3K/Akt signaling pathway with the inhibitor LY294002. Thus, RL-QN15 showed positive therapeutic effects on the distribution of epidermal nerve fibers and stimulated the recovery of sensory function after cutaneous injury. This study lays a solid foundation for the development of RL-QN15 peptide-based therapeutics against diabetic skin wounds.

MicroRNA-138 Regulates Spinal Cord Development by Activating the Shh in Fetal Rats.

INTRODUCTION: Dysregulation of spinal cord development can lead to serious neuronal damage and dysfunction, causing significant health problems in newborns. MiRNA-138 appears to be crucial for proliferation, differentiation, and apoptosis of cells. However, the regulation of miRNA-138 and downstream molecules in embryonic spinal cord development remain elusive. The aim of this experiment is to determine whether overexpression of miRNA-138 or RNA interference (RNAi) can regulate the development of spinal cord in fetal rats. METHODS: Two plasmid vectors including pLenti-III-mico-GFP (miRNA-138 open reading frame [ORF]) and pLenti-III-miR-Off (miRNA-138 short hairpin) were constructed and injected into the tail vein of rats on the 14th day of pregnancy. Hematoxylin-eosin (HE) staining was used to observe the cell morphology. QRT-PCR, Western blot, and immunostaining confirmed the regulatory relationship between miRNA-138 and downstream molecules sonic hedgehog (Shh). RESULTS: Overexpression of miRNA-138 increased neuron regeneration significantly and decreased neuronal apoptosis when compared with the control. Silencing of miRNA-138 increased neuronal apoptosis and spinal cord atrophy significantly. Furthermore, miRNA-138 ORF treatment effectively increased the expression level of miRNA-138 and also upregulated the level of Shh. Comparatively, knockdown of miRNA-138 downregulated Shh levels in myelodysplastic regions. CONCLUSION: These findings indicated that miRNA-138 overexpression could protect the spinal cord development of fetal rats, and the underlying mechanisms were associated with Shh expression. The present study provides a novel strategy to promote the molecular mechanism of embryonic spinal cord development.

A novel amphibian-derived peptide alleviated ultraviolet B-induced photodamage in mice.

Although the application potential of amphibian skin-derived active peptides in alleviating ultraviolet B (UVB)-induced damage has attracted increasing attention, research remains in its infancy. In this study, a new peptide (OM-GL15, GLLSGHYGRASPVAC) was identified from the skin of the green odorous frog (Odorrana margaretae). Results showed that OM-GL15 scavenged free radicals (2,2'-diazo-bis-3-ethylbenzothiazoline-6-sulfonic acid and 1,1-diphenyl-2-trinitrophenylhydrazine) and reduced Fe3+ to Fe2+. Moreover, topical administration of OM-GL15 significantly alleviated UVB-induced skin photodamage in mice. Exploration of the underlying mechanisms further showed that OM-GL15 exerted antioxidant potency. Specifically, the peptide reduced the levels of lipid peroxidation and malondialdehyde and protected epidermal cells from UVB-induced apoptosis by inhibiting DNA damage via down-regulation of p53, caspase-3, caspase-9, and Bax and up-regulation of Bcl-2. Our results highlight the potential application of amphibian skin-derived peptides in protection against UVB-induced photodamage and provide a novel peptide candidate for the development of anti-photodamage agents.

A novel peptide from the skin of amphibian Rana limnocharis with potency to promote skin wound repair.

In clinical trials, the healing of wounds remains a substantial physiological and financial incumbrance on patients. Therefore, the development of new drugs that can accelerate wound healing is vital. Based on genomic methods, we identified a new peptide (RL-RL10) with the amino acid sequence 'RLFKCWKKDS' from the skin of an amphibian frog species (Rana limnocharis). RL-RL10 promoted wound healing of human keratinocytes (HaCaT) in a concentration-dependent manner. RL-RL10 also had an effect on the migration and proliferation of HaCaT cells and promoted healing of a full-thickness wound in mice in a dose-dependent manner. In conclusion, we discovered RL-RL10 that promoted healing activity of cellular and animal wounds, thus providing a new peptide template for the development of novel wound-repairing drugs.

Effect and Molecular Mechanisms of Collateral Vessel Growth Mediated by Activation of Transient Receptor Potential Vanilloid Type 1.

Information on the function of transient receptor potential vanilloid 1 (TRPV1) in arteriogenesis is limited. We aimed to verify whether TRPV1 is involved in collateral vessel growth in rat hind limbs and elucidate the possible subcellular action mechanisms. Adult Sprague Dawley rats were chosen to establish the hind limb ischemic model and treatment with capsaicin. Angiographies were performed, and tissue was isolated for immunohistochemistry. In vitro, rat aortic endothelial cells (RAECs) were treated with capsaicin and antagonist capsazepine. The RAEC proliferation was determined, and the protein and mRNA levels of Ca2+-dependent transcription factors were assessed. In vivo, the collateral vessels exhibited positive outward remodeling characterized by enhanced inflammatory cell/macrophage accumulation in the adventitia and activated cell proliferation in all layers of the vascular wall and elevated endothelial NO synthetase expression in the rats with hind limb ligation. In RAECs, TRPV1 activation-induced Ca2+-dependent transcriptional factors, nuclear factor of activated T cells 1, calsenilin and myocyte enhancer factor 2C increase, and augmented RAEC proliferation could be a subcellular mechanism for TRPV1 in endothelial cells and ultimately contribute to collateral vessel growth. TRPV1, a novel candidate, positively regulates arteriogenesis, meriting further studies to unravel the potential therapeutic target leading to improved collateral vessel growth for treating ischemic diseases.