1.Identification of ITGAX in proteomics
We identified ITGAX, a protein that may be involved in the pathogenesis of epilepsy, in serum exosomes from patients with drug-resistant epilepsy, patients with seizure-free, and age - and sex-matched healthy individuals by proteomics. The manifestation of the corresponding peptide in the proteomic mass spectrometry is shown in Figure1. And the expression of ITGAX was significantly up-regulated in the drug-resistant epilepsy group compared with the other two groups.
2.Identification of serum exosomes
Serum exosomes were extracted from normal mice and epilepsy model mice by exosome extraction Kit, and characterized by transmission electron microscopy(TEM) and immunoblotting. As shown by TEM, these nanoscale vesicles showed typical exosomal "cup and plate" morphology (Figure 2a). Meanwhile, western blot analysis also verified the presence of the specific exosome markers of CD63 and CD9(Figure 2b).
3.ITGAX expression was up-regulated in serum exosomes of epileptic mice
To verify the expression of ITGAX in serum exosomes of epileptic models, western blotting and ELISA were used to detect the expression of ITGAX in serum exosomes of normal and epileptic groups. Compared with the normal group, the content of ITGAX was significantly up-regulated in serum exosomes of the epilepsy group(Figure 3 a-c), the difference was statistically significant, which was consistent with the previous proteomics results.
4.ITGAX expression was up-regulated in the hippocampus of epileptic mice
To verify ITGAX level in hippocampal tissues of epileptic models, western blotting was used to detect ITGAX expression in hippocampal tissues of normal and epileptic groups. The results showed that ITGAX was significantly upregulated in the hippocampus of the epilepsy group, compared with the normal group, and the difference was statistically significant(Figure 4 a-b).
5.Knocking down ITGAX expression in peripheral circulation can down-regulate ITGAX expression in hippocampus of epileptic mice
To explore whether the upregulation of ITGAX in the hippocampus after epilepsy comes from the central nervous system or the peripheral circulation, we treated mice with tail vein injection of ITGAX knockdown adeno-associated virus to knock down the expression of ITGAX in the peripheral circulation. First, the intervention was validated by western blotting and ELISA. The results are as follows: As shown in Figure 5a, tail vein injection of ITGAX knockdown adeno-associated virus intervention in healthy mice down-regulated ITGAX expression in the peripheral circulation, compared with the vehicle group. As shown in Figure 5b-c, western blotting evaluated the effect of tail vein injection of ITGAX knockdown adeno-associated virus on ITGAX content in the hippocampus before pilocarpine administration. There was no statistically significant difference between the vehicle group and the ITGAX- group. It was further verified that tail vein injection of ITGAX knockdown adeno-associated virus intervention could only down-regulate the expression of ITGAX in peripheral circulation, but could not cross the blood-brain barrier to interfere with the basic expression of ITGAX in the hippocampus. As shown in Figure 6a-b, compared with the Pilo+vehicle group, after ITGAX knockdown in the peripheral circulation, the up-regulated ITGAX in the hippocampus after epilepsy was also down-regulated, with statistically significant differences. In conclusion, the increase of ITGAX in the hippocampus after epilepsy may come from the peripheral circulation.
6.Inhibition of exosomes in the peripheral circulation can down-regulate the expression of ITGAX in the hippocampus after epilepsy
To further explore whether serum exosomes transfer ITGAX in the peripheral circulation to the hippocampus after epilepsy, we intraperitoneally injected GW4869 exosome inhibitor to intervene exosomes in the peripheral circulation of epileptic mice, and then detected the changes of ITGAX in the hippocampus after intervention by western blot. The results were as follows: As shown in Figure 7a, serum acetylcholinesterase activity test showed that compared with DMSO solvent group, serum exosome content was down-regulation after intraperitoneal injection of GW4869 exosome inhibitor, confirming the effectiveness of intervention. As shown in Figure 7b-c, compared with the Pilo+DMSO group, ITGAX expression was down-regulated in the hippocampus after inhibition of serum exosomes, with statistically significant differences. These results suggest that the increase of ITGAX in the hippocampus after epilepsy may be mediated by serum exosomes.
7.Effect of ITGAX expression on the latency of seizures in mice
To explore the effect of ITGAX expression level in the hippocampus on seizure phenotype, we examined the latency of epileptic seizure in each group after intervention. The latencies of the first epileptic seizure in the Pilo group, the Pilo+Vehicle group, the Pilo+ITGAX- group, the Pilo+DMSO group and the Pilo+GW4869 group were 13.78±0.84mins, 14.86±0.59mins, 27.25±1.97mins, 13.38±0.93mins, 20.43±1.55mins, respectively, as shown in Table 1. The latency of the first seizure was significantly delayed in the Pilo+ITGAX- group compared with the Pilo+Vehicle group, and similarly, the latency of the first seizure was significantly delayed in the Pilo+GW4869 group compared with the Pilo+DMSO group((Figure 8). And the difference was statistically significant. The results suggest that inhibition of ITGAX expression in hippocampus has protective effect on epileptic seizure in mice.
Table 1 Effect of ITGAX expression on the latency of seizures in mice
Groups Pilo Pilo+Vehicle Pilo+ITGAX- Pilo+DMSO Pilo+GW4869
|
Latency of 13.78±0.84 14.86±0.59 27.25±1.97 13.38±0.93 20.43±1.55
seizures (min)
|
8.Effect of ITGAX expression on blood brain barrier
8.1 Inhibition of central transport of ITGAX by peripheral circulation can improve the blood-brain barrier permeability disorder after epilepsy
In a study on ovarian cancer, ITGAX overexpression was found to activate the PI3k/Akt pathway and induce the upregulation of vascular endothelial growth factor (VEGFA)[20]. Coincidentally, VEGFA was significantly upregulated in the cerebral cortex of epileptic patients[21]. As a vascular regulator, increased VEGFA is associated with increased vascular permeability and blood-brain barrier dysfunction[22]. Therefore, we hypothesized that ITGAX may be involved in epilepsy through the regulation of the blood brain barrier. Based on the effect of the peripheral circulation on the expression of ITGAX in the hippocampus after epilepsy found by the above results, we first knocked down the expression of ITGAX in the peripheral circulation, then detected the expression of albumin in the hippocampus by western blot and the content of S100β in serum by ELISA to observe the changes of BBB permeability[23, 24]. So as to preliminarily explore the influence of ITGAX expression level on blood-brain barrier. The results were as follows: as shown in Figure 9a-b, the albumin content in the hippocampus of Pilo group was significantly up-regulated compared with the control group. Compared with the Pilo+Vehicle group, the expression of albumin in the hippocampus of Pilo+ITGAX- group was significantly down-regulated. As shown in Figure 9c, serum S100β levels in the Pilo group were significantly up-regulated compared with the normal group. And compared with the Pilo+Vehicle group, serum S100βlevel in Pilo+ITGAX- group was significantly down-regulated. The differences were statistically significant. Therefore, it is speculated that inhibition of central transport of ITGAX by peripheral circulation can improve the blood-brain barrier permeability disorder after epilepsy.
8.2 Inhibition of the central transport of ITGAX by serum exosomes can improve the blood-brain barrier permeability disorder after epilepsy
In order to further explore the effect of ITGAX on the blood-brain barrier, we interfered with the content of serum exosome, detected the expression of albumin in the hippocampus by western blot and detected the content of S100β in serum by ELISA to observe the changes of the blood-brain barrier permeability[23, 24], based on the above findings of serum exosome on the expression of ITGAX in the hippocampus after epilepsy. As shown in Figure 10a-b, compared with the control group, albumin content in the hippocampus of Pilo group was significantly up-regulated. And compared with the Pilo+DMSO group, albumin in the hippocampus of the Pilo+GW4869 group was significantly down-regulated. As shown in Figure 10c, compared with the control group, the serum S100β level in Pilo group was significantly up-regulated. Compared with Pilo+DMSO group, serum S100β level in the Pilo+GW4869 group was significantly down-regulated. The differences were statistically significant. In conclusion, inhibition of the central transport of ITGAX by serum exosomes may improve the blood-brain barrier permeability disorder after epilepsy.