Rhynchophylline Alleviates Hyperactivity and Cognitive Flexibility Impairment Associated With Inhibition of Inflammatory Responses in Mice That Partly Lack the Dopamine Transporter Protein

Abstract

Background and aims: Rhynchophylline (RHY) can alleviate some cognitive flexibility impairment and stereotyped behavior for attention-deficit hyperactivity disorder (ADHD) and Tourette syndrome (TS) patients as one of a key extract and an active ingredient in Ningdong granule (NDG), which is a Traditional Chinese medicine (TCM) preparation widely used in the treatment of ADHD and TS children in China; however, the underlying mechanism is not well understood. Therefore, this study aimed to evaluate how RHY alleviates hyperactivity and cognitive flexibility impairment while inhibiting inflammatory responses in mice that partly lack dopamine transporter protein (DAT- mice).

Methods: Male DAT- mice were randomly divided into the RHY group (n = 8) and administered RHY (30 mg/kg) in the DAT- group (n = 8) and administered saline (i.p., 10 mL/kg) in wild-type (WT) mice as the WT control group (n = 8). Hyperactivity and cognitive flexibility impairment were evaluated by the open field test (OFT) and the Morris water maze (MWM) test. The levels of the inflammatory factors of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) in cortical homogenates were tested by enzyme-linked immunosorbent assays (ELISA) after 8 weeks of treatment with RHY. In vitro, primary microglia and astrocytes extracted from the cortices of DAT- neonatal mice and WT neonatal mice were treated with lipopolysaccharide (LPS) (1 mg/mL) to induce neuroinflammatory responses and with RHY (20 mM) for 48 h. The levels of the inflammatory factors TNF-α, IL-1β, inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX2) in the culture medium were measured at 6 h, 24 h, and 48 h after treatment with LPS and RHY.

Results: RHY ameliorated hyperactivity and cognitive flexibility impairment in DAT- mice and inhibited the expression of the inflammatory factors TNF-α, IL-1β, iNOS, and COX-2 in microglia and astrocytes in vitro, and also inhibited the expression of TNF-α and IL-1β in cortical homogenates after 8 weeks of treatment.

Conclusion: RHY improved hyperactivity and cognitive flexibility impairment through inhibiting inflammatory responses in DAT- mice.

Keywords: DAT− mice; dopamine transporter; hyperactivity and cognitive flexibility impairment; inflammatory; rhynchophyllin.

FIGURE 1

(a) Chemical structure of RHY. (b) HPLC of the standard substance of RHY (Sigma, Japan) and RHY of the current experimental sample (Zelang Biotechnology, Nanjing, China).

FIGURE 2

DAT protein expression measured by western blotting for DAT− mice versus WT mice. The DAT mRNA band is shown in (a), and relative expression levels are shown in (b). **p < 0.01.

FIGURE 3

Spontaneous locomotor activity of RHY, DAT, and WT mice in the OFT. The results for the LPS group: (a) distance traveled; (b) movement duration; (c) speed; (d) and (e) number of crossings; **p < 0.01.

FIGURE 4

Spatial learning ability and memory in the MWM test: (a) Learning phase: latency to find the platform (**p < 0.01), (b) Probe test: time spent in the target quadrant, and (c) Probe test: distance traveled in the target quadrant, **p < 0.01.

FIGURE 5

Astrocytes and microglia remaining in the cell culture medium for the RHY + LPS, LPS, and WT groups: DAPI staining (a1–c1), GFAP immunostaining (a2–c2), CD11b staining (a3–c3), merged images (a4–c4), and the scale bar = 100 µm.

FIGURE 6

Content of TNF‐α and IL‐1β in cortical homogenates from the mouse groups: (a) TNF‐α and (b) IL‐1β; **p < 0.01.

FIGURE 7

Content of TNF‐α, IL‐1β, iNOS, and COX‐2 in the serum: (a) TNF‐α; (b) IL‐1β; (c) iNOS; (d) COX‐2; **p < 0.01.