Rhein Protects Against Neurological Deficits After Traumatic Brain Injury in Mice via Inhibiting Neuronal Pyroptosis

Figures

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  Figure 1. Rhein attenuated TBI-induced neurological functional impairment. The Neurological impact of TBI was tested by mNSS (A), rotarod (B), and open-field (C) behavioral task tests before and 8, 24, and 48 h after TBI. (D) TUNEL staining was applied to detect cortical harm in TBI. TUNEL-positive cells (%), the number of TUNEL-positive cells divided by the total cells per field, was assessed in 20 randomly selected fields. Data are presented as the mean ± SEM. *p < 0.05, ** p < 0.01 versus Sham group, #p < 0.05, ##p < 0.01 versus TBI group.

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  Figure 2. Rhein reduced levels of inflammatory mediator in the cortex after TBI. (A–C) Concentrations of pro-inflammatory cytokines IL-1β (A), IL-18 (B), and IFN-γ (C) were detected in the region of the contusion 8, 24, and 48 h after TBI by ELISA. (D) TLR4, MyD88, and NLRP3 mRNA expression were tested by qRT-PCR. GAPDH served as an internal control. Data are presented as the mean ± SEM. *p < 0.05 and **p < 0.01 versus Sham group, #p < 0.05 and ##p < 0.01 versus TBI group.

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  Figure 3. Rhein attenuated pyroptosis in the murine model of TBI. (A–D) Expression of pyroptosis-related proteins of caspase-1 (p45, p20, and p10), caspase-11, and GSDMD was assayed by Western blot 24 h after TBI (two randomly selected samples from each group were shown). (E) Representative photomicrographs at × 20 magnification of proteins immunostaining in the cortex 24 h after TBI. (F) Serum LDH was tested 24 h after TBI. Data are presented as the mean ± SEM. *p < 0.05 and **p < 0.01 versus Sham group, #p < 0.05, ##p < 0.01 versus TBI group.

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  Figure 4. Rhein reduced neuron injury-induced inflammatory mediator levels in vitro. The neurons were treated with stretch stimulation for 12 h in absence or presence of Rhein (10 µg/ml), then the expression of pro-inflammatory cytokines IFN-γ (A), IL-18 (B), and IL-1β (C) were tested by ELISA. mRNA expressions of TLR4, MyD88, and NLRP3 after stretch and Rhein treatments were detected by qRT-PCR (D). Data were presented as three independent experiments. *p < 0.05, **p < 0.01 versus control group; #p < 0.05, ##p < 0.01 versus stretch group.

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  Figure 5. Rhein ameliorated neuron injury-induced pyroptosis. The neurons were treated with stretch stimulation for 12 h in the absence or presence of Rhein (10 µg/ml), then cell lysate and culture supernatant were collected. (A–D) Pyroptosis-related protein expression was detected by Western blotting. The histogram was used to analyze protein expression. (E) Supernatant LDH concentration was detective after stretch and Rhein treatments. The statistics were based on at least three independent experiments. *p < 0.05, **p < 0.01 versus control group; #p < 0.05 versus Stretch group.