Salvia miltiorrhiza alleviates hypoxia-induced nerve injury associated with Ngb/Akt intracellular signaling pathway

Background: Salvia miltiorrhiza (Danshen), a traditional Chinese herbal medicine, can effectively improve the high-altitude adverse reactions of high-altitude patients. While, the mechanism of how they exert neuroprotective effect to intervene the hypoxic at high altitudes is still not well understood. Methods: The study established high altitude hypoxia mouse model and CoCl2 -induced PC12 cell hypoxia model , the protective effects of S. miltiorrhiza radix extract (SE), Tanshinone IIA (Tan IIA) and salvianic acid A sodium (SAS) on hypoxia model were studied in vitro and in vivo. Results: The results showed that SE, Tan(cid:0)A, and SAS are able to improve biochemical level in high altitude hypoxia mouse model, increase in Ca 2+ concentration, and decrease in MMP, inhibit apoptosis in CoCl 2 -induced PC12 cell hypoxia model by activating Akt signaling pathway, protecting neurons, thus improving the oxygen carrying capacity of brain tissue. Conclusion: This study conrms the ecacy of SE, Tan (cid:0) A, and SAS with respect to therapeutic treatment of hypoxia, shown that S. miltiorrhiza and its active monomers can protect neurons by activating Ngb/Akt intracellular signaling pathway, and attenuate cerebral anoxia and neuronal damage, subsequently nerve injury caused by hypoxia at high altitude. Providing important information for the clinical treatment of nerve injury caused by hypoxia at high altitude.


Introduction
The normal oxygen partial pressure of human tissue is 2-9% (14-65 mm Hg), whereas the oxygen partial pressure of intake air is 21% (160 mm Hg). Tissue hypoxia occurs when the outside air pressure is low or oxygen transport and use are blocked. Hypoxia is common stress reported in humans and animals [1]. Studies have shown that hypoxia can cause diseases of cardiovascular system, immune system, nervous system and many other physical disorders-even life-threatening pulmonary edema or cerebral edema [2]. These common diseases are closely related to tissue or cell hypoxia. Therefore, the pathogenesis of and interventions used in hypoxia-related diseases are currently hot research topics.
In hypoxic research, the study of anoxia injury of the nervous system is the most popular topic.
Neuroglobin (Ngb) is mainly distributed in the neurons and retina cells, and it has been of high concern since found in 2000 [3]. As the third kind of globins, Ngb has the ability of carrying oxygen similar to hemoglobin and myoglobin [4]. A large body of research has shown that the oxygen binding and neuronspeci c expression properties make neuroglobin a new target molecule for the study of neuronal hypoxia and ischemia [5]. Sun et al. showed that antisense-mediated knockdown of neuroglobin rendered cortical neurons more vulnerable to hypoxia, whereas overexpression of neuroglobin conferred protection of cultured neurons against hypoxia [6]. A similar effect was observed in neuroblastoma cell line SH-SY5Y in which neuroglobin over-expression enhanced cell survival under conditions of anoxia or oxygen and glucose deprivation [7]. Other studies demonstrated that Ngb expression increased signi cantly in neurons under hypoxic conditions, and Ngb has the ability of scavenging superoxide [8][9][10]. Therefore, Ngb can be an important target in the study of hypoxia in the nervous system.
It is of great signi cance to explore safe and effective drugs for the prevention of hypoxia at high altitudes. Recently, protective additives of hypoxia injury are mainly focused on natural extracts based on the free radical scavenging effects. The traditional Chinese Medicine Department of Tibet Military Region General Hospital has treated 181 patients with high-altitude sleep disorder syndrome since 2006. The clinical practice has proven that the traditional Chinese medicine which can promote blood circulation and remove stasis, could effectively improve the high-altitude adverse reactions of patients. According to the theory of traditional Chinese medicines, S. miltiorrhiza (Salvia miltiorrhiza) radix, also known as DanShen in Chinese, is a traditional Chinese medicine, which is used to promote blood circulation and relieve vessel stasis; it has been widely used in clinics in China for the treatment of cardiovascular diseases [11][12][13]. Tanshinone IIA (Tan IIA) and salvianic acid A sodium (SAS) are the main natural active ingredients puri ed from S. miltiorrhiza radix, SAS is a major water-soluble component extracted and Tan IIA is a diterpene quinone [14]. S. miltiorrhiza can inhibit the aggregation function of blood platelets, anticoagulation, improve the activity of brinolytic enzyme, reduce blood lipid and regulate blood. It can promote blood circulation and remove blood stasis, expand coronary artery, improve myocardial ischemia and improve hypoxia response at high altitudes [15].
The basic and clinical research has con rmed that S. miltiorrhiza and its active monomers have protect effect on hypoxic brain damage [16]. However, the mechanism of how S. miltiorrhiza and its active monomers exert the neuroprotective effect to directly intervene the hypoxic at high altitudes is still not well understood. Previous studies have puri ed Ngb in vitro and preliminarily explored the hypoxic neuroprotective effect of Ngb [17]. Therefore, this study takes Ngb as the key point, combining in vivo and in vitro research, to observe the intervention effect of S. miltiorrhiza radix and its active monomers on high-altitude hypoxia and to explain the pharmacological mechanism of S. miltiorrhiza radix and its active monomers on high-altitude hypoxia injury. Fifty grams of S. miltiorrhiza were extracted using deionized water and ethanol (1:1, 500 mL) for 1 h during microboiling under reflux. The extracts were ltered through three layers of gauze, and the drug extraction was then repeated. The ltrates were combined and were then concentrated to 250 mL at 60-70°C under reduced pressure.

Materials And Methods
For the in vivo experiments, samples were shaken, calibrated, and stored at 4℃. Based on a preliminary test, ACZ was prepared with normal saline according to 60 mg/mL. Tan A was prepared with soybean oil for injection at a concentration of 6 mg/mL. SAS was prepared with soybean oil for injection at 12 mg/mL.
For the in vitro experiments, SE was centrifuged at 1200 rpm for 3 min and ltered with 0.2 μM lter membrane to prepare 1 g/mL in Phosphate buffer solution (PBS), SAS, and Tan IIA dissolved in Dimethyl sulfoxide (DMSO). Through the CCK-8 test, we chose the dose of SE (1 mg/mL), Tan A (40 μM), and SAS (100 μM), which has no toxic effect on cell viability according to the follow-up test.

Animals and treatments
Seven-week-old male BALB/c mice weighing 20-25 g were obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd. and fed at the speci c pathogen free (SPF) level animal center of the Academy of Military Medical Sciences (Beijing, China). The mice were kept in an SPF animal facility at a constant temperature of 23°C and relative humidity of 40%-60%. The protocol was approved by the Cytotoxicity was assayed in PC12 cells grown in 96-well plates. Cells (1.5x10 5 cells/ml; 0.1 ml per well) were seeded into plates and allowed to grow overnight before replacement of the medium with serum-free medium supplemented with CoCl 2 1mM co-treated with drug for 24h. Subsequently, 10 μl CCK-8 was added into each well and incubated for 1-4 h. The absorbance was read at 450 nm with a PerkinElmer Victor X Microplate Reader (PerkinElmer, Inc., Waltham, MA, USA). Reductions in optical density (OD) due to drug treatment were used to assess cell viability and normalized against control incubated in medium (100% viability).
Measurement of the mitochondrial membrane potential (MMP) MMP was measured using ow cytometry, and the mitochondrial-speci c cationic dye, JC-1. PC12 cells (1.5x10 5 cells/ml) were plated in 12-well plates CoCl 2 1mM co-treated with drug for 24h. Cells were harvested, washed twice with PBS, and incubated with 0.5 mL JC-1 (25 μM) for 20 min at 37°C. MMP was assayed, and green (JC-1 monomer) and red (JC-1 aggregate) uorescence were monitored at emission wavelengths of 525 and 595 nm, respectively. Changes in the ratio between measurements were indicative of changes in the MMP.

Measurements of intracellular ROS and Ca 2+
PC12 cells (1.5x10 5 cells/ml) were plated in 12-well plates with CoCl 2 1mM co-treated with drug for 24h, and Intracellular ROS and cytosolic Ca 2+ were measured using the uorescent probes DCFH-DA and Fluo-3-AM, respectively, and a uorescence-activated cell sorter. DCFH-DA is converted into a uorescent compound in the presence of ROS. Fluo-3-AM was added to treated cells to measure Ca 2+ . After treatment with the indicated drugs, cells were incubated with DCFH-DA (10 μM) for 20 min at 37°C in the dark (for the ROS assay) or Fluo-3/AM (5 μmol/l) for 30 min at 37°C (the Ca 2+ assay), and the cells were then harvested and suspended in 500 μl HBSS. Intracellular ROS and Ca 2+ were measured using a ow cytometer (excitation wavelength, 488 nm; emission wavelength, 535 nm).

Cell apoptosis
Apoptosis was also measured using annexin V-FITC and PI. PC12 cells were plated (1.5x10 5 cells/ml; 0.1 ml) in 12-well plates with CoCl 2 1mM co-treated with drug for 24h. Cells were harvested, washed twice with ice-cold PBS, and then suspended in 200 μl ice-cold binding buffer. Subsequently, 10 μl HRP FITC-labeled annexin V and 5 μL PI were added to the cells. The cell suspension was gently mixed, and incubated for 15 min at room temperature in the dark. Apoptosis was monitored using ow cytometry (488 nm excitation wavelength), and the uorescence intensity was measured at 530 nm (emission wavelength). Annexin V+/PI-was used to document early apoptosis, whereas AnnexinV+/PI+ was used to assess the late apoptotic stages or necrotic cells.
Quantitative real-time PCR Total RNA was extracted from brain tissue and PC12 cells using Invitrogen® TRIzol reagent according to the manufacturer's instruction (Thermo Fisher Scienti c, Inc.). PC12 cells were maintained in 6-well plates until 80% confluence and then treated with Tan IIA SAS and SE in serum-free medium for various periods of time to assess the speci c induction of Ngb expression. CoCl 2 (1 mM) and dimethyl sulfoxide (DMSO) was used as the control. The quality of the RNA was con rmed by an A260/A280 ratio of 1.8 and an RNA integrity number ≥6.5.Complementary DNA (cDNA) was synthesized using a Transcriptor™ rststrand cDNA synthesis kit at 42°C for 30 min followed by 70°C for 5 min. qRT-PCR reactions were performed on an ABI StepOne Plus™ Real Time PCR instrument using SYBR Green PCR Master Mix. The ampli cation reactions were performed as follows: 20 seconds at 95°C, and 40 cycles at 95°C for 3 seconds, and 60°C for 30 seconds. The primers used in the current study are listed in Table 2. The quantity of each transcript was calculated as described in the instrument manual and was normalized to the amount of the housekeeping gene β-actin.

Preparation of total protein and Western blotting
Total protein was extracted from brain tissue and PC12 cells according to the instruction. PC12 cells (1.5x10 5 cells/ml) were plated in 6-well plates with CoCl2 1mM for 24h, and then treated with SE(1g/ml), SAS(50mM), Tan IIA(40mM) and LY294002(10mM) co-treated with SE(1g/ml), SAS(50mM), Tan IIA(40mM) for 24h. The protein expression of Ngb, Hif-1α, Akt and P-Akt were determined by Western blotting. Protein of brain and PC12 cells were extracted with ice-cold lysis buffer and centrifuged at 12,000 × g for 10 min., and the resultant supernatant assayed using BCA protein assay kit standardized to BSA. Equal amounts of total protein (40 μg) were loaded, separated by 12% SDS-PAGE, and transferred to polyvinylidene uoride (PVDF) membrane. The membranes were blocked in 5% TBST fat free milk (blocking buffer) for 2 hrs, brie y washed, and then incubated overnight at 4°C with speci c primary antibodies against human Ngb (PA5-76950; dilution 1:500), Hif-1α (dilution 1:500), Akt (YT0175; dilution 1:1000), P-Akt (YP0006; dilution 1:1000). The samples were subsequently incubated with monoclonal IgG (1:2,000) secondary antibody for 2 hours, and visualized on lm using a Santa Cruz ECL detection system. β-actin served as a loading control. The blank control group of mice and the DMSO solvent control group of PC12 cells served as the negative controls respectively. Densitometric analyses were performed to semiquantify protein expression.

Pharmacodynamic evaluation
During the drug administration, the rats of the control group were noted to be livelier and more energetic, with bright hair color and normal gains of weight. By contrast, the mice in entering the low pressure and low oxygen animal test cabin were mentally depressed, had dark hair color, and consumed less food. However, upon treatment of SE and Tan A, the mental state of the rats was gradually restored.
No signi cant differences in the weight of the rats were observed before entering the low pressure and low oxygen animal test cabin. However, the body weight of the mice in the model group were found to be lower than that of the control (P < 0.05). By the end of the treatment cycle, the body weights of the SE and Tan A groups mice were signi cantly higher compared with those of the model group (P < 0.05). The body weights in the ACZ group were lower compared with that of the model group, these results suggested that SE and Tan A enabled the mice to retain their weights, whereas the ACZ group had no improvement. (Figure. 1, Table 3)

Histological analysis
The control group appeared to have no observable nerve injury, whereas the other groups exhibited different degrees of lesions, especially in the model group. The Nissl body of the neuronal cells were turbid, the neurons were shrunken, and the coloring power decreased. And it can be seen the vasodilatation and congestion, cerebral edema. Cerebral softening and hemorrhage with microglia proliferation, and occasional neurophagic phenomenon. Although the SE, Tan A, and SAS groups also had similar pathological changes, these were signi cantly less severe compared with those of the model groups. The pathological HE staining results are shown in Figure 2A, and the Nissl stain results are shown in Figure 2B. The neurofrontal cortex image (indicated by the arrow) is seen in the frontal cortex of the brain, blood vessels are congested, and the peripheral space is widened. Neuron cytoplasmic nuclei are blurred, vertebral cells are reduced, and tinting is poor. The results showed the cerebral parietal cortex vascular congestion around the edema (indicated by the black arrow) and nerve phagocytosis (indicated by the green arrow).
Effects of the drug on the MDA level and activity of T-SOD, GSH-Px, and CAT In this experiment, the effects of SE, Tan A, and SAS on the activities of T-SOD, CAT, GSH-PX, and the content of MDA in brain tissue, and serum of mice were studied. As shown in Figure 3, the results showed that ACZ could increase the activity of CAT and T-SOD and decrease the content of MDA in serum and brain tissue compared with the model group. Also, SE, Tan A SAS could increase the content of CAT, T-SOD, and GSH-Px, decrease the content of MDA in serum and brain tissue.
Effect of SE, SAS, and Tan IIA on the HIF-1 α level It has been shown that HIF-1α is a key factor in the occurrence of hypoxia injury. In this study, The effects of SE, Tan A, and SAS on the content of HIF-1 α in serum and brain tissue of mice were detected by ELISA. As shown in Figure 4, low-pressure and low-oxygen chamber led to the increase of HIF-1α level in blood and brain tissue of mice. The content of HIF-1α in the serum and brain tissue of the model group was higher than that in the control group (P < 0.05). The content of HIF-1α of the ACZ, SE, Tan A and SAS groups were lower than that in the model group in the serum and brain tissue (P < 0.05).

Ngb expression in mouse brain
In this study, we investigated the effect of SE, Tan A, and SAS on the relative expression of Ngb mRNA and protein in mouse brain. As shown in Figure 5, the results showed that the relative expression of Ngb mRNA and protein in the control group was less than that in the model group (P < 0.05). Compared with the model group, the relative expression of Ngb mRNA and protein in ACZ, SE, and SAS groups were greater than that in the model group (P < 0.05).
The experimental results show that hypoxia has a signi cant inhibitory effect on PC12 cells. Different concentrations of CoCl 2 have hypoxia damage on PC12 cells. IC 50 is about 1 mm, as shown in Figure 6A.
Therefore, in this experiment, 1 mM concentration of CoCl 2 is selected as the condition of PC12 cell model. The model of hypoxia injury was successfully constructed.
Under the microscope, the morphology of PC12 cells was observed: in the normal condition, the process of PC12 cells was longer, cross-linked into a network, and the cells were prismatic and polygonal. After 24 h of culture, the cell membrane was smooth, round, and full, and synaptic connections were established between cells. Under the condition of hypoxia, PC12 cells shrunk into a round shape, the synapses between cells disappeared, and the membrane was folded, which was easy to fall off, as shown in Figure  6B.
In uence of SE, SAS, and Tan IIA on PC12 cell function under hypoxic conditions CoCl 2 is able to cause hypoxia injury to cells. In order to evaluate the effects of SE, Tan A, and SAS on the CoCl 2 -induced cell hypoxia model, the viability of the PC12 cells by the CCK-8 assay was rst investigated, and it was identi ed that SE, Tan A, and SAS led to a signi cant improvement in the PC12 cells' viability compared with the CoCl 2 model group (P < 0.05; Figure 7A).
In addition, annexin-V and PI staining were performed, and ow cytometry was used to distinguish living cells from apoptotic and necrotic cells. These experiments demonstrated that the number of apoptotic cells in the CoCl 2 group was signi cantly higher compared with those in the control group (P < 0.05), whereas the apoptosis of PC12 cells in the SE, Tan A, and SAS groups was signi cantly lower compared with that in the CoCl 2 group (P < 0.05). These results revealed that treatment with SE, Tan A, and SAS was able to protect the PC12 cells from CoCl 2 -induced apoptosis ( Figure 7B).
As a marker of oxidative stress, ROS participated in renal injury through oxidative stress and in ammatory reactions, and the levels of ROS are increased markedly in hypoxia injury. In the present study, the levels of intracellular ROS in the CoCl 2 group were found to be signi cantly higher compared with those of the control group (P < 0.05; Figure 7C). In addition, the ROS level in the SE, Tan A, and SAS treatment group was also signi cantly lower compared with that in the CoCl 2 group (P < 0.05). Therefore, ROS production was shown to be effectively inhibited by SE, Tan A, and SAS. When cells are stimulated by external stress, the mitochondrial function is compromised, with a decrease in the MMP, and an increase in the levels of ROS and the intracellular Ca 2+ concentration. In this study, after PC12 cells were stimulated by CoCl 2 , the intracellular Ca 2+ level increased signi cantly, whereas the MMP level decreased signi cantly (P<0.05, respectively). After administration of SE, Tan A, and SAS, the Ca 2+ concentration was reduced markedly, and the MMP level was elevated to an appreciable extent compared with that in the CoCl 2 , group (P<0.05). The decreases in Ca 2+ and MMP caused by cell damage were also found to be alleviated following treatment with SE, Tan A, and SAS. ( Figure 7D).

SE, SAS, and Tan IIA ameliorated hypoxic-induced nerve apoptosis via the Ngb/Akt intracellular signaling pathway
The mRNA expression level of Ngb was assessed by RT-qPCR in the CoCl 2 induced PC12 cells. In these experiments, the relative expression of Ngb mRNA in the hypoxia model group was higher than that in the control group (P < 0.05). The relative expression of Ngb mRNA in the drug group was signi cantly higher than that in the hypoxia model group (P < 0.05). To further investigate the correlation between Ngb/Akt intracellular signaling pathway and hypoxia, we introduced LY294002, LY294002 can penetrate cells, and speci cally inhibit PI3K/Akt signaling pathways, including the common inhibition of Akt phosphorylation. The PC12 cells were divided into 9 subgroups, including control groups, drug treated groups and LY294002 co-treated groups. Protein expression of Ngb, Hif-1α, Akt and P-Akt were determined by quantitative Western blot analysis. The results showed that CoCl 2 induced cell hypoxic injury and mechanism of hypoxia protection in PC12 cell re ected in the rise of Hif-1αand Ngb. SE, Tan A, and SAS effectively improve the protein expression level of Ngb in PC12 cells, and LY294002 co-treated groups substantially attenuated the effects of drug-induced upregulation of Ngb. Akt expression was not signi cantly changed, but phospho-Akt expression was increased by SE, Tan A, and SAS groups. Moreover, LY294002, an inhibitor of the AKT signaling pathway, could reverse this result. The phospho-Akt protein levels are compared with total Akt kinase protein, LY294002 co-treated groups decreased the P-Akt protein expression level of CoCl 2 induced PC12 cell hypoxic injury (Figure 8). These results indicated that SE, Tan A, and SAS can protected hypoxia induced PC12 from apoptosis via the NGB-AKT pathway Conclusion Hypoxia research plays an important role in military medicine and high altitude medicine. Safe and reliable traditional Chinese medicine research and development are of great signi cance to the prevention and treatment of diseases caused by high altitude hypoxia [18]. This paper com rmed the protective effects of SE, Tan A, and SAS on hypoxia mice model at high altitudes by in vitro and in vivo experiments. The results showed that S. miltiorrhiza and its active monomers can protect neurons by activating Ngb/Akt intracellular signaling pathway, and attenuate cerebral anoxia and neuronal damage caused by hypoxia at high altitude, and it can be used to prevent and treat the injury of hypoxia to nerve tissue and the occurrence of hypoxia-related diseases.

Discussion
S. miltiorrhiza, a traditional Chinese herbal medicine, is widely used in mainland China for the treatment of cardio/cerebrovascular disorders. Tan IIA and SAS are the main natural active ingredients puri ed from S. miltiorrhiza radix, The protective effects of Tan IIA and SAS have been well proven in various models of cerebral ischemia-reperfusion injury [35]. Its possible mechanism of the protective effect on the central nervous system involves calcium channel blockade, estrogen-like action, and antiperoxidation, which may inhibit cerebral nerve cell apoptosis and ameliorate mitochondrial dysfunction, etc. Hence, we hypothesized that treatment with S. miltiorrhiza and its active monomers might present bene cial effects against hypoxia response at high altitudes, and to bring insights into their mechanisms of action.
Body weight is an important non-speci c observation index in animal experiments, which mainly re ects the comprehensive metabolism and physical tness of animal body. ACZ is the rst choice for prevention of acute high-altitude reaction in foreign countries. But its side effects are signi cant, which can cause numbness, polyuria, thirst, gastrointestinal discomfort, and even lead to renal failure and toxic epidermal necrolysis [19]. The experiment also showed some adverse reactions of ACZ group mice; however, S. miltiorrhiza has signi cant e cacy and high safety compared with ACZ. Therefore, this experiment proves that S. miltiorrhiza can be used as a drug to prevent altitude hypoxia disease and provide experimental basis for clinical medication CAT, SOD, and GSH-Px play an important role in the balance of oxidation and antioxidation [20]. By producing superoxide, the body induces lipid peroxidation and forms lipid peroxides such as MDA, which indirectly re ects the degree of cell damage. ACZ, SE, Tan A, and SAS may increase the activity of CAT, T-SOD, GSH-Px and decrease the content of MDA to achieve the protective effect of hypoxia mice model at high altitudes.
Constructing the cell model of hypoxia by subjecting cells to CoCl 2 treatment is currently recognized and widely used. when the body is under oxidative stress conditions, the balance between oxidation and antioxidation is disrupted. The increases in ROS levels in the cells exceed their scavenging capacity. Mitochondria are the main site for the production of ROS, and also the main target of ROS. Numerous studies have shown that excessive ROS can cause oxidative damage to mitochondria, resulting in their abnormal function [21,22]. The abnormalities of this function are mainly manifested in a reduction of MMP, extracellular Ca 2+ in ux, mitochondrial DNA mutation, disruption of the mitochondrial respiratory chain, and so on. and they may therefore be convenient signal markers in oxidative damage, abnormal protein expression, and cell damage [23,24]. The present study has shown that SE, Tan A and SAS are able to inhibit CoCl 2 induced apoptosis of the PC12 cells. Although the intracellular ROS of PC12 cells were markedly increased upon CoCl 2 treatment, while the increase in Ca 2+ concentration, and concomitant decrease in MMP, caused by ROS were inhibited by SE, Tan A and SAS, indicating that SE, Tan A and SAS are able to inhibit cell injury caused by CoCl 2 .
Ngb, a novel neuroprotective protein, which has neuroprotective effect on hypoxia/ischemia injury in vivo and in vitro, and may participate in the pathophysiological process of ischemia and hypoxia injury. Increasing the content of Ngb in neurons can prevent and treat the injury of brain tissue caused by hypoxia and the occurrence of hypoxia-related diseases, which is helpful to reduce the occurrence of hypoxia symptoms at high altitudes [25,26]. Previous data have implied that the upregulated expression of Ngb could be an endogenous compensatory or protective mechanism in response to sublethal hypoxic/ischemic insults to brain neurons. At the cell level, inhibition of the expression of Ngb can reduce the survival rate of cells under hypoxia, on the contrary, increasing the expression of Ngb can enhance the survival rate of cells under hypoxia. At the animal level, the expression of Ngb was increased by intraventricular injection of adenovirus vector fused with Ngb gene and construction of transgenic mice [38]. After cerebral ischemia-reperfusion, the area of cerebral infarction in these animals was signi cantly smaller than that in normal animals. It is suggested that NGB can inhibit the brain injury caused by ischemia / hypoxia and has potential neuroprotective function. A large number of basic and clinical studies have shown that Ngb can stabilize MMP, affects mitochondrial related functions, including ATP and ROS production, play a neuroprotective role and reduce the number of toxic stimulation. In addition, Ngb can regulate apoptosis through direct interaction with VDAC, Gα and Cyt-C proteins, eventually trigger the downstream part of the apoptotic cascade [27][28][29]. In our study, S. miltiorrhiza and its active monomers were able to improve the expression of Ngb in brain neurons, eliminate superoxide, play the role of antioxidative stress. The regulation of SE, Tan A, and SAS on Ngb is fully demonstrated the certain correlation with their protection against hypoxia injury.
Ngb is favorably linked to HIF-1a and phosphorylated AKT. HIF-1α has emerged as a critical oxygensensitive transcription factor that orchestrates the body's protective response to hypoxia. The expression of HIF-1α under hypoxia has protective effects on astrocytes, thus playing an important role in cerebral protection [30]. Recent study has demonstrated that The role of HIF-1α in the regulation of oxygen homeostasis in tissue may be correlated with Ngb expression [31]. HIF-1α contributes to the upregulation of Ngb expression under hypoxic conditions in mice [32]. PI3K / Akt signal is involved in the regulation of promotion, differentiation, apoptosis and glucose transport. High concentration of PI3K inhibitor can block the phosphorylation of Akt, promote a series of tandem reactions caused by downstream molecules, accelerate the process of apoptosis, and inhibit the survival of neural stem cells. It reports that Ngb increases Akt phosphorylation, which can be antagonized by LY294002, the common inhibition of Akt phosphorylation. PI3K/Akt pathway is involved in Ngb inhibiting apoptosis and promoting cell growth. [33,34] Therefore, we suggest that S. miltiorrhiza and its active monomers is an inducer of Ngb /Akt intracellular signaling pathway, S. miltiorrhiza and its active monomers may protect neurons by activating Ngb/Akt intracellular signaling pathway and inhibiting the activity of downstream target gene.
In this experiment. The results showed that SE, Tan a, and SAS could signi cantly inhibit the decrease of MMP and apoptosis induced by CoCl 2 , and signi cantly improved the survival rate compared with the control group. Western blotting showed that SE, Tan A, and SAS could not only induce the expression of Ngb protein, but also promote the expression of Akt phosphorylation level, which can be inhibited by LY294002, a speci c inhibitor of PI3K / Akt signaling pathway, which indicates that SE, Tan A, and SA can signi cantly inhibit CoCl 2 induced PC12 cell injury, and the neuroprotective effects may be achieved by activating Ngb / Akt intracellular signaling pathway.
As shown in our study, SE, Tan A, and SAS are able to increase the activity of CAT, T-SOD, GSH-Px, and decrease the content of MDA, HIF-1 α in high altitude hypoxia mouse model, Also, they are able to Improve the response to hypoxia damage at high altitudes by increasing the expression of Ngb in brain neurons, increase in Ca2 + concentration, and decrease in MMP, inhibit apoptosis in CoCl 2 -induced PC12 cell hypoxia model by activating Akt signaling pathway,protecting neurons, thus improving the oxygen carrying capacity of brain tissue. This study con rms the e cacy of SE, Tan A, and SAS with respect to therapeutic treatment of hypoxia, shown that S. miltiorrhiza and its active monomers can protect neurons by activating Ngb/Akt intracellular signaling pathway, and attenuate cerebral anoxia and neuronal damage, subsequently nerve injury caused by hypoxia at high altitude. Finally, providing important information for the clinical treatment of nerve injury caused by hypoxia at high altitude Declarations Ethical Approval and Consent to participate: Ethical approval was obtained from the Ethics Committee of the Air Force Medical Center of PLA.

Consent for publication:
Publication consent was obtained from all authors.
Data and materials availability: All data needed to evaluate the conclusions in the paper are present in the paper.

Competing interests:
The author declares that he has no competing interests.    Figure 1 Effect of Salvia miltiorrhiza extract and its main components on weight gain of mice n=8 # Compared with the blank group, p <0.05; * compared with the model group, p <0.05 Histopathological changes in brain and heart tissue obtained from hypoxia mouse. (A) H&E staining; A.
Control group: In the parietal lobe, neuron cells in the DG region can be seen under the high magni cation of the hippocampus. The cell body is full and neatly arranged. The glial cells phagocytic neurons (black arrows), and cerebral blood vessels are basically normal. B. Model group: Edema around the parietal cortex of the brain (black arrow), Nerve phagocytosis (green arrow), vascular congestion in the frontal cortex of the brain (black arrow) shows blood vessel congestion, and the peripheral space widens. Neuron cytoplasmic nucleus is blurred, vertebral body cells are reduced, and tinting strength is poor. C. ACZ group:Hippocampal reticulum can be seen in the parietal lobe. An increase in the perivascular hyperemic space (black arrow) and nerve phagocytosis (green arrow) in the parietal lobe of the brain D. SE group: Edema around the frontal cortex of the brain (black arrow), neurons in the hippocampal DG region of the parietal lobe are normal E. Tan   The content of hypoxia inducible factor-1 α (HIF-1 α) of Salvia miltiorrhiza extract and its main components on hypoxia mouse model. The levels of HIF-1 α were investigated by ElSA in serum and brain. (n = 8)(compared with the blank group, P < 0.05; * compared with the model group, P < 0.05) Figure 5 Effect of Salvia miltiorrhiza and its main components on the relative expression of Ngb mRNA and protein in brain on hypoxia mouse model (n = 8) (compared with the blank group, P < 0.05; * compared with the model group, P < 0.05) Shown are (A) mRNA expression of Ngb; (B) western blot analysis of Ngb.
Each value is expressed as the mean ± S.E.M (n=8). *P<0.05 compared with the model group.