Polysaccharide-Rich Extract of Caesalpinia ferrea Stem Barks Modulates Inflammatory and Proliferative Phases Enhancing Diabetic Cutaneous Wound

MATERIALS AND METHODS

Animals: Female Wistar rats (180-220 g, 6-8 weeks, 6 animals/groups/experimental day/total number of animals used:60, study duration: 08.2018-06.2021) were maintained at 26±1°C under 12/12 hrs light/dark cycle, receiving food and water ad libitum. Animals were brought to the laboratory at least 1 hr before the experiments, which were conducted following protocols approved by the Animal Care and Use Committee (UECE n° 12784513-5), following the guidelines of the Brazilian Council of Control in Animal Experimentation (CONCEA). The efforts employed intended to minimize the number of animals used, as well as their suffering.

Plant material: Caesalpinia ferrea stem barks were collected in Custódio (Quixadá-CE, Brazil) and a plant sample was deposited in the Herbarium Prisco Bezerra of the Federal University of Ceará - UFC (exsiccate: 46085) and in the Faculty of Education, Science and Letters of Sertão Central (FECLESC). The name of the plant was verified in The Plant List at http://www.worldfloraonline.org.

Obtention of polysaccharide-rich extract from C. ferrea barks: Three bark pieces (5 g) were washed with distilled water, dried at 40°C and ground into fine particles. Five grams of powder was added to absolute MetOH (1:50, w/v,76°C) for depigmentation-the procedure was repeated twice. The polysaccharides were extracted from the extracellular matrix with 0.1 M NaOH at 97°C and the soluble portion was filtered and centrifuged (2496 g, 25°C) (the procedure was repeated three times). The alkaline supernatants, containing the polysaccharides, were pooled, neutralized (1 M HCl), precipitated with 4 volumes of absolute EtOH and re-centrifuged in the same conditions. The precipitate was dialyzed against distilled water under stirring for 72 hrs and re-centrifuged, being the final supernatant lyophilized and named polysaccharide-rich extract of Caesalpinia ferrea barks (PE-Cf: 3% yield), adapted from Yoon et al.²³.

Alloxan-induced diabetes mellitus: Animals received alloxan (45 mg kg^–1) by intravenous (i.v.) route for the destruction of pancreatic β cells and after 48 hours of administration, peripheral blood was collected for blood glucose assessment. Before alloxan administration, the blood glucose levels were also checked for normoglycemia. Rats within one month of induction that had a blood glucose of 200 mg dL^–1 or higher were selected for experiments. Before performing the glycemic test, animals were maintained fasting for 12 hrs but received water ad libitum³.

Excisional wound model: Animals were anaesthetized by intraperitoneal (i.p.) route with 10% ketamine (100 mg kg^–1) and 2% xylazine (10 mg kg^–1). After dorsum shaving and antisepsis (ethanol 70%), four circular full-thickness wounds were performed using a biopsy punch of 7 mm diameter (ABC instrumentos cirúrgicos, Brazil), followed by excision of epidermis, dermis and hypodermis to expose the panniculus carnosus²². Twenty-four hours after ulceration and recovery to anaesthesia, animals received topical treatment (the route used in traditional medicine) with 0.9% saline as control or PE-Cf (0.025-0.1%, dissolved in saline) for 14 days, twice a day. Wounds were excised using a scalpel and surgical scissors for analysis of inflammatory parameters present in the tissue, such as polymorphonuclear leukocyte infiltrate, fibroblasts and blood vessels²².

Wound area/closure: The wound area was measured with a 0.5 mm precision digital paquimeter (D = largest diameter, d = smaller diameter) to express the ulcerated area (A = π. D/2.d/2). The wound closure was calculated by the equation:

and expressed as a percentage²².

Inflammatory and scarring clinical signs: Hyperemia, edema, transudate, crust detachment wound scab and scar tissues were classified according to the following scores: (0) absence of edema, hyperemia and transudate, (1) mild edema, hyperemia and transudate, (2) moderate edema, hyperemia and transudate, (3) intense edema, hyperemia and transudate. Crust detachment (fissures, fragility) and scar tissue (rosy, pigmentation absence) were observed for presence or absence²².

Hyper-nociception was evaluated by a pressure transducer coupled to a digital analgesia meter (Insight, Brazil) that expressed mechanical pressure in grams (g). A mechanical stimulus (polypropylene tip-0.5 mm diameter) was applied to the ulcer edges and the result of the stimulus was interpreted as nociceptive behavior²².

Histopathology and histomorphometry: Tissues were immediately preserved in 10% neutral buffer formalin for histological analysis. Skin fragments were fixed in 10% formol for 24 hrs, subjected to dehydration in crescent alcoholic series, diaphanized in xylol and impregnated in paraffin (60°C). Fragments were put into paraffin-forming blocks, sectioned in microtome (5 μm thickness) and stained with hematoxylin and eosin (H&E). The histopathological parameters were scored from 0-4 according to Cavalcante et al.²⁴: (0) absence of ulcer/remodelled connective tissue, (1) absence of ulcer/fibrosis with mild chronic inflammation, (2) presence of ulcer/fibrosis with moderate chronic inflammation, (3) presence of ulcer/chronic inflammation (granulation tissue), (4) presence of ulcer/acute process (dilated vessels, mixed inflammatory infiltrate with neutrophils).

Five fields immediately below the ulcer/reepithelization of each animal were photographed (40× magnification, Nikon Eclipse H550S microscope, Japan) for quantification of polymorphonuclear, mononuclear and fibroblast/ myofibroblast cells and blood vessels using the Plugin “Cell Counter” ImageJ’s software (National Institutes of Health, EUA). The sample unit for each animal was the sum of the 5 fields per slide²².

Statistical analysis: Data were expressed as Mean±SEM and analyzed by two-way ANOVA followed by the Bonferroni Test and histopathological data was expressed as Median (maximum and minimum) and analyzed by the Mann-Whitney’s followed by the Dunn’s Test. Categorical data (absent/present) was expressed as relative frequency (%f) and analyzed by the Chi-Square Test. The p<0.05 was considered significant.

RESULTS

PE-Cf reduces the area of the diabetic cutaneous wounds and increases the healing rate: The treatment with PE-Cf (0.05-0.1%) reduced the wound area (Fig. 1a) from the 2^nd day (0.05%: 49.80±2.4, 0.1%: 43.33±2.8, saline: 59.76±1.4 mm²) until the 10^th day (0.05%: 5.02±0.4, 0.1%: 6.42±0.5, saline: 19.94±1.6 mm²). At 0.1% PE-Cf was more effective, reducing the wound area at all stages of the healing process. At 0.05 and 0.1%, PE-Cf increased the healing rate along the entire the time-course (0.05%: 349.9±77.9, 0.1%: 424.8±38.02, saline: 212.4±442.8 AUC), being more efficient at 0.1% and used for the following experiments (Fig. 1b).

1% PE-Cf reduces the inflammatory signs of skin wounds and accelerates diabetic cutaneous wound closure: The intensity of the inflammatory signs of inflammation (hyperemia, edema) of wounds treated with PE-Cf has been significantly reduced (Table 1). On the 5^th day after ulceration, there was a reduction in the intensity of hyperemia when compared to the moderate score of the saline-treated animals. In addition, on the 5^th day, transudate, when present in the animals treated with PE-Cf was slight. The intensity of edema, was also reduced on the 5^th day by 0.1% PE-Cf and PE-Cf increased the nociceptive threshold from the 5^th day until the 10^th day (Table 1). In the subsequent evaluation days, these parameters did not show any significant difference.

The treatment with PE-Cf reduced the time in which the crust remained on the wounds. On the 5^th day, it was observed that PE-Cf caused, partial or total, early crust detachment from the wound edges (48% vs. saline: 18%) and increased the frequency of scar tissue (69%), mainly, from the 10^th day compared to saline (28%). On the 14^th day of treatment 86% of ulcers had scar tissue compared to saline (62%) (Table 1).

1% PE-Cf reduces the inflammatory profile in histopathology and histomorphometry of diabetic cutaneous wound: In Fig. 2a-h, this semi-quantitative analysis of the healing process revealed that PE-Cf had promoted histological improvement in inflammatory parameters at the time course and reduced at 2^nd day the inflammatory profile (Table 2, Fig. 2a, e). Diabetes causes a persistent inflammatory phase associated with an impediment in the quantity and quality of the formation of mature granulation tissue. In the present study, the treatment of diabetic animals with PE-CF also reduced the inflammatory histopathological profile on the 2^nd day indicating a well-established proliferative phase, that is associated with a resolution of the acute inflammatory phase, as seen in the macroscopic analysis from the 2^nd day with an improvement in acute inflammatory clinical signs.

On the 2^nd day post-ulceration, the wound tissues treated with PE-Cf demonstrated ulcer with fibrosis and the presence of chronic inflammatory process, while the control animals showed an ulcer and mixed chronic inflammatory process, with granulation tissue and leukocyte infiltrate showing predominance of polymorphonuclear and mononuclear cells. On the 5^th day after ulceration, the ulcers presented a similar profile being, fibrosis and moderate chronic inflammatory process still present in the wound (Table 2, Fig. 2b, f).

Non-ulcerated tissue was predominant in both control and PE-Cf treated groups on the 10^th day (Fig. 2d, h). The treated tissues displayed discrete fibrosis, granulation tissue and mild chronic inflammatory process, as well as re-epithelialized tissues in some specimens, while control tissues showed persistent chronic inflammatory infiltration with the presence of mononuclear cells. Complete re-epithelization, mild inflammation, fibrosis and remodelled connective tissue, were found in both groups (Table 2, Fig. 2c, g).

Fig. 1(a-b):

0.1% PE-CF promotes wound closure and increases wound closure index in diabetic rats

Fig. 2(a-h):

Photomicrographs of excisional cutaneous wounds of diabetic rats treated with 0.1% PE-Cf

At any evaluation period was observed polymorphonuclear reduction. However, mononuclear cells were increased by 26% on the 2^nd day (242.4±48.2 vs. saline: 177.4±50.15) and reduced by 50-63% from the 5^th (117±27.93 vs. saline: 233±10.92) until the 7^th day (61.75±12.12 vs. saline: 163.6±53.77), respectively (Table 2, Fig. 2a-g). PE-Cf increased the number of fibroblasts on the 2^nd day by 60% (141.4±43.85 vs. saline: 57.2±37.9) until the 7^th day by 21% (232.6±31.79 vs. saline 181.4±35.9). The PE-Cf did not change the number of blood vessels on any day of treatment (Table 2, Fig. 2a-h).

DISCUSSION

The present study demonstrated that the polysaccharide-rich extract obtained from the stem barks of C. ferrea (PE-Cf), presented a 3.1% yield, high content of carbohydrate (35.3%, including 5.7% uronic acid), 3.0 mg g^–1 gallic acid equivalent of polyphenols and low content of protein (3.38%), used topically in the excisional wound in diabetic rats accelerates the healing process via reduction of the inflammatory clinical signs and wound area, an increase of healing rate and recruitment of mononuclear cells and fibroblasts.

In hyperglycemic animals, 0.1% PE-Cf was able to early reduction in transudate from the 2^nd-5^th day and inhibition of the persistent inflammatory state, seen by the reduction of edema and hyperemia on the 5^th day of treatment. Using the same wound model in normoglycemic rats, the treatment with 0.1% PE-Cf reduced the inflammatory clinical signs: edema (5^th day), hyperemia (2^nd day), transudate (2^nd day) and mechanical hyper-nociception (2^nd and 5^th days)²². The inflammatory pain, assessed by analgesimetry was also reversed by PE-Cf on the 5^th day. It was demonstrated that PE-Cf (1 mg kg^–1) administered in mice by intravenous route inhibited the number of abdominal contortions, peripheral mechanical hyper-nociception, polymorphonuclear migration and the formation of paw edema induced by zymosan²¹. These authors suggested an association of the anti-inflammatory effect of PE-Cf to its anti-oxidant effect since it reduced the formation of pro-oxidants and increased that of anti-oxidants.

Cells have several antioxidant defense mechanisms that prevent ROS formation or neutralize ROS after being generated and when the concentration of ROS overwhelms the capacity of these oxidative stress mechanisms causes damage in biomolecules, such as DNA, proteins, lipids and other cellular components⁷. In diabetes, high oxidative stress plays major role in complications and impairs the healing process. Therefore, it is suggested that the attenuation of the inflammatory phase by PE-Cf could also be related to its anti-oxidant activity, seen by the demonstration of the reduced formation of malondialdehyde (MDA) and the inhibition of the activity of myeloperoxidase (MPO) in the same model of excision cutaneous wounds in normoglycemic animals²².

Mononuclear assists in the healing process with immune system defense, debridement and regulatory functions. Its unbalanced polarization can be responsible for delayed wound healing^2,3,9. In diabetes, macrophages show delayed phenotype transition since they can develop inflammatory-oxidative (M1) or anti-inflammatory-healing (M2) phenotypes. Additionally, the histological profile on the 2^nd day already pointed to a well-established proliferative phase. PE-Cf may have recruited M2 macrophages attenuating the inflammatory response and stimulating fibroplasia (from the 2^nd-7^th-day post-wound) via modulation of Transforming Growth Factor beta (TGF-β)^3,22.

Probably due to the persistence of the inflammatory state caused by the condition of acute hyperglycemia, the administration of PE-Cf did not reduce neutrophil migration. In contrast, PE-Cf in normoglycemic rats reduced polymorphonuclear migration from the 2^nd-5^th day of treatment²².

It is known that the period of the healing process occurrence is important, since the larger area of the lesion and the higher probability of microorganism’s colonization may lead to infection and generate greater loss^25,26. Because of the data obtained in the present study, which used topical application of PE-Cf in wounds of diabetic animals and the data obtained from normoglycemic rats²² it was seen that the healing effect obtained after treatment with PE-Cf was accounted for the reduced wound area and the increased rate of healing (2^nd-10^th day), crust detachment (5^th day) and formation of scar tissue (10^th day) both in normoglycemic animals and in animals with acute hyperglycemia. It was previously suggested that the early closure of excisional cutaneous wounds was due to the modulation of the expression of the TGF-β on the 5^th day²².

It is known that TGF-β via the Smad pathway is responsible for the proliferation of fibroblasts, synthesis and maturation of extracellular matrix components²⁷. In animals with acute hyperglycemia, we can suggest that the migration of fibroblasts from the 5^th day and the increase in the healing rate from the 2^nd day of treatment may be influenced by the modulation of the inflammatory phase, possibly by TGF-β, released from macrophages, as suggested by de P. Pereira et al.²² using of the same polysaccharide extract.

The state of acute hyperglycemia induced by alloxan had been shown to slow the healing of oral mucosa ulcers and to increase the expression of pro-inflammatory cytokines such as Tumour Necrosis Factor-alpha (TNF-α and leukocyte migration, as it reduces the deposition of total collagen, the expression of fibroblast growth factor and the migration of fibroblasts³. The acute hyperglycemia process is also associated to the reduction of neoangiogenesis from the 5^th day after the formation of oral mucosa ulcers³. In contrast, in cutaneous wounds of normoglycemic animals treated with 0.1% PE-Cf the formation of new vessels was increased from the 5^th-7^th day of treatment²². However, this study following Kazemi-Darabadi et al.²⁸ in which the number of newly formed vessels is lower in diabetics in comparison with normoglycemic animals, is justified by the weakness of the formation of new vessels in the acute hyperglycemia induced by alloxan. Thus, the present study contributes to the suggestion of the use of C. ferrea polysaccharide-rich extract as an alternative healing therapy for diabetics, validating the popular use of C. ferrea barks in inflammatory processes.

CONCLUSION

In conclusion, PE-Cf accelerates the diabetic healing process acting both in the inflammatory phase by the reduction of clinical signs and leukocyte infiltration and proliferative phase via an increase of fibroblast/ myofibroblasts.

SIGNIFICANCE STATEMENT

Based on the healing effect previously demonstrated for the polysaccharide-rich extract of C. ferrea stem barks (PE-Cf) in normoglycemic rats, the purpose of this study was to test PE-Cf in the model of excisional cutaneous wounds in diabetic rats. The main findings obtained were the reduction of the wound area, hyper-nociception, crust detachment, scar tissue formation, leukocyte infiltration and increase in fibroblast/myofibroblast, suggesting that PE-Cf accelerates the healing process of diabetic rats, acting both in the inflammatory and proliferative phases. Thus, PE-Cf can be an alternative therapy to healing diabetic wounds.

ACKNOWLEDGMENTS

The authors would like to express profound thanks to ISCB, UFC, FECLESC and LAFFIN for the animal care and commitment to the data acquisition. This work was supported by grants from CNPq. AMS Assreuy is the senior investigator of CNPq.