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. 2020 Jan 22:2020:9049614.
doi: 10.1155/2020/9049614. eCollection 2020.

Salvianolic Acid D Alleviates Cerebral Ischemia-Reperfusion Injury by Suppressing the Cytoplasmic Translocation and Release of HMGB1-Triggered NF- κ B Activation to Inhibit Inflammatory Response

Wen Zhang  1   2 , Junke Song  1   2 , Wan Li  1   2 , Dewen Kong  2 , Yu Liang  2 , Xiaoyue Zhao  2 , Guanhua Du  1   2
Affiliations

Salvianolic Acid D Alleviates Cerebral Ischemia-Reperfusion Injury by Suppressing the Cytoplasmic Translocation and Release of HMGB1-Triggered NF- κ B Activation to Inhibit Inflammatory Response

Wen Zhang et al. Mediators Inflamm. .

Abstract

Inflammatory response participates in the overall pathophysiological process of stroke. It is a promising strategy to develop antistroke drugs targeting inflammation. This study is aimed at investigating the therapeutic effect and anti-inflammatory mechanism of salvianolic acid D (SalD) against cerebral ischemia/reperfusion (I/R) injury. A rat middle cerebral artery occlusion/reperfusion (MCAO/R) injury model was established, and an oxygen-glucose deprivation/reoxygenation (OGD/R) injury model was established in PC12 cells. Neurological deficit score, cerebral infarction, and edema were studied in vivo. Cell viability was achieved using the MTT method in vitro. The Bax, Bcl-2, cytochrome c, HMGB1, TLR4, TRAF6, NF-κB p65, p-NF-κB p65, and cleaved caspase-3 and -9 were tested via the Western blot method. Cytokines and cytokine mRNA, including TNF-α, IL-1β, and IL-6, were studied via ELISA and PCR methods. The translocation of HMGB1 and NF-κB were studied by immunofluorescence assay. The HMGB1/NeuN, HMGB1/GFAP, and HMGB1/Iba1 double staining was carried out to observe the localization of HMGB1 in different cells. Results showed that SalD alleviated neurological impairment, decreased cerebral infarction, and reduced edema in I/R rats. SalD improved OGD/R-downregulated PC12 cell viability. SalD also promoted Bcl-2 expression and suppressed Bax, cytochrome c, and cleaved caspase-3 and -9 expression. SalD decreased the intensity of TLR4, MyD88, and TRAF6 proteins both in vivo and in vitro, and significantly inhibited the NF-κB nuclear translocation induced by I/R and OGD/R. What's more, SalD inhibited HMGB1 cytoplasmic translocation in neurons, astrocytes, and microglia in both the cortex and hippocampus regions of I/R rats. In conclusion, SalD can alleviate I/R-induced cerebral injury in rats and increase the PC12 cell viability affected by OGD/R. The anti-inflammatory mechanism of SalD might result from the decreased nuclear-to-cytoplasmic translocation of HMGB1 and the inhibition on its downstream TLR4/MyD88/NF-κB signaling.

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Conflict of interest statement

The authors report no conflicts of interest.

Figures

Figure 1
Figure 1
SalD alleviated the neurological impairment, decreased cerebral infarction, and reduced edema in I/R rats. (a) Neurological deficit score (n = 12). (b) Representative TTC-stained slices. (c) Infarct volume (n=6). (d) Brain water content (n = 6). (e) Representative Evans blue-stained brains and slices. (f) Evans blue content (n = 5). Data were expressed as means ± SD. ##P < 0.01 versus the sham operation group; ∗P < 0.05 and ∗∗P < 0.01 versus the MCAO/R group.
Figure 2
Figure 2
SalD inhibited apoptosis induced by I/R injury in rats. (a) Representative TUNEL/NeuN immunofluorescence photograph. (b) The percent of TUNEL-positive cells. (c) The relative Bax/Bcl-2 ratio (n = 4). (d) The relative intensity of cleaved caspase-3 (n = 4). (e) The relative intensity of cleaved caspase-9 (n = 4). (f) The relative intensity of cytochrome c (n = 4). Proteins were acquired from the cerebral cortex region and assayed by the Western blot method. The intensity was normalized to β-actin. Data were expressed as means ± SD. ##P < 0.01 versus the sham operation group; ∗P < 0.05 and ∗∗P < 0.01 versus the MCAO/R group. Scale bar = 50 μm.
Figure 3
Figure 3
SalD reduced the inflammatory response induced by I/R injury in rats. (a) SalD inhibited the astrocytes and microglia activation in I/R rats. Representative figures showing GFAP and Iba1 staining. (b) SalD downregulated the generation of TNF-α, IL-1β, and IL-6 in I/R rats. (c) SalD attenuated the upregulated release of serum HMGB1 in I/R rats. Data are expressed as means ± SD. ##P < 0.01 versus the sham operation group; ∗P < 0.05 and ∗∗P < 0.01 versus the MCAO/R group. Scale bar = 50 μm.
Figure 4
Figure 4
SalD increased cell viability, reduced LDH release, and inhibited apoptosis and the inflammatory response induced by OGD/R injury in PC12 cells. (a) Cell viability (n = 4). (b) LDH release (n = 4). (c) The relative Bax/Bcl-2 ratio (n = 4). (d) The relative intensity of cleaved caspase-3 (n = 4). (e) The relative intensity of cleaved caspase-9 (n = 4). (f) The relative intensity of cytochrome c (n = 4). (g) The generation of TNF-α, IL-1β, and IL-6 cytokines (n = 6). (h) The transcription of TNF-α, IL-1β, and IL-6 mRNA (n = 4). Proteins were acquired from PC12 cells and assayed by the Western blot method. The intensity was normalized to β-actin. Data were expressed as means ± SD. ##P < 0.01 versus the control group; ∗P < 0.05 and ∗∗P < 0.01 versus the OGD/R group.
Figure 5
Figure 5
SalD inhibited HMGB1-triggered NF-κB activation in OGD/R-injured PC12 cells. (a) The relative intensity of TLR4 (n = 4). (b) The relative intensity of MyD88 (n = 4). (c) The relative intensity of TRAF6 (n = 4). (d) The relative p-NF-κB p65/NF-κB p65 ratio (n = 4). (e) The relative intensity of cytoplasmic HMGB1 (n = 4). (f) The relative intensity of nuclear HMGB1 (n = 4). (g) The immunofluorescence image of HMGB1 and p-NF-κB p65 double staining in PC12 cells. Proteins were acquired from PC12 cells and assayed by the Western blot method. Nuclear proteins were normalized to the intensity of Lamin B1, while other proteins were normalized to the intensity of β-actin. Data were expressed as means ± SD. ##P < 0.01 versus the control group; ∗P < 0.05 and ∗∗P < 0.01 versus the OGD/R group. Scale bar = 20 μm.
Figure 6
Figure 6
SalD inhibited HMGB1 cytoplasmic translocation and NF-κB nuclear translocation in the cortex and hippocampus of I/R rats. (a) The relative intensity of TLR4 (n = 4). (b) The relative intensity of MyD88 (n = 4). (c) The relative intensity of TRAF6 (n = 4). (d) The relative p-NF-κB p65/NF-κB p65 ratio (n = 4). (e) The relative intensity of cytoplasmic HMGB1 (n = 4). (f) The relative intensity of nuclear HMGB1 (n = 4). Protein samples were acquired from the cortex or hippocampus region and assayed by the Western blot method. Nuclear proteins were normalized to the intensity of Lamin B1, while other proteins were normalized to the intensity of β-actin. Data were expressed as means ± SD. ##P < 0.01 versus the sham operation group; ∗P < 0.05 and ∗∗P < 0.01 versus the MCAO/R group.
Figure 7
Figure 7
SalD inhibited HMGB1 cytoplasmic translocation in neurons, astrocytes, and microglial cells in the cortex and hippocampus of I/R rats. The immunofluorescent assay was conducted using the HMGB1/NeuN, HMGB1/GFAP, and HMGB1/Iba1 staining. Scale bar = 25 μm.
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