The effect of snakehead fish extract on acute wound healing process in hyperglycemic rats

Editorial note

Editorial Note (20^th October 2023): The F1000 Editorial Team has not yet received a new version of this article, as detailed in the Editorial Notes published on 16^th June and 4^th August 2023, despite repeated chasing. The F1000 Editorial Team will no longer be requesting a new version from the authors. Readers should be aware that the raw images for this article have been seen and verified by the F1000 Editorial Team, but have not been uploaded by the authors. Peer review activity remains suspended until the authors publish a new version of this article.

Editorial Note (4^th August 2023): The F1000 Editorial Team has not yet received a new version of this article, as detailed in the Editorial Note published on 16^th June 2023. The F1000 Editorial Team is actively contacting the authors to request the new version of the article. Peer review activity remains suspended until the authors publish a new version of this article.

Editorial Note (16^th June 2023): Since publication, it has been brought to the attention of the Editorial Team that raw macroscopic and histological images were missing from the underlying data of this article. As per our data availability policy, the Editorial Team requested these images from the authors in March 2023. The authors provided these images, which have been verified. The authors have been requested by the Editorial Team to upload the raw images to a data repository and create a new version of the article to include the data DOI. Peer review activity has been suspended until the authors publish a new version of this article.

Introduction

Prolonged hyperglycemic conditions can cause various complications, both macrovascular and microvascular. Patients tend to experience impaired wound healing processes, which can develop into chronically infected wounds. Chronically infected wounds will increase the risk of systemic infection leading to increase in the morbidity and mortality of patients. Therefore, it is necessary to have a good understanding of the wound healing process and appropriate interventions to support a good wound healing process.^1,2 Physiologically, wound healing is achieved through four phases: hemostasis, inflammation, proliferation, and remodeling. All these phases occur in an integrated way and require an optimal environment and conditions. In hyperglycemia conditions, systemic inflammation leads to increase oxidative stress, impairs migration of leukocytes to the wound area, and inhibit extracellular matrix synthesis.²

Protein plays an important role in regulating the body's homeostatic processes. They are the constituent components of immune cells, cytokines, tissues, and enzymes that are involved in the physiological process of wound healing. Neutrophils, monocytes, and macrophages release pro-inflammatory cytokines and growth factors such as platelet-derived growth factor (PDGF), transforming growth factor-β (TGFβ), and fibroblast growth factor (FGF), which are involved in the activation of fibroblasts and epithelial cells. PDGF stimulates fibroblast functions and collagenase expression. TGFβ controls the regulation signal of extracellular matrix deposits, increasing the production of matrix proteins and downregulation of protease enzymes. In the wound area, there is an increase in the level of hypoxia-inducible factor (HIF) in cells. HIF will bind to specific DNA and stimulate specific transcription genes such as vascular endothelial growth factor (VEGF). VEGF is the main vascular growth factor in the entire angiogenesis process.³

Snakehead fish (Channa striata), a native freshwater fish in South East Asia, contains high protein and albumin which act as antioxidants and constitutive tissue components. Snakehead fish also contains micronutrients such as zinc, iron, calcium, and magnesium which have role in the immune system and wound healing process. Administration of snakehead fish significantly increased fibroblasts in oral mucosal wounds of Wistar rats.⁴

Although there have been several studies regarding the benefits of snakehead fish extract on wound healing in diabetics, the wounds in those studies have different sizes and conditions. The difference in type of wounds, wound size, food intake factors, and metabolic condition can be confounding in assessing the effect of snakehead fish extract on wound healing.^4–6

We developed snakehead fish extract formula (Pujimin Plus^®) as a protein and micronutrients source with an aim to reduce hypoalbuminemia among patients and improve the wound healing process. However, the exact mechanism of how this extract improves wound healing remains unclear.

Therefore, this study was conducted to see the effect of snakehead fish extract on healing acute wound tissue in hyperglycemic rats by analyzing macroscopic and microscopic changes in wound tissue.

Methods

Results

Histological analysis of wound tissue

Microscopic observations for counting the number of neutrophils, fibroblasts, and vascular changes [or angiogenesis processes (the growth of new blood vessels from existing vessels)] were performed at 40× magnification. As noted in Table 5, the average number of neutrophils in extract group reached the highest value on day 3 (54.47±6.27) then declined on day 10 (3.6±2.43). Similar to extract group, the highest mean of neutrophils in control group were observed on day 3 (59±21.27) then declined on day 10 (5.58±2.63). There were no significant differences in neutrophil numbers in both the groups.

Table 5. Number of neutrophils, fibroblasts and vascular changes per field of view in experimental animals.

Histological view		Control Mean (SD)	Extract Mean (SD)	P value
Neutrophils	Day 0	21.00 (5.48)	19.67 (3.57)	0.661
	Day 3	59.00 (21.73)	54.47 (6.27)	0.666
	Day 10	5.58 (2.63)	3.60 (2.43)	0.279
Fibroblasts	Day 0	1.33 (0.71)	1.87 (0.18)	0.141
	Day 3	24.60 (10.25)	40.33 (10.13)	0.040*
	Day 10	44.83 (14.81)	42.07 (6.39)	0.715
Vascular changes	Day 0	6.33 (2.26)	5.67 (2.50)	0.670
	Day 3	13.53 (4.84)	14.80 (3.50)	0.648
	Day 10	4.08 (2.52)	3.87 (1.17)	0.868

* p<0.05.

The angiogenesis processes on day 3 increased to 14.80±3.50 in extract group and 13.53±4.84 in control group then decreased to 3.87± 1.17 in extract group and 4.08±2.52 in control group on day 10. There were no significant differences between both groups. Based on Table 5, the mean number of fibroblast cells in extract groups on day 0 and day 3 were higher than the control group. In extract group, observations on day 0 showed the mean number of fibroblast cells were 1.87±0.18, which then increased to 40.33±10.13 on day 3, and the highest number 42.07±6.39 was observed on day 10. The mean number of fibroblasts in extract group on day 3 had a significant difference compared to the control group (p=0.040). On day 0 and day 3, the mean number of fibroblasts in control group were lesser than the extract group with the value 1.33±0.71 and 24.60±10.25 respectively. On day 10, the mean number of fibroblasts in control group were higher than extract group but there was no significant differences (p=0.715).

Figure 1 shows the histological changes of the wound healing process over time. Histological images on day 0 were collected from wounded skin tissue of the rats after 2 hours of wound infliction. The clot was formed as a result of hemostasis process (Figures 1A-1B). On day 3, infiltration of inflammatory cells in wound tissue was seen, particularly neutrophils and several lymphocytes. More active fibroblasts migrated into the wound area of the extract group on day 3 compared to the control group (Figures 1C-1D). The proliferation phase represents a proliferation of both epithelial and dermal elements which results in re-epithelialization of the wound and laying of the primary extracellular matrix. On day 10, the number of inflammatory cells were less and the layer of the epidermis was thicker. The granular tissue was full of cells and arteries completely occupied the wound in both groups. The histological images showed a complete re-epithelialization with dense collagenous matrix (Figures 1E-1F).

Figure 1. Histological view of wound tissue of hyperglycemic rat.

(A) Control group on day 0; (B) Extract group on day 0. Clotting blood (square) in wound area; (C) Neutrophil infiltration (star), lymphocyte infiltration, and fewer active fibroblast (arrow) in control group on day 3, (D) More active fibroblasts (arrow) with neutrophil infiltration (star), lymphocyte infiltration seen in intervention group on day 3; (E) In control group on day-10; (F) Fibroblast (arrow) with dense extracellular matrix seen in intervention group on day-10. H&E stain, magnification 40×. Fibroblast is spindle-shaped cells (flat and elongated) with oval-shape nucleus. The cytoplasm is basophilic and is stained in purplish blue by H&E stain. During stress, fibroblasts adapt to their environment and have the ability to respond and send local signals. On day 3 after wound creation (Figs. C and D), fibroblasts transformed to active forms with euchromatic nuclei and obvious cytoplasm. This form plays role in wound healing to sends local signal, synthesizes the extracellular matrix, and produces the structural stroma for tissue. On day 10 (Figs. E and F), in remodeling phase, fibroblasts became to inactive forms with small cytoplasm and reduced amount of rough endoplasmic reticulum. The extracellular matrix and stroma became denser in this phase.

Discussions

Wound healing is the complex process that includes several phases such as hemostasis, inflammatory, proliferative, and remodeling. This whole phase requires optimal environment and conditions to be able to take place properly. In conditions of hyperglycemia, systemic inflammation occurs which increases the oxidative stress, impaired migration of leukocytes to the wound area, endothelial cell dysfunction, and decreased synthesis of extracellular matrices. This condition can interfere with the wound healing process.² Snakehead fish (C. striata) extract has high nutritional value and has shown various benefits in increasing albumin levels, lowering inflammatory status, and aiding the wound healing process.^12,15–17

In the inflammatory phase of the wound healing process, there is an increase in the number of neutrophils in the tissue. Neutrophils play a role in fighting infections through the phagocytosis of pathogens and foreign bodies as well as secreting inflammatory mediators such as Tumor Necrosis Factor Alpha (TNF-α) and Interleukin-1 (IL-1) that can attract and activate fibroblasts and epithelial cells. Neutrophils also produce high levels of protease (elastase and collagenase) that can clean up damaged extracellular matrix components.³ Generally, the number of neutrophils in a tissue begins to increase in the first 24–48 hours after injury, reaching a peak at 2–3 days and then decreasing thereafter through the process of apoptosis. In inflammatory conditions with infection, the number of tissue neutrophils can last longer and lead to an elongated wound healing process.¹⁸

Various studies have shown that micronutrients such as zinc, calcium, magnesium, manganese, and iron are required in the wound healing process. Micronutrient deficiency is associated with prolonged wound healing process and the occurrence of chronic wounds. Zinc plays a role in various stages of wound healing including platelet activation, polymorphonuclear influx, re-epithelial processes, angiogenesis, and extracellular matrix remodeling.¹⁹ Magnesium can improve insulin sensitivity and blood sugar regulation that play a role in the healing process of diabetic wounds.²⁰ Calcium is a micronutrient that plays a role in the homeostasis of skin tissue in mammals. Locally, calcium can modulate proliferation, maturation, and cell motility through transduction signals and gene expression in the process of epidermal regeneration and dermal reconstruction in wound healing.²¹ Iron plays a role in the process of angiogenesis in wound healing.²²

In this study, it was found that the number of tissue neutrophils in the control and groups increased on day 3 and decreased on day 10, although there were no significant differences in both groups, the average number of neutrophils in the control group on days 0, 3, and 10 were higher compared to the extract group.

In the proliferation phase, there are several processes that occur including matrix turnover to restore tissue structure and function, angiogenesis processes, and granulation tissue formations that lead to re-epithelialization processes. Migration and accumulation of fibroblasts in the wound area play a role in the movement from the extracellular matrix to the wound area.³

This study showed that the number of tissue fibroblasts in the control and extract group increased on days 3 and 10 with statistically significant differences on day 3 where the number of fibroblast cells of the extract group was higher than the control group. Similar results were obtained in a previous study that showed the administration of snakehead fish extract increased the number of fibroblasts and expression of fibroblast growth factor-2 (FGF-2) in the healing process of oral mucosa of the Wistar rats. Fibroblasts regulate angiogenesis via secretion of FGF-2.¹⁶ Snakehead fish extract contains zinc and calcium that can stimulate fibroblasts in tissue healing. Zinc plays an important role in the proliferation phase by stimulating fibroblast infiltration and extracellular matrix deposit.¹⁹ Calcium modulates proliferation and maturation of dermal structures in the wound area.²¹

The process of angiogenesis is stimulated by local factors including low oxygen pressure, low pH, and high lactate levels. VEGF is the primary vascular growth factor in the entire angiogenesis process.^3,23 Iron grade has a positive effect on wound healing by increasing VEGF and HIF1-α and contains functional proteins that play a role in collagen metabolism and extracellular matrix by procollagen-proline dioxygenase.²²

Our study showed that the number of vascular tissues in the control and extract groups increased on day 3 and decreased on day 10, despite no significant differences in both the groups. From this data, it can be concluded that snakehead fish extract does not affect the vascular tissue significantly in the wound healing process. Dose adjustment and assessment of anemia status in samples were required to assess iron requirements in rats, given the previous studies that assessed the effect of iron on wound healing using different doses, methods of application, and anemia status.^22,24

Macroscopically the extract group had a smaller erythema visual score than the control group, especially on day 3. Even though not significant, the formation of crust in the extract group on day 3, 5, and 7 had smaller visual score compared to control group. These macroscopic results showed that the extract group had lesser erythema on day 3 and fewer crust formation compared to the control group.

The limitation of this study is lack of tissue examination days (Microscopic changes in skin tissue can occur rapidly, especially during the inflammatory and proliferative phase, so histological examination should be done on a regular basis). Therefore, more routine tissue examination is needed to better assess histological changes in the wound healing process. The longer duration of snakehead fish extract intake can affect the result of the study.

Conclusion

In hyperglycemic rats, oral administration of snakehead fish extract significantly increased the number of fibroblasts in the proliferation phase of the acute wound healing process. Although insignificant, snakehead fish extract can also decrease the number of neutrophils of wound tissue in the inflammatory phase of acute wounds. Macroscopically, decreased erythema and fewer crust formation were seen in intervention group compared to control group. Therefore, snakehead fish extract has potential benefits and can be used to support the healing process of acute wounds in hyperglycemic conditions.

Data availability

Author contributions

Nurpudji Astuti Taslim: Conceptualization, Funding Acquisition, Investigation, Supervision, Validation, Writing – Review & Editing; Caroline Prisilia Marsella: Writing – Review & Editing; Agussalim Bukhari: Validation, Writing – Review & Editing; Muhammad Husni Cangara: Validation, Writing – Review & Editing; Andi Makbul Aman: Validation, Writing – Review & Editing; Aminuddin: Validation, Writing – Review & Editing; Mardiana Madjid: Validation, Writing – Review & Editing.