Analysis of global gene expression using RNA-sequencing reveals novel mechanism of Yanghe Pingchuan decoction in the treatment of asthma.

BACKGROUND: Yanghe Pingchuan decoction (YPD) has been used for asthma treatment for many years in China. We sought to understand the mechanism of YPD, and find more potential targets for YPD-based treatment of asthma. METHODS: An ovalbumin-induced asthma model in rats was created. Staining (hematoxylin and eosin, Masson) was used to evaluate the treatment effect of YPD. RNA-sequencing was carried out to analyze global gene expression, and differentially expressed genes (DEGs) were identified. Analysis of the functional enrichment of genes was done using the Gene Ontology database (GO). Analysis of signaling-pathway enrichment of genes was done using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. Real-time reverse transcription-quantitative polymerase chain reaction was undertaken to measure expression of DEGs. RESULTS: Pathology showed that YPD had an improvement effect on rats with asthma. RNA-sequencing showed that YPD led to upregulated and downregulated expression of many genes. The YPD-based control of asthma pathogenesis may be related to calcium ion (Ca2+) binding, inorganic cation transmembrane transporter activity, microtubule motor activity, and control of canonical signaling (e.g., peroxisome proliferator-activated receptor, calcium, cyclic adenosine monophosphate). Enrichment analyses suggested that asthma pathogenesis may be related to Ca2 + binding and contraction of vascular smooth muscle. A validation experiment showed that YPD could reduce the Ca2 + concentration by inhibiting the Angiopoietin-II (Ang-II)/Phospholipase (PLA)/calmodulin (CaM0 signaling axis. CONCLUSION: Control of asthma pathogenesis by YPD may be related to inhibition of the Ang-II/PLA/CaM signaling axis, reduction of the Ca2+ concentration, and relaxation of airway smooth muscle (ASM).

Zishen Yutai Pills Promote Angiogenesis at the Maternal-Fetal Interface in Recurrent Spontaneous Abortion Mice by Regulating miR-187/VEGF Axis.

Ethnopharmacological Relevance: Zishen Yutai pills (ZYP), a traditional Chinese patent medicine, was listed in China in 1981. It is composed of 15 traditional Chinese medicines and has the effects of regulating menstruation, helping pregnancy, and preventing abortion. In clinical practice, it is effective in preventing habitual and threatened miscarriages, and continuing to explore its mechanism of action is very meaningful research. Aim of the Study: To explore the possible mechanism of ZYP promoting angiogenesis at the maternal-fetal interface in recurrent spontaneous abortion (RSA). Materials and Methods: In vitro experiments, placental trophoblast cells (PTCs) were isolated from the placental tissue of RSA mice and divided into six groups: Control group, Model group, ZYP group, miR-187 inhibitor NC group, miR-18 7 inhibitor group, and miR-187 inhibitor+ZYP group. Cell viability and cell cycle were measured using CCK8 and flow cytometry, respectively. The expression levels of miR-187, VEGF, VEGF-R1, and VEGF-R2 were measured using RT-qPCR, WB, and IF staining. Animal experiments first establish an RSA mice model (CBA/J × DBA/2) and then randomly divide the mice into four groups (n=10): normal pregnancy group, RSA model group, ZYP group, and progesterone capsule group. Observed the changes in embryo absorption rate, pathological morphology of decidual tissue, and ultrastructure of vascular endothelial cells in each group of mice. RT-qPCR, WB, and IF staining methods were used to determine the expression of miR-187, VEGF, VEGF-R1, and VEGF-R2. Results: In vitro, ZYP promoted the viability of PTCs and regulated their cell cycle, and ZYP down-regulated miR-187, up-regulated VEGF, VEGF-R1 and VEGF-R2 levels. miR-187 inhibitor showed the same effects, and further ZYP intervention enhanced the effects. In vivo, ZYP remarkably reduced embryo resorption rates, and improved the pathological morphology of decidual tissues and ultrastructure of vascular endothelial cells. Moreover, ZYP down-regulated miR-187, up-regulated VEGF, VEGF-R1 and VEGF-R2. Conclusion: In summary, ZYP can regulate the expression of VEGF via miR-187, then promote the angiogenesis at the maternal-fetal interface, and playing a therapeutic role in RSA.

Yanghe Pingchuan granules mitigates oxidative stress and inflammation in a bronchial asthma rat model: role of the IKK/IκB/NF-κB signalling pathway.

Background: Bronchial asthma (BA) is a chronic inflammatory airway disease. Previous research has shown that Yanghe Pingchuan granules (YPG), among the granules formulated by the First Affiliated Hospital of the Anhui University of Chinese Medicine, exerts a precise therapeutic effect on BA. We previously showed that YPG improves airway inflammation in BA rats. Other studies have shown that the inhibitor of kappa-B kinase (IKK)/inhibitor of NF-κB (IκB)/nuclear factor kappa-B (NF-κB) signalling pathway plays a key role in inflammation mediation. Therefore, this study explored whether YPG could intervene in BA through the IKK/IκB/NF-κB signalling pathway. Methods: Ovalbumin-induced method was used to established BA rat model. After successful modelling, the authors used YPG to intervene the rats in BA rats. Hematoxylin-eosin (HE) staining was used to detect the bronchial pathological changes in BA rats, enzyme-linked immunosorbent assay (ELISA) was used to detect the changes of inflammatory factors (IL-1ß and IL-6) and oxidative stress indexes malondialdehyde (MDA), superoxide dismutase (SOD) and nitrogen monoxide (NO), Quantitative real-time polymerase chain reactionCR and western blot were used to detect the expression of IKK/IκB/NF-κB signalling pathway. Results: In BA model rats, YPG significantly improved the inflammatory response in bronchial tissues, reduced inflammatory factors IL-1ß and IL-6, alleviated oxidative stress, reduced MDA and NO, and increased SOD. Quantitative real-time polymerase chain reaction and western blot results showed that YPG could block the IKK/IκB/NF-κB signalling pathway. Conclusion: These findings showed that YPG had a definite therapeutic effect on BA, which may be related to blocking the IKK/IκB/NF-κB signalling pathway and improving inflammation and oxidative stress.

Investigating the mechanism of action of Yanghe Pingchuan Granule in the treatment of bronchial asthma based on bioinformatics and experimental validation.

Background: Yanghe Pingchuan Granule (YPG) is a patented Chinese medicine developed independently by the Anhui Provincial Hospital of Traditional Chinese Medicine. For many years, it has been used for the treatment of asthma with remarkable clinical effects. However, the composition of YPG is complex, and its potential active ingredients and mechanism of action for the treatment of asthma are unknown. Materials and methods: In this study, we investigated the potential mechanism of action of YPG in the treatment of asthma through a combination of bioinformatics and in vivo experimental validation. We searched for active compounds in YPG and asthma targets from multiple databases and obtained common targets. Subsequently, a protein-protein interaction (PPI) network for compound disease was constructed using the protein interaction database for Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. Finally, hematoxylin and eosin (H&E) staining, Masson staining, enzyme-linked immunosorbent assay (ELISA) analysis, immunofluorescence (IF) experiments, and Western blot (WB) experiments were performed to verify the possible mechanism of action of YPG for asthma treatment. Results: We obtained 72 active ingredients and 318 drug target genes that overlap with asthma. Serine/threonine-protein kinase (AKT1), tumor protein p53 (TP53), tumor necrosis factor (TNF), interleukin (IL)-6, IL-1ß, vascular endothelial growth factor-A (VEGFA), prostaglandin-endoperoxide synthase 2 (PTGS2), caspase-3 (CASP3), mitogen-activated protein kinase 3 (MAPK3) and epidermal growth factor receptor (EGFR) were the most relevant genes in the PPI network. KEGG analysis showed a high number of genes enriched for the nuclear factor kappa-B (NF-κB) signaling pathway. Animal experiments confirmed that YPG reduced inflammatory cell infiltration and down-regulated the expression of ovalbumin-induced inflammatory factors. Furthermore, YPG treatment decreased the protein expression of NFĸB1, nuclear factor kappa B kinase subunit beta (IKBKB), vascular endothelial growth factor (VEGF), and vascular endothelial growth factor receptor 2 (VEGFR2) in lung tissue. Conclusion: YPG has a positive effect on asthma by interfering with multiple targets. Furthermore, YPG may significantly inhibit the follicle-induced inflammatory response through the NF-ĸB signaling pathway.

Gandouling alleviates nerve injury through PI3K/Akt/FoxO1 and Sirt1/FoxO1 signaling pathway to inhibit autophagy in the rats model of Wilson's disease.

INTRODUCTION: Previous studies have shown that Gandouling (GDL) may alleviate the nerve damage caused by Wilson's disease (WD) by inhibiting the autophagy of nerve cell mitochondria. However, its mechanisms are still unclear. Revealing the therapeutic mechanism of GDL is beneficial for its clinical application and provides theoretical support for the development of new formulations for treating WD. METHOD: This time we found that the oxidative stress level in the body of the copper-overloaded WD rates increased, neurons in the hippocampus were damaged, and autophagy occurred. GDL reversed these situations and significantly improved the learning, memory, and spatial cognitive abilities of the high-copper-loaded WD rates. After GDL intervention, the expression of phosphatidylinositol-3 kinase (PI3K), phosphorylated serine-threonine protein kinase (AKT), and phosphorylated forkhead box protein O1 (FoxO1) significantly increased, whereas FoxO1 in the nucleus decreased and phosphorylated FoxO1 in the cytoplasm also significantly raised. In addition, the expression of Sirt1 significantly declined, and Ac-FoxO1 in the nucleus also significantly increased. RESULTS: These data indicated that GDL may promote the phosphorylation of FoxO1 and promote its nucleation by activating the PI3K/AKT/FoxO1 signaling pathway and inhibit Ac-FoxO1 hydrolysis in the nucleus through the Sirt1/FoxO1 signaling pathway to suppress the transcriptional activity of FoxO1. CONCLUSION: Furthermore, it inhibited the expression of autophagy genes Atg12 and Gabarapl1. In summary, our work provides new insights into the potential mechanisms of GDL repairing WD neuronal damage through autophagy pathways.

Integrated Metabolomics and Network Pharmacology to Reveal the Mechanisms of Gandouling Tablets Against Copper-Overload-Induced Neuronal Injury in Rats with Wilson's Disease.

Purpose: Gandouling Tablets (GDL), a proprietary Chinese medicine, have shown a preventive effect against Wilson's disease (WD)-induced neuronal damage in previous studies. However, the potential mechanisms need additional investigation. Combining metabonomics and network pharmacology revealed the GDL pathway against WD-induced neuronal damage. Methods: The WD rat model with a high copper load was developed, and nerve damage was assessed. Total metabonomics was used to identify distinct hippocampus metabolites and enriched metabolic pathways in MetaboAnalyst. The GDL's possible targets against WD neuron damage were then determined by network pharmacology. Cytoscape constructed compound metabonomics and pharmacology networks. Moreover, molecular docking and Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR) validated key targets. Results: GDL reduced WD-induced neuronal injury. Twenty-nine GDL-induced metabolites may protect against WD neuron injury. According to network pharmacology, we identified three essential gene clusters, of which genes in cluster 2 had the most significant impact on the metabolic pathway. A comprehensive investigation identified six crucial targets, including UGT1A1, CYP3A4, CYP2E1, CYP1A2, PIK3CB, and LPL, and their associated core metabolites and processes. Four targets reacted strongly with GDL active components. GDL therapy improved five targets' expression. Conclusion: This collaborative effort revealed the mechanisms of GDL against WD neuron damage and a way to investigate the potential pharmacological mechanisms of other Traditional Chinese Medicine (TCM).

LncRNA OIP5-AS1/miR-410-3p/Wnt7b axis promotes the proliferation of rheumatoid arthritis fibroblast-like synoviocytes via regulating the Wnt/ß-catenin pathway.

LncRNA OIP5-AS1 has a common gene imbalance in various cancers and tumours, which plays an important role in regulating its biological function. However, there are few studies on lncRNA OIP5-AS1 in rheumatoid arthritis (RA). The purpose of the present study was to investigate the role of lncRNA OIP5-AS1 in the pathogenesis of RA. In the present study, we established an adjuvant arthritis (AA) rat model to obtain primary fibroblast-like synoviocytes (FLSs);The subcellular localisation of lncRNA OIP5-AS1 was detected by fluorescence in situ hybridisation (FISH) assay; Cell proliferation of FLSs was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT) assay;IL-1ß, IL-6 and TNF-α concentrations were measured by enzyme-linked immunosorbent assay (ELISA);Quantitative real-time PCR (qRT-PCR), Western blots(WB) and immunofluorescence were used to detect the expression of lncRNA OIP5-AS1/miR-410-3p/wnt7b signal axis and Wnt/ß-catenin signal pathway related indicators in FLSs. FISH assay confirmed the presence of lncRNA OIP5-AS1 in the cytoplasm, suggesting that it acts as a competing endogenous RNA (ceRNA). qRT-PCR showed that the expression of lncRNA OIP5-AS1 was upregulated in FLSs, while the expression of miR-410-3p was downregulated in FLSs. We also found that lncRNA OIP5-AS1 knockdown inhibited the proliferation and inflammation of FLSs. Moreover, the expression of Wnt7b, the downstream target gene of miR-410-3p, and the activation of the Wnt/ß-catenin signalling pathway were also inhibited by lncRNA OIP5-AS1 knockdown. These results suggested that lncRNA OIP5-AS1 promotes the activation of the Wnt/ß-catenin signalling pathway by regulating the miR-410-3p/Wnt7b signalling axis, thereby participating in the occurrence and development of RA.

Metabolomic Analysis of Stephania tetrandra-Astragalus membranaceus Herbal Pair-Improving Nephrotic Syndrome Identifies Activation of IL-13/STAT6 Signaling Pathway.

The Stephania tetrandra−Astragalus membranaceus herbal pair (FH) is a classic herbal pair widely used in the treatment of nephrotic syndrome (NS). The effects of Stephania tetrandra (FJ) and Astragalus membranaceus (HQ) on NS have been reported, but the mechanism of their combination on the improvement of NS are still unclear. The NS model was established by injecting adriamycin into the tail vein. FH intervention reduced the levels of serum triglyceride, total cholesterol, interleukin-6 (IL-6), blood urea nitrogen (BUN), urinary protein, and the gene expression levels of aquaporin 2 (AQP2) and arginine vasopressin (AVP) in NS rats. In addition, FH improved kidney injury in NS rats by inhibiting the expression of interleukin 13 (IL-13), phospho-signal transducers, and activators of transcription 6 (p-STAT6), Bax, cleaved-caspase3, while promoting the expression of Bcl-2. By comprehensive comparison of multiple indexes, the effects of FH on lipid metabolism, glomerular filtration rate, and inflammation were superior to that of FJ and HQ. Metabonomic studies showed that, compared with FJ and HQ, FH intervention significantly regulated tricarboxylic acid (TCA) cycle, cysteine and methionine metabolism, and alanine, aspartic acid and glutamic acid metabolism. Pearson correlation analysis showed that succinic acid and L-aspartic acid were negatively correlated with urinary protein, cystatin C (Cys C) and BUN (p < 0.05). In summary, FH could reduce renal injury and improve NS through inhibiting the IL-13/STAT6 signal pathway, regulating endogenous metabolic pathways, such as TCA cycle, and inhibiting the expression of AQP2 and AVP genes. This study provides a comprehensive strategy to reveal the mechanism of FH on the treatment of NS, and also provides a reasonable way to clarify the compatibility of traditional Chinese medicine.

Induction mechanism of ferroptosis: A novel therapeutic target in lung disease.

Ferroptosis is a newly discovered form of non-apoptotic regulatory cell death driven by iron-dependent lipid peroxidation. Ferroptosis significantly differs from other forms of cell death in terms of biochemistry, genetics, and morphology. Ferroptosis affects many metabolic processes in the body, resulting in disruption of homeostasis, and is related to many types of lung disease. Although current research on ferroptosis remains in the early stage, existing studies have confirmed that ferroptosis is regulated by a variety of genes, mainly involving changes in genes involved in iron homeostasis and lipid peroxidation metabolism. Furthermore, the mechanism of ferroptosis is complex. This review summarizes the confirmed mechanisms that can cause ferroptosis, including activation of glutathione peroxidase 4, synthesis of glutathione, accumulation of reactive oxygen species, and the influence of ferrous ions and p53 proteins. In recent years, the mechanism of ferroptosis in the occurrence and development of many diseases has been studied; the occurrence of ferroptosis will produce an inflammatory storm, and most of the inducing factors and pathological manifestations of lung diseases are also inflammatory reactions. Therefore, we believe that the association between ferroptosis and lung disease deserves further study. This article aims to help readers to better understand the mechanism of ferroptosis, provide new ideas and targets for the treatment of lung diseases, and point out the direction for the development of new targeted drugs for the clinical treatment of lung diseases.

Yanghe Pingchuan Granules Alleviate Airway Inflammation in Bronchial Asthma and Inhibit Pyroptosis by Blocking the TLR4/NF-κB/NRLP3 Signaling Pathway.

Bronchial asthma (BA) is a chronic inflammatory disease of the airway. Previous research has shown that Yanghe Pingchuan granules (YPGs) exert a precise therapeutic effect on BA. In our previous work, we showed that YPGs improved inflammation of the airways in rat models of BA. Other studies have shown that the pathogenesis of BA is closely related to pyroptosis and that the TOLL-like receptor pathway plays a key role in the mediation of pyroptosis. Therefore, in the present study, we established a rat model of BA by applying the concept of pyroptosis and used the TLR4/NF-κB/NRLP3 signaling pathway as the target and YPGs as the treatment method. We evaluated the effects of YPGs on airway inflammation and pyroptosis in the model rats by HE staining, Masson's staining, AP-PAS staining, western blotting, and real-time quantitative PCR. The results showed that Yanghe Pingchuan granules could significantly improve the inflammatory response of bronchial tissue in BA rats, reduce the content of inflammatory factors IL-1ß and IL-18, and inhibit the expression of pyroptosis factor. Meanwhile, YPG can block the TLR4/NF-κB signaling pathway. These findings suggest that YPG may be an effective drug for the treatment of BA by blocking the TLR4/NF-κB signaling pathway and inhibiting pyroptosis.

Screening of different chemical components of sedative and hypnotic effects of Ziziphi Spinosae Semen before and after frying and determination of the Q-Marker.

Ziziphi Spinosae Semen (ZSS) is a traditional Chinese medicine used for sedation and hypnosis. Preliminary studies have shown that frying it could increase its sedative and hypnotic effects due to an increase in its chemical contents. However, the correlation between increased ZSS contents and therapeutic effects remains unclear. This study aimed to identify chemical components that change between ZSS and Fried Ziziphi Spinosae Semen (FZSS) and Q-markers related to these changed components' sedative and hypnotic effects. Differences between ZSS and FZSS were investigated using the UPLC fingerprint analysis. Components significantly different between ZSS and FZSS were screened using the UPLC-Q-TOF-MS analysis combined with a multivariate statistical method. In addition, ZSS and FZSS extracts were treated with diazepam in vitro to observe their differences in saturation competition between ZSS extract and diazepam, before and after processing, and diazepam on the GABA receptor in SD rats' brain tissue. Then, the chemical components of ZSS and FZSS that competed with diazepam to bind to the GABA receptor were identified by LC-MS/MS analysis. Finally, the binding efficiency of the different medicinal components was assessed using molecular docking technology. The results indicated significant differences in the content of various chemical components between ZSS and FZSS. Among them, the contents of adenosine, spinosin, 6'″-feruloylspinosin, jujuboside A and betulinic acid were found to be significantly increased after frying. LC-MS/MS and molecular docking analysis screened spinosin, 6'″-feruloylspinosin and betulinic acid as Q markers for the sedative and hypnotic effects of ZSS and FZSS. In summary, this study identified the changed sedative-hypnotic chemical components and Q-markers of ZSS before and after frying.

Total Saponins of Radix Clematis Regulate Fibroblast-Like Synoviocyte Proliferation in Rheumatoid Arthritis via the LncRNA OIP5-AS1/MiR-410-3p/Wnt7b Signaling Pathway.

Background: Rheumatoid arthritis (RA) is the most common autoimmune disease and affects multiple joints. Previous studies have shown that total saponins of Radix clematidis (TSC) have a clear therapeutic effect on RA, but the specific mechanism has not yet been clarified. Literature screening and previous research suggest that the lncRNA OIP5-AS1/miR-410-3p/Wnt7b signaling pathway exerts a regulatory effect on the pathogenesis of RA. In this study, we examined whether the TSC treatment of RA affects the lncRNA OIP5-AS1/miR-410-3p/Wnt7b pathway. Materials and Methods: Freund's complete adjuvant was used to create an adjuvant arthritis (AA) rat model with rat synovial cells being harvested and cultured. The experiment comprises a normal group, model group, TSC optimal-dose group, TSC optimal-dose group + lncRNA OIP5-AS1siRNA group, lncRNA OIP5-AS1 siRNA group, and lncRNA OIP5-AS1 siRNA + NC group. MMT was used to screen the optimal concentration of TSC. The level of lncRNA OIP5-AS1, miR-410-3p, Wnt7b, ß-catenin, c-Myc, cyclin D1, GSK-3ß, and SFRP4 mRNA were detected by real-time-qPCR, the expression of Wnt7b, ß-catenin, c-Myc, cyclin D1, GSK-3ß, and p-GSK-3ß (Ser9) protein were detected by immunofluorescence and Western blot. Results: We found that TSC inhibits the proliferation of RA FLS, TSC significantly reduced lncRNA OIP5-AS1, Wnt7b, ß-catenin, c-Myc, cyclin D1, and p-GSK-3ß/GSK-3ß mRNA/protein expression, whereas the miR-410-3p and SFRP4 mRNA/protein expression levels were significantly upregulated. Our data suggest that TSC can inhibit the excessive proliferation of FLS to treat RA, the mechanism of which may be closely related to regulation of the lncRNA OIP5-AS1/miR-410-3p /Wnt7b signaling axis and the Wnt signaling pathway.

Prediction of the potential mechanism of compound gingerol against liver cancer based on network pharmacology and experimental verification.

OBJECTIVES: To explore gingerol's potential mechanism for treating liver cancer using network pharmacology and molecular docking technology and to conduct in-vitro experiments of human liver cancer cell HepG2 to verify important signalling pathways. METHODS: We obtained potential targets of gingerol derivatives (6-gingerol, 8-gingerol and 10-gingerol) from PubChem and SwissTargetPrediction websites and collected related targets for liver cancer with the help of GeneCards. We performed Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis on key targets using the DAVID data platform and combined with Cytoscape 3.7.1 software to construct a component-target-signal pathway interaction map to study its mechanism of action. Subsequently, the components and key proteins were molecularly docked through Autodock Vina software. Finally, the important signal pathways were verified by HepG2 cell in-vitro experiments. KEY FINDINGS: A total of 318 drug targets were screened for gingerol derivatives, and 2509 gene targets related to liver cancer were collected. The Venn diagram showed that there were 104 intersection targets between gingerol derivatives and liver cancer. Module analysis results show that these intersection targets can be divided into 5 modules and 49 nodes. Bioinformatics analysis found that GO obtained 20 important functional items including cancer cell proliferation, protein kinase activity, phosphotransferase activity and kinase activity; KEGG enrichment analysis yielded a total of 20 key signal pathways including the PI3K-Akt signalling pathway. The results of molecular docking show that the binding energy of gingerol derivatives has good binding activity with PI3K and Akt. In-vitro experimental results show that gingerol derivatives and compound gingerol (compound gingerol is composed of 6-gingerol, 8-gingerol and 10-gingerol in a ratio of 7:1.5:1.5) can produce HepG2 cell proliferation inhibition, and each administration group can significantly increase the apoptosis rate of HepG2 cells and the fluorescence intensity of the nucleus and block the cell cycle in the S phase; the results of Western Blot and real-time quantitative PCR show that gingerol derivatives and compound gingerol can down-regulate the expression of Akt and p-Akt and up-regulate the expression of Bax/Bcl-2. And the effect of compound gingerol is more obvious than that of gingerol derivatives. CONCLUSIONS: The results of network pharmacology and experimental validation suggest that gingerol derivatives and compound gingerol can act against liver cancer by acting on the PI3K-Akt signalling pathway.

Emodin alleviates high glucose-induced oxidative stress, inflammation and extracellular matrix accumulation of mesangial cells by the circ_0000064/miR-30c-5p/Lmp7 axis.

Emodin has been shown to exert a renoprotective effect in diabetic nephropathy (DN). In this paper, we investigated whether circular RNAs (circRNAs) might be involved in the renoprotective mechanism of emodin in DN. The levels of malondialdehyde (MDA), reactive oxygen species (ROS), superoxide dismutase (SOD), interleukin-1ß (IL-1ß), IL-6 and tumor necrosis factor-α (TNF-α) were measured using the corresponding assay kits. The expression levels of circ_0000064, microRNA (miR)-30c-5p, large multifunctional protease 7 (Lmp7), fibronectin (FN), and collagen type I (Col.1) were gauged by quantitative real-time polymerase chain reaction (qRT-PCR) or western blot. Subcellular localization assay was used to assess the cellular localization of circ_0000064. Targeted relationships among circ_0000064, miR-30c-5p and Lmp7 were confirmed by dual-luciferase reporter, RNA pull-down and RNA immunoprecipitation (RIP) assays. Our data showed the alleviative effect of emodin on HG-induced oxidative stress, inflammation and extracellular matrix (ECM) accumulation in SV-MES13 cells. Circ_0000064 was an importantly downstream effector of emodin function in HG-induced SV40-MES13 cells. Moreover, circ_0000064 directly targeted miR-30c-5p, and circ_0000064 modulated Lmp7 expression through miR-30c-5p. Circ_0000064 silencing alleviated HG-induced cell oxidative stress, inflammation and ECM accumulation via up-regulating miR-30c-5p. The enforced expression of miR-30c-5p attenuated HG-induced oxidative stress, inflammation and ECM accumulation in SV40-MES13 cells by targeting Lmp7. Our findings identified that emodin alleviated HG-induced oxidative stress, inflammation and ECM accumulation in SV40-MES13 cells at least partially by the regulation of the circ_0000064/miR-30c-5p/Lmp7 axis.

An integrated approach to uncover quality markers of stir-baking Semen Cuscuta with salt solution preventing recurrent spontaneous abortion based on chemical and metabolomic profiling.

The previous research of clinical big data mining showed that stir-baking Semen Cuscuta with salt solution (YP) ranked the first in the usage rate of treating abortion caused by kidney deficiency. At the same time, pharmacodynamic studies also showed that YP has better effect on improving recurrent spontaneous abortion (RSA) compared to raw products of Semen Cuscuta (SP). However, there were few studies on the biomarkers of YP improving RSA. In this study, the chemical and metabonomic profiling were used to screen the quality markers of YP on improving RSA. Firstly, a metabolomics study was carried out to select representative biomarkers of RSA. The ultra-high performance liquid chromatography coupled with electrospray ionization-quadrupole-time of flight-mass spectrometry (UPLC-ESI-Q-TOF-MS) technique was used to investigate the components of exogenous and endogenous in serum of rats after administrated with YP and SP. As a result, 14 differential compounds were identified between the serum of rats administrated SP and YP. Compared to SP, there was an upward trend in YP of the compounds including kaempferol-3-glucuronide, iso-kaempferol-3-glucuronide, (1S) -11-hydroxyhexadecanoic acid and 3-phenylpropionic acid. Meanwhile, there was a reducing trend in YP of the compounds including kaempferol 3-arabinofuranoside, apigenin-3-O-glucoside, hyperoside, caffeic acid-ß-D glucoside, dicaffeoylquinic acid, linoleic acid, 3,4-dicaffeoylquinic acid, caffeic acid, palmitic acid and methyl myristate. 12 biomarkers for RSA indication were identified. SP and YP have a certain effect on the endogenous biomarker. The regulation effect of YP was higher than that of SP. The main metabolic pathways included phenylalanine, tyrosine and tryptophan biosynthesis, glycerophospholipid metabolism, fatty acid biosynthesis, sphingolipid metabolism, biosynthesis of unsaturated fatty acids. This study demonstrated a promising way to elucidate the active chemical and endogenous material basis of TCM.

Metabolomics study of dried ginger extract on serum and urine in blood stasis rats based on UPLC-Q-TOF/MS.

Blood stasis syndrome (BSS) is the pathological basis of many cardiovascular diseases. Ginger is often used as herbal medicine, condiment, and health food in China and Southeast Asia to improve some symptoms of cardiovascular disease, but its mechanism of efficacy and metabolic processes is not clear enough. In this study, a rat model of BSS was successfully established and treated with different doses of dried ginger extract. After the end of the administration period, the blood and urine of 5 groups of rats were collected for metabonomic analysis. Multivariate statistical analysis was used to explore metabolites and metabolic pathways, and the correlation between metabolites and pharmacodynamic indicators was further explored. The experimental results show that the pharmacodynamic indicators of dried ginger group (DG) extracts of different doses have different degrees of changes than model group (MG), and the high dose of dried ginger group (GJH) changes is the most significant (p < .05 or p < .01). Besides, 22 different metabolites were identified in the experiment. These metabolites mainly involve seven metabolism pathways in different impact value. DG has therapeutic effects on BSS rats by regulating multiple metabolic pathways. This study provides an effective method for understanding the metabolic mechanism of DG extracts on BSS.

[Changes in chemical compositions of Chrysanthemi Flos after frying and protective effects on CCl_4-induced acute liver injury in mice].

To reveal the processing mechanism of Chrysanthemi Flos from the changes of chemical compositions after frying and its effect on the efficacy of liver protection. Ultra performance liquid chromatography-quadrupole-time of flight-mass spectrometry(UPLC-Q-TOF-MS) and ultra high performance liquid chromatography(HPLC) were used for the qualitative and quantitative researches of chemical compositions before and after Chrysanthemi Flos frying. Progenesis QI and SPSS software were used for principal component analysis(PCA), partial least squares discriminant analysis(PLS-DA), variable importance projection(VIP) analysis and t-test to identify the compositions with significant changes. Pharmacodynamics experiment was used to investigate the protective effect of crude and fried Chrysanthemi Flos on CCl_4-induced acute liver injury in mice. According to mass spectrometry data, there were 28 chemical compositions in crude and fried Chrysanthemi Flos, mainly including flavonoids and organic acids. 13 compositions such as luteolin, apigenin and luteolin glycoside were increased significantly after frying, while 7 compositions such as chlorogenic acid, luteolin-7-O-glucuronide and apigenin-7-O-glucuronide were decreased significantly after frying. Through principal component analysis, crude and fried Chrysanthemi Flos products were divided into two categories, indicating that there were internal differences in quality. The results of liver injury protection experiment in mice showed that the AST, ALT and MDA contents were significantly decreased and SOD level was increased in mice with liver injury in both the high and medium dose groups. Histopathological examination showed that crude and fried Chrysanthemi Flos can protect the liver by reducing inflammatory cell infiltration, reducing steatosis, and repairing damaged liver cells. The results of this study showed that the chemical compositions had obvious changes after frying, and both crude and fried Chrysanthemis Flos had protective effects on CCl_4-induced acute liver injury in mice. In addition, in the range of high, medium and low doses, the liver protection effect of crude and fried Chrysanthemi Flos increased with the increase of dose. The experiment results provided reference for the mechanism of fried Chrysanthemi Flos and clinical selection of processed products.

The Study of Dried Ginger and Linggan Wuwei Jiangxin Decoction Treatment of Cold Asthma Rats Using GC-MS Based Metabolomics.

Dried ginger is the monarch drug in Linggan Wuwei Jiangxin (LGWWJX) decoction, which is used to treat cold asthma. The purpose of this study was to investigate and compare the effects of dried ginger and LGWWJX decoction for treatment of cold asthma rats at the metabolomics level using gas chromatography-mass spectrometry (GC-MS). OVA and ice water-induced cold asthma were induced in SD rats. The effects of dried ginger and LGWWJX decoction were evaluated by general morphological observation, hematoxylin and eosin staining, inflammatory cell count, IgE, IL-4, IFN-γ quantitation, and visceral index. GC-MS-based metabolomics was performed and analyzed using multivariate statistical analysis. Biomarker identification, pathway analysis, correlations between identified biomarker, and efficacy indices were performed. The results showed that dried ginger and LGWWJX decoction had obvious effects on cold asthma rats. Thirty-seven metabolites (15 in serum and 22 in urine) associated with cold asthma were identified. These metabolites were mainly carbohydrates, fatty acids and their products, organic acids, and others. Seven pathways were identified by MetaboAnalyst 4.0 metabolic pathway analysis. After intervention with dried ginger and LGWWJX decoction, the majority of altered metabolites and metabolic pathways returned to control levels. LGWWJX decoction regulated more metabolites of carbohydrates and fatty acids, which contribute to energy metabolism and oxidative stress in cold asthma, than dried ginger. We concluded that dried ginger and LGWWJX decoction both were effective for treatment of cold asthma. LGWWJX decoction was more effective than dried ginger for treatment of cold asthma. This study evaluated the effects of dried ginger and LGWWJX decoction on cold asthma at the metabolomics level. It provides a reference for the research on the compatibility of Chinese Medicine.

Rhizoma Paridis saponins ameliorates hepatic fibrosis in rats by downregulating expression of angiogenesis­associated growth factors.

Previously, we demonstrated that Rhizoma Paridis saponins (RPS), the major active component of Rhizoma Paridis, may exhibit hepatoprotective effects. The present study aimed to identify the potential mechanism of RPS on hepatic injury and improvement in hepatic fibrosis (HF). A HF model was created in Sprague­Dawley rats by administration of carbon tetrachloride. RPS was administered for treatment following creation of the HF model. The protein and mRNA expression of vascular endothelial growth factor (VEGF), platelet­derived growth factor (PDGF), extracellular signal­regulated kinase (ERK)1/2 and α­smooth muscle actin (SMA) was detected by reverse transcription quantitative polymerase chain reaction and western blot analysis. RPS was demonstrated to improve hepatic inflammation and decrease HF severity according to hematoxylin and eosin and Masson trichrome staining. Following RPS treatment, the level of alanine aminotransferase, aspartate aminotransferase and malondialdehyde, and expression levels of the mRNA and protein of VEGF, ERK1/2, PDGF and α­SMA in the model group was decreased. By contrast, the content of glutathione­PX and superoxide dismutase was increased. These data suggest that RPS may treat HF primarily through downregulation of the expression levels of the mRNA and phosphorylated VEGF, ERK1/2, PDGF and α­SMA proteins.

[Determination and pharmacokinetics of main components for Psoralea corylifolia-Myristica fragrants drug pair by using UPLC-MS/MS].

To conduct multiple-reaction monitoring(MRM) quantitative analysis with ultra-high performance liquid chromatography coupled with mass spectrometry method(UPLC-MS/MS), determine the concentrations of psoralen, isopsoralen, bakuchiol and dehydrodiisoeugenol in plasma under positive iron mode with chloramghenicol as internal standard, and investigate the pharmacokinetics process of the main components before and after oral administration of drug pair Psoralea corylifolia -Myristica fragrants. Thirty-six SD rats were randomly divided into three group(A, B, C) and received P. corylifolia extract, P. corylifolia-M. fragrants extract, and M. fragrants extract respectively by intragastric administration. The plasma samples were collected at different time points. In the plasma samples, psoralen, isopsoralen, bakuchiol and dehydrodiisoeugenol showed good linear relationship within concentration rages of 0.098 125 to 39.25, 0.084 37 to 33.75, 0.046 875 to 18.75, and 0.11 to 2.2 mg•L⁻¹ respectively. The precision and stability results showed that the determination method of plasma concentration for such compositions was stable and reliable. The pharmacokinetic parameters obtained by DAS 2.0 showed varying differences before and after compatibility. According to the experimental results, the compatibility of P. corylifolia and M. fragrants can significantly impact the pharmacokinetic process of main components, expand their distribution and accelerate their metabolism and elimination in vivo.