Combination of cell metabolomics and pharmacology: A novel strategy to investigate the neuroprotective effect of Zhi-zi-chi decoction
Introduction
Chronic stress has always been one of the most important reasons of public health concern. Long-term psychological stress can lead to psychological sub-health problems such as anxiety, depression, and insomnia, which can not be taken seriously at first because of no typical pathological features and gradually develop into major diseases, such as major depression. Excessive brain excitability and neuronal injury are increasingly considered to participate in the pathogenetic process of these mood and emotion disorders. Furthermore, several clinical studies have demonstrated the abnormal changes of excitatory neurotransmitters in some crucial brain regions. Therefore, preventing from inappropriate level of excitatory neurotransmitters and protecting neurons may be effective measures to cure these psychological sub-health diseases induced by chronic stress.
Zhi-zi-chi Decoction(ZZCD), a tradition Chinese medicine formula which consists of the dried ripe fruits of Gardenia jasminoides J.Ellis (Fructus Gardeniae, Chinese herbal name is “zhi zi”) and the fermented ripe seeds of Glycine max (L.) Merr. (Semen sojae praeparatum, Chinese herbal name is “dan dou chi”), has been extensively utilized for the treatment of febrile diseases, depression and agrypnia for over a thousand years in China (Zhang and Ai, 2010). The major active components of ZZCD are reported to be iridoid glycosides (Han et al., 2015) and isoflavones (Guo et al., 2018), which show various pharmacological activities, including benifit for cardiovascular disease (Sathyapalan et al., 2018), anti-inflammation (Koo et al., 2006), antioxidant and antipyretic effect (Debnath et al., 2011). Moreover, ZZCD is thought to improve neuronal function and anxiety state (Sun et al., 2013). However, there is no molecular mechanisms of neuroprotection of ZZCD which have been found.
Resembling sympathetic ganglion neurons in the phenotype, the adrenal phaeochromocytoma(PC12) cell line has been widely used as cellular model for neurobiological studies in vitro (Westerink and Ewing, 2010). Glutamate(Glu) is one of the major excitatory neurotransmitter, which is strongly associated with psychological and mental disease. Neuronal cells could be dead for excessive extracellular glutamate concentration (Kritis et al., 2015). Therefore, PC12 cells stimulated by glutamate are commonly utilized for studying the mechanisms of cytotoxicity and even neuroprotection of some active components in traditional Chinese medicine.
Weighing less than 2 kD, the full complement of small molecule metabolites found in a specific biological system such as a cell, organ, or organism is termed metabolome (German et al., 2005), which is a direct reflection of the systematic phenotype. Metabolomics is aimed to measure the concentration of these metabolites reliably and repeatably. The advanced modern analytical technologies including Nuclear magnetic resonance(NMR) and mass spectrometry(MS) coupled with chromatograhy can make this happen due to high sensitivity and throughput (Zhang et al., 2011). Cell metabolomics focus on the changes of intracelluar and extracellular molecule metabolites, which is helpful for comprehensive understanding its response to the environment.
In our study, PC12 cells treated glutamate were utilized to imitate neurotoxic cell model and gas chromatograhy coupled with mass spectrometry(GC-MS) based on cell metabolomics was performed to search for biomarkers and investigate the neuroprotective effect of ZZCD from the aspect of metabolism. However, considering the complexity of traditional Chinese medicine in system biology level, the knowledge and technologies of single subject reported in current literature cannot satisfy the exploration of the therapeutic mechanism. Thus an integrated and systemic molecular interpretation on the neuroprotective of ZZCD performed on multidiscipline platform is in urgent need.
Oxidative stress is an indicator of general stress and considered as one component of sub-health, which is associated with mitochondria dysfunction and increased reactive oxygen species(ROS). Oxidative stress commonly arises when the balance of redox is destroyed owing to the decreased antioxidants and antioxidant enzymes in tissue or organs. In addition, the induction of apoptosis-related proteins may also trigger the mitochondrial damage reaction, resulting in the inhibition of respiratory chain and a further incraease in ROS production (Susnow et al., 2009). Thus antioxidation plays an important role in the neuroprotective effect of traditional Chinese medicine.
In this study, in addition to cell metabolomics, pharmacological experiments based on oxidative stress were also performed to evaluate the protective effect of ZZCD. The combination of cell metabolomics and pharmacology was a novel strategy to investigate the neuroprotective effect of ZZCD systematically.
Section snippets
Materials and reagents
The dried ripe fruits of Gardenia jasminoides J.Ellis (Fructus Gardeniae, NO.180525) and the fermented ripe seeds of Glycine max (L.) Merr. (Semen sojae praeparatum, NO.180716-1) were obtained from Tong Han Chun Tang Chinese Herbal Factory (Shanghai, Chinese), identified by Prof. Lu-Ping Qin (Second Military Medical University).
PC12 cells were purchased from Cell Bank of the Chinese Academy of Sciences (Kunming, China). Dipsacoside B, Genipin-1-β-D-gentiobioside, Geniposide, Genipin, Glycitin,
Compound identification of ZZCD
The constituents in ZZCD was identified by comparing the mass spectrum and fragment information with literature. 39 substances were identified tentatively and shown in Table 1.
Content of key compounds in ZZCD
As shown in Fig. 1 and Table 2, Table 7 key compounds in ZZCD including Dipsacoside B, Genipin-1-β-D-gentiobioside, Geniposide, Genipin, Glycitin, Daidzein and Glycitein were determined by external standard method.
Cell viability assays
The effect of glutamate-induced cytotoxicity on the viability of PC12 cells was measured by MTT assay. As
Discussion
With the prevalent trend of chronic stress, psychological sub-health diseases have gradually become a huge obstacle to people's health. One of the most common pathogenic factors of psychological sub-health diseases is excitotoxicity where glutamate can depolarize neurons and change them to fire action potentials. PC12 cells are often used to study neuroprotection in vitro. Therefore, PC12 cells was utilized to study the neuroprotective effects of ZZCD against glutamate for the first time in our
Conclusion
In this study, a novel strategy of the combination of cell metabolomics and pharmacology was applied to systematically evaluate the protective effect of ZZCD on glutamate-induced cytotoxicity in PC12 cells (Fig. 21). Our study demonstrated that ZZCD not only recovered disturbed energy metabolism, amino acids metabolism and lipid metabolism but also exerted anti-oxidation and anti-apoptosis. As a consequence, ZZCD had a good neuroprotective effect and the novel strategy could be extended to more
Author contributions
Designed the study: Yin Zhang, Yuan Yao and Tingting Zhou.
Read and approved the manuscript: Jun Wen and Tingting Zhou.
Performed the experiments: Yin Zhang, Yuan Yao, Xiaolei Shi, Jun Fan and Taohong Huang.
Analyzed the data: Yin Zhang and Yuan Yao.
Wrote the paper: Yin Zhang.
Conflicts of interest
There are no conflicts of interest to declare.
Funding
This work was supported by the National Natural Science Foundation of China [81573584, 81773862].
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These authors contributed equally to this work.