Objective To explore the mechanism by which tumor necrosis factor alpha(TNF-α) induces RIP1 kinase-dependented apoptosis in L929-A fibroblastoma cells.Methods The sub-mitochondrial localization of receptor-interacting protein 1(RIP1),caspase-8 and Bid proteins was detected by dose-gradient trypsin digestion and Western blotting.The levels of reactive oxygen species (ROS),intracellular calcium concentration,mitochondrial membrane potential (MMP),and cellular adenosine triphosphate(ATP) content were determined by fluorescent probe labeling and flow cytometry assay.The mitochondrial respiratory chain complex Ⅰ and Ⅲ activities were detected by commercial kits.Nec-1,A RIP1 kinase specific inhibitor,and RIP1-/-or Bid-/-L929-A cells were used to detect the roles of RIP1 kinase and Bid protein in cell death.Results RIP1,caspase-8 and Bid proteins were co-located in the outer membrane of mitochondrial.TNF-α exposure for 3 h could induce Bid cleavage,inhibit mitochondrial respiratory chain complex Ⅲ activity and reduce MMP.Following these changes and after TNF-α exposure for 6-12 h,the intracellular calcium concentration and ROS were increased,whereas the ATP concentration was decreased,and the cells were killed.Inhibiting RIP1 kinase or knockdown RIP1 or Bid protein could suppress all the cytotoxic effects of TNF-α.Conclusion TNF-α treatment can result in RIP1 kinase-mediated Bid cleavage and inhibit mitochondrial respiratory chains and cell energy metabolism,which ultimately leads to the death of L929-A cells.

Twelve compounds were isolated from the ethanol extract of Fructus Gleditsiae Abnormalis by macroporous resin, silica gel, Sephadex LH-20, MCI and ODS column chromatographies. Their structures were identified on the basis of physicochemical properties and spectral data as gleditsioside A(1), gleditsioside B(2), gleditsioside H(3), gleditsioside I(4), gleditsioside J(5), gleditsioside K(6), gleditsia saponins C′(7), tamarixetin-7-O-β-D-glucopyranoside(8), neohesperidin(9), chrysoeirol-7-O-neohesperidoside(10); syringaresinol- O-β-D-glucopyranoside(11), liriodendrin(12). Compounds 8-12 were firstly isolated from this genus.

Twelve compounds were isolated and purified from ethyl acetatefraction of Cynanchum stauntonii by silica gel, Sephadex LH-20 and ODS column chromatography. Their structures were identified by spectral techniques and physicochemical properties as syringaresinol(1), (-)-(7R, 7′R, 7″R, 8S, 8′S, 8″S)-4′, 4″-dihydroxy-3, 3′, 3″, 5-tetramethoxy-7, 9′ ∶7′, 9-diepoxy-4, 8″-oxy-8, 8′-sesquineolignan-7″, 9″-diol(2), prinsepiol(3), 4-hydroxyacetophenone(4), baishouwubenzophenone(5), 2, 4-dihydroxyacetophenone(6), benzoic acid(7), 1, 4-benzenediol(8), 6-O-［E］-sinapoyl-α-D-glucopyranoside(9a), 6-O-［E］-sinapoyl-β-D-glucopyranoside(9b), 1-O-methyl-α-D-cymadropyranoside(10), β-daucosterol(11), and β-sitosterol(12). Compounds 1-3, 5 and 7-11 were firstly isolated from this plant.

ObjectiveTo employ the effective components and antiarrhythmic mechanism of Wenxin Granules （WXKL） by cell membrane chromatography （CMC） and ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry （UPLC-Q-TOF/MS）， combined with network pharmacology. MethodIn this study， the CMC/UPLC-Q-TOF/MS technique was employed to identify the components in WXKL that could specifically bind to myocardial cell membranes. By utilizing databases such as SwissTarget Prediction and GeneCards， the targets of WXKL's effective components and arrhythmia-related targets were mined. Cytoscape software was used to construct a "component-target-disease" network. Gene ontology（GO） function and Kyoto encyclopedia of genes and genomes（KEGG） pathway enrichment analyses were carried out， and molecular docking of key components and targets was performed. Finally， further verification was conducted through in vivo experiment of rats. ResultA total of 39 effective components were identified in WXKL. These included 13 components derived from Panax notoginseng， 15 components from Codonopsis pilosula， seven components from Glycyrrhizae Radix et Rhizoma， one component from Succinum， one component from Polygonatum odoratum， one component shared by both P. odoratum and C. pilosula， and one component shared by both Panax notoginseng and C. pilosula. Network pharmacology predicted that WXKL had 16 core antiarrhythmic targets and 79 related pathways， mainly involving adrenergic signaling in cardiomyocytes， cyclic guanosine monophosphate （cGMP）/protein kinase G （PKG）， calcium signal， cyclic adenosine monophosphate （cAMP）， interleukin （IL）-17， mitogen-activated protein kinase （MAPK）， and tumor necrosis factor （TNF） signaling pathways. The results of in vivo experiment of rats showed that WXKL significantly improved the expression of β₁-adrenergic receptor （β₁-AR）， cAMP， TNF-α， and calcium voltage-gated channel subunit alpha 1C （CACNA1C）. ConclusionWXKL can exert its antiarrhythmic effects through multiple components， multiple targets， and multiple pathways. This study provides a scientific basis for explaining the potential pharmacodynamic substance foundation and mechanism of action of traditional Chinese medicine in treating arrhythmia.