GPR124 induces NLRP3 inflammasome-mediated pyroptosis in endothelial cells during ischemic injury.

OBJECTIVE: G protein-coupled receptor 124 (GPR124) regulates central nervous system angiogenesis and blood-brain barrier (BBB) integrity, and its deficiency aggravates BBB breakdown and hemorrhagic transformation in ischemic mice. However, excessive GPR124 expression promotes inflammation in atherosclerotic mice. In this study, we aimed to elucidate the role of GPR124 in hypoxia/ischemia-induced cerebrovascular endothelial cell injury. METHODS: bEnd.3 cells were exposed to oxygen-glucose deprivation (OGD), and time-dependent changes in GPR124 mRNA and protein expression were evaluated using reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting. The effects of GPR124 overexpression or knockdown on the expression of pyroptosis-related genes were assessed at the mRNA and protein levels. Tadehaginoside (TA) was screened as a potential small molecule targeting GPR124, and its effects on pyroptosis-related signaling pathways were investigated. Finally, the therapeutic efficacy of TA was evaluated using a rat model of transient middle cerebral artery occlusion/reperfusion (tMCAO/R). RESULTS: During OGD, the expression of GPR124 initially increased and then decreased over time, with the highest levels observed 1 h after OGD. The overexpression of GPR124 enhanced the OGD-induced expression of NLRP3, Caspase-1, and Gasdermin D (GSDMD) in bEnd.3 cells, whereas GPR124 knockdown reduced pyroptosis. Additionally, TA exhibited a high targeting ability to GPR124, significantly inhibiting its function and expression and suppressing the expression of pyroptosis-related proteins during OGD. Furthermore, TA treatment significantly reduced the cerebral infarct volume and pyroptotic signaling in tMCAO/R rats. CONCLUSIONS: Our findings suggest that GPR124 mediates pyroptotic signaling in endothelial cells during the early stages of hypoxia/ischemia, thereby exacerbating ischemic injury.

Effect of 4-AP on MPP+/ MPTP-induced Parkinson's disease model.

To study the effect of 4-AP on Parkinson's disease (PD) cells and animal model. PD cells were pretreated with different concentrations of 4-AP for 24 hours, then PD cells were prepared by MPP+, and the cell activity was detected by CCK8 kit. PD mice were prepared by MPTP and then given 4-AP for 10 days. Finally, the behavioral changes of mice were detected by pole climbing test and open field test, and the expression of TH in the midbrain was detected by IHC and WB. 4-AP could increase the activity of PD cells induced by MPP+. In the field experiment, the total spontaneous activity distance of PD mice (1380.01 ± 151.84) cm was not different from that of 4-AP intervention (1228.65 ± 358.25) cm but was reduced than that of normal mice (2121.89 ± 235.95) (P<0.05). In the pole climbing test, the pole climbing time of PD mice was (7.95 ± 1.02) seconds, compared with that of PD mice treated with 4-AP, there was no difference between the two groups, but it was reduced than that of normal mice (P<0.05). IHC and Western blot showed that the mesencephalic TH of PD mice and drug-treated mice were reduced than that of normal mice (P<0.05), however, drug intervention could not reduce the expression of TH in mice with PD (P>0.5). 4-AP pretreatment can reduce the toxic and side effects of MPP+. 4-AP can not improve the motor function impairment of PD mice, nor can it reduce the toxic effect of MPTP on dopaminergic neurons. There are differences between pre-treatment and post-intervention in the treatment of MPP+/MPTP-induced PD. In order to better explore the drug treatment and target of PD, it is hoped that a cure for PD can be found in PD animals. The timing of intervention and cell and animal experiments should complement each other.

Effect of Different MPTP Administration Intervals on Mouse Models of Parkinson's Disease.

Objective: To research the effect of different 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) administration intervals on the behavior and pathology of mouse models of Parkinson's disease. Methods: Eighteen C57 male mice were divided into a control group, subacute model group, and chronic model group (6 mice per group). Animal models of Parkinson's disease were built according to MPTP administration. The behavior of mice was determined through an open-field test and pole test. Tyrosine hydroxylase expression in brain tissues was checked by immunohistochemistry and western blot. Result: In the open-field test, the total activity distance in the chronic model group (1271.05 ± 207.93 cm) was reduced significantly compared with that of the control group (1964.21 ± 379.77 cm), while the distance had no significant differences in the subacute model group (1950.57 ± 273.54 cm). At the same time, the number of times the mice crossed the center grid in the chronic model group (3.17 ± 1.17) was reduced compared with that in the control group (11.67 ± 6.65), while there were few differences in the subacute model group (9.33 ± 2.81). In the pole test, the climbing time (8.49 ± 1.44 s) and total rest time (103.64 ± 26.57 s) of mice in the chronic model group were longer than those in the control group, respectively (4.31 ± 0.70 s, 45.21 ± 14.36 s), while there were no significant differences in the subacute model group (4.51 ± 0.48 s, 52.44 ± 25.98 s). Besides, compared with the control group, TH expression in the subacute model group and chronic model group was reduced notably, and the changes of TH expression in the chronic model group were more significant. Conclusion: There is a little loss of midbrain dopaminergic neurons in the subacute Parkinson's disease mouse models induced by continuous MPTP administration, but there is no effect on the behavior. Long interval MPTP-induced chronic Parkinson's disease mouse models lose a lot of dopaminergic neurons, which is accompanied by anxiety-like behaviors in addition to motor dysfunction.