Effects of bioactive compound, Ginsenoside Rb1 on Burn Wounds Healing In Diabetic Rats: In�uencing M1 To M2 Phenotypic Trans

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-inammatory, bacteriostatic, and growth-promoting effectsoncells.The therapeutic bene�ts 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-1 β , 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 in�ammatory markers (IL-6) and anti-inammatory markers (IL-10), in�uence on hydroxyproline and hexosamine was observed. As indicated by increased CD163 (M2) and reduced CD68 (M1) on day 5, ginsenoside Rb1 effectively �ips 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-inammatory properties, antioxidant properties, increased tissue hexosamine and hydroxyproline levels. Skin tissue morphology was signi�cantly 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 in�ammatory phase of burn wound healing (IL-6), assisted the proliferation process (IL-10) and had considerably lower expression of IL-1 β 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.


Introduction
The healing of a burn wound is a multi-step process that begins with the in ammation 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 e cacy and fewer adverse effects than currently available treatments.Panax notoginseng is a traditional herbal medication used to treat in ammatory diseases, cardiovascular illnesses, traumatic injuries, and external and internal bleeding caused by damage.[9]Because of its potential anti-in ammatory, antioxidant, and cell growth-promoting activities, ginsenoside Rb1, an essential active ingredient of P. notoginseng, has gotten a lot of attention.[10][11][12][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 bene ts were investigated in this work to contribute to a scienti c foundation for the therapeutic use of ginsenoside Rb1 to treat burns in diabetic rats.

Ointment processing
Ginsenoside Rb1 (purity > 99.0%) was procured fromSelleckChem.A basic ointment base in the ratio of 1:6:3 will be prepared using liquid para n, 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.

Experimental Animals And Housing Conditions:
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.

Induction Of Diabetes:
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 de ned as those that have blood glucose level of > 200 mg/dl at fasting and were included in this investigation.[15] Wound Healing Activity Thermal burn wound model: The rats have split into ve groups of six ratsfor the thermal wound model.The rst 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 fth 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 nal 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 Biochemical Analyses: At the end of the test, the rats with burn wounds were sacri ced 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.

Estimation Of 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 pdimethylaminobenzaldehyde (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] Estimation Of Antioxidant Activity 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] Estimation Of Pro-in ammatory And Anti-in ammatory Cytokine Induction: On days 2 and 10, the blood samples were obtained from burn wound animals in each group following wounding.The amounts of pro-in ammatory (IL-6) and anti-in ammatory (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 xed in buffered formalin.Later, it is processed in a series of alcohol and xylene and embedded in para n 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. 18The 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 uorescence 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 lms 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 uoride or polyvinylidene di uoride 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 ndings were represented in standard units (Biotech Inc).The gene b-actin is employed as a housekeeping gene.[21] Statistical analysis: 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 signi cance, a substantial difference was considered.The mean and standard error of six animals' mean (SEM)is represented (n-6).

Result
Burn wound healing: In thermally produced burn wounds, the Ginsenoside Rb1showed a signi cant (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 signi cant (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.Hexosamine and hydroxyproline concentrations in healed wound tissue are shown in Table 2.The content of hydroxyproline and hexosamine in diabetic wound control was signi cantly (P < 0.05) reduced.
The treatment groups had signi cantly (P < 0.01) increased hydroxyproline and hexosamine concentrations than diabetic wound controls.
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.

Effect Ginsenoside Rb1on Protein Expression Of Cd68 And Cd163
In Experimental Rats: 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.Histopathologic Report: Ginsenoside Rb1 (0.5% w/w) treated group of burn wounds biopsy demonstrated virtually repaired skin architecture with normal epithelization, brosis 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 ndings revealed that wounds treated with Ginsenoside Rb1(0.5% w/w) showed minor in ammation, practically complete re-epithelialization, and well-organized collagen deposition.
All groups had varying degrees of in ammation on the 19th day after treatment, and the group control had signi cant 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 19thday post-treatment.

Discussion
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 in ammation.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 in ammation 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 broblast proliferation and collagen synthesis.Furthermore, ginsenoside Rb1 enhances skin keratinocyte movement and myo broblast transformation in senescent dermal broblasts 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 in ammatory phase to tissue healing.
Wound healing is a complicated biological process divided into four stages: haemostasis phase (0several hours after damage), in ammation 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 di cult-to-heal ulcers or extensive scarring, which has a signi cant and rising health and cost burden on our society.[27][28][29]The transition from the in ammatory stage to the regenerative stage of wound healing is vital, and evidence is growing that a faulty transition is associated to wound healing di culties.As a result, therapeutic developments focussing on this shift could be justi ed.[18]In order to protect from infections and removing dead tissues, the in ammatory phase is necessary as it brings haemostasis and activates innate immune system.[30]On the other hand, if the in ammation is prolonged, it may interfere with keratinocyte differentiation and activation, and obstruct wound healing from progressing through the usual stages.[28]Furthermore, persistent in ammation in chronic in ammatory 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 in ammation.[31] During wound healing, macrophages switch from a pro-in ammatory M1 phenotype to a tissue-repair M2 phenotype.This produces anti-in ammatory 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, broblast proliferation, and angiogenesis.[32,33]The transition from M1 to M2 is crucial for resolving in ammation and shifting the balance toward tissue healing.[24]In both animal and human wounds, continuous IL-1 β (pro-in ammatory 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.
[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, signi cantly 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-in ammatory properties and promotingmigration of dermal broblast to the lesion.In the wound, these broblasts multiply, creating extracellular matrix (ECM) biomaterials such as collagen to start the healing process.[38][39][40] The pro-in ammatory 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 in ammatory 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-in ammatory 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-in ammatory characteristics. 42The wound-healing environment appears to be altered by IL-10, which seems to reduce the expression of pro brotic/proin ammatory mediators, leading to a reduction in in ammatory 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 proin ammatory and anti-in ammatory 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, proin ammatory, and anti-in ammatory mediators behind the bene cial 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][45][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.

Conclusion
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-in ammatory mediators favoured the in ammatory phase of wound healing (IL-6).It also aided the proliferation process by enhancing anti-in ammatory 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 ndings.

Abbreviations
). 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 ndings 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).

Figure 2 The 3
Figure 2

Table 1
Effect of Ginsenoside Rb1 on thermal burn wound healing in diabetic rats

Table 2
Biochemical study of wound tissue in diabetic rats caused by streptozotocin The results are shown as mean ± S.E.M of six rats (n = 6).*P < 0.05 is the statistical difference from control

Table 4
Effect of Ginsenoside Rb1on CD68 and CD163 in diabetic burn wounds in rats