Sheng-Di-Da-Huang Decoction Inhibited Inflammation Expressed in Microglia after Intracerebral Hemorrhage in Rats

Min Cai, Zhonghai Yu, Wen Zhang, Li Yang, Jun Xiang, Jingsi Zhang, Zhennian Zhang, Ting Wu, Xiangting Li, Maodong Fu, Xuxia Bao, Xiaofei Yu, Dingfang Cai, Min Cai, Zhonghai Yu, Wen Zhang, Li Yang, Jun Xiang, Jingsi Zhang, Zhennian Zhang, Ting Wu, Xiangting Li, Maodong Fu, Xuxia Bao, Xiaofei Yu, Dingfang Cai

Abstract

Objects: Sheng-Di-Da-Huang Decoction was used as an effective hemostatic agent in ancient China. However, its therapeutic mechanism is still not clear. Inflammatory injury plays a critical role in ICH-induced secondary brain injury. After hemolysis, hematoma components are released, inducing microglial activation via TLR4, which initiates the activation of transcription factors (such as NF-κB) to regulate expression of proinflammatory cytokine genes. This study aimed to verify the anti-inflammatory effects of Sheng-Di-Da-Huang Decoction on ICH rats.

Materials and methods: Intracerebral hemorrhage was induced by injection of bacterial collagenase (0.2 U) in rats. Neurological deficits, brain water content, Evans blue extravasation, expression of TLR4, NF-κB, Iba-1 positive cells (activated microglia), tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β) were examined 1, 3, 7, and 14 days after collagenase injection. MR images were also studied.

Results: Sheng-Di-Da-Huang Decoction remarkably improved neurological function, reduced brain water content as well as Evans blue extravasation, downregulated expression of TLR4, NF-κB, TNF-α, and IL-1β, and inhibited microglial activation.

Conclusions: Sheng-Di-Da-Huang Decoction reduced inflammation reaction after ICH through inhibited inflammation expressed in microglia.

Figures

Figure 1
Figure 1
(a) Neurological deficit score (maximum total score is 13) of rats after injection of collagenase. (b) The result of corner turn test. Data were presented as mean ± SEM, n=5 rats for each group. ∗ P<0.05; ∗∗ <0.01 compared with ICH group.
Figure 2
Figure 2
Result of brain water contents for each group at different time points. Data were presented as mean ± SEM, n=5 rats for each group. ∗ P<0.05; ∗∗ <0.01 compared with ICH group.
Figure 3
Figure 3
Results of Evans blue extravasation in each group at different time points. Data were presented as mean ± SEM, n=5 rats for each group. ∗ P<0.05; ∗∗ <0.01 compared with ICH group.
Figure 4
Figure 4
Results of MRI study. The images, taken at the different time points, were from a single rat. (a) Representative T2-weighted MR images of intracerebral hematoma in rat brain at the level of the collagenase injection. (b) Representative SWI images of intracerebral hematoma in rat brains surrounding collagenase injection. n=5 rats for each group at each time point. The images were from different rats in the same group.
Figure 5
Figure 5
(a) Representative immunofluorescence images of Iba-1, ×200 magnified. (b) Iba-1 positive cells percentage (green fluorescence represents Iba-1 positive cell). Data were presented as mean ± SEM, n=5 rats for each group. ∗ P<0.05; ∗∗ <0.01 compared with ICH group.
Figure 6
Figure 6
Detection of TLR4 and NF-κB in the hemorrhagic area using Western blotting. (a) The bands of GAPDH, TLR4, and NF-κB at 1 day. (b) The bands of GAPDH, TLR4, and NF-κB at 3 days. (c) The bands of GAPDH, TLR4, and NF-κB at 7 days. (d) The bands of GAPDH, TLR4, and NF-κB at 14 days. (e) Quantitative results of the bands for TLR4 relative to GAPDH at each time point. (f) Quantitative results of the bands for NF-κB relative to GAPDH at each time point. Data are presented as mean ± SEM, n=5 rats for each group. ∗ P<0.05; ∗∗ <0.01 compared with ICH group. Results of TNF-α (g) and IL-1β (h) concentrations in each group at different time points. Data were presented as mean ± SEM, n=5 rats for each group. ∗ P<0.05; ∗∗ <0.01 compared with ICH group∗.

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