rhFGF-21 accelerates corneal epithelial wound healing through the attenuation of oxidative stress and inflammatory mediators in diabetic mice.

Diabetic keratopathy, commonly associated with a hyperactive inflammatory response, is one of the most common eye complications of diabetes. The peptide hormone fibroblast growth factor-21 (FGF-21) has been demonstrated to have anti-inflammatory and antioxidant properties. However, whether administration of recombinant human (rh) FGF-21 can potentially regulate diabetic keratopathy is still unknown. Therefore, in this work, we investigated the role of rhFGF-21 in the modulation of corneal epithelial wound healing, the inflammation response, and oxidative stress using type 1 diabetic mice and high glucose-treated human corneal epithelial cells. Our experimental results indicated that the application of rhFGF-21 contributed to the enhancement of epithelial wound healing. This treatment also led to advancements in tear production and reduction in corneal edema. Moreover, there was a notable reduction in the levels of proinflammatory cytokines such as TNF-α, IL-6, IL-1ß, MCP-1, IFN-γ, MMP-2, and MMP-9 in both diabetic mouse corneal epithelium and human corneal epithelial cells treated with high glucose. Furthermore, we found rhFGF-21 treatment inhibited reactive oxygen species production and increased levels of anti-inflammatory molecules IL-10 and SOD-1, which suggests that FGF-21 has a protective role in diabetic corneal epithelial healing by increasing the antioxidant capacity and reducing the release of inflammatory mediators and matrix metalloproteinases. Therefore, we propose that administration of FGF-21 may represent a potential treatment for diabetic keratopathy.

Two-hundred-liter scale fermentation, purification of recombinant human fibroblast growth factor-21, and its anti-diabetic effects on ob/ob mice.

Fibroblast growth factor-21 (FGF-21) is a potential cytokine for type II diabetes mellitus. This study aimed to optimize recombinant human FGF-21 (rhFGF-21) production in Escherichia coli BL21 (DE3) employing high cell density fermentation at a 200-L scale and pilot-scale purification. FGF-21 was eventually expressed in E. coli BL21 (DE3) using human FGF-21 synthetic DNA sequence via the introduction of vector pET-3c; the product is used as seed strain during the fermentation of rhFGF-21. Fermentation of rhFGF-21 was performed in a 30-L and 200-L fermenters. rhFGF-21 was primarily expressed in the form of inclusion bodies after IPTG induction. At the 200-L scale, the bacterial production and expression levels of rhFGF-21 were 38.8 ± 0.6 g/L and 30.9 ± 0.7%, respectively. Additionally, the high purification (98%) of rhFGF-21 was tested with HPLC analysis and reducing & non-reducing SDS-PAGE analysis. The final yield of purified rhFGF-21 was 71.1 ± 13.9 mg/L. The activity of rhFGF-21 stock solution reached at 68.67 ± 8.74 IU/mg. Blood glucose controlling and insulin sensitization were improved with treatment of rhFGF-21 in type II diabetic ob/ob mice. Our results showed that the relatively stable and time-saving pilot-scale production process was successfully established, providing an efficient and cost-effective strategy for large-scale and industrial production of rhFGF-21.

Design and Evaluation of Lyophilized Fibroblast Growth Factor 21 and Its Protection against Ischemia Cerebral Injury.

This study investigated the effect of the excipients, including glycine, mannitol, arginine, trehalose, sorbitol, and poloxamer188, on the stability of recombinant human fibroblast growth factor 21(FGF21) during the process of lyophilization and storage. The glass transition temperature (Tg), protein secondary structure, aggregation ratio, and the bioactivity of lyophilized FGF21 were measured. We furthermore investigated the effect of FGF21 against ischemia cerebral injury using the middle cerebral artery occlusion (MCAO) model in rats. The ischemia cerebral injury of MCAO rats was analyzed via 2,3,5-triphenyltetrazolium chloride and Nissl-staining. Endoplasmic reticulum (ER) stress related proteins were detected via Western blot. In this study, we found that aggregation was the primary mode of deterioration of lyophilized FGF21under accelerated storage conditions. Mannitol combined with trehalose and glycine formulations offers the most effective protein protection to reduce the aggregation. Administration of FGF21 protected cerebral ischemia and decreased ER stress related proteins in MCAO rats and PC12 cells.

Preparation of a recombinant collagen-peptide (RHC)-conjugated chitosan thermosensitive hydrogel for wound healing.

Thermosensitive chitosan hydrogels have been widely used in drug delivery and tissue repair, but further applications of these hydrogels have been limited by their weak mechanical strength and poor bioactivity. A thermoresponsive hydrogel formed by conjugating recombinant human collagen-peptide (RHC) with chitosan might be better suited for cell encapsulation and wound repair. RHC-chitosan hydrogels were prepared and tested, and the results showed that moderate RHC conjugation led to hydrogels with lower gelation temperature. The prepared RHC-containing hydrogels showed superior mechanical strength to chitosan-only hydrogels. Additionally, cells exhibited superior viability when cultured with RHC-modified hydrogels compared with hydrogels that had not been conjugated with RHC. Finally, RHC-chitosan hydrogels were injected onto the backs of rats with second-degree burns and promoted cell infiltration, vessel formation, and wound healing. Overall, the use of RHC-chitosan hydrogels is a promising and effective therapeutic approach for burn wound treatment.

Higher Biostability of rh-aFGF-Carbomer 940 Hydrogel and Its Effect on Wound Healing in a Diabetic Rat Model.

Hydrogels are excellent drug delivery carriers with excellent ductility. Here, we report the design of a higher biostability of a recombinant human acidic fibroblast growth factor (rh-aFGF) carbomer hydrogel formulation. To verify the optimality of this formula, we prepared various prescriptions and tested the resulting physical properties including micromorphology, long-term stability, accelerated stability, and destructive test. Furthermore, the efficacy for promoting wound healing in full-thickness injury and scald wound diabetic rat models was explored. We found that rh-aFGF-carbomer hydrogel had good physical properties. It was stable for 24 months at 5 ± 3 °C, and for 6 months at 25 ± 3 °C. In vivo, the rh-aFGF-carbomer 940 hydrogel achieved a remarkable promotion of skin wound healing in diabetic rats with full-thickness injuries or scald wounds. Our data suggest that rh-aFGF-carbomer hydrogel may have applications for the treatment of diabetic ulcers combined with other wounds.