Inhibition of curcumin on inflammation of BV-2 cells and primary microglia induced by Aβ1-42stimulation by enhancing autophagy / 中国药理学与毒理学杂志

ABSTRACT

Objective: To investigate the inflammatory effect and possible mechanism of curcumin in BV-2 cells and primary microglia by amyloid beta protein fragment 1-42 (Aβ1-42) stimulation. Methods: BV-2 cells were cultured with curcumin (0.1, 1,5, 10,15, 25 and 40 μmol·L-1) for 24 h, and cell viability was measured by MTT and ATP assay. After being pretreated with curcumin (5 and 10 μmol-L-1) for 2 h, BV-2 cells or primary microglia were co-cultured with curcumin and Aβ1-421 mg · L-1for 24 h. mRNA expressions of interleukin 6 (IL-6), tumor necrosis factor-α (TNF-α) and inducible nitric oxide synthase (/A/OS) in BV-2 cells, as well as, TNF-α and iNOS in primary microglia were detected by real time qPCR. After being pretreated with curcumin (5 and 10 pmol-L-1) for 2 h, BV-2 cells were co-cultured with curcumin and Aβ1-421 mg · L-1for 24 h. Protein expressions of iNOS, autophagy protein 5 (Atg-5), ubiquitin-binding protein 62 (P62) and protein ratio of microtubule-associated protein 1 light chain3- II (LC3-II) and LC3-1 were detected by Western blotting in BV-2 cells. After being pretreated with curcumin (5 and 10 Mmol - L-1) for 24 h, BV-2 cells or primary microglia were co-cultured with curcumin and Aβ1-42mg·L-1for 4 h. Ap,-42 protein were detected by Western blotting in BV-2 cells and primary microglia. After being pretreated with curcumin (5 and 10 μmol·L-1) for 24 h, primary microglia were co-cultured with curcumin and Aβ1-421 mg·L-1for 4 h. Protein levels of Aβ1-42and ionized calcium binding adapter molecule 1 (Iba1) were detected by immunofluorescence. After being pretreated with curcumin (5 and 10 μmol·L-1) for 2 h, BV-2 cells of knocked down Atg-5 gene were co-cultured with curcumin and Aβ1-421 mg·L-1for 24 h. Protein expressions of iNOS, Atg-5, P62 and protein ratio of LC3-II and LC3- I were detected by Western blotting. Results: Results of MTT and ATP assay showed that the curcumin concentration of less 25 μmol·L-1had no cytotoxicity to BV-2 cells. Compared with Ap,-42 group, curcumin 5 and 10 μmol·L-1reduced IL-6, TNF-α and iNOS mRNA expressions in BV-2 cells, as well as, TNF-α and iNOS mRNA expressions in primary microglia (P<0.01). Results of Western blotting showed that curcumin 10 μmol·L-1reduced protein expressions of iNOS and P62 (P<0.01) and increased the protein ratio of LC3-II and LC3- I and protein expression of Atg5 (P<0.01), compared with Aβ1-42group. Curcumin 10 μmol·L-1enhanced the phagocytosis to Aβ1-42protein in BV-2 cells and primary microglia (P<0.01), compared with Aβ1-42group. Results of immunofluorescence showed that curcumin 5 and 10 μmol·L-1increased the fluorescence intensity value of Ap,.42 in primary microglia (P<0.01), but decreased the fluorescence gray value of Iba1 in primary microglia (P<0.01), compared with Aβ1-42group. However, compared with Aβ group, curcumin 10 μmol·L-1did not change the protein ratio of LC3-II and LC3-1 or protein expressions of iNOS, P62 and Atg-5 in BV-2 cells of knocked down Atg-5 gene. Conclusion: Curcumin can inhibit the inflammatory activity of microglia stimulated with Aβ1-42by enhancing the expression of Atg-5 and increasing the capability of microglia for autophagy of Aβ1-42protein.