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. 2019 Nov 11:2019:7850154.
doi: 10.1155/2019/7850154. eCollection 2019.

Neuroprotective Effect of SCM-198 through Stabilizing Endothelial Cell Function

Qiu-Yan Zhang  1   2 , Zhi-Jun Wang  2   3 , Lei Miao  4 , Ying Wang  5 , Ling-Ling Chang  2 , Wei Guo  2 , Yi-Zhun Zhu  2   3
Affiliations

Neuroprotective Effect of SCM-198 through Stabilizing Endothelial Cell Function

Qiu-Yan Zhang et al. Oxid Med Cell Longev. .

Abstract

Leonurine, also named SCM-198, which was extracted from Herba leonuri, displayed a protective effect on various cardiovascular and brain diseases, like ischemic stroke. Ischemic stroke which is the leading cause of morbidity and mortality, ultimately caused irreversible neuron damage. This study is aimed at exploring the possible therapeutic potential of SCM-198 in the protection against postischemic neuronal injury and possible underlying mechanisms. A transient middle cerebral artery occlusion (tMCAO) rat model was utilized to measure the protective effect of SCM-198 on neurons. TEM was used to determine neuron ultrastructural changes. The brain slices were stained with Nissl staining solution for Nissl bodies. Fluoro-Jade B (FJB) was used for staining the degenerating neurons. In the oxygen-glucose deprivation and re-oxygenation (OGD/R) model of bEnd.3 cells treated with SCM-198 (0.1, 1, 10 μM). Then, the bEnd.3 cells were cocultured with SH-SY5Y cells. Cell viability, MDA level, CAT activity, and apoptosis were examined to evaluate the cytotoxicity of these treatments. Western blot and immunofluorescent assays were used to examine the expression of protein related to the p-STAT3/NOX4/Bcl-2 signaling pathway. Coimmunoprecipitation was performed to determine the interaction between p-STAT3 and NOX4. In the transient middle cerebral artery occlusion (tMCAO) rat model, we found that treatment with SCM-198 could ameliorate neuron morphology and reduce the degenerating cell and neuron loss. In the in vitro model of bEnd.3 cell oxygen-glucose deprivation and reoxygenation (OGD/R), treatment with SCM-198 restored the activity of catalase (CAT), improved the expression of Cu-Zn superoxide dismutase (SOD1), and decreased the malondialdehyde (MDA) production. SCM-198 treatment prevented OGD/R-induced cell apoptosis as indicated by increased cell viability and decreased the number of TUNEL-positive cells, accompanied with upregulation of Bcl-2 and Bcl-xl protein and downregulation Bax protein. The results were consistent with SH-SY5Y cells which coculture with bEnd.3 cells. The forthcoming study revealed that SCM-198 activated the p-STAT3/NOX4/Bcl-2 signaling pathway. All the data indicated that SCM-198 protected against oxidative stress and neuronal damage in in vivo and in vitro injury models via the p-STAT3/NOX4/Bcl-2 signaling pathway. Our results suggested that SCM-198 could be the potential drug for neuroprotective effect through stabilizing endothelial cell function.

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Conflict of interest statement

The authors declare that there is no conflict of interest.

Figures

Figure 1
Figure 1
The protection of SCM-198 on neuron morphology after ischemic stroke. (a) The representative TEM of neurons in the peri-ischemic region in the tMCAO model. SCM-198 diminished the changes in neuron morphology after I/R injury. Scale bar = 5 μm and 500 nm, n = 3. (b) Representative pictures of coronal sections from the ischemic rat brain stained with Nissl staining. SCM-198 reduced cell shrinkage and empty spaces. Scale bar = 20 μm (n = 5).
Figure 2
Figure 2
SCM-198 reduced neuron loss after I/R insult. (a) FJB staining of brain sections after reperfusion. Representative pictures of FJB staining of brain sections after reperfusion. No FJB-positive cells were found in the control group. Vast degenerating neurons were detected in the peri-ischemic regions of the tMCAO group. SCM-198 significantly reduced the number of degenerating neurons. (b) The quantitative analysis of the number of degenerating neurons. Scale bar = 50 μm. Values are expressed as mean ± SD. #p < 0.05 versus control group, ∗p < 0.05 versus tMCAO group (n = 5). (c) Immunofluorescence staining for NeuN after ischemia reperfusion. SCM-198 could reduce neuron loss in the ipsilateral brain cortex. Scale bar = 50 μm.
Figure 3
Figure 3
SCM-198 improved bEnd.3 cell antioxidative capacity in vitro. SCM-198 exhibited antioxidant abilities and improved the cell viability of bEnd.3. (a) Cell viability, evaluated by an MTT assay, was significantly reduced after OGD/R injury exposure, while 1 μM and 10 μM of SCM-198 could increase cell viability. (b) MDA level of the SCM-198 group was remarkably decreased as compared to the OGD/R group. (c) SCM-198 could predominantly increase intercellular antioxidative capacity by restoring the CAT activity. (d) SCM-198 obviously improved the expression of SOD1. (e) SCM-198 reduced the cell apoptosis in bEnd.3. OGD/R-induced cell apoptosis was determined by TUNEL staining. (f) The result showed that OGD/R obviously increased the apoptosis ratio, whereas treatment with SCM-198 reduced cell apoptosis. Values are expressed as mean ± SD. #p < 0.05 versus control group, ∗p < 0.05 versus OGD group (n = 3).
Figure 4
Figure 4
SCM-198 protected neurons via modulating BMECs in BMEC/neuron coculture system. bEnd.3 treated with SCM-198 and cocultured with SH-SY5Y exhibited protection against OGD/R injury by improving the cell viability and the antioxidative ability. (a) Treatment with SCM-198, especially 1 μM and 10 μM, increased bEnd.3 cell viability in OGD/R irritation. (b) SCM-198 could reduce the LDH leakage in SH-SY5Y cells. (c) SCM-198 decreased the production of MDA in SH-SY5Y cells after OGD/R injury. (d) SCM-198 increased the activity of CAT. (e) SCM-198 reduced cell apoptosis in SH-SY5Y. OGD/R-induced cell apoptosis was determined by TUNEL staining; the result showed that OGD/R obviously increased the number of apoptosis, whereas treatment with SCM-198 reduced cell apoptosis. (f) The quantitative analysis of apoptotic cells was calculated. Values are expressed as mean ± SD. #p < 0.05 versus control group, ∗p < 0.05 versus OGD group (n = 3).
Figure 5
Figure 5
The mechanism of SCM-198 inhibited apoptosis induced by OGD/R. (a–c) SCM-198 regulated the expression of apoptosis-related protein in bEnd.3. OGD/R increased the expression of Bax. OGD/R also decreased the expression of Bcl-2 and Bcl-xl, while SCM-198 could markedly improve the expression of Bcl-2 and Bcl-xl and reduce Bax expression. (d, e) SCM-198 regulated the expression of apoptosis-related protein in SH-SY5Ycells. OGD/R increased the expression of Bax and decreased the expression of Bcl-2 and Bcl-xl, while SCM-198 could obviously improve the expression of Bcl-2 and Bcl-xl and reduce Bax expression. (g–i) SCM-198 regulated the expression of p-STAT3, NOX4, and p-Akt in bEnd.3. SCM-198 protected against apoptosis through improving the level of p-STAT3 and inhibiting the expression of NOX4, then modulated p-Akt. Values are expressed as mean ± SD. #p < 0.05 versus control group, ∗p < 0.05 versus OGD group (n = 3).
Figure 6
Figure 6
SCM-198 inhibited apoptosis through the STAT3/NOX4/Bcl-2 pathway. (a) SCM-198 (10 μM) still observably decreased the overexpression of NOX4 induced by WP1066. (b) SCM-198 (10 μM) improved the expression of p-Akt. (c–e) SCM-198 (10 μM) could enhance the protection against apoptosis after inhibiting the activation of p-STAT3 in bEnd.3. (f) SCM-198 (10 μM) improved the expression of SOD1 after using WP1066. Values are expressed as mean ± SD. #p < 0.05 versus control group, ∗p < 0.05 versus OGD group (n = 3).
Figure 7
Figure 7
SCM-198 upregulates the interaction between p-STAT3 and NOX4. (a, b) The interactions between p-STAT3 and NOX4 were confirmed by immunoprecipitation. Values are expressed as mean ± SD. ∗p < 0.05 versus IgG group (n = 3).
Figure 8
Figure 8
SCM-198 could be the potential drug for neuroprotective effect through stabilizing endothelial cell function.
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