Isosteviol sodium injection improves outcomes by modulating TLRs/NF-κB-dependent inflammatory responses following experimental traumatic brain injury in rats

Abstract

Previous studies have shown that isosteviol sodium (STVNa) protects against permanent cerebral ischemia injury by inhibition of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB)-mediated inflammatory responses. Overwhelming evidence shows that toll-like receptors (TLRs) are the upstream regulators of NF-κB. On the basis of the similarity of the pathology caused by traumatic brain injury (TBI) and stroke, we speculated that STVNa may have a therapeutic effect against TBI through regulation of the TLRs/NF-κB signaling-mediated inflammatory response. Thus, we studied the potential therapeutic effects of STVNa and the underlying mechanisms. Male rats, subjected to controlled cortical impact (CCI) injury, were injected intraperitoneally with STVNa (5, 10, 20, 40, and 80 mg/kg, daily for 3 or 7 days) after trauma. Neurobehavioral scores, relative numbers of cortical lesions, and histology were examined. We also measured the mRNA and protein expression levels of TLRs/NF-κB signaling pathway-related genes including TLR2, TLR4, and NF-κB by quantitative real-time-PCR and western blotting, respectively, and concentrations of tumor necrosis factor-α and interleukin-1β by an enzyme-linked immunosorbent assay. The results indicated that STVNa (20 mg/kg) showed significant neuroprotective effects 3 and 7 days after TBI, including the reduction of cortical lesions, improvement of the neurological severity score, significantly increased number of restored neurons, decreased number of astrocytes, and lower concentrations of tumor necrosis factor-α and interleukin-1β. Results from quantitative real-time-PCR and western blotting also show that the mRNA and protein expression levels of TLR2, TLR4, and NF-κB were significantly lower in STVNa-treated rats compared with the vehicle-treated rats. The administration of STVNa attenuates the TLR/NF-κB signaling pathway-mediated inflammatory responses in the injured rat brain, and this may be the mechanism by which STVNa improves the outcome following TBI.

Fig. 1

Results of the dose–response relationship of isosteviol sodium (STVNa) were measured 3 and 7 days after traumatic brain injury (TBI) in rats. (a) The distribution of percent area of infarct in serial brain sections stained with cresyl violet-stain 3 days after TBI in vehicle-treated and STVNa-treated rats. (b) Neurological scores of Modified Neurological Severity Score (mNSS) 3 days after TBI in the study of the dose–response relationship of STVNa. (c) Neurological scores of mNSS at 3 and 7 days after TBI. (d) Neurological scores of the Bederson test at 3 and 7 days after TBI. Data were expressed as the mean±SEM (n=8/group). *P<0.05, **P<0.01 and ***P<0.001 versus the vehicle group by one-way analysis of variance with Tukey’s multiple-comparison test.

Fig. 2

Brain pathology after controlled cortical impact (CCI). (a) Representative photographs of cresyl-violet-stained brain sections from sham and CCI-injured wild type (WT) mice showing cortical cavitation and remaining hippocampal swelling in the areas 3 days after injury. (b, c) Quantitative analysis of relative hippocampal areas normalized to the contralateral side in saline-treated and isosteviol sodium (STVNa) (20 mg/kg)-treated rats at 3 days (b) and 7 days (c) after CCI or sham. Data are presented as mean±SEM, ***P<0.001, versus the saline-treated sham group, and ^###P<0.001, versus the saline-treated CCI group, one-way analysis of variance, followed by Tukey’s multiple-comparison test (n=7–9).

Fig. 3

Effects of isosteviol sodium (STVNa) on astrogliosis and neuron survival following controlled cortical impact (CCI). (a) Representative brain sections and microphotographs of selected brain regions from the same corresponding sections used for immunohistochemistry quantification 3 days after traumatic brain injury (TBI). (b) Effects of STVNa on glial fibrillary acidic protein (GFAP) immunoreactivity in TBI (n=3/group). Scale bar=50 m. (c) Effect of STVNa on neuronal immunoreactivity in the rats’ contusive brain (n=3/group). Scale bar=50 m (d) Quantitative analyses of GFAP and neuronal nuclei (NeuN) immunoreactivity in the ipsilateral brain regions. Data are represented as mean±SEM (n=3; ***P<0.001 vs. the sham group; ^##P<0.01 and ^###P<0.001 vs. the CCI/vehicle group).

Fig. 4

Isosteviol sodium (STVNa) inhibits the toll-like receptors (TLRs)/nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling pathway after traumatic brain injury (TBI) in vivo. (a) The mRNA expression levels of TLR2, TLR4, and NF-ΚB in the injured tissue were detected by RT-PCR at 3 days after trauma. (b) The protein expression levels of TLR2, TLR4, and NF-κB were detected by western blotting at 3 days after trauma. (c, d) Relative expression levels of TLR2, TLR4 (c), and NF-κB (d) were calculated by normalizing to that of GAPDH, respectively. Data are represented as mean±SEM (n=3; **P<0.01 vs. the sham group; ^#P<0.05 and ^##P<0.01 vs. the vehicle group).

Fig. 5

Isosteviol sodium (STVNa) treatment significantly downregulates the expression of inflammatory cytokines in the brain. (a, b) The expression levels of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) in the injured brains were determined by RT-PCR (a) and ELISA (b). Data are represented as mean±SEM (n=3; **P<0.01 vs. the sham group; ^#P<0.05 and ^##P<0.01 vs. the vehicle group).