ZhiShiXiaoPi tang inhibits autophagy induced by corticosterone and functional dyspepsia through blockade of the mTOR pathway
Graphical abstract
Introduction
Functional dyspepsia (FD) is a common and severe disease characterized by gastrointestinal dysfunction (Enck et al., 2017). Effective treatment of FD is constrained by a limited understanding of the underlying pathophysiology of the disease (Talley, 2017b). Psychological factors have been implicated in the development of FD and anti-depressants are frequently prescribed to treat it, but the molecular mechanism of their action is unknown (Ford et al., 2017; Holtmann et al., 2017; Talley et al., 2015). The brain–gut axis is of particular importance in the pathogenesis of depression-induced FD, because the physiological functions of the gastro-duodenal tract are generally achieved by signal transmission from the central nervous system to the enteric nervous system. Such signal transmission is mediated by the autonomic nervous system (Talley, 2017a). Furthermore, some of the brain's many pathways for controlling the perception of internal and external stimuli might be impaired in FD patients and cause somatic symptoms (Tack and Carbone, 2017). Depression causes abnormalities in the brain-gut axis and subsequently damages gastrointestinal function, causing FD (Lee et al., 2018). Increasing evidence has shown that neuron autophagy is significantly reduced during depression, subsequently leading to apoptosis and FD (Bockaert and Marin, 2015). Therefore, an increase in autophagy during depression could be a potential and effective strategy for the treatment of FD.
Autophagy is a cell survival mechanism that involves the degradation and recycling of cytoplasmic components (Strappazzon et al., 2011). If autophagic activity is insufficient, long-lived proteins and defective organelles accumulate, inducing cell death (Apel et al., 2009). In contrast, if autophagic activity exceeds a certain threshold, it can itself induce cell apoptosis and death (Apel et al., 2009). Autophagy is decreased during depression leading to increased neuronal damage and apoptosis during FD (Bockaert and Marin, 2015). Reductions in autophagy and subsequent apoptosis resulting from depression are accompanied by activation of the mammalian target of rapamycin (mTOR) signaling pathway (Price et al., 2018; Decuypere et al., 2012). Following mTOR activation, the expression of autophagy-related genes (Atg) that regulate the nucleation and elongation of autophagosomes, including Atg5, Atg7, and Atg8 (also known as microtubule-associated protein 1 light chain 3, LC3 in mammals), is reduced (Ly et al., 2018). Similarly, sequestosome 1 (SQSTM1, p62), which is one of the key autophagy substrates, decreasingly interacts with LC3 in response to mTOR activation (Shi et al., 2015). Thus, rescuing insufficient autophagy induced by depression may reduce neuronal damage and preserve gastrointestinal tract function in FD.
ZhiShiXiaoPi Tang (ZSXPT) originates from Lanshi Micang, a Chinese traditional medicine formulary. It contains 10 Chinese traditional medicine plants, including immature bitter orange, magnolia officinalis, coptis, pinellia ternate, rhizoma zingiberis, malt, rhizoma atractylodis macrocephalae, poria cocos, codonopsis pilosula, and liquorice, and has been widely used to treat FD in humans and in animal models (Lin et al., 1998; Oikawa et al., 2009; Cao et al., 2010). Previous studies have shown that ZSXPT significantly alleviates the clinical symptoms of FD characterized by the occurrence of early satiation, postprandial fullness, and epigastric pain (Lin et al., 1998; Oikawa et al., 2009). In addition, previous reports have shown that immature bitter orange, magnolia officinalis, pinellia ternate, rhizoma zingiberis, rhizoma atractylodis macrocephalae, and liquorice water extracts from ZSXPT also alleviate the clinical symptoms of FD (Oikawa et al., 2009; Wu et al., 2011). However, the molecular mechanisms by which ZSXPT protects neurons from reductions in autophagy and subsequent apoptosis in depression-induced FD remain elusive. In this study, we investigated the protective effect of ZSXPT on Cort-induced autophagy, ROS generation, MMP, and apoptosis. Our data reveal the molecular mechanism by which ZSXPT blocks the mTOR pathway in a PC12 cell model of depression and in a rat model of FD.
Section snippets
Materials and reagents
Hoechst 33,342 (B2261) was purchased from Sigma-Aldrich (St. Louis, MO, USA), dissolved in dimethyl sulfoxide (DMSO), and stored at −20 °C before use. Antibodies against mTOR (#2983), Phospho-mTOR (Ser2448, #5536), Bax (#2772), LC3I/II (#4108), Bcl-2 (#2870), p62 (#8025), Atg7 (#8558), Atg5 (#12994), PARP (#9532), and β-Actin (#3700) were purchased from Cell Signaling Technology (Beverly, MA, USA).
Preparation of ZSXPT and HPLC fingerprint analysis
ZSXPT was provided by the Department of Pharmacy, the Affiliated Hospital to Changchun University
Cort induces cell damage and apoptosis in NGF-induced PC12 cells
Cort treatment in NGF-induced PC12 cells is a well-known injury model of depression under in vitro conditions. To establish the model, PC12 cells were incubated in medium with Cort at varying concentrations. After Cort induction, cell viability, LDH release, and apoptosis were assessed using MTT and LDH assays as well as flow cytometry (FCM) to determine an optional condition. Cort decreased cell viability and increased LDH release in a concentration-dependent manner compared to the control
Discussion
In depression-induced FD, chronic inhibition of autophagy has been considered to be a main means of the pathogenesis of gastrointestinal symptoms (Fang et al., 2018; Gassen et al., 2014). Therefore, substantial efforts have been investigated to identify mediators with neuroprotective potential in FD therapy. Our results affirm the strong neuroprotective potential of ZSXPT against autophagy-mediated cell apoptosis and show that it occurs by blocking the mTOR pathway in Cort-induced neurons and
Conclusions
In summary, for the first time, we have demonstrated that ZSXPT pretreatment facilitates autophagy, increases mitochondrial membrane potential, and inhibits ROS generation and consequent apoptosis in Cort-treated neurons and in FD rats. Moreover, our results indicate that the neuroprotective effect of ZSXPT against autophagy-induced damage and apoptosis occurs mainly through the blockade of the mTOR signaling pathway (Fig. 8). The present investigation provides strong evidence that ZSXPT could
Author contributions statement
SG, QH, YX and JX conceived and designed the experiments. SG, QH, XT and XQ performed the research. YY and YZ analyzed the data. SG and QH wrote the paper. LT and PW drafted the manuscript, SG, JX and HG revised the manuscript. All authors gave the final approval and agreed to be accountable for all aspects of the work.
Conflicts of interest
The authors declare no competing interests.
Acknowledgements
This work was supported by the Jilin Province Pharmaceutical and Health Industry Development Special Guidance Fund Project, China (No. YYZX201705).
We thank LetPub (www.letpub.com) for its linguistic assistance during the preparation of this manuscript.
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Authors contributed equally to this work.