Fig. 3

BBR activated the pathway for dopa/dopamine synthesis in the gut microbiota. a The activity of TH and DDC increased after treating the gut bacteria of SD rats with BBR at 12 h (10 μg/mL, *P < 0.05, **P < 0.01), but weakened when TH inhibitor (BLMA5, 100 μM) or the DDC inhibitor (benserazide, 100 μM) was incubated with BBR as a whole (mean ± SD, *P < 0.05, **P < 0.01). The TH inhibitor BLMA or DDC inhibitor benserazide did not change the abundance of the intestinal bacteria at 50 or 100 μM. The activity of TH and DDC increased after treating the gut bacteria of SD rats with BBR at 12 h (10 μg/mL, *P < 0.05, **P < 0.01), but weakened when the TH inhibitor (BLMA5, 100 μM) or DDC inhibitor (benserazide, 100 μM) was incubated with BBR as a whole (mean ± SD, *P < 0.05, **P < 0.01). b Levels of BH₄ (the coenzyme of TH) increased significantly at 6 and 12 h after BBR or dhBBR treatment (10 and 20 μg/mL, respectively) in SD gut bacteria in vitro (mean ± SD, +23%↑, +31%↑ for BBR at 20 μg/mL at 6 and 12 h, **P < 0.01, ***P < 0.001; +26%↑, +34%↑ for dhBBR at 20 μg/mL at 6 and 12 h, **P < 0.01). In addition, the level of DDC coenzyme VB₆ was enhanced in the intestinal bacteria at 12 h (mean ± SD, +16%↑ for BBR at 20 μg/mL, ***P < 0.001; +19%↑ for dhBBR at 20 μg/mL, **P < 0.01). c The TH inhibitor (BLMA5, 100 μM) decreased the production of dopa in SD rat gut bacteria when treated with BBR for 6 and 12 h (*P < 0.05, **P < 0.01); DDC inhibitor (benserazide, 100 μM) also lowered the dopamine generation significantly when incubated with BBR (mean ± SD, **P < 0.01, ***P < 0.001). d The DDC inhibitor (benserazide, 100 μM) increased the generation of dopa in SD rat gut bacteria when treated with BBR (mean ± SD, *P < 0.05). e Levels of dopa/dopamine increased significantly at 6 hr after dhBBR treatment in the brain homogenate of mice in vitro (mean ± SD, **P < 0.01, ***P < 0.001). f Coincubation of mouse dopaminergic primary cells with BBR or dhBBR (10 μg/mL), significantly increased dopamine levels in the dopaminergic primary cells incubated with dhBBR (*P < 0.05). g Levels of TH/DDC increased significantly at 6 h after dhBBR treatment in the brain homogenate of mice in vitro (mean ± SD, **P < 0.01, ***P < 0.001). h Levels of BH₄/VB₆ increased significantly at 6 h after dhBBR treatment in the brain homogenate of mice in vitro (mean ± SD, **P < 0.01, ***P < 0.001). i BH₂ decreased significantly (*P < 0.05) with increasing BH₄ (*P < 0.05), when dhBBR was added to the mixture of BH₂ (as substrate) and dihydrobiopterin reductase at 37 °C for 2 h. j Levels of dopa/dopamine decreased significantly at 12 h after the treatment with DHFR inhibitor (methotrexate 100 μM) in the intestinal bacteria (mean ± SD, *P < 0.05)