Improved function of diabetic wound-site macrophages and accelerated wound closure in response to oral supplementation of a fermented papaya preparation

Abstract

Carica papaya Linn is widely known as a medicinal fruit. We sought to study a standardized fermented papaya preparation (FPP) for its effects on wound healing in adult obese diabetic (db/db) mice. FPP blunted the gain in blood glucose and improved the lipid profile after 8 weeks of oral supplementation. However, FPP did not influence weight gain during the supplementation period. FPP (0.2 g/kg body weight) supplementation for 8 weeks before wounding was effective in correcting wound closure. Studies on viable macrophages isolated from the wound site demonstrated that FPP supplementation improved respiratory-burst function as well as inducible NO production. Reactive oxygen species support numerous aspects of wound healing; NO availability in diabetic wounds is known to be compromised. Diabetic mice supplemented with FPP showed a higher abundance of CD68 as well as CD31 at the wound site, suggesting effective recruitment of monocytes and an improved proangiogenic response. This work provides the first evidence that diabetic-wound outcomes may benefit from FPP supplementation by specifically influencing the response of wound-site macrophages and the subsequent angiogenic response. Given that FPP has a long track record of safe human consumption, testing of the beneficial effects of FPP on diabetic wound-related outcomes in a clinical setting is warranted.

FIG. 1.

Oral supplementation with FPP improved blood hyperglycemia and dyslipidemia in diabetic mice. (A) Percentage increase of plasma glucose levels after the 8 weeks of FPP supplementation. Blood glucose was recorded before the start of supplementation (baseline) and then after the eighth week of supplementation. Data are presented as percentage change in 8 weeks compared with baseline. Data are expressed as mean ± SD (n = 10); *p < 0.05 compared with placebo. (B) Total triglycerides (TGLs), total cholesterol (TCHOL), HDL, and LDL levels in blood were determined after 8 weeks of FPP supplementation. Open bars, placebo supplemented; solid bars, the FPP-supplemented diabetic mice. Data are expressed as mean ± SD (n = 10) *p < 0.05. (C) FPP did not have any significant effect on overall weight gain throughout the 8 weeks of supplementation. Data are expressed as mean ± SD (n = 10).

FIG. 2.

Improved wound closure in FPP-supplemented diabetic mice. Full-thickness (skin and panniculus carnosum) dorsal wounds were created on the male diabetic (db/db) or nondiabetic (db/+) mice by using a 6-mm biopsy punch. The wounds were left to heal by secondary intention. Wounds were imaged on days 0, 3, and 7 after wounding, and the wound area was calculated by using digital planimetry. (A) Digital images of representative day 7 wounds from db/db and db/+ mice showing impaired closure in db/db mice. (B) Area measurements of wounds from db/db or db/+ mice. Data are expressed as mean ± SD (n = 5). *p < 0.05. (C) Digital images of representative day 7 wounds from FPP- or placebo glucose-treated db/db mice showing improved closure in FPP-treated db/db mice. (D) Area measurements of wounds from FPP- or placebo-treated db/db mice. Data are expressed as mean ± SD (n = 5). *p < 0.05. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article at www.liebertonline.com/ars).

FIG. 3.

Increased oxidant generation by wound macrophages. Wound macrophages were isolated from diabetic (db/db) animals supplemented with FPP or placebo for 8 weeks. Wound macrophage isolation was performed by using PVA sponges and CD11b magnetic-bead sorting. (A) Total oxidant production under nonstimulated (basal) or phorbol ester–stimulated (inducible) conditions was measured by using oxidant-sensitive fluorescence probe DCF and flow cytometry. Open bars, placebo supplemented; solid bars, the FPP-supplemented animals. Data are expressed as mean ± SD (n = 3), *p < 0.05. (B) Specifically to identify whether the oxidant species is superoxide, chemiluminescence-based assay for superoxide anion was performed on PMA-stimulated wound macrophages. Data are expressed as mean ± SD (n = 4), *p < 0.05.

FIG. 4.

Improved nitric oxide production by wound macrophages and elevated iNOS gene expression in wound tissue in FPP-supplemented diabetic mice. (A) Wound tissue was collected on day 3 after wounding from FPP- or placebo-supplemented db/db mice. NO production from wound macrophages was measured by using Greiss reagent. Wound macrophages were isolated from diabetic (db/db) animals supplemented with FPP or placebo for 8 weeks, as described earlier. Data are expressed as mean ± SD (n = 5), *p < 0.05. (B) iNOS transcripts were quantified in wound tissue by using real-time PCR. 18S was used as a reference housekeeping gene. Data are expressed as mean ± SD (n = 4), *p < 0.05.

FIG. 5.

Elevated abundance of CD68 and VEGF gene in wounds of diabetic mice supplemented with FPP. Wound tissue was collected on day 3 after wounding from FPP- or placebo-supplemented db/db mice, and total RNA was extracted. CD68 and VEGF transcripts were quantified by using real-time PCR. 18S was used as a reference housekeeping gene. Data are expressed as mean ± SD (n = 4); *p < 0.05.

FIG. 6.

Greater endothelial cell abundance in the wound tissue of FPP-supplemented diabetic mice. Day 7 wound tissue from FPP- or placebo-supplemented db/db mice was cryosectioned, and a marker of vascularization was estimated by staining for CD31 (red, rhodamine) and nuclei (blue, DAPI). The images in the right panels are magnifications of the white boxed area in the corresponding left panels. Scale bar, 50 μm (left panels). Scale bar, 20 μm (right panels). Bar graph presents image-analysis outcome (mean ± SD; n = 3); *p < 0.05. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article at www.liebertonline.com/ars).