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. 2022 Jan 21;22(1):33.
doi: 10.1186/s12886-022-02255-8.

Protection effect of thymosin β4 on ethanol injury in corneal stromal keratocyte

Jinghua Liu #  1   2 , Chen Guo #  3 , Peng Hao  2   4 , Peihong Wang  4 , Linghan Li  5 , Yuchuan Wang  2   4 , Xuan Li  6   7
Affiliations

Protection effect of thymosin β4 on ethanol injury in corneal stromal keratocyte

Jinghua Liu et al. BMC Ophthalmol. .

Abstract

Purpose: To investigate the protective effects of thymosin β4 (Tβ4) on ethanol injured human corneal keratocytes (HCKs).

Methods: HCKs and BALB/c mice were chosen as the study subject. Ethanol was used to treat the cells and corneal stroma of mice to build the ethanol injured model in vitro and vivo respectively. CCK-8 was used to evaluate the cell metabolic activity. DCFH-DA was used to detect the intracellular reactive oxygen species level. TUNEL was chose to detect the cell apoptosis rate. The cell proliferation and migration were investigated by using wound healing insert. Wound healing of corneal surface and stroma was observed by using fluorescein sodium eyedrop and HE stain. RT-qPCR, ELISA, and immunostaining were performed to detect gene and protein expression in keratocytes or corneal stroma tissue of mice.

Results: Ethanol induced oxidative stress injury and cell apoptosis on HCKs, and Tβ4 can alleviate it by up-regulating the expression of Bcl-2, catalase, and CuZnSOD, and inhibiting the expression of Caspase-3. Tβ4 promotes the proliferation of HCKs and the process of corneal wound healing. It may relevant to the up-regulated expression of Ki67.

Conclusions: Our study established an ethanol-injured corneal stroma model in both vitro and vivo. The present study confirmed that Tβ4 play a protective effect on the reconstruction process of ethanol-injured corneal stroma.

Keywords: Apoptosis; Cornea stroma; Ethanol; Oxidative stress; Thymosin β4.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
The results of CCK-8. A showed variation cell metabolic activity of HCKs in different groups. B showed the cell metabolic activity of HCKs with ethanol and/or Tβ4 treatment. *p < 0.05, **p < 0.01
Fig. 2
Fig. 2
The results of ROS. A showed the intracellular ROS of four groups, Scale bar: 1000 μm. B showed the fluorescence values (FV) of HCKs, IRL = FV/FVcontrl. C showed the expression of Catalase and CuZnSOD of HCKs. * p < 0.05(Catalase), # p < 0.05(CuZnSOD), ## p < 0.01(CuZnSOD) BL: bright light field
Fig. 3
Fig. 3
The results of cell apoptosis. A showed the cell apoptosis of four groups by TUNEL staining, Scale bar: 200 μm. B showed the result of positive cell (green) count. C showed the expression of Bcl-2 of HCKs detected by qPCR. D showed the expression of caspase-3 of HCKs detected by qPCR. E showed the protein level of caspase-3 of HCKs by ELISA. **p < 0.01
Fig. 4
Fig. 4
The results of wound healing in vitro. A showed the condition of gap at three time points (0 h, 24 h, 48 h), (10×). Scar bar: 200 μm. B showed the area of cells within the gap of four groups
Fig. 5
Fig. 5
The results of wound healing in vivo. A, B the fluorescence staining showing epithelial defect of cornea of four groups. The red circle means the baseline of corneal epithelial defects area. The green part means the epithelial defect area. C showed the HE staining of cornea of four groups (10×)
Fig. 6
Fig. 6
A The results of ki67 on corneal tissue. Red showed the expression of Ki67, blue = DAPI. Scale bar: 500 μm. B Positive cell count of corneal stroma of four groups. *p < 0.05
Fig. 7
Fig. 7
The results of cytokine correlating cell apoptosis on animal model. A showed the expression of Bcl-2 of BALB/c mice cornea tissue detected by qPCR. B showed the expression of caspase-3 of BALB/c mice cornea tissue detected by qPCR. **p < 0.01
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References

    1. Seo YS, Kwon JH, Yaqoob U, Yang L, De Assuncao TM, Simonetto DA, Verma VK, Shah VH. HMGB1 recruits hepatic stellate cells and liver endothelial cells to sites of ethanol-induced parenchymal cell injury. Am J Physiol Gastrointest Liver Physiol. 2013;305(11):G838–G848. doi: 10.1152/ajpgi.00151.2013. - DOI - PMC - PubMed
    1. Li Y, Wang S, Ni HM, Huang H, Ding WX. Autophagy in alcohol-induced multiorgan injury: mechanisms and potential therapeutic targets. Biomed Res Int. 2014;2014:498491. - PMC - PubMed
    1. Hoch K, Volk DE. Structures of Thymosin proteins. Vitam Horm. 2016;102:1–24. doi: 10.1016/bs.vh.2016.04.009. - DOI - PubMed
    1. Huff T, Müller CS, Otto AM, Netzker R, Hannappel E. beta-Thymosins, small acidic peptides with multiple functions. Int J Biochem Cell Biol. 2001;33(3):205–220. doi: 10.1016/S1357-2725(00)00087-X. - DOI - PubMed
    1. Stoeva S, Hörger S, Voelter W. A novel beta-thymosin from the sea urchin: extending the phylogenetic distribution of beta-thymosins from mammals to echinoderms. J Pept Sci. 1997;3(4):282–290. doi: 10.1002/(SICI)1099-1387(199707)3:4<282::AID-PSC119>3.0.CO;2-A. - DOI - PubMed