Corylin accelerated wound healing through SIRT1 and PI3K/AKT signaling: a candidate remedy for chronic non-healing wounds

Introduction: Chronic non-healing wound is a considerable clinical challenge and research into the discovery of novel pro-healing agents is underway as existing therapeutic approaches cannot sufficiently meet current needs.Method: We studied the effects of corylin in cell line fibroblasts and macrophages by Western blots, PCR, Flow cytometry assay, Immunofluorescence.Results: We showed that corylin, a main flavonoid extracted from Psoralea corylifolia L, reduced inflammatory responses, promoted collagen deposition, and accelerated the healing of full-thickness skin wounds in mice. Exploration of the underlying mechanisms showed that corylin activated the PI3K/AKT signaling, leading to fibroblasts’ migration, proliferation, and scratch healing. Corylin also activated sirtuin 1 (SIRT1) signaling, enhanced the deacetylation and cytoplasmic translocation of NF-κB p65, and therefore reduced lipopolysaccharide (LPS)-induced inflammatory responses in macrophages. Furthermore, inhibition of PI3K/AKT and sirtuin 1 pathway with LY294002 and EX527 prevent the therapeutic potency of corylin against chronic wounds.Conclusion: In summary, our results suggested that corylin may be a candidate for the development of novel pro-healing agents

Pedigree of this ADCC family.

<p>Cataract pedigree. Squares and circles symbolize males and females, respectively. Black and white lines denote affected status and unaffected status, respectively. A solid black arrow indicates the proband (III:1), and the diagonal lines indicate deceased family members.</p

Fluorescence microscopy and Western blot analysis.

<p>(A) pEGFP-N1, Cx46WT, Cx46T148I and Cx46G143R EGFP-tagged proteins were located in the cytoplasm and plasma membrane of normal-density HLECs. However, Cx46T148I as well as the reported Cx46-G143R showed aggregate signals from cytoplasmic inclusions in fluorescent images rather than the typical punctate staining in the WT proteins. (B) Cx46T148I and Cx46-G143R significantly increased the intracellular aggregation. (C) and (D) Western blot analysis of the cell lysates indicated a higher expression level of mutant Cx46/Flag than the WT or control lanes. Scale bar:20 μm.</p

Hydrophilicity analysis of the WT and mutant proteins as well as homologous modeling predicted by the Swiss-Model program.

<p>(A) The hydropathic character of the changes in the mutant protein indicated a higher hydrophobicity than that of the WT. (B) The conformation of mutant Cx46 underwent a significant change with an α-helix deletion in the C-terminal when compared with that of the WT model. The α-helix deletion was marked in red circle and the locations of amino acid 148 were marked in red.</p

Photographs of affected individuals in this family.

<p>(A) the proband (III:1), (B) her older brother (III:3) and (C) the proband’s son (IV:5) showed bilateral pulverulent nuclear cataracts.</p

T148 is a well-conserved amino acid of Cx46 and <i>GJA3</i>/p. T148I is a novel mutation with co-segregation in this family.

<p>(A) <i>GJA3</i>/p. T148I is located at the cytoplasmic loop (indicated by the blue square) domain of the Cx46 protein. The membrane topological structure of Cx46 was generated by TOPO2 software. This mutation (indicated by the red square) is located in the cytoplasmic loop domain; the black square indicates reported mutations associated with CCs (E: extracellular; M: membrane; I: intracellular). (B) Sanger sequencing results showed that this missense mutation was detected in all affected individuals and was not shown in unaffected family members or in the 100 unrelated control individuals. (C) Multiple protein sequence alignments. Multiple sequence alignments of <i>GJA3</i> from different species and <i>GJA</i> family members (including <i>GJA1</i>, <i>GJA5</i>, and <i>GJA8</i>) from a human revealed that codon 148, where the mutation (p. T148I) occurred, was located within a highly conserved region. The “mut.” sequence indicates the sequence with the mutation detected in this family.</p

Identification of a novel <i>GJA3</i> mutation in a large Chinese family with congenital cataract using targeted exome sequencing

<div><p>Autosomal dominant congenital cataract (ADCC) is a clinically and genetically heterogeneous ocular disease in children that results in serious visual impairments or even blindness. Targeted exome sequencing (TES) is an efficient method used for genetic diagnoses of inherited diseases. In the present study, we used a custom-made TES panel to identify the genetic defect of a four-generation Chinese family with bilateral pulverulent nuclear cataracts. A novel heterozygous missense mutation c.443C>T (p. T148I) in <i>GJA3</i> was identified. The results of the bioinformatic analysis showed that the mutation was deleterious to the structure and hemichannel function of Cx46 encoded by <i>GJA3</i>. Plasmids expressing wild-type and mutant human Cx46 were constructed and ectopically expressed in human lens epithelial cells (HLECs) or human embryonic kidney (HEK-293) cells. Fluorescent images indicated aggregated signals of mutant protein in the cytoplasm, and a higher protein level was also detected in T148I stable cell lines. In summary, we identified a novel mutation in <i>GJA3</i> for ADCC, which provided molecular insights into the pathogenic mechanism of ADCC.</p></div