Hesperidin Mitigates Skeletal Muscle Ischemia–Reperfusion Injury via Nrf2, NF-κB, and NLRP3 Pathways

Summary

This study demonstrates that hesperidin pretreatment protects rat skeletal muscle against tourniquet-induced ischemia–reperfusion injury by enhancing Nrf2-mediated antioxidant defenses while attenuating NF-κB-associated inflammation and NLRP3/caspase-1-associated pyroptotic and apoptotic responses.

Abstract

The application of tourniquets during limb surgery can lead to ischemia–reperfusion (IR) injury, a pathological process characterized by excessive oxidative stress, increased inflammatory signaling, and progressive muscle fiber damage. Hesperidin, a bioactive flavonoid derived from citrus fruits, is known for its antioxidant and anti-inflammatory properties. This study evaluated the protective effects of hesperidin against lower-limb skeletal muscle IR injury and investigated the molecular pathways potentially associated with these effects. Male Wistar rats were allocated to four groups: control, hesperidin alone, IR (2 h of ischemia followed by 2 h of reperfusion), and IR + hesperidin (100 mg/kg administered 30 min before ischemia). Histological examination of the gastrocnemius muscle was performed, and plasma markers of muscle injury, including creatine kinase (CK), lactate dehydrogenase (LDH), and alkaline phosphatase (ALP), were measured. Oxidative stress and antioxidant parameters (MDA, SOD, CAT, and GPx), components of the Nrf2/HO-1/NQO1 pathway, inflammatory mediators (NF-κB, TNF-α, IL-6, and IL-1β), pyroptosis-associated markers (NLRP3 and caspase-1), and apoptosis-associated proteins (Bax and caspase-3) were also assessed. IR resulted in pronounced muscle fiber disruption, elevated plasma injury markers, increased lipid peroxidation, and reduced antioxidant capacity. IR also decreased Nrf2, HO-1, and NQO1 levels while increasing NF-κB, NLRP3, caspase-1, pro-inflammatory cytokines, and apoptosis-associated proteins. Hesperidin pretreatment markedly attenuated these changes by preserving muscle structural integrity, reducing plasma injury markers and lipid peroxidation, restoring antioxidant enzyme activity, increasing Nrf2/HO-1/NQO1-associated antioxidant responses, and reducing NF-κB-associated inflammatory, NLRP3/caspase-1-associated pyroptotic, and apoptotic responses. Overall, hesperidin pretreatment protected skeletal muscle against tourniquet-induced IR injury and was associated with enhanced Nrf2-mediated antioxidant defenses and attenuation of NF-κB-associated inflammation and NLRP3/caspase-1-associated responses. These findings support further investigation of hesperidin as a potential prophylactic intervention for reducing tourniquet-associated lower-limb IR injury.

Introduction

In orthopedic and other extremity surgeries, tourniquets are commonly applied to create a bloodless operative field, typically for 1–2 h and occasionally for up to 3 h, thereby improving surgical accuracy. Although effective, tourniquet application inevitably induces localized ischemia followed by reperfusion, a sequence that can result in ischemia–reperfusion injury (IRI). Studies across multiple organ systems have demonstrated that IRI is characterized by an inflammatory response initiated upon restoration of blood flow to previously oxygen-deprived tissue. Notably, tissue damage occurring during reperfusion may exceed that sustained during the ischemic period itself^1,2. Clinically, tourniquet use has been associated with persistent postoperative pain lasting 2–6 weeks^3,4, increased limb edema at follow-up intervals of 2 and 6 weeks^5,6, and delayed functional recovery, including reduced joint mobility and quadriceps weakness that may persist for several weeks⁶.

During the ischemic phase, mitochondrial oxidative phosphorylation is suppressed, forcing cells to shift toward anaerobic metabolism and disrupting electron transport. As ATP stores decline, Na⁺/K⁺-ATPase activity becomes insufficient, resulting in the intracellular accumulation of Ca²⁺, H⁺, and Na⁺. Excess intracellular calcium destabilizes cellular membranes, impairs mitochondrial integrity, and activates inflammatory and apoptotic signaling cascades⁷. Upon restoration of blood flow, a surge in reactive oxygen species (ROS), complement activation, and leukocyte recruitment further exacerbates tissue injury^8,9. ROS promote leukocyte activation, chemotaxis, cytokine gene transcription, and NF-κB-associated signaling, thereby contributing to cellular damage. Complement activation and the release of pro-inflammatory mediators, including TNF-α, IL-1, and IL-6, further impair microvascular perfusion¹⁰. Infiltrating leukocytes subsequently release ROS, proteases, and elastases into the interstitial space, increasing vascular permeability and promoting edema, thrombosis, and parenchymal cell death¹¹.

Hesperidin, a flavanone glycoside abundant in citrus fruits, is widely recognized for its antioxidant, anti-inflammatory, and anticancer properties. Recent studies have shown that hesperidin can attenuate oxidative stress and inflammatory responses associated with IRI^12,13. Hesperidin has also demonstrated protective effects against chemically induced liver injury and has been reported to inhibit hepatocarcinogenesis by modulating oxidative stress, inflammation, and abnormal cell proliferation¹⁴. Based on this evidence, the hypothesis of the present study was that hesperidin pretreatment would attenuate skeletal muscle ischemia–reperfusion injury in a rat tourniquet model by enhancing Nrf2/HO-1/NQO1-associated antioxidant defenses while attenuating NF-κB-associated inflammation, NLRP3/caspase-1-associated pyroptotic responses, and apoptotic signaling. Accordingly, the present study evaluated the protective effects of hesperidin against lower-limb skeletal muscle ischemia–reperfusion injury and investigated the molecular pathways potentially associated with its antioxidant and anti-inflammatory effects, with particular emphasis on the Nrf2/HO-1/NQO1, NF-κB, and NLRP3/caspase-1 pathways.

Protocol

All experimental procedures involving animals were approved by the Ethics Committee of Umm Al-Qura University, Saudi Arabia (approval code: HAPO-02-K-012-2026-05-3459). All procedures were conducted in accordance with the institutional guidelines for the care and use of laboratory animals.

Animals

Adult male Wistar albino rats, 3–4 months of age and weighing an average of 322 ± 12 g, were used in the study. The animals were housed 10 per type IV cage on wood-shaving bedding and maintained at 24°C under a 12 h light/dark cycle, with unrestricted access to standard laboratory chow and water. Before the experimental procedures, all animals were fasted overnight but were allowed unrestricted access to water.

Experimental groups and induction of ischemia–reperfusion

Animals were randomly assigned to four experimental groups, with 10 rats in each group. Randomization was performed using a computer-generated random-number sequence. Anesthesia was induced by intraperitoneal administration of xylazine (8 mg/kg) and ketamine (60 mg/kg). Except for the control and hesperidin-only groups, ischemia was induced by placing a tightly secured rubber tourniquet around the proximal region of the left hind limb to occlude the common femoral artery and its collateral branches. A rubber tourniquet (width 5 mm) was applied around the proximal left hind limb at the level of the greater trochanter. Occlusion was standardized by applying a consistent tension using a spring tension gauge set at 2.5 N, ensuring complete occlusion of the common femoral artery and its collateral branches. Successful induction of ischemia was confirmed by limb cyanosis and a noticeable decrease in surface temperature. After 2 h of ischemia, the tourniquet was removed, and reperfusion was allowed to proceed for an additional 2 h. Reperfusion was confirmed by restoration of limb warmth and normal pink coloration, the presence of a palpable pulse, and a reduction in edema, consistent with previously described criteria^15,16. Subsequent experimental analyses were performed by evaluators blinded to the treatment conditions.

The control group (C; n = 10) received 0.50 mL of 0.5% carboxymethylcellulose (CMC) orally by gavage, 30 min before the sham procedure. These animals were anesthetized and handled in the same manner as the other groups, but were not subjected to tourniquet application, ischemia, or reperfusion. The hesperidin group (Hes; n = 10) received a single oral dose of hesperidin (100 mg/kg) dissolved in 0.5% carboxymethylcellulose (CMC) in distilled water by oral gavage 30 min before the sham procedure, following the regimen reported by Park et al.¹⁷ in a liver ischemia–reperfusion model. These animals underwent anesthesia and identical handling procedures but were not subjected to ischemia or reperfusion. This group was included to assess the baseline physiological effects of hesperidin alone.

The ischemia–reperfusion group (IR; n = 10) underwent 2 h of tourniquet-induced ischemia followed by 2 h of reperfusion according to the protocol described by Yildirim and Albayrak¹⁸. The IR + hesperidin group (IR + Hes; n = 10) received a single oral dose of hesperidin (100 mg/kg) by oral gavage 30 min before tourniquet application and was subsequently subjected to the same 2 h ischemia and 2 h reperfusion protocol used for the IR group.

At the end of the reperfusion period, the animals were euthanized, and whole blood was collected into lithium-heparin tubes. Plasma was separated by centrifugation at 1,400 × g for 10 min and stored at −25 °C until biochemical analysis. The left gastrocnemius muscle was excised and divided into three portions. One portion was fixed in 10% neutral-buffered formalin for histological examination; a second portion was snap-frozen and stored at −80 °C for RNA extraction; and a third portion was homogenized and stored at −25 °C for biochemical analyses.

Biochemical analyses

Blood samples were centrifuged at 1,400 × g for 10 min, and the resulting aliquots were stored at −25°C until analysis. Alkaline phosphatase (ALP), creatine kinase (CK), and lactate dehydrogenase (LDH) concentrations were quantified using commercially available enzyme-linked immunosorbent assay (ELISA) kits with detection ranges of 1.25–80 ng/mL, 31.25–2,000 pg/mL, and 1.563–100 ng/mL, respectively. All assays were performed according to the manufacturer's instructions.

Histological examination

Following euthanasia, the left gastrocnemius muscle was dissected and fixed in 10% neutral buffered formalin for a minimum of 24 h. The tissues were processed using standard histological procedures, embedded in paraffin, sectioned at 3 µm, and stained with hematoxylin and eosin for microscopic examination. For semiquantitative assessment of muscle injury, five randomly selected fields per section were examined at 400× magnification by two independent pathologists blinded to the experimental groups. Muscle injury was graded using a 5-point scoring system based on the degree of muscle fiber degeneration¹⁹, as follows: grade 0 (normal architecture), grade 1 (mild: isolated fibers with loss of striations), grade 2 (moderate: multiple degenerating fibers with interstitial widening), grade 3 (severe: widespread necrosis with marked edema and inflammation), and grade 4 (extremely severe: extensive necrosis with massive interstitial hemorrhage).

ELISA analysis of muscle homogenates

Gastrocnemius muscle samples were homogenized in ice-cold radioimmunoprecipitation assay (RIPA) lysis buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, and 0.1% sodium dodecyl sulfate (SDS). The lysis buffer was supplemented with a protease inhibitor cocktail containing 1 mM phenylmethylsulfonyl fluoride, 1 µg/mL aprotinin, 1 µg/mL leupeptin, and 1 µg/mL pepstatin A. The homogenates were centrifuged at 12,000 × g for 15 min at 4 °C, and the resulting supernatants were collected and stored at −80 °C until analysis.

Commercially available ELISA kits were used to quantify markers of oxidative stress, antioxidant capacity, inflammatory responses, and apoptosis. The oxidative stress and antioxidant parameters assessed were superoxide dismutase (SOD; 0.16–10 ng/mL), catalase (CAT; 0.79–50 ng/mL), malondialdehyde (MDA; 31.25–2,000 ng/mL), and glutathione peroxidase (GPx; 15.63–1,000 pg/mL). Components associated with antioxidant signaling included nuclear factor erythroid 2-related factor 2 (Nrf2; 0.16–10 ng/mL), heme oxygenase-1 (HO-1; 0.16–10 ng/mL), and NAD(P)H quinone dehydrogenase 1 (NQO1; 46.875–3,000 pg/mL).

The inflammatory mediators assessed were nuclear factor kappa B (NF-κB; 0.16–10 ng/mL), tumor necrosis factor alpha (TNF-α; 15.63–1,000 pg/mL), interleukin-6 (IL-6; 7.82–500 pg/mL), and interleukin-1 beta (IL-1β; 15.63–1,000 pg/mL). Apoptosis- and pyroptosis-associated proteins included caspase-3 (0.313–20 ng/mL), Bax (0.16–10 ng/mL), and NLR family pyrin domain containing 3 (NLRP3; 0.312–20 ng/mL). All measurements were performed according to the manufacturer's instructions.

Quantitative PCR for caspase-1

Total RNA was isolated from frozen gastrocnemius muscle homogenates using a phenol-guanidine-based RNA isolation reagent followed by purification with a silica membrane-based RNA purification method. Complementary DNA (cDNA) was synthesized from 8 µL of RNA using a reverse transcription system. Quantitative polymerase chain reaction (qPCR) was subsequently performed using a real-time PCR system, with β-actin serving as the internal reference gene. The reaction mixture (20 µL total volume) consisted of 10 µL of 2× SYBR Green PCR Master Mix, 2 µL of cDNA template, 1 µL each of forward and reverse primers (10 µM), and 6 µL of RNase-free water. The cycling protocol included an initial denaturation at 95 °C for 10 min, followed by 40 cycles of denaturation at 95 °C for 15 s, annealing at 60 °C for 30 s, and extension at 72 °C for 30 s. A melt curve analysis was performed from 65 °C to 95 °C in 0.5 °C increments to verify amplification specificity. Each sample was run in duplicate, and no-template controls (NTCs) and no-reverse-transcription controls (NRTs) were included in each run. Amplification efficiency for each primer pair was determined from a standard curve (fourfold dilution series of pooled cDNA) and ranged from 90–105%. Relative caspase-1 expression was calculated using the 2^−ΔΔCT method described by Livak and Schmittgen²⁰. The primer sequences used for qPCR were as follows: caspase-1 forward, 5′-GACCGAGTGGTTCCCTCAAG-3′; caspase-1 reverse, 5′-GACGTGTACGAGTGGGTGTT-3′; β-actin forward, 5′-CCGAGATCTCACCGACTACCTCA-3′; and β-actin reverse, 5′-TCAAAGTCCAGAGCGACATAGCA-3′.

Statistical analysis

All statistical analyses were performed using statistical analysis software. The group size of 10 animals per experimental condition was determined prospectively using dedicated power-analysis software, with an effect size of 0.8, a significance level of α = 0.05, and a desired statistical power of 0.80.

Before parametric analyses were performed, normality and homogeneity of variance were evaluated using the Shapiro–Wilk and Levene's tests, respectively. Both tests yielded p > 0.05, supporting the use of parametric statistical procedures. Comparisons among the four experimental groups were therefore performed using one-way analysis of variance (ANOVA), followed by Tukey's multiple-comparison test for post hoc pairwise comparisons. A p-value < 0.05 was considered statistically significant. All quantitative data were expressed as mean ± standard deviation (SD).

Results

Improvement of ischemia–reperfusion-induced histological alterations by hesperidin

Histological examination of the gastrocnemius muscle revealed regularly arranged longitudinal muscle fibers in the control and hesperidin groups (Figure 1A–D). In contrast, the ischemia–reperfusion (IR) group exhibited marked muscle fiber degeneration and increased intercellular spacing (Figure 1E,F). C...

Discussion

Tourniquet-induced ischemia remains a standard technique in many surgical specialties, particularly orthopedics, to obtain a bloodless operative field and improve visualization. Despite its utility, this approach inevitably produces localized ischemic stress followed by reperfusion injury, which can adversely affect several postoperative outcomes²¹. Although advances in tourniquet design and evidence-based application strategies have reduced certain complications²², no clin...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
ALP ELISA Kit (1.25–80 ng/mL)	ELK Biotechnology, China	ELK5657	https://www.elkbiotech.com/product/ELK5657
Aprotinin	Sigma-Aldrich, USA	A6279	https://www.sigmaaldrich.com/US/en/product/sigma/a6279
Bax ELISA Kit (0.16–10 ng/mL)	Novus Biologicals, USA	NBP2-69938	https://www.novusbio.com/products/nbp2-69938
β-actin Forward Primer (5′-CCGAGATCTCACCGACTACCTCA-3′)	Integrated DNA Technologies, USA	Custom	https://www.idtdna.com
β-actin Reverse Primer (5′-TCAAAGTCCAGAGCGACATAGCA-3′)	Integrated DNA Technologies, USA	Custom	https://www.idtdna.com
Carboxymethylcellulose (CMC), 0.5%	Sigma-Aldrich, USA	C5678	https://www.sigmaaldrich.com/US/en/product/sigma/c5678
Caspase-1 Forward Primer (5′-GACCGAGTGGTTCCCTCAAG-3′)	Integrated DNA Technologies, USA	Custom	https://www.idtdna.com
Caspase-1 Reverse Primer (5′-GACGTGTACGAGTGGGTGTT-3′)	Integrated DNA Technologies, USA	Custom	https://www.idtdna.com
Caspase-3 ELISA Kit (0.313–20 ng/mL)	Wuhan Fine Biotech Co., Ltd., China	ER0143	https://www.finebiotech.com/product/ER0143
CAT ELISA Kit (0.79–50 ng/mL)	ELK Biotechnology, China	ELK5986	https://www.elkbiotech.com/product/ELK5986
Centrifuge	Eppendorf, Germany	5810 R	https://www.eppendorf.com/us-en/lab-equipment/centrifuges/5810-5810-r-centrifuges
CK ELISA Kit (31.25–2000 pg/mL)	Abcam, UK	ab285275	https://www.abcam.com/products/ab285275
Dimethyl sulfoxide (DMSO), 5%	Sigma-Aldrich, USA	D8418	https://www.sigmaaldrich.com/US/en/product/sigma/d8418
Eosin Y	Sigma-Aldrich, USA	318906	https://www.sigmaaldrich.com/US/en/product/aldrich/318906
Ethanol (100%, 96%, 70%)	Sigma-Aldrich, USA	32205	https://www.sigmaaldrich.com/US/en/product/aldrich/32205
Formalin, 10% neutral buffered	Sigma-Aldrich, USA	HT501128	https://www.sigmaaldrich.com/US/en/product/sigma/ht501128
G*Power 3.1	Heinrich Heine University Düsseldorf, Germany	N/A	https://www.psychologie.hhu.de/arbeitsgruppen/allgemeine-psychologie-und-arbeitspsychologie/gpower
GPx ELISA Kit (15.63–1000 pg/mL)	ELK Biotechnology, China	ELK2222	https://www.elkbiotech.com/product/ELK2222
GraphPad Prism 8.0	GraphPad Software, USA	N/A	https://www.graphpad.com
Hematoxylin	Sigma-Aldrich, USA	HHS128	https://www.sigmaaldrich.com/US/en/product/sigma/hhs128
Hesperidin	Sigma-Aldrich, USA	H5254	https://www.sigmaaldrich.com/US/en/product/sigma/h5254
HO-1 ELISA Kit (0.16–10 ng/mL)	ELK Biotechnology, China	ELK1477	https://www.elkbiotech.com/product/ELK1477
IL-1β ELISA Kit (15.63–1000 pg/mL)	ELK Biotechnology, China	ELK1272	https://www.elkbiotech.com/product/ELK1272
IL-6 ELISA Kit (7.82–500 pg/mL)	ELK Biotechnology, China	ELK1158	https://www.elkbiotech.com/product/ELK1158
Ketamine	Sigma-Aldrich, USA	K2753	https://www.sigmaaldrich.com/US/en/product/sigma/k2753
LDH ELISA Kit (1.563–100 ng/mL)	Wuhan Fine Biotech Co., Ltd., China	ER0647	https://www.finebiotech.com/product/ER0647
Leupeptin	Sigma-Aldrich, USA	L2884	https://www.sigmaaldrich.com/US/en/product/sigma/l2884
Light microscope	Olympus, Japan	BX53	https://www.olympus-lifescience.com/en/microscopes/bx53/
Lithium heparin tubes	BD Vacutainer, USA	367886	https://www.bd.com/en-us/products/blood-collection
MDA ELISA Kit (31.25–2000 ng/mL)	ELK Biotechnology, China	ELK10920	https://www.elkbiotech.com/product/ELK10920
Microtome	Leica Biosystems, Germany	RM2255	https://www.leicabiosystems.com/us/histology-equipment/microtomes/
Microsoft Excel	Microsoft Corporation, USA	N/A	https://www.microsoft.com/en-us/microsoft-365/excel
NF-κB ELISA Kit (0.16–10 ng/mL)	ELK Biotechnology, China	ELK1693	https://www.elkbiotech.com/product/ELK1693
NLRP3 ELISA Kit (0.312–20 ng/mL)	Novus Biologicals, USA	NBP3-42294	https://www.novusbio.com/products/nbp3-42294
NQO1 ELISA Kit (46.875–3000 pg/mL)	MyBioSource, USA	MBS7606601	https://www.mybiosource.com/product/MBS7606601
Nrf2 ELISA Kit (0.16–10 ng/mL)	ELK Biotechnology, China	ELK8043	https://www.elkbiotech.com/product/ELK8043
Paraffin wax	Sigma-Aldrich, USA	327204	https://www.sigmaaldrich.com/US/en/product/aldrich/327204
Pepstatin A	Sigma-Aldrich, USA	P5318	https://www.sigmaaldrich.com/US/en/product/sigma/p5318
Protease inhibitor cocktail (PMSF)	Sigma-Aldrich, USA	P7626	https://www.sigmaaldrich.com/US/en/product/sigma/p7626
QIAzol Lysis Reagent	Qiagen, Germany	79306	https://www.qiagen.com/us/products/discovery-and-translational-research/dna-rna-purification/rna-purification/total-rna/qiazol-lysis-reagent
QuantiTect Reverse Transcription Kit	Qiagen, Germany	205311	https://www.qiagen.com
RIPA lysis buffer	Thermo Fisher Scientific, USA	89900	https://www.thermofisher.com/order/catalog/product/89900
RNeasy Mini Kit	Qiagen, Germany	74104	https://www.qiagen.com/us/products/discovery-and-translational-research/dna-rna-purification/rna-purification/total-rna/rneasy-mini-kit
Rotor-Gene Q	Qiagen, Germany	9001571	https://www.qiagen.com
Sodium chloride, 0.9%	Sigma-Aldrich, USA	S7653	https://www.sigmaaldrich.com/US/en/product/sigma/s7653
SOD ELISA Kit (0.16–10 ng/mL)	ELK Biotechnology, China	ELK8178	https://www.elkbiotech.com/product/ELK8178
TissueLyser II	Qiagen, Germany	85300	https://www.qiagen.com
TNF-α ELISA Kit (15.63–1000 pg/mL)	ELK Biotechnology, China	ELK1396	https://www.elkbiotech.com/product/ELK1396
Type IV cage	Bioscape, Germany	N/A	https://www.bioscape.de
Xylene	Sigma-Aldrich, USA	214736	https://www.sigmaaldrich.com/US/en/product/aldrich/214736
Xylazine	Sigma-Aldrich, USA	X1251	https://www.sigmaaldrich.com/US/en/product/sigma/x1251