Fig 1: Knockdown or pharmacological inhibition of G6PD protected DA neurons from LPS-elicited inflammatory insults. a Primary mouse microglia-enriched cultures were transfected with 10 nM scramble RNA (SS) or G6PD siRNAs (GS1 and GS2) for 30 h before examining the knockdown efficiency by Western blotting assay. b–d The reconstituted cultures, which were prepared by adding mouse microglia (5 × 104 microglia/well) with 30-h knockdown of G6PD by siRNAs onto mouse neuron-astrocyte layer, were treated with vehicle or LPS for 5–6 days. Western blotting assay (b), representative images (c), and cell counting (d) from three to four experiments revealed DA neuroprotection by knockdown of microglial G6PD. e–j Rat mesencephalic neuron-glia cultures were pretreated with vehicle, 6-AN, or DHEA for 30 min and treated with the vehicle or LPS. e Effects of 6-AN and DHEA on G6PD enzyme activity and NADPH levels at 2 days after LPS treatment. f–j Survival of DA neurons was determined by 3[H]DA uptake assay (f, g), western blotting assay (h, i), and representative images (j) at 7 days after LPS treatment. Results are mean ± SEM (a, d–g, i). *p < 0.05 compared with vehicle-treated controls. #p < 0.05 compared with LPS-treated cultures
Fig 2: Exacerbation between G6PD dysfunction and neuroinflammation mediated chronic neurodegeneration. The present study elucidated a pathophysiological role of microglial G6PD in PD neurodegeneration. LPS-induced aberrant upregulation of G6PD promoted cellular oxidative stress and NF-кB activation through increasing NADPH availability to NADPH oxidase. Pharmacological inhibition of G6PD activity or biological knockdown of G6PD attenuated oxidative stress, ameliorated inflammatory response of microglia, and prevented DA neurons from LPS-induced chronic degeneration. G6P, glucose-6-phosphate; G6PD, glucose-6-phosphate dehydrogenase; PPP, pentose phosphate pathway; NADPH, nicotinamideadenine-dinucleotide phosphate; NADP+, oxidized form of NADPH; R5P, ribose-5-phosphate; LPS: Lipopolysaccharide; 6-AN, 6-aminonicotinamide; DHEA, dehydroepiandrosterone; O2−, superoxide; H2O2, hydrogen peroxide; ROS, reactive oxygen species
Fig 3: Knockdown or inhibition of microglial G6PD dampened LPS-induced inflammatory response. a–f At 30 h after transfection with scramble siRNA (SS) and G6PD siRNAs (GS1 and GS2), mouse microglia-enriched cultures were used to examine knockdown efficiency (a) or were challenged with vehicle or LPS (b–f). a Significant knockdown of G6PD expression by G6PD siRNAs was detected by Western blotting assay. b TNFα secretion was measured by ELISA at 6 h after LPS treatment (the time point for maximal release of TNFα). TNFα levels in vehicle-treated control cultures transfected with SS, GS1, and GS2 were 69.36 ± 51.66, 74.67 ± 44.14, and 72.38 ± 44.44 pg/mL, respectively. c–f At 18 h after LPS treatment, immunochemistry (c, d) and Western blotting assay (e, f) showed significant suppression of microglial activation by G6PD knockdown. g–k Rat mesencephalic neuron-glia cultures were pretreated with vehicle or G6PD inhibitors 6-AN (10 μM) and DHEA (100 μM) for 30 min prior to the addition of LPS. g, h Western blotting results indicated that 6-AN and DHEA inhibited LPS-elicited Iba1 upregulation at 2 days after LPS treatment. The ratio of densitometry values of Iba1 normalized to GAPDH in (g) was analyzed (h). i TNFα secretion was measured by ELISA at 6 h after LPS treatment. TNFα levels in vehicle-treated control cultures with pretreatment with vehicle, 6-AN, and DHEA were 4.64 ± 8.21, 3.68 ± 5.40, and 3.39 ± 5.87 pg/mL, respectively. j, k The quantification of Iba1 staining and representative immunochemistry images. Results are mean ± SEM of three to four experiments performed in triplicate (a–c and f, h–j). *p < 0.05 compared with time-matched vehicle-treated controls. #p < 0.05 compared with LPS-treated cultures
Fig 4: Quercetin reduces oxidative stress in AOM/DSS-treated mice. (a) Quantification of the LPO expression level in control and experimental mice colon tissues. (b) Quantification of the NO expression level in control and experimental mice colon tissues. (c) Quantification of the CAT expression level in control and experimental mice colon tissues. (d) Quantification of the SOD expression level in control and experimental mice colon tissues. (e) Quantification of the G6PD expression level in control and experimental mice colon tissues. (f) Quantification of the GSH expression level in control and experimental mice colon tissues. Data are expressed as mean values ± SD (n = 10/group). ∗P < 0.05, Student's t-test.
Fig 5: Synthetic phenotypic change of VSMCs induced by conditional medium from CoCl2‐treated endothelial cells is ameliorated by NAC which is G6PD dependent. mRNA level of IL‐6 (A), iNOS (B), and MMP9 (C) was evaluated in A7r5 rat aortic VSMC cell line treated with conditional medium (CM, black bars) from HAECs treated by CoCl2 in the presence or absence of NAC or 6‐AN for 48 h, or in fresh medium (control, gray bars) supplemented with CoCl2 (400 μm), NAC (10 mm), or 6‐AN (50 μm) for 24 h. Data are expressed as mean ± SEM of independent experiments (n = 3). One‐way ANOVA test followed by Tukey's multiple comparison test was used. *P < 0.05 vs control group, #P < 0.05 vs CoCl2 group, &P < 0.05 vs NAC + CoCl2 group. [Colour figure can be viewed at wileyonlinelibrary.com]
Supplier Page from Abcam for Glucose 6 Phosphate Dehydrogenase Assay Kit (Colorimetric)