Vaccarin hastens wound healing by promoting angiogenesis via activation of MAPK/ERK and PI3K/AKT signaling pathways <i>in vivo</i>

Rats’ skin excision model to study the effects of vaccarin on wound healing in vivo . Hematoxylin and eosin staining was performed to evaluate Histopathologic characteristics. Immunohistochemistry was employed to assess the effects of vaccarin in accelerating angiogenesis. Western blot was used to evaluate relative protein expressed levels.

Results

Vaccarin could significantly promote wound healing and endothelial cells and fibroblasts proliferation in the wound site. Immunohistochemistry and Western blot studies showed that the nodal proteins and receptor (bFGFR) related to angiogenesis signaling pathway were activated, and the microvascular density in the wound site was markedly higher than that in the control group.

Conclusions

The present study was the first to demonstrate that vaccarin is able to induce angiogenesis and accelerate wound healing in vivo by increasing expressions of p-Akt, p-Erk and p-bFGFR. This process is mediated by MAPK/ERK and PI3K/AKT signaling pathways.

Vaccaria; Angiogenesis Inducing Agents; Wound Healing; Rats

Authorship

Bao Hou
* The authors contributed equally to this work.

Assistant Experimentalist, Department of Basic Medicine, Wuxi School of Medicine, Jiangnan University, Wuxi, China. Technical procedures, manuscript preparation.Jiangnan UniversityChinaWuxi, China Assistant Experimentalist, Department of Basic Medicine, Wuxi School of Medicine, Jiangnan University, Wuxi, China. Technical procedures, manuscript preparation.

http://orcid.org/0000-0003-0698-2765

Weiwei Cai
* The authors contributed equally to this work.

Experimentalist, Department of Basic Medicine, Wuxi School of Medicine, Jiangnan University, Wuxi, China. Histological examinations, manuscript preparation.Jiangnan UniversityChinaWuxi, China Experimentalist, Department of Basic Medicine, Wuxi School of Medicine, Jiangnan University, Wuxi, China. Histological examinations, manuscript preparation.

http://orcid.org/0000-0003-2183-7590

Ting Chen

Master, Department of Basic Medicine, Wuxi School of Medicine, Jiangnan University, Wuxi, China. Acquisition of data, statistics analysis.Jiangnan UniversityChinaWuxi, China Master, Department of Basic Medicine, Wuxi School of Medicine, Jiangnan University, Wuxi, China. Acquisition of data, statistics analysis.

http://orcid.org/0000-0001-9380-4953

Zhixuan Zhang

http://orcid.org/0000-0002-6662-7047

Haifeng Gong

http://orcid.org/0000-0003-4164-5676

Wei Yang

http://orcid.org/0000-0002-5611-5566

Liying Qiu

PhD, Professor, Department of Basic Medicine, Wuxi School of Medicine, Jiangnan University, Wuxi, China. Critical revision, final approval.Jiangnan UniversityChinaWuxi, China PhD, Professor, Department of Basic Medicine, Wuxi School of Medicine, Jiangnan University, Wuxi, China. Critical revision, final approval.

http://orcid.org/0000-0002-3684-792X

Correspondence: Prof. Liying Qiu Department of Basic Medicine, Wuxi School of Medicine Jiangnan University, Wuxi China qiulydoc@sina.com

*
The authors contributed equally to this work.

Conflict of interest: none

SCIMAGO INSTITUTIONS RANKINGS

Figures | Formulas

Figures (7)
Formulas (1)

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Figure 1
Chemical structure of vaccarin.

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Figure 2
Vaccarin promoted wound healing. The 1 cm diameter full-thickness skin excision wounds were made on both sides in SD rats’ back. 0.02 g of vehicle ointments was added to the left wound site and ointments with 0.1% vaccarin were added to the right wound site. (A) The wound closure percentage (%) post wounding. Vaccarin significantly contributed to the wound healing compared with the vehicle control group. Results are expressed as mean ± SD of eight independent experiments. (* P < 0.05, ** P < 0.01, versus control). (B) The wound appearance observed at different time points.

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Figure 3
Vaccarin ameliorated the pathological tissue of wound skin. (A) Representative images of H&E-stained histologic wound sections of control and vaccarin-treated rats on the 6th, 9th and 11th day after wounding (magnification x400). (B-D) Representative the inflammatory cells, fibroblasts and endothelial cell count from 10 randomly chosen high-power fields (magnification x400) from H&E-stained cutaneous wound sections.

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Figure 4
Vaccarin promoted angiogenesis. (A) Representative images of immunohistochemical PCNA staining of wound sections of control and vaccarin-treated rats on the 6th, 9th and 11th day after wounding (magnification x400). (B) Representative images of immunohistochemical CD31 staining of wound sections of control and vaccarin-treated rats on the 6th, 9th and 11th day after wounding (magnification x200). (C) Effect of vaccarin on the angiogenesis of rats was evaluated by MVD/CD31 compared with the blank control groups. (D-F) Representative of the inflammatory cells, fibroblasts and endothelial cell count from 10 randomly chosen high-power fields (magnification x400) from Immunohistochemistry stained cutaneous wound sections.

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Figure 5
Vaccarin activated bFGFR signaling. (A and B) Representative images of immunohistochemical p-VEGFR and p-bFGFR staining of wound sections of control and vaccarin-treated rats on the 6th, 9th and 11th day after wounding (magnification x400). (C and D) Representative the statistic IOD of p-VEGFR and p-bFGFR respectively. Data are presented as the mean ± standard error of the mean from six independent experiments. * P <0.05 compared with the blank control group.

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Figure 6
Vaccarin activated angiogenesis-associated channel proteins. (A and B) Representative images of immunohistochemical p-Akt and p-Erk staining of wound sections of control and vaccarin-treated rats on the 6th,9th and 11th day after wounding (magnification x400). (C and D) Compared with the vehicle control groupl p-Akt and p-Erk expression was higher following treatment with vaccarin. Data are presented as the mean ± standard error of the mean from six independent experiments.* P <0.05, ** P <0.01 compared with the vehicle control group.

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Figure 7
Effect of vaccarin on the expression of related proteins in skin of wound healing, compared with the vehicle control group* P <0.05, ** P <0.01.

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Figure 1 Chemical structure of vaccarin.

Figure 2 Vaccarin promoted wound healing. The 1 cm diameter full-thickness skin excision wounds were made on both sides in SD rats’ back. 0.02 g of vehicle ointments was added to the left wound site and ointments with 0.1% vaccarin were added to the right wound site. (A) The wound closure percentage (%) post wounding. Vaccarin significantly contributed to the wound healing compared with the vehicle control group. Results are expressed as mean ± SD of eight independent experiments. (* P < 0.05, ** P < 0.01, versus control). (B) The wound appearance observed at different time points.

Figure 3 Vaccarin ameliorated the pathological tissue of wound skin. (A) Representative images of H&E-stained histologic wound sections of control and vaccarin-treated rats on the 6th, 9th and 11th day after wounding (magnification x400). (B-D) Representative the inflammatory cells, fibroblasts and endothelial cell count from 10 randomly chosen high-power fields (magnification x400) from H&E-stained cutaneous wound sections.

Figure 4 Vaccarin promoted angiogenesis. (A) Representative images of immunohistochemical PCNA staining of wound sections of control and vaccarin-treated rats on the 6th, 9th and 11th day after wounding (magnification x400). (B) Representative images of immunohistochemical CD31 staining of wound sections of control and vaccarin-treated rats on the 6th, 9th and 11th day after wounding (magnification x200). (C) Effect of vaccarin on the angiogenesis of rats was evaluated by MVD/CD31 compared with the blank control groups. (D-F) Representative of the inflammatory cells, fibroblasts and endothelial cell count from 10 randomly chosen high-power fields (magnification x400) from Immunohistochemistry stained cutaneous wound sections.

Figure 5 Vaccarin activated bFGFR signaling. (A and B) Representative images of immunohistochemical p-VEGFR and p-bFGFR staining of wound sections of control and vaccarin-treated rats on the 6th, 9th and 11th day after wounding (magnification x400). (C and D) Representative the statistic IOD of p-VEGFR and p-bFGFR respectively. Data are presented as the mean ± standard error of the mean from six independent experiments. * P <0.05 compared with the blank control group.

Figure 6 Vaccarin activated angiogenesis-associated channel proteins. (A and B) Representative images of immunohistochemical p-Akt and p-Erk staining of wound sections of control and vaccarin-treated rats on the 6th,9th and 11th day after wounding (magnification x400). (C and D) Compared with the vehicle control groupl p-Akt and p-Erk expression was higher following treatment with vaccarin. Data are presented as the mean ± standard error of the mean from six independent experiments.* P <0.05, ** P <0.01 compared with the vehicle control group.

Figure 7 Effect of vaccarin on the expression of related proteins in skin of wound healing, compared with the vehicle control group* P <0.05, ** P <0.01.

Wound closure percentage (%) = [(area on day 0 - open area on day n) / area on day 0] \times 100 .

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