Neuroprotective effect of salvianolate on cerebral ischaemia-reperfusion injury in rats by inhibiting the Caspase-3 signal pathway

Journal Pre-proof Neuroprotective effect of salvianolate on cerebral ischaemia-reperfusion injury in rats by inhibiting the Caspase-3 signal pathway Pengwei Luan, Jiazhen Xu, Xinyue Ding, Qianfei Cui, Lixian Jiang, Yulan Xu, Yuying Zhu, Ruixiang Li, Guoqiang Lin, Ping Tian, Jiange Zhang PII:

S0014-2999(20)30036-4

DOI:

https://doi.org/10.1016/j.ejphar.2020.172944

Reference:

EJP 172944

To appear in:

European Journal of Pharmacology

Received Date: 15 September 2019 Revised Date:

12 January 2020

Accepted Date: 20 January 2020

Please cite this article as: Luan, P., Xu, J., Ding, X., Cui, Q., Jiang, L., Xu, Y., Zhu, Y., Li, R., Lin, G., Tian, P., Zhang, J., Neuroprotective effect of salvianolate on cerebral ischaemia-reperfusion injury in rats by inhibiting the Caspase-3 signal pathway, European Journal of Pharmacology (2020), doi: https:// doi.org/10.1016/j.ejphar.2020.172944. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier B.V.

Neuroprotective effect of Salvianolate on cerebral ischaemia-reperfusion injury in rats by inhibiting the Caspase-3 signal pathway Pengwei Luan a, b, c, Jiazhen Xu a, c, Xinyue Ding a, Qianfei Cui a, b, Lixian Jiang a, Yulan Xu a, b, Yuying Zhu a, Ruixiang Li a, Guoqiang Lin a, Ping Tian a, *, Jiange Zhang a, * a

The Research Center of Chiral Drugs, Innovation Research Institute of Traditional

Chinese Medicine (IRI), Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China. b

School of Pharmaceutical Sciences, Zhengzhou University, Zhengzhou 450001,

China. c

Equal contributions to this article

*

Corresponding author.

Corresponding Authors: Jiange Zhang, Innovation Research Institute of Traditional Chinese Medicine (IRI), Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China Tel.: +86-21-51323106; E-mail address: [email protected] Ping Tian, Innovation Research Institute of Traditional Chinese Medicine (IRI), Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China E-mail address: [email protected]

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Abstract Salvianolate has been widely used for the treatment of cerebrovascular diseases. However, the detailed molecular mechanism of how it alleviates cerebral ischaemia-reperfusion injury is not well understood. In the present study, we investigated the neuroprotective effects of salvianolate in acute cerebral infarction using the PC12 cell oxygen-glucose deprivation (OGD) model in vitro and the rat transient middle cerebral artery occlusion (MCAO) model in vivo. The results showed that the salvianolate significantly reduced the level of reactive oxygen species and inhibited the Caspase-3 signalling pathway in vitro; at the same time, in vivo experiments showed that salvianolate obviously reduced the infarct area (12.9%) and repaired cognitive function compared with the model group (28.28%). In conclusion, our data demonstrated that the salvianolate effectively alleviated cerebral ischaemia-reperfusion injury via suppressing the Caspase-3 signalling pathway. Key words: Salvianolate; Ischaemia-reperfusion injury; Reactive oxygen species; Caspase-3 signal pathway. 1. Introduction Stroke has become one of the most common disability diseases worldwide. According to the latest statistics of Lancet and other institutions, the incidence of stroke has risen sharply. Therefore, the treatment and prevention of stroke have gained increasing importance (Benjamin et al., 2017; Jeffrey et al., 2017). Ischaemic stroke is the most common and the main type of stroke, while reperfusion injury after ischaemia is the most harmful factor causing brain damage and dysfunction (Yin et al., 2015). Recent data support that neuroprotection after cerebral ischaemia-reperfusion injury may be an effective strategy (Vaidya et al., 2018; Albrecht et al., 2019; Luo et al., 2019). The occurrence of neuronal apoptosis usually involves many aspects. Research has indicated that cerebral ischaemia-reperfusion can cause blood-brain barrier (BBB) damage, and some substances will infiltrate into brain tissue and activate microglia. The microglia release many inflammatory factors and promote the 2/27

apoptosis of neuronal cells (Rahimian et al., 2019; Butturini et al., 2019). After cerebral ischaemia-reperfusion, the calcium ion is overloaded, and a large amount of glutamate is released to activate the N-methyl-D-aspartic acid (NMDA) receptor signalling pathway, while neuronal cells produce excitotoxicity and undergo apoptosis (Huang et al., 2017). At the same time, this process leads to nutritional deficiency, mitochondrial damage, energy metabolism hindrance and the production of reactive oxygen species after ischaemia-reperfusion, which can activate the neuronal apoptosis signalling pathway (Yu et al., 2015; Tang et al., 2019). Identifying some effective components to prevent neuronal apoptosis and promote neuroprotection should be an effective way to treat cerebral ischaemia-reperfusion injury. In recent years, an increasing number of studies have found that the monomer components of traditional Chinese medicine play an important role in the neuroprotection of stroke. For example, baicalin, salidroside, mangiferin, etc., all have good anti-apoptotic effects (Yang et al., 2016; Liu et al., 2018; Wang et al., 2018; Mu et al., 2017). Salvianolate is extracted from salvia and is widely used in the treatment of coronary heart disease in China (Song et al., 2013; Guo et al., 2018). Previously, many studies have indicated that salvianolate can promote angiogenesis, has anti-inflammatory and anti-apoptotic effects, alleviates myocardial cell damage and promotes the repair of cardiac function (Lin et al., 2017; Qiu et al., 2018; Xu et al., 2011). Because of the reparation effect of salvianolate on ischaemic heart injury, these studies will first determine the efficacy of salvianolate in the treatment of cerebral ischaemia-reperfusion injury and further explore its neuroprotective effect and possible mechanism. These studies may provide reliable evidence and ideas for the clinical application of salvianolate in the treatment of stroke. In conclusion, our major research direction is the neuroprotective mechanism of drug intervention after acute cerebral ischaemic reperfusion injury. 2. Materials and methods 2.1 Drugs and chemical compounds Cell Counting Kit-8 was purchased from Dalian Meilun Biotechnology Co., Ltd. (Shanghai, China). Salvianolate was purchased from Green Valley Pharmaceutical 3/27

(Shanghai, China), Roxithromycin ointment was purchased from Sinopharm Group Co., Ltd. (Shanghai, China). 2,3,5-triphenyltetrazolium chloride (TTC) was obtained from Sigma-Aldrich (St. Louis, MO, USA); total nitric oxide (NO, S0023), malondialdehyde (MDA, S0131), lactate dehydrogenase (LDH, C1007) and reactive oxygen species (ROS, S0033) assay kits were purchased from Beyotime Biotechnology Co., Ltd. (Shanghai, China). Hoechst 33342 assay kit and Annexin V-FITC/PI apoptosis double dyeing kit were from Dalian Meilun Biotechnology Co., Ltd. (Shanghai, China). Primary monoclonal Bax, Caspase-3, Caspase-9, Cleaved-caspase-3, β-actin and hypoxia-inducible factor-1α (HIF-1α) antibodies were purchased from Cell Signalling Technology (Danvers, MA , USA); Primary polyclonal Cleaved-caspase-9, MMP-2 and primary monoclonal MMP-9 were purchased from Affinity Biosciences (Cincinnati, OH, USA); Primary monoclonal Bcl-2 and NeuN were purchased from Abcam (Cambridge, USA). In addition, all other chemicals were of analytical grade and were obtained from Adamas-beta® (China). 2.2 Animal and acute cerebral ischaemia-reperfusion injury model Male Sprague-Dawley (SD) rats of approximately 250 g (seven-week-old) were obtained from Centre for Experimental Animals at Shanghai University of Traditional Chinese Medicine. The animal experiments were performed according to the experimental protocol approved by the Animal Experiment Ethics Committee of Shanghai University of Traditional Chinese Medicine in accordance with institutional guidelines (PZSHUTCM18120711). All animal experiments were also in accordance with the ARRIVE guidelines, the U.K. Animals (Scientific Procedures) Act, 1986 and associated guidelines, the EU Directive 2010/63/EU for animal experiments and the National Institutes of Health guide for the care and use of Laboratory animals (NIH Publications No. 8023, revised 1978). Acute cerebral ischaemia-reperfusion model was induced by MCAO, as previously described (Lu et al., 2012; Shen et al., 2006). The SD rats were fasted for 4 h before the cerebral ischaemia-reperfusion procedure was performed. The rats were anaesthetized with isoflurane inhalation, and a 4-0 silicon rubber-coated nylon 4/27

monofilament was inserted into the right internal carotid artery (ICA) through the common carotid artery (CCA). The monofilament was advanced approximately 18 mm past the CCA/ICA bifurcation to occlude the origin of the middle cerebral artery (MCA) at the junction of the circle of Willis. The right MCA was occluded for 120 min before the monofilament was removed. The suitable roxithromycin ointment (about 100mg / per rat) was applied to the wound site, and the rats were then allowed to recover in a warm box at 24 ± 1 °C by lamps. At the same time, Sham-operated rats received an identical surgery except that the intraluminal monofilament was not inserted (Longa et al., 1989; Huang et al., 2013). After the rats recovered, physiological saline, salvianolate and edaravone (30 mg/kg) were injected intraperitoneally (n = 5), and the drugs were injected every 24 h. 2.3 Evaluation of neurological deficits At 72 h after reperfusion, the neurological scores of rats from different groups were evaluated using the longa neural scoring method (Longa et al., 1989). The scoring system was as follows: 0, normal walk or no neurologic deficit; 1, failure to extend opposite forepaw fully or a mild focal neurologic deficit; 2, circling to the contralateral side or a moderate focal neurologic deficit; 3, falling to the contralateral side or a severe focal neurologic deficit; and 4, no spontaneous walking with depressed consciousness. The rats with neurological scores from 1 to 3 were chosen to receive treatment. 2.4 Laser speckle contrast imaging To further observe the changes of cerebral infarction in rats, the laser speckle contrast imaging (RWD Life Technology Co., Ltd, Shenzhen, China) was used to detect the infarction of rats from different groups at 72 h after reperfusion. The rats were anaesthetized by isoflurane followed by microscopic analysis to observe the infarct area and blood flow after cerebral ischaemia-reperfusion injury. 2.5 Morris water maze and Rota-Rod experiment The rats of different groups were tested by Morris water maze and Rota-Rod experiments after continuous treatment for two weeks. At the same time, Morris water maze (RWD Life Technology Co., Ltd, Shenzhen, China) and Rota-Rod (RWD Life 5/27

Technology Co., Ltd, Shenzhen, China) experiments were continuously conducted for five days. According to the instructions of the Morris water maze, the maximum duration of the escape incubation was set as 90 s. On the last day, the rats of different groups were evaluated using space exploration experiments. Long-term cognitive functions were also assessed by the Morris water maze. In the Rota-Rod experiment, the maximum crawl time on the rotary rod was set as 120 s. These rats were trained at the same time every day. The repair of motor function was assessed using Rota-Rod experiments. 2.6 Measurement of infarct rate and brain edema After evaluation neurological deficits, the rats were killed, and the brains were rapidly removed and continuously cut into six 2-mm-thick coronal slices that were then stained with 2% 2,3,5-triphenyltetrazolium chloride (TTC) for 10 min at 37 °C in a drying box (Jinghong, Shanghai, China). The slices were fixed with 4% paraformaldehyde for 5 min. Images of the stained sections were captured using a digital camera (Nikon, Japan) and recorded. The infarct area on each TTC-stained section was measured using Adobe Photoshop CS5.0 software (Adobe, USA). Infarction rate (%) = (the area of viable brain tissue in the right hemisphere - the area of viable brain tissue in the left hemisphere) / total area of the slice × 100. The cerebral edema is mainly determined by the classical method of wet and dry weight method. The sample to be tested is placed in the oven and baked at a certain temperature for a period of time. By measuring the wet weight and the dry weight, the formula is used, brain water content (%) = (wet weight - dry weight) / wet weight × 100 (LESLEY et al., 2008). 2.7 Nissl staining The brains were imbedded into paraffin and cut into 5-µm sections. The sections were de-paraffined by standard processes. The slices were soaked separately in xylene solution I, II, and III for 10 min. After that, the brain paraffin sections were soaked separately in a gradient ethanol solution for 5 min, and the sections were soaked in methyl violet solution (Solarbio, Beijing, China) for 20 min and washed in distilled water. The Nissl differentiation solution was added to different sections for 5 s. Then, 6/27

the Nissl bodies of each section were observed using an optical microscope (Nikon, Japan). 2.8 Immunofluorescence staining The immunostaining procedure in the cortex was performed as previously described. The sections were incubated with the primary antibody against neuronal nuclei antigen (NeuN, 1:200; Cambridge, MA, USA) at 4 °C overnight. Then, the sections were washed with PBS for 10 min followed by incubation with the fluorescent sary antibody (cy3, 1:6000; Shanghai, China). After washing with PBS for 10 min, DAPI was added to the slices for 5 min. Neutral gum solution (Solarbio, Beijing, China) was used to fix the sections, and the sections were observed by fluorescence microscopy (Nikon, Japan). 2.9 TUNEL assay Cell apoptosis was determined by fluorescence with the Situ Cell Death Detection Kit (Roche, Basel, Switzerland). Brain slices were de-paraffinized as described above. After incubation with the enzyme terminal deoxyribonucleotide transferase (TdT) at 37 °C for 1 h, the slices were subsequently washed with PBS and stained with DAPI. An inverted laser scanning confocal microscope (Nikon, Japan) was used to observe apoptotic cells. 2.10 Cell culture and oxygen-glucose deprivation model The PC12 cells was obtained from Zhong Qiao Xin Zhou Biotechnology Co., Ltd. (Shanghai China) and were maintained at 37 °C in RPMI 1640 Medium (GIBCO, Shanghai, China) supplemented with 10% (v/v) foetal bovine serum (FBS) (GIBCO, Shanghai, China) in a 5% CO2 humidified incubator at 37 °C. The oxygen-glucose deprivation (OGD) model was established as described previously (Wu et al., 2009). Briefly, PC12 cells were cultured in serum-free RPMI 1640 without glucose in a dedicated chamber (MCO-170MUVHL-PC, PHCbi, Japan) with 94% N2, 5% CO2 and 1% O2, at 37 °C for 4 h and then maintained in RPMI 1640 medium with 10% foetal bovine serum for 24 h to mimic reperfusion. The corresponding control group was incubated in conventional conditions. 2.11 Cell viability assay 7/27

Cell viability was determined using Cell Counting Kit-8 (CCK-8). PC12 cells were seeded in 96-well plates at a density of 5×103 cells/well. After culturing overnight, the PC12 cells were incubated in a hypoxic incubator for 4 h. Then, the culture medium was removed, and the cells were washed with phosphate buffer solution (PBS). Various concentrations of salvianolate (0.1-40 µM) were added to the cells for 24 h. After that, CCK-8 was added to each well and incubated for 4 h at 37 °C. Cell viability was determined by testing the absorbance at 450 nm using a spectrophotometer (Thermos Fisher, USA). 2.12 Measurements the levels of MDA, NO, LDH and ROS The cultured PC12 were seeded in 6-well plates and exposed to OGD for 4 h, then treated with 5 µM salvianolate for 24 h at 37 °C. At the end of treatment, the cells were washed twice with PBS and collected, lysed with cell lysis buffer, and centrifuged at 10,000 g for 15 min at 4 °C. Then, the clear supernatants were collected, and the assay was performed immediately. For MDA detection, the supernatants were reacted with thiobarbituric acid contained in the Lipid Peroxidation MDA Assay Kit, and the reaction products were measured spectrophotometrically at 532 nm (wang, et al. 2015). NO production was measured using a Total Nitric Oxide Assay Kit. Total NO production was estimated by spectrophotometric measurement of nitrite and nitrate concentrations in the cell culture supernatant fluid using Griess reagent according to the manufacturer's instructions. Optical density at 540 nm was measured using a microplate reader. Concentrations were calculated by comparing absorptions with a standard curve (hu, et al. 2016). LDH activity in a medium is related to the permeability of cell membranes. We detected LDH activity in culture supernatants using a commercial LDH Activity Assay kit in accordance with the manufacturer’s instructions (Guo, et al. 2017). The cultured PC12 cells were seeded in laser co-focus dishes (NEST, China) and exposed to OGD for 4 h and then to 5 µM salvianolate for 24 h at 37 °C. After treatment, the cells were washed with PBS and suspended in RPMI containing 10 µM DCFH-DA fluorescent probes at 37 °C for 20 min (Li, et al. 2016). The cells were washed again, and the co-focus dishes were observed by laser scanning confocal microscopy (Nikon, 8/27

Japan). 2.13 Cell apoptosis staining The apoptosis bodies of PC12 cells after OGD treatment were detected using the Hoechst 33342 assay kit. After culturing for 5 min, the culture medium was removed, and cells were washed three times with PBS. Then, the cells were fixed with warm paraformaldehyde for 30 min. After washing three times with PBS, Hoechst 33342 staining solution was added to the cells and incubated for 30 min. Finally, the slides were sealed with glycerine and observed by fluorescence microscopy (Nikon, Japan). 2.14 Flow cytometry PC12 cells were plated in 6-well culture plates at a density of 3 × 105/well; then, the cells were treated with OGD for 4 h and cultured with 5 µM salvianolate for 24 h. These cells were then collected and washed with PBS and re-suspended in 500 µl of 1× binding buffer. Then, the cells were stained with 5 µl of annexin V-FITC and 5 µl of PI in the dark for 15 min at room temperature. Immediately after Annexin-V/PI staining, the samples were analysed by flow cytometry (Beckman Cytoflex FCM, USA). 2.15 Western blot Total soluble proteins were extracted from the infarct tissue of the brain and from PC12 cells. The protein concentration was quantified using a BCA protein assay kit (Beyotime, Shanghai, China). Equal amounts of protein were separated using SDS-PAGE and transferred to PVDF membranes. PVDF membranes were probed with primary antibodies against HIF-1α (1:500), Bcl-2 (1:1000), Bax (1:1000), Cleaved-caspase-3 (1:1000), Caspase-3 (1:1000), Caspase-9 (1:1000), Cleaved-caspase-9 (1:1000), MMP-2 (1:1000), MMP-9 (1:1000), β-actin (1:1000) and NeuN antibody (1:1000). Immunoblotting analysis was performed by incubating the membrane overnight with corresponding primary antibodies. β-actin was used as an internal standard. Images were captured with Bioanalytical imaging system C600 (Azure, USA). 2.16 Statistical analysis Statistical differences were evaluated using an unpaired Student's t-test for the 9/27

comparison of two groups and a one-way ANOVA for multiple-group comparisons (Prism 7.0 for windows, Graph Pad Software, Inc., USA). The values are expressed as the mean ± S.E.M., and p value < 0.05 was considered significant difference. 3. Results 3.1 Salvianolate reduces cerebral infarction and nerve injury in a cerebral ischaemia-reperfusion injury model At 72 h after reperfusion, brain slices were stained by TTC to assess the extent of cerebral ischaemia. The experimental results revealed that the cerebral infarct rates in rats treated with 20 mg/kg or 30 mg/kg salvianolate were significantly reduced compared with the MCAO group treated with saline (3.23% ± 0.79 for the sham group; 28.28% ± 2.87 for the MCAO group, 24.10% ± 7.74 for the 5 mg/kg group; 23.36% ± 5.25 for the 10 mg/kg group; 11.24% ± 3.71 for the 20 mg/kg group; and 14.45% ± 3.22 for the 30 mg/kg group; Fig. 1A and Fig. 1B). Longa neurological score results indicated that rats treated with 20 mg/kg salvianolate exhibited the most significant neuroprotective effect compared with the MCAO group treated with saline (2.80 ± 0.44 for the MCAO group; 1.60 ± 0.54 for the 5 mg/kg group; 1.75 ± 0.50 for the 10 mg/kg group; 1.0 ± 0.70 for the 20 mg/kg group; and 1.40 ± 0.54 for the 30 mg/kg group, Fig. 1C). The results showed that salvianolate at 20 mg/kg could have a good protective effect in the rat cerebral ischaemia-reperfusion model. Therefore, 20 mg/kg salvianolate was used in subsequent experiments. The results revealed that salvianolate significantly reduced the infarction rate (28.28% ± 2.87 for the MCAO group; 15.38% ± 2.75 for the salvianolate group, Fig. 1D and Fig. 1E), and edaravone had a similar therapeutic effect (20.04%). Salvianolate had the same effect as edaravone in repairing neurological deficits (1.8 ± 0.44 for MCAO group; 0.8 ± 0.83 for salvianolate group, Fig. 1F). The dynamic parameter change of blood was monitored in real time by the laser speckle contrast imaging (LSCI) technique, and the change of infarct area could be clearly observed, as shown in Fig. 1G. The salvianolate group and the edaravone group exhibited an obviously reduced infarction area compared with the MCAO group. The results indicated that salvianolate had a significant therapeutic effect in the rat cerebral 10/27

ischaemia-reperfusion injury model that was similar to the effect by the drug edaravone. (Fig. 1G). 3.2 Salvianolate attenuates the damage of blood-brain barrier The Evans blue (2%, i.v.) was used to observe the effect of salvianolate on the blood-brain barrier after cerebral ischaemia-reperfusion injury in rats. It was found that salvianolate could protect blood-brain barrier to reduce permeability of Evans blue in the brain compared with the MCAO group (Fig. 2A). The absorbance of Evans blue content was measured by microplate reader at 620 nm wavelength after the brain homogenate was extracted with dimethyl sulfoxide (Fig. 2B). The amount of Evans blue in salvianolate group was obviously less than that in the MCAO group. Salvianolate was also found to reduce the occurrence of cerebral edema by measuring water content (Fig. 2C). Thus, salvianolate could repair damage to the blood-brain barrier to a certain extent after cerebral ischaemia-reperfusion injury. Proteins associated with the blood-brain barrier, such as MMP-2 and MMP-9, were also detected by western blotting, and the quantitative analysis indicated the salvianolate could reduce the expression of both proteins (Fig. 2D-G). Cerebral ischaemia-reperfusion injury can increase the expression of matrix metalloproteins, decrease the tight junctions between endothelial cells, and increase the permeability of the blood-brain barrier, leading to the outflow of intravascular substances and causing brain edema. Our results suggest that reducing damage to the blood-brain barrier may also be one of the mechanisms of salvianolate in alleviating cerebral ischaemic injury. 3.3 Salvianolate alters the pathological changes of cerebral ischaemic tissue Nissl staining and NeuN immunofluorescence staining were used to observe the pathological changes of brain tissue after cerebral ischaemia-reperfusion injury. The Nissl staining results showed that the Nissl body was significantly reduced, and there were many vacuoles in the cortex and striatum in the MCAO group (Fig. 3A). Immunofluorescence staining was also performed; anti-NeuN was applied to mark the neuronal nuclei. The fluorescence density of neuronal nuclei in the salvianolate group was obviously higher than that in MCAO group (Fig. 3B and Fig. 3C). To further confirm the protective effect of salvianolate on neurons, we found that NeuN protein 11/27

expression in the salvianolate group was significantly higher than that in MCAO group (Fig. 3D and Fig. 3E). These results suggest that salvianolate could significantly improve pathological changes and reduce the enlargement of ischaemic penumbra. 3.4 Salvianolate resists apoptosis on cerebral ischaemia tissue All of the above results showed that salvianolate could protect neurons and reduce brain damage after cerebral ischaemia-reperfusion injury. TUNEL staining of brain slices was performed to study whether the neuroprotective mechanism of salvianolate was related to cell apoptosis. The experimental results shown in Fig. 4A and Fig. 4B showed that there was a large number of apoptotic cells in the MCAO group, while the number of apoptotic cells in the salvianolate group was decreased significantly. To confirm our hypothesis, the expression of apoptosis-related proteins (Bax, Caspase-3 and Caspase-9) was determined by western blotting. These proteins were defined as pro-apoptotic proteins and were elevated in the MCAO group after cerebral ischaemia-reperfusion injury; however, the expression of these proteins was decreased in the salvianolate group (Fig. 4C-E). These results suggested that salvianolate could reduce the expression of pro-apoptotic proteins and inhibit the apoptotic pathway after cerebral ischaemia-reperfusion injury. In contrast, the expression of the anti-apoptotic protein Bcl-2 was increased (Fig. 4F). Cerebral ischaemia-reperfusion injury can lead to DNA damage, which can subsequently activate the apoptosis signalling pathway and damage brain neurons. Therefore, it could be inferred that salvianolate can block the activation of the apoptosis signalling pathway through the action of apoptosis pathway-related proteins, which would be further confirmed in the following cell experimental studies. 3.5 Salvianolate treats cerebral ischaemia-reperfusion injury over a long period of time After cerebral ischaemia-reperfusion injury, we conducted a long-term administration of salvianolate in rats and explored the neurocognitive and motor function repair ability of rats. The experimental scheme was designed as shown in Fig. 5A. The MCAO model was established in rats of different groups. Reperfusion was 12/27

performed 2 h later, and administration was continued for 14 days. The neurological deficit scores were recorded during this period. Morris water maze and Rota-rod tests were applied to detect long-term neurocognitive and motor function repair. In the Morris water maze experiment, typical swimming paths and the neurological defect scores were recorded (Fig. 5B and Fig. 5C). The results indicated that the latency of the hidden platform was decreased in the salvianolate group (Fig. 5D), and the times of passing the platform were increased in the salvianolate group (Fig. 5E). In the Rota-rod experiment, motor function repair was determined in the different groups (Fig. 5F). The results indicated that the long-term learning and memory deficits after cerebral ischaemia-reperfusion injury were significantly ameliorated in the salvianolate group. Taken together, the results of the behavioural assays demonstrated that salvianolate could repair neurocognitive and motor dysfunction. 3.6 Salvianolate mitigates the damage to nerve cells in an oxygen-glucose deprivation model Using in vivo experiments, we found that the neuroprotective effects of salvianolate may be related to the apoptotic pathway. To further study the anti-apoptotic mechanism of salvianolate, we used an in vitro OGD injury cell model, and the CCK-8 assay indicated that the drug concentration below 10 µM had negligible damage to normal PC12 cells (Fig. 6A). Further research showed that 5 µM salvianolate was most effective at increasing the viability of OGD-PC12 cells compared with other concentration (Fig. 6B). With edaravone (10 µM) as the positive control, the results indicated that the protective effect of 5 µM salvianolate on PC12 cells was similar to that of edaravone (Fig. 6C). In the OGD model, we observed the effects of salvianolate on these oxidative stress factors after hypoxic injury. Reactive oxygen species expression was detected by DCFH-DA fluorescent probes in the PC12 cells after OGD to investigate the effect of hypoxia on mitochondrial damage. The results showed that salvianolate significantly reduced the production of reactive oxygen species compared with the model group, which was equivalent to the effect of edaravone (Fig. 6D and Fig. 6E). Reactive oxygen species production can increase the content of nitric oxide and accelerate the production of lipid peroxidation in cells, 13/27

while MDA can damage cell membranes and lead to lactate dehydrogenase exposure. Therefore, the expression of these oxidative damage-related factors (such as NO, MDA and LDH) were also detected. Compared with the model group, concentrations of NO, MDA and LDH in the salvianolate and edaravone groups were significantly lower, as shown in Fig. 6F-H. At the same time, studies revealed that once glycolysis is blocked, the production of intracellular energy is affected, thereby reducing the activity of the NADH dehydrogenase complex in the electron respiratory transmission chain. Thus, a large amount of reactive oxygen species would be produced in cells, which eventually leads to mitochondrial damage and triggers the activation of the Caspase-3 apoptotic signalling pathway (Park et al., 2019). 3.7 Salvianolate reduces apoptosis after oxygen-glucose deprivation Hoechst 33342 staining was used to observe the effect of salvianolate on the apoptosis of PC12 cells after OGD. A large number of apoptotic bodies was found in the model group. However, few apoptotic bodies were found in the salvianolate group and the edaravone group, as shown in Fig. 7A and Fig. 7B. In addition, flow cytometry was used to detect the apoptotic rate after OGD. Salvianolate was found to significantly inhibit apoptosis (Fig. 7C and Fig. 7D). 3.8 Salvianolate reduces the activation of the Caspase-3 pathway in vitro Under hypoxic conditions, the anti-apoptosis mechanism of salvianolate was further explored in vitro. We found by western blotting that salvianolate could obviously downregulate the expression of HIF-1α in neurons after OGD (Fig. 8A). On the other hand, treatment with salvianolate caused an increase in the expression of the anti-apoptotic factor Bcl-2 protein (Fig. 8B) and a decrease in the expression of the pro-apoptotic factor Bax protein (Fig. 8C), which can activate the apoptosis signalling pathway. Our results also showed that salvianolate could reduce the expression of the apoptotic proteins Caspase-3 and Caspase-9 (Fig. 8D and Fig. 8E). In addition, salvianolate reduced the expression of the activating apoptotic proteins (Fig. 8F and Fig. 8G). In general, salvianolate reduced the expression of HIF-1α, inhibited the production of reactive oxygen species, and prevented the activation of apoptotic signalling pathways, to protect neurons. 14/27

4. Discussion A certain period of time after cerebral ischaemia-reperfusion, brain function cannot be restored, and there is more serious brain dysfunction, known as cerebral ischaemia-reperfusion injury (CIR). CIR is associated with the formation of free radicals, intracellular calcium overload, toxicity of excitatory amino acids, high leucocyte aggregation and a lack of high-energy phosphoric acid (Masato et al., 2019). However, the pathogenesis is not yet clear. CIR is multifactorial. For instance, damage to the blood-brain barrier, inflammation and neuronal apoptosis occur in the brain. Thus, researchers are attempting to overcome this problem, but the outcomes are far from satisfactory; many other factors remain to be identified. At present, there is still no highly effective drug to treat CIR. It is reported that salvianolate can protect CIR by reducing damage to the blood-brain barrier and inhibiting the activation of JAK/STAT1 induced by IFN-gamma in endothelial cells (Liu et al., 2017). Salvianolate can also suppress inflammation, counter LPS-induced behavioural deficits and reduce the number of NLRP3 inflammasome bodies (Jiang et al., 2017). Salvianolate can inhibit mitochondrial dysfunction by upregulating mortalin and repair mitochondrial damage caused by H2O2. Previous studies have found that the blood-brain barrier plays an important role in CIR (Bar-Klein et al., 2017). Cerebral ischaemia-reperfusion triggers endothelial cell damage and leads to the brain micro-vascular rupture, which finally increases permeability and edema (Zhang et al., 2018). It is well known that when cerebral edema occurs, it is often accompanied by the release of pro-inflammatory factors, such as TNF-α, IL-6, and reactive oxygen species, which cause neuronal damage (Sweeney et al., 2018; Jiang et al., 2018). These substances can cause some pathological responses, such as microglial polarization induced by cytokines and chemokines, neuronal damage caused by haemoglobin spillover, etc. Our study indicated that of the different salvianolate doses screened in a rat MCAO model, salvianolate at 20 mg/kg effectively reduced cerebral infarction and improved neurological function. At the same time, we searched the literature and reached the same conclusion. Fan et al. found that a dose of salvianolate less than 10 15/27

mg/kg could not reduce the infarction, but the infarction was reduced with doses greater than 20 mg/kg. Therefore, considering the increase in dose may bring certain side effects, we believe that salvianolate at 20 mg/kg could have a good protective effect in rat cerebral ischaemia-reperfusion models (Fan et al., 2018).Evans blue staining showed that salvianolate could reduce the permeability of the BBB and decrease the levels of proteins related to the BBB, such as MMP-2 and MMP-9, suggesting that salvianolate could also repair damage to the BBB and inhibit harmful substances from entering the brain. We focused our experiments on neuronal apoptosis. The OGD model was established to explore the mechanism of how salvianolate reduces brain neuronal damage after cerebral ischaemia-reperfusion injury. After screening different doses of salvianolate, we found that the protective effect of salvianolate on neuronal injury after OGD was dose dependent. We further found that salvianolate could reduce the increase in NO and MDA levels in PC12 cells induced by OGD, suggesting that the protective effect of salvianolate on neuronal injury might be related to oxidative stress. Mitochondria are the main source of reactive oxygen species. In an oxygen-glucose-deprived PC12 cell model, glucose deficiency prevents cells from entering the tricarboxylic acid cycle (TCA cycle) and cannot provide nicotinamide adenine dinucleotide (NADH). The absence of NADH reduces the activity of mitochondrial complex enzymes and hinders the electron transfer on the mitochondrial membranes. OGD leads to a decrease in the activity of mitochondrial complex I and II, which causes the production of a number of reactive oxygen species (Zhu et al., 2019; Fernandez et al., 2019). According to our study, the neuroprotective effect of salvianolate is achieved by reducing the level of reactive oxygen species, increasing the expression of Bcl-2 protein and inhibiting the activation of apoptotic signalling pathway (Yan et al., 2010; Chen et al., 2011). We know that Caspase-3 protein plays an important role in apoptosis, which is closely related to DNA damage and mitochondrial destruction (Meng et al., 2019). Hypoxia in cells induces a series of cellular oxidation processes. These processes can destroy cell glycolysis; that is, glucose deprivation can induce adenine nucleoside 16/27

triphosphate (ATP) to convert to adenosine diphosphate (ADP), ultimately leading to cell apoptosis. We found the salvianolate could improve cell viability after OGD; we thus suspected that salvianolate exerted an anti-apoptosis effect by inhibiting the Caspase-3 signalling pathway (Zhang et al., 2019). We found that salvianolate could improve the survival rate of cells after OGD, and because our research found that salvianolate could also inhibit the expression of Caspase-3 and Caspase-9 proteins, we believe that salvianolate played an anti-apoptotic role by inhibiting the Caspase-3 signalling pathway. At the same time, in the MCAO model of ischaemia-reperfusion injury, we found that intraperitoneal injection of salvianolate could protect rats from acute cerebral ischaemia-reperfusion injury, decrease neurological deficits and attenuate the incidence of cerebral infarction. These results suggest that salvianolate has the potential to repair nerve damage and treat neurological impairment by inhibiting the Caspase-3 signalling pathway in rats after cerebral ischaemia-reperfusion injury. 5. Conclusions Our results demonstrated that salvianolate protected against acute ischaemia-reperfusion-induced injury in vitro and in vivo. The protective effect of salvianolate in the nervous system was confirmed using MCAO and OGD models. We provide evidence that salvianolate can prevent neuronal apoptosis and reduce the infarct area. The cognitive and motor activities of rats were also enhanced by salvianolate treatment. In addition, the reactive oxygen species level of PC12 cells was increased by hypoxia; at the same time, these results indicated that the Caspase-3 signalling pathway can be triggered by reactive oxygen species. Last, we also showed that salvianolate can effectively reduce the expression of reactive oxygen species and apoptotic proteins, revealing a potential for salvianolate to be a possible therapeutic for acute cerebral ischaemia-reperfusion injury. Conflict of interests The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. 17/27

Acknowledgements This work was supported by grants from the Shanghai Municipal Education Commission (2019-01-07-00-10-E00072), the State Administration of Traditional Chinese Medicine of People's Republic of China (GZYYGJ2019059) and Science and Technology Commission of Shanghai Municipality (18401933500).

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Scheme. 1. Schematic illustration of the mechanism of salvianolate in the Caspase-3 signalling pathway. The rats were induced ischaemia-reperfusion injury by MCAO, which may induce decreases in mitochondrial complex enzyme activity in neurons. This will then cause a lot of reactive oxygen species production. The salvianolate can reduce production of the reactive oxygen species and inhibit activation of Caspase-3 signalling pathway.

Fig. 1. Effect of salvianolate in rat cerebral ischaemia-reperfusion model. To evaluate the protection effect, different doses of salvianolate was administered for 72 h after rat cerebral ischaemia-reperfusion. The TTC staining of brain tissue in different dose groups (A). The results of infarction rate and neurological deficit score in different dose groups. Afterwards, the 20 mg/kg dose of salvianolate was used subsequent experimental (B-C). The TTC staining of brain tissue in different groups (D). The results of infraction rate and neurological deficit score in different groups (E-F). Brain blood flow image was observed by using a laser speckle system in different groups (G). The date is expressed as the means ± S.E.M. (n = 5 rats in each group). *P < 0.05, **P < 0.01 and ***P < 0.001 vs MCAO group.

Fig. 2. Effect of salvianolate on blood brain barrier disruption and the MMP-2 and MMP-9 expression. The staining and content determination of Evans blue in different groups (A-B). The change of cerebral edema rate in different groups (C). Representative protein (MMP-2and MMP-9) expression in different groups by western blotting analysis (D-G). The date is expressed as the means ± S.E.M. (n = 3 rats in each group). *P < 0.05, **P < 0.01 and ***P < 0.001 vs MCAO group.

Fig. 3. Detection of brain tissue injury in different groups. The histopathological changes in the cortex and striatum area in different groups by Nissl staining method (A). NeuN stain were detected according to immunofluorescence assay (B-C). Expression of NeuN proteins in different groups by western blotting analysis (D-E). The date is expressed as the means ± S.E.M. (n = 3 rats in every groups). *P < 0.05, 25/27

**P < 0.01 and ***P < 0.001 vs MCAO group.

Fig. 4. Effect of salvianolate on tissue apoptosis and expression of related apoptotic proteins. Fluorescence intensity of apoptosis expression in different groups by TUNEL assay (A-B). Representative protein (Bax, Caspase-3, Caspase-9 and Bcl-2) expression in different groups by Western blotting analysis (C-F). The date is expressed as the means ± S.E.M. (n = 3 rats in every groups). *P < 0.05, **P < 0.01 and ***P < 0.001 vs MCAO group.

Fig. 5. Long-term effect of salvianolate in cerebral ischaemia-reperfusion model. Long-term experimental arrangement and progress diagram (A). The typical swim path in different groups by Morris water maze (B). The change of continues neurological deficit score in different groups (C). The times of escape latency in different groups (D). The times of passed platform in different groups (E). Time to retention in different groups by Rota-Rod experiment (F). The date is expressed as the means ± S.E.M. (n = 5 in every groups, n = 3 in the edaravone group). *P < 0.05, **P < 0.01 and ***P < 0.001 vs MCAO group.

Fig. 6. Effect of salvianolate on PC12 cells after OGD. The cell viability of different doses (0-40 µM) salvianolate on the PC12 cells (A). The cell viability of suitable doses range (0-10 µM) in the PC12 cell after oxygen-glucose deprivation for 4 h (B). The cell viability of in different groups after treatment (C). Fluorescence images of reactive oxygen species expression in the different groups by laser scanning confocal microscope (D). Fluorescence intensity of reactive oxygen species expression in different groups by fluorescent microplate reader (E). Levels of NO, MDA and LDH concentration in different groups (F-H). The date is expressed as the means ± S.E.M. (n = 5 in every groups). *P < 0.05, **P < 0.01 and ***P < 0.001 vs model group.

Fig. 7. Anti-apoptosis effect of salvianolate on PC12 cells after OGD. The changes 26/27

of apoptotic bodies in different groups by Hoechst 33342 staining (A-B). The apoptotic rate in different groups by flow cytometry (C-D). The date is expressed as the means ± S.E.M. (n = 5 in every groups). *P < 0.05, **P < 0.01 and ***P < 0.001 vs model group.

Fig. 8. Effect of salvianolate on the Caspase-3 pathway in vitro. The PC12 cells were treated with salvianolate or edaravone for 24 h after OGD 4 h. Representative protein expression levels in different groups (HIF-1α, Bcl-2, Bax, Caspase-3, Caspase-9, Cleaved-caspase-3 and Cleaved-caspase-9) by western blotting analysis (A-F). The date is expressed as the means ± S.E.M. (n = 5 in every groups). *P < 0.05, **P < 0.01 and ***P < 0.001 vs model group.

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Highlights


Salvianolate (salvia) significantly reduced infarct area and neurological deficit score after cerebral ischaemia-reperfusion injury (CIR).




Long-term administration of Salvia improved neurocognitive and motor function after CIR.




Salvia increased Bcl-2 protein and decreased Bax protein expression.




Salvia reduced the expression of oxidative damage related factors (NO, MDA, LDH).




Salvia inhibited the activation of ROS-mediated apoptotic signaling pathway.

Author Agreement All authors have read and approved to submit it to your journal. There is no conflict of interest of any authors in relation to the submission. This paper has not been submitted elsewhere for consideration of publication. Author Contributions Section Pengwei Luan, Jiazhen Xu, Guoqiang Lin, Ping Tian and Jiange Zhang conceived and coordinated the study. Pengwei Luan, Jiazhen Xu, Xinyue Ding and Jiange Zhang designed and did the pharmacology experiments; while Jiange Zhang provided technical assistance. Pengwei Luan, Qianfei Cui, Lixian Jiang, Yulan Xu analyzed the data. Yuying Zhu, Ruixiang Li and Jiange Zhang provided language modification. All authors reviewed the results and approved the final version of the paper.