Resveratrol Attenuates Acute Inflammatory Injury in Experimental Subarachnoid Hemorrhage in Rats via Inhibition of TLR4 Pathway

Abstract

Toll-like receptor 4 (TLR4) has been proven to play a critical role in neuroinflammation and to represent an important therapeutic target following subarachnoid hemorrhage (SAH). Resveratrol (RSV), a natural occurring polyphenolic compound, has a powerful anti-inflammatory property. However, the underlying molecular mechanisms of RSV in protecting against early brain injury (EBI) after SAH remain obscure. The purpose of this study was to investigate the effects of RSV on the TLR4-related inflammatory signaling pathway and EBI in rats after SAH. A prechiasmatic cistern SAH model was used in our experiment. The expressions of TLR4, high-mobility group box 1 (HMGB1), myeloid differentiation factor 88 (MyD88), and nuclear factor-κB (NF-κB) were evaluated by Western blot and immunohistochemistry. The expressions of Iba-1 and pro-inflammatory cytokines in brain cortex were determined by Western blot, immunofluorescence staining, or enzyme-linked immunosorbent assay. Neural apoptosis, brain edema, and neurological function were further evaluated to investigate the development of EBI. We found that post-SAH treatment with RSV could markedly inhibit the expressions of TLR4, HMGB1, MyD88, and NF-κB. Meanwhile, RSV significantly reduced microglia activation, as well as inflammatory cytokines leading to the amelioration of neural apoptosis, brain edema, and neurological behavior impairment at 24 h after SAH. However, RSV treatment failed to alleviate brain edema and neurological deficits at 72 h after SAH. These results indicated that RSV treatment could alleviate EBI after SAH, at least in part, via inhibition of TLR4-mediated inflammatory signaling pathway.

Keywords: early brain injury; inflammation; resveratrol; subarachnoid hemorrhage; toll-like receptor 4.

Figure 1

Effects of resveratrol (RSV) treatment on pro-inflammatory cytokine release and microglia activation at 24 h post subarachnoid hemorrhage (SAH). (A,B) Western blot analysis showing that RSV administration significantly suppressed Iba-1 (microglia marker) expression after SAH; (C,G) Immunofluorescence
staining indicted that RSV could evidently reduce the number of activated microglia; (D–F) RSV markedly alleviated the expression of IL-1β, IL-6, and TNF-α in the brain cortex after SAH. Bars represent the mean ± SD. ** p < 0.01, * p < 0.05, and ns means non-significant. Scale Bars = 50 μm.

Figure 2

Effects of resveratrol (RSV) on the expression and activation of toll-like receptor 4 (TLR4)-related pathway 24 h post subarachnoid hemorrhage (SAH). (A) Representative Western blot images to detect the effects of RSV on the expressions of high-mobility group box 1 (HMGB1), TLR4, myeloid differentiation factor 88 (MyD88), and nuclear factor-κB (NF-κB); (B–E) Quantitative analyses of HMGB1, TLR4, MyD88, and NF-κB among all experimental groups. RSV treatment significantly reduced HMGB1, TLR4, MyD88, and NF-κB protein levels as compared with SAH + vehicle group. Bars represent the mean ± SD. *** p < 0.001, ** p < 0.01, * p < 0.05, and ns means non-significant.

Figure 3

Effects of resveratrol (RSV) on high-mobility group box 1 (HMGB1), toll-like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88), and nuclear factor-κB (NF-κB) distribution at 24 h after subarachnoid hemorrhage (SAH). (A–E) RSV treatment could significantly reduce the immunoreactivity of HMGB1, TLR4, MyD88, and NF-κB in the cerebral cortex when compared with SAH + vehicle group. Bars represent the mean ± SD. *** p < 0.001, ** p < 0.01, * p < 0.05, and ns means non-significant. Scale Bars = 50 μm.

Figure 3

Effects of resveratrol (RSV) on high-mobility group box 1 (HMGB1), toll-like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88), and nuclear factor-κB (NF-κB) distribution at 24 h after subarachnoid hemorrhage (SAH). (A–E) RSV treatment could significantly reduce the immunoreactivity of HMGB1, TLR4, MyD88, and NF-κB in the cerebral cortex when compared with SAH + vehicle group. Bars represent the mean ± SD. *** p < 0.001, ** p < 0.01, * p < 0.05, and ns means non-significant. Scale Bars = 50 μm.

Figure 4

Effects of resveratrol (RSV) on neural apoptosis, brain edema, and neurological function at 24 h post subarachnoid hemorrhage (SAH). (A–D) RSV significantly reduced the elevated levels of cleaved caspase-3 and pro-apoptotic protein Bax, and enhanced the diminished level of Bcl2; (E,F) RSV administration markedly decreased the number of TUNEL-positive neural cells compared with the SAH + vehicle group; (G,H) RSV ameliorated brain edema and neurological behavior impairment at 24 h post SAH. Bars represent the mean ± SD. *** p < 0.001, ** p < 0.01, * p < 0.05, and ns means non-significant. Scale Bars = 50 μm.

Figure 5

Effects of resveratrol (RSV) on neuronal survival, brain edema, and neurological function at 72 h post subarachnoid hemorrhage (SAH). (A,B) RSV treatment significantly increased the proportion of survived neurons compared with the SAH + vehicle group; Higher magnification of Nissl staining was shown in the red box for all groups; (C,D) RSV failed to alleviate brain edema and the impairment of neurological behavior compared with SAH + vehicle group at 72 h post SAH. Bars represent the mean ± SD. *** p < 0.001, * p < 0.05, and ns means non-significant. Scale Bars = 50 μm.

Figure 5

Effects of resveratrol (RSV) on neuronal survival, brain edema, and neurological function at 72 h post subarachnoid hemorrhage (SAH). (A,B) RSV treatment significantly increased the proportion of survived neurons compared with the SAH + vehicle group; Higher magnification of Nissl staining was shown in the red box for all groups; (C,D) RSV failed to alleviate brain edema and the impairment of neurological behavior compared with SAH + vehicle group at 72 h post SAH. Bars represent the mean ± SD. *** p < 0.001, * p < 0.05, and ns means non-significant. Scale Bars = 50 μm.

Figure 6

Schematic illustration of the experiment design. Resveratrol (RSV) was administered intraperitoneally (ip) at 2 h and 12 h after initial bleeding. In the first set of experiments, neurological scores, brain edema, Western blot analysis, immunohistochemistry, and TUNEL apoptosis were evaluated at 24 h after subarachnoid hemorrhage (SAH). In another set of experiments, neuronal survival, brain edema, and neurological function were evaluated at 72 h after SAH to determine the possible long-term benefits of RSV.