Weina Li's research while affiliated with Shandong Academy of Sciences and other places

Leucine-rich α-2-glycoprotein-1 (LRG1) is a novel pro-fibrotic factor that modulates Transforming Growth Factor-β (TGF -β) signaling. However, its role in the corneal fibrotic response remains unknown. In the present study, we found that the LRG1 level increased in alkali-burned mouse corneas. In the LRG1-treated alkali-burned corneas, there were higher fibrogenic protein expression and neutrophils infiltration. LRG1 promoted neutrophils chemotaxis and CXCL-1 secretion. Conversely, LRG1-specific siRNA reduced fibrogenic protein expression and neutrophil infiltration in the alkali-burned corneas. The clearance of neutrophils effectively attenuated the LRG1-enhanced corneal fibrotic response, while the presence of neutrophils enhanced the effect of LRG1 on the fibrotic response in cultured TKE2 cells. In addition, the topical application of LRG1 elevated Interleukin-6 (IL-6) and p-Stat3 levels in the corneal epithelium and in isolated neutrophils. The clearance of neutrophils inhibited the expression of p-Stat3 and IL-6 promoted by LRG1 in alkali-burned corneas. Moreover, neutrophils significantly increased the production of IL-6 and p-Stat3 promoted by LRG1 in TKE2 cells. Furthermore, the inhibition of Stat3 signaling by S3I-201 decreased neutrophil infiltration and alleviated the LRG1-enhanced corneal fibrotic response in the alkali-burned corneas. S3I-201 also reduced LRG1 or neutrophils induced fibrotic response in TKE2 cells. In conclusion, LRG1 promotes the corneal fibrotic response by stimulating neutrophils infiltration via the modulation of the IL-6/Stat3 signaling pathway. Therefore, LRG1 could be targeted as a promising therapeutic strategy for patients with corneal fibrosis.

Aims: Corneal nerve fibers are derived from the ophthalmic division of the trigeminal ganglion (TG). Here, by sequencing of microRNAs (miRNAs) and messenger RNAs (mRNAs) from diabetic and normal TG tissues, we aimed to uncover potential miRNAs, mRNAs, and the network of their interactions involved in the pathogenesis of diabetic corneal neuropathy. Main methods: We performed RNA sequencing to systematically screen out differentially expressed miRNAs and mRNAs in TG tissues from diabetic and normal mice. Functional enrichment analyses were performed to illustrate the biological functions of differentially expressed mRNAs (DEmRNAs). Following this, miRNA-mRNA regulatory networks were built by means of bioinformatics methods to suggest regulatory role for miRNAs in the pathogenesis of diabetic corneal neuropathy. Finally, the credibility of the sequencing-based results was validated using qRT-PCR. Key findings: Sequencing analyses disclosed that 68 miRNAs and 114 mRNAs were differentially expressed in diabetic TG tissues compared with normal TG samples. The functional analyses showed that DEmRNAs participated in diabetes-related biological processes. After applying an optimized approach to predict miRNA-mRNA pairs, a miRNA-mRNA interacting network was inferred. Subsequently, the expression and correlation of miR-350-5p and Mup20, miR-592-5p and Angptl7 as well as miR-351-5p and Elovl6 were preliminarily validated. Significance: Our study provides a systematic characterization of miRNA and mRNA expression in the TG during diabetic corneal neuropathy and will contribute to the development of clinical diagnostic and therapeutic strategies for diabetic corneal neuropathy.

The insulin and Wnt signaling pathways are involved in cell proliferation, tissue homeostasis, and tumorigenesis. However, their interrelationship in the pathophysiological process of diabetic corneal injury remains unclear. In this study, the role of insulin in the diabetic cornea was investigated in vitro, using cultured TKE2 cells and trigeminal ganglion neurons, and in vivo, by assessing corneal wound-healing responses in diabetic mice. A selective Wnt antagonist (XAV-939) and activator (BML-284) were used to regulate the interactions between insulin and the Wnt pathway. The results demonstrated that insulin promoted corneal epithelial wound healing and sensation recovery, whereas the expression of molecules involved in the Wnt/β-catenin pathway was also upregulated in the injured corneal epithelium. However, XAV-939 limited the insulin-induced epithelial and corneal nerve repair. By contrast, BML-284 treatment promoted the healing of the corneal epithelium and corneal nerve repair in diabetic mice. These results indicate that insulin, via Wnt signaling, contributes to diabetic corneal epithelial wound healing and nerve injury recovery and is therefore, a potential protective factor for diabetic corneal epithelial wounds and nerve injury.

Leucine-rich α-2-glycoprotein-1 (LRG1) is involved in several pathophysiological processes, including angiogenesis, cutaneous wound repair and cancer metastasis. In this study, we investigated the potential role and mechanism of LRG1 in corneal re-epithelialisation and nerve regeneration in streptozotocin-induced diabetic mice. We found decreased levels of LRG1 in the corneal epithelium after wounding in diabetic mice compared to normal controls. Hyperglycaemia downregulated the LRG1 expression in the corneal epithelium in vivo, as well as in vitro in a cultured mouse corneal epithelial stem/progenitor cell line (TKE2 cells) exposed to high glucose (HG; 30 mM) in the culture medium. Exogenous application of LRG1 accelerated corneal re-epithelialisation and nerve regeneration in normal mice and diabetic mice. LRG1 also overcame the suppression of wound healing in TKE2 cells by HG conditions, and it activated repair-related signalling by JAK2/STAT3, AKT, epidermal growth factor receptor (EGFR) and transforming growth factor (TGF)-β3. We also found that LRG1 treatment overcame the hyperglycaemia-suppressed expression of matrix metalloproteinase 3 (MMP3) and metalloproteinase 13 (MMP13) in the regenerated corneal epithelium. The promoted effects of LRG1 on corneal re-epithelialisation and nerve regeneration were blocked by inhibitors of MMP3 and MMP13. Subconjunctival injection of 0.5 μg MMP inhibitors did not cause any obvious toxic damage in corneal epithelial cells. Immunoprecipitation and proximity ligation assay experiments confirmed that endogenous LRG1 coprecipitated with MMP3 and MMP13 in TKE2 cells. These results indicate that LRG1 promoted wound repair and nerve regeneration in the diabetic corneal epithelium by regulation of MMPs. Our findings reveal a new function and mechanism for LRG1 in the cornea, and they provide new insights for a better understanding of diabetic keratopathy.

Mesencephalic astrocyte-derived neurotrophic factor (MANF) is a neurotrophic factor widely expressed in mammalian tissues, and it exerts critical protective effects on neurons and other cell types in various disease models, such as those for diabetes. However, to date, the expression and roles of MANF in the cornea, with or without diabetic keratopathy (DK), remain unclear. Here, we demonstrate that MANF is abundantly expressed in normal corneal epithelial cells; however, MANF expression was significantly reduced in both unwounded and wounded corneal epithelium in streptozotocin-induced type 1 diabetic C57BL/6 mice. Recombinant human MANF significantly promoted normal and diabetic corneal epithelial wound healing and nerve regeneration. Furthermore, MANF inhibited hyperglycemia-induced endoplasmic reticulum (ER) stress and ER stress-mediated apoptosis. Attenuation of ER stress with 4-phenylbutyric acid (4-PBA) also ameliorated corneal epithelial closure and nerve regeneration. However, the beneficial effects of MANF and 4-PBA were abolished by an Akt inhibitor and Akt-specific small interfering RNA (siRNA). Finally, we reveal that the subconjunctival injection of MANF-specific siRNA prevents corneal epithelial wound healing and nerve regeneration. Our results provide important evidence that hyperglycemia-suppressed MANF expression may contribute to delayed corneal epithelial wound healing and impaired nerve regeneration by increasing ER stress, and MANF may be a useful therapeutic modality for treating DK.

Purpose: To investigate the contribution of phosphatase and tensin homologue (PTEN) on the delayed epithelial regeneration and impaired Akt activation in diabetic mice. Methods: The expression of PTEN on cornea was compared between normal and diabetic mice. The corneal epithelial and nerve regeneration rate was evaluated in diabetic mice after the treatment with PTEN small interfering RNA (siRNA), PTEN inhibitors, or Akt inhibitor. The reactivation of epithelial regeneration-related signaling, including phosphorylated (p)-Akt, p-Stat3, Sirt1, and Parkin, were assessed with Western blot and immunofluorescence staining. The effects of PTEN inhibition on cellular proliferation and migration were further evaluated in cultured mouse corneal epithelial cells. Results: PTEN messenger RNA and protein levels exhibited up-regulation in diabetic cornea. Upon central epithelial debridement, the epithelial regeneration rate was significantly promoted in diabetic mice with the treatment of PTEN inhibition than that of vehicle control (P < 0.05), which accompanied with the recovered levels of p-Akt, p-Stat3, Sirt1, and Parkin. However, the promotion of diabetic corneal epithelial regeneration rate and Akt reactivation was completed reversed by Akt inhibitor. In vitro, PTEN inhibition promoted their migration, but not the proliferation capacity. In addition, PTEN inhibitor treatment also improved the recovery of corneal nerve fiber density and sensitivity that was impaired in diabetic mice. Conclusions: Elevated PTEN expression contributes to the impaired corneal epithelial regeneration and Akt activation in diabetic mice, which can be improved with PTEN inhibition. Translational relevance: Our study suggests that PTEN inhibition may serve as a new strategy for restoring the impaired corneal epithelial regeneration ability in patients with diabetes.