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. 2025 Mar 31;28(1):11–23. doi: 10.3831/KPI.2025.28.1.11

Ameliorating Effect of Red Fruit (Pandanus conoides Lamk) Oil in Streptozotosin-Induced Diabetic Peripheral Neuropathy Rat Model: role of oxidative and neuroinflammatory pathways

Indah Tri Lestari 1, Kusnandar Anggadiredja 1,*, Afrillia Nuryanti Garmana 1, Ratna Annisa Utami 2
PMCID: PMC11933913  PMID: 40165881

Abstract

Objectives

Diabetic peripheral neuropathy (DPN) is the most common cause of neuropathy worldwide, with oxidative and inflammatory being the pathways involved in the disease pathology. Red fruit (Pandanus conoides Lamk) oil (RFO) is known to have potent antioxidants and anti-inflammatory activities. To investigate the effects of RFO in a rat model of streptozotocin-induced DPN.

Methods

Male Wistar rats were induced with 55 mg/kg streptozotocin (STZ) intraperitoneally. Rats with fasting blood glucose ≥ 200 mg/dL were assigned into groups treated with vehicle, pregabalin 30 mg/kg, and 0.3, 0.6, or 1.2 mL/kg RFO, respectively. The treatments were given for 3 weeks, six weeks after STZ induction. Neuropathic pain was evaluated by cold allodynia and thermal hyperalgesia tests. Histopathological changes of the pancreas and sciatic nerve were evaluated by the Gaussian adaptive threshold method. Feed and drinking intake, blood glucose levels, body weight, and sciatic nerve proinflammatory cytokines were also measured.

Results

There were increases in blood glucose levels, feed and drink intake, levels of MDA, TNF-α, IL-6, NFκB, and iNOS; decrease in catalase level; and damage to the sciatic nerve in the vehicle-treated DPN rat model compared with normal rats (p < 0.05). Meanwhile, significant decrease in weight, number of pancreatic β cells, latency times for thermal hyperalgesia as well as cold allodynia were observed in this group of rats. RFO administration at 0.6 and 1.2 mL/kg significantly improved all of the measured indices, except for iNOS, where only decreasing trend was observed. Pregabalin did not affect blood glucose levels, body weight or pancreatic β cells but improved thermal hyperalgesia as well as cold allodynia, the sciatic nerve cell counts, TNF-α IL-6, NFκB, and iNOS levels.

Conclusion

RFO improves DPN by neuroprotective effect through the involvement of the oxidative and neuroinflammatory pathways.

Keywords: diabetic peripheral neuropathy, neuroiflammatory, Pandanus conoides Lamk, oxidative, rat, red fruit oil

INTRODUCTION

Diabetes is a global epidemic and the International Diabetes Federation estimates there were 537 million diabetes cases in 2021 and forecasts that cases will rise to 783 million in 2045 [1]. This rise will also increase diabetes problems. Diabetic peripheral neuropathy (DPN), the most common neuropathy worldwide, affects half of diabetics, causing high morbidity, poor quality of life, and mortality [2], and it is characterized by high sensitivity to harmless or noxious stimuli (allodynia or hyperalgesia). Neuropathic pain can be tingling, searing, acute, stabbing, or an electric shock sensation [3].

Although DPN pathophysiology is multifaceted, it is dominated by oxidative stress and neuroinflammation. Hyperglycemia drives the production of reactive oxygen species, which cause oxidative damage, neuroinflammation, and nerve injury. Fabiyi-Edebor [4] found that malondialdehyde (MDA), an oxidative stress biomarker, is elevated in the sciatic nerves of rats with DPN. Additionally, proinflammatory factors, such as NF-κB, TNF α, IL-6, and iNOs can also contribute to DPN pain, and increased NF-κB, TNF α, and IL-6 levels have been observed in rat models of DPN [5].

In DPN, the role of oxidative stress and inflammation, and the safety and tolerability of current medicines, make natural products an intriguing treatment option. Natural diabetes treatments reduce oxidative stress and inflammation. Red fruit (Pandanus conoides Lamk), an antioxidant herb from Papua, is used as food and medicine, and phytochemical screening indicates that it contains carotenoids like tocopherol, beta-carotene, and α-tocopherol, as well as fatty acids like oleic, linoleic, and linolenic acids [6].

In various animal models of metabolic diseases, red fruit has been demonstrated to have potential health benefits via its effect on oxidative and inflammatory pathways. The fruit extract or oil reduces TNF-α [7], normalizes elevated MDA [8], and increases GPx levels [9]. However, no studies have reported the effects of red fruit oil (RFO) in DPN models or its possible physiological targets.

Here, we developed a streptozotocin (STZ)-induced rat model of DPN, in which RFO’s effects were tested by evaluating behavioral, histological, and biochemical indices.

MATERIALS AND METHODS

1. Animals, diabetes induction, and treatment

A total of 25 male Wistar rats (weight: 250-300 g, age: 12-15 weeks) were used. The rats were maintained at a temperature of 23 ± 2℃, a relative humidity of 55% ± 10%, and a 12-hour/12-hour light/dark cycle. Before the experiment, rats were acclimatized for seven days. Diabetes was induced via intraperitoneal STZ injection at 55 mg/kg. Rats with fasting blood glucose levels of ≥ 200 mg/dL were assigned into the vehicle, pregabalin-, or RFO (0.3, 0.6, or 1.2 mL/kg) groups. RFO was purchased from PT. Hujan Bahagia, West Papua, Indonesia. Treatments were administered for three weeks six weeks after STZ induction. The animal experiment protocol was approved by the Animal Ethics Committee of Bandung Institute of Technology (approval number 08/KEPHP-ITB/3-2022).

2. Food and water intake

At week nine, before the rats were sacrificed, food and water consumption was measured by providing each cage with 250 g of food and 1,000 mL of water and then subtracting the quantity of food and water remaining the next day from the initial amount.

3. Blood glucose and body weight measurements

To determine blood glucose levels, blood samples were collected via tail puncture, and glucose concentration was measured using a portable glucometer (Autogluco, DR). Glucose measurements were done at week 0 (baseline), week 6 (start of treatment), and week 9 (end of treatment).

4. Thermal hyperalgesia and cold allodynia pain threshold test

The test was performed by measuring tail flick latency in response to heat (45 ± 2℃, cutoff time: 12 s) and cold (10 ± 2℃, cutoff time: 15 s) stimuli. The procedure was repeated thrice at five-minute intervals between observations [10].

5. Oxidative pathway analysis

MDA levels were measured as described previously [11]. The sample (0.5 mL) was mixed to homogeneity with 20% trichloroacetic acid (0.5 mL) and 0.67% thiobarbituric acid (1 mL). The mixture was then heated for 10 minutes at 95℃-100℃. Upon cooling, it was centrifuged for 10 minutes at 4,000 rpm. The supernatant was then recovered and its absorbance was measured at 532 nm.

Catalase activity was measured using the Sinha technique with modifications [12]. Samples were treated with 65 mM H2O2 in 50 mM phosphate buffer (pH 7.4) with 0.1% bovine serum albumin. K2Cr2O7 in glacial acetate. Absorbance changes were measured at 570 nm.

6. Effect on inflammatory biomarkers

Elisa kits were used to measure the levels of the proinflammatory cytokines, TNF-α and IL-6 (Elebsains, China), in rat sciatic nerve tissue, as well as NFκB and iNOS (BT Lab, China) according to the kit manufacturers’ protocols. Absorbance was read at 450 nm using an ELISA reader (Tecan).

7. Histopathological examination

At the end of the study (week 9), the rats were sacrificed through exposure to CO2 in a gas chamber. Next, the pancreas and sciatic nerve were extracted and fixed using 10% buffered formalin, followed by histopathological examination as previously described [13]. Briefly, tissues were embedded in paraffin and sectioned at 5 μm thickness using a rotary microtome, followed by hematoxylin and eosin staining.

8. Histopathological analysis using the Gaussian adaptive threshold method

The Gaussian adaptive threshold method was used to further analyze the histopathological results by converting the images into the variable of grayscale 1 with kernel (5.5), maxValue 255, THRESH BINARY as threshold type and block size 3, C:2. Objects were calculated using SciPy library’s label function and stored as a label array. The number of objects was calculated as ‘particle count’.

9. Data analyses

Behavioral and neurochemical analysis data were expressed as mean ± standard deviation. Data were analyzed using a one-way analysis of variance, followed by LSD using the Statview statistical software. p < 0.05 indicated statistically significant differences.

RESULTS

1. Diabetes parameters

Successful diabetes induction was indicated by increased blood glucose levels, reduced body weight, and increased food and water intake (Fig. 1).

Figure 1.

Figure 1

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The effects of RFO on diabetes parameters as measured by (A) blood glucose level, (B) body weight, (C) food intake and (D) water intake. Six weeks following STZ induction, the rats were given the treatments for three weeks. Data represents the average ± SD of 4 rats. a,bp < 0.05 vs DPN and normal, respectively, ANOVA post hoc LSD.

All rats had normal blood glucose levels (72.75-84.75 mg/dL) after acclimatization for one week. After seven days of STZ injection, all rats had significantly increased glucose levels (range: 380-475 mg/dL), which remained high after six weeks (range: 409-476 mg/dL). RFO administration at 0.6 and 1.2 mL/kg for three weeks following diabetes induction significantly reduced blood glucose levels (p < 0.05). The average glucose level in rats treated with 0.3 mL/kg RFO was 482.75 mg/dL, compared with 550 mg/dL in the vehicle-treated group. The rats’ initial body weight ranged from 262-280 g after acclimatization and decreased significantly in all STZ-injected groups (p < 0.05). At week six, the normal group had an average weight of 277 ± 8.16 g, while the weights of the STZ-treated group ranged from 214-232 g. In the vehicle- and PGB-treated rats, weight loss (214.75 ± 12.13 g and 215 ± 14 g, respectively) occurred until week nine, while the 0.3 mL/kg RFO-treated group exhibited no change in body weight. The groups treated with 0.6 and 1.2 mL/kg RFO exhibited increased body weight (247.5 ± 18.20 g and 234.75 ± 2.48 g, respectively). Calculation of food and water consumption at week nine revealed that the vehicle-treated diabetic group had the highest food intake (37.28 ± 2.19 g), followed by the PGB (36.83 ± 1.95 g) and 0.3 mL/kg RFO (33.45 ± 1.66 g) groups. RFO administration for three weeks at 0.6 and 1.2 mL/kg lowered food consumption significantly (p < 0.05), to 28.8 ± 1.62 g and 29.58 ± 0.94 g, respectively, although consumption was still higher than in the control group (19.08 ± 0.68 g). Water intake results had a similar pattern to food intake. The quantities consumed by the vehicle PGB, RFO 0.3, 0.6, and 1.2 mL/kg groups were 131.29 ± 3.74, 128.42 ± 7.88, 95.80 ± 7.09, 92.58 ± 8.56, and 89.79 ± 6.32 g, respectively.

2. Cold allodynia and thermal hyperalgesia

Analysis of changes in response to cold allodynia and thermal hyperalgesia pain stimuli indicated the successful establishment of the rat model of DPN (Fig. 2).

Figure 2.

Figure 2

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The effect of RFO on diabetic peripheral neuropathy parameters as represented by the latency time to (A) cold allodynia and (B) thermal hyperalgesia. Six weeks following STZ induction, the rats were given the treatments for three weeks. Data represents the average ± SD of 4 rats. a,bp < 0.05 vs DPN and normal, respectively, ANOVA post hoc LSD.

In the first week after STZ injection, the latency ranged from 15.35-14.71 s. At week six, the latency in the treated diabetic groups decreased and ranged from 4.58-4.06 s. At week nine, the latencies of the vehicle-, PGB-, and 0.3, 0.6, and 1.2 mL/kg RFO-treated groups were 4.15, 9.39, 4.39, 6.46, and 7.14 s, respectively. Despite the increase, these values were not comparable to the normal group (latency: 15.09 s). In the first week after STZ injection, the latency time to thermal hyperalgesia ranged from 10.98-11.11 s. At week six, the latency decreased and ranged from 4.90-4.05 s. Upon RFO administration at 0.3, 0.6, and 1.2 mL/kg, the latencies increased dose-dependently to 4.35, 6.5, and 6.83 s, respectively, although they were still lower than in the normal and PGB groups (10.94 and 9.38 s, respectively).

3. Histopathological examination of the pancreas

Fig. 3 shows pancreatic tissue photomicrographs. It was observed that vehicle-treated diabetic rats had smaller cells, with some cells showing acidophilic degeneration with colored pyknotic nuclei (red arrows), which indicates degeneration. Light congestion (yellow arrow) was also visible. The RFO-treated diabetic group exhibited a partial restoration of normal cellular distribution in the islets of Langerhans, as well as increased cellularity.

Figure 3.

Figure 3

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Effects of RFO on histopathological presentation of pancreas. Groups; (A) Normal, (B) DPN, (C) Pregabalin, (D) RFO dose 1, (E) RFO dose 2, (F) RFO dose 3. Arrows; green: normal, red: acidophilic degeneration, yellow: light congestion (stained with Hematoxylin – Eosin, magnification 400×).

4. Histopathological examination of the sciatic nerve

Fig. 4 presents nerve tissue histology, which revealed that the axons of normal sciatic nerves were surrounded by a centrally located myelin sheath, Schwann cell nuclei, and normal blood capillaries. However, in the hyperglycemic group, the myelinated nerve fibers were swollen and pale, and they exhibited histopathological changes (red arrows). Moreover, the axons had a degenerative appearance (green arrows) and the Schwann cells were hypertrophied and hyperplastic (yellow arrows).

Figure 4.

Figure 4

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Effects of RFO on histopathological presentation of the sciatic nerve. Groups; (A) Normal, (B) DPN, (C) Pregabalin, (D) RFO dose 1, (E) RFO dose 2, (F) RFO dose 3. Arrows; green: normal, red: swollen myelinated, green: degenerated axon, yellow: hypertrophied and hyperplastic Schwan cells (stained with Hematoxylin – Eosin, magnification 400×).

5. Histopathological analysis using the Gaussian adaptive threshold method

In this study, diabetes severity was quantified by counting the number of normal pancreatic β cells, while the level of DPN damage was assessed by counting the number of damaged cells in the sciatic nerve tissue. Both calculations were based on the same principle and were done using Gaussian adaptive threshold analysis (Fig. 5).

Figure 5.

Figure 5

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The effects of RFO on cellular changes in pancreatic beta cells (represented by observed beta cells, A) and sciatic nerves (represented by ceural cell damage, B). Quantitative calculations were carried out using the Gaussian adaptive threshold method. a,bp < 0.05 vs DPN and normal, ANOVA post hoc LSD.

Analysis of the histopathological results using the Gaussian adaptive threshold method revealed more measurable pancreatic damage. In pancreatic histopathology, the method was used to determine the number of remaining β cells. The results revealed that the vehicle-treated hyperglycemic group had the lowest number of β cells (183 ± 10.70). RFO treatment at the escalating doses improved the parameter, and the numbers of cells were 222.25 ± 30.40, 297.50 ± 36.30, and 404.50 ± 49.30, respectively. However, the number did not return to normal (525.25 ± 46.47 particles). Pregabalin did not show improvement, and it was associated with 200.50 ± 13.27 β cell particles.

Gaussian adaptive threshold analysis showed that the number of damaged sciatic nerve cells was highest in the vehicle-treated hyperglycemic group (604.91 ± 31.67 particles). Treatment with RFO at 0.3, 0.6, and 1.2 mL/kg decreased the number of damaged cells dose-dependently to 577.50 ± 72.02, 357.08 ± 32.69, and 355.5 ± 50.67 particles, respectively, although the decrease did not reach the normal level (179.41 ± 45.12 particles). Pregabalin, the reference drug, lowered the number to 340.166 ± 16.89 particles.

6. Oxidative pathway analysis

In this study, the oxidative stress markers, MDA and catalase, were evaluated in the sciatic nerve tissues of rats with DPN (Fig. 6).

Figure 6.

Figure 6

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The effects of RFO on oxidative parameters in the sciatic nerve, represented by the levels of (A) MDA and (B) catalase. Six weeks following STZ induction, the rats were given the treatments for three weeks. Data represent the mean ± SD of 4 mice. a,bp < 0.05 vs DPN and normal, ANOVA post hoc LSD.

Compared with the normal control, STZ injection significantly increased MDA levels in the sciatic nerve (94.41 ± 11.25 mM vs 177.25 ± 10.18 mM, p < 0.05). RFO administration at the escalating doses significantly decreased MDA levels to 135.37 ± 12.81, 117.50 ± 15.21, and 128.04 ± 12.03 mM, respectively. However, pregabalin did not significantly decrease MDA levels when compared with the vehicle-treated hyperglycemic group (177.25 ± 10.18 mM vs 172.83 ± 3.93 mM, p > 0.05). STZ injection significantly decreased catalase levels to 2.24 ± 0.12 U/mL when compared with the normal value (3.14 ± 0.42 U/mL). Treatment with RFO at 0.3, 0.6, and 1.2 mL/kg maintained catalase at normal levels, i.e., 2.60 ± 047, 2.89 ± 0.27, and 3.00 ± 0.53 U/mL, respectively.

7. Effect on inflammatory biomarkers

In this study, the inflammation markers, TNFa, IL-6, NF-κB, and iNOS were evaluated in the sciatic nerve tissue of rats with DPN (Fig. 7). This analysis revealed that when compared with the normal control, STZ injection increased the sciatic neural levels of TNF-α (21.18 ± 0.95 pg/mL vs 35.20 ± 4.25 pg/mL, p < 0.05). Treatment with RFO at 0.3, 0.6, and 1.2 mL/kg significantly reduced the levels to 23.17 ± 4.07, 21.25 ± 2.33, and 19.07 ± 0.40 pg/mL, respectively. This effect was most potent upon pregabalin treatment, which reduced the level to 18.30 ± 1.56 pg/mL.

Figure 7.

Figure 7

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The effect of RFO on the inflammatory pathway in the sciatic nerve, represented by the levels of (A) TNF-α, (B) IL-6, (C) NFκB, and (D) iNos. Six weeks following STZ induction, the rats were given the treatments for three weeks. Data represents average ± SD of 4 rats. a,bp < 0.05 vs DPN and normal, respectively, ANOVA post hoc LSD.

When compared with the normal controls, IL-6 levels were significantly elevated in STZ-treated rats (68.97 ± 5.40 pg/mL vs 113.00 ± 7.03 pg/mL, p < 0.05). RFO administration at 0.3, 0.6, and 1.2 mL/kg significantly lowered the IL-6 levels to 97.34 ± 7.83, 96.31 ± 4,366, and 95.65 ± 3.72 pg/mL (p < 0.05), respectively. Pregabalin, the reference drug, also lowered IL-6 levels significantly (p < 0.05), although not as potently as RFO.

When compared with the normal control, STZ treatment increased NFκB levels (4.47 ± 0.17 ng/mL vs 5.60 ± 0.53 ng/mL, p < 0.05). Treatment with RFO at 0.6 and 1.2 mL/kg significantly lowered NFκB levels to 4.33 ± 0.50 and 4.33 ± 0.44 ng/mL/kg, respectively. NFκB levels were also lowered by pregabalin and 0.3 mL/kg RFO (4.73 ± 0.422 and 4.59 ± 0.49 ng/mL, respectively), although not significantly.

When compared with the normal rats, iNOS levels were significantly increased in STZ-injected rats treated with a vehicle (50.99 ± 1.74 ng/mL vs 60.18 ± 2.72 ng/mL, p < 0.05). Treatment with pregabalin significantly lowered iNOS levels to 53.37 ± 2.40 ng/mL (p < 0.05). RFO administration at 0.3, 0.6, and 1.2 mL/kg caused a gradual, but not significant, decrease in iNOS levels to 57.98 ± 6.38, 55.87 ± 5.60, and 53.97 ± 5.18 ng/mL, respectively.

DISCUSSION

Neuropathy is a common diabetes consequence. STZ is used in long-term diabetes induction experiments because of its toxic effects on pancreatic β cells, including protein carboxylation, DNA alkylation, free radical release, and glycoside hydrolase inhibition [13]. These effects resemble several clinical, physiological, and pathological features seen in patients [14].

In vitro experiments have demonstrated that RFO increases the levels of nitric oxide [6], an endogenous substance with antioxidant activity [15]. A study on lipopolysaccharide-induced murine macrophages found that RFO exerts anti-inflammatory effects by regulating reactive oxygen species and NF-κB [16]. This preliminary research suggests that the biological effects of RFO are partly due to its effects on oxidative balance and the inflammatory pathway. No comprehensive animal model studies have examined the anti-neuropathic effects of RFO or its mechanisms of action. This study examined RFO’s antioxidant and anti-inflammatory effects.

STZ-induced diabetic rats lose weight because of enhanced structural protein and fat catabolism via glycogenolysis and lipolysis. A previous study found that chronic catabolism reduces muscle and fat mass [17]. Unlike the vehicle-treated diabetic group, the groups administered RFO at 0.6 and 1.2 mL/kg had steady body weights after three weeks. RFO fatty acids may help maintain a stable body weight. According to Li et al. [18], long-chain monounsaturated fatty acids suppress food intake by regulating proopiomelanocortin neuronal activity, while long-chain saturated fatty acids increase food intake without changing body weight, fat mass, or muscle mass. Pregabalin did not reduce weight when compared with hyperglycemic rats. A study found that by regulating hypothalamic dopaminergic regulation, pregabalin stimulates hunger but does not affect glucose metabolism [19].

Hyperglycemic rats had greater eating and drinking rates, as well as high blood glucose levels and low body weights. Consistent with previous findings, this study also found that STZ-induced rats had increased food (polyphagia) and drink (polydipsia) intake [20]. RFO reduced food and water consumption in hyperglycemic rats when compared with STZ induction, probably because of the effect of fatty acids, a major RFO component, on the increment of rodent food reward-related behavior [18].

Pancreatic histology revealed β cell destruction in the hyperglycemic group. Histopathological analysis showed cell necrosis, degeneration, moderate congestion, and mononuclear inflammatory cell infiltration. According to Szkudelski and Szkudelska [17], β cell necrosis causes the production of hydrogen peroxide, hydroxyl radicals, and lethal nitric oxide levels. RFO can prevent cell damage via its antioxidant properties. Here, the Gaussian threshold approach revealed that RFO reduced cell damage by increasing the number of cells with perfectly round nuclei. Adaptive thresholding breaks the image into smaller areas. The Gaussian adaptive approach uses light intensity or picture pixel differences. When compared with the control group, a decrease in the number of β cells and the islet of Langerhans diameter was seen in vehicle-induced STZ-induced hyperglycemic group. The hyperglycemic groups treated with RFO at 0.6 and 1.2 mL/kg exhibited a considerable β cell count recovery.

This investigation confirmed previous observations that STZ-induced diabetic rats develop DPN after six weeks [21]. Jain et al. [10] discovered that faster tail withdrawal in diabetics occurred after six to ten weeks. Through a heat immersion test, we found that diabetic rats had a reduced tail-flicking latency, indicating thermal hyperalgesia. Studies have demonstrated hyperalgesia in diabetic rats [22], although thermal hypoalgesia has been observed in long-term diabetes (up to 32 weeks post-STZ induction). These findings show that hyperalgesia is more common early in diabetes, while hypoalgesia is common later. A study supporting this idea revealed mechanical and thermal hyperalgesia at week five post-STZ and hypoalgesia at week nine [23]. Previous diabetic rat studies reported hyperalgesia at eight weeks and hypoalgesia at twelve weeks [24]. Here, the three-week RFO plus pregabalin treatment enhanced latency and reduced thermal hyperalgesia. In STZ-induced animal models of DPN, hyperalgesia is caused by increased oxidative stress and the activity of protein kinase C, aldose reductase, poly (ADP-ribose) polymerase, ACE, and toll-like receptor 4 [25].

Several human and animal studies indicate that hyperglycemia causes DPN by damaging unmyelinated C-fibers, small-myelinated Aδ-fibers, and large-myelinated Aβ-fibers [26]. Although the mechanisms are complex, nociception in diabetic rats is modulated by hyperglycemia’s direct peripheral nervous system toxicity, increased primary afferent fiber activity, increased glutamate release, reduced opioidergic activity, inhibitory GABAergic systems that alter dopaminergic receptor sensitivity and responsiveness, and changes in L-type calcium channels [27].

Pregabalin, the first-line DPN treatment, occupies the α2δ subunit of voltage-dependent calcium channels in the central nervous system and it reduces hyperalgesia and allodynia by targeting the CaVα2δ-1 subunit. Among other effects, reduced calcium influx in nerve terminals blunts glutamate release [28].

Histopathological examination of STZ-induced diabetic rats indicated sciatic nerve fiber damage, axonal degeneration, and myelin sheath degradation. Sciatic nerve function was partially improved by PGB and RFO at 0.6 and 1.2 mL/kg. Histopathological tests showed that RFO improved axon structure and myelinated nerve density when compared with diabetic rats. Histopathological damage analysis using the Gaussian adaptive approach showed that the cells of diabetic rats were damaged more than those of the normal and RFO groups. Inflammatory cells feature vacant cells and smaller nuclei in the middle. Adaptive thresholds can segregate nuclei from core groups with imprecise boundaries [29]. Korzynska et al. [30] demonstrated that this approach accurately detects and distinguishes nuclei. Here, the Gaussian adaptive threshold method revealed that compared with the normal group, the DPN model had more injured sciatic nerve cells, and this condition was improved by RFO and PGB.

MDA, a function and indicator of lipid peroxidation [31], which cross-links and polymerizes cell membranes, is often utilized to assess oxidative stress. Changes influence membrane characteristics, ion transport, enzyme activity, membrane normalization, and cell surface aggregation. Antioxidants are crucial because they reduce oxidative tissue damage [11]. Previous research found that RFO decreases MDA and protects from oxidative stress [8]. RFO is rich in tocopherol [32], which may reduce free radicals. In this study, pregabalin decreased MDA levels but did not vary from hyperglycemic rats. This is consistent with the findings by Shinouchi et al. [33] that pregabalin does not significantly affect STZ-induced MDA elevation.

Our investigation revealed decreased catalase levels in the sciatic nerve, as previously reported [20]. According to Kanj et al. [34], reduced catalase levels promote oxidative stress. Higher oxidative stress decreases catalase levels and increases DM complications. The enzyme catalase, which is present in most mammalian tissues, is an antioxidant, and it converts hydrogen peroxide into water and oxygen. Decreased sciatic nerve catalase levels reflect a changed oxidant–antioxidant equilibrium, which reduces nerve tissue protection from DPN.

Numerous DPN pathways involve inflammation and the activation of NF-κB, which modulates proinflammatory mediators like IL-6, TNF-α, and iNOS, and causes their transcription [35]. Our investigation found that compared with normal controls, NF-κB, IL-6, and TNF-α levels were considerably elevated in DPN rats. RFO’s influence on NF-κB levels may be because of its beta-carotene concentration, which is proven to suppress this proinflammatory cytokine [36].

During chronic inflammatory processes, iNOS is the main active factor. In the hours after injury, TNF-a and IL-1 upregulate iNOS via the NF-kB pathway. DPN experiments involving iNOS inhibition show reduced mechanical and thermal hyperalgesia. This study found that RFO lowered iNOS levels in DPN rats. NO production can be reduced and DPN damage repaired by iNOS downregulation [37].

This investigation demonstrated increased IL-6 levels, as reported by Li et al. [38]. IL-6 is also a pleiotropic cytokine with a variety of physiological roles, including regulation of immunity, metabolism, endothelial function, and neuronal activity [39]. It is unclear how IL-6 helps DPN development. Peripheral nerve injury causes pathological pain, although, in experimental diabetes, IL-6 restoration improves sensory nerve function, somatic motor conduction velocity, tactile allodynia, and thermal hyperalgesia [40]. Decreased IL-6 levels after RFO treatment have been previously reported by Xia et al. [6], and RFO may reduce IL-6 levels because of its omega-3 PUFA content [32].

TNF-α, a potent proinflammatory cytokine mainly secreted by macrophages, can regulate inflammatory responses and immune cells. Previous research shows that TNF-α levels are elevated in rat models of DPN [33]. In patients with DPN, increased plasma TNF-α levels are linked to neuropathic pain. TNF-α causes COX-2 overexpression, causing inflammation and DPN. TNF-α activates miR-146a expression and inhibits TRAK1 and TRAF6 activation, resulting in the generation of proinflammatory cytokines, including IL-1 and IL-6. RFO reduced TNF-α and IL-6 levels, as seen in the results. Based on this observation, RFO may act via the physiological machinery [40].

CONCLUSION

RFO significantly improves the behavioral index in a rat model of DPN, probably via a mechanism that interferes with oxidative and inflammatory pathways. This study’s results highlight an avenue for the development of potential alternatives for combating DPN.

Funding Statement

FUNDING This research was supported by a grant from Research, Community Service, and Innovation at the Bandung Institute of Technology, Indonesia (SF.PPMI-1-19-2023).

Footnotes

ETHICAL APPROVAL

This research was approved by the Animal Ethics Committee of Institut Teknologi Bandung (certificate no. 08/KEPHP-ITB/3-2022, 25th March 2022).

CONFLICTS OF INTEREST

The authors declare that they have no conflicts of interest.

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