Journal of Surgical Research

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Journal of Surgical Research Combined effect of substance P and curcumin on cutaneous wound healing in diabetic rats Vinay Kant, PhD, Dinesh Kumar, PhD, Raju Prasad, PhD, Anu Gopal, MVSc, Nitya N. Pathak, PhD, Pawan Kumar, PhD, Surender K. Tandan, PhD Journal of Surgical Research Volume 212, Pages 130-145 (May 2017) DOI: 10.1016/j.jss.2017.01.011 Copyright © 2017 Elsevier Inc. Terms and Conditions

Fig. 1 Effect of combination therapy on cutaneous wound closure. (A) Representative photographs of wounds of control, gel-treated, and SP + curcumin (Cum)-treated diabetic rats on days 3, 7, 11, 14, and 19 postwounding. (B) Wound contraction (percentage) in control, gel-treated and SP + Cum-treated groups on different days. Data are expressed as means ± standard error of the mean (n = 5). *P < 0.05, **P < 0.01, and ***P < 0.001 versus other group(s) on the same day. Color version of the figure is available online. Journal of Surgical Research 2017 212, 130-145DOI: (10.1016/j.jss.2017.01.011) Copyright © 2017 Elsevier Inc. Terms and Conditions

Fig. 2 Relative mRNA expressions of TNF-α (A) and IL-10 (B) in healing tissue of excision wounds of control, gel-treated, and SP + curcumin (Cum)-treated diabetic rats on days 3, 7, 14, and 19 postwounding. The mRNA expressions were normalized by β-actin at each time point, and data are expressed as means ± standard error of the mean fold change (n = 4). ELISA analysis of TNF-α (C) and IL-10 (D) in healing tissue of rats of different groups on days 3, 7, 14, and 19 postwounding. Data are expressed as mean ± standard error of the mean (n = 5). *P < 0.05, **P < 0.01, and ***P < 0.001 versus other group(s) on the same day. Journal of Surgical Research 2017 212, 130-145DOI: (10.1016/j.jss.2017.01.011) Copyright © 2017 Elsevier Inc. Terms and Conditions

Fig. 3 Relative mRNA expressions of VEGF (A), TGF-β1 (B) in healing tissue of control, gel-treated, and SP + curcumin (Cum)-treated diabetic rats. The mRNA expressions were normalized by β-actin at each time point, and data are expressed as mean ± standard error of the mean fold change (n = 4). (C) Representative Western blots of β-actin, VEGF, and TGF-β1 from the homogenate samples of control (C), gel- and combination (Comb)-treated rats on days 3, 7, 14, and 19 postwounding. Semiquantitative protein expressions of VEGF (D) and TGF-β1 (E) in different groups. Data are expressed as mean ± standard error of the mean (n = 4). *P < 0.05, **P < 0.01, and ***P < 0.001 versus other group(s) on the same day. Journal of Surgical Research 2017 212, 130-145DOI: (10.1016/j.jss.2017.01.011) Copyright © 2017 Elsevier Inc. Terms and Conditions

Fig. 4 Relative mRNA expressions of IL-1β (A), MMP-9 (B), HIF-1α (C), SDF-1α (D), HO-1 (E), and eNOS (F) in healing tissue of control, gel-treated and SP + curcumin (Cum)-treated diabetic rats on days 3, 7, 14, and 19 postwounding. The mRNA expressions were normalized by β-actin at each time point. Data are expressed as mean ± standard error of the mean (n = 4). *P < 0.05, **P < 0.01, and ***P < 0.001 versus other group(s) on the same day. Journal of Surgical Research 2017 212, 130-145DOI: (10.1016/j.jss.2017.01.011) Copyright © 2017 Elsevier Inc. Terms and Conditions

Fig. 5 (A-C) ELISA analysis of SOD (A), catalase (B) and GPx (C) in healing tissue of control, gel treated and substance P (SP) + curcumin (Cum)-treated diabetic rats on days 3, 7, 14, and 19 postwounding. Data are expressed as mean ± standard error of the mean (n = 5). *P < 0.05, **P < 0.01, and ***P < 0.001 versus other group(s) on the same day. Journal of Surgical Research 2017 212, 130-145DOI: (10.1016/j.jss.2017.01.011) Copyright © 2017 Elsevier Inc. Terms and Conditions

Fig. 6 (A) Representative images of H&E-stained histologic wound sections of control, gel-treated, and SP + curcumin (Cum)-treated diabetic rats on days 3, 7, 14, and 19 postwounding (40× magnification and scale bar 50 μm). Low magnification (10×) images of wounds are shown in inset boxes in the left upper corner. I = inflammatory cells; F = fibroblasts; BV = blood vessels; C = collagen; E = epithelial layer. (B) Histologic scoring (score from 1-15) of H&E-stained cutaneous wound sections of control, gel- and substance P (SP) + curcumin (Cum)-treated diabetic rats. SP + Cum-treated group had significantly higher score during the entire experiment, compared with control group. Data are expressed as mean ± standard error of the mean. *P < 0.05, **P < 0.01, and ***P < 0.001 versus other group(s) on the same day (n = 10). Color version of the figure is available online. Journal of Surgical Research 2017 212, 130-145DOI: (10.1016/j.jss.2017.01.011) Copyright © 2017 Elsevier Inc. Terms and Conditions

Fig. 7 (A) Representative immunohistochemical CD31 staining of wound sections of control, gel-treated, and SP + curcumin (Cum)-treated diabetic rats on days 3, 7, 14, and 19 postwounding (40× magnification and scale bar 50 μm). Arrow indicates the positive reaction for CD31 on the endothelial cells. (B) Semiquantitative analysis of MVD in wound sections of control, gel-treated, and SP + curcumin (Cum)-treated diabetic rats on days 3, 7, 14, and 19 postwounding. MVD was assessed by counting the number of microvessels in 20 randomly chosen high-power fields (HPFs; 40×) in wounded dermis and hypodermis. Data are expressed as mean ± standard error of the mean. *P < 0.05, **P < 0.01, and ***P < 0.001 versus other group(s) on the same day (n = 20). Color version of the figure is available online. Journal of Surgical Research 2017 212, 130-145DOI: (10.1016/j.jss.2017.01.011) Copyright © 2017 Elsevier Inc. Terms and Conditions

Fig. 8 (A) Representative immunohistochemical GAP-43 staining of wound sections of control, gel-treated, and SP + curcumin (Cum)-treated diabetic rats on days 3, 7, 14, and 19 postwounding (40× magnification and scale bar 50 μm). Arrow indicates the positive reaction for GAP-43 on the nerve fibers. (B) Semiquantitative analysis of GAP-43–positive nerve fibers in wound sections of control, gel-treated, and SP + curcumin (Cum)-treated diabetic rats on days 3, 7, 14, and 19 postwounding. It was assessed by counting the number of GAP-43–positive nerve fibers in 30 random high-power fields (HPFs; 40×) in wounded dermis and hypodermis. Data are expressed as mean ± standard error of the mean. *P < 0.05, **P < 0.01, and ***P < 0.001 versus other group(s) on the same day (n = 30). Color version of the figure is available online. Journal of Surgical Research 2017 212, 130-145DOI: (10.1016/j.jss.2017.01.011) Copyright © 2017 Elsevier Inc. Terms and Conditions

Fig. 9 (A) Picrosirius red-stained histologic sections of cutaneous wound showing collagen fibers under polarized light on days 3, 7, 14, and 19 postwounding in control, gel-treated, and SP + curcumin (Cum)-treated diabetic rats. Scale bar 100 μm and magnification 20×. (B) Total collagen fraction (percentage) per high-power field in picrosirius red-stained histopathologic wound sections of control, gel-treated, and SP + curcumin (Cum)-treated diabetic rats on days 3, 7, 14, and 19 postwounding. Data are expressed as mean ± standard error. *P < 0.05, **P < 0.01, and ***P < 0.001 versus other group(s) on the same day (n = 8). Color version of the figure is available online. Journal of Surgical Research 2017 212, 130-145DOI: (10.1016/j.jss.2017.01.011) Copyright © 2017 Elsevier Inc. Terms and Conditions