INTRODUCTION:

Wound healing is a complex and dynamic process that involves a variety of processes, namely biochemical, immunological and physiological which act as repairs.¹ Wound healing occurs immediately after an injury, through several phases, namely the inflammatory, proliferation and remodeling phases. disturbance in these phases can cause healing delay or even wound healing does not occur.²

Diabetes mellitus (DM) can cause wound healing disorders because it can affect one or more biological processing mechanisms.³ Multiple and complex pathophysiological factors also play a large role in the failure of the wound healing process.⁴ Excessive inflammatory cytokines in people with DM cause a prolonged inflammatory response, resulting in uncontrolled activation that can cause tissue injury and can even increase susceptibility to pathogenic microorganisms and poor healing.⁵

Healing wounds with DM occurs longer than normal wound healing. Under normal condition, the wound healing process has been completed on day 13. Whereas in diabetic wound healing by day 13 still in the accumulation of granulation tissue.⁶ In diabetic wound healing, proliferation and angiogenesis decreased, which causes scar tissue decreased neovascularization and wound healing becomes longer.⁷ It is caused by a decrease in the secretion of growth factors, such as Platelet Derived Growth Factor (PDGF) due to the process of resolution of inflammation by inhibition macrophages M2.^8,9

According to previous studies, the expression of PDGF in wound healing with diabetes mellitus continues to decline on days 3,5 and 7 as compared to the expression of PDGF normal wound healing due to a decrease in the activity and function of macrophages. Decreased expression of PDGF on days 3,5 and 7 will disrupt proliferation and angiogenesis so that healing becomes delayed.^10,11 Several previous studies using recombinant PDGF to accelerate wound healing, but the therapy is costly and has the effect of burning sensation in the wound area.⁵ Utilization of natural ingredients like fruit extracts as treatment increasingly in demand by the public, because it has fewer side effects compared to chemical drugs.¹²

Okra fruit extract has anti-inflammatory effects, antiviral, plays a role in wound healing process, antioxidants, and also serves as antidiabetes.^13-15. In this study, okra fruit extracts obtained from Batu, Malang. With the provision of okra fruit extracts in the inflammatory phase to improve the function and activity of macrophages, so the expression of PDGF maybe increased.^13,15,16 Accordingly, the aimed of this study was to know the effect of the okra fruit extract in increasing the expression of PDGF in the wound healing process.

MATERIAL AND METHODS:

Study Design:

This study was an analytical experimental study, post-test only control group design. This research has been done after received an ethical clearance approval letter from Health Research Ethical Clearance Commission with number of ethics 228/HRECC.FODM/V/2019.

Okra’s fruit Extract Preparation:

Okra fruit liquid extract derived from fresh okra dried fruit of Materia Medika Batu, Malang. Okra fruit macerated using ethanol 70% for 24 hours, then filtered and evaporated using a rotary evaporator at a temperature40-60ᴼC, Then diluted with 5% DMSO to produce a concentration of 100% at dose 250mg/kgBW. Then do the dose adjustment in Wistar rats into25 mg/100grBB/1cc.¹⁷

Animal Subjects Preparations:

24 male Wistar rats (Rattus novergicus) aged 2-3 months, 110-150gram, were taken from the Biochemistry Laboratory of Airlangga University's Faculty of Medicine, acclimated for 7 days, and fed regular rats meal. Wistar rats were starved for 4hours before receiving a single 45mg/kgBW (6.75mg/150 g/BW) dose of STZ 2 percent intraperitoneally (Nacalai Tesque, Inc.).¹⁸ After 3days, rats with random blood glucose≥ 200mg/dlwere used as subjects in further procedures.¹⁹

24 male Wistar rats suffering from DM were extraction the lateral incisor teeth, then divided into two groups, control groups (days 3, 5, 7) and treatment groups (day 3, 5, 7). In the treatment group after the extraction were given okra fruit extract at a dose of 250mg/kgBW orally once daily. Each control and treatment groups on day 3, 5, and 7 sacrificed with lethal doses of ketamine injection given intraperitoneally. Incisor region of the mandible socket was cut vertically and then continued technical preparation of paraffin on all groups.²⁰

Procedures Immunohistochemistry:

Preparations socket mandibular incisor region that have been taken do decalcification and proceed with the preparation of immunohistochemistry. Immunohistochemistry using immunohistochemistry kit (Scy Tek Laboratories) and PDGF-BB monoclonal antibody (sc 365 805: Santacruz Biotechnology). Immunohistochemical observation by observing macrophage cells which express PDGF using magnification microscope 400x.²⁰ Data of the results for each group were tabulated.

Statistical analysis:

The SPSS 16 software for Windows was used to examine the data collected (SPSS Inc., Chicago, USA). The Kolmogorov-Smirnov test was used to evaluate the normality distribution of the data. Given that all variables were normally distributed, a one-way ANOVA test was used to find differences between groups, followed by Tukey's HSD test. The average and standard deviation of the results are shown (SD). The threshold for significance was set at 0.05.

RESULTS:

The results showed an increase in the number of average expression of PDGF in the treatment group were given extracts of okra compared with the control group.The expression of PDGF on the socket mandibular incisor region from Wistar rats with diabetes mellitus, with immunohistochemical staining was observed using a light microscope magnification of 400x. Expression of PDGF will be seen as positive immunoreactive cells with brown DAB chromogen detection by the macrophage cell nucleus. Based on the observations (Figure 1), we can see a significant difference to the amount of the mean expression of PDGF treatment group than the control group.

Figure 1: The expression of PDGF in the control group on day 3(A). The expression of PDGF in the treatment group on day 3 (B). The expression of PDGF in the control group on day 5 (C). The expression of PDGF in the treatment group on day 5 (D). The expression of PDGF in the control group on day 7 (E). The expression of PDGF in treatment group on day 7 (F).

The results mean of the expression of PDGF, and test homogeneity and normality, showed that each group is normally distributed data and homogeneous. Then proceed with the analysis of Independent T-tests on each day between the two groups and found significant differences in the mean number of PDGF expression (P <0.05) (Table 1).

Table 1: The mean value and standard deviation of PDGF expression in treatment and control group day 3, 5, and 7

Days-	Control group (x ± SD)	P value (Anova)	treatment group (x ± SD)	P value (Anova)
3	3.72 ± 1.65^b	0.000*	10.92 ± 0.81^b	0.000*
5	7.95 ± 0.52^a		15.72 ± 1.32^a
7	7.89 ± 0.62^a		17.72 ± 1.24^a

*a significant difference (p <0,05)

Different letter shows significant difference

According to the analysis of Oneway ANOVA followed by analysis of Tukey to determine profile analyzes the amount of expression of PDGF in each group, also found significant differences (P<0.05) (Table 2) in the control group day 3 to 5 and day 3 to 7, but there is no significant difference in the control group day 5 to 7 and the average amount showing decreased PDGF expression. In the treatment group of 3 to 5 days, days 3 to 7 there are significant differences (p<0.05).

Table 2: Tukey HSD test results in the control and treatment groups

	Control group
Days -	3	5	7
3		0.001*	0.001*
5			0.997
7
	Treatment group
Days-	3	5	7
3		0.001*	0.000*
5			0.083
7

*Different letter shows significant difference (p<0.05)

DISCUSSION:

The results are consistent with the hypothesis that has been created that okra fruit extract (Abelmoschus esculentus) can increase the expression of PDGF on the wound healing process of tooth extraction Wistar rats with diabetes mellitus. Itcan be observed through a number of expressions of PDGF. In the control group, the number of PDGF expression was significantly lower than the treatment group, this is due to the induction of Streptozotocin (STZ), that is diabetogenic agent that can affect blood glucose levels. Induction of STZ affect blood glucose levels through the destruction of pancreatic beta cells and the regulation of GLUT 2 that decreases receptor sensitivity insulin.²¹

STZ-induced hyperglycemic causes the formation of oxidative stress due to increased free radicals and AGEs, which led to the issuance of proinflammatory cytokines (IL-6, IL-1, TNF-α) in large numbers and dysfunction of inflammatory cells, such as macrophages in the healing process.¹⁸ This causes the late inflammatory phase become longer and occurs proliferation process due to the amount of expression of PDGF on the state of diabetes mellitus decreased. This is consistent with results of previous studies that say that the expression of PDGF in rats with diabetes mellitus were observed in macrophage cell secretion is lower and tends to decrease until the 15th day compared to mice that were not diabetic melitus.^10,22

In the control group, the amount of expression of PDGF on day 3 compared to day 5 showed a significant increase, while on day 5 compared to day 7 showed an increase but not significantly. It is caused by the change in the function and activity of inflammatory cells that secrete PDGF due to prolonged inflammatory phase on the condition diabetes mellitus. The biggest source secretion of PDGF, macrophage, was change in the function and activities.¹⁹

The transitional period towards inflammatory to proliferative phase disruption caused of dysfunction and activity of macrophages that cause inflammation and dysregulation phase of granulation tissue formation associated with impaired wound healing process Wistar rats with diabetes mellitus. Macrophages will release proinflammatory cytokines such as, IL-6, IL-1, TNF-αin large numbers and activation of NF-αB by AGEs increase the polarization phenotype of macrophages into M1 as inflammatory agents and reduce the function of macrophages efferocytosis. These three things are causing elongation of the inflammatory phase and disrupt changes phenotype of macrophages M1 to M2 as an agent for resolution of the inflammation and secretion of growth factor PDGF in the control group of more decreased.^23,24 In addition, inhibition of inflammatory resolution process results in fibroblast and keratinocyte migration which also play a role in PDGF secretion. It is causing on the results of the study are the control group decreased expression of PDGF day 5 towards the day to 7. If PDGF secreted declining and inadequate, then the proliferation process of angiogenesis can be inhibited so that the healing of wounds that occur prolonged.²⁵

In the treatment group showed a mean expression of PDGF higher and statistically significant differences compared to the control group. This is likely due to the intervention of the oral administration of okra fruit extracts are supress the elongation inflammatory phase in Wistar rats with diabetes mellitus. With an effective dose of drug action allows functions to be optimal, so that oral administration can reach higher target receptor than the topical administration.²⁶ Okra fruit extract of this study using a dose of 250 mg/kgBW and converted to a dose of 37.5 mg/150grBW, which is the dose optimal based previous studies.²⁷ This is also supported by the results of this study which showed a significant increase in the expression of PDGF in the treatment group.

In the treatment group, the highest expression of PDGF obtained on day 7, it is in line with the theory that the expression of PDGF secreted by macrophages highest in the 7th day as the culmination process of angiogenesis. There are significant differences in the mean expression of PDGF day 3 to day 5 in the treatment group. It shows okra fruit extract is able to regulate the M2 macrophage function as an agent of resolution of the inflammation so that the expression of PDGF on day 5 increased compared with the 3rd day. There are an increasing number of PDGF expression at day 7 compared to day 5 in the treatment group and the statistically there is no difference in the expression of PDGF.

Okra’s fruit extract (Abelmoschus esculentus) can increase the amount of expression of PDGF consistently on day 3 to day 7 in the treatment group compared with the control group. This caused okra fruit extracts have potential in the wound healing process as an anti-inflammatory agent, an antioxidant, immunomodulatory activity and macrophage function and antidiabetic.^28,29 Okra fruit extract contains important compounds such as flavonoids, tannins, saponins, alkaloids and quercetin.³⁰ Flavonoids can inhibit the cyclooxygenase and lipooksigenaseenzyme in inflammatory cascade reaction so can reduce the production of prostaglandin and leukotriene. Decreased proinflammatory prostaglandins as mediators can limit the inflammatory cells in the wound area. In addition, okra fruit extracts can inhibit NF-kB activation induced by binding of AGEs and receptor on macrophages. So the production of pro-inflammatory cytokines such as TNF-a and IL-1b can be supressed.²⁷^,31

The decrease of proinflammatory cytokines can also be caused by the potential antidiabetic okra fruit extract (Abelmoschus esculentus) through regulations controlling blood glucose levels. Quercetin its role as an inhibitor of α-glucosidase enzyme in the intestinal epithelial wall. The decline in blood glucose levels play a role as an anti-oxidative stress through the barriers ROS activity that causes a decrease in the activity of secreting mediators proinflammatory. In addition, the high reactivity of the hydroxyl groups from flavonoid will bond directly with the component reactive free radicals (ROS) so terminating radical chain reaction.^32-34 Termination of radical chain reactions that prevent free radical damage to the tissue, lowering the excessive proinflammatory cytokines and enhance the function of macrophages efferocytosis against apoptosis neutrophil cells.³⁵

Flavonoids also act as an immunomodulatory function of macrophages by binding directly to the estrogen receptor in the nucleus of the macrophages resulting in biological activity that enhances the function of macrophages and secreting growth factor.³⁶ Increased efferocytosis function and reduction of proinflammatory cytokines causing the shifting process M1 into M2 macrophage phenotype in inflammatory transition phase towards proliferation. Macrophage (M2) will release PDGF and anti-inflammatory cytokines such as IL-10, which immediately will stop the inflammatory process and stimulate the proliferation wound healing process including angiogenesis.³⁷

CONCLUSION:

The oral administration of okra fruit extract (Abelmoschus esculentus) can boost the expression of Platelet Derived Growth Factor (PDGF) in Wistar rats with diabetes mellitus throughout the wound healing process following tooth extraction.

ACKNOWLEDGMENT:

The authors would like to express gratitude to all the involved team and Faculty of Dental Medicine, Universitas Airlanggafor the invaluable support in conducting this research.

CONFLICTS OF INTERESTS:

The authors declare that they have no conflicts of interest.

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Received on 17.03.2022 Modified on 30.12.2022

Research J. Pharm. and Tech 2023; 16(11):5329-5333.

DOI: 10.52711/0974-360X.2023.00863