Skeletal Muscle Ischemia-reperfusion Research Articles

Possible protective effect of quercetin on lung injury induced by skeletal muscle ischemia reperfusion (IR) injury of adult male albino rats: Histological and biochemical study.

When a lower limb is injured, the most delicate organs that are at risk of harm are the lungs. Among the flavonoids, quercetin is a significant component that is found in apples and onions in the highest proportions. Numerous biological actions, including as anti-inflammatory, antioxidant, and anti-cancer properties, have been linked to quercetin. To investigate the impact of quercetin on lung injury induced by skeletal muscle ischemia reperfusion (IR) injury. Three equal groups of twenty-four adult rats were used: control, Ischemia-reperfusion (IR) group and IR group treated with quercetin. Rats in (IR) group were exposed to ischemia by ligation of femoral artery for 2h thenafter removal of the clamp, reperfusion was estabilished for another 24h. IR group treated with quercetin, rats were underwent hind limb IR as described in group II then were given quercetin that was administered at a dose of 20mg/kg intraperitoneally. Measurement of cytokinesin serum, MDAin tissue homogenate and VEGF in serumand tissue homogrnate in addition to mRNA expression level and detection of protein levelof bothsirtuin-1(SIRT1) and NF-κB were assessed at the end of experiment. Histological and immunohistochemical assessment of the lungs were also carried. IR group showed notable rise of inflammatory cytokines such as IL-1β, IL-6 and TNF-α in addition to high level of VEGF and MDA in IR group when compared to the IR group treated with quercetin. Also, gene expressionand protein level of SIRT1 were reduced while NF-κB mRNA expressionand protein level weresignificantly upregulated in IR group compared to IR group treated with quercetin. Histologically, IR group indicated marked histological alterations of lung tissue. Also, IR showed strong brownish cytoplasmic immunostaining for iNOS and abundance of Ki67-positive cells. These alterations were significantly reversed in IR group treated with quercetin. Biochemical and immunohistochemical findings of this study demonstrate that quercetin administration have protective effects against lung injury induced by lower limb IR.

Targeting CD36 With EP 80317 Reduces Remote Inflammatory Response to Hind Limb Ischemia-Reperfusion in Mice.

Reperfusion of ischemic skeletal muscle triggers oxidative stress and an immediate inflammatory reaction, leading to damage of distant organs such as the lungs. The inflammatory process implicates numerous mediators, including cytokines, chemokines, and arachidonic acid metabolites. In the orchestration of the inflammatory cascade, a critical role is played by the cluster of differentiation-36 receptor (CD36), a scavenger receptor class B protein (SR-B2) which is expressed on macrophages and functions as a Toll-like receptor coreceptor. A mouse model of hind limb ischemia-reperfusion has been used to investigate the interplay between CD36 signaling and remote inflammation: leukocyte recruitment, regulation of the nucleotide-binding domain leucin-rich repeat and pyrin-containing receptor 3 (NLRP3) inflammasome, and release of nuclear factor-kappa B (NF-ĸB) and arachidonic acid metabolites. Levels of reactive oxygen species, inflammatory mediators, and gene expression were measured in blood and lung tissue samples collected from anesthetized mice on which unilateral hind limb ischemia was induced by rubber band constriction for 30 min followed by reperfusion for 3 h. The CD36 modulator EP 80317, a member of the growth hormone releasing peptide 6 family, was employed as a pharmacological agent to mitigate distant lung injury following skeletal limb ischemia-reperfusion. Targeting CD36 on monocytes/macrophages, EP 80317 abated pro-inflammatory signaling and transcriptional activity encompassing lipid and cytokine mediators. Targeting CD36 was shown to offer promise for curtailing tissue injury following hind limb ischemia-reperfusion.

Inhibition of CTR1 expression improves hypoxia/reoxygenation-induced myoblast injury by blocking cuproptosis

Skeletal muscle ischemia-reperfusion injury (IRI) is a common severe disease with a complex pathological process. This study found that copper chloride (CuCl2) inhibited cell viability in a concentration dependent manner, increased intracellular copper levels and downregulated copper transporter 1 (CTR1) expression. CTR1 upregulation promoted copper uptake by myoblasts and then enhanced cuproptosis, leading to a significant increase in the levels of dihydrolipoamide S-acetyltransferase (DLAT) oligomers, while a significant decrease in the levels of lipoylated (Lip)-dihydrolipoamide S-succinyltransferase (DLST) and Lip-DLAT, ultimately inhibiting cell viability and inducing cell injury. Inducing cuproptosis with elesclomol plus CuCl2 (ES + Cu) further confirmed that “ES + Cu” treatment significantly reduced the contents of adenosine triphosphate (ATP) and glutathione (GSH), decreased the activities of mitochondrial complex I and III, and increased the contents of lactate (LA), malondialdehyde (MDA), creatine kinase (CK) and lactate dehydrogenase (LDH); when tetrathiomolybdate (TTM) was added to inhibit cuproptosis, myoblast injury was recovered significantly. Meanwhile, hypoxia/reoxygenation (H/R) induced CTR1 expression, increased the levels of intracellular copper, DLAT oligomers, LA, MDA, CK and LDH, reduced the levels of Lip-DLST, Lip-DLAT, ATP and GSH, and weakened the activities of mitochondrial complex I and III; after knocking down CTR1 expression, the levels of intracellular copper and the activation of cuproptosis pathway were decreased, and cell viability, injury and inflammation levels were significantly improved. Therefore, cuproptosis can promote myoblast injury, while H/R enhances copper uptake by inducing CTR1 expression, thereby enhancing cuproptosis and inducing cell injury, indicating that cuproptosis is a new mechanism of H/R-induced myoblast injury.

Carbon Dot Nanozyme Ameliorating Ischemia-Reperfusion-Induced Muscle Injury by Antioxidation and Downregulating iNOS/COX-2 Pathway.

Skeletal muscle ischemia-reperfusion (IR) injury is a prevalent type of muscle injury caused by events, such as trauma, arterial embolism, and primary thrombosis. The development of an IR injury is associated with oxidative stress and an excessive inflammatory response. Nanozymes, which have exceptional free radical scavenging activities, have gained significant attention for treating oxidative stress. This study demonstrates that carbon dot (C-dot) nanozymes possess superoxide dismutase (SOD)-like activity and can act as free radical scavengers. The carbon dot nanozymes are presented to mitigate inflammation by downregulating the iNOS/COX-2 pathway and scavenging reactive oxygen-nitrogen species to reduce oxidative stress, thereby suppressing inflammation. In the IR injury of skeletal muscle mice, we demonstrate that C-dots can effectively reduce inflammatory cytokines and tissue edema in skeletal muscle following IR injury in the limb. These findings suggest that C-dots have potential as a therapeutic approach for IR injury of skeletal muscle with negligible systemic toxicity. This offers a promising strategy for clinical intervention.

2-Substituted (N)-Methanocarba A3 Adenosine Receptor Agonists: In Silico, In Vitro, and In Vivo Characterization.

2-Arylethynyl (N)-methanocarba adenosine 5'-methylamides are selective A3 adenosine receptor (AR) agonists containing a preestablished receptor-preferred pseudoribose conformation. Here, we compare analogues having bulky 2-substitution, either containing or lacking an ethynyl spacer between adenine and a cyclic group. 2-Aryl compounds 9-11, 13, 14, 19, 22, 23, 27, 29, 31, and 34, lacking a spacer, had human (h) A3AR K i values of 2-30 nM, and others displayed lower affinity. Mouse (m) A3AR affinity varied, with 2-arylethynyl having a higher affinity than 2-aryl analogues (7, 8 > 3c, 3d > 3b). However, 2-aryl-4'-truncated derivatives had greatly reduced hA3AR affinity, even containing affinity-enhancing N 6-dopamine-derived substituents. Molecular modeling, including molecular dynamics simulation, predicted stable poses in the canonical A3AR agonist binding site, but 2-aryl (ECL2 interactions) and 2-arylethynyl (TM2 interactions) substituents have different conformations and environments. In a hA3AR miniGαi recruitment assay, 31 (MRS8062) was (slightly) more potent compared to a β-arrestin2 recruitment assay, both in engineered HEK293T cells, and its maximal efficacy (E max) was much higher (165%) than reference agonist NECA's. Thus, in the 2-aryl series, A3AR affinity and selectivity were variable and generally reduced compared to the 2-arylethynyl series, with a greater dependence on the specific aryl group present. Selected compounds were studied in vivo in an ischemic model of peripheral artery disease (PAD). Rigidified 2-arylethynyl analogues 3a-3c were protective in this model of skeletal muscle ischemia-reperfusion injury/claudication, as previously shown only for moderately A3AR-selective ribosides or (N)-methanocarba derivatives. Thus, we have expanded the A3AR agonist SAR for (N)-methanocarba adenosines.

Benzoylaconine Protects Skeletal Muscle Against Ischemia-Reperfusion Injury Through Activation of IF1-Dependent AMPK/Nrf2 Axis.

Aconitum carmichaelii (Fuzi) has been conventionally used to cure a variety of ailments, such as pain, cold sensations, and numbness of limb muscles (Bi Zheng) in China. Our prior investigations identified Benzoylaconine (BAC) as a bioactive alkaloid derived from Aconitum carmichaelii, with other studies also demonstrating its significant pharmacological potential. This study aimed to explore the potential of BAC as a protective agent against skeletal muscle ischemia-reperfusion (I/R) injury and to elucidate the underlying mechanisms. In vivo models involved subjecting Sprague-Dawley rats to I/R through femoral artery ligation followed by reperfusion, while in vitro models utilized C2C12 cells subjected to hypoxia/reoxygenation (H/R). CCK-8 assay was used to assess cell viability. TUNEL staining and flow cytometric analysis were used to measure cell apoptosis. Biochemical assay was used to assess skeletal muscle injury and oxidative stress. Immunofluorescence and Western blot were performed to determine protein levels. BAC effectively protected muscle tissue from I/R injury, enhancing cell viability (p<0.01), elevating SOD levels (p<0.05), and reducing CK (p<0.01), LDH (p<0.01), ROS (p<0.01), MDA (p<0.01), and apoptosis-related molecules in vivo and in vitro (p<0.05, p<0.01). Mechanistically, BAC increased the expression of IF1, phosphorylated AMPK, facilitated the translocation of nuclear Nrf2, and induced the expression of HO-1 (p<0.01). Notably, AMPK inhibitor Compound C significantly hindered the ability of BAC to ameliorate H/R-induced cell injury (p<0.05), oxidative stress(p<0.01), and apoptosis (p<0.05), as well as promote Nrf2 nuclear translocation (p<0.01). Moreover, silencing of IF1 with siRNA abolished BAC-induced activation of AMPK/Nrf2 axis (p<0.01). Our study provides novel evidence supporting the potential of BAC as a myocyte-protective agent against I/R injury, and we establish a previously unknown mechanism involving the activation of the IF1-dependent AMPK/Nrf2 axis in mediating the protective effects of BAC.

Anti-oxidative and anti-inflammatory effects of Ginkgo biloba extract (EGb761) on hindlimb skeletal muscle ischemia-reperfusion injury in rats.

In posterior spine surgery, retractors exert pressure on paraspinal muscles, elevating intramuscular pressure and compromising blood flow, potentially causing muscle injury during ischemia-reperfusion. Ginkgo biloba extract (EGb 761), known for its antioxidant and free radical scavenging properties and its role in treating cerebrovascular diseases, is investigated for its protective effects against muscle ischemia-reperfusion injury invitro and invivo. Animals were randomly divided into the control group, receiving normal saline, and experimental groups, receiving varying doses of EGb761 (25/50/100/200 mg/kg). A 2-h hind limb tourniquet-induced ischemia was followed by reperfusion. Blood samples collected pre-ischemia and 24 h post-reperfusion, along with muscle tissue samples after 24 h, demonstrated that EGb761 at 1000 μg/mL effectively inhibited IL-6 and TNF-α secretion in RAW 264.7 cells without cytotoxicity. EGb761 significantly reduced nitric oxide (NO) and malondialdehyde (MDA) levels, myeloperoxidase (MPO) activity, and increased glutathione (GSH) levels compared to the control after 24 h. Muscle tissue sections revealed more severe damage in the control group, indicating EGb761's potential in mitigating inflammatory responses and oxidative stress during ischemia-reperfusion injury, effectively protecting against muscle damage.

In vitro modeling of skeletal muscle ischemia-reperfusion injury based on sphere differentiation culture from human pluripotent stem cells

Skeletal muscle ischemia-reperfusion (IR) injury poses significant challenges due to its local and systemic complications. Traditional studies relying on two-dimensional (2D) cell culture or animal models often fall short of faithfully replicating the human in vivo environment, thereby impeding the translational process from animal research to clinical applications. Three-dimensional (3D) constructs, such as skeletal muscle spheroids with enhanced cell-cell interactions from human pluripotent stem cells (hPSCs) offer a promising alternative by partially mimicking human physiological cellular environment in vivo processes. This study aims to establish an innovative in vitro model, human skeletal muscle spheroids based on sphere differentiation from hPSCs, to investigate human skeletal muscle developmental processes and IR mechanisms within a controlled laboratory setting. By eticulously recapitulating embryonic myogenesis through paraxial mesodermal differentiation of neuro-mesodermal progenitors, we successfully established 3D skeletal muscle spheroids that mirror the dynamic colonization observed during human skeletal muscle development. Co-culturing human skeletal muscle spheroids with spinal cord spheroids facilitated the formation of neuromuscular junctions, providing functional relevance to skeletal muscle spheroids. Furthermore, through oxygen-glucose deprivation/re-oxygenation treatment, 3D skeletal muscle spheroids provide insights into the molecular events and pathogenesis of IR injury. The findings presented in this study significantly contribute to our understanding of skeletal muscle development and offer a robust platform for in vitro studies on skeletal muscle IR injury, holding potential applications in drug testing, therapeutic development, and personalized medicine within the realm of skeletal muscle-related pathologies.

The Effect of "Proanthocyanidin" on Ischemia-Reperfusion Injury in Skeletal Muscles of Rats.

Background and Objectives: Lower limb skeletal muscle ischemia-reperfusion (IR) injury is associated with increased morbidity and mortality, and it is common in several clinical situations such as aortic aneurysms repairment, peripheral arterial surgery, vascular injury repairment, and shock. Although it is generally accepted that oxidative stress mediators have a significant role in IR injury, its precise mechanism is still unknown. Anecdotally, it is sustained not only by structural and functional changes in the organ it affects but also by damage to distant organs. The purpose of this report is to illustrate the effect of proanthocyanidin on IR injury. Materials and Methods: In our study, 18 male Wistar albino rats were used. The subjects were divided into three groups containing six mice each (control, C; ischemia-reperfusion, IR; ischemia-reperfusion and proanthocyanidin; IR-PRO). Intraperitoneal proanthocyanidin was given to the IR and proanthocyanidin groups 30 min before laparotomy, and 1 h ischemia led to these two groups. After one hour, reperfusion started. Muscle atrophy-hypertrophy, muscle degeneration-congestion, fragmentation-hyalinization, muscle oval-central nucleus ratio, leukocyte cell infiltration, catalase enzyme activity, and TBARS were all examined in lower-limb muscle samples after one hour of reperfusion. Results: When skeletal muscle samples were evaluated histopathologically, it was discovered that muscle atrophy-hypertrophy, muscle degeneration-congestion, fragmentation-hyalinization, and leukocyte cell infiltration with oval-central nucleus standardization were significantly higher in the IR group than in the C and IR-P groups. Oval-central nucleus standardization was significantly higher in the IR and IR-PRO groups than in the control group. TBARS levels were significantly higher in the IR group than in the control and IR-PRO groups, while catalase enzyme activity was found to be significantly lower in the IR group than in the control and IR-PRO groups. Conclusions: As a consequence of our research, we discovered that proanthocyanidins administered before IR have a protective impact on skeletal muscle in rats. Further research in this area is required.

UCHL1 Negatively Regulates Key Factors and Signals Involved in Skeletal Muscle Myogenesis

Ubiquitin C-terminal hydrolase L1 (UCHL1), a deubiquitinating enzyme that was originally discovered in neurons, is also expressed in skeletal muscle, but its functions remain to be fully understood. Skeletal muscle injury is prevalent in trauma and surgical procedures, and skeletal muscle ischemia-reperfusion (IR) injury is a common and significant public health problem. Myogenesis is a critical process involved in embryonic development, growth, and regeneration following skeletal muscle injury. In this study we report that UCHL1 negatively affects some key factors that are involved in myogenesis and mitophagy, both during muscle development as well as during the regeneration process following IR injury; we hypothesize that UCHL1 is a negative regulator of myogenesis. First, we observed that UCHL1 knockdown in C2C12 myoblasts resulted in increased differentiation and myotube formation. Furthermore, western blot showed that UCHL1 KD consistently upregulated myogenin and MyoD protein levels, key proteins involved in myogenesis, at multiple time points throughout differentiation of the myotubes. Consistent with this in vitro result, skeletal muscle specific knockout (smKO) of UCHL1 increased muscle fiber size in both 1- and 2-month-old mice (p=1.77x10−17, p=6.51x10−13). Additionally, 1- and 2- month-old UCHL1 smKO mice also exhibited increased protein levels of various myosin heavy chain isoforms. To induce IR injury, a tourniquet was placed on the hindlimb for 90 minutes and then removed, allowing for reperfusion for 3-12 days with the contralateral hindlimb serving as a control. Following skeletal muscle IR injury, myogenin and MyoD protein expression was upregulated in injured muscle from UCHL1 smKO mice. Functionally, UCHL1 smKO mice had increased muscle performance after injury compared to control mice when subjected to in situ contractile testing. Interestingly, several mitophagy markers including Parkin and DRP1 were upregulated in IR injured muscle from UCHL1 smKO mice compared to WT mice (p=0.0003, p=0.029). This data suggests that skeletal muscle UCHL1 may function as a negative regulator of myogenesis, both during growth, and repair following injury, possibly by decreasing mitochondrial activity. This work was funded by NIH grant R01HL147105. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Inflammatory Responses of the Remote Organs After Tourniquet-Induced Ischemia-Reperfusion in Mouse Hindlimb

Tourniquet use is the most effective tool for controlling life-threatening extremity hemorrhage before other treatments and for creating bloodless operating fields in surgical procedures. However, tourniquet-induced ischemia-reperfusion (IR) also causes skeletal muscle injury and associated secondary end-organ damage. Our previous studies have demonstrated that there are involvements of inflammatory cytokines in tourniquet-induced skeletal muscle IR injuries. The objective of this study is to investigate the inflammatory responses of end-organs after tourniquet-induced mouse hindlimb IR. We hypothesized that tourniquet use would result in an inflammatory response in the remote organs. The unilateral hindlimbs of mice were subjected to 3 hours of tourniquet application by placing a rubber band at the hip joint. Then the rubber bands were released for reperfusion over different periods of time (1, 3, 7, and 14 days). The mRNA levels of IL-1β and TNFα (two inflammatory cytokines) in the lungs, liver, kidneys, and heart were measured using real-time RT-PCR analysis. In the lungs, the levels of IL-1β and TNFα mRNA were significantly elevated at day 1 after tourniquet-induced IR, then decreased at days 3 and 7, and returned to a high level at day 14. In the liver, the levels of IL-1β and TNFα mRNA were increased at day 1 and remained at a high level until day 14. However, tourniquet-induced hindlimb IR did not change the levels of IL-1β or TNFα mRNA in the kidneys or heart during the whole period of tourniquet-induced IR. In conclusion, these results suggest that the inflammatory response to tourniquet-induced hindlimb IR is different among distant organs, which will help to develop therapeutic strategies for different end-organs after tourniquet-induced hindlimb IR. NIH/NIGMS 1R01GM145736 to Y-LL. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Evaluation of the effect of enriched hydrogen saline solution on distant organ (lung) damage in skeletal muscle ischemia reperfusion in rats.

Ischemia-reperfusion (IR) injury is a major concern that frequently occurs during vascular surgeries. Hydrogen-rich saline (HRS) solution exhibits antioxidant and anti-inflammatory properties. This study aimed to examine the effects of HRS applied before ischemia in the lungs of rats using a lower extremity IR model. After approval was obtained from the ethics committee, 18 male Wistar albino rats weighing 250-280 g were randomly divided into three groups: control (C), IR and IR-HRS. In the IR and IR-HRS groups, an atraumatic microvascular clamp was used to clamp the infrarenal abdominal aorta, and skeletal muscle ischemia was induced. After 120 min, the clamp was removed, and reperfusion was achieved for 120 min. In the IR-HRS group, HRS was administered intraperitoneally 30 min before the procedure. Lung tissue samples were examined under a light microscope and stained with hematoxylin-eosin (H&E). Malondialdehyde (MDA) levels, total sulfhydryl (SH) levels, and histopathological parameters were evaluated in the tissue samples. MDA and total SH levels were significantly higher in the IR group than in the control group (p < 0.0001 and p = 0.001, respectively). MDA and total SH levels were significantly lower in the IR-HRS group than in the IR group (p < 0.0001 and p = 0.013, respectively). A histopathological examination revealed that neutrophil infiltration/aggregation, alveolar wall thickness, and total lung injury score were significantly higher in the IR group than in the control group (p < 0.0001, p = 0.001, and p < 0.0001, respectively). Similarly, alveolar wall thickness and total lung injury scores were significantly higher in the IR-HRS group than in the control group (p = 0.009 and p = 0.004, respectively). A statistically significant decrease was observed in neutrophil infiltration/aggregation and total lung injury scores in the IR-HRS group compared to those in the IR group (p = 0.023 and p = 0.022, respectively). HRS at a dose of 20 mg/kg, administered intraperitoneally 30 min before ischemia in rats, reduced lipid peroxidation and oxidative stress, while also reducing IR damage in lung histopathology. We believe that HRS administered to rats prior to IR exerts a lung-protective effect.

Carbon Monoxide Alleviates Post-ischemia-reperfusion Skeletal Muscle Injury and Systemic Inflammation.

Restoration of blood flow in skeletal muscle after a prolonged period of ischemia induces muscular ischemia-reperfusion injury, leading to local injury/dysfunction in muscles followed by systemic inflammatory responses. However, preventive/curative agents for skeletal muscle ischemia injury are unavailable in clinics to date. Increasing evidence has validated that carbon monoxide (CO) prevents the progression of ischemia-reperfusion injury in various organs owing to its versatile bioactivity. Previously, we developed a bioinspired CO donor, CO-bound red blood cells (CO-RBC), which mimics the dynamics of RBC-associated CO in the body. In the present study, we have tested the therapeutic potential of CO-RBC in muscular injury/dysfunction and secondary systemic inflammation induced by skeletal muscle ischemia-reperfusion. The results indicate that CO-RBC rather than RBC alone suppressed elevation of plasma creatine phosphokinase, a marker of muscular injury, in rats subjected to both hind limbs ischemia-reperfusion. In addition, the results of the treadmill walking test revealed a significantly decreased muscular motor function in RBC-treated rats subjected to both hind limbs ischemia-reperfusion than that in healthy rats, however, CO-RBC treatment facilitated sustained muscular motor functions after hind limbs ischemia-reperfusion. Furthermore, CO-RBC rather than RBC suppressed the production of tumour necrosis factor (TNF)-α and interleukin (IL)-6, which were upregulated by muscular ischemia-reperfusion. Interestingly, CO-RBC treatment induced higher levels of IL-10 compared to saline or RBC treatments. Based on these findings, we suggest that CO-RBC exhibits a suppressive effect against skeletal muscle injury/dysfunction and systemic inflammatory responses after skeletal muscle ischemia-reperfusion.

Ultrasound-Mediated Destruction of P-Selectin-Targeted and Cannabinoid Type 2 Receptor Agonist-Loading Microbubbles Ameliorates Skeletal Muscle Ischemia-Reperfusion Injury in Rat

Current study aims to explore the ameliorative effects of ultrasound in combination with P-selectin-targeted/AM1241-loaded microbubbles (AM1241 MBs) in rats with skeletal muscle ischemia/reperfusion (I/R) injury. AM1241 MB was prepared via rotary evaporation and mechanical shaking. Histological lesions and cellular apoptosis in skeletal muscle were detected by H&amp;E staining and TUNEL staining, respectively. Changes in antioxidant capacity and inflammatory response in skeletal muscle tissue were analyzed by ELISA method. Changes of Nfr2/HO-1 pathway-related proteins were measured via the western blot. As a result, ultrasound-mediated destruction of AM1241 MBs significantly improved the degree of damage and edema in skeletal muscle tissue of rats with I/R injury. Inflammatory response and cellular apoptosis in skeletal muscle tissue were also significantly improved, while the antioxidant capacity was significantly enhanced. Western blot analysis exhibited that ultrasound in combination with AM1241 MBs treatment activated the Nrf2/HO-1 pathway. Nevertheless, selective knockdown of Nrf2 significantly reversed the beneficial effects of ultrasound combined with AM1241 MBs treatment on I/R injuries of the skeletal muscle. Ultrasound-mediated disruption of AM1241 MBs could effectively ameliorate the I/R injuries of skeletal muscle tissues in rats via activating the Nrf2/HO-1 pathway.

Necrostatin-1 prevents skeletal muscle ischemia reperfusion injury by regulating Bok-mediated apoptosis.

Receptor interacting serine/threonine kinase 1 (RIPK1) mediates apoptosis by regulating the classic proapoptotic effectors Bcl-2-associated X protein (Bax) and Bcl-2 homologous antagonist/killer (Bak). Although Bcl-2-related ovarian killer (Bok) is structurally similar to Bak and Bax, it is unclear whether it mediates apoptosis in skeletal muscle ischemia reperfusion (IR) injury. We hypothesized that by regulating Bok-mediated apoptosis, inhibiting RIPK1 with necrostatin-1 would reduce skeletal muscle IR injury. Rats were randomized into four groups: sham (SM), IR, IR treated with necrostatin-1 (NI), or vehicle dimethyl sulfoxide (DI). For the IR group, the right femoral artery was clamped for 4 hours and then reperfused for 4 hours, and for the NI and DI groups, necrostatin-1 (1.65 mg/kg) and the equal volume of dimethyl sulfoxide were intraperitoneally administered prior to IR induction. The structural damage of muscle tissue and protein expression of Bok, Bcl-2, and cleaved caspase-3 were investigated, and apoptotic cells were identified with terminal dUTP nick-end labeling (TUNEL) staining. In vitro, human skeletal muscle cells (HSMCs) were exposed to 6 hours of oxygen-glucose deprivation followed by normoxia for 6 hours to establish an oxygen-glucose deprivation/reoxygenation (OGD/R) model. To determine the role of Bok, cell viability, lactate dehydrogenase (LDH) release, and flow cytometry were examined to demonstrate the effects of necrostatin-1 and Bok knockdown on the OGD/R insult of HSMCs. Necrostatin-1 pretreatment markedly reduced IR-induced muscle damage and RIPK1, Bok, and cleaved caspase-3 expression, whereas upregualted Bcl-2 expression (p < 0.05). Furthermore, necrostatin-1 prevented mitochondrial damage and decreased TUNEL-positive muscle cells (p < 0.05). In vitro, HSMCs treated with necrostatin-1 showed reduced Bok expression, increased cell viability, and reduced LDH release in response to OGD/R (p < 0.05), and Bok knockdown significantly blunted the OGD/R insult in HSMCs. Necrostatin-1 prevents skeletal muscle from IR injury by regulating Bok-mediated apoptosis.

Beneficial Effects of Insulin on Ischemia Reperfusion Injury in Human Skeletal Muscle.

PurposeExaggerated leucocyte activity is a crucial step in the pathophysiology of skeletal muscle ischemia-reperfusion injury (IRI). We tested the hypothesis that insulin, via its’ anti-leukocyte activity, attenuates skeletal muscle IRI in humans.MethodsMaterials and This randomized, blinded, placebo-controlled trial was conducted in patients with skeletal muscle ischemia who required revascularization. Treatment protocols were similar among them except for the insulin group, which received an infusion of insulin at 2.5 U/h. The degree of endothelial adhesiveness; leukocyte activity and pro-inflammatory status via P-selectin, tumor necrosis factor (TNF)-alpha, and myeloperoxidase (MPO) levels in the venous effluent; and clinical outcomes were measured.ResultsTwenty-four consenting patients were randomized to the insulin or control group. There were no significant differences between the two groups except for the median serum insulin level, which was higher in the insulin group (P<0.01). No serious intervention-related adverse events were observed. P-selectin (55.04-99.86 pg/mL; P<0.001), MPO (110.8-160.6 pg/mL; P<0.001), and TNF-alpha (12.16-36.01 pg/mL; P<0.001) levels demonstrated a significant increase post-reperfusion in the ‘control’ group, reaching a peak value at 2 hours post-reperfusion. The increase in all three markers from baseline was significantly diminished in the insulin group at the two-hour (P-selectin, P=0.001; MPO, P=0.001; TNF-alpha, P=0.005) and four-hour (P-selectin, P=0.003; MPO, P=0.002; TNF-alpha, P=0.01) intervals. The differences in clinical outcomes between the insulin and control groups were not statistically significant.ConclusionIn clinical practice, insulin has the potential to attenuate the severity of skeletal muscle IRI inhibiting P-selectin, MPO, and TNF-alpha levels.

Resolution of Inflammation after Skeletal Muscle Ischemia-Reperfusion Injury: A Focus on the Lipid Mediators Lipoxins, Resolvins, Protectins and Maresins.

Skeletal muscle ischemia reperfusion is very frequent in humans and results not only in muscle destruction but also in multi-organ failure and death via systemic effects related to inflammation and oxidative stress. In addition to overabundance of pro-inflammatory stimuli, excessive and uncontrolled inflammation can also result from defects in resolution signaling. Importantly, the resolution of inflammation is an active process also based on specific lipid mediators including lipoxins, resolvins and maresins that orchestrate the potential return to tissue homeostasis. Thus, lipid mediators have received growing attention since they dampen deleterious effects related to ischemia–reperfusion. For instance, the treatment of skeletal muscles with resolvins prior to ischemia decreases polymorphonuclear leukocyte (PMN) infiltration. Additionally, remote alterations in lungs or kidneys are reduced when enhancing lipid mediators’ functions. Accordingly, lipoxins prevented oxidative-stress-mediated tissue injuries, macrophage polarization was modified and in mice lacking DRV2 receptors, ischemia/reperfusion resulted in excessive leukocyte accumulation. In this review, we first aimed to describe the inflammatory response during ischemia and reperfusion in skeletal muscle and then discuss recent discoveries in resolution pathways. We focused on the role of specialized pro-resolving mediators (SPMs) derived from polyunsaturated fatty acids (PUFAs) and their potential therapeutic applications.

RIP1 Regulates Mitochondrial Fission during Skeletal Muscle Ischemia Reperfusion Injury

Background Dynamin related protein-1 (Drp1)-mediated mitochondrial fission relates to ischemia reperfusion (IR) injury, and its association with necroptosis is implied. We hypothesized that receptor-interacting protein 1 (RIP1), a key kinase in necroptosis, acted as an upstream of Drp1-mediated mitochondrial fission during skeletal muscle IR. Methods Thirty rats were randomized into the SM, IR, NI, MI, and DI group (n = 6). The rats in the SM group were shamly operated, and those in the IR group were subjected to 4-hour ischemia of the right hindlimb that was followed by 4-hour reperfusion. Intraperitoneal administration of Nec-1 1 mg/kg, Mdivi-1 1.2 mg/kg and same volume of DMSO were given before ischemia in the NI, MI and DI groups, respectively. Upon reperfusion, the soleus muscles were harvested to determine morphological changes and the expression of RIP1, total Drp1 and p-Drp1 (Ser616). Moreover, the muscular oxidative stress indicators and plasma muscle damage biomarkers were detected. Results IR led to impaired histopathological structures and mitochondrial fragmentation in the soleus muscle tissue, accompanied with increased muscular oxidative stress and muscle injury biomarkers, which could be similarly alleviated by Mdivi-1 and Nec-1 (p < 0.05). RIP1 and p-Drp1 (Ser616) protein levels were significantly upregulated in the soleus muscle subjected to IR injury, this upregulation was attenuated in the NI group, and Mdivi-1 downregulated the protein expression of p-Drp1 (Ser616) but not of RIP1 (p < 0.05). Conclusion RIP1 functions as an upstream of Drp1-mediated mitochondrial fission in the execution of necroptosis during skeletal muscle IR.

Histological and immunohistochemical study of the effect of ozone versus erythropoietin on induced skeletal muscle ischemia-reperfusion injury in adult male rats

ABSTRACT Ischemia reperfusion (IR) injury of skeletal muscles is a serious problem because of its local and systemic complications. Previous studies reported that ozone and erythropoietin could alleviate IR effect on several organs. The current research is established to evaluate the possible protective role of ozone versus erythropoietin following IR injury of the gastrocnemius muscle. Fifty rats were equally divided into five groups: I control, II ischemia reperfusion (IR), III post-reperfusion ozone treated, IV post-reperfusion erythropoietin-treated, and V recovering post-reperfusion without treatment groups. The right femoral arteries of all rats were clamped for three hours to induce ischemia then clamps were released to allow reperfusion for two hours. Rats of group II were scarified immediately after reperfusion period. Rats of group III were injected with ozone just after reperfusion for 14 days. Animals of group IV were injected with erythropoietin just after reperfusion for 14 days. Rats of group V rats were kept for 2 weeks following reperfusion without treatment. Blood samples were obtained to estimate lactate dehydrogenase (LDH) and creatine kinase (CK) enzymes. Gastrocnemius muscle was processed for measurement of tissue malondialdehyde (MDA), as well as examination by light and electron microscopes. iNOS and PCNA immunohistochemistry and statistical analysis were applied. The current results indicated that both ozone and erythropoietin could be used as protective agents reducing the muscular damage induced by IR injury.

Reducing effects of ginkgo biloba (egb761) extract on skeletal muscle ischemia-reperfusion injury in rats

Aim: The restoration of circulation following a period of ischemia results in the release of the metabolites that accumulate in the ischemic tissue into circulation with untoward effects on the organs. Many studies have been conducted to date into the prevention of such ischemia-reperfusion injuries. Ginkgo Biloba extract is known to have antioxidant effects, although there is a lack of data on its effects on a skeletal muscle ischemia-reperfusion model. The present study investigates the use of Ginkgo biloba extract (egb761) for the reduction of experimentally-induced skeletal muscle ischemia-reperfusion injury. Material and Methods: A total of 32 Sprague-Dawley rats were divided into four groups as follows: Group 1 (n=8), control group; Group 2 (n=8), limb ischemia model; Group 3 (n=8), ischemia-reperfusion model; and Group 4 (n=8), Ginkgo biloba group. After the models were created in each group, soleus muscle, lung and liver tissues were removed. Tissue malondialdehyde (MDA) levels were measured biochemically to demonstrate lipid peroxidation. The percentage of viable cells in the soleus muscle was calculated through a histochemical examination. Results: The comparison of soleus muscle MDA levels revealed no statistically significant difference between Group 1 and Groups 2 and 3, whereas the levels were significantly lower in Group 1 than those in Group 4 (p&lt;0.05). Similarly, there was a significant reduction in Group 4 than in Group 3 (p&lt;0.05). The analysis of lung tissue MDA levels revealed no significant difference between Group 1 and Group 2, whereas MDA levels were significantly increased in Group 3 than in Group 1 (p&lt;0.05), while there was no difference between Group 1 and Group 4. The analysis of liver tissue MDA levels showed significantly increased levels in Groups 3 and 4 than in Group 1 (p&lt;0.05). The percentage of viable cells was significantly decreased in Groups 2 and 3 than in Group 1 (p&lt;0.05). There was a significant reduction in Group 4 than in the control group. Conclusion: The results of the present study suggest that Ginkgo biloba (EGb761) extract may be used to reduce the local and systemic effects occurring after the restoration of blood flow following acute arterial occlusion. There is, however, an apparent need for large-scale, prospective, randomized trials to demonstrate the efficacy of Ginkgo biloba treatment.