Abstract
Purpose: To use label-free proteomic method to investigate the mechanism of action of nuanggong zhitong decoction (NZD) on primary dysmenorrhea (PD).
 Methods: A mouse model of PD was established through oxytocin administration. The mice were divided into control group (normal mice), model group (PD mice administered normal saline), and treatment group (mice given NZD). The serum levels of PGE2 and PGF2α in the mice were measured by ELISA. The differentially expressed proteins (DEPs) among the three groups were revealed by identifying the proteins that were up-regulated (or down-regulated) in model group and down-regulated (or up-regulated) in the treatment group. The DEPs in the three groups were identified using Nano- HPLC-MS/MS, and their functions were investigated using bioinformatics analyses. The accuracy of proteomics was verified with western blot analysis.
 Results: Thirty-eight up-regulated and 66 down-regulated DEPs were identified. Bioinformatics analysis revealed that the DEPs were related to immune response, signal conduction, protein binding, and metabolism. STRING analysis indicated a total of 53 DEPs have direct or indirect functional links. Western blot results revealed that levels of Stat1, Rock1, vinculin and vaveolin-1 were consistent with the results of proteomic analysis.
 Conclusion: These findings provide further insights into the mechanism underlying the protective effects of NZD.
 Keywords: Primary dysmenorrhea, Uterus, Nuangong zhitong decoction, Vinculin, Caveolin, Differentially expressed proteins (DEPs), Bioinformatics
Highlights
Primary dysmenorrhea (PD) refers to recurrent menstrual cramps that are not due to other diseases
Administration of Nuangong zhitong decoction (NZD) induced a remarkable decrease in the levels PGE2 and PGF2α (p < 0.01)
These findings suggest that NZD can significantly relieved PD
Summary
Primary dysmenorrhea (PD) refers to recurrent menstrual cramps that are not due to other diseases. Nuangong zhitong decoction (NZD) has been used for clinical treatment of PD for many years in China It was developed from the traditional Chinese prescription of Wen jing decoction which has been used clinically treating dysmenorrhea for decades. Nuangong zhitong decoction (NZD) is composed of Cinnamomi ramulus, evodiamine, asarum, Radix linderae and rhizoma corydalis at the ratio of 1:2:1:2:2. Label-free quantitative proteomics has been employed to explore the mechanisms of medicine, including traditional Chinese Medicine (TCM) [11,12,13,14] It emerged as a powerful approach for large-scale protein analysis with quantifying peptides and proteins with the use of a peptide’s response as a quantitative measure [15,16]. On the 12th day, oxytocin (20 U/kg) was injected intraperitoneally to the mice to induce severe uterine contraction.
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