Plasma proteins as potential targets of abnormal Savda syndrome in asthma patients treated with unique Uighur prescription

Abstract

The therapeutic effect of Uighur prescription on abnormal Savda in asthma patients was evaluated using plasma proteomics in order to elucidate the biological mechanism and identify potential therapeutic targets of abnormal Savda. In the present study, 40 asthma patients with abnormal Savda including abnormal Savda Munziq and Savda Mushil were enrolled and treated with Uighur prescription. The effect of Uighur prescription on protein expression and potential targets was investigated by isobaric tags for relative and absolute quantitation (iTRAQ)-based proteomics and bioinformatics analysis. Expression of candidate proteins was verified by an enzyme‑linked immunosorbent assay. Following treatment with the Uighur prescription, 22 proteins were differentially expressed in the plasma of patients with asthma and abnormal Savda. The majority of these proteins were localized in intermediate filaments and the cytoskeleton and acted as antioxidant enzymes and binding proteins. Furthermore, they participated in the defense and inflammatory response, and the response to oxidative stress and wound healing. Peroxiredoxin 2 and carboxypeptidase B2 expression was significantly upregulated, whereas S100A7 was considerably downregulated in the whole plasma of patients (all P Introduction Asthma is a chronic disease of airway inflammation that presents with varying and recurrent symptoms, including cough, chest tightness and shortness of breath (1). Progress has been made in understanding the pathogenesis, diagnosis and treatment of asthma (2), and novel drugs, formulations and dosing instruments have been applied to treat asthma (3,4). However, asthma still presents a considerable threat to health, due to its high morbidity and mortality rates (5). Therefore, the development of novel drugs and complementary therapies, possibly in the form of traditional medicine, such as traditional Uighur medicine, is urgently required. Uighur medicine is a well-established branch of medicine comprised of unique types of medicines, including munziq and mushily of abnormal savda. It is currently practiced by physicians and clinicians from the Xinjiang Uighur autonomous region in China (6,7). Uighur medicine shares an origin with Greco-Arab medicine and describes the incidence of illnesses associated with abnormal Hilits (representing syndromes), which are caused by an imbalance amongst four normal Hilits (representing humors), known as Safra, Kan, Phlegm and Savda (6,8). Quantitative or qualitative changes in any Hilit and the resulting disturbance of dynamic homeostasis of these Hilits may result in the development of corresponding symptoms, including increased quantity of urine, facial edema and weak pulse. Among these, abnormal Savda is the dominant syndrome in disease progression and often develops in conjunction with other abnormal Hilits (8). In traditional Uighur medicine, Savda is the major syndrome responsible for almost all complex diseases, including asthma, type II diabetes mellitus, cancer, and various cardiovascular and neurodegenerative illnesses (8). Previous studies have demonstrated that abnormal Savda is associated with relatively consistent biological changes in complex diseases that are manifested holistically within a population (9–11). Abnormal Savda may be treated with a unique Uighur prescription, comprised of ten herbal ingredients mixed in specific proportions. These are C. dichotoma (10.6%), A. italic (10.6%), G. Uralensis (7.1%), A. capillus-veneris (4.9%), E. humifusa (4.9%), Z. jujuba (4.9%), L. angustifolia (4.9%), F. vulgare (4.9%), M. officinalis (4.9%) and A. pseudoalhagi (42.3%) (7). A number of studies have demonstrated that Uighur prescription may mitigate oxidative stress associated with abnormal Savda, possibly by protecting cells from mitochondrial oxidative damage (12). Uighur medicine may also modulate abnormal changes in the neuroendocrine-immunity network and prevent carcinogenesis in murine models (13). Furthermore, flavonoids isolated from abnormal Savda Munziq are capable of inducing cell-cycle arrest and apoptosis of tumor cells (14). The results of these studies indicate that the biological basis of Savda may change as abnormal Savda develops, but is restored following Uighur prescription treatment. In the disease state, protein expression is dynamically altered in a spatiotemporal manner and modulated by post-translational processing and chemical modifications. Along with the application of emerging proteomics techniques, serum/plasma has become a biomedium for the study of disease etiology, diagnostic biomarkers and drug targets of asthma in contemporary medicine (15). In particular, proteomics analysis has improved knowledge on the allergens of asthma and the effects of clinical therapy, and has even guided personalized therapy (16). In addition, abnormal changes in the whole regulatory network of protein expression are associated with the overall pathological state of patients with asthma (13). This holistic concept of understanding disease etiology through understanding of biological systems is shared by the theories of traditional Uighur medicine. The present study assessed the effect of Uighur prescription of abnormal Savda on the regulatory network of relevant plasma proteins in asthma patients using proteomics. It also identified differentially expressed proteins that are potentially targeted by Uighur prescription. The aim of the present study was to provide evidence for the role of Uighur prescription in treating abnormal Savda at the proteomics level and to contribute to the scientific interpretation and application of Uighur medicine. Materials and methods Uighur prescription Abnormal Savda Munziq is a traditional Uyghur medicinal herbal preparation that consists of the following (10,12,13): 15 g Cordia dichotoma fruit and Ziziphus jujube fruit, 7 g Anchusa italic plant, Dracocephalum moldavica L, Lavandula angustifolia, Adiantum capillus-veneris, Foeniculum vulgare Mill, Euphorbia humifusa Willd, 10 g Glycyrrhiza uralensis Fisch and 15 g Alhagi pseudalhagi. Abnormal Savda Mushil preparations contain: 45 g Alhagi pseudalhagi and Fructus Cassiae fistulae, 15 g Pogonatherum crinitum, Terminalia chebula Retz. and Tenninlia chebula, 12 g Rosa rugosa, 6 g Polypodium vulgare, Glycyrriza Uralensis Fisch. and Foeniculum vuLgare Mill., 10 g Lavandula angustifolia, Adiantum capillus-veneris, Euphorbia humifusa Willd, Anchusa italic, Iberis pectilata L., Viola tianschanica Maxim, Nymphaea L. and Amygdalus communis L., 18 g Raisin, 30 g Cordia dichotoma fruit and Qizil Guliqent and 21 g Cassia angustifolia. All ten herbs were originally grown in Xinjiang, China and collected by the Chi Kang Barbour Pharmaceutical Co. (Xinjiang, China) by professional herbal growers. Patient data In total, 40 patients diagnosed with bronchial asthma between August 2013 and January 2015 were selected for the present study according to the diagnostic criteria of the Guide for Prevention and Treatment of Bronchial Asthma established by the Chinese Medical Association in 2013 (17) and the Global Initiative for Asthma in 2010 (revised in 2012) (18). The present study consisted of 23 males and 17 females, with a mean age of (36.93±12.14) years old (age range, 20 and 60 years old). Abnormal Hilits, including abnormal Kan, Phlegm, Safra or Savda, were identified by an independent diagnosis of two experienced physicians specialized in Uighur medicine. The classification outcome as abnormal Savda was further defined according to the established criteria of Uighur medicine (6,7) and using the assessment of symptom scores for abnormal Savda, including slow pulse ( Peptide detection Nos. Protein information Rec. Symbol Uniprot ID MW (kDa) Calc. pI Peptide score Peptide coverage Unique peptides Fold-change^a 1 Keratin, type II cytoskeletal 6A KRT6A P02538 60 8 1,803.12 55.32 3 6.346 2 Calmodulin-like protein 3 CALL3 P27482 16.9 4.42 37.41 8.05 1 4.897 3 Keratin, type II cytoskeletal 1 K2C1 P04264 66 8.12 1,883.75 53.73 34 4.878 4 Keratin, type II cytoskeletal 72 K2C72 Q14CN4 55.8 6.89 177.81 7.24 1 4.511 5 Keratin, type I cytoskeletal 27 K1C27 Q7Z3Y8 49.8 5.05 44.26 6.54 1 3.927 6 Annexin A1 ANXA1 P04083 38.7 7.02 65.14 8.96 2 3.499 7 InaD-like protein INADL Q8NI35 196.2 4.94 18.24 0.33 1 2.748 8 Keratin, type I cytoskeletal 10 KRT10 P13645 58.8 5.21 1,061.74 44.18 21 2.664 9 Thioredoxin THIO P10599 11.7 4.92 56.05 8.57 1 2.065 10 Carboxypeptidase B2 CPB2 Q96IY4 48.4 7.71 54.12 3.55 2 1.841 11 Serum amyloid A-2 protein SAA2 P0DJI9 13.5 9.14 161.27 31.97 1 1.697 12 Peroxiredoxin-2 PRDX2 P32119 21.9 5.97 48.54 9.09 2 1.502 13 Myeloperoxidase MPO P05164 83.8 8.97 30.51 1.61 1 1.501 14 Keratin, type II cytoskeletal 2 epidermal KRT2 P35908 65.4 8 718.83 28.01 10 1.390 15 Zinc finger and BTB domain-containing protein 18 ZBT18 Q99592 58.3 5.69 28.1 1.34 1 1.345 16 Transforming growth factor beta-1 TGFβ1 P01137 44.3 8.53 35.31 7.69 2 1.263 17 Thrombospondin-4 TSP4 P35443 105.8 4.68 142.57 6.35 4 0.764 18 Protein S100-A7 S100A7 P31151 11.5 6.77 50.31 10.89 1 0.701 19 Retinol-binding protein 4 RBP4 P02753 23 6.07 123.03 9.95 2 0.692 20 Platelet factor 4 variant PF4VL P10720 11.5 9.1 193.28 48.08 2 0.679 21 Lysozyme C LYSC P61626 16.5 9.16 115.72 18.24 3 0.642 22 Protein CASC1 CASC1 Q6TDU7 83.1 5.29 24.76 1.40 1 0.581 a For a candidate protein, the fold-change (ratio) of >1.2 (base 2 logarithm values) or Plasma protein level (ng/ml)^a Protein Prior to treatment (mean ± SD) Following treatment (mean ± SD) Paired t-test P-value PRDX2 379.170±40.978 419.180±48.579 CBP2 1,693.570±114.878 1,783.030±194.455 0.030^b MPO 253.900±46.917 273.606±46.263 0.065 TGFβ1 0.410±0.314 0.445±0.306 0.531 S100A7 1.747±0.115 1.686±0.111 0.026b KRT6A 0.592±0.128 0.557±0.131 0.027^b a Samples from 40 patients b P Yoder M, Zhuge Y, Yuan Y, Holian O, Kuo S, van Breemen R, Thomas LL and Lum H: Bioactive lysophosphatidylcholine 16:0 and 18:0 are elevated in lungs of asthmatic subjects. 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