Cerebroprotective effects of verbascoside in mice with intracerebral hemorrhage and its relationship with Toll-like receptor 4-mediated signaling pathway
Objective To investigate the cerebroprotective effect of verbascoside(VB)in mice with intracerebral hemorrhage(ICH)and whether it is related to the inhibition of Toll-like receptor 4(TLR4)-mediated acute inflammatory response.Methods Eight-week-old C57BL/6 male mice were used for the study,including 40 wild-type and 20 TLR4 knockout(TLR4-/-)mice.The wild-type mice were divided into sham group,ICH group,and post-ICH VB treatment group(ICH+VB 15 mg/kg or 30 mg/kg)according to the complete randomization method,and the TLR4-/-group mice were divided into TLR4-/-+ICH group and TLR4-/-+ICH+VB(30 mg/kg)group,with 10 mice in each group.The ICH model was constructed using the method of type Ⅷ collagenase injection in the right striatum.After the model was successfully established(modified Garcia score 10),the mice were injected intraperitoneally with VB(15,30 mg/kg)or an equal volume of saline once a day for 3 consecutive days.The modified Garcia score was used to evaluate the damage of brain function,and the area of cerebral hematomas was calculated to assess the hemorrhage volume after slicing,the water content of brain tissues was calculated by the wet/dry method,and the rate of neuronal apoptosis was detected by TUNEL staining.Immunofluorescence staining(IF)was used to detect the relative expression of Iba-1 in brain tissues.Real-time quantitative fluorescence polymerase chain reaction(qRT-PCR)assay and Western blot(WB)assay were used to detect the expression of TLR4 and its downstream inflammatory mediators,including nuclear factor-κB,tumor necrosis factor alpha(TNF-α),interleukin(IL)1β and IL-6.Differences in the above indexes between groups of wild-type mice and between groups of TLR4-/-were compared.Cortical tissues of TLR4-/-mice within 2 hours of birth were removed to culture primary neurons,and the neurons were co-cultured with mouse microglia cell lines(BV2 cells)using Transwell chambers,and were treated with red blood cell(RBC)lysates or RBC lysates+VB(50 μmol/L)respectively for 24 h.Then,the expression of NeuN protein in neurons was observed using IF.Results Compared with the ICH group,the modified Garcia scores of mice in the VB-treated groups(particularly in the ICH+VB 30 mg/kg group)were significantly higher(P＜0.05).Brain hemorrhage volume in the sham group,ICH group,ICH+VB 15 mg/kg group,and ICH+VB 30 mg/kg group were 0 mm3,7.33±1.99 mm3,3.19±0.47 mm3,1.66±0.26 mm3,the water content of brain tissue was 79.00±0.50％,85.00±0.75％,81.50±0.50％,80.25±0.25％,the apoptosis rate of neurons was 4.8±2.0％,38.7±6.8％,21.5±6.9％,9.8±4.2％,and the differences were statistically significant(P＜0.05).IF results showed that the relative expression of Iba-1 in brain tissues of the ICH group was significantly higher than that of the VB-treated group(P＜0.05).The qRT-PCR and WB results showed that,compared with those in the the ICH group,the mRNA,NF-κB,TNF-α,IL-1β,and IL-6 mRNA and protein expression levels in the VB-treated group were reduced(P＜0.05).The differences in Garcia score,brain tissue water content,and neuronal apoptosis rate were not significantly different between TLR4-/-+ICH+VB group and TLR4-/-+ICH group(all P＞0.05).Meanwhile,in the in vitro experiments,there was no statistically significant difference in the survival of neurons co-cultured with BV2 cells after treatment with RBC lysate or RBC lysate+VB(50 µmol/L)(P=0.872).Conclusion VB can improve the neural function deficit in mice after ICH,reduce the volume of hemorrhage,degree of brain edema and apoptosis of neurons,which might be related to the mechanism of inhibiting TLR4 signaling pathway,reducing the activation of microglia and release of pro-inflammatory cytokines.
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