OBJECTIVE: To examine the therapeutic effect of Fangji Fuling Decoction (FFD) on sepsis through network pharmacological analysis combined with in vitro and in vivo experiments. METHODS: A sepsis mouse model was constructed through intraperitoneal injection of 20 mg/kg lipopolysaccharide (LPS). RAW264.7 cells were stimulated by 250 ng/mL LPS to establish an in vitro cell model. Network pharmacology analysis identified the key molecular pathway associated with FFD in sepsis. Through ectopic expression and depletion experiments, the effect of FFD on multiple organ damage in septic mice, as well as on cell proliferation and apoptosis in relation to the mitogen-activated protein kinase 14/Forkhead Box O 3A (MAPK14/FOXO3A) signaling pathway, was analyzed. RESULTS: FFD reduced organ damage and inflammation in LPS-induced septic mice and suppressed LPS-induced macrophage apoptosis and inflammation in vitro (P<0.05). Network pharmacology analysis showed that FFD could regulate the MAPK14/FOXO signaling pathway during sepsis. As confirmed by in vitro cell experiments, FFD inhibited the MAPK14 signaling pathway or FOXO3A expression to relieve LPS-induced macrophage apoptosis and inflammation (P<0.05). Furthermore, FFD inhibited the MAPK14/FOXO3A signaling pathway to inhibit LPS-induced macrophage apoptosis in the lung tissue of septic mice (P<0.05). CONCLUSION FFD could ameliorate the LPS-induced inflammatory response in septic mice by inhibiting the MAPK14/FOXO3A signaling pathway.
Mice ; Animals ; Mitogen-Activated Protein Kinase 14/metabolism* ; Wolfiporia ; Lipopolysaccharides/pharmacology* ; Sepsis/complications* ; Signal Transduction ; Inflammation/drug therapy* ; Oxygen Radioisotopes

To provide experimental basis and theoretical guidance for further research on the molecular mechanism of notoginsenoside R_1(NGR_1) in the treatment of depression, the present study analyzed the potential mechanism of NGR_1 in the treatment of depression through network pharmacology and verified it by molecular docking and animal experiments. PharmMapper, SwissTargetPrediction, and GeneCards were used to predict the related targets of both NGR_1 and depression to obtain the potential targets of NGR_1 in the treatment of depression. The database for annotation, visualization and integrated discovery(DAVID) was used for GO functional annotation and KEGG pathway enrichment analysis to screen the possible mechanisms of NGR_1 exerting antidepressant effect. Cytoscape 3.9.0 was adopted to construct a protein-protein interaction(PPI) network, and the topological analysis was performed to obtain the core targets. The binding activity of NGR_1 to core targets was tested by molecular docking. The depression model was prepared by injecting lipopolysaccharide(LPS) into the lateral ventricle in mice, and intervened with NGR_1. The antidepressant effect of NGR_1 was detected by behavioral tests and RT-qPCR. The results showed that by network pharmacology, 56 common targets of NGR_1 and depression were predicted, and GO enrichment analysis determined 13 related biological processes, mainly involving G protein-coupled receptor signaling pathway, positive regulation of transcription from RNA polymerase Ⅱ promoter, cytokine-mediated signaling pathway, gene expression, apoptosis, cell proliferation, and signal transduction. In addition, KEGG pathway enrichment analysis identified ten potential pathways, including neuroactive ligand-receptor interaction signaling pathway, lipid and atherosclerosis signaling pathway, cAMP signaling pathway, PI3 K-AKT signaling pathway, and lipid and atherosclerosis signaling pathway. PPI analysis revealed that the core targets included CASP3, VEGFA, IGF1, STAT3, MAPK1, PPARG, MTOR, MAPK14, NR3 C1 and AR, and molecular docking demonstrated that NGR_1 had desirable binding activity to these target proteins. In animal experiments, the results showed that NGR_1 improved the disease behavior of depressed mice, significantly inhibited the neuroinflammatory response(reducing the mRNA expression of Iba-1, TNF-α, IL-1β, and IL-6), and regulated the mRNA expression of lipid and atherosclerosis signaling pathway-related targets(CASP3, STAT3, MAPK1 and MAPK14). This indicated that the antidepressant mechanism of NGR_1 may be related to the regulation of lipid and atherosclerosis signaling pathway. In conclusion, network pharmacology was used to reveal the core targets and pathways of NGR_1, and some of them were verified in animal experiments, which provided the basis for in-depth exploration on the mechanism of NGR_1 in the treatment of depression.
Mice ; Animals ; Caspase 3 ; Animal Experimentation ; Mitogen-Activated Protein Kinase 14 ; Network Pharmacology ; Molecular Docking Simulation ; Antidepressive Agents/pharmacology* ; Lipopolysaccharides ; Atherosclerosis ; RNA, Messenger ; Drugs, Chinese Herbal/pharmacology*

OBJECTIVETo investigate the protective effects of hydrogen peroxide preconditioning (HPP) on the pheochromocytoma (PC12) cells treated with 1-methyl-4-phenylpyridinium (MPP(+)) and to explore the potential mechanisms.

METHODSThe viability and apoptosis of PC12 cells were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and 4',6'-diamidino-2-phenylindole (DAPI) staining, respectively. The expressions of 14-3-3 protein and phosphorylated p38 mitogen-activated protein kinase (MAPK) were determined by Western blot. Enzyme-linked immunosorbent assay (ELISA) was used to measure the activity of extracellular signal-regulated protein kinase 1/2 (ERK1/2).

RESULTSThe cell viability decreased and the number of apoptotic cells increased dramatically in MPP(+) group compared with that in Control group. HPP induced a significant increase in cell viability and a marked decrease in population of apoptotic cells of the MPP(+)-treated PC12 cells, accompanied with up-regulation of 14-3-3 protein and increase of ERK1/2 and p38 MAPK activities. The 14-3-3 protein expression was positively correlated with the phosphorylation of ERK1/2. Furthermore, inhibition of the ERK1/2 with PD98059 abolished the 14-3-3 protein up-regulation in PC12 cells induced by HPP.

CONCLUSIONHPP protects PC12 cells against MPP(+) toxicity by up-regulating 14-3-3 protein expression through the ERK1/2 and p38 MAPK signaling pathways.

1-Methyl-4-phenylpyridinium ; toxicity ; 14-3-3 Proteins ; biosynthesis ; Animals ; Apoptosis ; drug effects ; physiology ; Blotting, Western ; Cell Survival ; drug effects ; Enzyme-Linked Immunosorbent Assay ; Hydrogen Peroxide ; pharmacology ; Mitogen-Activated Protein Kinase 1 ; metabolism ; Mitogen-Activated Protein Kinase 3 ; metabolism ; Neurons ; drug effects ; metabolism ; pathology ; PC12 Cells ; Phosphorylation ; Rats ; Signal Transduction ; drug effects ; physiology ; Up-Regulation ; p38 Mitogen-Activated Protein Kinases ; metabolism

OBJECTIVETo investigate the role of p38α mitogen-activated protein kinases (MAPK) in human esophageal squamous cell carcinoma cell line Eca109.

METHODSSpecific short hairpin (shRNA) vector as well as eukaryotic expression vector harbouring full length cDNA of human p38α MAPK were transfected into Eca109 cells. Cell proliferation after transfection was detected by MTT, cell cycle and apoptosis were assayed by flow cytometry. The variation of migration and invasion after transfection was determined using wound healing assay and Transwell assay, respectively.

RESULTSThe proliferation of Eca109 cells after knock-down for 48 h (0.951 ± 0.086) was significantly increased (t = 3.20, P < 0.05) compared with control (0.811 ± 0.012), Sphase was increased but not significantly. Cell apoptosis rate after knock down for 48 h (17.400 ± 5.495) was significantly increased (t = 40.06, P < 0.01) compared with control(1.000 ± 0.721) . Migration after knock down for 72 h (0.034 ± 0.031) were enhanced pronouncedly (t = -5.79, P < 0.01) compared with control (0.278 ± 0.021) and invasive ability also increased; whereas the proliferation of Eca109 cells after over-expression for 48 h (0.472 ± 0.089) was inhibited significantly (t = -7.50, P < 0.01) compared with control(0.811 ± 0.012), cells arrested at G1 phase (t = 4.80, P < 0.01). Cell apoptosis rate (32.233 ± 1.457) were decreased significantly (t = 17.20, P < 0.01) compared with control (1.000 ± 0.721) mm, migration after overexpression for 72 h ((0.770 ± 0.054) mm) was suppressed pronouncedly compared with control groups of (0.278 ± 0.021) mm(t = 11.00, P < 0.01).Invasion after overexpression was inhibited.

CONCLUSIONSp38α MAPK plays an anti-oncogenic role in the pathogenesis of esophageal squamous cell carcinoma cell line Eca109.

Carcinoma, Squamous Cell ; genetics ; metabolism ; pathology ; Cell Division ; Cell Line, Tumor ; Cell Proliferation ; Esophageal Neoplasms ; genetics ; metabolism ; pathology ; Humans ; Mitogen-Activated Protein Kinase 14 ; metabolism ; RNA, Small Interfering ; Transfection

OBJECTIVETo study the effects of antisense p38α mitogen-activated protein kinase (hereinafter referred to as p38α) on myocardial cells exposed to hypoxia and burn serum.

METHODSThirty adult SD rats were inflicted with 40% TBSA full-thickness burn on the back to obtain burn serum. The myocardial cells were isolated from 80 neonatal SD rats and cultured, then they were divided into 4 groups according to the random number table: normal control group (N, ordinary culture without any treatment), hypoxia+burn serum group (HB, exposed to hypoxia after being treated with 10% burn rat serum), hypoxia+burn serum+infection group (HBI, exposed to hypoxia and 10% burn rat serum after being infected with antisense p38α gene-carrying adenovirus), hypoxia+burn serum+empty vector infection group (exposed to hypoxia and 10% burn rat serum after being infected with adenovirus empty vector). At post hypoxia hour (PHH) 1, 3, 6, and 12, mRNA and protein expression levels of p38α in the latter 3 groups were determined by RT-PCR and Western blotting, cell viability was determined by methylthianolyldiphenyl-tetrazolium bromide assay, and lactate dehydrogenase (LDH) activity was assayed at the same time point. At PHH 1, 6, and 12, apoptosis rate of myocardial cells was assessed by annexin V staining method. The indexes of group N were determined with the methods mentioned-above. Three wells were set at each time point in each group. Data were processed with one-way analysis of variance and LSD- t test.

RESULTS(1) At PHH 1, 3, and 6, the p38α mRNA level was higher in group HB than in group N and group HBI (with t values from 2.725 to 4.375, P values all below 0.05). (2) At PHH 1, 3, and 6, the p38α protein level was higher in group HB than those in group N and group HBI (with t values from 5.351 to 7.981, P values all below 0.01). (3) At PHH 3, 6, and 12, the cell viability in group HB (0.115 ± 0.007, 0.104 ± 0.006, 0.094 ± 0.005) was lower than that in group N (0.141 ± 0.014) and group HBI (0.136 ± 0.009, 0.124 ± 0.010, 0.112 ± 0.007, with t values from 2.357 to 6.812, P values all below 0.05). (4) The LDH activity was up-regulated in group HB as compared with that in group N and group HBI at each time point (with t values from 22.753 to 201.273, P values all below 0.01). (5) At PHH 1, 6, and 12, the apoptosis rate of myocardial cells in group HB [(5.4 ± 0.7)%, (8.7 ± 1.1)%, (13.6 ± 1.7)%] was higher than that of group N [(3.1 ± 0.3)%] and group HBI [(4.3 ± 0.5)%, (5.1 ± 0.7)%, (7.2 ± 0.9)%, with t values from 2.345 to 9.700, P < 0.05 or P < 0.01].

CONCLUSIONSAntisense p38α can protect the myocardial cells from the injury of hypoxia and burn serum.

Animals ; Antisense Elements (Genetics) ; genetics ; Apoptosis ; Cell Hypoxia ; Cells, Cultured ; Female ; Male ; Mitogen-Activated Protein Kinase 14 ; genetics ; metabolism ; Myocytes, Cardiac ; metabolism ; pathology ; Rats ; Rats, Sprague-Dawley ; Serum ; Transfection

Epidemic diseases have caused huge harm to the society. Traditional Chinese medicine(TCM) has made great contributions to the prevention and treatment of them. It is of great reference value for fighting diseases and developing drugs to explore the medication law and mechanism of TCM under TCM theory. In this study, the relationship between the TCM theory of cold pestilence and modern epidemic diseases was investigated. Particularly, the the relationship of coronavirus disease 2019(COVID-19), severe acute respiratory syndrome(SARS), and influenza A(H1 N1) with the cold pestilence was identified and analyzed. The roles of TCM theory of cold pestilence in preventing and treating modern epidemic diseases were discussed. Then, through data mining and textual research, prescriptions for the treatment of cold pestilence were collected from major databases and relevant ancient books, and their medication laws were examined through analysis of high-frequency medicinals and medicinal pairs, association rules analysis, and cluster analysis. For example, the prescriptions with high confidence levels were identified: "Glycyrrhizae Radix et Rhizoma-Bupleuri Radix-Paeoniae Radix Alba" "Glycyrrhizae Radix et Rhizoma-Pinelliae Rhizoma-Bupleuri Radix", and TCM treatment methods with them were analyzed by clustering analysis to yield the medicinal combinations: "Zingiberis Rhizoma-Aconiti Lateralis Radix Praeparata-Ginseng Radix et Rhizoma" "Poria-Atractylodis Macrocephalae Rhizoma" "Cinnamomi Ramulus-Asari Radix et Rhizoma" "Citri Reticulatae Pericarpium-Perillae Folium" "Pinelliae Rhizoma-Magnoliae Officinalis Cortex-Atractylodis Rhizoma" "Paeoniae Radix Alba-Angelicae Sinensis Radix-Glycyrrhizae Radix et Rhizoma-Bupleuri Radix-Scutellariae Radix-Rhizoma Zingiberis Recens" "Ephedrae Herba-Armeniacae Semen Amarum-Gypsum Fibrosum" "Chuanxiong Rhizoma-Notopterygii Rhizoma et Radix-Angelicae Dahuricae Radix-Platycodonis Radix-Saposhnikoviae Radix". Then, according to the medication law for cold pestilence, the antiviral active components of medium-frequency and high-frequency medicinals were retrieved. It was found that these components exerted the antiviral effect by inhibiting virus replication, regulating virus proteins and antiviral signals, and suppressing protease activity. Based on network pharmacology, the mechanisms of the medicinals against severe acute respiratory syndrome coronavirus(SARS-CoV), 2019 novel coronavirus(2019-nCoV), and H1 N1 virus were explored. It was determined that the key targets were tumor necrosis factor(TNF), endothelial growth factor A(VEGFA), serum creatinine(SRC), epidermal growth factor receptor(EGFR), matrix metalloproteinase 9(MMP9), mitogen-activated protein kinase 14(MAPK14), and prostaglandin-endoperoxide synthase 2(PTGS2), which were involved the mitogen-activated protein kinase(MAPK) pathway, advanced glycation end-products(AGE)-receptor for AGE(RAGE) pathway, COVID-19 pathway, and mTOR pathway. This paper elucidated the medication law and mechanism of TCM for the prevention and treatment of epidemic diseases under the guidance of TCM theory of cold pestilence, in order to build a bridge between the theory and modern epidemic diseases and provide reference TCM methods for the prevention and treatment of modern epidemic diseases and ideas for the application of data mining to TCM treatment of modern diseases.
Aconitum ; Antiviral Agents ; COVID-19/epidemiology* ; Calcium Sulfate ; Communicable Disease Control ; Communicable Diseases/virology* ; Creatinine ; Cyclooxygenase 2 ; Drugs, Chinese Herbal/therapeutic use* ; Endothelial Growth Factors ; Epidemics/prevention & control* ; ErbB Receptors ; Humans ; Matrix Metalloproteinase 9 ; Medicine, Chinese Traditional ; Mitogen-Activated Protein Kinase 14 ; Pinellia ; SARS-CoV-2 ; TOR Serine-Threonine Kinases ; Tumor Necrosis Factors ; COVID-19 Drug Treatment