BV2 cells were treated with 10µM MPP + for 24h After treatment cells were collected and expression of Pin1 was checked by western blotting B BV2 cells were treated with 1 µg/ml

LPS for 24 h. After treatment, cells were collected and expression of Pin1was checked by western blotting. C. BV2 cells were treated with 3 different doses (10ng/ml, 20ng/ml and 30ng/ml) of TNF-α for 18 h. After treatment, cell lysates were made and expression of Pin1was checked by western blotting. D. BV2 cells were treated with 10ng/ml dose of TNF- α for different time points (6h, 12h, 18h and 24h).

Figure 4

Figure 4. Pin1 associates with p65 in BV2 cells. A. BV2 cells were grown in T25 flasks. When cell density reached approximately 80%, the cells were treated with either 10µM MPP+ for 24h or 1µg/ml LPS for 24h or 10ng/ml TNF-α for 18h. After treatment, cells were lysed in modified RIPA buffer and lysates from each sample was then subjected to immunoprecipitation (IP) using antibodies specific for Pin1. IP samples were probed with antibodies to p65 to demonstrate a specific nteraction between Pin1 and p65. B. BV2 cells were grown in T25 flasks. When cell density reached approximately 80%, the cells were treated with either 10µM MPP+ for 24h or 1µg/ml LPS for 24h or 10ng/ml TNF-α for 18h. After treatment, cells were lysed in modified RIPA buffer and lysates from each sample was then subjected to immunoprecipitation (IP) using antibodies specific for p65. IP samples were probed with antibodies to Pin1. Results are representative of two separate experiments.

Figure 5. Pin1 inhibitor Juglone attenuates MPP+ or LPS or TNF-α induced Pin1 expression and cytotoxicity in BV2 and primary microglial cells. BV2 cells were treated with 10ng/ml TNF-α for 18 hrs in the presence or absence of different doses (1µM, 3µM, 10µM and 20µM) of Juglone. A. Cytotoxicity of BV2 cells during 10M MPP+ treatment in the presence or absence of above mentioned four different doses of Juglone. Cytotoxicity was determined by Sytox green nucleic acid stain by measuring the fluorescence at regular intervals, as described in Materials and Methods. B. The two panels demonstrate phase contrast images (right) and fluorescence micrographs (left) to show the extent of Sytox green staining of cells in the field of view. C. BV2 cells were treated with10M MPP+ for 24h in the presence or absence of different doses (1µM, 3µM and 10µM) of Juglone. After treatment, cell lysates were prepared and immunoblotted for Pin1. D. BV2 cells were treated with 1g/ml LPSfor 24h in the presence or absence of 3µM Juglone. After treatment, cell lysates were prepared and immunoblotted for Pin1. E. BV2 cells were treated with 10ng/ml TNF-α for 18h in the presence or absence of 3µM Juglone. After treatment, cell lysates were prepared and immunoblotted for Pin1. F. Primary glial cells from C57BL/6 postnatal day 1 (P1) mouse pups were immunolabelled for IBA-1(marker of microglia) and Pin1 (upper panel) and for GFAP (marker of astrocytes) and Pin1. Data represent the mean ± SEM of three different experiments. **, p < 0.01 vs. TNF-α; ***, p < 0.001 vs. TNF-α.

Figure 6. Juglone inhibits TNF-α or LPS or MPP+ induced activation of p65 in BV2 cells.A. BV2 cells were treated with 10ng/ml TNF-α in presence or absence of 3µM Juglone. 18h after treatment, cells were immunostained for Pin1 and p65. B. BV2 cells were treated with10M MPP+ for 24h in the presence or absence of different doses (1µM, 3µM and 10µM) of Juglone. After treatment, cell lysates were prepared and immunoblotted for p65. C.

Bar graph showing means Western blot p65/β-actin ratios ± SEM from 3 different experiments. D. BV2 cells were treated with 1g/ml LPSfor 24h in the presence or absence of 3µM Juglone. After treatment, cell lysates were prepared and immunoblotted for p65. E.

Bar graph showing means Western blot p65/β-actin ratios ± SEM from 3 different experiments. F. BV2 cells were treated with 10ng/ml TNF-α for 18h in the presence or absence of 3µM Juglone. After treatment, cell lysates were prepared and immunoblotted for p65. G. Bar graph showing means Western blot Pin1/β-actin ratios ± SEM from 3 different experiments. ***, p < 0.001 vs. control; **, p < 0.01 vs. control; @, p < 0.05 vs. MPP+ or TNF-α; #, p < 0.01 vs. MPP+; $, p < 0.05 vs LPS.

Figure 7

Figure 7. Juglone attenuates generation of nitric oxide in BV2 and primary microglial culture.A. BV2 cells were treated with LPS (1µg/ml) in presence or absence of 3 different doses (1µM, 3µM and 10µM) of Juglone. After 24 h of treatment nitric oxide production was

determined indirectly by quantifying the nitrite levels in the supernatant using the Griess reagent and a sodium nitrite standard curve. B. BV2 cells were treated with TNF-α (10ng/ml) in presence or absence of 4 different doses (1µM, 3µM, 5µM and 10µM) of Juglone. After 18 h of treatment nitric oxide production was determined indirectly by quantifying the nitrite levels in the supernatant using the Griess reagent and a sodium nitrite standard curve. C.

Primary microglial cells from C57BL/6 one day old pups were treated with LPS (100ng/ml) in presence or absence of 3µM of Juglone. After 12 h of treatment nitric oxide production was determined indirectly by quantifying the nitrite levels in the supernatant using the Griess reagent and a sodium nitrite standard curve. D. BV2 cells were treated with10M MPP+ for 24h in the presence or absence of different doses 3µM of Juglone. After treatment, cell lysates were prepared and immunoblotted for iNOS. E. Bar graph showing means Western blot iNOS/β-actin ratios ± SEM from 3 different experiments. F. BV2 cells were treated with 1g/ml LPSfor 24h in the presence or absence of 3µM Juglone. After treatment, cell lysates were prepared and immunoblotted for iNOS. G. Bar graph showing means Western blot iNOS/β-actin ratios ± SEM from 3 different experiments. H. BV2 cells were treated with 10ng/ml TNF-α in presence or absence of 3µM Juglone. 18h after treatment, cells were immunoblotted for iNOS. I. Bar graph showing means Western blot iNOS/β-actin ratios ± SEM from 3 different experiments. ***, p < 0.001 vs control; **, p < 0.01 vs control; *, p < 0.05 vs TNF-α or LPS; #, p < 0.01 vs LPS; @, p < 0.001 vs TNF-α

Figure 8

Figure 8.Juglone inhibits LPS induced cytokine production in BV2 cells and primary microglia culture. BV2 cells were seeded in equal numbers on poly-d-lysine coated plates. The cells were treated with LPS (300ng/ml) for 24 h in the presence or absence of 3µM Juglone. After treatment, cytokine levels in the supernatant were quantified using the luminex immunoassay system. Levels of IL-6 (A), TNF-α (B) and IL-12 (C) in the supernatant. Primary microglia culture was initiated from C57BL/6 one day old pupand seeded in equal numbers on poly-d-lysine coated plates. The cells were then treated wilth LPS (100ng/ml) for 24 h in the presence or absence of 3µM Juglone. After treatment, cytokine levels in the supernatant were quantified using the luminex immunoassay system. Levels of IL-6 (D), TNF-α (E) and IL-12 (F) in the supernatant. Data represent the mean ± SEM of three different experiments. ***, p < 0.001 vs. control; **, p < 0.01 vs. control; *, p < 0.05 vs. LPS.

Figure 9

Figure 9. Increased expression of Pin1 in glial cells of MPTP-treated mice. C57BL/6 mice were treated with 4 doses of MPTP (18mg/kg/dose) at 2 h interval via i.p. and 24 h after MPTP treatment mice were sacrificed. A. Double labeling of GFAP and Pin1 in substantia nigra (SN) region of brain. A single enlarged GFAP positive asatroglial cells expresses Pin1 in the cytosol (inset). B. Double labeling of IBA-1 and Pin1 in SN region of brain. A single enlarged IBA-1 positive microglial cell expresses Pin1 in the cytosol (inset).

Figure 10

Figure 10. Activation of p65 in nigrostriatum of MPTP-treated mice. Mice were treated with 4 doses of MPTP (18mg/kg/dose) at 2 h interval via i.p. and 24 h after MPTP treatment mice were sacrificed. A. Western blotting of p65 in substantia nigra. B. Western blotting of p65 in striatum. Mice receiving PBS as a control, MPTP (18mg/kg/dose) or MPTP and Juglone (3mg/kg/day) were sacrificed 24 h after the last dose of MPTP administration and ventral midbrain region was dissected out. C. The mRNA expression of TNF-α , IL-1β, IL-6 and iNOS was analyzed by quantitative real-time PCR. Data represent the mean ± SEM of six mice per group. **, p < 0.01 vs. control; ***, p < 0.001 vs. control; @, p < 0.05 vs MPTP; a < 0.01 vs MPTP.

Figure 11

Figure 11. Juglone attenuates MPTP-induced iNOS expression in glial cells. Mice were treated with MPTP (18mg/kg/dose) at 2 h interval via i.p. in the presence or absence of Juglone (3mg/kg/day). 24 h after the last dose of MPTP injection mice were sacrificed and substantia nigra (SN) tissue sections were processed for immunostaining. A. Double labeling of GFAP and iNOS in SN. B. Double labeling of IBA-1 and iNOS in SN. Inset pictures demonstrated colocalization of GFAP/IBA-1 and iNOS. Images were captured at 20X and 60X (insets) magnifications.

CHAPTER IV

ANTI-INFLAMMATORY AND NEUROPROTECTIVE EFFECTS OF A NOVEL