ABSTRACT
OBJECTIVE: To explore the mechanisms of macrophage migration inhibitory factor (MIF)/nucleus factor-κB (NF-κB) in mediating 1-methyl-4-phenylpyridinium iodide (MPP +)/1-Methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP)-induced activation of Nod-like receptor protein 3 (NLRP3) inflammasome in microglials and the its effects on neurons. METHODS: Murine microglial cell line Bv-2 was infected with a lentivirus carrying MIF shRNA for MIF knockdown and then treated with MPP+. The total protein levels of NLRP3, caspase-1, p65 and p65 in the cell nuclei and cytoplasm were detected. ELISA was used to detect the levels of IL-1ß and IL-18 in the culture supernatant, which served as the conditioned culture medium for MN9D cells, whose TH expression level was detected using Western blotting. The effect of stereotactic injection of an adeno-associated virus (AAV) carrying MIF shRNA on behaviors was assessed in a C57BL/6 mouse model of Parkinson disease (PD) induced by intraperitoneal MPTP injection. TH and Iba-1 immunohistochemistry was used to evaluate the number of substantia nigra neurons and the activation of microglia cells, and the protein expressions of MIF, NLRP3 and TH in the substantia nigra were detected with Western blotting. RESULTS: MPP+ significantly increased NLRP3 and MIF expressions in Bv-2 cells (P < 0.05). MIF knockdown in Bv-2 cells significantly lowered NLRP3 and caspase-1 protein expressions and IL-1ß and IL-18 levels in the culture supernatant (P < 0.05) without affecting total protein level of p65. Bv-2 cells with MIF knockdown showed significantly lowered p65 protein expression in the nuclei but increased p65 expression in the cytoplasm (P < 0.05). The conditioned medium derived from Bv-2 cells with MIF knockdown, as compared with that from than MPP +-treated Bv-2 cells, significantly increased TH expression in MN9D cells (P=0.01). Compared with those in MPTP group, the mice receiving injections of AAV-MIF-shRNA had higher scores in pole test and open field test with lower scores in traction test, and showed increased TH-positive neurons, decreased Iba-1 microglia cell activation, reduced expressions of MIF and NLRP3, and increased expression of TH in he substantia nigra (all P < 0.05). CONCLUSION: Inhibition of MIF can reduce the expression of NLRP3 inflammasomes and inflammatory factor caused by MPP+ in microglia cells to relieve the damage of dopaminergic neurons and alleviate microglia cell activation, thus offering protection against neuroinflammation in Parkinson's disease.
Subject(s)
Inflammasomes , Macrophage Migration-Inhibitory Factors , 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine , 1-Methyl-4-phenylpyridinium , Animals , Disease Models, Animal , Dopaminergic Neurons , Macrophage Migration-Inhibitory Factors/genetics , Male , Mice , Mice, Inbred C57BL , Microglia , Mitochondrial Permeability Transition Pore , NLR Family, Pyrin Domain-Containing 3 Protein/genetics , NLR ProteinsABSTRACT
The present paper reports the contents of ursolic acid in Crataegus pinnatifida var. major before and after processing determined by CS-920 TLC scanner: 0.274% and 0.265%, coefficients of variation 1.180% and 1.150% respectively. This method is simple, fast, reproducible and highly sensitive.