Salvianolate lyophilized injection induced autophagy against neuronal apoptosis through the Akt/mTOR pathway in Neuro-2a cells

Background: Stroke remains a worldwide health problem. Salvianolate lyophilized injection (SLI) is one of the most widely-used in clinics in China for treating stroke. Our previous research found that SLI protects against stroke by inhibiting oxidative stress and inammation. Whether SLI has other biological effects on stroke remains unknown. Methods: Neuro-2a (N2a) cells were treated with SLI (10, 25, 50μg/ml) and exposed to oxygen-glucose deprivation/reoxygenation (OGD/R) as an in vitro model of ischemic stroke. Then, cell apoptosis was assessed using ow cytometry, apoptosis and autophagy related proteins were investigated by western blotting. Autophagy inhibitor 3-methyladenine (3-MA) pretreatment was used to detect the apoptosis effect of SLI on N2a cells. Results: In this work, we veried that SLI promoted cell proliferation, inhibited cell injury and apoptosis in OGD/R-induced N2a cells. The increase the expression of autophagy markers LC3 and Beclin-1, and decrease autophagy substrate protein p62 demonstrated the induction of autophagy by SLI. Additionally, we found that SLI activation of autophagy signicantly preceded inhibition of apoptosis in N2a. Pretreatment with autophagy inhibitor 3-MA could antagonize the protective effect of SLI on inhibiting apoptosis, which may be related to the Akt/mTOR signalling pathway. Conclusions: The present study indicated that SLI has potential as a novel therapy for ischemic stroke, and its possible mechanism is to induced autophagy and inhibit apoptosis through blocking Akt/mTOR signaling pathway in OGD/R-induced N2a cells, providing new insights into the mechanism of SLI in the treatment of stroke. The present study indicated that SLI has potential as a novel therapy for ischemic stroke, and its possible mechanism is to induced autophagy and inhibit apoptosis through blocking Akt/mTOR signaling pathway in OGD/R-induced N2a cells, providing new insights into the mechanism of SLI in the treatment of ischemic stroke.


Background
Ischemic stroke is a common cerebrovascular disorder disease, not only seriously affects people's physical and mental health, but also causes a huge economic burden on families and society [1,2]. The previous study revealed that ischemic stroke is characterized by multiple kinds of pathological and physiological changes, including oxidative stress, in ammatory reaction and increased vascular permeability [3][4][5]. Recent studies have shown that autophagy and apoptosis are simultaneously involved in the pathogenesis of ischemic stroke, where exists interaction between each other [6,7].
Autophagy, a catabolic process, is a vital phenomenon existing in eukaryotic cells. Autophagy dysfunction is closely associated with certain disease, including nervous system diseases, immune responses, cancer and diabetes [8][9][10][11]. Accumulating evidence indicates that autophagy contributes to cerebral ischemic stroke and is involved in ischemic brain injury [12,13]. Autophagy is activated in various cell types in the brain such as neurons, glia cells, and brain microvascular cells upon ischemic stroke [14]. During early reperfusion in rats, activating autophagy can provide a rmative neuroprotection against the cerebral ischemia/reperfusion (I/R)-induced injury [15]. Mild hypothermia can reduce the cerebral ischemic injury by activating autophagy and inhibiting pyroptosis [16]. However, autophagy is a 'double-edged sword [17]'. In some special circumstances, excessive autophagy could lead to cell death, playing a deleterious role in the etiology and progression of ischemic stroke [18]. Therefore, following ischemic stroke, how to intervene autophagy development is a key to tissue repair and functional recovery.
Recent studies have shown apoptosis and autophagy share common stimuli and pathways, which creates a complex network between the two pathways [19]. In certain instance, autophagy can block the induction of apoptosis by inhibiting the caspase-dependent pathway, thereby reducing cellular injury [20].
Whereas in other instance, autophagy mechanism leads to increased apoptotic cell death [21]. Autophagy can occur in parallel to apoptosis or the former can be dominant rst, yet ultimately leading to autophagy-associated apoptosis [22]. In short, the interplay and crosstalk between autophagy and apoptosis is complex but important.
Salvianolate lyophilized injection (SLI) is the water-soluble extracted from Salvia Miltiorrhiza, and is mainly used for the clinical treatment of mild to moderate cerebral infarction [23]. We previously demonstrated that SLI has signi cant protection on cerebral I/R injury in rats, which may be related to reduce oxidative stress, suppress in ammatory responses [24]. However, as far as we know, these is no report on how SLI regulates both autophagy and apoptosis, and what is the interplay between them induced by SLI. Therefore, the aim of this study was to explode the effects of SLI on autophagy and apoptosis, and to further investigate the potential molecular mechanisms in the OGD/R-induced N2a cells injury in vitro.

Materials And Methods
Drug and cells culture treatment SLI, provided by Tianjin Tasly Pharmaceutical Co, Ltd (Tianjin, China), is approved by CFDA (China Food and Drug Administration, GuoYaoZhunZi-Z20110011) in 2011. The ngerprint chromatogram of SLI contains ve major bioactive components, namely Sal B, Sal D, Sal Y, alkannic acid and RA [25]. Mouse neuroblastoma N2a cells (ATCC, Manassas, USA) were cultured in Minimum Essential Medium (MEM; Hyclone, USA) containing 10% fetal bovine serum (FBS; Gibco, USA), and antibiotic-antimycotic (Gibco) at 37 °C temperature in a humidi ed incubator under 5% CO2 and 95% air. The cells were divided into control group, oxygen-glucose deprivation/reoxygenation (OGD/R) group, and SLI groups with different concentration gradients (10, 25, or 50 µg/ml). The OGD/R model was performed as our previously described [26]. N2a cells were cultured in balanced salt solution and then transferred to a special chamber that was closed and included a gas mixture with consisting of 95% N2 and 5% CO2. The special chamber was placed in an incubator at 37 °C for 4 h. After OGD, N2a cells were maintained in complete medium in a normoxic incubator for 4, 6, or 24 h. Control cells were treated identically without undergoing OGD conditions.

Cell Viability And LDH Release Analysis
Cell viability was analyzed using CCK-8 assay. N2a cell suspension (100 µl/well, 1.0 × 106/ml) was cultured in 96-well microplates until the cells adhered to the wells and grew evenly, which can be used in experiments. After cell grouping treatment, remove the cell supernatant, add 100 µl MEM containing 10% CCK-8 (Dojindo, Japan) into each well of the culture plate, and incubate at 37℃ for 30 min. The absorbance was measured at 450 nm using a microplate reader (TECAN, Switzerland).
Lactate dehydrogenase (LDH) release assay was detected by an LDH Cytotoxicity Assay Kit (Dojindo, Japan). After the cells were incubated in different groups, 25 µl cell supernatant was collected from each well and transferred to the new 96-well microplates and added CytoTox-ONETM Reagent, which were prepared in advance. The new 96-well plate was incubated 20℃ for 30 min, then 50 µl of stop solution was added to the wells, and mixed for 30 s while shaking. The wavelength absorbance at 560/590 nm was measured for the cell viability test (TECAN, Switzerland).

Detection Of Apoptosis By Flow Cytometry
Apoptosis was evaluated by Annexin V-FITC/PI Apoptosis Detection Kit (Invitrogen, USA). After treatment, N2a cells were trypsinized, washed with cold PBS, and then resuspended in 150 µl binding buffer containing 5 µl Annexin-V-FITC and 1 µl propidium iodide (PI). After incubation at 37 °C for 15 min in dark, the uorescence level was quanti ed by ow cytometry (BD Biosciences, USA).

Determination Of Cytochrome C Release
Cytochrome c (Cyt-c) release was examined by using the Cytochrome-c ELISA Assay Kit according to manufacturer's instructions (JingKangBio, Shanghai, China). After drug intervention, N2a cells were collected and washed twice. 50 µl of samples and standards were added to the wells of microplate, which were pre-coated with cytochrome c speci c antibody. Then 100 µl of a streptavidin-HRP conjugate is added to all wells and incubated at 37 °C for 60 min. Washed with washing buffer and added chromogenic agent A (50 µl) and B (50 µl) into the microwells, and then incubated at 37 °C in the dark for 15 min. After adding stop solution (50 µl), the absorbance was measured using a microplate reader (TECAN, Switzerland).

Statistical analysis
The data were analyzed by SPSS 23.0 statistical software, which were expressed as mean ± S.D. The comparison between different groups was analyzed by one-way analysis of variance, followed by Bonferroni's multiple comparison tests. The differences were statistically signi cant at P < 0.05.

Effects of SLI on OGD/R-induced loss of cell viability
The effects of SLI on OGD/R-induced cytotoxicity were detected with CCK-8 assay and LDH release. As shown in Fig. 1, treatment of SLI (10, 25, 50 µg/ml) signi cantly increased cell viability and decreased LDH release against OGD/R-mediated cytotoxicity.

Effects of SLI on OGD/R-induced cellular apoptosis and apoptosis-related proteins
Annexin V-FITC and PI double staining were used to detect apoptosis of N2a cells after OGD4h/R24h. As shown in Fig. 2A, B, apoptotic rate was much higher in OGD/R group than in control group. Treatment with SLI (25 and 50 µg/ml) down-regulated apoptotic rate signi cantly, suggesting that it possesses antiapoptotic property. In addition, it was found that the release of Cyt-c was signi cantly decreased in cells incubated with 50 µg/ml SLI (Fig. 2C).
In order to further evaluate apoptosis following OGD/R, apoptosis-related proteins were detected using western blot. The results revealed that the expression of Cleaved-caspase-3 increased, while antiapoptotic protein Bcl-2 decreased (Fig. 2D, E) in the OGD/R group compared to the control group. SLI treatment signi cantly reduced the expression of Cleaved-caspase-3, and increased Bcl-2 compared to OGD/R group.

SLI enhances autophagy following OGD/R-induced N2a cells
To evaluate the effect of SLI on the autophagy, the autophagy related proteins were assessed by western blot. First, we measured the autophagic protein LC3 for 4 h OGD followed by 0-24 h reoxygenation. As it can be seen from Fig. 3A, B, across reoxygenation, the LC3 level rst decreased and then increased compared with that of the control, and the lowest point at 6 h after reoxygenation. Therefore, we further assessed the effect of SLI on the autophagy proteins expression at OGD4h/R6h. As presented in Fig. 3C-F, LC3 and Beclin-1 protein expression was signi cantly reduced, whilst p62 expression was signi cantly increased in the OGD/R group compared with the control group. After SLI treatment, especially at 50 µg/ml dose, signi cantly up-regulated expression of LC3 and Beclin-1, and down-regulated expression of p62. These results indicated that SLI enhanced the activation of autophagy in N2a cells following OGD/R, and we found that autophagy alteration precedes apoptosis.

SLI inhibites apoptosis on N2a cells after OGD/R by activating autophagy
To investigate whether inhibition of apoptosis process by SLI is associated with an activation of autophagy in OGD/R-induced N2a cells, the autophagy inhibitor 3-MA was used in this part of the experiment. First, we directly observed the cell viability and LDH release of OGD/R-induced N2a cells (Fig. 4A, B). The results suggested that the SLI-induced protection was reversed by 3-MA. Then, we also observed the apoptosis rate and Cyt-c release ( Fig. 4C-E). Similar results, apoptosis rate and Cyt-c release showed an enlargement in SLI + 3-MA group than SLI group. Collectively, our results suggested activating autophagy may play a key role in SLI-mediated inhibition of apoptosis.

SLI Regulates Akt/mTOR Signaling Pathway In N2a Cells
The Akt/mTOR pathway has been described as an important autophagy regulatory signaling pathway.
Akt can activate its downstream mTOR to inhibit the occurrence of autophagy. Therefore, we examined the phosphprylation of Akt and mTOR level in OGD/R induced N2a cells in vitro. Western blot analysis revealed that both the levels of phosphorylation of Akt and mTOR were increased at OGD4h/R6h. After administration treatment, SLI signi cantly decreased the ratio of p-Akt/Akt and mTOR/β-actin compared with the OGD/R group (Fig. 5A-C).

Discussion
Ischemic stroke is severe brain disease, which refers to cerebral vascular stenosis or occlusion, ultimately leading to hypoxia or even necrosis of brain tissue [27]. In Chinese traditional medicine, ischemic stroke is considered to belong to the category of blood stasis syndrome, and "huo xue hua yu" is the most common method. Salvia miltiorrhiza is one of the most commonly used drugs for "huo xue hua yu". Salvianolate lyophilized injection (SLI) is separated from water-soluble extract of Salvia miltiorrhiza by freeze-drying. It has been widely used to treat acute cerebral infarction in clinical in China. We previously reported that SLI can promote the recovery of nerve function post stroke rats in MCAO/R or diabetic model [28,29]. The mechanism may be related to reduce oxidative stress [29], repress in ammatory responses [30] and strengthen BBB function [31]. However, the underlying molecular mechanism is not comprehensively clear and warrants further investigation. In this study, we detected the protection mechanism of SLI in vitro using an OGD/R model in N2a cells. And we demonstrated that SLI confers an extraordinary neuro-protection against OGD/R-induced N2a cells disruption and the mechanism be veri ed that reducing neuronal apoptosis by activating Akt/mTOR dependent autophagy pathway.
Increasing evidence con rms that apoptosis is an important mechanism of secondary injury, which increases signi cantly during brain I/R injury [32][33][34]. During apoptosis, Cyt-c is released from the mitochondria intermembrane space into the cytosol. Subsequently recruiting and resulting in cascade reaction of caspase. Ultimately, caspase-3 e ciently cleaves, leading to apoptosis [35]. Bcl-2 family mainly involved in mitochondrial mediated apoptosis pathway [36]. Bcl-2 is an important anti-apoptotic protein expressed in surviving neurons. The protein bcl-2 can exert its antiapoptotic effect by binding to adenosine translocation factor which competes with Bax. Our results showed that SLI signi cantly reduced apoptosis rate by ow cytometry, inhibited the release of Cyt-c, decreased Cleaved-caspase-3 expression, and increased Bcl-2 level in OGD/R N2a cells (Fig. 2). Evidence suggested that the SLI served as an anti-apoptosis element in vitro. However, there are many questions remain to be further elucidated, especially regarding the role and the underlying mechanisms of SLI in the anti-apoptosis effect of ischemic stroke.
Autophagy is a dynamic process that decomposes the damaged organelles, abnormal proteins, and metabolic waste and then maintains the normal physiological microenvironment [37]. It is worth noting that autophagy is considered to be the self-protection mechanism of cells, but in some cases, autophagy may promote cellular demise through excessive self-digestion or stimulation of activation of other cell death pathways [38]. Recently, increasing evidence has supported that regulating autophagy is a mechanism of cerebral I/R injury [39-41]. Luo et al. found that inhibition of autophagy via activation of PI3K/Akt pathway contributes to against neuronal death caused by ischemic insults [42]. In contrast, Yu's group also suggested that moderate activation of autophagy can increase autophagy of hippocampal neurons by regulating mTOR, reduce reperfusion injury, and protect neurons [43]. Actually, autophagy is a double-edged sword [44]. Under certain conditions, moderate autophagy can elimination defective organelles and maintain the normal cellular homeostasis and cell vitality [45]. However, long-term excessive autophagy brings redundant degradation of cell contents, leading to cell injury and apoptosis [46]. There is con icting report as to the role of autophagy in ischemic stroke, which may be related to the degree of damage and the effect time. Therefore, whether autophagy is bene cial or detrimental in ischemic brain remains to be further discussed. In this experiment, we found that the level of autophagy rst decreased and then increased with increasing duration of reoxygenation. And it will restore to control level until reoxygenation for 24 h. That is, autophagy is impaired in early stage of cerebral ischemia, in keeping with previous reports [47]. In the present our experiment, we found that treatment with SLI during hypoxia-reoxygenation can abrogate OGD/R-induced autophagy inhibition, increased the expression of LC3 and Beclin-1, decrease the accumulation of p62, and ultimately ameliorate cell viability reduction. In short, SLI showed obvious protection of auto-phagocytosis in vitro.
Autophagy and apoptosis are two important processes of self-destruction, which interact with each other in cell death and survival [48]. Emerging evidence demonstrated that many stress pathways sequentially elicit autophagy and apoptosis within the nerve cells. The interplay between them is also complex but important. Luo et al. con rmed that upregulated autophagy may present as an anti-apoptotic mechanism by eliminating of apoptosis-associated molecules [49]. However, He et al. studies suggested that apoptosis activation is often related to increased autophagy processes [50]. In many cases autophagy precedes apoptosis and several proteins (e.g. BECN1) play important roles in both pathways [51,52]. Sirois et al. observed that autophagy predated development of apoptosis and persisted while apoptosis was activated [53]. In our present study, the autophagy inhibition was at its lowest point at reoxygenation 6 h, before rising gradually. And apoptosis rate was much higher than that of the control group at reoxygenation 24 h. It is indicated that autophagy precedes apoptosis occur, which is consistent with literatures. The limitation of this study is no time-dependent analysis of apoptosis occurs, and autophagy detection only persisted to reoxygenation 24 h. Whether autophagy will continue to rise and lead to excessive autophagy is unclear. However, in Fig. 2, 3, it is clear that SLI able to reverse OGD/R-induced autophagy inhibition and occurrence of apoptosis. And the time of activating autophagy is earlier than that of SLI inhibiting apoptosis. As expected, when autophagy was suppressed by 3-MA, an autophagy inhibitor, the protection capacity of SLI on cells viability, apoptosis, and Cyt-c leakage were attenuated, suggesting that SLI inhibits cell apoptosis by up-regulating autophagy (Fig. 4).
The Class PI3K/Akt/mTOR signaling pathway is a well-known autophagy pathway [54]. mTOR is a major negative regulator of autophagy and it is downstream to the PI3K/Akt pathway [55]. It has been recently reported that the neuroprotective effect of neurotrophic factor, is mediated by autophagy through the PI3K/Akt/mTOR pathway in cortical neurons [56]. Meanwhile, PI3K/Akt/mTOR pathway is a key intracellular signaling transduction pathway that drives cellular growth and represses cell apoptosis [57].
The Akt/mTOR pathway maybe associated with both autophagy and apoptosis and possible common upstream pathway [58,59]. The inhibition of the PI3K/Akt/mTOR signal transduction pathway is consistently associated with triggering autophagy and reducing neuronal apoptosis [60]. Based on this pathway, we observed the p-Akt and mTOR expressions in N2a cells treated by OGD/R with or without SLI in vitro (Fig. 5). The results revealed that SLI can down-regulated the expressions of p-Akt and mTOR, thereby promoting autophagy. This indicated that the neuroprotective effect of SLI on ischemic stroke can be explained by regulating the autophagic activity via Akt/mTOR pathway. Of course, it should be pointed out that the Akt/mTOR signal pathway is not the only regulator of also modulate the autophagy. To further clarify whether SLI could regulate the Akt pathway, Ly294002, an Akt phosphorylation inhibitor, should be used in future research.
In this study, we focused on the relationship between autophagy and apoptosis after OGD/R induced N2a cells and investigated the role of the Akt/mTOR signaling pathway in the regulation of them. Our study demonstrated that SLI can induce autophagy and suppress apoptosis after OGD/R injury, at least in part, through Akt/mTOR-dependent pathway.

Conclusions
The present study indicated that SLI has potential as a novel therapy for ischemic stroke, and its possible mechanism is to induced autophagy and inhibit apoptosis through blocking Akt/mTOR signaling pathway in OGD/R-induced N2a cells, providing new insights into the mechanism of SLI in the treatment of ischemic stroke.

Declarations
Ethics approval and consent to participate Not applicable.

Consent for publication
The manuscript is approved by all authors for publication.

Availability of data and materials
The data and materials generated or analyzed during this study are available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no competing interests.