Shenmai Injection Exerts Neuroprotective Functions by Down-regulating MicroRNA-19a in H 2 O 2 induced PC12 Cells

Background: Acute ischemic stroke (AIS) and following reperfusion therapy-induced cerebral ischemia reperfusion (I/R) injury have been recognized as an important subject of cerebrovascular disease with high mortality. Oxidative stress is an important pathological process of cerebral I/R injury. microRNA-19a (miR-19a) is involved in I/R. As the organ protectant agent, Shenmai Injection (SMI) is widely used in the clinical treatment of cerebral infarction. Purpose: This study aims to explore whether SMI can reduce oxidative stress by regulating miR-19a, thereby treating I/R injury. Methods: The oxidative stress state of PC12 cells was induced by H 2 O 2 , and then the cells were cultured with SMI. The therapeutic effect of SMI was evaluated by detecting cellular superoxide dismutase (SOD), malondialdehyde (MDA) and other oxidative markers with the kit. Western blot, PCR, immunouorescence and other techniques were used to elucidate the potential mechanism of SMI. Results: Cell viability assay results showed that SMI could improve the viability of PC12 cells stimulated by H 2 O 2 . Compared with the H 2 O 2 group, after SMI treatment, the contents of MDA and reactive oxygen species (ROS) were signicantly reduced, while the activity of SOD was signicantly increased, and SMI could reduce apoptosis by increasing the content of adenosine 5'-triphosphate (ATP) in cells and enhancing the mitochondrial membrane potential ( ∆ Ψm). Western blot and qRT-PCR results showed that these effects were partially achieved through the AMPK/Sirt1/PGC-1α pathway. The level of miR-19a was signicantly increased in H 2 O 2 group, and SMI could protect the cells by reducing miR-19a. Further investigated the target of miR-19a, and transfected cells with miR-19a mimic and inhibitor respectively. We found that AdipoR2 was a direct target of miR-19a, and miR-19a could inhibit AdipoR2/PI3K/Akt/mTOR pathway.


Introduction
AIS is an important contributor to mortality and severely affects human health [][] . Timely restoration of the blood supply is considered the main therapy for AIS [] . However, the reperfusion process following ischemic attack may further aggravate brain injury, known as cerebral I/R injury [][] .
Although the exact mechanisms of I/R injury remain not fully clari ed, it is increasingly recognized that a variety of pathophysiologic processes, including oxidative stress, in ammation, mitochondrial dysfunction, and apoptosis play a key role in the occurrence and development of I/R injury [][] . Therefore, treatments based on the above mechanisms are considered as a promising strategy for alleviating the outcome of cerebral I/R injury. SMI, as a traditional Chinese medicine injection, was approved by the China Food and Drug Administration (CFDA) in 1995 and has been widely used as an organ protectant in China [][] . Consisting of aqueous extracts from two eminent Chinese Traditional medicine-Red ginseng (Hong Shen) and Ophiopogonis Radix (Mai Dong). Although Shenmai injection is widely used in the clinical treatment of cerebral infarction [] , but due to the lack of in-depth molecular biology studies and the complexity of its chemical components, the mechanism of action of SMI remains unclear. Taken together, this study aimed to explore the effect of SMI on miR-19a, its role in anti-oxidative stress and its possible related targets. To provide more evidence for the diagnostic markers of AIS and provide more methods for its treatment. Peroxidation MDA Assay Kit, Reactive Oxygen Species (ROS) Assay Kit, ATP Assay Kit, and Mitochondrial membrane potential assay kit with JC-1 were purchased from Beyotime (Shanghai, China). Anti-AMPK alpha 1, Anti-SIRT1, Anti-PGC1 alpha, Anti-Bax, and Anti-Bcl-2 were from Abcam (Cambridge, MA, USA).

Cell Cultures and Cell Treatment
The neuron-like rat pheochromocytoma cell line PC12 cells were obtained from Zhong Qiao Xin Zhou Biotechnology Co (Shanghai, China). The cells were cultured in DMEM medium containing 10% FBS in a humidi ed incubator (5% CO2, 37℃). The stimulating method of oxidative stress was to add H 2 O 2 to the glucose-free medium, and cultured the cells for 24 h, then added SMI and continue to cultured the cells for 24h.

Cell transfection
The miR-19a mimic, inhibitor and their negative control were synthesized by GenePharma Co., Ltd.
(Shanghai, China). Small interference RNA against AMPKα1 (si-AMPKα1) was purchased from Santa Cruz Biotechnology, Inc. (California, USA). Before H 2 O 2 , PC12 cells were seeded in 6-well plates and transfected with mimic, mimic-NC, inhibitor, inhibitor-NC, Si-AMPKα1, or empty vector respectively. All these transfection procedures were performed for 24h using Lipofectamine 2000 (ThermoFisher Scienti c, Shanghai, China) following the manufacturer's protocols. Then harvested the cells to perform follow-up experiments as indicated.

CCK-8 Assay.
To determine the appropriate concentration of H 2 O 2 and SMI for further investigation in the subsequent experiments, cell viability was measured by the CCK-8 test. Brie y, 10µL CCK-8 was added into each well and incubated for 2 h. At last, the absorbance measurements were detected at 450 nm using an automatic microplate reader (Bio-Tek, USA).

TUNEL Staining
PC12 cells were seeded into 6-well culture plates. After treatment, cells were xed with 4% paraformaldehyde for 30 minutes. Then use 0.3% Triton X-100 PBS incubated for 5 minutes. After incubated with TUNEL detection solution at 37°C for 1 hour. Images were captured using a uorescence microscope (BX71, Olympus, Tokyo, Japan).

ROS Generation and Mitochondrial membrane potential assays
Brie y, the cells were seeded in 6-well plates, using the corresponding commercial kits according to the manufacturer's protocols. Images were captured using a uorescence microscope as previously described.

Determination of MDA, SOD, and ATP
The activities of MDA, SOD and ATP in cells were assessed using the corresponding commercial kits according to the manufacturer's protocols, using a Microplate Reader to measure the value. Levels were standardized using total cellular protein determined by BCA assay.
2.9 Immuno uorescence staining PC12 cells were xed with 4% paraformaldehyde for 30 min and then permeabilized with 0.3% Triton X-100. After blocking with 5% BSA Blocking Buffer, the cells were incubated with AMPK alpha 1 antibody or AdipoR2 antibody overnight at 4°C. Next, cells were incubated with CoraLite 488-conjugated secondary goat anti-mouse IgG antibody at 37°C for 1 h and DAPI for 5 min. Fluorescence images were acquired by a uorescence microscope.

Western Blotting Analysis
After corresponding treatments, protein extracts were isolated from each group of cells using RIPA protein lysis buffer containing protein phosphatase inhibitors. The concentration of total protein was measured via the BCA Protein Quanti cation Kit according to the manufacturer's protocol. Equal amounts of protein samples (30 µg) were separated by SDS-PAGE and transferred to the polyvinylidene di uoride (PVDF) membranes. The corresponding membranes were blocked for 1 h and then incubated with primary antibodies and subsequently incubated with secondary antibodies for 1 h at room temperature. The ECL developer was added, then the protein-antibody complexes were photographed and analyzed by Image J software.

qRT-PCR Experiment
Total cellular RNA was isolated by TRIzol reagent (Invitrogen, USA). Used the Applied Biosystems 7300 Fast Real-Time PCR System (Thermo Fisher Scienti c, USA), according to the manufacturer's protocols, in the 20ul reaction volume, rst reversely transcribed RNA into complementary DNA (cDNA), and then carried out the quantitative real-time PCR (qRT-PCR). All primers were designed and synthesized by Shanghai Sangon Biotech in China. miR-19a expression was determined using Hairpin-itTM miRNAs RT-PCR Quantitation Kit (GenePharma, China) with U6 as an internal reference. The speci c sequences of every pair of primers were available in Table S1. The relative amount of gene expression was calculated by 2 −△△CT . All experiments were repeated three times to intensify the credibility.

Statistical Analysis
The data were presented as mean±SD. Data were analyzed using GraphPad Prism software (version 8.0.1). The P value was calculated using one-way analysis of variance (ANOVA). P < 0.05 was considered statistically signi cant.

Chemical characterization of SMI
The quality control of the ngerprint of Shenmai Injection requires that 16 characteristic peaks corresponding to the standard ngerprint of Shenmai injection should be presented within 8-90 min. And clarify the three characteristic peak components, namely Ginsenoside Rb1, Ginsenoside Rg1 and Ginsenoside Re ( gure 1).

SMI Protects the Cell Viability of H 2 O 2 -Stimulated PC12 Cells
The CCK-8 method was used to detect the effects of different concentrations of H 2 O 2 and SMI on the cells. As shown in gure 2(a,b), the higher concentration of H 2 O 2, the lower cell survival rate, while the SMI of 0-100ul/ml has no signi cant effect on the cell survival rate. When the concentration of H 2 O 2 is 100uM, the cell survival rate is 45.56%. Therefore, in the following experiments, 100uM H 2 O 2 is used to stimulate the cells as a pathological model of oxidative stress. After stimulation with 100uM H 2 O 2 , treatment was performed with different concentrations of SMI. After 20ul/ml SMI was applied, the cell survival rate was the highest ( gure 2(c)), so 20ul/ml SMI was used for subsequent experiments.

SMI Ameliorated cells Injury by Enhancing AMPKα1 Level
To clarify the mechanism of SMI, the expression of AMPKα1 was measured by immuno uorescence method, and to further investigate whether AMPKα1 plays the key role in SMI treatment, we used AMPKα1-speci c siRNA to signi cantly decrease AMPKα1. As shown in gure 3, SMI signi cantly enhanced the decreased uorescence intensity due to H 2 O 2 stimulation, and this effect was reversed by  4(a,b,c)) and western blot ( gure 4(d,e,f,g,)) showed the same trend of results, H 2 O 2 signi cantly decreased the expression of AMPKα1, SIRT1, and PGC-1α, compared with the control group. SMI treatment could signi cantly upregulate the expression of AMPKα1, SIRT1, and PGC-1α in PC12 cells. However, the trend of SMI increasing AMPKα1, SIRT1, and PGC-1α was reversed to varying degrees after the cells were transfected with Si-AMPKα1. These results showed that SMI may have a protective effect on H 2 O 2 -induced PC12 cells via the AMPKα1/Sirt1/PGC-1α pathway.

SMI Attenuated H 2 O 2 -Induced PC12 Cells Apoptosis through AMPKα1
TUNEL assay was performed to detected cell apoptosis. Compared with the control group, the proportion of TUNEL-positive cells (red uorescence) treated with H 2 O 2 were signi cantly increased ( gure 5(a)).
Treatment with SMI signi cantly reduced the apoptotic index compared to the H 2 O 2 group.
Bax and Bcl2, as proapoptotic protein and antiapoptotic protein, played crucial roles in regulating cell apoptosis. In the H 2 O 2 group, the protein expression of Bcl2/Bax was markedly lower compared to the control group, and were ameliorated by treatment with SMI ( gure 5(b,c)). These results were consistent with the TUNEL assay.
Furthermore, the effects of SMI on PC12 cell apoptosis were blocked by Si-AMPKα1. These data further supported the hypothesis that the AMPKα1 played an essential role in the antiapoptosis mechanism of SMI.

SMI Attenuated H 2 O 2 -Induced PC12 Cells Oxidative Stress through AMPKα1
To explore the possible mechanism of SMI on H 2 O 2 -induced injury in PC12 cells, we examined the biochemical markers of oxidative stress, including MDA, ROS, and SOD activities. H 2 O 2 could dramatically increase the MDA, ROS levels and decrease the activity of SOD, compared with the control group ( gure 6). However, SMI treatment could signi cantly increase the SOD activity and decrease the levels of MDA, ROS. In contrast, the improvement of SOD activity and MDA, ROS level was suppressed by Si-AMPKα1. All the above results demonstrated that SMI could markedly alleviate H 2 O 2 -induced oxidative abnormalities in PC12 cells, whereas the antioxidative activity was attenuated by Si-AMPKα1.

SMI Attenuated H 2 O 2 -Induced PC12 Cells Mitochondrial damage through AMPKα1
Assess cell mitochondrial function by detecting adenosine 5'-triphosphate (ATP) and mitochondrial membrane potential (∆Ψm) levels. SMI treatment could signi cantly change the JC-1 uorescent probe from green uorescence to red uorescence in PC12 cells, and increase the ATP levels ( gure 7), compared with the H 2 O 2 group. But the effect of SMI was partially reversed by Si-AMPKα1. The results showed that SMI could signi cantly alleviate the mitochondrial dysfunction of PC12 cells via increasing ATP and ΔΨm, and the mechanism of action is related to AMPKα1.

SMI declined the miR-19a expression in H 2 O 2 -Induced PC12 cells
Considering that miR-19a was identi ed as a sign of nerve injury, we investigated whether miR-19a was associated with H 2 O 2 induced cells. qRT-qPCR analysis showed that the H 2 O 2 exposed PC12 cells signi cantly induced miR-19a expression compared with the control group ( gure 8(a)). By contrast, SMI overturned the acceleration of H 2 O 2 on miR-19a expression. These results manifested that miR-19a might either be involved in or serve as an effector of H 2 O 2 injury, and SMI could restrain miR-19a expression in H 2 O 2 -stimulated PC12 cells.

SMI Regulated the Effects of AMPKα1 in H 2 O 2 -Induced PC12 cells Through miR-19a
The above experimental results showed that the therapeutic effect of SMI was related to both AMPKα1 and miR-19a. Therefore, to verify whether miR-19a had a regulatory effect on AMPKα1, we transfected cells with miR-19a mimic and inhibitor respectively, and observed the effect of increased and silenced miR-19a expression on AMPKα1 by immuno uorescence method.
As shown in Figure 8( 3.6.2 SMI activated AMPKα1/Sirt1/PGC-1α signaling pathway via reducing miR-19a expression To further clarify the relationship between miR-19a and AMPKα1/Sirt1/PGC-1α pathway, cells were transfected with miR-19a mimic and inhibitor, respectively, to observe the effect of increased and decreased expression of miR-19a on AMPKα1/Sirt1/PGC-1α pathway. The results showed that the increased expression of miR-19a could offset the activation of AMPKα1/Sirt1/PGC-1α pathway by SMI ( gure 9), while the decreased expression of miR-19a had similar activation of AMPKα1/Sirt1/PGC-1α pathway by SMI. The above results indicated that SMI improved cell oxidative stress injury through miR-19a/AMPKα1/Sirt1/PGC-1α pathway.

Down-Regulation of miR-19a Caused by SMI Inhibited Cell Apoptosis
As mentioned above, the apoptosis level was assessed by tunnel assay and the expression of Bcl2/Bax protein. Compared with the cells directly treated with H 2 O 2 and SMI, the cells transfected miR-19a with mimic showed higher uorescence intensity and lower ratio of Bcl2/Bax ( gure 10), indicating that mimic reversed the protective effect of SMI. However, cells transfected with inhibitor and then stimulated with H 2 O 2 , the apoptosis rate was lower than that of untransfected cells, indicating that inhibitor could reduce the apoptosis induced by H 2 O 2 , but its protective effect was lower than that of SMI.

Down-Regulation of miR-19a Caused by SMI Inhibited Cell Oxidative Stress
Compared with untransfected cells, the MDA content and ROS uorescence intensity of mimictransfected cells were signi cantly increased after H 2 O 2 and SMI ( gure 11(a,c)), and the SOD content was signi cantly decreased ( gure 11(b)), indicating that the oxidative stress of cells was enhanced. Inhibitor and SMI have similar effects on reducing cellular oxidative stress, but SMI was more effective.

Down-Regulation of miR-19a Caused by SMI Inhibited Cell Mitochondrial damage
Compared with cells transfected with mimic, more JC-1 uorescent probes changed from red light to green light in ∆Ψm detection, and the ATP content was also reduced ( gure 12). The cells were transfected with inhibitor and then stimulated with H 2 O 2 , compared with the cells directly stimulated with H 2 O 2 without transfection, the JC-1 uorescent probe changed more from green to red, and the ATP content also increased. The results showed that miR-19a had a protective effect on Mitochondria function.

SMI Regulated the Effects of AdipoR2 in H 2 O 2 -Induced PC12 cells Through miR-19a
To further investigate the mechanisms miR-19a silencing modulated apoptosis, we predicted the putative gene targets of miR-19a silencing by using the TargetScan software. Among all of the predicted gene targets, encoding for adiponectin receptor 2 (AdipoR2) was chosen as a candidate for two reasons. First, in the brain, adiponectin signaling directly affects important brain functions, such as energy balance, synaptic plasticity, and hippocampal neurogenesis, and through its receptors AdipoR1 and AdipoR2 [] . Secondly, despite the association between serum adiponectin levels and stroke risk or stroke rehabilitation is unclear, several studies have suggested that adiponectin mediated mechanisms have a protective effect on atherosclerosis and stroke pathogenesis [][] . Hence, we used immuno uorescence to con rm whether AdipoR2 is a direct target of miR-19a.
As shown in Figure 13, SMI can signi cantly increase the uorescence intensity of AdipoR2 weakened by H 2 O 2 , while the effect of SMI on AdipoR2 was reversed by miR-19a mimic. On the other hand, miR-19a inhibitor can increase the uorescence intensity of AdipoR2 just like SMI. The above results indicate that AdipoR2 is one of the direct targets of 19a, and SMI can increase the expression of AdipoR2 by reducing the expression of miR-19a. 3.8.2 SMI activated AdipoR2/PI3K/Akt/mTOR signaling pathway via reducing miR-19a expression After con rmed that SMI can regulate AdipoR2 through miR-19a, the western blot method was used to further determine whether SMI can regulate AdipoR2/PI3K/Akt/mTOR pathway through miR-19a ( gure 14). Compared with H 2 O 2 group, SMI signi cantly increased the expression of genes in the AdipoR2/PI3K/Akt/mTOR pathway, and this effect was signi cantly reversed by miR-19a mimic. After H 2 O 2 stimulation, SMI and miR-19a inhibitor had similar effects, both activating the AdipoR2/PI3K/Akt/mTOR pathway, but SMI seemed to have a better effect, suggesting that AdipoR2/PI3K/Akt/mTOR may be one of the pathways through which SMI passes through miR-19a.

Discussion
SMI is a Chinese patent medicine injection widely used in the clinic. In previous studies, SMI and its main constituents, ginsenosides Rb1 and Rg1, reduced the release of lactate dehydrogenase (LDH) and improved the survival rate of cultured neurons, vascular endothelial cells, and astrocytes under hypoxia/hypoglycemia/reoxygenation injury [] ; helping maintain the integrity of the blood-brain barrier (BBB) function during focal cerebral ischemic injury [13] ; protecting mitochondria from oxidative stress by increasing the level of PDH, suggesting that it may improve the energy metabolism of cardiomyocytes [] .
In this study, SMI treatment obviously improved cell viability. Further experiments con rmed that SMI exerted an antioxidation effect by regulating MDA, SOD, and ROS levels or activities, affecting mitochondrial membrane potential and ATP contents, to prevented cell apoptosis as evidenced by increased Bcl2/Bax ratio, and decreased TUNEL-positive rate.
The AMP-activated protein kinase (AMPK), known as an "energy sensor" or "gauge," is expressed in all types of cells. Previous studies have reported that AMPK has a protective effect on global cerebral Compared with previous studies, this study evaluated the effect of H 2 O 2 stimulation on miR-19a in PC12 cells. The results showed that SMI obviously reduced miR-19a expression, thereby attenuating PC12 damage in H 2 O 2 injured. Overexpression of miR-19a can reduce the protective effect of SMI on PC12, while silencing miR-19a can also have the same protective effect on cells as SMI. Collectively, these data indicated that SMI is an effective treatment that ameliorated oxidative stress damage by downregulating miR-19a in H 2 O 2 -induced apoptosis of PC12 cells, and this protective effect is associated with the AMPKα1/Sirt1/PGC-1α pathway.
Based on the obtained results that SMI protected PC12 against apoptosis by regulating miR-19a, further experiments to identify the potential target of SMI were conducted. As adiponectin is associated with many cerebrovascular risk factors, such as type II diabetes, hypertension and changes in lipid Declarations Ethics approval and consent to participate Not applicable.

Consent to publish
Not applicable.

Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
Competing interests metabolism, a link between adiponectin and stroke is expected [] . AdipoR2 inhibits the induction of intracellular cell adhesion molecule-1 and vascular cell adhesion molecule-1 [22] , which typically bind to leukocytes and initiate atherosclerosis following endothelial cell injury [

Conclusions
Taken together, we have con rmed that SMI protected PC12 against H 2 O 2 injury and suppressed oxidative stress, mitochondrial dysfunction and apoptosis. More importantly, we found that downregulation of miR-19a inhibits PC12 apoptosis and might attenuate H 2 O 2 injury. All these results indicated that the protective effect of SMI on PC12 is at least partially mediated by down-regulation of miR-19a, which activates AMPKα1/Sirt1/PGC-1α and AdipoR2/PI3K/Akt/mTOR pathways and thus inhibits apoptosis. These ndings demonstrated that miR-19a, AMPKα1/Sirt1/PGC-1α pathway, and AdipoR2/PI3K/Akt/mTOR pathway might be potential therapeutic targets for H 2 O 2 injury treatment, and it might also lay the foundation for the further development of new drugs for AIS.

Page 13/30
The authors declare no con ict of interest.     Immuno uorescence images showed the effect of SMI on AMPKα1 expression. Scale bar =20μM.