AO and AI Rat
Social isolation combined with the resident-intruder paradigm is a classic modeling method in the rat model of AO and AI [46, 47]. Social isolation can induce aggressive behavior in rats [58]. The prolonged isolation of resident rats makes them irritable and aggressive. They can instinctively attack invaders, to protect their territory and generate hostile psychology and behavior. Previous studies have reported that social isolation combined with the resident-intruder paradigm could successfully prepare a scientific and stable rat model of high-aggressive and low-aggressive behavior [37]. Meanwhile, AO and AI rat models were successfully replicated in this paper, laying the foundation for subsequent experiments. However, part of the aggression is because rats, like humans, are a social species. Therefore, isolation-induced aggression likely has a different etiology than resident-intruder aggression and other types of aggression expressed in humans (e.g., rage and impulsiveness,). This may be a flaw in the current study.
Aggression or aggressive behavior, which may be triggered by anger [59], has various associations with anger. Aggression may also be viewed as the behavioral expression of anger [60]. Both anger and aggression could be seen as strong and lasting personal traits [61, 62], individuals with characteristics of aggression are often accompanied by a trait of anger and are more likely to generate anger and enmity in the face of offensive or irritating events [63]. Anger is characterized by increased physiological arousal, and an increased predisposition toward aggressive behavior [64, 65]. In the close association between anger and aggression, many neurochemical and genomic studies have included Spielberger’s Anger-Hostility-Aggression as a research object, in addition to anger itself [66].
ABT is the main indication of the AO and AI rat models. OFT is often used to measure anxiety-like behavior in rats [67]. OFT and SPT testified that emotional changes induced aggressive behavior and the complexity of emotions in rats. The generation of anger is often accompanied by a certain degree of anxiety and depression [68, 69]. In our study, after modeling, AO rats exhibited higher scores of aggressive behaviors, shorter attack latency, and a higher total distance of OFT than AI rats. The SPC of AO and AI rats was significantly lower than that of the control group. After BXD treatment, the score of AO rats’ aggressive behavior was decreased, the latency of aggression was prolonged, the total distance of OFT was shortened, and the SPC was increased. After SY treatment, the score of attack behavior of AI rats was increased, the latency of attack was shortened, the total distance of OFT was prolonged, and the SPC was increased. This finding suggests that BXD can effectively correct the abnormal behavior of AO rats, and SY can effectively correct the abnormal behavior of AI rats. These three evaluation methods have been also used in previous studies on attack behavior evaluation [68, 69]. The results of the present study also confirmed that these three evaluation methods could effectively evaluate the rat model of AO and AI, that BXD could effectively improve abnormal AO tendencies, and that SY could effectively improve abnormal AI tendencies by changing the score of the ABT, OFT, and SPT, and that the intervention effect was good. This result is consistent with the results of relevant reports [70, 71].
Possible action targets and mechanism of BXD against AO and SY against AI
Many studies have demonstrated that 5-HT is a key neurotransmitter that regulates anger attacks, and depression patients with anger are more prone to 5-HT imbalance than patients without anger [34, 37, 72, 73]. Decreased levels of 5-HT or 5-HIAA in brain tissue can increase the occurrence of aggressive behavior and violence [74]. The activity of 5-HT neurons in the DRN increased aggressive behaviors among male mice [75]. By contrast, studies have also found that overexpression of 5-HT1A receptors reduces the activity of 5-HT neurons in the DRN and enhances the aggressive behavior of mice [76]. Studies in knockout mice have also reported that increased aggressive behavior was accompanied by decreased 5-HT levels or reduced activity [77–79]. Some scholars have revealed that different basal levels of 5-HT and phasic changes may have a different roles in different types of aggression [80]. In our study (Figure 5B), both AO and AI rats have a lower level of 5-HT expression in the DRN than normal rats, and AI rats have a lower level of 5-HT expression than AO rats. Furthermore, BXD and SY could treat the abnormally decreased 5-HT level in the DRN.
The PFC, hypothalamus and hippocampus are the key brain areas involved in emotion regulation [81, 82]. The cell body of 5-HT neurons is mainly in the DRN. The DRN has nerve fiber projections with the PFC, hippocampus, and hypothalamus [83], as evidenced by our preliminary study [37]. The results revealed that in AO and AI rats, 5-HT levels in the PFC, hypothalamus, and hippocampus were decreased in tandem with those in the DRN. Our previous research on high and low aggression yielded similar results [37]. Similarly, BXD and SY could treat the abnormal decrease of 5-HT levels in the PFC, hypothalamus, and hippocampus (Figures 3A–3C), and then regulate AO and AI, which require 7 days to achieve a curative effect (Figure 2C).
The release of 5-HT from 5-HT neurons in the DRN was significantly decreased in AO and AI rats, as were the levels of 5-HT projected to the PFC, hypothalamus, and hippocampus via fibers. BXD could relieve AO and SY could relieve AI by treating the aforementioned abnormalities.
GABA is an essential inhibitory neurotransmitter [39]. GABA can seriously affect the function of the PFC [84] and participate in the regulation of emotional disorders [40]. 5-HT and GABA levels have synaptic connections and regulate each other [37, 85–87]. Various pathological processes, including chronic pain, epilepsy, and schizophrenia, are often accompanied by changes in GB1 expression and function [88, 89]. Studies have confirmed that GABA can regulate the level of 5-HT in the DRN through GABABR and GABAAR [90]. The decreased expression of GB1 and GABABR2 in the hippocampus of rats may be related to the generation of AO, and the central mechanism of BXD for calming the liver and regulating qi may be related to the recovery of the expression and function of GABABR in the hippocampus [91]. BXD can also increase the abnormally decreased GABA in the cerebral cortex [92]. Depression model rats can induce the up-regulation of GABABR2 protein expression in rat hippocampal neurons. SY may play an antidepressant role by reducing the expression of GABABR2 protein in hippocampal neurons [93]; SY may also inhibit the decrease of the ratio of Glu to GABA in the hippocampus [94]. In this study, we found that GABA concentration in the DRN was increased and GB1 expression was up-regulated, BXD and SY could effectively reduce GABA concentration (Figure 3D) and down-regulate the expression of GB1 (Figure 5B), then regulate the AO and AI, with a 7-day treatment period required to achieve the curative effect (Figure 2D). This finding suggests that BXD can improve AO by reducing the GABA content in the DRN and down-regulating the expression of GB1, and the best time for improvement is 7 days after treatment. SY can also improve AI in the same way. This finding is consistent with that of relevant research. The intervention of BXD and SY could significantly reduce the scores of anger and depression expression and regulate the contents of 5-HT and GABA in rhesus monkeys [95]. SY can correct behavioral abnormalities and GABA abnormalities in the forehead, hypothalamus, and hippocampus in depressed rats [96]. However, some research results demonstrate that [97] the GABA content in each brain area of rhesus monkeys with liver-qi depression syndrome induced by anger is significantly reduced, whereas the GABA content in each brain area of rhesus monkeys with liver-qi depression syndrome induced by depression is significantly increased. This result is not consistent with that of this study. There may be differences in the models, and the changes in their internal biological indicators and mechanisms are not completely consistent. Furthermore, the expression of GB1 and GABABR2 in the hippocampus neurons of AO rats has decreased [91], which is negatively correlated with the results of the present study because the expression of GB1 is different due to different detected brain regions.
Furthermore, the results of the current study suggested that the GB1 immunoreactivity was increased in both AI and AO compared with that of control rats, whereas, the GB1 immunoreactivity in AI rats was higher than that in AO rats. However, for 5-HT immunoreactivity, no difference was found between AI and AO rats, suggesting that GB1 change is more sensitive to the main difference between AI and AO. The GABA terminal is in close contact with GABABR/5-HT double-standard neurons [37, 98]; GABA may regulate the activity of 5-HTergic neurons through GABABR and participate in the regulation of nociceptive information transmission [98]. The expression of GB1 in 5-HT neurons in the DRN of rats from each group was confirmed using the immunofluorescence double-labeling technique (Figure 4). We determined the objective conditions for GB1-mediated GABA regulation of the existence of 5-HT neurons in the DRN. Although having more the brain regions of histogram and fluorogram will provide more intuitive and in-depth insights, we have obtained the fluorogram and histogram of 5-HT and GB1 in key brain regions (Figures 5, 6), which could explain the problem. Of course, if conditions permit, adding more histograms and fluorograms of brain regions will be more perfect, providing a good guide for our future research work.
Therefore, GABA levels were increased significantly in the DRN of angry-out and AI rats, which further mediated the synaptic effect and exerted an inhibitory function by binding with GB1 on 5-HT neurons in the DRN, thus reducing the release of 5-HT in the DRN and leading to a significant decrease of 5-HT in the PFC, hypothalamus, and hippocampus. This is also the intervention target and way of BXD and SY.
Baclofen is a GB1-specific agonist (Ali Shah et al., 2013), which can bind to GB1 [99]; whereas CGP35348 is a GABABR-specific inhibitor that can specifically bind to GABABR on a presynaptic membrane and/or postsynaptic membrane. GABABR activation in the DRN would enhance the aggressive behavior of male mice, and microinjection of baclofen into the DRN could increase the level of extracellular 5-HT in the prefrontal region [75]. Baclofen can promote the expression of GB1, improve the binding ability of GABA and its receptor, enhance the inhibitory effect of GABA, and thus inhibit the release of 5-HT in the DRN [100]. The current study demonstrated that the levels of 5-HT in the PFC, hippocampus, and hypothalamus in each group were decreased significantly when the DRN was injected with baclofen, while the reverse was observed after the DRN was injected with CGP35348. Therefore, from both positive and negative aspects, GB1-mediated GABA regulates 5-HT levels in the PFC, hippocampus, and hypothalamus, which plays an essential role in AO and AI, and is also the intervention target of BXD and SY.
In conclusion, BXD and SY can act on GB1 in the DRN, and then affect the effect of GABA, thus affecting the levels of 5-HT in the PFC, hypothalamus, and hippocampus of AO and AI rats. Thus, GB1 in the DRN mediates GABA regulation of 5-HT levels in the PFC, hypothalamus, and hippocampus, which is the target of BXD in regulating AO and SY in regulating AI. However, some scholars [101] have found that the extracellular 5-HT content of the medial prefrontal cortex increased after microinjection of baclofen into the DRN, which may be because the total content of 5-HT in and out of the cells in the prefrontal region was not synchronized with the change in extracellular 5-HT content. Furthermore, baclofen injection into the DRN at night can decrease the level of 5-HT in the DRN [100], whereas daytime injection can increase the level of 5-HT in the DRN [102]. Therefore, different injection times of baclofen may lead to the opposite expression of 5-HT.
Notably, BXD can correct the abnormal GB1-mediated GABA regulation of 5-HT levels in the PFC, hypothalamus, and hippocampus in AO, whereas SY can correct the aforementioned changes in AI, and the correction direction is the same. However, BXD can correct the abnormal behavior in AO rats, and SY can correct the abnormal behavior in AI rats, but the correction direction is the opposite. BXD is composed of Paeoniflorin, the volatile oil of Rhizoma Cyperi, and Paeonol, the main effective component of Paeonia lactiflora pall, Rhizoma Cyperi, and Cortex moutan. SY is composed of Bupleurum saponin, Paeoniflorin, and volatile oil of Cyperus, the main effective component of Radix Bupleuri, Radix Paeoniae Alba, and Rhizoma Cyperi. Thus, both BXD and SY contain paeoniflorin and the volatile oil of Rhizoma Cyperi. Paeoniflorin could ameliorate symptoms and improve the functional capability of post-stroke depression rats [103], and it can also protect against cognitive impairment [104]. The volatile oil of Cyperus rotundus can inhibit depression by regulating the content of 5-HT in the brain [105]. However, BXD contains paeonol, whereas SY contains Bupleurum saponin. Paeonol is capable of not only calming hypnosis but also significantly improving learning memory and anxiety [106]. Saikosaponin can increase the 5-HT level in rat brain, and effectively improve depression-like behavior [107]. Saponin A can improve depression-like behavior and regulate the expression of 5-HT and NE in the hippocampus of rats [108]. Paeoniflorin and Cyperus volatile oil may be inclined to correct the micro mechanism of action; Paeonol may be inclined to behavioral correction of anger; Bupleurum saponin may be inclined to behavioral correction of depression. These findings provide an indication for further exploration in the future.
As basic research, the results provide a new scientific basis for in-depth exploration of the brain’s central mechanism of “AO and AI”, clarify the respective corresponding action targets of BXD and SY, and thus open up new ways and new ideas for revealing the essence of Traditional Chinese Medicine (TCM) theory “anger harms the liver”, as well as for basic research and clinical treatment of TCM emotional diseases.