Sodium tanshinone IIA sulfonate protects against Aβ‐induced cell toxicity through regulating Aβ process

Abstract Sodium tanshinone IIA sulfonate (STS) has been reported to prevent Alzheimer's disease (AD). However, the mechanism is still unknown. In this study, two in vitro models, Aβ‐treated SH‐SY5Y cells and SH‐SY5Y human neuroblastoma cells transfected with APPsw (SH‐SY5Y‐APPsw cells), were employed to investigate the neuroprotective of STS. The results revealed that pretreatment with STS (1, 10 and 100 µmol/L) for 24 hours could protect against Aβ (10 µmol/L)‐induced cell toxicity in a dose‐dependent manner in the SH‐SY5Y cells. Sodium tanshinone IIA sulfonate decreased the concentrations of reactive oxygen species, malondialdehyde, NO and iNOS, while increased the activities of superoxide dismutase and glutathione peroxidase in the SH‐SY5Y cells. Sodium tanshinone IIA sulfonate decreased the levels of inflammatory factors (IL‐1β, IL‐6 and TNF‐α) in the SH‐SY5Y cells. In addition, Western blot results revealed that the expressions of neprilysin and insulin‐degrading enzyme were up‐regulated in the SH‐SY5Y cells after STS treatment. Furthermore, ELISA and Western blot results showed that STS could decrease the levels of Aβ. ELISA and qPCR results indicated that STS could increase α‐secretase (ADAM10) activity and decrease β‐secretase (BACE1) activity. In conclusion, STS could protect against Aβ‐induced cell damage by modulating Aβ degration and generation. Sodium tanshinone IIA sulfonate could be a promising candidate for AD treatment.

treatment were reported failed, such as solanezumab, a monoclonal antibody targeting Aβ peptide. 10 Thus, finding new therapeutic drugs is urgent.
Natural products are large potential sources of compounds for AD treatment. 11 Sodium tanshinone IIA sulfonate (STS) is a derivative of Tanshinone IIA, which extracted from the dried roots of Danshen (Salvia miltiorrhiza). A large number of studies have shown that STS could protect against cardiovascular diseases. [12][13][14][15] Besides the well-known cardioprotective effect, STS possesses neuroprotective activity against neural dysfunction. [16][17][18] Previous studies suggested that STS have some pharmacological actions, such as anti-oxidative stress, 19,20 anti-inflammation. 13,21 However, STS has not yet been reported to have any Aβ-regulation effect. Considering during AD process, Aβ aggregation can damage and cause neuronal death by inducing oxidative stress and neuroinflammation. 22,23 Therefore, it was hypothesized that STS could display the neuroprotective effects through modulating Aβ process.
In this study, two in vitro models, Aβ-treated SH-SY5Y cells and SH-SY5Y human neuroblastoma cells transfected with APPsw (SH-SY5Y-APPsw cells), were employed to investigate the neuroprotective of STS. Different doses (1, 10 or 100 µmol/L) of STS were used to treat cells. We revealed that STS could obviously protect against Aβ-induced cell toxicity through modulating Aβ degration and generation.

| Cell viability
The SH-SY5Y cells were seeded in the 96-well plates. The cell viability was measured by MTT assay.

| Reactive oxygen species (ROS) level
The cells were collected and centrifuged. The 2′7′-dichlorofluorescein diacetate (DCFH-DA) fluorescent dye method (Invitrogen) was used to measure the ROS level. The presence of ROS can convert non-fluorescent DCFH-DA to fluorescent dichlorofluorescein (DCF).

| Malondialdehyde (MDA) level
The cells were collected to detect the level of MDA by using the kit (Nianjing Jiancheng Bioengineering Institute) according to the manufacturer's instructions.

| Superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities
The cells were collected to detect the activities of SOD and GSH-Px by using the kits (Nianjing Jiancheng Bioengineering Institute) according to the manufacturer's instructions.

| Nitric oxide (NO) level
The cells were collected to detect the level of NO by using the kit (Abcam) according to the manufacturer's instructions.

| ELASA
The SY5Y cells were collected. The concentrations of IL-1β, IL-6, TNF-α and iNOS were measured by using the ELISA kits (Thermo Fisher Scientific) according to the manufacturer's instructions.
The SH-SY5Y-APPsw cells were collected. The levels of Aβ1-40 and Aβ1-42 were measured by using the ELISA kits (Invitrogen) according to the manufacturer's instructions. The activities of α-, β-and γ-secretase were measured by using the ELISA kits (R&D Systems) according to the manufacturer's instructions.

| Western blot analysis
The cells were collected and lysed in RIPA buffer. The lysate was collected and extracted the protein.

| Statistical analysis
The data were analysed by using Student's t test and ANOVA by SPSS 19.0 statistical software (IBM). The results were expressed as the mean ± SEM. The differences were considered as statistically significant at P < .05.

| STS ameliorates Aβ-induced cell toxicity in SH-SY5Y cells
In order to prove whether STS had the neuroprotective effect, the Aβ-

| STS ameliorates oxidative stress, nitrosative stress and neuroinflammation in Aβ-treated SH-SY5Y cells
Oxidative and nitrosative stress statements are observed in the AD patients' brain. 24,25 As shown in Figure 3, oxidative stress (increased levels of ROS and MDA, decreased activities of SOD and GSH-Px) and nitrosative stress (increased levels of NO and iNOS) were observed in Aβ-treated SH-SY5Y cells. While, STS pretreatment decreased the levels of ROS, MDA, NO and iNOS, and increased the activities of SOD and GSH-Px significantly. In addition, Aβ accumulation can also induce neuroinflammation in the AD patients' brain. 26,27 In this study, Aβ-treatment increased the neuroinflammatory factors (IL-1β, IL-6 and TNF-α) in SH-SY5Y cells. Sodium tanshinone IIA sulfonate pretreatment significantly decreased these neuroinflammatory factors (Figure 4). These findings suggested that the neuroprotective effects of STS might be in connection with its anti-oxidative and nitrosative stress and antiinflammatory abilities.

| STS improves the expressions of Aβ-degrading enzymes in Aβ-treated SH-SY5Y cells
NEP and IDE are two important Aβ-degrading enzymes in the cell. 28,29 As shown in Figure 5

| STS inhibits Aβ generation in SH-SY5Y-APPsw cells
Furthermore, we also studied the effect of STS on Aβ generation.
A SH-SY5Y cell line overexpressing the human APP Swedish mutant (SH-SY5Y-APPsw) was used for investigation. The levels of Aβ1-40 and Aβ1-42 were significantly decreased after STS treatment in a dose-dependent manner ( Figure 6A,B). Western blot result also showed the same effect ( Figure 6C,D). We next studied the effect of STS on APP cleavage process. Amyloid precursor protein is mainly cleaved by three enzymes, α-, β-and γ-secretases. ELISA results showed that STS increased α-secretase activity and decreased β-secretase activity, while did not affect the γ-secretase activity ( Figure 7A-C). Inconsistently, qPCR results indicated that STS increased ADAM10 and decreased BACE1 mRNA expression.

| D ISCUSS I ON
In this study, we studied the neuroprotective effect of STS against  Amyloid precursor protein (APP) is an integral membrane protein, which can be cleaved by α-(the extracellular region), β-(the extracellular region) and γ-secretase enzymes. Aβ is the cleaved product of APP by β-and γ-secretase under pathological conditions. Amyloid precursor protein is mainly cleaved by α-secretase and γ-secretase under normal physiological conditions. 42 The cleavage by α-secretase can prevent the generation of Aβ. Thus, increasing ADAM10 (α-secretase), or inhibiting of BACE1 (β-secretase), can avoid Aβ generation. In our study, STS could decrease the levels of Aβ1-40 and Aβ1-42 in SH-SY5Y-APPsw cells. Both ELISA and qPCR results found that STS increased the activity of α-secretase and decreased the activity of β-secretase. However, STS did not affect the activity of γ-secretase. In addition, Aβ clearance is another important pathway to protect against AD process. Aβ can be cleared through the intracellular pathway or the extracellular pathway. Blood-brain barrier (BBB) transport redominates the extracellular pathway. 43 Intracellular pathway is mainly occurred in neurons or glia. 44 The enzymatic breakdown-induced degradation clearance is the major pathway, including NEP and IDE. [45][46][47] In this study, STS increased the protein expressions of NEP and IDE in SH-SY5Y cells. The above results demonstrated that STS could protect against Aβ-induced cell toxicity through modulating Aβ degration and generation.
In conclusion, the study provides some evidence that STS can alleviate Aβ-induced cell toxicity by inhibiting oxidative stress and neuroinflammation. This neuroprotective effect of STS might be through regulating Aβ degration and generation. Sodium tanshinone IIA sulfonate might be developed as a new anti-AD drug. However, whether STS could affect Aβ transport is still unknown. Further studies are needed.

ACK N OWLED G EM ENTS
This work was supported by National Natural Science Foundation of China (No. 81774103).

CO N FLI C T O F I NTE R E S T
The authors declare that they have no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data used to support the findings of this study are available from the corresponding author upon request.