Research into the anti-pulmonary fibrosis mechanism of Renshen Pingfei formula based on network pharmacology, metabolomics, and verification of AMPK/PPAR-γ pathway of active ingredients.

ETHNOPHARMACOLOGICAL RELEVANCE: Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive disease with limited therapy. Renshen Pingfei Formula (RPFF), a classic Chinese medicine derivative formula, has been shown to exert therapeutic effects on IPF. AIM OF THE STUDY: The study aimed to explore the anti-pulmonary fibrosis mechanism of RPFF through network pharmacology, clinical plasma metabolomics, and in vitro experiment. METHODS: Network pharmacology was used to study the holistic pharmacological mechanism of RPFF in the treatment of IPF. The differential plasma metabolites for RPFF in the treatment of IPF were identified by untargeted metabolomics analysis. By integrated analysis of metabolomics and network pharmacology, the therapeutic target of RPFF for IPF and the corresponding herbal ingredients were identified. In addition, the effects of the main components of the formula, kaempferol and luteolin, which regulate the adenosine monophosphate (AMP)-activated protein kinase (AMPK)/peroxisome proliferator-activated receptor γ (PPAR-γ) pathway were observed in vitro according to the orthogonal design. RESULTS: A total of 92 potential targets for RPFF in the treatment of IPF were obtained. The Drug-Ingredients-Disease Target network showed that PTGS2, ESR1, SCN5A, PPAR-γ, and PRSS1 were associated with more herbal ingredients. The protein-protein interaction (PPI) network identified the key targets of RPFF in IPF treatment, including IL6, VEGFA, PTGS2, PPAR-γ, and STAT3. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis acquired the main enriched pathways, and PPAR-γ involved in multiple signaling pathways, including the AMPK signaling pathway. Untargeted clinical metabolomics analysis revealed plasma metabolite variations in patients with IPF versus controls and before versus after RPFF treatment for patients with IPF. Six differential metabolites were explored as differential plasma metabolites for RPFF in IPF treatment. Combined with network pharmacology, a therapeutic target PPAR-γ of RPFF in IPF treatment and the corresponding herbal components were identified. Based on the orthogonal experimental design, the experiments showed that kaempferol and luteolin can decrease the mRNA and protein expression of α-smooth muscle actin (α-SMA), and the combination of lower dose can inhibit α-SMA mRNA and protein expression by promoting the AMPK/PPAR-γ pathway in transforming growth factor beta 1 (TGF-ß1)-treated MRC-5 cells. CONCLUSIONS: This study revealed that the therapeutic effects of RPFF are due to multiple ingredients and have multiple targets and pathways, and PPAR-γ is one of therapeutic targets for RPPF in IPF and involved in the AMPK signaling pathway. Two ingredients of RPFF, kaempferol and luteolin, can inhibit fibroblast proliferation and the myofibroblast differentiation of TGF-ß1, and exert a synergistic effect through AMPK/PPAR-γ pathway activation.

[Effect of Maxing Shigan Decoction and dissembled prescriptions against airway inflammation in RSV-aggravated asthma and mechanism of regulating TRPV1].

This study investigated the effect of Maxing Shigan Decoction(MXSGD) and its disassembled prescriptions against the airway inflammation in respiratory syncytial virus(RSV)-aggravated asthma and the regulation of transient receptor potential vanilloid-1(TRPV1). To be specific, ovalbumin(OVA) and RSV were used to induce aggravated asthma in mice(female, C57BL/6). Then the model mice were intervened by MXSGD and the disassembled prescriptions. The eosinophil(EOS) in peripheral blood, inflammatory cells in bronchoalveolar lavage fluid(BALF), enhanced pause(Penh) variation, and lung pathological damage in each group were observed, and the changes of interleukin(IL)-4, IL-13, substance P(SP), and prostaglandin E2(PGE2) in BALF were mea-sured by enzyme-linked immunosorbent assay(ELISA). Quantitative real time polymerase chain reaction(qPCR) and Western blot were used to detect mRNA and protein of TRPV1 in mouse lung tissue. In the in vitro experiment, 16 HBE cells were stimulated with IL-4 and RSV. Then the changes of TRPV1 expression after the intervention with the serum containing MXSGD and its disassembled prescriptions were observed. Besides, the intracellular Ca~(2+) level after the stimulation with TRPV1 agonist was evaluated. The results showed that the mice in the model group had obvious asthma phenotype, the levels of various inflammatory cells in the peripheral blood and BALF and Penh were significantly increased(P<0.05, P<0.01), and the lung tissue was severely damaged compared with the control group. Compared with the model group, the levels of EOS in the peripheral blood and BALF were significantly decreased in the MXSGD group, the SG group and the MXC group(P<0.05, P<0.01). The levels of WBC and neutrophils in BALF were significantly decreased in the MXSGD group and SG group(P<0.01), the levels of neutrophils in BALF were decreased in the MXC group(P<0.05). The improvement effect of the MXGSD on the level of inflammatory cells in peripheral blood and BALF was better than that of two disassembled groups(P<0.05, P<0.01). After 50 mg·mL~(-1) acetylcholine chloride stimulation, the Penh values of the MXSGD group and the MXC group significantly decreased(P<0.01), and the Penh value of the SG group decreased(P<0.05). The levels of IL-4, IL-13, PGE2 and SP in BALF could be significantly decreased in the MXSGD group(P<0.05, P<0.01), the levels of IL-13 and PGE2 in BALF could be decreased in the MXC group(P<0.05, P<0.01), and the levels of IL-13, PGE2 and SP in BALF could be decreased in the SG group(P<0.05, P<0.01). MXSGD could down-regulate the protein and mRNA expression of TRPV1 in lung tissue(P<0.05, P<0.01). The serum containing MXSGD and its disassembled prescriptions could down-regulate TRPV1 expression in 16 HBE cells stimulated by IL-4 combined with RSV and inhibit the inward flow of Ca~(2+) induced by TRPV1 agonist, especially the serum containing MXSGD which showed better effect than the serum containing disassembled ones(P<0.05). In vivo and in vitro experiments verified the protective effect of MXSGD and its disassembled prescriptions against airway inflammation in RSV-exacerbated asthma, the whole decoction thus possessed synergy in treating asthma, with better performance than the dissembled prescriptions. Different groups of prescription had made contributions in improving airway hyperresponsiveness, anti-allergy and anti-inflammation. The mechanism is the likelihood that it regulates TRPV1 channel and levels of related inflammatory mediators.

Effect of Houpo-Mahuang Decoction on aggravated asthma induced by cigarette smoke and the expression of TRPA1 and tight junctions in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Cigarette smoke (CS) is a common environmental irritant and a risk factor for asthma, as it induces as well as aggravates asthmatic attacks. The injured airway epithelial tight junctions (TJs) aggravate asthma. CS can aggravate asthma by activating the transient receptor potential ankyrin A1 (TRPA1) channel and enhancing TJs destruction. Houpo Mahuang decoction (HPMHD) is a classic traditional Chinese prescription for the treatment of asthma. However, its underlying action mechanism is unclear. AIM OF THE STUDY: The present study aimed to evaluate the effect of HPMHD on the asthma phenotype and the regulation of TRPA1 and TJs in a CS-induced mouse model of aggravated asthma. MATERIALS AND METHODS: Under optimized chromatographic and mass spectrometry conditions, the ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS) technique was used to detect and analyze the major chemical components of HPMHD. C57BL/6 female mice were randomly divided into seven groups, viz, normal saline (NS) group, ovalbumin (OVA) + CS group, dexamethasone group, HPMHD high-dose group and low-dose groups, n-butanol extract group, and ethyl acetate extract group, with 10 mice in each group. OVA sensitization and challenge, and CS exposure were used to establish the aggravated asthma model. As the main indices to evaluate the protective effect of HPMHD, the eosinophils count in peripheral blood, percentages of inflammatory cells classified and the levels of interleukin (IL)-4, IL-5, IL-13 in the bronchoalveolar lavage fluid (BALF), airway responsiveness enhanced pause (Penh), and changes in lung histopathology were determined and compared among the groups. The mRNA and protein expression of TRPA1 and TJs in lung tissue was also examined. RESULTS: Using UPLC-QTOF-MS, the chemical components of HPMHD, including ephedrine, pseudoephedrine, laetrile, and amygdalin amide, were identified by 51 signal peaks. Compared with those in the NS group, the eosinophil number in the peripheral blood and the eosinophils and neutrophils percentages in BALF of the OVA + CS group were remarkably increased. Following the inhalation of 50 µl of acetylcholine chloride (ACH) at doses of 25 and 50 mg/mL, the Penh increased significantly (p < 0.01). Moreover, in the OVA + CS group, hematoxylin and eosin (H&E) staining of lung tissue showed a significant number of infiltrated inflammatory cells, increased mucus secretion in the lumen, damaged bronchial mucosa, increased thickness of tracheal wall, and increased score of lung damage (p < 0.01). The IL-4/5/13 levels were also remarkably increased (p < 0.01). The protein as well as gene expression of both ZO-1 and occludin decreased markedly in the lung tissue, while the expression of TRPA1 and claudin-2 was increased (p < 0.05, p < 0.01). Next, the OVA + CS group and the treatment groups were compared. The inflammatory cells, Penh value, and levels of IL-4/5/13 were significantly reduced, and less lung injury was observed in the treatment groups. The gene and protein levels of TRPA1 and TJs were corrected (p < 0.05, p < 0.01); the effects on the HPMHD high-dose and ethyl acetate extract groups were particularly remarkable. CONCLUSIONS: HPMHD reduced airway hyperresponsiveness, inflammatory cell recruitment and Th2 cytokine secretion in CS-induced aggravated asthma mice, in a manner potentially dependent on regulation of the expression of TRPA1 and TJ proteins. Both the n-butanol and ethyl acetate extracts contained the active ingredients, especially the ethyl acetate extract.

Effects of cigarette smoke on the aggravation of ovalbumin-induced asthma and the expressions of TRPA1 and tight junctions in mice.

The occurrence of asthma is closely related to environmental factors such as cigarette smoke (CS), one of the common risk factors. Environmental stimuli have the potential to activate transient receptor potential ankyrin 1 (TRPA1) and cause or aggravate asthma. The destruction of tight junctions (TJs) between airway epithelial cells by environmental stimuli in asthma has been researched. It is worth exploring whether CS can injury TJs and aggravate asthma by activating TRPA1. The objective of this study was to investigate the aggravation of CS on ovalbumin (OVA)-induced asthma related phenotypes and TJs expression in mice, and to explore the relationship between TRPA1 and the expression of TJs protein. Female wild type (WT) C57BL/6 mice, induced by OVA, CS and OVA plus CS (OVA + CS) respectively, were used to establish a 42-day asthma model, and mice with TRPA1 knockout (TRPA1-/-) were treated in the same way. This study detected the number of inflammatory cells in peripheral blood and bronchoalveolar lavage fluid (BALF), the levels of IL-4, IL-5, IL-13 in BALF, enhanced pause (Penh) of lung function, pathological changes and the gene and protein expressions of TRPA1 and TJs (including ZO-1, Occludin and Claudin-2) in lung tissues. Compared with normal saline (NS) group, WT mice in the OVA group and OVA + CS group were significantly higher in asthma related phenotypes. The WT-OVA + CS group also showed higher Penh value, levels of IL-5 and IL-13 in BALF and lung tissue injury scores when compared with the WT-OVA group and WT-CS group. However, WT-OVA + CS group mice had significantly larger number of neutrophils in BALF than the WT-OVA group, and had larger number of eosinophils in peripheral blood and higher levels of IL-4 in BALF than the WT-CS group. Meanwhile, compared with the WT-NS group, the expressions of TRPA1 and Claudin-2 in lung tissues increased in other three groups while their expressions of ZO-1 and Occludin decreased, among which, the WT-OVA + CS group showed more remarkable changes. Compared with the WT-OVA + CS group, mice in the TRPA1-/--OVA + CS showed a significant decrease in the number of inflammatory cells, levels of IL-4, IL-5 and IL-13 in BALF, Penh value and lung tissue injury score, and a downregulation of Claudin-2 expression while an upregulation of ZO-1 and Occludin expressions. In addition, the airway inflammation and injury, and the expressions of ZO-1, Occluding and Claudin-2 expressions were found with no statistic differences between TRPA1-/--OVA group and TRPA1-/--OVA + CS group. These results suggest that CS has aggravated the airway inflammation, pathological damage and destruction of TJs in airway epithelium of OVA-induced asthmatic mice, the processes of which are related to the increase of TRPA1 expression.

[Effect of San'ao Decoction on ovalbum induced asthmatic mice and expression of TRPV2 in lung].

To observe the efficacy of San'ao Decoction(SAD) in diffusing the lung and relieving asthma, and its intervention effect on the expression of transient receptor potential V2(TRPV2) during alleviating asthma, this study replicated an ovalbumin(OVA)-induced asthmatic mice model, and investigated the intervention effect of SAD on the airway inflammation and airway hyperresponsiveness. The regulatory mechanisms of SAD on the mRNA and protein expressions of TRPV2 in lung tissues and the levels of interleukin-4(IL-4),-10(IL-10), nerve growth factor(NGF), prostaglandin D_2(PGD_2) in bronchoalveolar lavage fluid(BALF) were discussed. Compared with the control group, the model group showed typical asthmatic phenotype, the level of eosinophils(EOS) in peripheral blood and BALF as well as the airway hyperresponsiveness were increased(P<0.01), and pathological damage in lung tissue was serious. The mRNA and protein expressions of TRPV2 in lung tissue were increased significantly, while the levels of IL-4, IL-10, NGF and PGD_2 in BALF were elevated(P<0.05,P<0.01). SAD could relieve bronchial asthma manifested as repaired lung patholo-gical changes(P<0.05), reduce the level of EOS in blood and BALF(P<0.05, P<0.01), and improve pulmonary resistance and lung compliance(P<0.05, P<0.01). SAD could also regulate the inflammatory cytokine levels of IL-4, IL-10, NGF, PGD_2 in BALF, and reduce the gene and protein expression of TRPV2 in the lung tissue(P<0.05, P<0.01). It is verified that SAD could reduce the lung inflammation, and improve lung function in asthmatic mice. The regulatory mechanism of SAD on asthma induced by OVA might be related to the regulation of TRPV2 expression and the induced decrease of Th2-related cytokines and neuropeptides, which provides the evidences for the treatment of asthma with SAD.

[Research on compatibility of prescriptions including Ginseng Radix et Rhizoma and Trogopterus Dung based on complex network analysis].

The applications of prescriptions including Ginseng Radix et Rhizoma and Trogopterus Dung in contemporary literatures from 1949 to 2016 are compiled and the data mining techniques containing scale-free complex network method are utilized to explore its practical characteristics, with comparison between modern and ancient ones. The results indicate that malignant neoplasms, coronary heart disease which present Qi deficiency and blood stasis type are the main diseases treated by prescriptions including Ginseng Radix et Rhizoma and Trogopterus Dung according to the reports during 1949 to 2016. The complex network connection shows that Glycyrrhizae Radixet Rhizoma, Angelicae Sinensis Radix, Astragali Radix, Typhae Pollen, Salviae Miltiorrhizae Radix et Rhizoma are the primary drugs related to Ginseng Radix et Rhizoma and Trogopterus Dung. The next are Paeoniae Radix Alba, Atractylodis Macrocephalae Rhizoma, Persicae Semen, Foria, et al. Carthami Flos, Notoginseng Radix et Rhizoma, Cyperi Rhizoma, Bupleuri Radix are the peripheral ones. Also, Ginseng Radix et Rhizoma-Glycyrrhizae Radixet Rhizoma, Trogopterus Dung-Glycyrrhizae Radixet Rhizoma, Ginseng Radix et Rhizoma-Angelicae Sinensis Radix, Trogopterus Dung-Angelicae Sinensis Radix, Ginseng Radix et Rhizoma-Astragali Radix, Trogopterus Dung-Astragali Radix are the main paired drugs. The paired drugs including Ginseng Radix et Rhizoma-Trogopterus Dung-Glycyrrhizae Radixet Rhizoma, Ginseng Radix et Rhizoma-Trogopterus Dung-Angelicae Sinensis Radix, Ginseng Radix et Rhizoma-Trogopterus Dung-Astragali Radix, Ginseng Radix et Rhizoma-Trogopterus Dung-Typhae Pollen have a higher support degree. The main compatible drugs are different in ancient and modern prescriptions including Ginseng Radix et Rhizoma and Trogopterus Dung. Notoginseng Radix et Rhizoma, Typhae Pollen, Salviae Miltiorrhizae Radix et Rhizoma, Astragali Radix are utilized frequently in modern prescriptions while less used in ancient ones. It is also shown that more attentions are paid to the drugs contributing to invigorating Qi and promoting blood circulation in modern times with comparative results between modern and ancient prescriptions.

[Compatible principles of Xiebai decoction family based on complex network analysis].

In order to explore the compatible principles of Xiebai decoction family, formulae from ancient and modern Xiebai decoction family were collected and sorted in this study. The compatible characteristics, core herbs, as well as the relativity of herbs nature in Xiebai decoction family were analyzed based on scale free network and other data-mining methods such as association rules, clustering analysis and correspondence analysis. The scale free network results showed that in Xiebai decoction family, Mori Cortex-Lycii Cortex-Glycyrrhizae Radix et Rhizoma was used as the core compatible group and formed the complicated compatible network with other additional herbs; association rules results showed that the core herbs in such formulae included Mori Cortex, Lycii Cortex, Glycyrrhizae Radix et Rhizoma, scutellaria root, Platycodon root, Anemarrhena, and almond, which formed corresponding herbal pairs and compatibility; clustering analysis showed that Mori Cortex was the core herb in Xiebai decoction family, and Mori Cortex-Lycii Cortex-Glycyrrhizae Radix et Rhizoma was its main combination unit, which was always compatible with herbs of clearing heat, reducing phlegm, supplementing Qi and nourishing Yin to form the series prescriptions. The results indicated that the core compatibility features of Xiebai decoction family were clearing heat in lung and relieving cough and asthma, providing a basis for the clinical application of Xiebai decoction family.

Effect of Renshen Pingfei Decoction, a traditional Chinese prescription, on IPF induced by Bleomycin in rats and regulation of TGF-ß1/Smad3.

AIM OF THE STUDY: Idiopathic pulmonary fibrosis (IPF), one of the clinical common diseases, shares similar pathogenesis with ancient disease "Feibi" in Chinese medicine, Renshen pingfei decoction (RPFS), a classical prescription, was commonly used in treating Feibi. In the current study, the protective role of RPFS in rats model of IPF and the mechanism via regulation of TGF-ß1/Smad3, were evaluated and explored. METHODS: The chemicals of RPFS were analyzed by UPLC-QTOF-MS. Under the optimized chromatographic and MS condition, the major components in RPFS were well separated and detected. An IPF model was established in rats which were induced with Bleomycin (BLM). After treated with corresponding medicine for 7 days, 14 days, 21 days and 28 days respectively, lung function of rats were measured; peripheral blood and bronchoalveolar lavage fluid (BALF) were assessed; histopathological changes and homogenate of lung tissue were detected; TGF-ß1 and Smad3 mRNA and protein expressions in lung tissue were examined as well. RESULTS: 43 signal peaks of chemical components in RPFS were identified by UPLC-QTOF-MS method. Compared with model group, RPFS group exerted significant effects on IPF model rats in improving lung function and decreasing HYP content of lung tissue (P<0.01), reducing the level of TGF-ß1 and NFκB in BALF (P<0.05), decreasing SOD and MDA level in serum (P<0.01), as well as down-regulating TGF-ß1 and Smad3 mRNA and protein expressions of lung tissue (P<0.01). CONCLUSION: RPFS could reduce the lung injury and fibrosis degree and improve lung function of IPF model rats. The protective role might mediated by down-regulating TGF-ß1/Smad3 signaling pathway.

[Research on application status of Chinese herbal decoction pieces based on clinical survey].

Chinese herbal decoction pieces are the basic approaches for clinical traditional Chinese medicine (TCM), reflecting the features and advantages of TCM. In order to investigate the clinical application status and features of Chinese herbal decoction pieces, the questionnaire on application of commonly used Chinese herbal decoction pieces was designed in this study for analysis of the application situations of Chinese herbal decoction pieces from 56 medical institutions in 10 provinces. The results showed 549 varieties of Chinese herbs and 801 varieties of decoction pieces were used on clinic. They can be classified into 19 categories according to their effects. The varieties of Gancao (Glycyrrhizae Radix et Rhizoma), Huangqi (Astragali Radix), Dihuang (Rehmanniae Radix), Chuanxiong (Chuanxiong Rhizoma), Baizhu (Atractylodis Macrocephale Rhizima), Huangqin (Scutellariae Radix), Danggui (Angelicae Sinenses Radix), Baishao (Paeoniae Radix Alba) and Maidong (Ophiopogonis Radix) were most common ones; the application of Chinese herbal decoction pieces from different medical institutions was differentiated from areas to areas. The survey results reflected the general situation about application of decoction pieces, providing the basic data for recording and completing Chinese herbal decoction pieces in essential drug system, with certain reference significance for the production of Chinese medicinal materials and the allocation of the varieties of Chinese herbal decoction pieces.

[Effect of Xianxiong decoction on acute lung injury mice induced by lipopolysaccharide].

OBJECTIVE: To investigate the effect of Xianxiong decoction on the mice with acute lung injury induced by lipopolysaccharide. METHOD: Eighty female ICR mice were randomly divided into 8 groups: model group, Xianxiong decoction group, Daxianxiong decoction group, Xianxiong decoction group without Kansui Radix group, Xianxiong decoction group without Glycyrrhizae Radix et Rhizoma group, Glycyrrhizae Radix et Rhizoma and Kansui Radix group, normal group and control group. Animals of each group, except normal group, were undertaken intraperitoneal injection and intranasal inhalation of lipopolysaccharide (LPS) on day 1, 2, 3 to establish acute lung injury (ALI) model. 30 min after modeling, 0.2 mL corresponding drugs were administrated to each mice, dexam ethasone and normal saline were given to the mice of control group and normal group respectively. White blood cell in blood, neutrophil percentage of blood and bronchoalveolar lavage fluid (BALF) supernatant, the ratio of wet and dry lung tissue ( W/D), histopathological changes of lung tissue were estimated. Sixty ICR mice were randomly divided into normal, model, control, high, middle and low dose Xianxiong decoction groups and were modeled in the same way. ELISA was applied to detect the level of NF-kappaB, TNF-alpha and IL-6 in BALF, PCR for NF-kappaB and TNF-alpha mRNA in lung tissue, and Western blot for NF-kappaB and TNF-alpha. Half of 20 ICR mice were administrated with Xianxiong decoction of its maximum tolerant normal saline. RESULT: Compared with model group, the number of WBC in blood of Xianxiong decoction group mice decreased (P < 0.01), percentage of neutrophils in both blood and BALF decreased as well (P < 0.01, P < 0.05); it also significantly reduced the ratio of W/D (P < 0.01); and found the alveolar wall, the number of inflammatory cells infiltrating improved, compared with model group. Xianxiong decoction reduced the level of NF-kappaB, TNF-alpha and IL-6 in BALF (P < 0.01, P < 0.01, P < 0.05); its high and low dose groups only found TNF-alpha level declined. Five mice died 24 h after administration of Xianxiong decoction which indicated its toxicity when other influential factors were considered. CONCLUSION: Xianxiong decoction is effective on the ALI mice induced by LPS, but it is of toxicity at 3 g x mL(-1).

[Study on incompatibility of traditional Chinese medicines].

The incompatibility of traditional Chinese medicines is related to the clinical medication safety, so has attracted wide attentions from the public. With the deepening of studies on the incompatibility of traditional Chinese medicines represented by 18 incompatible herbs, the incompatibility of theory traditional Chinese medicines has raised to new heights. From the origin of incompatibility theory of traditional Chinese medicines, relationship of herbs, harms of incompatible herbs and principle of prevention to toxic effects of specific incompatible medicines, the innovation and development of the traditional Chinese medicine incompatibility theory was explored. Structurally, the incompatibility of traditional Chinese medicines refers to the opposition of two herbs based on seven emotions and clinical experience. The combination of incompatible herbs may lead to human harms, especially latent harm and inefficacy of intervention medicines. The avoidance of the combination of incompatible herbs and the consideration of both symptoms and drug efficacy are the basic method to prevent adverse reactions. The recent studies have revealed five characteristics of incompatible herbs. Toxicity potentiation, toxication, efficacy reduction and inefficacy are the four manifestations of the incompatible relations. The material changes can reflect the effects of toxicity potentiation and toxication of opposite herbs. The accumulation of toxicity and metabolic changes are the basis for latent harms. The antagonistic effect of main efficacies and the coexistence of positive and negative effects are the distinctive part of the incompatibility. The connotation of incompatible herbs plays an important role in the innovation of the traditional Chinese medicine incompatibility theory.

[Clinical study on aconite prescriptions with incompatible herbs in different areas based on association rules and analysis on compatibility features].

OBJECTIVE: To explore the current application and features of Aconite prescriptions with incompatible herbs in grade A class three hospitals in east China and central China through a clinical study and comparative analysis. METHOD: Clinical prescriptions containing Aconite with incompatible herbs were collected. Association rules were utilized to analyze the compatible features of these herbs. RESULT: This analysis found that the frequently used incompatible herba; pairs are Aconiti Lateralis Radix Praeparata-Pinelliae Rhizoma, with the support rate of 44.45%, occupying nearly half of the surveyed prescriptions; Pinelliae Rhizoma is the most frequently used herb in the two areas, with support rate up to 76.24%. Among the top 10 herbal pairs in the support rate, except for Aconiti Lateralis Radix Praeparata and Pinelliae Rhizoma, the top 10 herbs in Central China were mostly for warming the middle jiao and tonifying qi, such as Zingiberis Rhizoma, Atractylodis Macrocephalae Rhizoma and Codonopsis Radix; Whereas those in east China were mostly for activating and nourishing blood, such as Angelicae Sinensis Radix, Chuanxiong Rhizoma, and Salviae Miltiorrhizae Radix et Rhizoma. Among the top 10 herbal pairs in the support rate, except for Aconiti Lateralis Radix Praeparata-Pinelliae Rhizoma, the core herbal pairs applied in central China were mainly for resolving phlegm and warming the middle jiao, such as Pinelliae Rhizoma-Glycyrrhizae Radix et Rhizoma, Pinelliae Rhizoma-Zingiberis Rhizoma; Whereas those in east China were principally for activating blood and tonifying qi, like Atractylodis Macrocephalae Rhizoma and Pinelliae Rhizoma, Angelicae Sinensis Radix and Pinelliae Rhizoma. Among the core herbal groups in the two areas, the most frequently used herbal groups in the two areas are Aconiti Lateralis Radix Praeparata, Glycyrrhizae Radix et Rhizoma and Pinelliae Rhizoma with the support rate of 59.73%, accounting for the highest proportion among all of herbal groups. CONCLUSION: There are the combined clinical application of Aconite with incompatible herbs, mostly with Aconiti Lateralis Radix Praeparata-Pinelliae Rhizoma, but with differences in the combined application in east China and central China.

[On the Commonness of San'ao Decoction and Its Analogous Formulas in Facilitating Fei].

San'ao Decoction (SD) and its analogous formulas derived in the following generations are common used prescriptions for treating pulmonary diseases with principal symptoms such as cough and asthma. They are usually compatible with Chinese herbs for facilitating Fei, dispelling wind, resolving phlegm and fluid retention. Material bases in these formulas are mainly derived from Chinese drugs, but dissolution contents of active components are changed and new components are produced after compatibility. By multilevel effect evaluation, these analogous formulas all have commonness in ventilating Fei and superiorities of evidence-based derivation. The effect pathway of commonness was involved in cell structure protection, anti-inflammation, antioxidant, and immunoregulation.

CD4⁺CD25⁺FOXP3⁺ T cells, Foxp3 gene and protein expression contribute to antiasthmatic effects of San'ao decoction in mice model of asthma.

OBJECTIVE: San'ao decoction (SAD) is a commonly used traditional combinatorial formula composed of Herba Ephedrae, Radix Glycyrrhizae and Amygdalus Communis Vas. Early studies showed that in the OVA sensitization asthmatic mice model its compatibility could lower airway reactivity and airway inflammatory cell infiltration. Based on the above results, this study mainly discussed San'ao decoction's immunomodulatory effects on Tregs. METHODS: UPLC-PDA-TOF-MS was applied to analyze chemicals of SAD, and under the optimized chromatographic and MS condition, the major components in SAD were well separated and detected within 22 min. An asthma model was established in BALB/c mice that were sensitized and challenged with ovalbumin. After 2 weeks' treatment, peripheral blood and bronchoalveolar lavage fluid (BALF) were assessed for inflammatory cell counts; histological change of lung tissue were detected; flow cytometry detection of splenic CD4(+)CD25(+)Foxp3(+) Treg cells of the mice were counted; Foxp3 expression in lung tissues were examined as well. RESULTS: 22 Peaks signal chemical components in SAD were identified by UPLC-QTO-MS method. In terms of the percentage of eosinophile in peripheral blood and bronchoalveolar lavage fluid (BALF), SAD groups were significantly lower (p<0.01) than model group. Compared with model group, lung histological changes of SAD groups were reduced; the proportion of CD4(+)CD25(+)Foxp3(+) Treg cells in CD4(+) cells of asthmatic mice also decreased; SAD significantly increased the proportion of CD4(+)CD25(+)Foxp3(+) Treg cells and promoted Foxp3 expression in a mouse model of asthma. CONCLUSION: These results suggest that the antiasthmatic effects of SAD are at least partially associated with CD4(+)CD25(+)Foxp3(+) Treg cells.

The effect of Qi'ao Decoction on ovalbumin induced and lipopolysaccharide enhanced severe asthma mice and its mechanism.

AIM: To evaluate the effect of Qi'ao Deocoction (QAD) on the inflammation and hyperresponsiveness of asthma mice. METHODS: 120 Balb/C mice were randomly divided into six groups: normal group, model group, dexamethasone group, high dose QAD group, medium dose QAD group and low dose QAD group. The asthma model was reproduced in Balb/C mice sensitized by ovalbumin, challenged by OVA and LPS. The mice of the normal group were sensitized, challenged and intranasally instilled by PBS. On day 28-34, 6.7, 13.4 and 26.8 g · kg(-1) Qi'ao Decoction were administrated; 0.002 4 g · kg(-1) dexamethasone solution was given to the dexamethasone group; normal and model groups were given the same amount of normal saline. Bronchoalveolar lavage fluid, airway hyperresponsiveness, lung histopathology and cytokines were then collected and analyzed. RESULTS: Compared with normal group, total cellular score, the number of macrophages, lymphocytes, eosinophils and neutrophils of model group significantly increased (P < 0.01). Compared with model group, the administration of dexamethasone induced a significant decrease in eosinophils and neutrophils (P < 0.05, P < 0.01). The number of eosinophils, which plays an important role in airway inflammatory reaction of asthma, of the three QAD groups all decreased (P < 0.01). RL before and after Ach (5 mg · mL(-1)) stimulation in the model group both overtook that in the normal group (P < 0.01). Compared with model group, dexamethasone group, high dose QAD group, medium dose QAD group and low dose QAD group groups all had significantly lower RL before and after Ach stimulation (P < 0.01). Normal pulmonary histopathology was found in the normal group. In the model group, mice exhibited marked increases in inflammatory cell infiltration, mostly including neutrophils and macrophages, perivascular inflammation and thickened alveolus wall (P < 0.01). Dexamethasone application mitigated inflammation around the bronchi (P < 0.05). These histopathological changes were ameliorated in the three decoction groups (P < 0.01, P < 0.05). In addition, alveolus and airway wall lesions of medium dose QAD group and high dose QAD group were reduced, the number of inflammatory cells infiltrated around the walls decreased, no clear degeneration of bronchial epithelial cells was found, and exudates in bronchi declined in different degrees. Compared with normal group, IFN-γ and IL-12 of model group significantly decreased, while IL-4 increased, showing statistic difference (P < 0.05). Compared with model group, IFN-γ and IL-12 level of dexamethasone group went up too, but IL-4 declined (P < 0.05). The level of IFN-γ of medium dose QAD group and high dose QAD group both increased; IL-4 and IL-12 of medium dose group were found significant differences (P < 0.05); but none of the cytokines of low dose QAD group showed statistical significance (P > 0.05). CONCLUSION: QAD can significantly inhibit airway inflammation and airway hyperresponsiveness of mice with severe asthma induced by ovalumin and lipopolysaccharide, adjust the balance of cytokines, and improve lung histopathological condition. So, it exhibits great effect on severe asthma.

[Research on the origins and development of the verse "eighteen antagonism" in Chinese material medica].

The verse "eighteen antagonism" of Chinese materia medica is a kind of describing of drug nature concerning the incompatibility of drugs in compound prescription. Through organizing the medical books in different historical periods, it is found that the drugs in this verse basically coming from TAO Hong-jing's ben cao jing ji zhu (Variorum of Shennong's Classic of Materia Medica), while the style of verse mostly appeared during the Song-Jin-Yuan period was made. The formed verse was explained and supplemented further in the Ming Dynasty, the basis of which appeared in the Song-Jin-Yuan Dynasty, resulting in the increase of the number of antagonistic drugs and lengthy verse. The origin of the verse seen in Ru men shi qin (Confucians' Duties to Their Parents) and those in other books was not identical and was the most popular one after the Qing Dynasty, becoming the most popularly known even today.

[Study on evaluation system for gynecological disease model characterized by Qi stagnation and blood stasis syndrome with female SD rats].

OBJECTIVE: To establish an evaluation system for animal model with gynecological disease characterized by Qi stagnation and blood stasis syndrome, in order to disprove syndrome characteristics of the model by classic clinical prescriptions, and evaluate the specificity and reliability of the model with macroscopic biological signs and symptoms. METHOD: The model characterized by Qi stagnation and blood stasis syndrome was established by injecting adrenaline into female SD rats and conducting unpredictable chronic stimulus such as reversal of day and night, swimming in cold water, thermal stimulation in oven, noise and tail suspension for two weeks. They were also orally administered with Xiangfu Siwu Tang, Shaofu Zhuyu Tang and positive control drug aspirin in groups. A comprehensive evaluation was conducted for the model on the basis of haemorheology, four blood coagulation indexes, four diagnostic information (digital imaging of tongue, paw and tail, temperature, weight, ingestion, electrocardiograph, and open filed test), and syndrome rating. RESULT: Compared with the normal group, the model group showed obvious changes in haemorheology, four blood coagulation indexes, animal behavior, weight, ingestion, syndrome rating and heart rate. Their tongue and paw pictures were analyzed with Photoshop 7.0, showing significant difference in red, green and blue percentage composition from the normal group. Groups given aspirin and Xiangfu Siwu Tang showed notable changes in haemorheology, four blood coagulation indexes, animal behavior, weight, ingestion, heart rate, syndrome rating, and red, green and blue percentage composition in tongue and paw pictures, whereas the group given Shaofu Zhuyu Tang showed no remarkable improvement. CONCLUSION: The evaluation system for the animal model with gynecological disease characterized by Qi stagnation and blood stasis syndrome is established to provide reference for studies on the evaluation system for qi stagnation and blood stasis syndrome models.

[Research on Chinese medicine pairs (I)--Their formation and development].

Chinese medicine pair (CMP) was frequently applied in traditional Chinese medicine (TCM) clinic, and its significance was shown in long-term clinical practices and many accumulated experiences. It is the unique combination of two relatively fixed Chinese medicines in TCM clinic with the basic feature and principle of TCM compatibility, is the most fundamental and the simplest form of TCM formulae with certain theory basis and combinatory reason, which is proven effective. And the unique combination is frequently used for achieving mutual reinforcement or detoxication. CMP is an intermediate point between single herb and many TCM formulae, reflecting the regularity of TCM formulae compatibility and connotation of differential treatment. This paper analyzed and summarized the basic characteristics, development process and research significance of CMP, which aims to lead the modern basic and applied research on compatibility theory of CMP.

[Research on Chinese medicine pairs (II)--Their data mining].

Data mining technology has become a powerful tool in traditional Chinese medicine (TCM) research. In this paper, based on the principle and basic requirements of data mining, the mining methods and procedures were described. And then the application of data mining technology in Chinese medicine pair research was classified and summarized, such as the compatibility characters, characteristic pairs, dosage-effect relationship and property compatibility, which provide the direction and data base for modern research of Chinese medicine pair.

[Molecular docking of chlorogenic acid, 3,4-di-O-caffeoylquinic acid and 3,5-di-O-caffeoylquinic acid with human serum albumin].

OBJECTIVE: To investigate the mechanism of binding of human serum albumin (HSA) with potential sensitinogen, including chlorogenic acid and two isochlorogenic acids (3,4-di-O-caffeoylquinic acid and 3,5-di-O-caffeoylquinic acid). METHODS: By using the docking algorithm of computer-aided molecular design and the Molegro Virtual Docker, the crystal structures of HSA with warfarin and diazepam (Protein Data Bank ID: 2BXD and 2BXF) were selected as molecular docking receptors of HSA sites I and II. According to docking scores, key residues and H-bond, the molecular docking mode was selected and confirmed. The molecular docking of chlorogenic acid and two isochlorogenic acids on sites I and II was compared based on the above design. RESULTS: The results from molecular docking indicated that chlorogenic acid, 3,4-di-O-caffeoylquinic acid and 3,5-di-O-caffeoylquinic acid could bind to HSA site I by high affinity scores of -112.3, -155.3 and -153.1, respectively. They could bind to site II on HSA by high affinity scores of -101.7, -138.5 and -133.4, respectively. In site I, two isochlorogenic acids interacted with the key apolar side-chains of Leu238 and Ala291 by higher affinity scores than chlorogenic acid. Furthermore, the H-bonds of isochlorogenic acids with polar residues inside the pocket and at the entrance of the pocket were different from chlorogenic acid. Moreover, the second coffee acyl of isochlorogenic acid occupied the right-hand apolar compartment in the pocket of HSA site I. In site I, the second coffee acyl of isochlorogenic acid formed the H-bonds with polar side-chains, which contributed isochlorogenic acid to binding with site II of HSA. CONCLUSION: The isochlorogenic acids with two coffee acyls have higher binding abilities with HSA than chlorogenic acid with one coffee acyl, suggesting that isochlorogenic acids binding with HSA may be sensitinogen.