Molecular Changes in Diabetic Wound Healing following Administration of Vitamin D and Ginger Supplements: Biochemical and Molecular Experimental Study.

BACKGROUND: Circulating micro-RNAs are differentially expressed in various tissues and could be considered as potential regulatory biomarkers for T2DM and related complications, such as chronic wounds. AIM: In the current study, we investigated whether ginger extract enriched with [6]-gingerol-fractions either alone or in combination with vitamin D accelerates diabetic wound healing and explores underlying molecular changes in the expression of miRNA and their predicted role in diabetic wound healing.
METHODS: Diabetic wounded mice were treated with [6]-gingerol-fractions (GF) (25 mg/kg of body weight) either alone or in combination with vitamin D (100 ng/kg per day) for two weeks. Circulating miRNA profile, fibrogenesis markers, hydroxyproline (HPX), fibronectin (FN), and collagen deposition, diabetic control variables, FBS, HbA1c, C-peptide, and insulin, and wound closure rate and histomorphometric analyses were, respectively, measured at days 3, 6, 9, and 15 by RT-PCR and immunoassay analysis.
RESULTS: Treatment of diabetic wounds with GF and vitamin D showed significant improvement in wound healing as measured by higher expression levels of HPX, FN, collagen, accelerated wound closure, complete epithelialization, and scar formation in short periods (11-13 days, (P < 0.01). On a molecular level, three circulating miRNAs, miR-155, miR-146a, and miR-15a, were identified in diabetic and nondiabetic skin wounds by PCR analysis. Lower expression in miR-155 levels and higher expression of miR-146a and miR-15a levels were observed in diabetic skin wounds following treatment with gingerols fractions and vitamin D for 15 days. The data showed that miRNAs, miR-146a, miR-155, and miR-15a, correlated positively with the expression levels of HPX, FN, and collagen and negatively with FBS, HbA1c, C-peptide, and insulin in diabetic wounds following treatment with GF and /or vitamin D, respectively.
CONCLUSION: Treatment with gingerols fractions (GF) and vitamin D for two weeks significantly improves delayed diabetic wound healing. The data showed that vitamin D and gingerol activate vascularization, fibrin deposition (HPX, FN, and collagen), and myofibroblasts in such manner to synthesize new tissues and help in the scar formation. Accordingly, three miRNAs, miR-155, miR-146a, and miR-15, as molecular targets, were identified and significantly evaluated in wound healing process. It showed significant association with fibrin deposition, vascularization, and reepithelialization process following treatment with GF and vitamin D. It proposed having anti-inflammatory action and promoting new tissue formation via vascularization process during the wound healing. Therefore, it is very interesting to consider miRNAs as molecular targets for evaluating the efficiency of nondrug therapy in the regulation of wound healing process.

1. Introduction

Diabetes is one of the diseases characterized by multiple metabolic disorders, significantly attributed to many extensive skin damages [1-3]. More than 20% of diabetic patients showed severe and persistent complications of diabetes such as ulcer of lower extremity and poor wound healing during their lifetime which seriously affect their quality of life [4, 5].

Impair or poor in diabetic wound healing may lead to several complications particularly chronic infections, neuropathy, increase in the levels of collagen-degrading matrix metalloproteinases (MMPs), a decline in collagen synthesis, and finally a disturbance in microvascularization which subsequently produce serious diagnostic and therapeutic problems among diabetic wounds [6-9]. Previously, it was reported that microbes produce toxins in infected diabetic wounds which produce a significant concomitant killing of regenerating cells and formation of unpleasant exudates and leading finally to poor or lack of healing process [10, 11].

Although various strategies such as microbial infection control, wound offloading, endovascular treatment, moist dressings, and surgical repair are more effective for wound healing, slower wound healing has been reported in diabetic wounds, and an amputation was the end-up solution for diabetic foot ulcer in 7%-20% of patients [12, 13].

Therefore, new therapeutic strategies based upon adjuvant therapies of herbal origin that promote diabetic wound healing are continuously being evaluated to reduce the morbidity and mortality among diabetic wounded patients. About 80% of herbal plants were shown to provide globally medical use in treating many diseases [14-18].

In many countries, ginger (Zingiber officinale) as a widely used spice and condiment has a variety of effects on the skin, including improvement of wound healing. In particular, this may be due to both antioxidant and anti-inflammatory properties of ginger [19-23], which significantly helps in or promotes the reformation of new blood vessels [20].

Recent studies support the efficiency of ginger ethanolic extracts to produce a significant antimicrobial and wound healing capability in models of experimental animals [24, 25]. In addition, these studies support the use of plants such as ginger in herbal medicine and as a base for the development of new drugs [21, 22]. It was proposed previously that ginger extracts in combination with curcumin might provide structure and function in skin and consequently reduce the possibility of the formation of nonhealing wounds [26].

Several bioactive compounds were estimated in different types of ginger rhizomes [27], including gingerols, shogaols, phenylbutenoids, diarylheptanoids, flavonoids, diterpenoids, and sesquiterpenoids, which proved their beneficial effects against the symptoms of many diseases, parasites, and insects and in poor wound healing [27-30]. The most active phenolic compounds present in ginger are [6]-gingerol, [8]-gingerol, and [10]-gingerol; these compounds are considered as the main components responsible for ginger pharmacological effects [14, 15].

Also, the biological effects of vitamin D supplementation were extended recently far beyond usual calcium metabolism. Significantly accelerated wound healing and improved healing quality were reported in diabetic mice following vitamin D supplementation [31-33].

The data of that study showed that vitamin D ameliorates impaired wound healing by suppressing endoplasmic reticulum (ER) stress [31-33]. In addition to that, it was reported previously that the promising effects of vitamin D supplementations on wound healing proceed through stimulating the phagocytosis and killing the bacteria by macrophages [34], suppressing interferon-g-mediated macrophage activation [35], activating insulin receptor expression, and downregulating cytokine generation [36].

Although both ginger and vitamin D contribute in improving impaired or delayed wound healing in diabetes [19-40], little is known about their effects either alone or in combination on the molecular changes accompanying healing process in diabetic wounds, especially micro-RNAs.

MicroRNAs (miRNAs) are endogenous noncoding, single-stranded RNAs~22 nucleotides in length [41] which have been identified in a considerable number to regulate posttranscriptional gene expression of about one-third of human genes [41, 42]. It was reported in the diagnosis of many diseases particularly T2DM, cancer, and neurological diseases [43-45], whereas many biological processes, such as cell proliferation, apoptosis, differentiation, and metabolism were shown to be controlled by miRNAs [46]. It mainly is paired with target-specific bases on genes causing target mRNA degradation or inhibiting of its translation process [46, 47].

Previous studies suggested an alternative pre-mRNA splicing mechanism and fibrin markers deposition, fibronectin, collagen, and hydroxyproline, in wound healing; these two mechanisms play a role in wound healing [48, 49]. Also, the expression of miRNAs such as miR-146a and miR-125b improved diabetes-associated nonhealing wounds via epigenetic regulation mechanisms and suppression for inflammatory genes such as TNFα within the wounds [50-52].

From the aforementioned data [20-52], concerning the role of both ginger and vitamin D in wound healing via providing structure, function, and vascularity of skin tissues, the epigenetic regulation role of miRNAs greatly supports the proposal of this study.

Thus, in the current study, we investigated whether ginger extract enriched with gingerol-fractions either alone or in combination with vitamin D accelerates diabetic wound healing and explores underlying molecular changes in the expression of miRNAs and their predicted role in diabetic wound healing.

2. Materials and Methods

2.1. Preparation of the Gingerol Fractions (GF)

Fresh ginger rhizomes (9gm) were fractionated into 14 fractions (F1 to F14) with gradients of n-hexane and ethyl acetate by column chromatography (18.0 cm by 4.2-cm internal diameter [i.d.]) on silica gel (70-230 mesh) [53]. Gingerol fractions (GF) were identified in F3 by high-pressure liquid chromatography (HPLC) analysis as shown in the flow chart (Figure 1). In addition to that, further fractionation and quantification of gingerols were estimated with a semipreparative HPLC system. In this step, a sample of 50 mg of GF was injected in the HPLC system and gingerol was collected from each fraction separated and further weighed according to previous methods [53]. The analyzed amount of GF contains 42.6%  [6]-gingerol (the main ginger compound), 4.9%  [8]-gingerol, 3.8%  [10]-gingerol, and 48.7% other minor compounds. Upon using in the treatment process, GF has diluted in a2% Tween 80 solution and was orally administered in a maximum volume of 2.5 to 3.5 ml per rat as mentioned previously [53].

Figure 1

Flow chart of the fractionation of ginger, analysis of the chemical composition of major active fractions (gingerol).

2.2. Animals and Study Design

A total of 60 healthy Wistar male rats, six-week-old, were used in this study. The animals were housed and subjected to normal feeding, drinking, and health care mechanism according to the guidelines of the experimental animal care center, College of Applied Medical Sciences, King Saud University, Riyadh, Saudi Arabia [54, 55]. The experiment and the procedures were approved by the Ethics Committee of the Experimental Animal Care Society, Rehabilitation Research Chair (RRC), College of Applied Medical Sciences, King Saud University, Riyadh, Saudi Arabia, under file number ID: RRC-2018-059. Animals had no history of surgery, infection, and other medical interventions, not to take any nutritional supplements that may affect vitamin D, were included in this study. The animals were randomly assigned to five groups of 12 each [54, 55].

2.3. Induction of Diabetes

Mice were injected intraperitoneally with only one dose of STZ (100 mg/kg, in 0.01M sodium citrate, pH4.3–4.5). A single large dose of STZ for seven days is used for attempting to severe T1DM by direct toxicity to β cells with little or no measurable insulin production [56, 57]. Nondiabetic mice were injected with only a saline vehicle. After 7 days, levels of fasting blood sugar (FBG) were assayed by the Hitachi 7060 C automatic. Sandwich biotin-avidin enzyme-linked immunosorbent assay (BA-ELISA) was used to measure serum insulin and C-peptide as previously reported [58]. Insulin assay had an interassay coefficient of variation (CV) of < 9.0% and no cross-reactivity to proinsulin (< 0.05%). Also, Hemoglobin A1c (HbA1c) levels in the whole blood were quantified by Glycomet kit (Biocode Hycel, Massy, France) using the method of exchange chromatography. Mice with fasting blood glucose levels higher than 250 mg/dl were considered diabetic [58].

2.4. Excision Wound Healing Assessments

Before wounding process, all animals were anesthetized with ketamine hydrochloride (50 mg/kg, i.p., body weight) in combination with xylazine hydrochloride (10 mg/kg) of body weight and shaved at the predetermined area, dorsum portion using depilatory cream (Reckitt Benckiser, Inc., UK) as previously reported [54]. Then, a circular wound was inflicted by cutting away approximately 2 cm of the diameter of the predetermined area on the anterior-dorsal side of each mouse using a sterile surgical blade as described previously [59]. To avoid any disturbance, the animals were then placed in separate cages and the bedding was changed daily.

2.5. Wound Treatments

Rats were divided into five groups, as follows. (1) Normal group: nondiabetic mice received a saline vehicle for 15 days; (2) diabetic Mellitus group (DM group): diabetic mice were injected with a saline vehicle for 15 days; (3) vitamin D treatment group (VD treatment group): diabetic mice were treated with vitamin D (100 ng/kg per day) for 15 days; (4) diabetic mice received gingerol-enriched solution (25 mg/kg of body weight) through oral route for 15 days; (5) diabetic mice received combinations with vitamin D (100 ng/kg per day) plus gingerol-enriched solution (25 mg/kg of body weight) for 15 days as mentioned before [60]. Periods spent for complete wound contraction and epithelialization were significantly calculated as a percentage of a reduction in wound area as shown elsewhere [60]. A controlled camera (Sony Cyber-Shot, Dscw80) was used to monitor the progressive changes in wound area on a wounding day, followed by measurements on 3rd, 6th, and 9th day. Later on, the contraction in the wound area was evaluated by using the ImageJ program [60]. At the end of the treatment period, rats were anesthetized and a specimen sample of tissue was isolated from the healed skin of each group of mice for wound healing and histochemical analysis [60].

2.6. Assessment of Wound Healing and Epithelialization Rate

After surgery, wound area was measured by tracing the wounds with the help of transparent sheet on days 3, 6, and 9 as previously reported [61]. In addition to that, the percent of wound contraction was significantly calculated from the measurements of wound size taken at both the time of surgery and the time of biopsy according to the following equation [62]:(see [62]), where A0 is the original wound area, and At is the area of wound at the time of biopsy [62].

Epithelialization period was calculated as the number of days required for falling off the dead tissue remnants of the wound without any residual raw wound [63].

2.7. Assessment of Hydroxyproline

Dried wound tissues were subjected to estimate hydroxyproline (HPX) at 60°C as previously reported [64]. In this experiment, the tissues were hydrolyzed with adding 5 mL of 6N HCl for three hours at 130°C. Neutral hydrolysates (pH 7.0) were subjected to Chloramine-T oxidation for 20 min in room temperature. After 10 min, 0.4 M perchloric acid was added as solution of stop reaction, termination of chloramine T oxidation. Finally for color development, 1 mL of Ehrlich's reagent was added to tubes, shook, and placed in water bath (60°C /20 min). Hydroxyproline concentration was measured in cooled solutions colorimetrically at 557 nm by using ultraviolet (Systronics-2203) spectrophotometer.

Hydroxyproline and collagen concentrations were measured according the following equations: (see [65]).(see [65]).

2.8. Assessment of Fibronectin (FN) Levels

Excised tissues were subjected to estimate fibronectin (FN) levels as previously mentioned [66]. In this method, excised tissues were rinsed in 5-10 mL of ice-cold PBS (0.01mol/L, pH 7.0-7.2) and homogenized by using a glass homogenizer on ice. To further break cell membranes, the resulting suspension was sonicated with an ultrasonic cell disrupter. Then, the produced homogenates were centrifuged for 5 minutes at 5000×g and the resulting residue was used to estimate the concentration of fibronectin using immune assay ELISA kit (ABIN1874233, Atlanta, GA30338, USA) at a wavelength of 450 nm using a spectrophotometer [66]. The concentration of fibronectin was then determined by comparing the O.D. of the samples to the standard curve as previously reported [66].

2.9. Molecular Assessments of microRNAs from Skin Wounds

2.9.1. Extraction and Purification of Circulating RNA

First, total RNA was extracted from skin wound tissues by using TRIzol reagent (Clontech Laboratories Inc., Mountain View, CA, USA) according to the manufacturer's protocol [58, 65]. Then both integrity and quantity of total RNA were assessed by using an Agilent 2100 Bioanalyzer (Agilent Technologies) as previously reported [45, 54]. Finally, cDNA of miR-146a, miR-155, and miR-15a were then synthesized using the Mir-X miRNA First-Strand Synthesis Kit (Clontech Laboratories Inc.) [58, 65].

2.9.2. Real-Time qPCR Analysis of microRNAs

Mir-X miRNA qRT-PCR SYBR Kit (Clontech Laboratories Inc.) with the applied biosystems 7300 Quantitative Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) was used to measure the expression of miR-146a, miR-155, and miR-15a in skin wound tissue [56, 58]. In this experiment, ready-made solutions containing the primers and probes for miR-146a, miR-155, and miR-15a (Applied Biosystems, Foster City, CA) were used to estimate the molecular levels of miR-146a, miR-155, and miR-15a by using a real-time RT–PCR analysis (ABI 7300 system, Applied Biosystems) [58, 67]. The expression levels of the analyzed miRNAs were significantly measured in relation to a normalized internal quantitative control, U6 snRNA levels and the 2−ΔΔCt system. All reactions were run in duplicate to avoid errors and to exactly determine values of cycle threshold mean for each sample including both amplified miRNAs and endogenous control [58, 68].

2.10. Statistical Analysis

The data obtained were statistically analysed using a statistical software SPSS version 17. Among groups, the results obtained were expressed as Mean and standard deviation. Kruskal–Wallis one-way ANOVA and post-hoc (Tukey HSD) test were used to compare the mean values of the studied variables. Additionally, Spearman rank correlation analysis was performed to assess the relationship between various study parameters [58, 68]. The data obtained were deemed significant at P < 0.05.

3. Results

Table 1 shows established type 1 diabetic model induced by one dose of STZ (100 mg/kg). The rats with blood glucose greater than 250 mg/dl were defined as diabetic. Higher levels of FBS, HbA1C, and C-peptide (ng/mL) along with reduced levels of insulin were significantly (p=0.05) reported in diabetic rats compared to those of controls. Compared to diabetic rats, gingerols fraction (GF) and /or vitamin D treated rats showed significant improvement (p=0.001) in the levels of FBS, HbA1c, C-peptide, and insulin, respectively (Table 1). Also, body weight was reported as a marker of diabetes. In diabetic rats treated with GF along with vitamin D, body weight was significantly restored (P< 0.01; p< 0.001), respectively, towards normal ranges compared to nontreated diabetic rats (Table 1).

Table 1

Diabetic control variables and body weight changes in control and diabetic mice following established STZ-induced diabetic mice.

Variables	Control	Diabetic	Vitamin D	GF group	GF+Vitamin D
Body weight (g)	42±3.7	28±1.8∗	29.7±2.3∗∗	31.7±1.5∗∗∗	34.1±3.7∗∗∗
FBG (mg/dl)	86±2.4	286±3.7∗	240±3.4∗∗	210±6.3∗∗∗	186±2.1∗∗∗
HbA1C	3.6±1.1	7.9±3.7∗	5.1±2.5∗∗	4.8±2.4∗∗∗	4.8±3.4∗∗∗
Insulin (mU/L)	9.6±1.6	4.6±1.3∗	5.2±1.6∗∗	7.6±2.8∗∗∗	8.3±2.4∗∗∗
C-peptide (ng/mL)	0.6±0.25	2.85±0.75∗	2.3±0.68∗∗	2.1±0.71∗∗∗	1.85±0.9∗∗∗

Data are expressed as mean ± SD; ∗p<0.05 (compared with control), ∗∗p<0.01 (vitamin D vs. diabetic group), and ∗∗∗p<0.001 (diabetic group vs. GF/ or GF+Vitamin D group). GF: gingerols fraction, FBG: fasting blood sugar, and HbA1C: glycated hemoglobin A1c.

Effect of GF and vitamin D administration on fibrogenesis markers was evaluated in this study (Figure 2(a)). The levels of HPX, FN, and collagen were significantly higher in GF- (P < 0.001) and vitamin D-treated (P < 0.01) mice than in normal control and diabetic rats. Moreover, it was observed that the effects of GF and vitamin D were dose-dependent (Figure 2(a)). Compared to the normal control rats, the diabetic rats showed a significant reduction (P < 0.05) in the levels of HPX, FN, and collagen as the fibrogenesis markers in their wounds (Figure 2(a)).

Figure 2

Molecular changes in fibrogenic markers (HPX, collagen, and fibronectin) (a), miRNAs (miR-146a, miR-155, and miR-15a) (b), and percentage of skin wound healing (c) in diabetic and control mice following treatment with gingerols fractions either alone or in combination with vitamin D supplements. There was significant increase in the expression levels of HPX, collagen, and fibronectin in diabetic mice that received gingerols in combination with vitamin D in comparison with diabetic mice only (P=0.001) (a). Also, expression of miRNAs, miR-146a and miR-155, was significantly downregulated in comparison with miR-15a significantly upregulated in diabetic wound tissues following treatment with both gingerols fractions and vitamin D (P=0.001) (b). Maximum wound contraction (90 %-97 %) was reported in diabetic skin wound treated with vitamin D, gingerols fractions, and a combination of both gingerols and vitamin D, respectively (c).

Changes in the expression of miRNAs as molecular markers were reported in skin wound tissues of diabetic and nondiabetic rats (Figure 2(b)). Compared to the normal control rats, the diabetic rats showed a significant upregulation (increase, P < 0.05) in the expression levels of miR-155 and a significant downregulation (reduction, P < 0.05) in the expression levels of miR-146a and miR-15a supporting significant association of miRNAs as molecular markers in diabetic wounds (Figure 2(b)). However, in diabetic treated rats, skin wound tissues showed significant decline in the expression levels of miR-155 and increase in the levels of miR-146a and miR-15a following treatment with GF (P< 0.001), vitamin D(P< 0.0.1), and GF+ vitamin D (P< 0.001), respectively (Figure 2(b)). The data showed that miRNAs, miR-146a, miR-155, and miR-15a, as molecular targets for diabetic wound healing, were significantly regulated following treatment with gingerols fractions either alone or in combination with vitamin D. miR-155 was significantly downregulated in comparison with miR-15a and miR-146a significantly upregulated in diabetic wound tissues following treatment with both gingerols fractions and vitamin D (P=0.001).

Gingerols fractions and Vitamin D treatments significantly accelerated diabetic wound healing as shown in Table 2 and Figures 2(c) and 3. Wound closure, complete epithelialization, and scar formation were examined in 6 mm diameter wounds and measured by digital camera every 3 days until day 15. Delayed wound healing was observed in diabetic wounds (P<0.01) compared to normal wounds, whereas wound closure, complete epithelialization, and scar formation without any raw wound residues were significantly reported at day 19 while most of the diabetic wounds remained open with a low average closure rate of 50% (Table 2, Figure 2(c)).

Table 2

Effect of vitamin D and gingerols fraction supplements on wound contraction and epithelialization period of wound in excision wound model.

Group	Wound contraction					Epithelialization period (days)
	0 day	3rd day	6th day	9th day	Scar area (mm2)
Control	196.1 ± 0.45	156.2± 0.2 (25%)	96.7 ± 3.7 (48%)	86.4 ±1.6 (68%)	93.4±2.5	14.6 ± 0.45
Diabetes	198.7 ± 0.6a	185±2.1a (16.9 %)	112.3± 1.2a (29.2 %)	108 ±3.8 (52.5 %)a	78.3±3.2a	18.8 ± 0.65b
Vitamin D	196.6 ± 0.65	128.3±2.3 (39.6%)a	75 ± 3.6 (71 %)b	65 ±4.8 (86.5%)c	96.8±3.1c	13.0± 0.52c
GF	198.6 ± 0.56	115 ± 2.1 (42 %)a	61± 1.1 (81.8%)b	35.4 ±0.4 (92.6 %)c	98.6±1.4c	12.0 ± 0.35c
GF+VitaminD	197.9 ± 0.98	102 ± 0.31 (51.4 %)	56 ± 3.1 (87.5 %)	26.4 ±0.3 (96%)c	89.3±2.3c	11.3 ± 2.3c

aP < 0.05, bP < 0.01, and cP < 0.001. All values are represented as mean ± SD, n = 12, animals in each group. Data were analyzed by one-way ANOVA, followed by Tukey-Kramer Multiple Comparisons Test. GF: gingerols fraction group.

Figure 3

Photographs represent the percentage of wound closer rates on different postexcision days (0-15 days). Vitamin D treated group: diabetic mice received vitamin D (100 ng/kg per day) for 15 days; GF group: diabetic mice received gingerol-enriched solution (25 mg/kg of body weight) through oral route for 15 days; GF+Vitamin D group: diabetic mice received combinations with vitamin D (100 ng/kg per day) plus gingerol-enriched solution (25 mg/kg of body weight).

In diabetic wound treated with GF and/or vitamin D, wound closure, complete epithelialization, and scar formation were significantly (P< 0.001) improved in short periods (11-13 days) compared to both normal and diabetic cases. The data obtained during the proposed periods confirmed that treatment with gingerol fraction along with vitamin D accelerating epithelialization improved diabetic wound closure. Maximum wound contraction (90%-97%) was reported in diabetic skin wounds treated with vitamin D, gingerols fractions, and a combination of both gingerols and vitamin D, respectively, and increased the healing effect by 38%-40% (Figure 2(c)).

Correlation analysis showed that miRNAs, miR-146a, miR-155, and miR-15a, as molecular biomarkers, correlated positively with (P < 0.001) the expression levels of HPX, FN, and collagen and negatively with (P < 0.001) FBS, HbA1c, C-peptide, and insulin in diabetic wounds following treatment with GF and /or vitamin D, respectively (Table 3).

Table 3

Correlation between circulating miRNAs expression with fibrogenic markers (HPX, collagen, and fibronectin), diabetic controls, and wound healing of diabetic skin wound tissues of mice following treatment with gingerols fraction and/or vitamin D for three weeks.

Variables	Whole cohort (qRT-PCR) of miRNAs concentrations  (a)
	miR-146a		miR-155		miR-15 a
	R	P	R	P	R	P
HPX (μg/g of tissues)	0.52	0.001	0.22	0.001	0.57	0.001
Collagen	0.31	0.001	0.39	0.001	0.49	0.001
Fibronectin	0.25	0.001	0.31	0.001	0.35	0.001
Diabetes ( HbA1c)	-0.46	0.001	-0.51	0.001	-0.54	0.001
Insulin	-0.39	0.001	-0.31	0.001	-0.38	0.001
C-peptide (ng/mL)	-0.23	0.001	-0.42	0.001	-0.53	0.001
Epithelialization period (days)	0.47	0.01	0.33	0.03	0.41	0.01
wound closer (% contraction)	0.25	0.01	0.48	0.01	0.52	0.01
Scar formation	0.26	0.01	0.21	0.01	0.32	0.01

 aData are R (Spearman). HbA1C: glycated hemoglobin A1c, miR: microRNA, and HPX: hydroxyproline.

Also, significant positive association was reported between the expression of miR-146a, miR-155, and miR-15a in treated wound tissues and healing activity of both GF and vitamin D as measured by accelerated wound closure, complete epithelialization, and scar formation in short periods (11-13 days, (P < 0.01)) as shown in Table 3.

4. Discussion

In this study, delayed diabetic wound healing in rats treated with STZ was significantly improved following treatment with vitamin D and gingerols fractions (GF) for two weeks.

In this current study, diabetic control variables, FBS, HbA1C, C-peptide, and insulin, were significantly improved following treatment with GF and vitamin D, respectively.

Previous research studies suggested an inverse lower vitamin D status in patients with DM and significant association between hypovitamin D and the progression of diabetes [69, 70]. Also, patients with type 2 DM were in risk with lower 25(OH) D concentrations [12, 71] and need the administration of vitamin D together with calcium to improve their glycemic status. It was reported that treatment with vitamin D together with calcium significantly has effects on glucose homeostasis in diabetic and nondiabetic patients [72, 73].

Recent studies also confirmed our data that gingerols plays an integral part in maintaining glucose homeostasis [73, 74], glucose uptake in adipocytes [73], and skeletal muscle cells [75]. The mechanistic action of gingerols against diabetes goes through an increase in the expression of Glucose transporter type 4 (GLUT4) on the cell membrane [76] and activation of the AMP-activated protein kinase (AMPK) pathway [77]. Thus, it showed antihyperglycemic and antihyperlipidemic effects in type 2 diabetic subjects [78].

In uncontrolled diabetes, persistent complications such as ulcer of lower extremity and poor wound healing were significantly reported. Also, slower diabetic wound healing was observed even with the best conventional treatment and more than 15% of patients need an amputation despite undergoing standard care treatment [76].

In this study, diabetic wounds showed a significant decrease in the expression levels of HPX, FN, and lower deposition of collagen compared to normal controls. Consistent with the data of this study, previous reports suggested that delayed diabetic wound healing may lead to chronic infections, neuropathy, increase in collagen-degrading matrix metalloproteinases (MMPs), decrease in collagen synthesis, and microvascular disorders which subsequently produce serious diagnostic and therapeutic problems [6-9].

Thus in this current study we examined the effects of a ginger extract enriched with gingerol-fractions either alone or in combination with vitamin D in accelerating diabetic wound healing. Significant increase was reported in the levels of fibrogenesis markers, hydroxyproline (HPX), fibronectin (FN), and collagen, significantly observed in diabetic wound tissues treated with gingerol-fractions and vitamin D. The increase in HPX, FN, and collagen significantly improved wound healing in short periods (11-13 days) with complete wound closure, epithelization, and scar formation with increased healing effect by 38%-40% compared to diabetic and control groups.

Our data support the role of increasing of HPX, FN, and collagen as parameters of fibrin deposition which are significantly important in skin epithelialization and elasticity during the wound healing process; also vitamin D and gingerol have been shown to activate vascularization and myofibroblasts in such manner to synthesize new tissue and help in the scar formation [79-81]. In addition, the improved wound healing results in our study matched with those recorded in diabetic wound healing following treating with vitamin D [30–34, 82] and ginger extracts or its active constituents [83-86].

Topical application of 1,25(OH)2D was shown to produce positive wound healing effects in diabetic and nondiabetic wounds. It was proposed that vitamin D increases reepithelialization of wounds via calcium and the global vitamin D receptor (VDR) which significantly is very important in the regulation of epidermal and hair follicle differentiation [78, 79]. Also, anti-inflammatory vitamin D supplements significantly enhance NF-κB activation and signaling mechanisms in various cell types which in turn decreases inflammation and promotes wound healing [87-89]. Recently, it was suggested that vitamin D treatment may improve impaired wound healing in diabetic mice by suppressing endoplasmic reticulum (ER) stress [90, 91].

While several studies have evaluated the positive effects of vitamin D as a wound healing agent [78, 79, 82, 87–91], little information on the value of 6-gingerol as a wound-preventative was recorded [92]; only combination of curcumin and ginger extracts showed a novel approach in improving structure, function in skin, and concurrently nullifying the formation of nonhealing wounds [83, 84].

In this study, we suggest for the first time assessing biological activities of vitamin D and gingerols in combination for effects on skin wounds in STZ-induced diabetic rat. The data showed a significant increase in the levels of HPX, FN, and collagen and reduction in diabetic controls and overall improvement in wound healing in short time (11.3 day) following treatment with a combination of gingerols and vitamin D. This may be due to antioxidant, anti-inflammatory, and fibrogenesis activities [79, 88, 90–94], which significantly support the possibility of considering vitamin D and gingerols as potential therapeutic agents for diabetic wound healing.

In this study, the role of miRNAs in wound healing was evaluated in diabetic and nondiabetic skin tissues.

In diabetic wound tissues, we identified three circulating miRNAs, including increased concentrations of miRNAs, miR-155, and decreased concentrations of miR-146a and miR-15a, which were strongly linked to outcome measures of HPX, FN, collagen, and diabetic markers such as FBS, insulin, C-peptide, HbA1c, and impaired wound closure, epithelization, and complete scar formation.

After treating skin wounds with gingerols fractions and vitamin D, lower expression in miR-155 levels and higher expression of miR-146a and miR-15a levels were observed. The data showed that miRNAs, miR-146a, miR-155, and miR-15a, correlated positively with the expression levels of HPX, FN, and collagen and negatively with FBS, HbA1c, C-peptide, and insulin in diabetic wounds following treatment with GF and /or vitamin D, respectively. Also, miR-146a, miR-155, and miR-15a showed a positive association with healing activity of both GF and vitamin D as measured by accelerated wound closure, complete epithelialization, and scar formation in short periods (11-13 days, (P < 0.01). The data showed that miRNAs, miR-146a, miR-155, and miR-15a, as molecular targets, may have a role in healing wounds in diabetes and nondiabetes skin tissues during vascularization, fibrin deposition, and reepithelialization process.

Micro-RNAs are likely to be important regulators differentially expressed in various tissues and could be considered as potential biomarkers for T2DM and related complications [22]. Several miRNAs were reported to regulate adipose tissues, control glucose metabolism, and develop T2DM in experimental animals and humans [23]. Up- and downregulation changes of miRNAs expression in different tissues and body fluids significantly reflect disease pathology, especially in diabetes-related complications including microvascular complications [95-100].

PCR results confirmed that miR-155 expression was lower, and miR-146a and miR-15 expression was higher after treatment with both gingerols fractions and vitamin D on days 3, 6, 9, and 15. Also, faster healing was observed in treated diabetic skin wounds compared to normal and diabetic nontreated wounds. Regulation of these miRNAs by gingerols fractions and vitamin D occurred via antioxidant and anti-inflammatory mechanisms as previously reported [101-103].

It was suggested that inhibition of miR-155 and improving of miR-146a expression promote downregulation of inflammatory cells, including neutrophils and macrophages (CD68-positive), and upregulation of fibrogenesis markers including collagen deposition, HPX, and fibronectin which ultimately improves or reduces inflammatory process and produces potential effects in accelerating wound healing process [104-107].

More additional recognized role of vitamin D in wound healing process is to regulate miRNA expression levels via miRNA signaling or apoptotic mechanism which increases fibrin deposition and reepithelialization process [108, 109]. Supporting the anti-inflammatory action of our identified miRNAs, miR-155 expression was lower, and miR-146a and miR-15, previously miR-146a as a negative regulator of the innate immune response [102, 103], were downregulated in skin wounds of the diabetic mouse, which was associated with enhanced NF-kB signaling and inflammation [110]. Thus, in order to improve diabetic wound healing, an increase in miR-146a expression was needed to activate mesenchymal stem cells which repress proinflammatory genes within diabetic wounds [50, 111–113].

In conclusion, treatment with vitamin D and gingerols fractions (GF) for two weeks significantly improves delayed diabetic wound healing. The data showed that vitamin D and gingerol activate vascularization, fibrin deposition (HPX, FN, and collagen), and myofibroblasts in such manner to synthesize new tissues and help in the scar formation. Accordingly, three miRNAs, miR-155, miR-146a, and miR-15, as molecular targets, were identified and significantly evaluated in wound healing process. It showed significant association with fibrin deposition, vascularization, and reepithelialization process following treatment with GF and vitamin D. It proposed having anti-inflammatory action and promoting new tissue formation and vascularization during the wound healing process. Therefore, it is very interesting to consider miRNAs as molecular targets for evaluating the efficiency of nondrug therapy in the regulation of wound healing process.