DOI: https://doi.org/10.21203/rs.3.rs-2395798/v1
Panax notoginseng (P.notoginseng) has been used traditionally to treat traumatic injuries.Ginsenoside Rb1, a key active ingredient derived from Panax notoginseng, has received a lot of interest due to its anti-inflammatory, bacteriostatic, and growth-promoting effectsoncells.The therapeutic benefits of ginsenoside Rb1 on burn wounds in STZ-induced diabetic rats, as well as the probable underlying processes, were investigated in this work. The skin wound healing effect of ginsenoside Rb1 (0.25% and 0.5% w/w) in a rat model of burn wounds in diabetic rats was observed at various time points after treatment. On days 5 and 19 following treatment,immunohistochemistry and Western blot analysis forIL-1β, TNF-α, CD68 and CD163 of biological tissues were done. The macroscopic observation was used to track the healing of skin wounds at various periods. The protein expression of CD68 and CD163, which serve as M1 and M2 macrophage markers, was examined in detail. More notably, the ability of ginsenoside Rb1 to alter inflammatory markers (IL-6) and anti-inflammatory markers (IL-10), influence on hydroxyproline and hexosamine was observed. As indicated by increased CD163 (M2) and reduced CD68 (M1) on day 5, ginsenoside Rb1 effectively flips the M1 to M2 phenotypic transition at the right time to improve burn wound healing in diabetic rats.Ginsenoside Rb1(0.5% w/w) treatment showed higher tensile strength, anti-inflammatory properties, antioxidant properties, increased tissue hexosamine and hydroxyproline levels. Skin tissue morphology was significantly improved following 19 days of ginsenoside Rb1 (0.5% w/w) therapy, according to hematoxylin-eosin and Masson's trichrome staining. Furthermore,Ginsenoside Rb1 (0.5% w/w) favoured the inflammatory phase of burn wound healing (IL-6), assisted the proliferation process (IL-10) and had considerably lower expression of IL-1β and TNF-α on the later stage of wound healing.Overall, the data showed that ginsenoside Rb1(0.5% w/w) accelerates burn wound healing in diabetic rats through a mechanism that may be linked to the M1 to M2 phenotypic shift.
The healing of a burn wound is a multi-step process that begins with the inflammation and terminates with epithelialization.[1] Diabetes may impact the length of a burn patient's hospital stay.[2] Moreover, hyperglycemia is linked to a higher risk of overall morbidity in burn patients.[3]
Furthermore, as diabetes people can't distinguish between hot and warm due to a loss of sensation in their lower extremities, they are more likely to get foot burns from electric heating, pads, water baths, and foot spas.[5] Burn damage treatment is considered an unmet clinical need, with no satisfactory solution available to date.[5]Because diabetes is a global epidemic,healthcare practitioners will face more challenges in treating diabetic burn victims.[6]The usage of Ginsenoside Rb1 in a diabetic thermal wound rat model was examined in this work.
In treating second-degree burns, silver sulfadiazine (SSD) is routinely used for the prevention of burn wound infections and helps to reduce symptoms.[7] However, considering the side effects of these medications (antibacterial activity-related side effects, cytotoxicity, and so on),[8]the prognosis of some patients remains bleak. As a result, new local topical medications for treating wounds and scalds are urgently needed, with proven therapeutic efficacy and fewer adverse effects than currently available treatments. Panax notoginseng is a traditional herbal medication used to treat inflammatory diseases, cardiovascular illnesses, traumatic injuries, and external and internal bleeding caused by damage.[9]Because of its potential anti-inflammatory, antioxidant, and cell growth-promoting activities, ginsenoside Rb1, an essential active ingredient of P. notoginseng, has gotten a lot of attention.[10–13]But, the role of Ginsenoside Rb1has not been investigated in diabetic burn wounds. Thus, the effects of ginsenoside Rb1(0.25% and 0.5% w/w) onhealing of burn wounds in diabetic rats and the processes behind these benefits were investigated in this work to contribute to a scientific foundation for the therapeutic use of ginsenoside Rb1 to treat burns in diabetic rats.
Ginsenoside Rb1 (purity > 99.0%) was procured fromSelleckChem. A basic ointment base in the ratio of 1:6:3 will be prepared using liquid paraffin, propyleneglycol, and glycol stearate, respectively.[14]Adequate levels of test substance will be added to the base ointment for preparing two doses of test ointments-Ginsenoside Rb1 (High Dosage (0.5% w/w) and low dose (0.25% w/w).The ointment base will be applied topically to the vehicle group. For positive control, 1% silver sulphadiazine ointment will be used as a reference drug. During the treatment period, 0.5 g of the test ointments or reference drug will be administered to the wound sites topically once a day.
Wistar albino rats of both sexes weighing 150–250 g were used to test burn wound healing capabilities. The animals were housed in poly-propylene cages with optimum humidity, light, and temperature [Temp: 25 ± 2ºC, 75% relative humidity, andlight/dark cycles (12/12 h)]. Theanimals were fed a commercial pellet diet for rats and adequate water for at least one week before testing. The Institutional Animal Ethics Committee (IAEC) of Erode College of Pharmacy, Erode, Tamilnadu, India (565/02/CA/18/CPCSEA) approved all experimental procedures. Experiments were carried out as per the guidelines for laboratory animal care and use.
Animals were kept starving overnight. Nicotinamide (HiMedia Labs Pvt. Ltd.) was given at a 110 mg/kg body weight dose 15 minutes before streptozotocin (STZ) was given. A 65 mg/kg dose of STZ (Sigma, USA) solution dissolved in a citrate buffer with a pH of 4.5 was given intraperitoneally (i.p). Further, to minimize hypoglycemia caused by increased pancreatic insulin secretion, a 10% glucose solution s were given to rats for an additional 24 hours following STZ treatment. Blood was drawn from the rats' tail veins 72 hours after they received STZ injections. Diabetic rats were defined as those that have blood glucose level of > 200 mg/dl at fasting and were included in this investigation.[15]
The rats have split into five groups of six ratsfor the thermal wound model. The first group is non-diabetic (normal control), and the second group is diabetic (diabetic control) receiving simple ointment. The third received silver sulfadiazine (1% w/w). The fourth and fifth groups receivedginsenoside Rb1 (0.25% w/w and 0.5% w/w) respectively. The third set was utilized to assess wound closure and to do further biochemical testing. The dorsal skin hairs were carefully removed one day before the burn. For 24 hours, the animals were monitored to check if shaving had caused any irritation. A metal rod of 2.5 cm diameter was heated to a temperature of 80–85°C, and for 20 s, it was pressed on the dorsal skin of rats to create thermal burn injuries. When the animals had recovered from the anaesthetic, the wound was dressed with a clean, sterile gauge, and they were maintained separately. The burn was treated daily with drugs. The wound closure rate was recorded using transparent paper and a permanent marker on the 5th, 10th, 14th, 17th and 19th post-wounding days.[16] The percentage of wound closure was calculated using the method below for the final analysis of the data.[17–18]
% Wound closure = [(Day 0 wound area - Day “n” wound area)/ Day 0 wound area] x 100
Where n = 5th day, 10th day, 14th, 17th and 19th post wounding days
At the end of the test, the rats with burn wounds were sacrificed to analyze the healing process in terms of the biochemical characteristics. The burn wounds area of experimental rats was excised to assess tissue hydroxyproline and hexosamine.
Hydroxyproline, the most crucial indicator of collagen turnover, was evaluated in the burn wounds granulation tissue. Tissues were dried at 60–70°C in a hot air oven to a consistent weight, then hydrolyzed in 6 N HCl in a sealed tube for 4 hours at 130°C. After neutralization to pH 7.0, the hydrolysate was subjected for 20 minutes to chloramine-T oxidation before being halted by the addition of 0.4 M perchloric acid. The colour was made at 60°C using Ehrlich reagent and detected with a UV–Vis spectrophotometer at 557 nm (Shimadzu, Columbia, MD).[19]
The weighed granulation tissues were subjected to hydrolysis for 8 hours at 98°C in 6 N HCl, neutralization donewith 4 N NaOH at pH 7, and further diluted with distilled water for the measurement of hexosamine. After mixing with acetylacetone solution for 40 minutes, the diluted solution was heated to 96°C. Ethanol (96%) was added after cooling the mixture, and then a solution of p-dimethylaminobenzaldehyde (Ehrlich's reagent) was added. After the solution had been well mixed and allowed to cool for 1 hour, at 530 nm, the absorbance was measured using a Shimadzu double beam UV–Vis spectrophotometer. The amount of hexosamine was determined using a standard curve. Hexosamine concentration was determined in milligrams per gram of dry tissue weight. [19]
On day 8, blood was taken from the retro-orbital plexus of burn wound animal models and centrifuged for 10 minutes at 506.11 rpm (Microcentrifuge) to separate plasma to test antioxidant activity. The serum was used to perform the antioxidative enzyme test. To assess the degree of lipid peroxidation (LPO), thiobarbituric acid reactive substances quantity was tested using the Uchiyama and Mihara procedure The Sedlak and Lindsay methodology was used to assess the reduced glutathione (GSH)levels, whereas the Kono method evaluated the superoxide dismutase (SOD)activities. Aebi's standard procedure was used to assay catalase (CAT).[19]
On days 2 and 10, the blood samples were obtained from burn wound animals in each group following wounding. The amounts of pro-inflammatory (IL-6) and anti-inflammatory (IL-10) cytokines were assessed using commercially available enzyme-linked immunosorbent assays (ELISAs). The tests were carried out according to the instructions of the manufacturer. The concentrations of cytokine were determined in pg/ml by drawing the curve for the standard. Each experiment was done three times to ensure that the results were correct.[20–21]
Histopathology, Immunohistochemistry and Western blot analysis for IL-1β TNF-α, CD68 and CD163.
The wound-healing tissue was removed and then fixed in buffered formalin. Later, it is processed in a series of alcohol and xylene and embedded in paraffin blocks on the 5th and 19th days. The repair effect was assessed by examining the stained sections using an optical microscope using hematoxylin-eosin (H&E) and Masson's trichrome staining.18 The sections were treated with primary antibodies to IL-1β and TNF-α for immunohistochemistry. The manufacturer's protocol was followed for all phases of immunohistochemical staining. A fluorescence microscope was used to examine the immunohistochemistry samples.
Wound skin samples were fully homogenized in the presence of lysis buffer (PBS, pH 7.4) before being centrifuged at 10,000 rpm for 10 minutes. The proteins that were prepared were electrophoresed on 10% sodium dodecyl sulfate (SDS)-polyacrylamide gels. After that, the proteins were moved to PVDF Western blot membranes for 2 hours at 40 V, primary anti-bodies were overnight incubated at 4°C. After that, the membrane would be incubated for 1 hour at 22°C with the HRP-conjugated anti secondary antibody. The membrane was then examined using an X-ray films and an improved chemiluminescent reagent.
Western blot analysis to examine CD68 and CD163:
A 30 lb protein sample electrophoresed on a 10% SDS-PAGE gel was used to examine CD68 and CD163. For 1 hour, the gel was then blocked at room temperature using a blocking solution over a polyvinylidene fluoride or polyvinylidene difluoride membrane (5% skim milk powder TBST). After that, primary antibodies were used to probe the membranes (Abcam, Cambridge, UK, 1:1000). The next day, the membranes were cleaned and incubated with secondary antibodies (horseradish peroxidase-conjugated antimouse or antirabbit at 1:2000). Chemiluminescence substrate was used to enhance proteins, and the Chemidoc XRS plus system was used to scan them (Bio-Rad). The findings were represented in standard units (Biotech Inc). The gene b-actin is employed as a housekeeping gene.[21]
Dunnett's test was used to analyse differences between means after data were submitted to analysis of variance (ANOVA). At a P < 0.05 threshold of significance, a substantial difference was considered. The mean and standard error of six animals' mean (SEM)is represented (n-6).
In thermally produced burn wounds, the Ginsenoside Rb1showed a significant (P < 0.05) improvement in wound contraction percentage compared to the control group. In most post-wounding days, the Ginsenoside Rb1 ointment (0.5% w/w) rats had a more prominent and significant (P < 0.05) proportion of wound contraction than the normal control rats (Table-1). On the 19th day of the study, the Ginsenoside Rb1 ointment (0.5% w/w) and Silver sulfadiazine treated groups had the highest percentage of wound closure, with 99.23 ± 3.41 71.36 ± 3.21, respectively (Table 1). Consequently, ginsenoside Rb1 ointment (0.5% w/w) was shown to be the most effective treatment.
| % Wound Closure | |||
|---|---|---|---|
| Treatment Group | 5th Day | 12th Day | 19th Day |
| Normal Control | 2..94 ± 0.12 | 14.75 ± 1.46 | 31.08 ± 1.22 |
| Diabetic Control | 2.36 ± 0.31 | 9.55 ± 1.45 | 22.47 ± 1.36 |
| Silver sulfadiazine (1% w/w) | 8.26 ± 1.43* | 59.34 ± 4.16* | 71.36 ± 3.21* |
| Ginsenoside Rb1(0.5% w/w) | 8.11 ± 1.14* | 71.33 ± 3.45* | 89.17 ± 3.12* |
| Ginsenoside Rb1(0.25% w/w) | 9.88 ± 1.34* | 80.23 ± 3.25* | 99.23 ± 3.41* |
The results are shown as mean ± S.E.M of six rats (n = 6). *P < 0.05 is the statistical difference from control
Hexosamine and hydroxyproline concentrations in healed wound tissue are shown in Table 2. The content of hydroxyproline and hexosamine in diabetic wound control was significantly (P < 0.05) reduced. The treatment groups had significantly (P < 0.01) increased hydroxyproline and hexosamine concentrations than diabetic wound controls.
When diabetes control wounds were compared to normal wound controls, malondialdehyde (MDA) levels in diabetic control mice were significantly higher. Furthermore, diabetic control mice had substantially lowered GSH, SOD, and CAT levels than normal control animals (P < 0.05). Ginsenoside Rb1(0.5% w/w) therapy was shown to be superior to diabetic control wounds (P < 0.05).
| Treatment groups | Hexosamine content (mg/100 mg tissue) | Hydroxyproline content (mg/g tissue) | MDA (nmol/mg of protein) | SOD (µg/mg of protein) | Catalase (µg/mg of protien) | GSH (ng /mg of protein) |
|---|---|---|---|---|---|---|
| Normal control | 0·711 ± 0·036 | 12·46 ± 0·44 | 3·87 ± 0·39 | 71·29 ± 2·16 | 42·45 ± 3·24 | 19·11 ± 2·31 |
| Diabetic control | 0·182 ± 0·02* | 2·62 ± 0·41* | 12·23 ± 1·03* | 39·22 ± 3·42* | 12·42 ± 1·49* | 7·22 ± 0·64* |
| Silver sulfadiazine (1% w/w) | 0·39 ± 0·04* | 11·14 ± 0·54* | 11·16 ± 1·36* | 30·43 ± 1·87* | 12·05 ± 1·58* | 8·03 ± 0·73* |
| Ginsenoside Rb1(0.25% w/w) | 0·581 ± 0·032* | 17·15 ± 0·66* | 9·25 ± 1·42* | 59·54 ± 2·24* | 29·64 ± 3·67* | 15·14 ± 1·26* |
| Ginsenoside Rb1(0.5% w/w) | 0·631 ± 0·041* | 21·06 ± 0·76* | 7·32 ± 1·51* | 68·72 ± 2·14* | 34·72 ± 3·89* | 18·24 ± 1·36* |
The results are shown as mean ± S.E.M of six rats (n = 6). *P < 0.05 is the statistical difference from control
After creation of thermal wounds, in the diabetic control-treated arm,the IL-10 level was considerably (P < 0.05) lower (2nd Day: 432.4 ± 21.6pg/ml; 10th day: 528.4 ± 26.6pg/ml) compared tosilver sulfadiazinetreated arm (2nd Day: 609.5 ± 30.5pg/ml; 10th day: 785.4 ± 40.2 pg/ml)(Table 3).In the Ginsenoside Rb1(0.5% w/w)treated group, IL-10 levels increased significantly (P < 0.05) on the second and tenth days after being wounded, to 860.0 ± 22.6pg/ml and 1342.6 ± 49.6 pg/ml respectively.The IL-6 level in Ginsenoside Rb1(0.5% w/w)treated rats (92.3 ± 10.4pg/ml) was less than that in the diabetic control(110.2 ± 13.5pg/ml), at 48 h after wounding. At the same time, the IL-6 levels were greatly reduced (P < 0.05) to 39.6 ± 9.8pg/ml in the Ginsenoside Rb1(0.5% w/w)treated group on day ten after wounding. On the other hand, on the 10th day following wounding, diabetic control rats had a high IL-6 level (97.4 ± 15.1pg/ml). The level of IL-6 in the Silver sulfadiazine treated group (101.0 ± 14.4pg/ml) on 2nd Dayafter wounding was substantially (P < 0.05) less compared to the animals in the diabetic control group, and the drop in IL-6 levels was maintained at 70.5 ± 11.5pg/ml, on10th dayafter wounding.
| IL-10 (pg/ml) | IL-6 (pg/ml) | |||
|---|---|---|---|---|
| Day 2 | Day 10 | Day 2 | Day 10 | |
| Normal control | 426.2 ± 19.7 | 502.2 ± 21.4 | 99.4 ± 10.2 | 89.2 ± 14.4 |
| Diabetic Control | 432.4 ± 21.6 | 528.4 ± 26.6 | 110.2 ± 13.5 | 97.4 ± 15.1 |
| Silver sulfadiazine (1% w/w) | 609.5 ± 30.5* | 785.4 ± 40.2* | 101.0 ± 14.4* | 70.5 ± 11.5* |
| Ginsenoside Rb1 (0.25% w/w) | 825.0 ± 25.4* | 1211.7 ± 44.2* | 97.3 ± 11.6* | 42.9 ± 10.7* |
| Ginsenoside Rb1 (0.5% w/w) | 860.0 ± 22.6* | 1342.6 ± 49.6* | 92.3 ± 10.4* | 39.6 ± 9.8* |
The results are shown as mean ± S.E.M of six rats (n = 6). *P < 0.05 is the statistical difference from control
CD68 and CD163 protein expression in the wound skin region of burn wounds was detected using Western blotting (Table-4). When comparing Group II (diabetic control rats) to Group 1 (normal control rats), there was an up-regulation of both CD68 and CD163 levels after the therapy. In diabetic rats treated with Ginsenoside Rb1 (0.5% w/w), however, there was a substantial (P < 0.05) downregulation of CD68 and an increase in CD163 levels. All treatment groups had higher levels of CD163 by day 5, with Ginsenoside Rb1 (0.5% w/w) having the most considerable rise.
| Duration in days | ||||
|---|---|---|---|---|
| Day 5 | Day 19 | |||
| Values (Units/ β actin) | ||||
| CD68 | CD163 | CD68 | CD163 | |
| Normal Control | 0.65 ± 0.02 | 0.24 ± 0.04 | 0.51 ± 0.04 | 0.42 ± 0.06 |
| Diabetic control | 2.6 ± 0.18 | 0.98 ± 0.07 | 2.6 ± 0.17 | 1.7 ± 0.16 |
| Silver sulfadiazine (1% w/w) | 2.1 ± 0.05* | 1.3 ± 0.07* | 1.4 ± 0.16* | 1.7 ± 0.16* |
| Ginsenoside Rb1 (0.25% w/w) | 1.5 ± 0.07* | 1.9 ± 0.14* | 0.8 ± 0.01* | 1.8 ± 0.14* |
| Ginsenoside Rb1 (0.5% w/w) | 1.2 ± 0.09* | 2.1 ± 0.12* | 0.4 ± 0.01* | 2.4 ± 0.11* |
The results are shown as mean ± S.E.M of six rats (n = 6). *P < 0.05 is the statistical difference from control
Ginsenoside Rb1 (0.5% w/w) treated group of burn wounds biopsy demonstrated virtually repaired skin architecture with normal epithelization, fibrosis within the dermis and restitution of the adnexa (Fig. 1), compared to the reference standard Silver sulfadiazine (1% w/w).
Masson's trichrome staining could be used to assess the quantity of new collagen deposition (Fig. 2). The Ginsenoside Rb1(0.5% w/w)group showed mature and well-developed collagen depositions. Finally, the findings revealed that wounds treated with Ginsenoside Rb1(0.5% w/w) showed minor inflammation, practically complete re-epithelialization, and well-organized collagen deposition.
All groups had varying degrees of inflammation on the 19th day after treatment, and the group control had significant expressions of IL-1β and TNF-α, as shown in Figure-3. The SSD and Ginsenoside Rb1 (0.25%, 0.5% w/w) groups had lower expression than the control group; however, the Ginsenoside Rb1 (0.5% w/w) group had lower expression considerably lower expression of IL-1β and TNF-α on the 19th-day post-treatment.
Even in physically healthy people, burn injuries are frequently accompanied by consequences. People with diabetes may have a higher risk of complications and death. Wound healing failures and infections are also common in people with diabetes.[22]Because numerous components of wound healing physiology are disrupted in diabetes wounds, resulting in delayed wound healing and persistent inflammation. Less endothelial progenitor cells, lower endothelium-derived nitric oxide synthase activity, a growth factor shortage, reduced macrophage activity, reduced collagen deposition, increased ECM proteolysis, and the switch from M1 to M2 phenotype are all examples of molecular imbalances.[23]For resolving inflammation and changing the balance toward tissue repair, this transition from M1 to M2 is critical.[24]
Ginsenoside Rb1 (0.5% w/w) has been reported to have a potent healing effect on burn wounds by several mechanisms, including enhanced vascularization in the surrounding tissue, production of Interleukin 1 beta (IL-1β) and vascular endothelial growth factor (VEGF) from the burn wound. The stimulation of VEGF synthesis and increases in expression of hypoxia-inducible factor (HIF)-1 in kerationcytes and an increase in IL-1 owing to macrophage buildup in the burn site are all contribute to angiogenesis. Also, by promoting the bio-active substances (histamine, SP, and MCP-1),ginsenoside Rb1(0.5% w/w) facilitate burn wound healing.[25]
Ginsenosides was also reported to promote wound healing by activating the mitogen-activated protein kinase pathway, stimulating intracellular cAMP levels and associated protein expression in the nucleus, enhancing the dermal fibroblast proliferation and collagen synthesis. Furthermore, ginsenoside Rb1 enhances skin keratinocyte movement and myofibroblast transformation in senescent dermal fibroblasts of human skin by stimulating the production of growth factors, including a sequence of SASP factors. [26]In addition to the above mechanism,M1 to M2 transition is crucial, as it shifts the wound from the inflammatory phase to tissue healing.
Wound healing is a complicated biological process divided into four stages: haemostasis phase (0–several hours after damage), inflammation phase (1–3 days), proliferation phase (4–21 days), and remodelling phase (21 days–1 year). [27] Any of these interrupted stages leads to poor healing, such as chronically difficult-to-heal ulcers or extensive scarring, which has a significant and rising health and cost burden on our society.[27–29]The transition from the inflammatory stage to the regenerative stage of wound healing is vital, and evidence is growing that a faulty transition is associated to wound healing difficulties. As a result, therapeutic developments focussing on this shift could be justified.[18]In order to protect from infections and removing dead tissues, the inflammatory phase is necessary as it brings haemostasis and activates innate immune system.[30]On the other hand, if the inflammation is prolonged, it may interfere with keratinocyte differentiation and activation, and obstruct wound healing from progressing through the usual stages.[28] Furthermore, persistent inflammation in chronic inflammatory situations, such as diabetic wounds, is expected to raise metalloproteinases and other proteases, which degrade ECM components and growth factors essential for healing.[23]Furthermore, a lot of scarring has been associated with persistent inflammation.[31]
During wound healing, macrophages switch from a pro-inflammatory M1 phenotype to a tissue-repair M2 phenotype. This produces anti-inflammatory mediators like decoy IL-1 receptor type II, IL-10, and IL-1R antagonist, as well as bioactive molecules like VEGF, IGF1 and TGF that promote ECM synthesis, fibroblast proliferation, and angiogenesis.[32, 33]The transition from M1 to M2 is crucial for resolving inflammation and shifting the balance toward tissue healing.[24]In both animal and human wounds, continuous IL-1 β (pro-inflammatory cytokines) blocked the upregulation of proliferator-activated receptor (PPAR)γ activity, which is essential for macrophage phenotypic transformation. As a result, it was discovered that diabetes induces a faulty M1–M2 transition, which delays wound healing.[34]As a result, regulation of the above pathways is required for optimal wound healing.
In addition to the burn wound repair investigation, we included two additional experimental groups (Groups I to V) to investigate the differences in CD68 (M1 phenotype) and CD163 protein expression (M2 phenotype). On day 5, the current study found that ginsenoside Rb1 (0.5% w/w) had elevated CD163 (2.1 ± 0.12) and lowered CD68 (1.2 ± 0.09), compared to diabetic control ratsthat had decreased CD163 (0.98 ± 0.07) and increased CD68 (2.6 ± 0.18)(Table-4). CD68 and CD163 are glycoproteins and markers of wound healing macrophages. This transition from M1 to M2 phenotype is crucial in diabetic wounds, and the findings highlight the mechanism behind enhanced wound healing of ginsenoside Rb1(0.5% w/w) in diabetic animals with burn wounds.Furthermore, the reduced concentrations of TNF-α and IL-1β on day 5 in the ginsenoside Rb1(0.5% w/w) group (Figure-3) further supports the transition from M1 to M2 phenotype. The low TNF-α and IL-1β level are sustained throughout the healing period in ginsenoside Rb1groups (Figure-3).
Further, the current research findings haverecorded erythema, thickness, reduced collagen and inflammation in control group animals (Fig. 1&2), which were practically recovered to normal in ginsenoside Rb1 (0.5% w/w)treated groups, with maximal burn wound closure (99.23 ± 3.41) suggesting considerable (P < 0.05) burn wound healing activity. This might be related to ginsenoside Rb1 anti-inflammatory, antioxidant, and cell growth-promoting properties.[35]Improved tensile strength may be aided by collagen production, angiogenesis, maturation, and fibre stability.[36] The levels of hydroxyproline and hexosamine in the tissue were examined since they are directly related to collagen production and extracellular matrix development, respectively.[37] Whenginsenoside Rb1(0.5% w/w) treated burn wounds were compared to untreated diabetic control rats, significantly higher levels of hydroxyproline and hexosamine (Table-2) were found (P < 0.05). Ginsenoside Rb1 activates macrophages, releasing cytokines and growth factors with antibacterial and anti-inflammatory properties and promotingmigration of dermal fibroblast to the lesion. In the wound, these fibroblasts multiply, creating extracellular matrix (ECM) biomaterials such as collagen to start the healing process.[38–40]
The pro-inflammatory mediator IL-6 (Table-3) was noticed as soon as 12–24 hours after cutaneous damage, and these ingredients promote angiogenesis, which is essential in the inflammatory stage of wound healing.[41] More intriguingly, the outcomes of this investigation revealed that ginsenoside Rb1(0.5% w/w) did not affect IL-6 levels on day two samples (Table-3). This shows that during the early phases of recovery, ginsenoside Rb1(0.5% w/w) did not affect pro-inflammatory cytokines produced by macrophages. On the other hand, Ginsenoside Rb1(0.5% w/w) therapy increased IL-10 levels on day ten following burn injury. It's worth mentioning that IL-10 is a cytokine generated by T cells and macrophages with anti-inflammatory characteristics.42The wound-healing environment appears to be altered by IL-10, which seems to reduce the expression of profibrotic/proinflammatory mediators, leading to a reduction in inflammatory cell recruitment to the wound.[42, 43] Treatment with ginsenoside Rb1(0.5% w/w) increased serum IL-10 levels while decreasing IL-6 expression, especially on day ten after burn injury. As a result, ginsenoside Rb1 regulates proinflammatory and anti-inflammatory cytokines and the systemic immunological pathways that relate them to cellular proliferation.
Biochemical analysis of plasma samples was performed to determine the function of anti-oxidants, pro-inflammatory, and anti-inflammatory mediators behind the beneficial effect of ginsenoside Rb1. In our research, ginsenoside Rb1(0.5% w/w) shown extraordinary antioxidant activity by substantially (P < 0.05) boosting the levels of antioxidant enzymes like SOD, CAT, and glutathione (GSH), suggesting that ginsenoside Rb1 could aid in the prevention of oxidative damage and the improvement of the healing process (Table-2).
SOD-1 catalyzes the dismutation of superoxide radicals into dioxygen and hydrogen peroxide (H2O2), which are both potentially hazardous. The CAT activity of the ginsenoside Rb1(0.5% w/w) treated group was much higher, suggesting that elevated CAT may effectively neutralize H2O2 accumulated due to enhanced SOD activity. [44–46] GSH is also a critical endogenous thiol antioxidant that acts as a supporting factor for glutathione peroxidase (GPx) in removing lipid hydroperoxide.[46]Furthermore, when reactive oxygen species destroy polyunsaturated lipids, MDA, a secondary metabolite of LPO,is utilized to determine the level of osmotic damage in an organism.[47]In this study, ginsenoside Rb1(0.5% w/w) significantly lowered blood MDA levels (7·32 ± 1·51) when compared to diabetic controls (12·23 ± 1·03). Notably, the diabetic control animals in this study had lower anti-oxidant levels and greater MDA levels, which might explain why their burn wounds took longer to heal.
The current work demonstrates the therapeutic potential of the ginsenoside Rb1(0.5% w/w) for treating diabetic burn wounds, as it ingeniously alters the transition from M1 to M2 phenotype at the right time to improve diabetic burn wound healing. On day 5, there was an increase in CD163 (M2) and a reduction in CD68 (M1). Furthermore, ginsenoside Rb1 (0.5% w/w) increased tissue hydroxyproline and hexosamine levels, which improved collagen production and extracellular matrix formation in diabetic burn wounds. Similarly, not interfering with the generation of pro-inflammatory mediators favoured the inflammatory phase of wound healing (IL-6). It also aided the proliferation process by enhancing anti-inflammatory mediator synthesis (IL-10). Overall, our data point to ginsenoside Rb1 (0.5% w/w) therapeutic potential as a stand-alone therapy or in conjunction with other standard burn care medicines for the successful treatment of diabetic burn wounds. Additional study is needed, however, to corroborate the current findings.
Silver sulfadiazine
Streptozotocin
lipid peroxidation
Glutathione
Superoxide dismutase
Catalase
Vascular endothelial growth factor
proliferator-activated receptor
Acknowledgments
The authors extend their sincere appreciation to the Deanship of Scientific Research at King Khalid University for funding support for this review through the Large Research Group Project under grant number "RGP 2/89/43".
Ethical Approval
The Institutional Animal Ethics Committee (IAEC) of Erode College of Pharmacy, Erode, Tamilnadu, India (565/02/CA/18/CPCSEA) approved all experimental procedures. Experiments were carried out as per the guidelines for laboratory animal care and use.
Competing Interest
Author declare that there is no potential conflict of interest in this paper
Author Contribution
KrishnarajuVenkatesan,Yahia Alghazwani, and Yahya I.Asiri conceived and designed research, conducted experiments, and Kousalya Prabahar, Jamal Moideen Muthu MohamedDurgaramaniSivadasananalyzed data and wrote the manuscript, Rajalakshimi Vasudevan,Noohu Abdulla Khan, Kumar Venkatesan, Premalatha Paulsamy, and Kalpana Krishnaraju supervision of the work. All authors read and approved the manuscript.
Funding statement
This research was funded by the Deanship of Scientific Research at King Khalid University; Grant number "RGP 2/89/43".
Availability of data and materials: None