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Tanshinone inhibits neuronal cell apoptosis and inflammatory response in cerebral infarction rat model

We aimed to investigate the effect and mechanisms of tanshinone (TSN) IIA in cerebral infarction. The cerebral infarction rat model was established by middle cerebral artery occlusion (MCAO). After pretreatment with TSN, cerebral infarct volume, cerebral edema, and neurological deficits score were evaluated, as well as cell apoptosis in hippocampus and cortex of the brain was examined with terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and the levels of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and C-reactive protein (CRP) were determined by Enzyme-Linked Immunosorbent Assay (ELISA). In addition, rat primary neuronal cells were isolated and cultured in oxygen-glucose deprivation (OGD) conditions. After pretreatment with TSN, cell viability and apoptosis were observed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and flow cytometry analysis, respectively. The expressions of Bax and B-cell lymphoma 2 (Bcl-2) were detected by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and western blotting. Compared with untreated cerebral infarction rat, TSN treatment significantly reduced cerebral infarct volume, cerebral edema, and neurological deficits score (P < 0.05). Cell apoptosis as well as the levels of IL-6, TNF-α, and CRP in hippocampus and cortex of cerebral infarction rat were inhibited after pretreatment with TSN (P < 0.05). Furthermore, TSN remarkably increased cell viability and inhibited cell apoptosis ratio (P < 0.05) in OGD-induced rat neuronal cells. Besides, TSN significantly downregulated the expression of Bax and upregulated Bcl-2 (P < 0.05). TSN IIA has a preventive effect on cerebral infarction by inhibiting neuronal cell apoptosis and inflammatory response in vitro and in vivo.

Cerebral infarction is a severe cardiovascular and cerebrovascular disease, which often occurs due to brain hypoxic-ischemic necrosis, resulting in embolism and thrombosis. 1 Although thrombectomy 2 and drug therapy 3 have been widely applied to clinical therapy of cerebral infarction, the therapeutic effect is still limited. It has been well known that accelerated neuronal cell apoptosis and inflammatory response have been proved to be associated with the cerebral infarction. 4,5 Accordingly, drugs target to cell apoptosis and inflammatory response may be an effective treatment strategy in cerebral infarction.
Tanshinone (TSN) IIA is a lipophilic chemical found in Salvia miltiorrhiza. 6 TSN has been revealed to be associated with cell lipid peroxidation, methyl guanine transferase activity, cell apoptosis, and anti-inflammatory effects. 7,8 Previous study showed that TSN was able to protect against brain injury. 6 However, the mechanisms of TSN in cerebral infarction remain to be fully investigated.
Thus, this study established cerebral infarction rat model and oxygen-glucose deprivation (OGD) neuronal cell model and then evaluated the effects of pretreated TSN on cerebral infarct volume, cerebral edema, neurological deficits, cell apoptosis, and inflammatory response in cerebral infarction rat, which aimed to uncover the effects and mechanisms of TSN in cerebral infarction in vitro and in vivo.

Animal model
This study was approved by the local Animal Ethics Committee of the Animal Laboratory Center. A total of 45 healthy Sprague-Dawley male rats (250-280 g) were acclimatized under appropriate condition for a week before the experiment and then were randomly and equally assigned to three groups (n = 15 for each group): sham group, middle cerebral artery occlusion (MCAO) group, and TSN group. Before the MCAO operation, TSN (5 mg/kg) was intraperitoneally injected daily into rats in the TSN group for 7 days. Then, MCAO was performed in rats of MCAO and TSN groups. In brief, the rats were anesthetized using 5% chloral hydrate by intraperitoneal (i.p.) injection, and then an incision was cut in the cervical midline. The exposed external carotid arteries were closed by sterile suture, and the vascular clamp was used to clamp internal carotid artery. Subsequently, an incision in the common carotid artery was made, and then a silicone-coated suture (4-0) was inserted for ligation. After 2 h, the rats underwent reperfusion. Once the symptoms such as left hemiparesis, contralateral forelimb sagging, and standing instability were appeared in rats, the cerebral infarction model rats were considered to be successfully constructed. All the procedures of MCAO were performed in rats of the sham group but without the ligation. All the rats were sacrificed after the cerebral infarction model was induced. The hippocampus and cortex were collected.

Evaluation of cerebral edema
The brain water content was measured to evaluate cerebral edema. After reperfusion for 24 h, brain tissue samples were collected from cerebral infarct areas in rats of the MCAO and TSN groups and from corresponding areas in rats of the sham group. The wet weight was obtained from untreated samples and the dry weight was obtained from dried sample using a desiccating oven. Brain water content was measured with (wet weight − dry weight) × 100/wet weight.

Assay of cerebral infarct volume
After reperfusion for 6 h, 24 h, 3 days, 7 days, and 14 days, the rats were sacrificed by chloral hydrate and the brains were cut rapidly. Then forebrains were dissected into six sections in coronal plane (2-mm thick), and each section was stained with 2% 2,3,5-triphenyltetrazolium chloride (TTC) (Sigma, St. Louis, MO, USA) for 5 min. Image J analysis software (V1.6; NIH, Bethesda, MD, USA) was used to measure the cerebral infarct volume in each slice.

Neurological evaluation
After reperfusion for 24 h, neurological deficits were evaluated according to the previously described procedure. 9 Score was defined as following-0: no neurologic deficit; 1: fail to fully extend left forepaw; 2: circle to the left forepaw; 3: fall to the left forepaw; and 4: fail to walk spontaneously and have a depression.

TUNEL assay
The rat hippocampus and cortical tissues were fixed in 4% paraformaldehyde and then paraffinembedded. The sections were then dewaxed and dehydrated. The TUNEL method was used to detect DNA fragmentation in tissue cells by In Situ Cell Death Detection Kit (Roche, Mannheim, Germany). In brief, the sections were reacted with reaction mixture in dark at 37°C for 60 min and fluorescence microscope (Olympus, Tokyo, Japan) was used to observe fluorescence. The cells with positive signals in each sample were counted in 10 randomly selected visual fields.

Detection of inflammatory factor levels
The cortical tissue was placed in ice-cold homogenization buffer and then supernatants were obtained by centrifugation at 3000 r/min for 15 min. The levels of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and C-reactive protein (CRP) were detected using the corresponding Enzyme-Linked Immunosorbent Assay (ELISA) Kits (Shanghai Kexing Biochemical Reagent Co., Ltd, China) according to the manufacturer's instructions.

Cell culture and treatment
Five newborn rats, approximately 1-day old, were used to isolate the primary neuronal cells. Briefly, rats were anesthetized and sacrificed by cervical dislocation. The hippocampal tissues were collected and then digested using 0.05% trypsin. After centrifugation, the cell deposits (neuronal cells) were cultured in Dulbecco's Modified Eagle Medium (DMEM) containing 5% fetal bovine serum (Gibco, Carlsbad, CA, USA). The neuronal cells were divided into control group, OGD group, and TSN group. Cells in the TSN group were cultured in DMEM with 5 µg/mL TSN for 5 days. Then, OGD was performed in the OGD and TSN groups. In brief, the medium was changed with glucose-free medium to induce the final glucose concentration <1 mM. Both cells in the OGD and TSN groups were cultured under the condition of 95% N 2 and 5% CO 2 , while cells in the control group were cultured in 5% CO 2 . After culturing for 4 h, the medium was changed with the original conditions.

Cell apoptosis assay
Cell apoptosis was evaluated using Annexin V-FITC Apoptosis Detection kit (BD, San Jose, CA, USA). Briefly, after 72 h of treatment, cells were collected and washed with cold phosphatebuffered saline (PBS). Then, the cells were added into Binding Buffer, and then Annexin V-FITC and propidium iodide (PI) were incubated with cells in dark for 15 min. Cells apoptotic rate was calculated according to flow cytometer (BD).

Western blotting
The cells in the three groups were placed on ice for 30 min in lysis buffer (Beyotime). Supernatant was acquired by centrifugation at 12,000 r/min for 20 min at 4°C. Protein sample was separated on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel and then transferred onto polyvinylidene fluoride (PVDF) membranes. Defatted milk (5%) was used to block the membranes at room temperature for 1 h and then the membranes were incubated with goat anti-rat GAPDH monoclonal antibody (1:1000, Santa Cruz, Santa Cruz, CA, USA), goat anti-rat BCL-2 polyclonal antibody (1:500, Santa Cruz), and goat anti-rat Bax polyclonal antibody (1:500, Santa Cruz) at 4°C overnight. After being washed for three times with PBS, the membranes were incubated with donkey anti-goat IgG (H+L)-horseradish peroxidase (HRP) (1:5000, Santa Cruz) for 2 h at 25°C. Ultimately, the proteins were detected with enhanced chemiluminescence (ECL; Applygen Technologies Inc., Beijing, China).

Statistical analysis
SPSS 19.0 statistical analysis software (SPSS Inc., Chicago, IL, USA) was used to analyze data. Continuous variables were expressed as the mean ± standard deviation (SD) and analyzed by t test and one-way analysis of variance (ANOVA). A value of P < 0.05 was considered statistically significant.

TSN alleviates cerebral infarction in rat
To investigate the effect of TSN on cerebral infarction in rat, cerebral infarct volume, cerebral edema, and neurological deficits were evaluated. The results showed that compared with the sham group, brain water content, cerebral infarct volume, and neurological deficits scores were significantly increased in the MCAO group (all P < 0.01; Figure 1(a)-(c)). Pretreatment with TSN remarkably reduced brain water content (P < 0.05), cerebral infarct volume (P < 0.01), and neurological deficits scores (P < 0.05) than those in the MCAO group.

TSN inhibits neuronal cell apoptosis in brain of cerebral infarction rat
To investigate the protection mechanism of TSN in cerebral infarction rat, neuronal cell apoptosis in brain was evaluated using TUNEL method. The results revealed that MCAO operation significantly promoted neuronal cell apoptosis in hippocampus and cortical tissues compared with the sham group (all P < 0.01; Figure 2(a) and (b)), while cell apoptosis was significantly lower in the TSN group than in the MCAO group in hippocampus and cortical tissues (P < 0.05 or P < 0.01). Figure 2(c) shows the representative images of TUNEL assay in cortical tissues.

TSN inhibits cell apoptosis in primary rat neuronal cells
In addition to in vivo experiments, OGD cell model was performed to mimic hypoxic-ischemic environment in primary rat neuronal cells. MTT results found that after 24, 48, or 72 h of treatment, cell viability was significantly lower in the OGD group than in the control group (P < 0.05 or P < 0.01; Figure  3(a)), while pretreatment with TSN obviously improved cell viability compared with the OGD group (P < 0.05 or P < 0.01; Figure 3(a)). Flow cytometry results showed that compared with the control group, cell apoptotic rates were significantly increased in the OGD group (P < 0.01; Figure 3(b) and (c)), while TSN remarkably inhibited in the TSN group in comparison with the OGD group (P < 0.01; Figure 3(b) and (c)). In addition, both the mRNA and protein levels of anti-apoptotic protein Bcl-2 were significantly inhibited and pro-apoptotic protein Bax was obviously increased in the OGD group compared with the control group (P < 0.01; Figure 3(d)-(f)), while TSN remarkably reversed these changes in the expressions of Bcl-2 and Bax compared with the OGD group (P < 0.05 or P < 0.01; Figure 3(d)-(f)).

TSN inhibits the levels of inflammatory factors in brain of cerebral infarction rat
Furthermore, we evaluated the effect of TSN on inflammatory response in cerebral infarction rat.
ELISA results revealed that TNF-α, CRP, and IL-6 levels were all higher in the MCAO group than in the sham group (P < 0.01), while reduced levels of TNF-α, CRP, and IL-6 were found in the TSN group than in the MCAO group (P < 0.05 or P < 0.01; Figure 4(a)-(c)).

Discussion
Previous studies had shown the neuroprotective effects of TSN on mice focal cerebral ischemia and focal cerebral ischemic/reperfusion injury. 10 Similarly, in the study, TSN was proved to protect against rat cerebral infarction, including cerebral infarct volume, cerebral edema, and neurological deficits, both in vitro and in vivo in this study.  It has been well acknowledged that cell apoptosis and inflammation are critical regulators of cerebral infarction. Bcl-2 family plays a vital role in regulating cell apoptosis, which included various cell apoptosis-related factors, such as Bcl-2 and Bax. 11 The Bcl-2/Bax ratio is important to regulate cell survival and death. 11,12 Our study suggested that the downregulated Bax and upregulated Bcl-2 were consistent with the antiapoptotic role of TSN in hippocampus and cortex of cerebral infarction rat and OGD neuronal cells. Triterpenoid-enriched extract of S. miltiorrhiza also reported to reduce the aortic atherosclerotic lesion, which might be associated with downregulated CRP and monocyte chemotactic protein. 13 In addition, TSN could inhibit the expressions of TNF-α, IL-1β, and IL-6 in activated macrophages induced by lipopolysaccharide. 14 Similarly, our study found that the pretreatment with TSN could inhibit the levels of IL-6, TNF-α, and CRP in hippocampus and cortex of cerebral infarction rat, prompting that the protective role of TSN against cerebral infarction might be closely associated with inflammation reaction. In conclusion, TSN IIA has a preventive effect on cerebral infarction by inhibiting neuronal cell apoptosis and inflammatory response in vitro and in vivo. These data may provide potential therapeutic strategies for cerebral infarction.