Background: Febrile seizure (FS) is the most common pediatric neurological emergency, associated with inflammation. High-mobility group box 1 (HMGB1) enhanced febrile seizure in developing rats as a pro-inflammatory molecule, and displayed an increased level in serum and cerebrospinal fluid of children with febrile seizure. However, the mechanism of its contribution in febrile seizure is unclear.

Purpose: Although HMGB1 was reported to participate in the pathogenesis of seizures in adult mice through the HMGB1-TLR4 signaling pathway, the research about HMGB1-TLR4 in the pathogenesis of FS is limited. Together with the evidence that the levels of the downstream inflammatory factors of TLR4/NF-κB pathway were significantly increased in children with FS, it is of great importance to explore whether the activation of TLR4/NF-κB signaling pathway is involved in HMGB1 associated inflammation in FS.

Methods: The animal model of FS was made successfully, during which seizures were provoked every four days by hot water (45℃), and repeated ten times totally. The latency to seizure, seizure duration, grade of seizure were recorded. After forty days' modeling, rats were divided into different groups according to their grades of seizures (FS (0) - FS (V)). Reverse transcription-polymerase chain reaction (RT-PCR) was applied to investigate mRNA expression of HMGB1, TLR4 and NF-κB in hippocampus, Western-blot (WB) and immunohistochemistry (IHC) were applied to evaluate protein expressions of them in hippocampus, and enzyme-linked immunosorbent assay (ELISA) was applied to detect the protein expressions of them in peripheral blood.

Results: The mortality rate of our FS modeling was 28.125%. The mean seizure latency was 2.48±0.45min, and the mean duration of seizure was 1.91±0.37min. There were 2 rats in FS (0-II) group, 11 rats in FS (III) group, 18 rats in FS (IV) group, and 15 rats in FS (V) group. The mRNA level of HMGB1, TLR4 and NF-κB in the hippocampus of the FS (V) and FS (IV) groups were significantly higher than those in WT, while there was no difference between FS (III) and WT. Based on this data, the comparisons of HMGB1, TLR4 and NF-κB protein levels in the hippocampus and serum were done for FS (V), FS (IV) and WT. Although increments for HMGB1, TLR4, NF-κB levels in FS (V) were observed with a good consistency between WB and IF, no significant upregulation was shown in FS (IV). Additionally, our ELISA data showed that the significances of the increments in these three proteins between FS (V) and WT were smaller in the serum than those in the hippocampus, indicating that the inflammation was more severe in the hippocampus.

Conclusion: In summary, our study detected a seizure degree-related upregulation of HMGB1 and TLR4/NF-κB signaling pathway both in the hippocampus and serum of rats with FS, indicating the involvement of TLR/NF-κB pathway in inflammation promoted by HMGB1 during FS. Our work also displayed the relationship between seizure degree and inflammatory response. In the future, more work will be done about the molecule mechanism of FS, and the antibody of HMGB1 will be also investigated as a novel therapy to treat the disease.

1.Animal model construction

FS was provoked once every four days, and repeated ten times totally by hot water (45 ℃) [1]

2. Real-time PCR

qPCR was done following the instructions from DBI-2044 Bestar SybrGreen qPCR master mix, with the reaction program: 50 °C for 2 min; 95 °C for 10 min; 40 cycles of 95 °C for 5 s, 55 °C for 30 s, and 72 °C for 30 s.

3. Western Blot

The primary antibodies used were rabbit anti-HMGB1(1:1000, 6893S, CST), rabbit anti-TLR4 (1:300, ab217274, abcam), rabbit anti-NF-κB (1:1000, 8242S, CST), mouse anti-β-actin (1:5000, E021020, EARTHOX). The secondary antibodies were anti-rabbit IgG, HRP-linked (1:10000, E030120, EARTHOX) and anti-mouse IgG, HRP-linked (1:10000, E030110, EARTHOX).

4. Immunofluorescence

The primary antibody used were rabbit anti-HMGB1(1:300, AF7020, Affinity), rabbit anti-TLR4 (1:300, AF7017, Affinity), rabbit anti-NF-κB (1:300, AF5006, Affinity), The secondary antibodies were Alexa Fluor anti-rabbit 594 (1:500, EARTHOX, USA). We used a fluorescence microscope (OlympusBX51, NIKON, Japan) at excitation/emission wavelengths of 547/570 nm (Cy3, red), and 360/460 nm (DAPI, blue). Images were taken at 200 × magnification.

5. ELISA

ELISA was carried out to analyze the concentrations of HMGB1 (ELR-HMGB1, FANKEWEI), TLR4 (ELR-TLR4, FANKEWEI) and NF-κB (ELR-NF-κB, FANKEWEI) in the serum

1. Animal model construction and group division for FS group. A total of 64 rats were modeled with FS, after forty days’ modeling, 46 of them survived and the mortality rate was 28.125%. The proportions of each FS group were shown in Table 1.

Grade of seizure: 0, no seizure; I, facial clonus; II, nodding; III, forelimb clonus; IV, rearing; V, rearing and falling back.

2. FS rose the mRNA levels of HMGB1, TLR4 and NF-κB in the hippocampus of rats in FS (IV) and FS (V). Our data indicated that only the FS of grade IV or V changed the mRNA levels of these inflammation-related molecules (Figure 1).

3. FS rose the protein levels of HMGB1, TLR4 and NF-κB in the hippocampus of rats in the FS (V) group. Although increments for HMGB1, TLR4, NF-κB levels in FS (V) were observed with a good consistency between WB and IF, no significant upregulation was shown in FS (IV) (Figures 2-3).

4. FS increase the protein expression levels of HMGB1, TLR4 and NF-κB in serum of peripheral blood for FS (V). The expression levels of three proteins in the serum behaved similarly with WT for FS (IV), while higher levels were displayed for FS (V) (Figure 4). 

In summary, our study detected a seizure degree-related upregulation of HMGB1 and TLR4/NF-κB signaling pathway both in the hippocampus and serum of rats with FS, indicating the involvement of TLR/NF-κB pathway in inflammation promoted by HMGB1 during FS. Our work also displayed the relationship between seizure degree and inflammatory response. In the future, more work will be done about the molecule mechanism of FS, and the antibody of HMGB1 will be also investigated as a novel therapy to treat the disease.