Gynura procumbensin vitro adventitious root nanohydrogel improves inflammation-proliferation transition in second-degree burn healing

Nano-hidrogel de raiz adventícia in vitro de Gynura procumbens melhora a transição de inflamação-proliferação na cicatrização de queimaduras de segundo grau

Abstract

Inflammation-proliferation transition plays a key role in the successful healing of a common burn type, second-degree burn. Gynura procumbens in vitro adventitious root nanohydrogel is currently being studied for its immunomodulatory to improve reparative environment. Root production and nanohydrogel preparation was done respectively by in vitro propagation and emulsion/ solvent diffusion with carbomer as a polymer. Characterization of nanohydrogel was performed afterward. An in vivo study was carried out in Balb/c mice having second-degree burns at different time and space. In vitro adventitious root was induced from leaf explant under indole butyric acid plant growth regulator (PGR). Rapid propagation was done through liquid culture. Upon formation into nanohydrogel (Gr-nh), it had a sphere shape with 6.88 nm in size and a PDI value of 0.16. Interaction among extract and polymer was proven by shifting in hydroxyl, alkene, and vinyl ether groups. Loading efficiency and loading amount were 99.99% and 56.39%, respectively. The percentage of release increased in line with pH (4-6). The pH of Gr-nh was 6.58 with spread diameter 8.83 ± 0.29 cm. Four days after the burn, Gr-nh showed a swift decline in local IL6 (116.1 pg/ml, p=0.0005), reduced levels of local (18.97 ng/l, p=0.69) and systemic (8.83 ng/l, p=0.0028) PGE2, and elevated local VEGF (343.6 ng/l, p=0.0005) compared to the control. By day 10, systemic IL6, as well as local and systemic PGE2 levels in Gr-nh, were notably lower compared to all treatments. Meanwhile, local VEGF remained elevated and significantly different from all other groups. Gr-nh showed better IL6 and PGE2 reduction than free extract during the inflammation stage and better VEGF enhancement than free extract in the proliferation stage. Pearson correlation analysis suggests that Gr-nh aids in coordinating IL6, PGE2, and VEGF spatially and temporally to optimize burn healing.

Keywords:
burn; Gynura procumbens; health; nanohydrogel; reparative environment

Resumo

A transição inflamação-proliferação desempenha um papel fundamental na cicatrização bem-sucedida de um tipo comum de queimadura, a queimadura de segundo grau. O nano-hidrogel de raiz adventícia in vitro de Gynura procumbens está atualmente em estudo por suas propriedades imunomoduladoras para melhorar o ambiente reparador. A produção da raiz e a preparação do nano-hidrogel foram realizadas respectivamente por propagação in vitro e difusão/emulsão de solvente, usando carbômero como polímero. A caracterização do nano-hidrogel foi realizada posteriormente. Um estudo in vivo foi realizado em camundongos Balb/c com queimaduras de segundo grau em diferentes tempos e espaços. A raiz adventícia in vitro foi induzida a partir de explantes foliares sob nutrição hormonal de ácido indolbutírico (PGR). A propagação rápida foi feita através de cultura líquida. Após a formação em nano-hidrogel (Gr-nh), apresentou forma esférica com tamanho de 6,88 nm e um valor de PDI de 0,16. A interação entre o extrato e o polímero foi comprovada pela mudança nos grupos hidroxila, alceno e éter vinílico. A eficiência de carga e a quantidade de carga foram de 99,99% e 56,39%, respectivamente. A porcentagem de liberação aumentou de acordo com o pH (4-6). O pH do Gr-nh foi de 6,58 com diâmetro de dispersão de 8,83 ± 0,29 cm. Quatro dias após a queimadura, Gr-nh mostrou uma rápida redução local de IL6 (116,1 pg/ml, p=0,0005), níveis reduzidos de PGE2 local (18,97 ng/l, p=0,69) e sistêmico (8,83 ng/l, p=0,0028) e aumento local de VEGF (343,6 ng/l, p=0,0005) em comparação com o controle. No dia 10, o IL6 sistêmico, assim como os níveis de PGE2 local e sistêmico em Gr-nh, foram consideravelmente mais baixos em comparação com todos os tratamentos. Enquanto isso, o VEGF local permaneceu elevado e significativamente diferente de todos os outros grupos. Gr-nh mostrou melhor redução de IL6 e PGE2 do que o extrato livre durante a fase inflamatória, e melhor aumento de VEGF do que o extrato livre na fase de proliferação. A análise de correlação de Pearson sugere que Gr-nh ajuda a coordenar espacial e temporalmente IL6, PGE2 e VEGF para otimizar a cicatrização de queimaduras.

Palavras-chave:
queimadura; Gynura procumbens; inflamação; nano-hidrogel; ambiente reparador

1. Introduction

A second-degree burn affects the entire epidermis and part of the dermis layer. Hot liquids, flames, or other intense heat sources mostly cause this type. Blisters, redness, and pain result from tissue damage (Jeschke et al., 2020). The healing process covers overlapping stages: inflammation, proliferation, and remodeling. Inflammation happens immediately to 1 week, and proliferation is characterized by re-epithelialization upon 48 h of post-burn and continues until 2 weeks while remodeling begins after 2 weeks and can undergo months (Jeschke et al., 2020). These stages are tightly regulated, especially the switch from inflammation to proliferation stage. Interleukin (IL)-6 signaling is the key to switching to a reparative environment. Aberrant of this signaling causes unresolved wound and risk of infection (Johnson et al., 2020). Unresolved burns cost burn care since they cause pain distress and require careful management while the death toll is near 200,000 cases worldwide (Greenhalgh, 2019). On the other hand, uncontrolled proliferation leads to scar formation and even fibrosis (Johnson et al., 2020).

Tissue injury generates damage associated molecular pattern (DAMP) – molecules that attract neutrophils and monocytes. Monocytes differentiate into M1 (proinflammatory) macrophages that produce IL6 while neutrophils introduce soluble IL-6-receptor α (sIL-6Rα). Upon binding, an inflammatory environment is perpetuated to clean the wound. IL6 causes Th2 to produce IL4 and it in turn upregulates the expression of receptor IL4 in M1. M1 polarizes into M2 (anti-inflammatory) with the help of IL4. IL6 induces M2 to support angiogenesis by secreting vascular endothelial growth factor (VEGF), a marker of proliferation (Johnson et al., 2020). Besides IL6, prostaglandin E2 (PGE2) also promotes the transition of inflammation-proliferation and enhances tissue remodeling (Landén et al., 2016). However, they can activate pain perception during inflammation (Hassanshahi et al., 2022). Establishment of an ideal burn dressing that can properly orchestrate an immune response is compulsory to prevent non-healing second-degree burns and chronic pain.

Gynura procumbens also known as longevity spinach has immunomodulatory effects. Phenolics promote immune orchestration to regulate inflammation by achieving homeostasis between proinflammatory and anti-inflammatory cytokines (Boshtam et al., 2017). Moreover, α-pinene, 3-carene, and limonene act as antinociceptive (Huang et al., 2019). Application of G. procumbens in injury was reported in several findings (Huang et al., 2019; Kim et al., 2021; Sutthammikorn et al., 2021; Zahra et al., 2011). The abundance of bioactive compounds is high in root over other organs (Krishnan et al., 2015). Rapid biomass acquisition with enhanced metabolites can be achieved through in vitro propagation that produces in vitro adventitious roots (Kusuma et al., 2021; Manuhara et al., 2019, 2017; Muthoharoh et al., 2019; Faizah et al., 2018; Lestari et al., 2018; Kusuma et al., 2017; Noviyanti et al., 2017). The use of G. procumbens adventitious root as burn medication is considerable.

Phytomedicine is gaining popularity but low solubility in water hampers its echo. Formulation of drug delivery with nanotechnology tackles the issue and also controls delivery (Gunasekaran et al., 2014). Nanoencapsulation is among the robust methods to produce nano-size drugs with enhanced activity to resolve diseases (Wardana et al., 2020, 2023). In term of burn treatment, nanohydrogel stands out as an optimal choice due to its ability to maintain a moist environment, crucial for effective healing. Nanohydrogels, which are essentially hydrogels—polymer networks capable of absorbing and retaining substantial biological fluids—engineered at the nanoscale, offer significant promise (Kamoun et al., 2017; Liu et al., 2022). Additionally, the incorporation of active ingredients, such as medicinal herbs, can further augment the healing process. Previous research has demonstrated the efficacy of incorporating substances like curcumin, ciprofloxacin, and lidocaine into hydrogels, which facilitated burn healing by modulating inflammation and pain while stimulating tissue proliferation (Karri et al., 2016; Sanchez et al., 2018).

The aim of this recent study is to evaluate the immunomodulatory effects of nanohydrogel derived from G. procumbens adventitious roots in expediting the healing of second-degree burns and managing pain perception.

2. Materials and methods

G. procumbens fresh leaves, Murashige and Skoog medium (Murashige and Skoog, 1962), indole butyric acid hormone, methanol, filter paper Whatman no 1 (Sigma-Aldrich), carbomer (polyacrylic acid) (Merck), propylene glycol (PG) (Merck), triethanolamine (TEA) (Merck), double distilled water, dialysis tube (Ward’s Science), acetate buffer 4 and 5, KH₂PO₄·NaOH buffer 6, quercetin (Nitra Kimia), IL6 ELISA kit (E0049Mo, Biotech, Shanghai, China), PGE2 ELISA kit (EA0028Mo, Biotech, Shanghai, China), and VEGF ELISA kit (E0114Mo, Biotech, Shanghai, China), Bioplacenton (Kalbe).

2.1. Production and extraction of adventitious root

The method for producing G. procumbens adventitious roots in vitro, as outlined by Manuhara et al. (2017), involved two main steps: inducing adventitious roots from leaf explants and propagating these roots in liquid culture using shake flasks (Figure 1). All procedures were carried out under aseptic conditions. Initially, leaves (3 – 5 from the tip) were washed, treated with detergent, sterilized with 20% Clorox, and then rinsed with sterile distilled water. These leaves were subsequently cut into pieces approximately 1 – 1.5 cm² and cultured on solid MS medium supplemented with indole butyric acid hormone (5 mg/L), sucrose (30 g/L), and agar (8 g/L) for adventitious root induction, with harvest taking place after 21 days.

Figure 1
Production of G. procumbens in vitro adventitious root: (A) G. procumbens, (B) induction of in vitro adventitious root from leaf, (C) liquid culture to obtain root with high biomass at relatively short time, (D) the dried G. procumbens in vitro adventitious root, (E) G. procumbens in vitro adventitious root extract.

For the subsequent liquid culture, the induced adventitious roots served as inoculum. After being retrieved from the induction medium, these roots were sterilized with 20% Clorox, washed thrice with sterile distilled water, and divided into three parts before being cultured. The inoculum was then placed into 100 mL of liquid culture in 250 mL shake flasks containing MS medium supplemented with indole butyric acid hormone (5 mg/L) and sucrose (30 g/L). Harvesting of the in vitro adventitious roots from the liquid culture was conducted after 28 days.

2.2. Extraction

The extract preparation followed the method outlined by Wang et al. (2013) with some modificatios. In vitro adventitious roots were washed with tap water and sun-dried. Once dried, they were ground into semi-fine powder. Each 50 g of in vitro adventitious root was soaked in 500 mL of methanol PA overnight, repeated in triplicate. The resulting extraction was concentrated using a rotary evaporator until a thick extract was obtained.

2.3. Nanohydrogel formulation

G. procumbens adventitious root nanohydrogel was formulated using emulsion/ solvent diffusion method described by Elegbede et al. (2020) with the assistance of ultrasonication as a modification. Water phase was made by dissolving carbomer into double distilled water (120^oC, 30 min) while organic phase was made by mixing extract, methanol, and propylene glycol. Water phase was added to organic phase then performed ultrasonication (Biosafer, Nanjing Gengchen Scientific Instrument) for 5 min. TEA was added during ultrasonication. The nanohydrogel was collected and stored in the refrigerator for further use. G. procumbens adventitious root then stated as Gr-nh.

2.4. Physicochemical characterizations

Characterization of physicochemical properties comprised polydispersity index (PDI) and particle size (Particle Size Analyzer, Biobase). The morphology of nanohydrogel was examined by scanning electron microscope (SEM) (Thermo Fisher Scientific). Meanwhile, FTIR (Shimadzu IRTracer-100) was used to study functional groups of nanohydrogel.

2.5. Loading and release

Loading and release assays were performed in vitro (Wardana et al., 2023). First and second equations were used to calculate loading efficiency (LE) and loading amount (LA) respectively. Meanwhile, the release profile was conducted in a buffer with a pH similar to the natural pH of the skin and calculated using third equation. The absorbance was subsequently measured using a UV spectrophotometer (Thermo Scientific). The quantity of drug released was expressed in terms of quercetin (µg) (Equations 1, 2 and 3).

$LE=\frac{totaladdeddrug-freedrug}{totaladdeddrug}\times 100$ (1)

$LA=\frac{totaladdeddrug-freedrug}{totalweightgel}\times 100$ (2)

$C{t}^{\prime }=Ct+\frac{v}{V}{{\displaystyle \sum }}_0^{i-t}Ct$ (3)

Where:

Ct '= correction concentration at time t (µg/ml)

Ct = measured concentration at time t (µg/ml)

v = volume of aliquots (ml)

V = total buffer volume (ml)

2.6. pH test and rheological studies

Both characterizations were done based on a study by Yandri and Setyani (2021) and Fenny and Safitri (2021) with modifications. The pH test was performed by pH meter. The electrodes were submerged in the gel solution until got a constant number. The fixed number was recognized as pH value of nanohydrogel preparation. In addition, rheological study comprising spreadability test was also done. As much as 0.5 g of nanohydrogel was placed in the middle of flat glass then transparent material with 150 g mass was added on top of the gel. It is allowed to sit for 60 s. The spread diameter was measured afterward.

2.7. In vivo study

Male Balb/C mice aged 8 weeks were subjected to suffer second-degree burns (number of ethical clearance: 0117/HRECC.FODM/II/2024). Study by Wasef et al. (2020) with modification was used to initiate second-degree burn (1% total body surface area, TBSA). A total of 16 mice with burn were randomly divided into 4 groups as follows.

C = control, second-degree burn + double distilled water.

C+ = control positive, second-degree burn + Bioplacenton

T1 = treatment 1, second-degree burn + Gr-nh

T2 = treatment 2, second-degree burn + extract

The dose of both Gr-nh and extract was 0.5% and given once every 48 h according to Sutthammikorn et al. (2021) with modification. The respective mice were sacrificed at day 4 and 10 of post-burn. Blood and burned skin tissue were collected for ELISA analysis.

3. Statistical analysis

The in vivo study data were meant to be performed statistical analysis using GraphPad Prism. Blood serum and tissue homogenate data were presented as mean ± SD. A normality test was first done then followed by analysis of significance based on data distribution. Multiple comparisons were carried out afterward. Subsequently, Pearson correlation was performed to draw a correlation between parameters. A significant difference was considered when p value was less than 0.05.

4. Result

4.1. Production and extraction of G. procumbens in vitro adventitious root

An adventitious root is a root that grows from non-root tissue and arises in response to environmental stimuli or developmental cues (Steffens and Rasmussen, 2016). In the recent study, formation of adventitious root from leaf explant was under exogenous indole butyric acid orchestration. Adventitious roots had a slender morphology and were surrounded by numerous fine hairs (Figure 1B). These roots grew well on leaf blades and petioles, but many roots were found primarily on the petioles. The roots first appeared on days 5 – 7 of the induction process and continued to elongate until they covered the entire surface of the medium and were propagated in liquid culture on day 21. The total dry weight of in vitro adventitious roots obtained was 51.9 g. Meanwhile, the maceration yield was 1.07 g. The extract was brownish-yellow in color with a slightly thick consistency (Figure 1E).

4.2. Physicochemical properties of Gr-nh

G. procumbens adventitious root nanohydrogel (Gr-nh) was G. procumbens adventitious root extract encapsulated by carbomer and engineered into nano-size. The average size of Gr-nh was 5.5 nm with a polydispersity index (PDI) value of 0.16, indicating a monodisperse distribution (Mudalige et al., 2019). In addition, nanohydrogel had a sphere form in SEM imaging (Figure 2B). The interaction between extract and carbomer was proven by FTIR (Figure 1C). The key spectral bands in the extract appeared in the absorption range: 3268.77 cm^-1, 2846.64 cm^-1, and 1040.32 cm^-1, corresponding to OH (hydroxyl), CH (alkane), and C-O (vinyl ether) stretching, respectively. This is indicating flavonoid group of compounds that majority found in plants (Catauro et al., 2015; Nandiyanto et al., 2019). Upon interaction, all these regions were detected but exhibited shifts. Specifically, the hydroxyl, alkene, and vinyl ether were identified in the absorption spectrum at: 3252.17 cm^-1, 2842.68 cm^-1, and 1031.62 cm^-1, respectively. Carbonyl (C=O) was evident in carbomer at 1703.55 cm^-1, with a shifted band observed upon interaction with the extract, at 1675.09 cm^-1.

Figure 2
Physicochemical properties of G. procumbens in vitro adventitious root nanohydrogel (Gr-nh): (A) distribution of particle size, (B) morphology of Gr-nh through SEM imaging, (C) FTIR spectra evidenced nanoencapsulation of Gr-nh.

4.3. Loading and release

Gr-nh was successfully established with 99.99% of LE and 56.39% of LA. Release of Gr-nh was intended to skin’s natural pH (4,5, and 6) (Luebberding et al., 2013). Percentage of release increased in line with pH but the amount was extremely low. Gr-nh release profile was 3.28%, 5.13%, and 4.20% at pH 4,5, and 6, respectively after 180 minutes, while 0.91%, 0.77%, and 0.25% of the drug was released within 5 minutes at each pH level. The release pattern corresponds to the increasing pH of the medium, but anomalies in the data persist, particularly noticeable in Gr-nh (as shown in Figure 3). These discrepancies are likely attributable to the dynamic nature of the polymeric system during the formulation of nanohydrogels (Priya James et al., 2014).

Figure 3
Release profile of Gr-nh: percentage release shows an increasing trend as the pH becomes higher.

4.4. pH and spreadability study

The pH assessment aimed to ascertain the acidity of Gr-nh to ensure it would not cause skin irritation. Gr-nh exhibited a pH of 6.58, falling within the range of human skin pH, which typically ranges from pH 4.5 to 7.0 (Nurman et al., 2019). Spreadability test was conducted to determine the gel preparation's thickness-associated property. Gr-nh could spread to a diameter of 8.83 ± 0.29 cm after being loaded for 1 minute. This value slightly exceeded the optimal spreadability range of 5-7 cm (Nurman et al., 2019).

4.5. In vivo study

The levels of local and systemic IL6 and PGE2, as well as local VEGF, were measured at the peak of inflammation, which occurred on day 4 after burn (Figure 4). The Gr-nh group was able to significantly decrease local IL6 compared to both the control (C) and positive control (C+) groups. The extract group (T2) exhibited better local IL6 inhibition than the Gr-nh group. However, both preparations showed the same effect in maintaining high systemic IL6 levels (Figure 4B). This suggests that IL6 trans-signaling likely induces keratinocytes and macrophages to express VEGF for angiogenesis (Song et al., 2018). Another inflammation marker, PGE2, was not significantly reduced in Gr-nh compared to all treatments at the local level. Interestingly, the systemic levels of PGE2 for all treatments were observed to be lower than the local levels. Notably, Gr-nh treatment (8.84 ± 0.45 ng/l) resulted in a significant reduction in systemic PGE2 compared to the control (C: 13.16 ± 1.61 ng/l; p = 0.0028), Bioplacenton (C+: 14.89 ± 0.93 ng/l; p = 0.0003), and free extract (T2 : 11.64 ± 0.37 ng/l; p = 0.0323) groups. The elevated local PGE2 levels are presumably attributed to its role as one of the important mediators of proliferation during the resolution of inflammation (Zhang et al., 2018). Meanwhile, local VEGF of Gr-nh group was the highest (343.6 ± 9.84 ng/l) and significantly different with other treatments. The result indicates proliferation begins even during inflammation.

Figure 4
Level of local IL6 (A), systemic IL6 (B), local PGE2 (C), systemic PGE2 (D) and local VEGF (E) after 4 days post burn. C = 2^nd degree burn + dd water, C+ = 2^nd degree burn + Bioplacenton, T1 = 2^nd degree burn + Gr-nh, T2 = 2^nd degree burn + G. procumbens adventitious root extract. Number of replication: 3, significant difference among groups are assessed by Tukey test. P > 0.05 = non significant (ns), P ≤ 0.05 = *, P ≤ 0.01 = **, P ≤ 0.001 = ***, P ≤ 0.0001 = ****.

Proliferation assessment was conducted on day 10 (Figure 5). Following the elevated systemic IL6 levels observed at day 4, systemic IL6 was notably suppressed in the Gr-nh group (50.22 ± 2.72 pg/ml). The Gr-nh group also exhibited the lowest recorded level of local PGE2 (8.01 ± 0.22 ng/l), which was significantly lower compared to the control (C: 10.12 ± 0.61 ng/l; p = 0.0064), positive control (C+: 9.54 ± 0.80 ng/l; p = 0.035), and free extract (T2: 14.81 ± 0.32 ng/l; p < 0.0001) groups. Additionally, systemic PGE2 levels in the Gr-nh group were the lowest, measured at 10.22 ± 3.05 ng/l, and significantly lower compared to all treatments, especially the free extract group (18.68 ± 1.89 ng/l; p = 0.0024). Meanwhile, the local level of VEGF in the Gr-nh group was lower than that observed at day 4 but subsequently became significantly higher than that of all other groups (306.6 ± 7.03 ng/l). Proliferation in the Gr-nh group appears to have been sustained until day 10 post-burn, although it was expected to be controlled, given the reductions in IL6, PGE2, and local VEGF compared to the levels observed at day 4. However, further research is needed to confidently support this assumption. Proliferation should be regulated at the appropriate time, as excessive levels of VEGF are known to contribute to scar formation and fibrosis (Johnson et al., 2020; Landén et al., 2016). Regarding the extract group, systemic IL6 and PGE2 levels were higher than those in the Gr-nh group, but the local VEGF level was lower. In this group, it is suspected that the inflammatory phase is still ongoing. These results suggest the enhanced ability of Gr-nh to regulate the immune response at the appropriate time, thereby optimizing the healing process.

Figure 5
Level of systemic IL6 (A), local PGE2 (B), systemic PGE2 (C), and local VEGF (D) after 10 days post burn. C = 2^nd degree burn + dd water, C+ = 2^nd degree burn + Bioplacenton, T1 = 2^nd degree burn + Gr-nh, T2 = 2^nd degree burn + G. procumbens adventitious root extract. Number of replication: 3, significant difference among groups are assessed by Tukey test. P > 0.05 = non significant (ns), P ≤ 0.05 = *, P ≤ 0.01 = **, P ≤ 0.001 = ***, P ≤ 0.0001 = ****.

4.6. Correlation analysis

Correlation analysis was carried out by Pearson correlation (Table 1). Crosstalk among cytokines during wound healing is time and spatially regulated. At day 4, the correlation of both local and systemic IL6 and PGE2 was positive, indicating a supportive role in the inflammatory stage. However, this correlation is stronger in local (r = 0.778) than systemic (r = 0,527). Local IL6 and VEGF showed a strong negative correlation but moderate positive correlation showed by systemic IL6 and local VEGF. A strong positive correlation (r = 0.667) was exhibited by local PGE2 and VEGF. Those results support the effect of IL6 trans-signaling and PGE2 to modulate proliferation. Systemic PGE2 and IL6 still positively correlated upon day 10. Interestingly, the correlation of local PGE2 and VEGF that was previously positive at day 4 became negative at day 10. It can be assumed that PGE2 no longer regulates VEGF expression in later proliferation. However, further study should be done to prove this assumption.

Table 1
Correlation analysis between IL6, PGE2, and VEGF during day 4 and 10 post burn.

Time (day post burn)	Local Cytokines	r Coefficient
4	IL6 vs PGE2 (local)	0.778
	IL6 vs PGE2 (systemic)	0.527
	IL6 vs VEGF (local)	-0.619
	IL6 (systemic) vs VEGF (local)	0.466
	PGE2 vs VEGF (local)	0.667
10	IL6 vs PGE2 (systemic)	0.593
	PGE2 vs VEGF (local)	-0.786

5. Discussion

The healing of second-degree burns is a complex and context-dependent immune orchestration process, wherein aberrations can lead to unresolved healing and excessive pain. A critical aspect of this healing process is the transition between the inflammation and proliferation stages. Treatment with anti-inflammation and antinociceptive agents is believed to promote a reparative environment (Yu et al., 2023).

Inflammation is mainly characterized by an influx of inflammatory leucocytes such as neutrophils and monocytes/ macrophages to eliminate harmful stimuli. Tissue injury drives IL6 signaling. Neutrophils come to clear cell debris and release soluble IL6R. IL6 from intact endothelial cells binds to their receptor causing monocyte influx and differentiation into M1. The M1 produces more IL6 and perpetuates inflammation by activating Th17 and mediates pathogen clearance. Expression inflammatory cytokine IL6 commonly peaks at day 3 and returns to normal at day 10 (Johnson et al., 2020). On day 4, both T1 and T2 groups decreased local IL6 to the lowest level compared to the C and C+ groups. It assumed that G. procumbens adventitious root metabolites show rapid anti-inflammation in the injured tissue. Interestingly, systemic IL6 was maintained high in Gr-nh and free extract. Besides having pro-inflammatory properties, IL6 also helps tissue repair. First, IL6 supports macrophage polarization from M1 to M2 by upregulation of IL4 receptors. Antiinflammation macrophage, M2 cells, are found in abundance in the late stage of inflammation (Bosurgi et al., 2017). IL6 trans-signaling regulates migration of fibroblasts for re-epithelialization, as well as keratinocytes and macrophages to express VEGF for angiogenesis. The formation of new vessels keeps tissue nourished (Song et al., 2018). This reveals the specificity of G. procumbens to regulate IL6 signaling during the inflammation stage.

It is commonly known that inflammation correlates to nociception. Release of proinflammatory mediators from M1 cells such as IL6 and PGE2, activates their receptor in nociceptive neurons. This activation results in Ca²⁺ influx and activates cyclic AMP/protein kinase A (PKA). Activation of cyclic AMP/PKA signals increases the activity of transient receptor potential vanilloid 1 (TRPV1), a pro-nociceptive cation channel that contributes to pathological pain (Domoto et al., 2021). Compared to local IL6 at day 4, the Gr-nh group showed a slight downregulation in local PGE2; however, no significant difference was reported among the groups. This was due to PGE2 acting as a modulator of fibroblast migration to support proliferation stages, as well as inducing macrophage polarization (Zhang et al., 2018). Zhang et al. (2018) reported chitosan chitosan-loaded PGE2 hydrogel improves tissue repair and prevents scar formation. The high PGE2 helps to suppress IL6, so peripheral pain perception can be compromised. Later the high PGE2 was diminished as evidenced by both local and systemic levels of PGE2 at day 10. PGE2 is also known to support angiogenesis by upregulation of VEGF. The correlation between local PGE2 and VEGF at day 10 was positive, with a coefficient of 0.667. However, controlling the expression of VEGF is important to prevent scar formation and skin fibrosis (Johnson et al., 2020). By day 10, the levels of these two markers had decreased. Taken together, the sustained release of Gr-nh offers temporally and spatially controlled pain perception without compromising the proliferative environment.

Proliferation is the second stage following inflammation, yet it can overlap each other. The objective is to regenerate the damaged tissue. It happens under M2 orchestration by which VEGF is an important mediator. The major population of M2 produces VEGF to induce keratinocyte migration and form new epithelial tissue. Besides promoting keratinocytes, VEGF attracts and activates endothelial cells to form new vessels. The new vessels appear as early as 3 days when inflammation still occurs (Johnson et al., 2020; Landén et al., 2016). The Gr-nh group exhibited the highest level of local VEGF, significantly surpassing all other treatments on day 4. PGE2 acts as a proangiogenic factor during the resolution of inflammation, further amplifying VEGF to accelerate angiogenesis (Landén et al., 2016). This finding is supported by the observation that PGE2 and VEGF levels tended to be higher than IL6 in all groups by day 4. Local VEGF remained elevated until day 10, while both local and systemic PGE2 were significantly diminished. Additionally, the correlation between local PGE2 and VEGF was 0.667 at day 4 but became negative at day 10 (-0.786). The immunomodulatory of Gr-nh to improve VEGF expression was better than a free extract because it has a small size (5.5 nm) and water soluble property resulting in accumulation in injured tissue (Khan et al., 2019; Liu et al., 2022). Moreover, VEGF can be maintained at a high level until day 10 post-injury because of controlled release (Khan et al., 2019; Liu et al., 2022). Previously, the administration of nanocomposite containing M2 with sustained release could prolong M2 excretion for up to day 7 and exhibited a broad proliferative effect in diabetic tissue (Jiang et al., 2024).

Acknowledgements

The authors would like to acknowledge the support received from the Ministry of Education, Culture, Research, and Technology Republic of Indonesia through the Master’s Thesis Research Funding (PPS-PTM) for the fiscal year 2023 under contract number SP DIPA-023.17.1.690523/2023.

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Publication Dates

• Publication in this collection
  10 Jan 2025
• Date of issue
  2024

History

• Received
  28 May 2024
• Accepted
  01 Oct 2024