Neuroprotective Potential of Hygrophila auriculata Targeting Oxidative Stress-Mediated Deficits in Streptozotocin-Induced Sciatic Nerve Injury

Abstract Objective  Diabetic neuropathy, a microvascular complication of diabetes, affects 50% of individuals. Addressing this challenge is challenging due to its poorly understood origin and existing therapeutic approaches. This study used a methanolic extract from Hygrophila auriculata (MEHA) to treat oxidative stress-induced sciatic nerve injury in diabetic rats. Materials and Methods  A study was conducted to assess the nociceptive reflex after a single streptozotocin (STZ) (45 mg/kg intraperitoneal.) injection. The rats were divided into six groups ( n  = 6 rats per group). Group I nondiabetic (ND) rats received oral gavage of 1% carboxymethyl cellulose (CMC). The diabetic rats in groups II to VI were given 1% CMC, 100, 200, and 400 mg/kg of MEHA, and 180 mg/kg of metformin (MET). The freshly prepared 1% (w/v) CMC suspension of both MEHA and MET was administered over a 4-week period, commencing from the 28th day through the 56th day post-STZ injection. The impact of STZ-induced sciatic nerve injury was analyzed through the estimation of serum glucose and glycohemoglobin levels, paw withdrawal and tail-flick latencies, oxidative stress markers, and neural histoarchitecture. Results  Diabetic (STZ) control group II showed significantly altered serum glucose and glycohemoglobin levels, a reduced paw withdrawal threshold, and reduced paw withdrawal and tail-flick latencies in contrast to ND group I. Furthermore, increased oxidative stress in the sciatic nerve correlates with a reduced nociceptive threshold and disrupted neural histoarchitecture in diabetic rats. These behavioral, biochemical, and molecular changes were markedly and dose-dependently reduced by MEHA and MET treatments. Conclusion  The antioxidant efficacy of MEHA modulated oxidative stress in STZ-sensitized diabetic rats and corrected neuropathic pain by attenuating hyperglycemia.


Introduction
Diabetes mellitus is a global health concern, with projections of a 366 million global diabetic population by 2030. 1 Prolonged hyperglycemia causes structural and functional abnormalities in the peripheral and central nervous systems, leading to diabetic neuropathy. 2 This condition is characterized by symptoms such as algesia, neuronal atrophy, diminished nerve conduction velocity, and sensory deficits. 3,4The etiology of diabetic neuropathy is multifaceted, involving pathways like heightened glucose flux, hexosamine shunt activity, and oxidative stress. 5,6The prevalence of diabetic neuropathy can reach 7.0% despite intensive insulin therapy, and under standard care, it can escalate to 16.1%. 7Current pain management approaches include tricyclic antidepressants, anticonvulsants, and opioids, but these offer only partial relief and are constrained by potential side effects. 8ygrophila auriculata, also known as "Kokilaksha" in Ayurveda, is a plant belonging to the Acanthaceae family with bioactive components like alkaloids, steroids, tannins, proteins, flavonoids, terpenoids, and phenolic compounds, which are used as antioxidants and antidiabetic agents. 9The ethanolic extracts from H. auriculata aerial parts show antidiabetic and antioxidant properties in streptozotocin (STZ)-induced diabetic rats. 10Hygrophila leaves extracts increase pain threshold in mice and inhibit abdominal constriction, indicating their analgesic activity through central and peripheral mechanisms. 113][14] These findings suggest that mitigating oxidative stress could potentially impede the progression of diabetic neuropathy.Leveraging the known bioactive constituents and neuroprotective capabilities of H. auriculata in ameliorating alloxan-induced diabetic neuropathy 15 and transient global cerebral ischemia, 16 this study explored the neuroprotective potential of H. auriculata, specifically targeting oxido-nitrosative stress in STZinduced diabetic neuropathy.

Plant Procurement, Authentication, and Extraction
The Botany Department at GES's HPT Arts and RYK Science Institution in Nashik, India, authenticated and certified H. auriculata (K.Schum) Heine, a plant native to the Matori-Makhamalabad region.The specimen sample was assigned voucher number HPTRYK/342/2021-22.Using Soxhlet's extractor, methanol was used to make an extract of coarsely powdered aerial parts of H. auriculata.Methanol is an effective solvent for extracting phenolic, flavonoid, alkaloid, and terpenoid compounds with high yields and high content.It is well known that the methanolic extract of H. auriculata (MEHA) aerial parts can help Wistar rats with alloxan-induced diabetic neuropathy. 15The extract was then concentrated using a rotary evaporator under reduced pressure.The freeze-dried MEHA was then analyzed to identify bioactive components. 17

Animals
Standardized conditions for young adult male Wistar albino rats (150-200 g, 6-12 weeks) and Swiss albino mice (20-25 g, 6-8 weeks) included keeping the temperature at 25°C AE 1°C, the relative humidity between 45 and 55%, and free access to food pellets and filtered water.An adaptation period of 7 to 10 days preceded the commencement of the experimental protocol.The ethical review committee at Bhupal Noble's University, Udaipur, Rajasthan, approved Proposal 24/BNCP/IAEC/2021 in accordance with the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) criteria for animal testing. 18

Drugs and Reagents
STZ (Product No. S0130) and metformin (MET; Product No. 317240) were purchased from Sigma (St. Louis, Missouri, United States).Span Diagnostic Chemicals, India, provided the glucose oxidase-peroxidase (GOD-POD) and hemoglobin A1c (HbA1c [glycated]) kits.The reagents required for conducting the antioxidant assays were prepared in-house prior to the estimation process.

Acute Oral Toxicity Study
The study followed Organization for Economic Cooperation and Development (OECD) Guideline 425 to investigate the acute oral toxicity of MEHA. 19Mice were given varying doses of a 1% carboxymethyl cellulose (CMC) suspension of MEHA, and toxicity or mortality signs were observed for up to 72 hours.The aim was to determine the median lethal dose (LD 50 ) and establish appropriate doses for further research.Neuroprotective Potential of Hygrophila auriculata in STZ-Induced Sciatic Nerve Injury Jadhav, Vaghela

Diabetes Induction and Assessment
A single intraperitoneal dose of 45 mg/kg of STZ in citrate buffer (pH 4.4, 0.1 M) was used to make male Wistar albino rats diabetic.Concurrently, age-matched control rats received an equivalent volume of citrate buffer.Blood collection via the tail vein took place 48 hours post-STZ administration, and serum glucose levels were colorimetrically assessed following cold centrifugation of blood samples at 2,500 rpm for 15 minutes at 4°C, utilizing the GOD-POD method.Rats exhibiting serum glucose values ÃÃ p < 0.01 and ÃÃÃ p < 0.001 for groups IV, V, and VI compared to group II.### p < 0.001 for group II compared to group I. Neuroprotective Potential of Hygrophila auriculata in STZ-Induced Sciatic Nerve Injury Jadhav, Vaghela exceeding 250 mg/dL were confirmed to be diabetic and subsequently included in the study. 20

Experimental Protocol
After baseline assessment of the nociceptive reflex using tail-flick test on the 28th day post-STZ injection, rats were randomly assigned to six groups (n ¼ 6 rats per group).Group I nondiabetic (ND) rats received oral gavage of 1% CMC.Group II to VI diabetic rats received oral gavage of 1% CMC, MEHA (100, 200, and 400 mg/kg), and MET (180 mg/kg), respectively.The freshly prepared 1% (w/v) CMC suspension of both MEHA and MET was administered over a 4-week period, commencing from the 28th day through the 56th day post-STZ injection. 20

Assessment of Diabetic Neuropathy
Body Weight, Food and Water Intake, and Urine Output At the conclusion of the 56th day, the body weight changes (g) of the animal were quantified.Daily pellet consumption served as the basis for calculating food intake (g), while calibrated water bottles facilitated the estimation of water intake (mL).Urine output (mL) was measured using a calibrated 250 mL container attached to metabolic cages. 21

Serum Glucose and Glycohemoglobin (HbA1c) Levels
On the 56th day, blood samples were obtained through tail venepuncture and collected in Eppendorf tubes.Following cold centrifugation at 3,200 rpm for 10 minutes at 4 °C, the serum was separated and stored at À20°C for subsequent analysis.Serum glucose and glycohemoglobin (HbA1c) levels were quantified using a biochemical analyzer with the GOD-POD method and the HbA1c (glycated) kit, respectively.

Behavioral Assessment
A behavioral assessment of hyperalgesia and allodynia was conducted at intervals on day 0, 28, 35, 42, 49, and 56.

Assessment of Thermal Hyperalgesia
Thermal hyperalgesia was evaluated through the Eddy's hot plate test 22 and the tail-flick test. 23The former, utilizing Eddy's hot plate analgesiometer, gauged impaired temperature perception by measuring the paw withdrawal latency of rats subjected to a 55 AE 0.5°C heated plate.A 15-second cutoff period was implemented to prevent tissue harm.The latter involved a tail-flick device (TFA-01) from Orchid Scientific, India, employing an intensity-controlled laser beam focused on the distal region of the tail to measure the tail-flick latency.

Assessment of Mechanical Hyperalgesia
Mechanical hyperalgesia was assessed using the Randall-Selitto paw pressure test, employing the Randall-Selitto algesimeter (UGO Basile, SRL Biological Research Apparatus, Italy) to apply linearly increasing pressure to the dorsum of the rat's hind paw until a vocalization threshold was reached, indicating the nociceptive withdrawal threshold. 24

Assessment of Mechanotactile Allodynia
Mechano-tactile allodynia was evaluated through Von-Frey hair (IITC, Woodland Hills, United States) test, utilizing calibrated bending forces of varying strengths applied with nylon microfilaments.The withdrawal threshold was determined as the slightest force required to elicit a withdrawal reflex in at least one of five trials, excluding locomotion-assisted voluntary movement as a withdrawal response.A significant decrease in withdrawal thresholds to von Frey microfilament application indicated mechanotactile allodynia. 25

Biochemical Estimations Oxidative Stress Markers
For the assessment of oxidative stress markers, rats were euthanized under deep diethyl ether anesthesia on the 56th day, and the left-sided sciatic nerves were surgically removed.The sciatic nerves were homogenized in 0.1 M icecold phosphate-buffered saline (pH 7.4) using a probe homogenizer (Polytron PT 2500E, Kinematica AG, Switzerland) and subsequently frozen at À20°C for the analysis of neural malondialdehyde (MDA), 26 reduced glutathione (GSH), 27 and superoxide dismutase (SOD) 28 levels.Neuroprotective Potential of Hygrophila auriculata in STZ-Induced Sciatic Nerve Injury Jadhav, Vaghela

Neural Histoarchitecture Investigation
For neural histoarchitecture investigation, the sciatic nerves were meticulously dissected, embedded in paraffin wax, and preserved in a neutral formalin buffer.Sections of 5 μm thickness were cut using a Leica Biosystems Microtomefrom Germanyand stained with Mayer's hematoxylin and eosin.Pathological alterations were examined under 400x light microscopy.

Statistical Analysis
The datasets were presented as mean AE standard error of mean.Two-way analysis of variance (ANOVA) analyzed behavioral assessments with a posthoc Bonferroni test, and biochemical estimations were analyzed by one-way ANOVA with a posthoc Tukey's multiple range test in Graph Pad Prism 9.0 software (San Diego, United States).Statistical significance was considered at p-value less than 0.05.

Acute Oral Toxicity Study
The study found no mortality or toxicity in the oral administration of MEHA suspension in a 1% CMC solution for 72 hours and recommended a maximum tolerated dose of 2,000 mg/kg with dosages of 100, 200, and 400 mg/kg for further investigation.
Effect of MEHA on Body Weight, Food, and Water Intake, and Urine Output The study found that diabetic group II had a lower body weight, more food and water intake, and more urine output after 4 weeks of intraperitoneal STZ administration.However, persistent administration of MEHA-treated groups IV and V and MET-treated group VI improved these factors compared to group II (►Table 1).

Effect of MEHA on Serum Glucose and Glycohemoglobin (HbA1c) Levels
STZ administration led to increased serum glucose and HbA1c levels in diabetic group II after 4 weeks, but groups IV, V, and VI were shown to improve these levels (►Table 2).Fig. 4 Effect of chronic treatment of methanolic extract from Hygrophila auriculata (MEHA) on mechanotactile allodynia in von Frey microfilament test.The data are expressed as mean AE standard error of mean (n ¼ 6) and analyzed by two-way analysis of variance with posthoc Bonferroni test.Ã p < 0.05 for group III, Ã p < 0.05 and ÃÃ p < 0.01 for group IV, and Ã p < 0.05, ÃÃ p < 0.01 and ÃÃÃ p < 0.001 for group V and VI, compared to group II.### p < 0.001 for group II compared to group I. MET, metformin; ND, nondiabetic; STZ, streptozotocin.

Effect of MEHA on STZ-Induced Thermal and Mechanical Hyperalgesia, and Mechanotactile Allodynia
The study found no significant difference in the nociceptive threshold of paw withdrawal and tail-flick reaction between ND group I and diabetic group II on day 0, but after 4 weeks of STZ injection, group II had a significant decrease in the threshold.2][3][4].

Effect of MEHA on Neural MDA, GSH, and SOD
The diabetic group II had significantly higher levels of neural MDA besides lower levels of GSH and SOD in the sciatic nerve compared to ND group I on the conclusion of 56th day.However, groups IV, V, and VI had significantly decreased MDA, and restored GSH and SOD levels compared to group II (►Figs.5-7).

Neural Histoarchitectural Investigation
The study found that ND group I (►Fig.8A) did not show signs of neural necrosis, congestion, neutrophil or macrophage infiltration, or abnormalities in sciatic neuroarchitecture.However, diabetic (STZ) control group II (►Fig.8B) showed significant nerve cell death, necrotizing neuronal edema, disintegration of myelin and axons, and infiltration of neutrophils and macrophages.Neural vacuolization and necrosis were also observed, with distended myelinated and unmyelinated nerve fibers.MEHA-treated groups III (►Fig.8C) and IV (►Fig.8D) showed a reduction in neutrophilic and macrophagic infiltration, edematous, necrotic, congested, and vacuolated nerve cells, distended myelinated and unmyelinated nerve fibers, and axonal degeneration.MEHAtreated group V (►Fig.8E) showed reduced counts of infiltrated neutrophils and macrophages, decreased necrotizing neural congestion, and improved nerve fiber edema.In METtreated group VI (►Fig.8F), a restored neural histoarchitecture was observed.

Discussion
Neuropathy, characterized by spontaneous pain, allodynia, and hyperalgesia, develops in about 50% of diabetic patients throughout the disease. 29A STZ-induced diabetic rat model is utilized to imitate chronic neuropathic pain with hyperalgesia and allodynia experienced by diabetic patients. 30STZ causes hyperglycemia and thereby altering the nociceptive response. 31,32n this study, rats that were sensitized to STZ had significantly higher levels of serum glucose and glycohemoglobin (HbA1c), lower body weight, and higher amounts of food, water, and urine intake than ND rats.Along with mechanotactile allodynia, diabetic animals also show signs of mechanical and thermal hyperalgesia in the Randall-Selitto, von Frey hair, Eddy's hot plate, and tail-flick tests.These findings indicate a considerable drop in the nociceptive threshold of diabetic rats.The outcomes are consistent with earlier findings. 33,34When diabetic rats were treated with the MEHA and MET, their serum glucose and HbA1c levels, food and water consumption, and urine output were all significantly   lower than those of the diabetic control group.The daily administration of MEHA and MET effectively reduced the dose-dependent mechanotactile allodynia and thermal and mechanical hyperalgesia produced by diabetes.
One of the main factors causing diabetic neuropathy is oxidative stress.Superoxide and hydroxyl radicals are produced due to the autoxidation of monosaccharides caused by chronic hyperglycemia.The generation of reactive oxygen species is recognized to be responsible for the transmission of pain. 35Nitric oxide and superoxide anions are essential mediators of glucose-induced oxidative damage.Superoxide anions have the capability to react with nitric oxide, leading to the production of peroxynitrite, which promptly induces protein nitrosylation, lipid peroxidation, DNA impairment, and eventual cell death. 36Superoxide anions are also thought to be responsible for several oxidative alterations, such as an increase in the activity of protein kinase C and aldose reductase, both of which are further linked to pain perception. 37The administration of MEHA and MET protected the diabetic animals' sciatic nerve by reducing noticeably elevated lipid peroxidation levels.
The first line of defense against free radicals includes GSH and SOD.9][40] The concomitant decrease in GSH and SOD activity increases the sciatic nerve's susceptibility to oxidative stress caused by hyperglycemia.Administration of MEHA and MET signifi-cantly restored GSH and SOD levels.These findings are consistent with previous investigations of H. auriculata, indicating its neuroprotective and antioxidant potential against transient global cerebral ischemia in rats. 16reliminary phytochemical analysis of MEHA showed the presence of flavonoids, terpenoids, and saponins, which supports its potential to scavenge free radicals and counteract STZ-augmented oxidative stress and the development of diabetic neuropathy.These findings are compatible with past studies demonstrating the antioxidant and antihyperglycemic efficacies of flavonoids, 41 terpenoids, 42 and saponins. 43hen the histoarchitecture of the sciatic nerve in the diabetic control group was studied, it showed a lot of necrotizing neural congestion, edematous myelinated and unmyelinated nerve fibers, and a lower mass of myelinated fibers because of neural necrosis and vacuolization.Instead, MEHA and MET protect neurons by lowering the amount of swollen, unmyelinated, and myelinated nerves as well as neural necrotizing congestion.This helps the sciatic nerve's structure get back to normal.

Conclusion
In conclusion, this study's findings suggest that MEHA has substantial antinociceptive, antioxidant, and neuroprotective potential in managing painful diabetic neuropathy caused by STZ.More research is necessary to understand precisely the mechanism underlying H. auriculata actions.

Fig. 1
Fig. 1 Effect of chronic treatment of methanolic extract from Hygrophila auriculata (MEHA) on thermal hyperalgesia in Eddy's hot plate test.The data are expressed as mean AE standard error of mean (SEM; n ¼ 6) and analyzed by two-way analysis of variance with posthoc Bonferroni test.Ã p < 0.05 for group III, Ã p < 0.05 and ÃÃ p < 0.01 for group IV, and Ã p < 0.05, ÃÃ p < 0.01, and ÃÃÃ p < 0.001 for group V and VI, compared to group II.### p < 0.001 for group II compared to group I. MET, metformin; ND, nondiabetic; STZ, streptozotocin.

Fig. 2
Fig. 2 Effect of chronic treatment of methanolic extract from Hygrophila auriculata (MEHA) on thermal hyperalgesia in tail-flick test.The data are expressed as mean AE standard error of mean (n ¼ 6) and analyzed by two-way analysis of variance with posthoc Bonferroni test.

Fig. 3
Fig.3Effect of chronic treatment of methanolic extract from Hygrophila auriculata (MEHA) on mechanical hyperalgesia in Randall-Selitto paw pressure test.The data are expressed as mean AE standard error of mean (n ¼ 6) and analyzed by two-way analysis of variance with posthoc Bonferroni test.ÃÃ p < 0.01 for group IV, and Ã p < 0.05, ÃÃ p < 0.01, and ÃÃÃ p < 0.001 for group V and VI compared to group II.### p < 0.001 for group II compared to group I. MET, metformin; ND, nondiabetic; STZ, streptozotocin.

Fig. 5
Fig.5Effect of methanolic extract from Hygrophila auriculata (MEHA) on diabetes-induced alteration in neural malondialdehyde (MDA) extent, the marker of lipid peroxidation (LPO).The data were expressed as mean AE standard error of mean (n ¼ 6) and analyzed by one-way analysis of variance with posthoc Tukey's multiple range test.ÃÃ p < 0.01 for group IV, and ÃÃÃ p < 0.001 for groups V and VI compared to group II.### p < 0.001 for group II compared to group I. MET, metformin; ND, nondiabetic; STZ, streptozotocin.

Fig. 6
Fig.6Effect of methanolic extract from Hygrophila auriculata (MEHA) on diabetes-induced alterations in glutathione (GSH) level.The data were expressed as mean AE standard error of mean (n ¼ 6) and analyzed by one-way analysis of variance with posthoc Tukey's multiple range test.ÃÃ p < 0.01 for group IV, and ÃÃÃ p < 0.001 for groups V and VI compared to group II.### p < 0.001 for group II compared to group I. MET, metformin; ND, nondiabetic; STZ, streptozotocin.

Fig. 7
Fig.7Effect of methanolic extract from Hygrophila auriculata (MEHA) on diabetes-induced alterations in superoxide dismutase (SOD) level.The data were expressed as mean AE standard error of mean (n ¼ 6) and analyzed by one-way analysis of variance with posthoc Tukey's multiple range test.ÃÃ p < 0.01 for group IV, and ÃÃÃ p < 0.001 for groups V and VI compared to group II.### p < 0.001 for group II compared to group I. MET, metformin; ND, nondiabetic; STZ, streptozotocin.

Table 1
Effect of MEHA on body weight, food, and water intake, urine output .01 and ÃÃÃ p < 0.001 for groups IV, V, and VI compared to group II.### p < 0.001 for group II compared to group I.Journal of Health and Allied Sciences NU © 2024.The Author(s).

Table 2
Effect of MEHA on serum glucose and HbA1c levels Abbreviations: ANOVA, analysis of variance; HbA1c, hemoglobin A1c; MEHA, methanolic extract from Hygrophila auriculata; MET, metformin; ND, nondiabetic; SEM, standard error of mean; STZ, streptozotocin.The data were expressed as mean AE SEM (n ¼ 6) and analyzed by one-way ANOVA with posthoc Tukey's multiple range test.