Elsevier

Journal of Ethnopharmacology

Volume 213, 1 March 2018, Pages 221-229
Journal of Ethnopharmacology

Determination of the effect of Pinellia ternata (Thunb.) Breit. on nervous system development by proteomics

https://doi.org/10.1016/j.jep.2017.11.014Get rights and content

Abstract

Ethnopharmacological relevance

Banxia (BX) is the dried tuber of Pinellia ternata (Thunb.) Breit., a commonly prescribed Chinese medicinal herb for the treatment of cough, phlegm, and vomiting in pregnant women. However, raw BX has been demonstrated to exert toxic effects on reproduction and the precise and comprehensive mechanisms remain elusive.

Aim of the study

We applied an iTRAQ (isobaric tags for relative and absolute quantitation, iTRAQ)-based proteomic method to explore the mechanisms of raw BX-induced fetal toxicity in mice.

Materials and methods

The mice were separated into two groups, control mice and BX-treated mice. From gestation days 6–8, the control group was treated with normal saline and the BX group was exposed to BX suspension (2.275 g/kg/day). Gastrulae were obtained and analyzed using the quantitative proteomic approach of iTRAQ coupled to liquid chromatography-tandem mass spectrometry (LC-MS/MS). A multi-omics data analysis tool, OmicsBean (http://www.omicsbean.cn), was employed to conduct bioinformatic analysis of differentially abundant proteins (DAPs). Quantitative real-time PCR (qRT-PCR) and western blotting methods were applied to detect the protein expression levels and validate the quality of the proteomics.

Results

A total of 1245 proteins were identified with < 1% false discovery rate (FDR) and 583 protein abundance changes were confidently assessed. Moreover, 153 proteins identified in BX-treated samples showed significant differences in abundance. Bioinformatics analysis showed that the functions of 37 DAPs were predominantly related to nervous system development. The expression levels of the selected proteins for quantification by qRT-PCR or western blotting were consistent with the results in iTRAQ-labeled proteomics data.

Conclusion

The results suggested that oral administration of BX in mice may cause fetal abnormality of the nervous system. The findings may be helpful to elucidate the underlying mechanisms of BX-induced embryotoxicity.

Introduction

Pinelliae rhizoma (Banxia in Chinese) is the dried root of Pinellia ternata (Thunb.) Breit.; the first recorded use in Shen Nong Ben Cao Jing ( a classical work of traditional Chinese medicine [TCM]) was approximately 2000 years ago. It is a commonly used Chinese medicinal herb for the treatment of cough, phlegm, vomiting, and cancer (Kao et al., 2012, Wu et al., 2011). Pharmacological studies have shown that BX has suitable antiemetic properties for the management of severe pregnant vomiting (Grobetal et al., 2014, Kurata et al., 1998). However, raw BX has been reported as an irritant, teratogen, carcinogen, mutagen, and, in particular, are productive toxin (Kao et al., 2012, Wu et al., 2011). It is reported that pregnant rats exposed to high doses of BX extract (0.2 and 2 g/kg) may induce fetal abnormalities (Shin et al., 2007). Our previous study also indicated that a suspension of raw BX powder (6 g/kg) caused embryotoxicity in mice (Xu et al., 2013). Subsequently, we confirmed that raw BX suspension (2.275 g/kg) may affect fetal body length (Xu et al., 2017). Nevertheless, the mechanism of raw BX-induced toxicity is still not fully understood.

As Chinese medical herbs contain multiple chemical components and act on multiple targets (Yu et al., 2012), it is difficult to elucidate the mechanism of herbal toxicity using only conventional research methods such as segment II teratology and histological analyses. For the past few years, high-throughput methods such as proteomics and metabolomics with bioinformatics have emerged as effective approaches to extend drug safety knowledge (Ahuja and Sharma, 2014, Robinson and Piersma, 2013). We previously used a metabolomic approach to evaluate the changes of endogenous metabolites in the placental and amniotic fluid of pregnant rats exposed to BX and 20 significantly different metabolic perturbations were identified (Xie et al., 2016).

Proteomics is a systematic approach for performing proteins studies on a large-scale. It is also useful in revealing the potential targets or biomarkers by studying the targets of pharmaceutical products and analyzing major components of physiological pathways (Suo et al., 2016). The role of proteomics in TCM research has increased insignificance and its use in the study of TCM has experienced a steady increase over many years (Fan et al., 2017, Lao et al., 2014, Liu and Guo, 2011). Among the many proteomics technologies, iTRAQ has become popular for protein identification and quantification owing to its sensitivity, accuracy, and high throughput (Wiese et al., 2007). Herein, an iTRAQ-based proteomic method was used to explore the mechanisms of raw BX-induced fetal toxicity in mice.

Section snippets

Plant material and preparation

BX (batch number 20140210), the Chinese medicinal herb was purchased from Anhui Fengyuan Tongling Herbal Pieces Co., Ltd and authenticated by Professor Shengjin Liu (Nanjing University of Chinese Medicine). The dried raw BX were pulverized and sieved through a 100-mesh sieve in order to prepare a powder. A suspension of 0.17 g/mL BX was prepared by the addition of 51 g BX powder into 300 mL water.

Drugs, animal, groups, and treatments

ICR mice of 10 weeks old were obtained from Vital River Laboratory Animal Technology Co., Ltd

Proteomic expression patterns in gastrulae of BX-treated mice

A shotgun quantitation approach based on iTRAQ was used to obtain an overall summary of proteomic changes in the BX group compared with the control group. A total of 1245 DAPs were identified with < 1% FDR in two biological replicates; 583 protein abundance changes were confidently assessed using at least two unique peptides. When the fold-change threshold cutoff was set at 1.5-fold for increased accumulation and < 0.67-fold for decreased accumulation, 153 proteins with differential

Discussion

Raw BX is a typical herbal medicine, which has been widely used in Chinese clinical practice for thousands of years. However, previous reports showed that BX exerted significant toxicity in pregnant mice and embryos (Grobetal et al., 2014). Shin (2007) revealed that BX might cause fetal abnormalities by influencing the expression of genes related to antioxidants, growth factors, apoptosis, and tumors. Metabolomic studies revealed the mechanisms of BX cardio-toxicity were the inhibition of the

Conclusions

In summary, a shotgun quantitation approach based on iTRAQ was applied to assess protein expression changes caused by BX in mice gastrulae. A total of 1245 proteins were identified with < 1% FDR, and 583 protein abundance changes were confidently assessed. Moreover, 153 proteins identified in BX-treated samples showed significant differences in abundance. Among the DAPs, the functions of 37 proteins were predominantly related to nervous system development, including brain development and neuron

Acknowledgments

This study was financially supported by the National Natural Science Foundation of China (Grant number 81303299).

Conflict of interest

All the authors declare that there are no conflicts of interest.

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