Several researchers have found that stress is vital for the subsequent development of depressive symptoms (Bai et al., 2014; Li et al., 2017; Miura et al., 2008; Seo et al., 2017). A suitable stress model should therefore be designed for a more effective simulation of the pathophysiology of depression. Because the physiological state that is assumed to be experienced by the CUMS-exposed mice is similar to the clinical symptoms of depression, a method to induce CUMS was applied in our experiment to simulate the pathophysiology of depression (Li et al., 2020). In the behavioral experiments, the BW, SPT, NSFT, and FST indexes were employed to assess the degree of lost weight, pleasure deficiency, despair, and anxiety in the mice, respectively. The results (Fig. 2B-F and Fig. 6B-F) show that AOVO effectively mitigated depressive symptoms in the CUMS-exposed mice, and TAK242 was found to play a similar role. Furthermore, the result of the OFT (Fig. 2G and Fig. 6G) indicated that treatments involving the administration of AOVO, fluoxetine, or TAK242 with CUMS-exposure did not remarkably influence the locomotor activities of the animals. In brief, similar to TAK242, AOVO appears to also exert a significant antidepressant effect on CUMS-exposed mice.
In the first stage of the experiment (Fig. 2A), four different doses were selected to determine whether AOVO had an anti-depression effect. As revealed by the results of the behavioral tests, AOVO mitigated depressive symptoms in the CUMS-exposed mice. The results of HE and ELISA (Fig. 3A-D) jointly revealed that in the hippocampal samples of the CUMS-exposed animals, AOVO was found to inhibit damage in the CA1 and DG regions of the hippocampus and downregulate several inflammatory factors (IL-6, IL-1β, and TNF). Interestingly, in contrast to the lower dose AOVO group (1.00 mL/kg), the higher dose AOVO group (2.00 mL/kg) did not show a significant effect. Thus, in the subsequent experiment, only lower-dose AOVO (0.25 mL/kg, 0.50 mL/kg, and 1.00 mL/kg) was investigated for further analysis.
The pathogenesis of depression has been correlated with central nervous system (CNS) inflammation (Park, 2019). The CNS inflammatory response is accompanied by the overproduction of proinflammatory factors and neuronal damage (Beurel et al., 2020). Some researchers have also reported that IL-1β, IL-6, and TNF are positively correlated with MDD (He et al., 2020; Vancassel et al., 2018). In the present study, a method used to induce CUMS in mice was adopted to simulate a model of chronic inflammation congruent with the inflammation-related encephalopathy. Consistent with the above reports, the results of HE and ELISA (Fig. 3A-B and Fig. 7A-B) confirmed that CUMS could cause damage to the CA1 and DG hippocampal regions, while also increasing the contents of IL-1β, IL-6, and TNF in the hippocampus. In contrast, AOVO could significantly attenuate this phenomenon. The above findings indicate that AOVO protected mice against CUMS-induced CNS inflammation by decreasing the levels of IL-1β, IL-6 and TNF in the hippocampus. Accordingly, pathways associated with the production of the production of these inflammatory factors may be viable candidates to mitigate the symptoms experienced by MDD patients.
Both pre-clinical and clinical research has indicated that the expression of TLR is related to depression (Qiu et al., 2021; Xu et al., 2020). TLR4, the most representative component of the TLR family, plays a predominant role in the neuroinflammatory process (Cheng et al., 2021; Gao et al., 2021). The activation of TLR4 facilitates the release of inflammatory factors (IL-1β, IL-6, and TNF) (Fu et al., 2019). Subsequently, these deteriorate depressive symptoms. In addition, it is generally known that stress is capable of activating TLR4 to modulate the protein expression of the related TLR4/MyD88/NF-κB signaling pathway (Zhou et al., 2019). Thus, inhibiting the TLR4 signaling pathway might represent a viable treatment method to improve inflammation-associated depressive symptoms. As revealed by this study, CUMS-exposure upregulated the expression of TLR4 and subsequently affected the protein expression of the TLR4/MyD88/NF-κB signaling pathway, thus inducing the production of inflammatory factors (IL-1β, IL-6, and TNF). These results were consistent with those reported previously. In contrast, AOVO significantly ameliorated the CUMS-induced upregulation of TLR4 and MyD88 (Fig. 4A), which was also confirmed by the results of TLR4 immunofluorescence (Fig. 4B). In addition, AOVO could significantly reduce the phosphorylation of IKKα/β and IκBα and prevent the nuclear translocation of NF-κB p65 in CUMS-exposed mice (Fig. 5A-D). The results of NF-κB immunofluorescence (Fig. 5E) confirmed the above results. Accordingly, AOVO downregulated the protein expression of the TLR4/MyD88/NF-κB signaling pathway correlated with CUMS-exposed mice.
To further investigate the antidepressant mechanism of AOVO and TAK242, a TLR4 inhibitor was applied at the second stage of the experiment (Fig. 6A). TAK242 can exert a neuroprotective effect through the TLR4-mediated MyD88/NF-κB signaling (Zhong et al., 2020). Similar to the above reports, in this study, TAK242 was found to protect against neuroinflammation in CUMS-exposed mice by inhibiting TLR4-mediated MyD88/NF-κB signaling. As revealed by the results of the behavioral experiments (Fig. 6B-F), coadministration with AOVO increased the antidepressant effects of TAK242 on the CUMS-exposed mice. The above results suggest that AOVO may synergize with TAK242 to mitigate depression-like symptoms in CUMS-exposed mice. Whether AOVO alters the effects of TAK242 on TLR4 downstream-related proteins warrants further exploration. Previous studies have reported that TAK242 can suppress MyD88 protein expression by reducing TLR4 protein expression. The coadministration of AOVO and TAK242 could further downregulate MyD88 expression (Fig. 8A). Thus, all the above data indicate that AOVO mitigates depression-like symptoms in CUMS-exposed mice via the TLR4/MyD88 signaling pathway.
Relevant studies have shown that NF-κB is pivotal for modulating the TLR4-mediated proinflammatory cascade (Chen et al., 2021; Xu et al., 2020). Moreover, the NF-κB activation is correlated with IKK-dependent IκBα phosphorylation and the translocation of cytosolic NF-κB p65 to the nucleus (Qu et al., 2021). Subsequently, several proinflammatory factors (IL-6, IL-1β, and TNF) are released. In this study, the results obtained by WB indicated that AOVO and TAK242 could inhibit the phosphorylation levels of IKKα/β and IκBα (Fig. 5A-B and Fig. 9A-B). Next, cytosolic NF-κB p65 was inhibited from being translocated to the nucleus (Fig. 5C-D and Fig. 9C-D), which was verified by the results obtained by NF-κB immunofluorescence (Fig. 5E and Fig. 9E). This was followed by an increase in the levels of IL-6, IL-1β, and TNF (Fig. 3B-D and Fig. 7B-D). In addition, the coadministration of AOVO and TAK242 could further enhance the above effects (Fig. 7B-D). It has been reported that the upregulated expression of inflammatory factors (IL-6, IL-1β, and TNF) leads to a significant increase in IDO1 (indoleamine-2,3-dioxygenase). When the IDO1 was activated by neuroinflammation, it could decrease the levels of the 5-HT, thus leading to depression. The results of Fig. 10A-B are consistent with the above report. In brief, AOVO was found to inhibit neuroinflammation in CUMS-exposed mice by inhibiting the TLR4-mediated MyD88/NF-κB signaling pathway..
A total of 74 chemical compositions were isolated from AOVO by GC/MS (Fig. 1) and 73 among the 74 were identified. To increase the accuracy of the GC/MS results, compounds with a score above 80 were selected (Table 1). Interestingly, the above 73 compounds were all small molecule compounds, and most were terpenes. Of all the above compounds, nootkatone was the most abundant (18.41%). It has been reported that terpenes, especially nootkatone, can improve depression-like behavior (Abd Rashed et al., 2021; Yan et al., 2021). Furthermore, nootkatone exerts neuroprotective effects. Accordingly, nootkatone may be an essential component in AOVO that is responsible for its antidepressant-like effect.
In this study, we conducted a composition analysis of the AOVO and demonstrated that its administration could reduce CUMS-induced depressive behaviors and neuroinflammation via suppressing the TLR4-mediated MyD88/NF-κB signaling pathway. However, which precise component of AOVO possesses antidepressant activities remains unclear. Thus, a follow-up study should be performed to investigate the mechanism underlying the antidepressant effect of AOVO and the specific functional components involved.