2022 Volume 45 Issue 9 Pages 1269-1275
Edaravone, an antioxidant protective agent, has anti-cerebral ischemic reperfusion injury (CIRI) effects, but its anti-CIRI mechanism is unclear. The aim of this study is to investigate the anti-CIRI mechanism of edaravone based on the nuclear factor-E2-related factor 2 (Nrf2)/ferroportin (FPN) pathway that regulates ferroptosis-mediated cerebral ischemia–reperfusion injury. We evaluated the brain injury by constructing a middle cerebral artery occlusion and reperfusion (MCAO/R) model in rats. The results showed that cerebral infarct volume and neurological impairment scores were increased in cerebral ischemia–reperfusion rats, with impaired sensorimotor ability; furthermore, brain tissue glutathione (GSH) content was decreased, Fe2+, malondialdehyde (MDA) and lipide peroxide (LPO) content were increased, and the expression level of glutathione peroxidase 4 (GPX4), a key protein of ferroptosis, was also decreased. Meanwhile, the Nrf2 expression level was increased and the FPN expression level was decreased after cerebral ischemia–reperfusion, while the levels of interleukin (IL)-6, IL-1β, tumor necrosis factor (TNF)-α, and myeloperoxidase (MPO) were increased. However, edaravone exhibited a protective effect on cerebral infarct and neurological and sensorimotor function in relevant tests. In addition, we also found that edaravone decreased the contents of Fe2+, MDA, and LPO in the brain tissue of MCAO/R rats and increased GSH content to inhibit ferroptosis. Furthermore, Western blot showed that after treatment with edaravone, the expression of Nrf2, GPX4, and FPN was up-regulated, the nuclear location of Nrf2 was increased, and the levels of inflammation-related indicators IL-6, IL-1β, TNF-α, and MPO were lower than in the MCAO/R group. Our results demonstrated that edaravone inhibits ferroptosis to attenuate CIRI, probably through the activation of the Nrf2/FPN pathway.
Ischemic stroke is a global concern that aggravates the cost of health care services. Currently, its main treatment involves thrombolysis and thrombus extraction; however, the reperfusion caused by sudden recovery of blood flow often triggers cerebral ischemic reperfusion injury (CIRI). CIRI is the most common cause of disability in ischemic stroke, and the mechanism of injury is complex. Neuroprotective therapy plays an important role in mitigating CIRI,1,2) and is a hot topic in CIRI treatment research.
Ferroptosis is a newly discovered form of regulated cell death characterized by the continuous depletion of glutathione peroxidase 4 (GPX4) and glutathione (GSH), as well as the disruption of body iron homeostasis, iron-dependent accumulation of lipid reactive oxygen species, and the production and accumulation of lipid peroxides that induce cell death.3) Research indicates that in cerebral ischemia–reperfusion, ferroptosis occurs mainly in neurons and exacerbates CIRI,4,5) and inhibition of ferroptosis attenuates CIRI.6)
The mechanisms regulating the occurrence of ferroptosis in cerebral ischemia–reperfusion are related to multiple pathways, such as iron metabolism, amino acid metabolism, and lipid metabolism. Nuclear factor E2-related factor 2 (Nrf2) is a key factor that negatively regulates ferroptosis, and it has been shown that Nrf2 activation increases the levels of key factors in ferroptosis, including ferroportin (FPN), GPX4, and GSH, to inhibit ferroptosis.7) FPN is the only known intracellular iron transport export protein, and Nrf2 can inhibit ferroptosis by upregulating FPN levels and suppressing iron overload.8)
Edaravone is an important antioxidant that plays a protective role in ischemic stroke and significantly improves clinical symptoms. Clinical and experimental studies have shown that edaravone acts as a free radical scavenger, scavenging many free radicals, such as hydroxyl radicals and peroxyl radicals, thereby inhibiting lipid peroxidation and reducing CIRI.9,10) However, the mechanism of edaravone is not fully understood. Studies have revealed that edaravone inhibits ferroptosis in amyotrophic lateral sclerosis (ALS) and depression,11,12) but whether ferroptosis mediates the anti-injury mechanism of edaravone in cerebral ischemia–reperfusion needs to be investigated.
Therefore, in this study, we used the middle cerebral artery occlusion and reperfusion (MCAO/R) method to establish a cerebral ischemia–reperfusion model in rats and investigated the anti-CIRI mechanism of edaravone based on the regulation of ferroptosis by Nrf2.
Seventy-three specific-pathogen-free (SPF)–grade healthy male Sprague Dawley (SD) rats, weighing 240 ± 20 g, were purchased from Hunan Slake Jingda Experimental Animal Co., Ltd., China (animal certificate number SCXK (Xiang) 2013-0004). The animals were reared in an SPF animal laboratory, and the ambient temperature was maintained at 23 ± 1 °C. All protocols followed the ARRIVE guidelines in terms of study design, sample size, randomization, outcome measures, data analysis, experimental procedures, and reporting of results. This study was approved by the Animal Ethics Committee of the Hunan University of Chinese Medicine (HUCM; Changsha, China; Approval No. LLBH-202103290002).
ReagentsEdaravone (6 mg/kg) was purchased from Jilin Huinan Changlong Biochemical Pharmaceutical Co., Ltd. (China); a wire plug (head diameter 0.36 ± 0.02 mm) was purchased from Beijing Xinong Technology Co., Ltd. (China); 2,3,5-triphenyte-trazolium-chloride (TTC) dye was purchased from Sigma; Western blot-based immunoprecipitation (IP) cell lysate, along with the nuclear and cytoplasmic protein extraction kit, was purchased from Beyotime (China); the malondialdehyde (MDA) kit, GSH kit, tissue iron (Fe2+) kit, and lipid peroxide (LPO) kit were purchased from Nanjing Jiancheng Bioengineering Institute (China); a rat interleukin 6 (IL-6) kit, rat IL-1β kit, and rat tumor necrosis factor α (TNF-α) kit were purchased from Wuhan Huamei Bioengineering Co., Ltd. (China); a rat myeloperoxidase (MPO) kit was purchased from Shanghai Enzyme Linked Biological Technology Co., Ltd. (China); mouse anti-β-actin monoclonal antibody was purchased from Sigma; Nrf2 antibody, GPX4 antibody, and mouse Lamin B1 monoclonal antibody were purchased from Affinity Biosciences; FPN antibody was purchased from Proteintech; goat anti-rabbit secondary antibody and goat anti-mouse secondary antibody were purchased from Merck Millipore (Burlington, MA, U.S.A.); a sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) gel preparation kit was purchased from Beyotime.
Cerebral Ischemia–Reperfusion Model Construction and Drug AdministrationThe MCAO/R model was established using the modified Longa method with reference to a previous study.13) Rats were anesthetized with 2% pentobarbital sodium (40 mg/kg) intraperitoneally and fixed on a constant-temperature operating table, and the skin of the neck was fully exposed. The anterior median carotid incision was made, and the right common carotid arteries (CCA), external carotid artery (ECA), and internal carotid artery (ICA) were separated. A small incision was made in the stump of the ECA, and the wire was inserted into the ICA via the bifurcation of the ECA. The arterial clamp was released from the distal part of the ICA, and the wire was adjusted to a depth of approximately 18 ± 2 mm, at which point a slight resistance to wire insertion occurred, and the wire was inserted right into the anterior cerebral artery. The middle cerebral artery was blocked. The CCA artery clip was released to restore blood flow, and the ECA was tied tightly with the wire plug to fix the wire plug and prevent bleeding. The skin of the neck was sutured, and the incision was disinfected. After blocking blood flow for 2 h, the suture was removed from the ECA for reperfusion for 24 h for subsequent testing. The rats were randomly divided into the sham group, MCAO/R group, and edaravone intervention group, with 10 rats in each group. In the sham-operated group, we only separated the blood vessels and did not perform operations such as cutting and inserting wires. Edaravone was administered intraperitoneally 3 d before the experiment at a dose of 6 mg/kg.14) Equal volumes of saline were given to the sham-operated group and the MCAO/R group. All groups were fed postoperatively at 23 ± 1 °C and resumed eating and drinking after awakening.
Neurological Deficit Score and Behavioral TestsThe neurological deficit score and each behavioral test were performed after 2 h of ischemia and 24 h of reperfusion in rats. The interval between tests was greater than 5 min.
Neurological Deficit ScoreThe neurological deficit scoring criteria were as follows: 0 points if there were no signs of neurological deficits and normal activity; 1 point if the contralateral forelimb of the lesion could not be fully straightened when the tail was lifted; 2 points if a turn to the contralateral side occurred when crawling; 3 points if the body fell to the contralateral side when walking; 4 points if the animal could not walk on its own and lost consciousness.15) A score between 1 and 4 was considered successful modeling, with higher scores indicating more severe behavioral disorders.
Balance Beam TestThe balance beam and the black box were fixed so that one end of the balance beam led to the black box, and one end was blocked by a baffle to prevent the rats from escaping. The balance beam and black box were suspended 50 cm above ground, and a protective pad was placed underneath to prevent the rats from falling off the balance beam. The rats were placed in the center of the balance beam and allowed a time limit of 2 min. Each rat was scored according to whether it passed the balance beam and the time it took: 6 = passed the balance beam without any slipping; 5 = passed the balance beam with only one slip; 4 = passed the balance beam with a hind limb slipping rate <50%; 3 = passed the balance beam with a hind limb slipping rate ≥50%; 2 = incapable of passing the balance beam but able to sit on it; 1 = unable to pass the balance beam and fell when sitting on it.16) The test was repeated 3 times at each interval greater than 5 min, and each rat’s score was recorded.
Cylinder ExperimentThe rats were placed in a transparent glass cylinder 20 cm in diameter and 30 cm in height with the top open and observed closely. After adaptation of the rats to the environment, we observed and recorded the rats’ left and right forelimbs and the number of simultaneous wall touches with the bilateral forelimbs (if one limb touched the wall first and the other limb touched the wall immediately afterward, it was counted as a simultaneous wall touch). We scored the utilization of the left forelimb of each rat as follows: (number of left forelimb touches + 1/2 number of bilateral touches)/(number of left forelimb touches + number of right forelimb touches + number of bilateral touches) × 100%.17) The test was repeated three times intervals greater than 5 min, and the score of left forelimb use was recorded for each rat.
Rope Climbing Muscle Strength TestThe ends of the 50 cm-long and 0.15-mm-diameter wire rope were fixed and tensioned, and suspended 40 cm above ground, and a protective pad was placed underneath to prevent the rats from being injured by falling. The rats were placed flat on the wire rope, and the suspension time was measured while the rats’ climbing posture was closely observed and scored: 0 = immediate fall; 1 = hanging on with both front paws; 2 = hanging on with both front paws but trying to climb on the rope; 3 = both front paws and one or both rear paws hanging on and trying to climb the rope; 4 = hanging on with all four paws and tail wrapped around the rope; 5 = moving on the rope independently and trying to escape.18,19) The test was repeated 3 times at intervals greater than 5 min, and hanging time and hanging score were recorded for each rat.
Adhesive Patch ExperimentThe rats were moved to another quiet cage to adapt to the environment. After 5 min, the rats were captured, and a piece of sticky patch with a circular diameter of 1 cm was stuck on the distal radius of the wrist of each forelimb bilaterally; the rats were then placed back into the quiet cage. The rats were observed closely, and a timer was used to record the total time to remove the sticky patch from the left and right limbs from the time the rats perceived and started trying to remove the sticky patch.20) The test was repeated three times at intervals greater than 5 min.
Measurement of Cerebral Infarct VolumeAfter 24 h of reperfusion, 5 rats in each group were decapitated, and the brains were frozen at −20 °C for 15 min. The brains were then evenly cut into 5 continuous 2 mm coronal slices from the frontal pole. The slices were placed in 2% TTC phosphate buffer, transferred to a constant-temperature water bath at 37 °C, and incubated in the dark for 15 min. The brain slices were turned over once every 5 min with gentle movements to prevent damage. After staining, the brain slices were fixed with 4% paraformaldehyde for 24 h. The tissue in the non-ischemic area was rose-red, and that in the infarcted area was white. The volume of cerebral infarction was calculated with reference to a previous study.13)
Detection of Fe2+, GSH, MDA, and LPO Contents in Brain TissueAfter 24 h of reperfusion, the right ischemic cortical tissue of rats was snap-frozen in liquid nitrogen and then placed in a pre-cooled grinding bowl and ground into a white powder; 10% brain tissue homogenate was made by adding pre-cooled saline at a ratio of 1 : 9 by weight and volume. After the homogenization, the brain tissue was centrifuged for 10 min at 3500 r/min on a centrifuge, and the supernatant was taken after centrifugation for the assay. The specific experimental procedures were performed in strict accordance with the instructions of each reagent.
Detection of Inflammation-Related Indexes IL-6, IL-1β, TNF-α and MPO Content in Brain TissueAfter 24 h of reperfusion, the right ischemic cortex tissues of rats were snap-frozen in liquid nitrogen, then placed in a pre-cooled grinding bowl and ground into a white powder; 10% homogenate was made by adding pre-cooled phosphate buffered saline (PBS) at a ratio of 1 : 9 by weight and volume and then placed in a refrigerator at −20 °C overnight. The tissue homogenate was centrifuged at 5000 r/min on a centrifuge after freeze-thawing twice to destroy the cell membrane. The supernatant was extracted for 5 min for the assay. The specific experimental procedures were performed in strict accordance with the instructions of each reagent.
Western Blot Detection of Nrf2, FPN, and GPX4 Expression Levels in Brain TissuesAfter 24 h of reperfusion, the right ischemic cortical tissue of rats was lysed with lysis solution, and the supernatant was collected. The nuclear and cytoplasmic protein extraction kit was used to obtain nuclear extract for Western blot analysis. The bicinchonininc acid (BCA) kit was used to detect the concentration of each group of proteins separately, leveled at the lowest concentration, added to the loading buffer, and then heated in a metal bath at 100 °C for 10 min to fully denature the proteins. After electrophoresis, membrane transfer, and closure, the membranes were incubated with primary antibodies (Nrf2 [1 : 1000], FPN [1 : 1000], GPX4 [1 : 1000], Lamin B1 [1 : 1000] and β-actin [1 : 8000] antibodies) overnight at 4 °C. After the membranes were washed 5 times with TBST, the secondary antibodies (1 : 10000) were incubated with the membranes on a shaker at room temperature for 1 h. Enhanced chemiluminescence (ECL) was developed and exposed, and the images were analyzed using the Quantity One grayscale analysis software. The relative integrated optical density (IOD) value of the target protein was determined using β-actin as the internal reference protein, and the ratio of IOD of the target protein to IOD of the internal reference protein was calculated to express the relative protein content.
Statistical AnalysisStatistical analysis was performed using SPSS 23.0 software, and all experimental data were expressed as mean ± standard deviation (S.D.) for units of measurement. One-way ANOVA was used for comparison of means between multiple groups obeying normal distribution, and the least significant difference (LSD) method was used for further two-by-two comparison of those with equal variance. The Dunnet T3 test was used for those with uneven variances; if they did not obey normal distribution, the rank sum test was used, and the difference was considered significant at p < 0.05.
To reveal the anti-CIRI effect of edaravone, we observed the effect of edaravone on sensorimotor function and cerebral infarct volume in the MCAO/R rat model. The behavioral results showed a significant increase in the neurological deficit score (Fig. 1A), a significant decrease in the balance beam score (Fig. 1B), a significant decrease in the utilization of the contralateral limb of cerebral infarction in the cylinder test (Fig. 1C), a significant decrease in the grip test score and time (Figs. 1D, E), and a significant increase in sticker removal time (Fig. 1F) in rats after cerebral ischemia–reperfusion. Edaravone intervention significantly reversed these sensorimotor effects. The TTC staining results showed that the infarct volume in the MCAO/R group was significantly increased, and edaravone significantly reduced the infarct volume in MCAO/R rats (Figs. 1G, H). These results revealed that edaravone attenuated CIRI in rats.
All rats were randomly assigned to the sham group (natural saline, 10 mL/kg), MCAO/R group (rats given normal saline), or edaravone group (6 mg/kg). (A) The neurological deficit score. (B) Balance beam score. (C) Utilization of the affected limb in the cylinder experiment. (D) Rope climbing muscle strength score. (E) Rope climbing time. (F) Sticker removal time. (G) TTC staining to observe cerebral infarct volume; white is the infarct area, red is the normal area. (H) Quantitative analysis of cerebral infarct volume. Data is expressed as mean ± standard deviation, n = 5, ** p < 0.01 vs. sham group, ## p < 0.01 vs. MCAO/R group.
To investigate the modulating effect of edaravone on ferroptosis in the brain tissue of MCAO/R rats, we examined the contents of Fe2+, GSH, MDA, LPO, and other characteristic indicators of ferroptosis in the brain tissue of rats, and the results showed that compared with the sham group, the brain tissue of MCAO/R rats had significantly higher Fe2+, MDA, and LPO contents (Figs. 2A, C, D) and significantly lower GSH content (Fig. 2B), while edaravone significantly decreased Fe2+, MDA, and LPO contents and increased GSH content in the brain tissue of MCAO/R rats (Figs. 2A–D). In addition, we detected the expression level of GPX4 protein, a key protein of ferroptosis in brain tissue. As seen in Fig. 2F, the GPX4 protein level in the MCAO/R group was significantly reduced, and the expression level of GPX4 was significantly increased with the intervention of edaravone (Figs. 2E, F). Therefore, the outcomes demonstrated that edaravone may protect against ferroptosis in MCAO/R rats.
(A) Detection of Fe2+ content in rat brain tissue. (B) Detection of GSH content in rat brain tissue. (C) Detection of MDA content in rat brain tissue. (D) Detection of LPO content in rat brain tissues. (E) Western blot assay of the GPX4 protein expression level in rat brain tissue. (F) Quantitative analysis of the GPX4 protein expression level. Data are presented as mean ± standard deviation, n = 6, ** p < 0.01 vs. sham group, ## p < 0.01 vs. MCAO/R group.
Total Nrf2, nuclear Nrf2 (nNrf2), and FPN protein expression levels were measured, and the results showed that total Nrf2 and nNrf2 protein levels had a significant increase, while FPN levels decreased considerably in the brain tissue of MCAO/R rats. Edaravone intervention further increased total Nrf2 and nNrf2 protein expression levels and FPN expression levels in the brain tissue of MCAO/R rats (Fig. 3).
(A) Western blot was used to detect the effect on total Nrf2, nNrf2, and FPN protein expression levels in rat brain tissue. (B) Quantitative analysis of total Nrf2 protein expression levels. (C) Quantitative analysis of nNrf2 protein expression levels. (D) Quantitative analysis of FPN protein expression levels. Data are presented as mean ± standard deviation, n = 6, * p < 0.05, ** p < 0.01 vs. sham group, ## p < 0.01 vs. MCAO/R group.
In addition, we observed the effects of edaravone on the contents of inflammation-related indexes IL-6, IL-1β TNF-α, and MPO in the brain tissue of MCAO/R rats. The enzyme-linked immunosorbent assay (ELISA) results showed that the contents of IL-6, IL-1β, TNF-α, and MPO were significantly increased in brain tissue after MCAO/R, and edaravone could appreciably reduce the IL-6, IL-1β, TNF-α, and MPO contents compared with the MCAO/R group (Fig. 4).
(A) IL-6. (B) IL-1β. (C) TNF-α. (D) MPO. Data are presented as mean ± standard deviation, n = 6, ** p < 0.01 vs. sham group, ## p < 0.01 vs. MCAO/R group.
This study was mainly focused on the mechanism of edaravone in CIRI. The findings showed that, after edaravone intervention, the cerebral infarction size, neurological impairment score, and sensorimotor dysfunction of rats were significantly lower than those of rats without intervention. Furthermore, the key indicators of ferroptosis, such as the levels of Fe2+, MDA, and LPO, were appreciably improved in the edaravone group. We also found that GSH and GPX4 were increased after edaravone intervention, which suggested that the effects of edaravone in the treatment of CIRI may be related to ferroptosis. To explore this in greater depth, we detected the ferroptosis regulatory proteins Nrf2 and FPN, and found that the rat treated with edaravone was less serious than the rat treated with cerebral ischemia–reperfusion injury, and that the nuclear location of Nrf2 was increased. The IL-6, IL-1β, TNF-α, and MPO contents were also lower than in the MCAO/R group, which suggested that the mechanism of edaravone that improved CIRI may occur through the inhibition of ferroptosis via the Nrf2/FPN pathway.
As a clinically used oxidative radical scavenger, edaravone has been shown to exert neuroprotective effects in CIRI through mechanisms related to inhibition of oxidative stress, scavenging of oxidative radicals, and inhibition of apoptosis.21,22) Recent studies have shown that edaravone injection in patients with clinical cerebral ischemia–reperfusion injury significantly delays the progression of cerebral edema and infarction and reduces mortality in the acute phase.23) Our experiment revealed that rats had obvious symptoms of sensorimotor impairment and a significant increase in cerebral infarct volume after cerebral ischemia–reperfusion, and edaravone intervention could improve sensorimotor function and reduce cerebral infarct volume, which is consistent with the above-mentioned findings. Our findings indicate that edaravone can reduce cerebral ischemia–reperfusion injury.
Ferroptosis, a new type of cell death, is involved in and exacerbates CIRI; it is therefore an important target for CIRI intervention.6,24) Our results showed that after cerebral ischemia–reperfusion in rats, Fe2+, MDA, and LPO contents were significantly increased, GSH content was significantly decreased, and GPX4 expression levels were significantly decreased in brain tissue, which is consistent with previous findings and suggests that ferroptosis plays an important role in the pathophysiology and development of cerebral ischemia–reperfusion injury7,25) Meanwhile, edaravone intervention significantly decreases Fe2+, MDA, and LPO content and increases GSH content and GPX4 expression levels. Studies have found that edaravone can inhibit ferroptosis to intervene in the progression of ALS, depression, and other diseases,11,12) suggesting that edaravone can also inhibit ferroptosis to reduce CIRI.
Nrf2 is a key factor in the negative regulation of ferroptosis. After activation, Nrf2 binds to the original antioxidant reaction to regulate the expression of a variety of target genes involved in the regulation of ferroptosis, such as GSH synthesis enzymes, GPX4, and FPN related to iron metabolism.26,27) FPN, the only known membrane iron output protein, plays an important role in maintaining iron homeostasis. Appropriate activation of FPN can reduce intracellular iron overload and inhibit ferroptosis-mediated cerebral ischemia–reperfusion injury. In addition, studies have shown that Nrf2 can be directly combined with the FPN promoter region, which was further confirmed by chromatin immunoprecipitation (CHIP).28) The activation of Nrf2 promotes the expression of FPN. The Nrf2/FPN pathway is an important regulator of ferroptosis, promoting iron efflux to inhibit iron overload and ferroptosis.8) Our data showed that after cerebral ischemia–reperfusion, the expression of Nrf2 increased and the expression of FPN decreased. Meanwhile, edaravone intervention could increase the expression of Nrf2 and FPN protein in rat brain tissue. In addition, studies have reported that increasing the expression of Nrf2 and FPN protein can reduce iron overload to inhibit ferroptosis and alleviate myocardial ischemia–reperfusion injury.8) These findings suggest that edaravone can activate the Nrf2/FPN pathway to inhibit ferroptosis and alleviate CIRI.
Furthermore, the inflammatory response is an important pathological process in CIRI, and ferroptosis has a strong pro-inflammatory effect.29) It has been reported that edaravone can inhibit the inflammatory response to reduce CIRI.30) We also found that edaravone can reduce the levels of IL-6, IL-1β, TNF-α, MPO, and other inflammation-related indicators to inhibit the inflammatory response in cerebral ischemia–reperfusion rats, but whether edaravone reduces the inflammation via inhibiting ferroptosis requires further investigation.
The findings of the present study suggest that edaravone may alleviate CIRI injury by activating the Nrf2/FPN pathway to inhibit ferroptosis and inflammation (Fig. 5). Although edaravone may alleviate CIRI by scavenging reactive oxygen species and superoxide anions, and inhibiting ferroptosis may also be an important approach to reducing CIRI, the application of edaravone may alleviate iron-dependent lipid peroxidation via the activation of the Nrf2/FPN pathway. However, the specific mechanism behind the activation of Nrf2 by edaravone is unclear and needs to be explored further.
The results of this study revealed that edaravone activation of the Nrf2/FPN pathway inhibited ferroptosis and inflammation to attenuate cerebral ischemia–reperfusion injury.
This study was supported by the Hunan Provincial Natural Science Foundation of China (Grant No. 2021JJ30505), the Scientific Research Fund of the Hunan Provincial Education Department (Grant Nos. 19B436 and 19C1409), the Hunan Administration of Traditional Chinese Medicine Science Foundation (Grant No. 2021215), the Scientific Research Foundation of Hunan University of Chinese Medicine (Grant No. 202024), the Excellent Teaching Team of Postgraduates in Hunan Province [Teaching Team of Postgraduates in Basic Medicine; Grant No. (2019)370-118]. The funding agencies had no role in the study design; collection, analysis, or interpretation of the data; writing of the report; or decision to submit the article for publication.
The authors declare no conflict of interest.
