Background
Limb ischemia/reperfusion (I/R) injury is a life-threatening syndrome that is often caused by trauma, primary thrombosis, arterial embolism, limb or flap reattachment, artery transplantation, prolonged tourniquet application, and abdominal compartment syndrome [
1]. Mild I/R injury can lead to skeletal muscle fibrosis, persistent damage and necrosis, which can affect limb function; in severe cases, patients may require amputation. Patients with severe limb I/R injury can develop multiple organ dysfunction syndromes that threaten their lives [
2].
Though the mechanisms of skeletal muscle I/R injury are diverse, a growing body of evidence has proved that oxidative stress and inflammatory responses have important roles in the progress of skeletal muscle I/R injury [
3,
4]. Overproduction of reactive oxygen species (ROS) has been observed in I/R-injured organs. Excessive production of ROS can initiate lipid peroxidation, inactivate antioxidative stress-related proteins, and aggravate I/R injury [
4]. Thus, injury can stimulate lipid peroxidation of biological membranes. Notably, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity and the levels of F
2-isoprostanes and malondialdehyde (MDA) are often used as indicators of oxidative stress [
3‐
6]. Various defense mechanisms are induced by ROS-induced injury, and the levels of antioxidants such as catalase (CAT), glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) are often used as indicators of oxidative stress [
3,
7]. In addition, ROS-induced injury can promote the formation and release of many inflammatory cytokines, such as tumor necrosis factor alpha (TNF-α), interleukin 6 (IL-6) and interleukin 1 beta (IL-1β) [
4‐
9]. Under oxidative stress or inflammatory conditions, the nuclear factor kappa-B (NF-κB) and mitogen-activated protein kinase (MAPK) pathways can be activated [
10]. Many I/R injury models have been reported to elicit activation of the p38, JNK, ERK1/2 and p65 pathways [
8,
9]. Specifically, it has been reported that activation of p38 and ERK1/2 is involved in renal I/R injury [
11]. Furthermore, blockade of P38 α and β may protect the lungs from acute I/R injury by reducing the expression of IL-1β [
12]. Moreover, 6-gingerol exerts protective effects against I/R-induced intestinal mucosal injury by inhibiting the formation of ROS and the activation of p38 and NF-κB [
13]. It has also been reported that gypenoside protects cardiomyocytes against I/R injury through inhibition of MAPK pathway signaling and NF-κB p65 translocation into nuclei [
14].
Based on this information, inhibition of the production and release of ROS and inflammatory cytokines is considered one of the strategies for addressing limb I/R injury.
Ginkgo biloba can alleviate injury associated with stroke or myocardial infarction through its powerful antioxidant and anti-inflammatory properties [
15,
16]. Bilobalide is one of the major pharmacological components of
Ginkgo biloba. It has been reported that bilobalide can protect neurons and endothelial cells from oxidative and inflammatory stress injury [
15,
16]. However, at present, little is known about whether bilobalide can protect skeletal muscle from I/R injury. In this study, we hypothesized that bilobalide could alleviate skeletal muscle damage caused by I/R injury by relieving oxidative stress and systemic inflammatory responses. To test this hypothesis, we examined the effects of bilobalide on skeletal muscle using methods including hematoxylin and eosin (H&E) staining and assessment of the wet weight/dry weight ratio of muscle tissue. Then, we measured lipid peroxidation, antioxidant activity, and inflammatory cytokine levels using test kits. Finally, we examined the activation of members of the MAPK and p65 NF- κB pathways via Western blotting.
Discussion
In this study, we initially found that bilobalide partially restored the morphology of the gastrocnemius after I/R injury and reduced tissue edema in skeletal muscle. Furthermore, we demonstrated that bilobalide could relieve oxidative stress and systemic inflammatory responses caused by I/R injury. In addition, our findings suggested that bilobalide suppressed the activation of the p38, ERK1/2, JNK, and NF- κB p65 pathways in the I/R injured skeletal muscle, suggesting that the protective effects of bilobalide might be partly mediated by suppressing the activation of MAPK/NF- κB pathway.
Previous studies have indicated that skeletal muscle I/R injury is an important clinical problem that should not be ignored [
1‐
3]. At present, there are different ways to treat limb I/R injury, including physical therapy and chemical therapy. It has been reported that ischemic preconditioning [
9], ischemic postconditioning [
21], controlled reperfusion [
22], hypothermia [
23], light-emitting diode therapy [
24] and some other physical therapies can relieve skeletal I/R injury [
25]. In addition, several agents, such as dexamethasone [
10], curcumin [
26], salvianolic acid [
8], silibinin [
27], simvastatin [
28], cyclosporine A [
29], hydrogen-rich saline [
30] and lipoxin A4 [
4], have been shown to be effective in attenuating skeletal I/R injury. In cases of traumatic injuries in which I/R is not predictable and early intervention is desired, such strategies are not as relevant. This is especially true for severe extremity injuries. Lifesaving surgical procedures are performed in these cases with the objective of stopping hemorrhage for the preservation of vital organ function; the extremities are not the primary focus. Moreover, even though the strategies and agents mentioned above have shown some benefits in the laboratory, none have been established to have any clinical benefits. Hence, there is still a need to discover novel substances with antioxidant and anti-inflammatory capacities that can be utilized for the treatment of skeletal I/R injury.
Bilobalide can be extracted from
Ginkgo biloba leaves, which are widely used in traditional Chinese medicine. EGb 761, a standardized extract from
Ginkgo biloba leaves, has various pharmacological functions and has been used worldwide. Bilobalide accounts for approximately 3% of EGb 761 [
31]. Otani et al. reported that bilobalide can prevent cytotoxic brain edema caused by triethyltin [
32]. Others have shown that bilobalide can protect against ischemia or I/R injury-induced edema formation and that it is a potential antiedema drug [
33]. In the present study, we found that bilobalide attenuated I/R injury in a skeletal I/R injury model. Histological findings indicated that there were fewer histopathologic changes in bilobalide-treated animals subjected to I/R than in untreated animals subjected to I/R. Moreover, we demonstrated that bilobalide significantly reduced edema in I/R-injured skeletal muscle.
Oxidative stress is considered to play important roles in the process of limb I/R injury [
8]. Our data showed that bilobalide significantly ameliorated skeletal muscle oxidative stress induced by I/R injury by decreasing NADPH oxidase activity and F
2-isoprostane and MDA levels and by enhancing CAT, GSH-Px and SOD activity. Meanwhile, antioxidative effects of bilobalide have been reported in the context of neuronal degeneration in Alzheimer’s disease [
34]. Parimoo et al. showed that bilobalide can be used as a promising hepatoprotectant due to its antioxidative effects [
35]. Lu et al. demonstrated that bilobalide protects melanocytes from H
2O
2-induced oxidative damage by increasing antioxidant expression [
36]. These results indicate that the antioxidative effects of bilobalide may be important in cases of I/R-injured skeletal muscle.
Inflammatory responses are generally considered to be major causes of skeletal muscle I/R injury [
4]. Puntel and colleagues have illustrated that MPO activity is an indicator of neutrophil infiltration in I/R injury in skeletal muscle [
37]. Infiltrating neutrophils can also release a variety of inflammatory cytokines, such as TNF-α, IL-1β and IL-6, which play roles in inflammatory reactions [
38]. Several studies have demonstrated that bilobalide acts as an anti-inflammatory mediator in cerebral I/R injury and many other inflammatory response-related diseases [
39‐
41]. In this study, we found that bilobalide could relieve systemic inflammatory responses in the skeletal muscle caused by I/R injury.
Under oxidative stress or inflammatory response conditions, the MAPK and NF- κB pathways can be activated [
10]. Recent studies have shown that in some I/R injury models, including the skeletal I/R injury model, the p38, ERK1/2, JNK and p65 pathways are active [
4,
8]. In the present study, we found that inhibition of these four specific cellular pathways with their specific inhibitors (P38 MAPK: SB203580; ERK: ravoxertinib; JNK: SP600125; P65 NF-κB: SC75741) alleviated apoptosis and local oxidative stress and decreased inflammatory cytokine levels in IR-injured skeletal muscle (Supplementary Figure
2). Additionally, we demonstrated that the protein levels of phosphorylated p38, phosphorylated ERK1/2, phosphorylated JNK, and p65 was significantly increased in skeletal muscle after I/R injury and that bilobalide partially inhibited p38, ERK1/2, JNK, and p65 activation, suggesting that activation of the corresponding signaling pathways might mediate some of the protective effects of bilobalide in this model. These observations are consistent with the findings of previous studies. Priyanka and colleagues showed that bilobalide abates inflammation induced by hypoxia in adipocytes via reducing activation of the NF- κB and JNK signaling pathways [
40]. Zhou and colleagues showed that bilobalide inhibits the secretion of inflammatory factors in BV2 microglia in response to oxygen/glucose deprivation and reoxygenation by controlling the activation of TLR/MyD88/NF- κB pathways [
41]. In other studies, however, bilobalide has failed to inhibit increases in p-ERK1/2 expression. For example, Jiang et al. proved that bilobalide could play an important role in neuroprotection against cerebral I/R injury through a mechanism related to downregulation of JNK and p38 activation but not ERK1/2 activation [
42]. Previous studies have also revealed that bilobalide cannot regulate the expression of p-ERK1/2 in SH-SY5Y cells [
43]. These variations in results may be associated with the different species and sources of cells or the different animal and disease models used.
There were some limitations to our study. In this study, we treated the rats with bilobalide after 3 h of ischemia and then reperfused them for 24 h. However, in some clinical cases (for example, during reattachment of a limb or flap), there is not enough time for bilobalide treatment between ischemic injury and reperfusion. We did not test whether administering bilobalide treatment after I/R injury had the same protective effects as administering it between ischemia and reperfusion. In addition, we observed involvement of the p38, ERK1/2, JNK and p65 pathways in the protective effects of bilobalide on skeletal muscle. However, the specific effects of these pathways in limb I/R injury remain to be investigated.
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