Part 2 Antidepressant-Like Effects Of Cistanche Tubulosa Extract On Chronic Unpredictable Stress Rats Through Restoration Of Gut Microbiota Homeostasis

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DISCUSSION

Identification of Anti-depression Activity of CTE and Its Clinical Advantages

Cistanche is anti-oxidant and has neuroprotective effects

This study evaluated the antidepressant activity of CTE in CUS rats, confirmed the efficacy of in vivo models such as FST, SPT, OFT, and NSFT, and confirmed the anti-depressive activity of C. tubulosa in CUS rats which is frequently used for the treatment of kidney deficiency, impotence, and female infertility in TCM. Currently, the most commonly prescribed antidepressants are the serotonin and norepinephrine reuptake inhibitor (SNRI) venlafaxine, followed by the selective serotonin reuptake inhibitor (SSRI) FLX (Thase et al., 2001; Mcintyre, 2017). However, these antidepressants have been shown to possess severe side effects including cardiac toxicity, blood pressure, sexual dysfunction, and sleep disorders (Ferguson, 2001; Jin et al., 2015). Among all classes of antidepressants, SSRI and SNRI are correlated with the highest incidence of sexual dysfunction (Montejogonzalez et al., 1997; Clayton et al., 2014). Of particular note is the overall incidence of sexual dysfunction, which is at 59.1% (604/1,022) when all antidepressants are considered as a whole; and about 40% of patients show low tolerance to sexual dysfunction, thus drug compliance is severely affected (Clayton, 2001). In view of the drawbacks of current depression treatment, Cistanches Herba shows great potential for clinical application, not only because of its potent antidepressantlike activity but also because Cistanches Herba has been traditionally used to treat impotence, which should at least eliminate one concern of the most common side effects caused by other antidepressants (Fu et al., 2017). In addition, Cistanches Herba shows no signs of toxicity and treatment-related changes in rats, making us believe it has less harmful side effects and is very safe when used therapeutically (Gao et al., 2016).

CTE Modulates Neurotransmitters and Neurotrophins

Results from this study indicate that CTE administration can significantly increase CUS-induced hippocampus 5-HT level and BDNF expression, along with 5-HT level in the colon. This could well explain the antidepressant activities that CTE exerts. More than 90% of 5-HT in the body is synthesized by specialized endocrine cells in the gut called enterochromaffin cells (Gershon and Tack, 2007; Yano et al., 2015). Previous research demonstrated that spore-forming bacteria in mouse and human microbiota promoted 5-HT biosynthesis from colonic enterochromaffin cells, which supply 5-HT to the mucosa, lumen, and circulating platelets for hosting 5-HT regulation (Yano et al., 2015). So it would be critical to clarify the association between altered gut microbiota structure and 5-HT concentration in the colon after oral administration of CTE, which will shed light upon the pharmacological mechanism of action for the antidepressant-like activity of CTE. Although CTE administration led to significant gut microbiota changes and 5-HT amount increases in the colon, no correlation was found between the altered gut microbiota and the changed 5-HT level in the colon based on Pearson's correlation coefficients. Therefore, it remains unclear whether the homeostasis of gut microbiota led to 5-HT biosynthesis in the colon after CTE administration of the CUS rats. Our ongoing experiment of microbiota transplantation in germ-free mice is to further investigate this.

It has been reported that the aqueous extract of C. tubulosa exhibited an antidepressant effect in the mouse model, modulation of the monoamine system and HPA axis both contributes to the antidepressant effect of C. tubulosa (Wang D. et al., 2017). In our present study, CTE significantly improved depression-like behaviors in rats under CUS by regulating neurotransmitters and neurotrophins in the hippocampus. This well-defined extract, which is composed of 48.6% PhGs, 6.9% iridoid glycosides, and 20.0% total saccharides suggests this mixture might exert the antidepressant effect in multiple ways. For example, PhGs can increase DA levels in the striatum (Tian and Pu, 2005; Geng et al., 2007), iridoids can restore HPA axis dysfunctions and upregulate BDNF expression (Cai et al., 2015; Wang et al., 2015). However, the exact target of the antidepressant effect of CTE on the molecular level, and the contributions from the main constituents in the extract remain unknown, further study is needed to elucidate the comprehensive antidepressant mechanism of CTE.

Cistanche can resist apoptosis

CTE Restores Gut Microbiota Composition and SCFAs Production

In this study, the homeostasis of gut microbiota composition was restored by CTE in the CUS model. An abundance of Bacteroides, strict anaerobes with high importance in the gut from early life (Arboleya et al., 2015), was elevated after CTE administration. Previous studies indicate that Bacteroides fragilis could reverse autism-like behaviors in mice (Hsiao et al., 2013). Our results demonstrate that Bacteroides was positively associated with 5-HT in the hippocampus, and negatively associated with SCFAs, which is correlated with the observed anti-depressive effect of CTE. Moreover, changes in microbial composition have recently been linked to host immunity (Round and Mazmanian, 2009). For example, various Bacteroides spp. can expand the Treg cell population, bias the TH1/TH2 phenotype, or suppress host inflammatory responses by SCFAs (Samuelson et al., 2015). The immune system provides an additional connection between gut microbiota and depression (Miller et al., 2009). Hence, whether CTE affects the Bacteroides population, further regulates the immune system, and then exerts its anti-depressive effects on the host, needs to be investigated in the future. A previous publication indicated prebiotic administration resulted in a significant increase in the abundance of Bacteroides and Parabacteroides, and a decrease in the abundance of Ruminococcus (Burokas et al., 2017). CTE administration also led to a similar increase of Bacteroides and Parabacteroides, as well as a decrease of Ruminococcus. This is probably because the oligosaccharides and polysaccharides in CTE are potential sources of prebiotics. Low abundance of Deinococcus was only detected in the CUS-induced model group, but not detected in the control and CTE administration group. This suggests that the role of these non-dominant bacteria in depression should not be ignored.

Deinococcus was first discovered in 1956 and is known for its remarkable resistance to damage caused by a range of factors一ionizing radiation, desiccation, UV radiation, and oxidizing agents (Gerber et al., 2015). Additionally, Deinococcus possesses the ability to degrade and metabolize sugars due to the presence of genes encoding sugar-metabolizing enzymes (Gerber et al., 2015). In this study, a dramatic increase in the relative abundance of Deinococcus was observed in the CUS-induced group, and following CTE administration the level of Deinococcus dropped significantly to the level of the control group. This implies that CUS might lead to a disordered sugar metabolism level in gut microbiota, and an increase in Deinococcus might attribute to the stress response in CUS rats. After long-term treatment of CTE, CUS rat's sugar metabolism levels could become normal, and the abnormal growth of Deinococcus would then disappear. Based on this, non-dominant bacteria such as the genus Deinococcus can serve as a diagnostic marker for the onset of depression and demonstrate the practicability for the study of the regulation of gut microbiota as a therapeutic target.

Many species from genus Weissella have been isolated and used as probiotic lactic acid bacteria (LAB) due to their beneficial anti-inflammatory, immunomodulatory, and anti-oxidation effects (Ojekunle et al., 2017; Park et al., 2017; Sandes et al., 2017). There is no publication so far that focuses on the connection between the genus Weissella and depression. Our results show that 28 days stress procedure caused a significant reduction in the relative abundance of Weissella in the CUS model group compared to the control non-stressed rats, and daily oral administration of CTE caused a significant increase in the relative abundance of Weissella compared to CUS rats. Interestingly, W. beninensis at species level showed consistent results as Weissella did at the genus level. This confirms a substantial relationship between Weissella and the application of CTE for the treatment of depression. Thus, the combinational use of Weissella probiotics and CTE would be even more beneficial for depression patients.

Short-chain fatty acids are the key molecules that modulate microglia maturation and function, as well as depression (Erny et al., 2015; Dinan and Cryan, 2017). In this study, it was found that CTE could reverse the disordered concentration of acetate and hexanoic acid to a reasonable level. Previous research demonstrated that acetate directly interacts with the hypothalamic mechanisms in the brain (Frost et al., 2014), implying that CTE could regulate disordered acetate and HPA axis for anti-depression. However, there is no published study focusing on the association between hexanoic acid and depression development, which we will further investigate in the future.

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CONCLUSION

In conclusion, CTE exerted potent antidepressant activities via restoring the level of 5-HT, BDNF, and SCFAs, and modulating the relative abundance of gut microbiota in genus level in CUS rats. Correlation analysis revealed that altered gut microbiota genera were also substantially with changed neurotransmitters, neurotrophins, and SCFAs levels. Therefore, CTE was identified as a potential therapeutic agent for depression targeting the microbiota-gut-brain axis.

Archive under accession number SRP128788. The authors declare that all other data supporting the findings of this study are available within the article and its Supplementary Material, or are available from the corresponding author on request with no restrictions.

AUTHOR CONTRIBUTIONS

YL, YP, PT, and XL designed the experiments. YL, PM, HY, and HX carried out the experiments. YL, YP, MW, CP, and LX analyzed the data. YL, YP, and XL wrote the manuscript.

FUNDING

This work was supported by grants from the National Key Research and Development Program of China (SQ2017YFC170458) and the Shanghai Key Laboratory of Psychotic Disorders (16-K02).

SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fphar. 2018.00967/full#supplementary- material

Cistanche is anti-oxidant and has neuroprotective effects

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Clayton, A. H., El, H. S., Iluonakhamhe, J. P., Ponce, M. C., and Schuck, A. E. (2014). Sexual dysfunction associated with major depressive disorder and Archive under accession number SRP128788. The authors declare that all other data supporting the findings of this study are available within the article and its Supplementary Material, or are available from the corresponding author on request with no restrictions.