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. 2023 Jan 4:13:1104929.
doi: 10.3389/fphys.2022.1104929. eCollection 2022.

Bioengineered silkworm model for expressing human neurotrophin-4 with potential biomedical application

Wenchang Zhang  1   2   3 , Zhiqing Li  1   2   3 , Weiqun Lan  1   2   3 , Hao Guo  1   2   3 , Feng Chen  1   2   3 , Feng Wang  1   2   3 , Guanwang Shen  1   2   3 , Qingyou Xia  1   2   3 , Ping Zhao  1   2   3
Affiliations

Bioengineered silkworm model for expressing human neurotrophin-4 with potential biomedical application

Wenchang Zhang et al. Front Physiol. .

Abstract

Neurotrophin-4 (NT-4) is a neurotrophic factor that plays important roles in maintaining nerve cell survival, regulating neuronal differentiation and apoptosis, and promoting nerve injury repair. However, the source of sufficient NT-4 protein and efficient delivery of NT-4 remain a challenge. This study aims to express an activated human NT-4 protein in a large scale by genetically engineering silk gland bioreactor of silkworm as a host. We showed that the expression of human NT-4-functionalized silk material could promote proliferation of mouse HT22 cells when compared to the natural silk protein, and no obvious cytotoxicity was observed under the conditions of different silk materials. Importantly, this functional silk material was able to induce the potential differentiation of HT22 cells, promote peripheral neural cell migration and neurite outgrowth of chicken embryo dorsal root ganglion (DRG). All these results demonstrated a high bioactivity of human NT-4 protein produced in silk gland. Therefore, based on the silkworm model, the further fabrication of different silk materials-carrying active NT-4 protein with good mechanical properties and great biocompatibility will give promising applications in tissue engineering and neurons regeneration.

Keywords: biomedical application; bombyx mori; human neurotrophic-4; silk gland bioreactor; silk material.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Generation of transgenic silkworm for human NT-4 expression. (A) A schematic diagram of piggyBac [DsRed,NT-4] transgenic expression vector in MSG of silkworm. (B) Screening for the positive G1 eggs and moth exposed under RFP fluorescence (red), scale bar is 500 µm and 5 mm, respectively. (C) The mRNA expression of NT-4 by qRT-PCR analysis. (D) Protein analysis of NT-4 by Western blotting. The upper and lower bands were predicated as full size and mature peptides of NT-4, respectively.
FIGURE 2
FIGURE 2
Quantitative analysis of NT-4 expression in transgenic silkworm. (A) SDS-PAGE analysis of the crude extracts from NT-4 and WT silkworm cocoon. BSA standard with different contents of 125 ng, 250 ng, and 500 ng were used to quantitate the recombinant NT-4 concentration. The red and black arrowheads point to the full size and mature peptides of NT-4, respectively. (B) Western blotting analysis of the crude extracts from NT-4 silkworm cocoon. Active NT-4 standard with different contents of 200 ng, 300 ng, and 400 ng were used to quantitate the recombinant NT-4 concentration.
FIGURE 3
FIGURE 3
NT-4 promoted the proliferation of HT22 cells. (A) CCK-8 assay of WT and NT-4 extracts on HT22 cell proliferation after 24 h treatment. For the significant analysis: *p < 0.05. (B) EdU-488 staining was used to analyze HT22 cells undergoing DNA replication and proliferation by different treatments (green). The nuclei DNA was counterstained with DAPI (blue). Scale bar is 400 μm.
FIGURE 4
FIGURE 4
NT-4 promoted the expression of neuronal marker genes in HT22 cells. The mRNA expression of neuronal marker genes including NSE, TUBB3, and MAP2 in HT22 cells were analyzed by qRT-PCR after the treatments with WT and NT-4 extracts. GADPH was used as the internal control. For the significant analysis: **p < 0.01 and ***p < 0.001.
FIGURE 5
FIGURE 5
NT-4 contributed to myelination process in HT22 cells. (A) The mRNA expression of myelin genes including MPZ and MBP in HT22 cells were analyzed by qRT-PCR after the treatments with WT and NT-4 extracts. GADPH was used as the internal control. For the significant analysis: **p < 0.01 and ***p < 0.001. (B) The protein expression of MBP in HT22 cells were analyzed by western blotting after the treatments with WT and NT-4 extracts. GADPH was used as the internal control.
FIGURE 6
FIGURE 6
NT-4 induced the potential differentiation of HT22 cells. (A) The mRNA expression of astrocyte specific gene GFAP in HT22 cells was analyzed by qRT-PCR after the treatments with WT and NT-4 extracts. GADPH was used as the internal control. For the significant analysis: ***p < 0.001. (B) Immunofluorescence staining of HT22 cells by GFAP antibody after the treatments with WT and NT-4 extracts (red). The nuclei DNA was counterstained with DAPI (blue). Scale bar is 200 μm.
FIGURE 7
FIGURE 7
NT-4 promoted peripheral neural cell migration and neurite outgrowth of DRG. (A) Chicken embryo was dissected from the egg. (B) DRG neurons was isolated from the spinal cord of chicken embryo and observed under a microscope. Scale bar is 200 μm. (C) DRGs were cultured in RPMI-1640 complete medium and the peripheral migration cells of DRG were stained by GFAP antibody (red). The nuclei DNA was counterstained with DAPI (blue). Scale bar is 200 μm. (D) The peripheral migration progression and neurite outgrowth of DRGs after WT and NT-4 treatments at different time points were analyzed and observed under a microscope. The magnified images showed the distinct speed of DRG outgrowth. Scale bar is 200 μm.
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