In a previous study, we hypothesized that baicalin would have therapeutic effects in patients with ADHD [23]. To investigate this hypothesis, we investigated if baicalin decreased ADHD symptoms and its underlying mechanisms in 4-week-old SHRs in this study, as 4-week-old rats are developmentally equivalent to the beginning of human childhood [28]. SHRs are one of the most frequently used animal models of ADHD worldwide because they display symptoms similar to the core symptoms of ADHD, such as hyperactivity, impulsivity, and poorly sustained attention compared with WKY control rats [29, 30]. For drug safety considerations during the course of the study, we measured changes in body weight and food intake in rats to evaluate the effect of baicalin on the growth and development of rats. The results showed that baicalin did not affect the normal eating and weight gain of rats, demonstrating that baicalin was safe for gavage.

The core clinical symptoms of ADHD are hyperactivity, impulsivity, and inattention. Whether baicalin can control the core symptoms of ADHD is the basic condition for verifying our hypothesis. In previous studies, moving distance and moving speed were often recorded by the open-field test method to evaluate the hyperactivity behavior in ADHD [31]. The MWM can be used to test spatial learning in rodents, and it has been widely used to test the attention and learning abilities of SHR rats [32]. We evaluated the effects of baicalin on the regulation of behavior in SHRs. In accordance with previous studies, saline-treated SHRs showed increased moving distance and moving speed, which represents typical hyperactivity symptoms, compared with WKY rats, as shown in Tables 3 and 4. After treating the SHRs with baicalin and MPH, these rats showed significant reductions in locomotion. In tests of the spatial learning abilities of the SHR rats, the SHRs treated with MPH and baicalin (especially 150 mg/kg and 100 mg/kg) were faster to find the platform and showed increased ratios in the target quadrant than the saline-treated SHRs (Tables 5 and 6). The typical trajectories showed that the SHRs treated with MPH and baicalin (especially 150 mg/kg and 100 mg/kg) presented significant thigmotaxis movement loci, which is a tendency to move closer to the wall, compared to the WKY rats, while the saline-treated SHRs displayed a chaotic movement trajectory (Figs. 1 and 3) [33]. The behavioral tests showed that treatment with MPH and baicalin (especially 150 mg/kg and 100 mg/kg) significantly reduced the locomotion and increased the spatial learning abilities of SHRs compared with saline-treated SHRs, thus showing effects on the regulation of SHRs behavior.

During the MWM test, WKY rats often floated on the water at the moment they were released into the water and during the experiment. Previous studies have also reported this phenomenon [34]. This phenomenon contrasts with rats’ typical fear of water. Thus, some scholars have suggested that WKY rats have abnormal behaviors and exhibit different degrees of depressive symptoms [35]. One possible reason is the presence of sensory abnormalities in WKY rats. In the study of hypertension, WKY rats are not sensitive to sound stimuli compared with SHRs [36]. Further research is needed to be done to prove whether WKY rats prefer swimming more than other rats or if there is a tactile abnormality in WKY rats.

According to the DA deficit theory, ADHD symptoms might be related to disturbed DA neurotransmission; therefore, we examined the DA system. DA is a monoamine neurotransmitter in the brain; the entire process of dopamine synthesis, release, and removal requires the synaptic vesicles in the synaptosomes. Therefore, the synaptosome is a key structure in the study of the DA system [37, 38]. In synaptosomes, abnormalities in the function of dopamine transporters and D1/D2 receptors in the brain have been reported [39, 40]. Recent research found that DA synthesis, vesicular localization and release are dynamically regulated by several factors, including tyrosine hydroxylase (TH), vesicular monoamine transporter 2 (VMAT2), synaptosomal-associated protein 25 (SNAP25), and syntaxin 1a [41, 42]. TH is the rate-limiting enzyme in the conversion of L-tyrosine to 3,4-dihydroxy-L-phenylalanine (L-DOPA), which is ultimately converted to DA. DA is then transported from the cytoplasm into synaptic vesicles by VMAT2, which is expressed in presynaptic terminals. Syntaxin 1a and SNAP-25 mediate the anchoring and fusion of vesicles and presynaptic membranes and the release of DA into the synaptic cleft. The released DA can bind to DA receptors in the presynaptic and postsynaptic membranes. DA is then taken back up into dopaminergic terminals by the dopamine transporter in the presynaptic membrane and then degraded by monoamine oxidase. An alteration in any of these factors may ultimately disturb the metabolism, transport, and utilization of DA and lead to DA deficits in the brain. As shown in this study, saline-treated SHRs showed significantly decreased protein and mRNA levels of TH, SNAP25, VMAT2, and syntaxin 1a compared with WKY rats. After treatment with MPH and baicalin (especially the 150 mg/kg and 100 mg/kg doses), SHRs showed significantly increased protein and mRNA levels of TH, SNAP25, VMAT2, and syntaxin 1a compared with saline-treated SHRs (Figs. 5 and 6). These results indicated that the pharmacological effects of baicalin were largely associated with DA synthesis, vesicular localization, and release and suggested that baicalin might significantly influence DA levels in the brain. We, therefore, measured the DA levels in the PFC and striatum, which are two key brain regions closely related to the dopamine system and the onset of ADHD [43, 44]. As shown in Fig. 7, MPH can simultaneously increase DA content in the prefrontal and striatum regions, while baicalin at the doses of 150 mg/kg and 100 mg/kg only significantly increased DA levels in the striatum and had little influence on the PFC. The biochemical action of MPH is to block the dopamine transporter (DAT) and norepinephrine transporter (NET), which results in an elevated concentration of dopamine (DA) and norepinephrine (NA) at synapses to control the symptoms of ADHD [45, 46]. The PFC is a key brain region mediating cognitive and executive functions, such as working memory, sustained attention, inhibitory response control, and cognitive flexibility [43, 47]. ADHD patients have shown delayed maturation in the PFC [48], dysfunction of the frontostriatal circuitry [49], and hypoactivation in the frontal cortex [50, 51]. Thus, researchers found that the PFC is identified as the primary target of MPH [52]. Commonly, MPH acts on attention and cognition through an increase in D in the striatum; the conventional dose of MPH has an almost marginally significant increase in the PFC [53]. However, some researches has found that a therapeutic dose of MPH acutely improves cognitive functions by modifying the function of SNAP25 and glutamate receptors in the PFC, while an overdose of MPH inhibited this function and induced psychosis; the PFC is the primary target of a therapeutic dose of MPH worked on [54].

In this study, MPH increased the DA content in both regions, meanwhile, with activation of SNAP25/VMAT2. We speculate that administration of MPH for 4 consecutive weeks may affect the PFC since MPH has long-lasting metaplastic effects in the PFC [55]. Though no glutamate receptor was detected in this study, the activation status of SNAP25/VMAT2 is clearly and the state of glutamate receptors cannot be ruled out. A previous study from Lujun Zhang [56] proved that after treatment with baicalin, baicalin passed through the blood–brain barrier and distributed within the brain tissue, specifically in the hippocampus, striatum, cortex, and thalamus, although the exact mechanism was not reported [57]. Research has found that baicalin can regulate the GABA receptor and inflammatory pathway in the PFC, suggesting that the PFC may also be the target region for baicalin [58, 59]. However, recent researches on the pharmacokinetics of baicalin showed the baicalin content in brain’s target was higher in the striatum and cerebellum, the striatum is the preferential distribution location of baicalin [60] and this maybe the reason why baicalin showed little influence on the DA concentration in the prefrontal cortex region. Thus, we must examine whether the striatum region is the “target region” of baicalin. This topic will be a new direction in further research. We will use more animal experiments to prove our theory in the further. We will use more animal experiments to prove it in the future. Besides, a recent research by our group showed that baicalin has the potent to depress the expression of DAT mRNA as well as protein in SHR rat, to block the reuptaking DA process performed by presynaptic neuron, and then to increase the DA concentration to improve the conduction of DA in the synaptic gap. [Rongyi Zhou, et al. (submitted) Effect of baicalin on the expression of dopamine transporter in SHR rats in striatum synaptosome]. All of the above results indicated that baicalin affected the core symptoms of ADHD by modifying the regulation of DA synthesis, vesicular localization, and release and increased DA levels in the striatum.

This study provides evidence that baicalin regulates DA synthesis and release, increases DA levels in the striatum, and controls the core symptoms of ADHD [61,62,63]. However, the results of this study raise many questions that need to be resolved. First, further research is needed on the optimal dose of baicalin for controlling the core symptoms of ADHD and regulating DA synthesis, vesicular localization, and release. Second, during the MWM test, the WKY rats usually floated on the water, which is contrary to the physiological characteristics of rats, which are afraid of water [64, 65]. Therefore, this phenomenon needs to be examined further in future research. Third, we need to further investigate why baicalin had little effect on the DA levels in the PFC, while it significantly increased the DA levels in the striatum. Is the target of baicalin action located in the striatum? Other synaptic-associated proteins might be useful in these future investigations. Synaptophysin, which is a synaptic vesicle glycoprotein expressed in neuroendocrine cells and most neurons in the CNS, is a hallmark of synaptic vesicle maturation that is considered an indirect marker of synaptogenesis in the developing brain [66]. Similarly, postsynaptic density protein 95, which is involved in the maturation of excitatory synapses and brain-derived neurotrophic factor, which is related to synaptic plasticity [67], are associated with synaptosomal development and the onset of ADHD [68, 69]. Thus, the roles of these proteins should be examined in future studies.

We know that the current research content and mechanism explanation is superficial. However, we aim to publish the research so that it can be seen by more people for continued research that may be helpful for ADHD children. A better understanding of ADHD can help with treatment of children with ADHD and improve quality of life for these children and their families.