RETRACTED ARTICLE: Isoginkgetin attenuates endoplasmic reticulum stress-induced autophagy of brain after ischemic reperfusion injury

Isoginkgetin is characterized by properties of potent anticancer and anti-inflammation. To explore its effect on ischemic stroke, a rat model of ischemia/reperfusion (I/R) injury was established and induced by transient middle cerebral artery occlusion/reperfusion (MCAO/R). Different doses of isoginkgetin were intraperitoneally injected into each rat. Expressions of ER stress activation-related makers including phosphorylated inositol-requiring enzyme 1 (IRE1), phosphorylated protein kinase RNA-like endoplasmic reticulum kinase (p-PERK), activating transcription factor-6 (ATF6), and two autophagy markers (ratio of LC3II/I and Beclin-1) were detected by western blot. Infarct volume, neurological deficits, and brain water content were detected. The results showed that ER stress and autophagy were activated by cerebral (I/R) injury, which could be effectively attenuated following pre-ischemia isoginkgetin administration. Moreover, autophagy induced by ER stress was triggered by the activation of PERK and IRE1 pathways. ER stress inhibitor (4-PBA) and ER related signaling inhibitors including PERK, GSK, IRE1, and DBSA markedly inhibited ER stress and autophagy induced by I/R. In addition, isoginkgetin markedly mitigated cerebral infarction, edema, neuronal apoptosis as well as neurological impairment induced by I/R injury, while tunicamycin (ER stress activator TM) and rapamycin (autophagy activator RAPA) could eliminate these lesions. This research identified a novel therapeutic agent isoginkgetin, which could effectively attenuate I/R injury by blocking autophagy induced by ER stress.


Introduction
As a primary cause of disability and death, ischemic stroke has been considered a serious human health challenge [1]. So far thrombolysis is the main therapy for ischemic stroke due to its ability to reduce infarct size in the brain [2]. Unfavorably, cerebral I/R injury has been frequently encountered in ischemic brain tissues following the restoration of blood perfusion and the injury is irreversible for patients thereby severely hindering their rehabilitation after ischemic stroke [3]. Despite some potential neuroprotective agents such as curcumin and tetrandrine have been identified [4,5], it is of great significance to develop more effective neuroprotective agents in treating cerebral I/R injury and to explore its underlying molecular mechanisms.
Isoginkgetin is a natural compound that is extracted from Ginkgo biloba tree leaves [6]. Increasing studies have demonstrated that isoginkgetin has potent features of anticancer and anti-inflammation. For instance, it suppresses cell invasion by regulating the expression of phosphatidylinositol 3-kinase/Akt-dependent matrix metalloproteinase-9 in breast carcinomas and melanoma [7]. Isoginkgetin has also been reported to sensitize cancer cells to apoptosis by disrupting protein clearance and lysosomal homeostasis in multiple myeloma [6]. Isoginkgetin is identified to inhibit the expressions of cytokines such as tumor necrosis factorα and IL-6 in RAW264.7 macrophages induced by LPS in a dose-dependent manner [8], confirming the nature of potential anti-inflammation. One previous study has found that isoginkgetin exhibits a stronger neuroprotective effect against cytotoxic insults induced by amyloid-β, a kind of strong oxidative stress inducer [9], suggesting a potential neuroprotective role in ischemic stroke, whereas its practical function and molecular mechanisms remain unclear.
As a crucial organelle in eukaryotic cells, endoplasmic reticulum (ER) has multiple biological features namely lipid formation, Ca 2+ storage, protein synthesis, and signaling transduction [10,11]. Increasing evidence has determined that ER stress is not generated until a series of intracellular and extracellular stimuli interfere with certain homeostatic properties of ER [12]. Previous studies have found that ER stress plays a vital role in the development of brain I/R injury [13]. Autophagy as a self-preservation mechanism in eukaryotic cells represents a self-devouring cellular catabolic process by degrading long-lived proteins, damaged organelles, and apoptotic cells so that the homeostasis and normal functions of various cells can be maintained [14]. LC3 has been recognized and employed as a specific autophagy marker. Meanwhile, the number of LC3-II, as a conjugated form of LC3, is intimately related to the number of autophagosomes. Accumulating evidence has demonstrated that autophagy is able to be activated in different brain cell types namely neurons, glial cells, and cerebral microvascular cells during the progression of ischemic stroke [15]. Taken together, the inhibition of ER stress is a promising research orientation to figure out a satisfactory therapeutic strategy for patients suffering from cerebral I/R injury. ER stress is also known as UPR, which plays a role in eliminating either unfolded or misfolded proteins for the maintenance of cellular homeostasis during this process. Three UPR signaling sensors PERK, IRE1, and ATF6 are once activated, the process indicates UPR [16]. Hence, the regulatory mechanism of the PERK and IRE1 pathway might contribute to understanding ER stresstriggered autophagy in ischemic stroke.
This project explored the effects of isoginkgetin in cerebral I/R injury using an MCAO/R rat model. We supposed that isoginkgetin could protect the nervous system function and reduce I/R damage. In addition, we attempted to explore the molecular regulation mechanism of isoginkgetin and its regulation of marker genes and signaling pathways. The results revealed that isoginkgetin could effectively alleviate cerebral I/R injury by decreasing nerve function damage, the volume of infarction, and brain water content. Furthermore, our results demonstrated that isoginkgetin attenuated cerebral I/R injury by suppressing autophagy induced ER stress. Our research identified a potential neuroprotective agent, isoginkgetin, which might be applied in treating ischemic stroke.

Animal model of MCAO/R
Male Sprague-Dawley (SD) rats (5 rats per group) weighing approximately 240-260 g were obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd (Beijing, China). MCAO/R treatment was conducted to induce cerebral I/R injury in rats following previous research [17]. Briefly, after anesthesia through the intraperitoneal injection with 42 mg/kg sodium pentobarbital, fixation of the rats was conducted using an adhesive tape. Subsequently, an incision was made along the ventral midline of the neck so that the right common carotid artery was exposed. After that, a 0.235 mm diameter nylon filament was inserted into the end of the internal carotid artery to obstruct the starting point of the right middle cerebral artery. Following occlusion for 2 h, the nylon filament was removed allowing reperfusion for 6, 12, and 24 h, respectively. In the sham group, identical procedures were performed using rats other than the MCAO. The surgical port was cleaned up of blood clots in the neck, closed layer by layer, and the skin was sutured with No. 1 silk thread and the excess obstruction thread was removed so that it could be left about 1.5 cm outside the skin. The neat skin was disinfected twice with medical alcohol. In order to ensure that the mice were alive and not infected due to cerebral edema, intramuscular injection of 2.5 mg/kg gentamicin sulfate and 2.0 mg/kg intramuscular furosemide were required immediately after surgery. As the animals were unconscious after surgery, they needed to be monitored carefully. The rats were removed from the operating table and placed in a double-layer dry cotton squirrel cage, then covered with a dry surgical drape, and placed in an air-conditioned room at 26°C. Following 120 min, the blocking thread was pulled out about 0.5 cm. After the rats were awake, the cotton cloth was removed from the squirrel cage and glucose was added to its drinking water to ensure that the rats had sufficient energy after surgery. In the future breeding process, litter was changed frequently to make the living environment dry to reduce the chance of infection. Assessment of the nerve function scores was made after the completion of reperfusion. Under deep anesthesia, the rats were put to death through cervical dislocation, then the brain tissues were collected and kept in a fridge at −80°C for measuring the volume of infarction and being used for subsequent experiments. All of the laboratory animals employed in this experiment were approved by the Animal Ethical Committee of the Affiliated Hospital of Youjiang Medical University for Nationalities.

Evaluation of infarct volume
After I/R for 6, 12, or 24 h, respectively, the animals were anesthetized with sodium pentobarbital and brain sections were prepared at 2.0 mm thickness. Staining to the five sections was subsequently performed using 1% of 2, 3, 5-triphenyltetrazolium chloride (TTC, Sigma-Aldrich) for 30 min. Images were then captured using a Bio-Rad Quantity One. Infarction volumes were calculated using the Image-Proplus 6.0 software (Media Cybernetics, USA).

Neurological deficit assessment
Neurological deficit was assessed as per neurologic deficit scores following I/R for 6, 12, or 24 h using the scales by Longa et al [19]. 0 point represented no observable neurological deficits; 1 point represented a failure to extend the left forepaw; 2 points represented circling to the left; 3 points represented falling to the left; 4 points represented unable to walk spontaneously.

Evaluation of brain water content
The evaluation of the brain water content in rats was conducted according to the previously mentioned procedures [20]. The wet weight of infarct brain hemispheres was measured by an electronic scale, dried in a desiccating oven set at 105°C for 24 h, and the dry weight was measured. Based on the data collected after measurement, the total brain water content was obtained using the following formula: brain water content = [(wet weightdry weight)/wet weight] × 100%.

Western blot
Total protein of the brain tissues was extracted using RIPA buffer. Protein samples with the R E T R A C T E D same volume were isolated using 10% SDS-PAGE and subsequently delivered to a PVDF membrane (Millipore, USA). The membrane was blocked with 5% skimmed milk and incubated with primary antibodies at 4°C overnight. These primary antibodies were listed as follows: anti-ATF6 (1:500 Then the proteins of interest were determined using an enhanced chemiluminescence (ECL) kit (Millipore, Shanghai, China) and band intensity was quantified by Image-Pro 6.0 software (Media Cybernetic).

Double immunofluorescence staining assay
Following permeated in 0.1% Triton X-100 solution for 0.5 h, the sections of brain tissues were sealed by 5% goat serum for an additional 0.5 h and supplemented with primary antibodies including anti-p-PERK, anti-p-IRE1, anti-LC3, and anti-Beclin-1 for incubation at 4°C overnight. By adding secondary antibody the following day, the sections were incubated again at room temperature for 2 h and then counterstained using 4,6-diamidino-2-phenylindole dihydrochloride (DAPI) for 5 min after a cycle of washing with PBS. Photographs were ultimately taken using a laser confocal microscope. The fluorescence intensities were analyzed using ImageJ.

TUNEL staining assay
Neuronal apoptosis of the cerebral tissues was evaluated using TUNEL assays following previous studies [21]. The tissue sections were supplemented with a methanol solution containing 0.2% H 2 O 2 , incubated 0.5 h so that endogenous peroxidase activity could be blocked, and then incubated with a mixture of TUNEL reaction (Millipore, Shanghai, China) at 37°C for 1 h. The staining results were observed using a laser confocal microscope.

Examination of transmission electron microscopy (TEM)
Following the fixation of the brain tissues with 1% OsO 4, the same buffer was supplemented containing 7.5% sucrose and incubated at 4°C for 2 h. The staining was subsequently performed using a 2% aqueous solution of uranyl acetate for 1 h. After dehydration in graded ethanol, the sections were embedded in epoxy resin. The ultramicrotome method was employed to collect the coronal slice in the cortex penumbra area, which was then stained with lead citrate and uranyl acetate. Finally, observation of the sections was conducted under JEM-1011 electron microscopy.

Statistical analysis
All data collected were expressed as means ± standard deviation (SD) and quantitative analysis was conducted by SPSS 21.0 software (SPSS, USA). Pairwise comparison was determined using Student's t-tests and multiple-group comparison was analyzed via one-way ANOVA. The values of P < 0.05 were regarded as the significant threshold.

Results
This study used the MCAO/R rat model to explore the role of isoginkgoin in brain I/R injury. We assumed that isoginkgo could protect the nervous system function and reduce I/R damage. We further explored the molecular regulation mechanism of isoginkgol and its regulation of marker genes and signal pathways. Cerebral I/R induced severe brain tissue injury by activating ER stress and autophagy. We firstly assessed neurological scores using Longa score 5-point rating scale. The results revealed that the neurological scores were markedly increased following reperfusion (p < 0.01, Figure 1(a)). Meanwhile, the TTC staining assay indicated that the cerebral infarct volume was markedly elevated after reperfusion compared with the sham group (p < 0.01, Figure 1(b)). In addition, cerebral I/R injury greatly elevated brain water content in comparison to the sham group (p < 0.05, Figure 1(c)). Neuronal apoptosis of the cerebral tissues was evaluated according to the TUNEL assay, indicating an increase of TUNEL positive cells following 24 h reperfusion compared with the sham group (p < 0.01, Figure 1(d)).
Western blot was subsequently performed to detect expressions of the three major markers of ER stress activation, the results revealed that MCAO treatment markedly elevated the expression of ATF6 following reperfusion at several time points (p < 0.05, Figure 1(e) and (f)), as well as phosphorylated-PERK (p-PERK) (p < 0.01, Figure 1(e) and (g)) and phosphorylated-IRE1 (p-IRE1) (p < 0.01, Figure 1(e) and (h)) when compared with the sham group. Meanwhile, western blot results also showed that MCAO treatment followed by reperfusion at different times elevated the ratio of LC-3II/I (p < 0.05, Figure 1(i) and (j)) and Beclin-1 expression (p < 0.01, Figure 1(i) and (k)) compared with the sham group. Moreover, the development of auto-phagosome in the ischemic penumbra of the cerebral cortex was also evaluated using TEM and the findings indicated the presence of moderate neuronal vacuolation, mitochondrial fragmentation, and a large number of lysosomes and autophagosomes after reperfusion for 24 h compared with the sham group (Figure 1(l)). It was therefore that 24 h reperfusion was to be selected for subsequent experiments. These results suggested that a rat model of cerebral I/R injury was successfully constructed and I/R injury could lead to severe brain tissue injury by activating ER stress and autophagy.

Isoginkgetin effectively attenuated cerebral I/R injury
To explore how isoginkgetin acted on cerebral I/ R injury, isoginkgetin, at the doses of 6.25, 12.5, 25, 50, and 100 mg/kg, was intraperitoneally injected into each rat and the chemical structure of isoginkgetin was shown in Figure 2(a). We found that cerebral I/R markedly improved neurological scores (p < 0.01) and effectively attenuated at the doses of 12.5-100 mg/kg (p < 0.05), instead of 6.25 mg/kg of isoginkgetin (p > 0.05) (Figure 2(b)). Hence, we selected the dose of isoginkgetin (100 mg/kg) with maximum inhibitory effect for the subsequent experiments. TTC staining assay indicated an apparent increase in infarct volume of cerebral I/R in comparison to the sham group (p < 0.01). However, administration of 100 mg/kg isoginkgetin effectively declined the infarct volume compared with the I/R group at 24 h (p < 0.01) (Figure 2(c)). Meanwhile, the brain water content of cerebral I/R was also largely increased, which was markedly attenuated following the treatment using 100 mg/kg isoginkgetin (p < 0.05, Figure 2(d)). Additionally, cerebral I/R substantially improved cell apoptosis in the ischemic region cortex of rat brains (p < 0.01), which was effectively attenuated by the administration of 100 mg/kg isoginkgetin (p < 0.01, Figure 2(e)). The findings of the experiments suggested that isoginkgetin could effectively alleviate cerebral I/R injury in rats.

Isoginkgetin administration significantly attenuated cerebral I/R-induced ER stress and autophagy
Then, we undertook a further investigation on the effect of isoginkgetin on cerebral IR-induced ER stress using the rat model. Cerebral I/R indicated an evidently increase in expressions of ATF6 (p < 0.01, Figure 3(a) and (b)), p-PERK (p < 0.01, Figure 3(a) and (c)), and p-IRE1 (p-IRE1) (p < 0.01, Figure 3(a) and (d)) in brain tissues in contrast to the sham group, while 100 mg/kg isoginkgetin effectively attenuated expressions of ATF6, p-PERK and p-IRE1 induced by cerebral I/R (Figure 3(a-d)). Evaluation of neuronal apoptosis in cerebral cortex using TUNEL assay. (e-h) Expressions of ATF6, p-PERK, and p-IRE1 in brain tissues were subjected to western blot and quantitative analysis with β-actin as the internal reference. (i-k) The expressions of LC3 and Beclin-1 in brain tissues were determined using western blot and quantitative analysis with β-actin as the internal reference. (l) Rats were induced by MCAO and reperfusion for 24 h. The auto-phagosome formation in the ischemic penumbra of the cerebral cortex was observed by TEM. # P < 0.05, ## P < 0.01 vs sham group. Scale bar = 100 μm (×200 magnification).

R E T R A C T E D
Meanwhile, a double immunofluorescence staining assay was performed and showed that I/R injury elevated expression levels of p-PERK and p-IRE1 (p < 0.01), and the administration of 100 mg/kg isoginkgetin significantly attenuated cerebral I/R-induced elevation of the fluorescence intensity of p-PERK (p < 0.05) and p-IRE1 (p < 0.01) (Figure 3(e)). Western blot results indicated that cerebral I/R markedly increased LC-3II/I ratio (p < 0.01) and the Beclin-1 expression (p < 0.01) in the cerebral tissue of rats compared with the sham group, whereas administration of 100 mg/kg isoginkgetin effectively attenuated the elevation of LC-3II/ I and Beclin-1 induced by cerebral I/R (p < 0.05) ( figure 3(f-h)). Furthermore, a double immunofluorescence staining assay was conducted indicating an elevation in the number of LC-3 and Beclin-1 positive cells following cerebral I/R injury compared with the sham group (p < 0.01). Meanwhile, 100 mg/kg isoginkgetin administration markedly attenuated the fluorescence intensity elevation of LC-3 and Beclin-1 induced by cerebral I/R(p < 0.01) (Figure 3(i)). Taken together, isoginkgetin administration substantially attenuated cerebral I/R-induced autophagy, indicating that isoginkgetin administration could effectively attenuate cerebral I/R-induced ER stress autophagy in rats.

Isoginkgetin administration attenuated apoptosis induced by I/R injury through multiple ER related signaling pathways
The action of isoginkgetin on several ER-related signaling pathways was subsequently evaluated. Western blot results revealed that I/R injury markedly elevated expressions of ATF4,

R E T R A C T E D
CHOP, Atg5, and Atg12 (all p < 0.01) in brain tissues in contrast to that of the sham group, and this effect was greatly attenuated following isoginkgetin administration at 100 mg/kg (p < 0.05) (Figure 4(a) and (b)). In addition, I/R injury substantially elevated expressions of p-JNK, Bax, and Bim (all p < 0.01) whereas decreased Bcl-2 expression in the brain tissues of rats in contrast to that of the sham group, while this effect was reversed after administrating 100 mg/kg isoginkgetin (p < 0.05) (Figure 4 (c) and (d)). The findings revealed that isoginkgetin could reduce I/R injury-induced apoptosis via regulating several ER associated signaling pathways.

R E T R A C T E D
Conversely, 3-MA exerted no apparent effect on ER stress ( Figure 5(a-c)) and inhibited autophagy activity ( Figure 5(a,d), and (e)) compared with I/ R-24 h group. Further, the specific PERK inhibitor GSK2656157 and specific IRE1 inhibitor DBSA were applied. The findings suggested that GSK significantly decreased the expression of p-PERK (p < 0.01, Figure 5(a) and (b)) whereas p-IRE1 expression remained the same (Figure 5(a) and (c)) when compared with the I/R-24 h group. DBSA did not affect p-PERK expression ( Figure 5(a) and (b)) but significantly downregulated p-IRE1 expression (p < 0.05, Figure 5(a) and (c)) in comparison with that of the I/R-24 h group. Moreover, both GSK and DBSA significantly decreased the ratio of LC-3II/I (p < 0.05, Figure 5(a) and (d)) and Beclin-1 expression (p < 0.01, Figure 5(a) and (e)). These results demonstrated that the PERK and IRE1 pathways were closely involved in autophagy induced by ER stress during cerebral I/R injury.

Isoginkgetin administration protected against cerebral I/R injury via suppressing ER stress and autophagy
TM, the specific ER stress activator tunicamycin and RAPA, specific autophagy activator rapamycin were ultimately employed. We found that I/R injury significantly elevated neurological scores, infarct volume, and brain water content (all p < 0.01) in contrast to the sham group. Meanwhile, the administration of 100 mg/kg isoginkgetin obviously decreased neurological scores, infarct volume, and brain water content (all p < 0.01) compared with the I/R-24 h group, whereas additional TM and RAPA markedly attenuated inhibitory effects of isoginkgetin on neurological scores, infarct volume and brain water content (all p < 0.05) (Figure 6(a-c)). Neuronal apoptosis in brain tissues was assessed using TUNEL assay and the findings revealed that I/R injury markedly elevated TUNEL positive cell count in contrast to the sham group (p < 0.01). Conversely, following 100 mg/kg isoginkgetin administration, the number of TUNEL positive cells was decreased compared with the I/R-24 h group (p < 0.01), and additional TM and RAPA substantially attenuated inhibitory effects of isoginkgetin on neuronal apoptosis (p < 0.05) ( Figure 6(d)). Moreover, I/R injury greatly elevated expressions of Cleaved Caspase-3 and Cleaved Caspase-9 (both p < 0.01) in cerebral tissue of rats in contrast to that of the sham group, and the administration of 100 mg/kg isoginkgetin effectively attenuated I/R injury-induced elevation of Caspase-3 and Cleaved Caspase-9  (Figure 6(e)). Generally speaking, the findings demonstrated that isoginkgetin could effectively prevent cerebral I/R injury through suppressing ER stress and autophagy.

Discussion
As ischemic stroke poses a severe threat to individual health, the identification of effective neuroprotective agents becomes increasingly urgent [22]. The current research found that cerebral I/R injury substantially triggered the markers of both ER stress and autophagy, and confirmed that ER stress-induced autophagy after the activation of the PERK and IRE1 pathway. Moreover, isoginkgetin could effectively attenuate cerebral I/R injury. Furthermore, either specific signalingrelated inhibitors or activators were used and determined that isoginkgetin effectively attenuated cerebral I/R injury following blocking ER stressinduced autophagy. Several potential neuroprotective agents have been identified to protect against cerebral I/R injury in mouse models in the last decades, namely atazanavir [23], gastrodin [24], and xuesaitong [25]. However, the protective effects of neuroprotective agents are obtained in an animal model and (d) Evaluatioin of neuronal apoptosis in brain tissues using TUNEL assay. Scale bar = 100 μm (×200 magnification). (e) Protein expressions of Cleaved Caspase-3 and Cleaved Caspase-9 in brain tissues were determined using western blot. ## P < 0.01 vs sham group; * P < 0.05, ** P < 0.01 vs I/R-24 h group; & p < 0.05 vs 100 mg/kg isoginkgetin group.

R E T R
A C T E D even exhibited certain limits. For example, one previous study found that pre-administration of a low dose of atazanavir exhibited a better neuroprotective role in the ischemic penumbra of MCAO/R rats and effectively reduced the infarct volume [23]. However, the rate of venous thrombolysis of cerebral infarction in China was only 1.3% as per the survey of the 'Eleventh Five-Year Plan' key projects supported by National Science and Technology [26]. Hence, it is of great significance to identify more effective neuroprotective agents that may contribute to clinical selection. The present study revealed that isoginkgetin could markedly attenuate cerebral I/R injury induced by MCAO administration in rats, characterized by neurological damage, decreased infarct volume, and reduced brain water content. These results provided potential therapeutic strategies for protecting against ischemic stroke. Brain I/R injury has been reported to promote ER stress production, which marks a critical process in the progression of ischemic stroke [27]. Appropriate autophagy can protect cells in ischemic nerve tissues whereas apoptosis can also occur under excessive autophagy [28]. To investigate how isoginkgetin affects ER stress and autophagy, expressions of ER stress activation-related makers ATF6, p-PERK, and p-IRE1 [29] and ratios of LC3II/I and Beclin-1 [30] were detected. The results indicated that isoginkgetin could markedly reduce the expressions of ER stress-related makers and autophagy markers induced by cerebral injury. TM and RAPA were subsequently administrated for further confirmation and the results demonstrated that both TM and RAPA could attenuate isoginkgetin protection on cerebral I/R injury.
As ischemic stroke involves series of complex processes associated with I/R injury including apoptosis and autophagy, more efforts are required for the treatment of this disorder [31]. Autophagy is a continuous physiological process that may exacerbate cell apoptosis via overdegradation [32]. The expressions of markers Cleaved Caspase-3 and Caspase-9 associated with apoptosis were determined subsequently to explore the effect of isoginkgetin on autophagy-caused apoptosis [33]. We found that isoginkgetin could substantially alleviate cerebral I/R injury-induced elevation of both markers. Meanwhile, TM and RAPA attenuated isoginkgetin inhibition of cell apoptosis, further suggesting that isoginkgetin inhibited autophagy-caused apoptosis. Increasing evidences have revealed that the development of cerebral I/R injury can be influenced by ER stress through modulating multiple signaling pathways namely activating transcription factor 4 (ATF4) and CCAAT/enhancer binding protein homologous protein (CHOP) signaling pathways [34]. Activation of CHOP is vital to apoptosis induced by ER stress via downregulating the expression of anti-apoptotic factor B cell lymphoma-2 (Bcl-2) [35]. Moreover, the JNK signaling pathway is also essential which is involved in processes of apoptosis and inflammation induced by ER stress of cerebral I/R injury [36]. Hereof, the present research investigated the effects of isoginkgetin on ER stress related signaling pathways, and the findings demonstrated that isoginkgetin could effectively suppress ER stress induced apoptosis factors including ATF4, CHOP, Atg5, Atg12, P-JNK, Bax, Bcl-2, and Bim. These data indicated that isoginkgetin significantly prevented cerebral I/ R injury through blocking autophagy induced by ER stress following the regulation of multiple signaling pathways. Our study provided that isoginkgetin might be considered a promising agent for neuroprotection in treating ischemic stroke.

Conclusion
Taken together, the present study demonstrated that isoginkgetin produced an effective protection role in cerebral I/R injury by blocking ER stressdependent autophagy. Meanwhile, it provided a novel therapeutic strategy of administration of isoginkgetin in treating cerebral ischemic stroke.

Ethical approval and consent to participate
Informed consent was obtained from all individual participants included in the study. All producers were approved by the Animal Ethical Committee of the Affiliated Hospital of Youjiang Medical University for Nationalities. Procedures operated in this research were completed in keeping with the standards set out in the Announcement of Helsinki and laboratory guidelines of research in China.

Data availability statement
The data that support the findings of this study are available on request from the corresponding author: * Xuebin Li, Center for Clinical Research, School of Preclinical Medicine, Youjiang Medical College for Nationalities, Baise City, Guangxi Province, 5,33,000, PR. China.Email address: xue-binliclinical@163.com The data are not publicly available due to their containing information that could compromise the privacy of research participants.

Disclosure statement
No potential conflict of interest was reported by the author(s).

Funding
The author(s) reported there is no funding associated with the work featured in this article.