ABSTRACT
This study aimed to investigate the effect and the possible mechanism of Shenlian( SL) extract on tumor necrosis factor-α( TNF-α)-induced ECV304 injury. After the establishment of TNF-α-induced ECV304 cells injure model,MTT assay was used to detect cell viability and the level of reactive oxygen species( ROS) was measured by flow cytometry. The contents of superoxide dismutase( SOD),malondialdehyde( MDA),nitric oxide( NO),endothelin-1( ET-1) and interleukin-1β( IL-1β) in the supernatant were detected by biochemical method and enzyme linked immunosorbent assay( ELISA). The expression levels of apoptosis-related proteins B-lymphoma-2 gene( Bcl-2),Bcl-2 associated X protein( Bax),caspase-3,caspase-9 and nuclear factor E2 associated factor2( Nrf2)/Kelch like epichlorohydrin associated protein-1( Keap1) signaling pathway related proteins Nrf2,Keap1,quinone oxidoreductase( NQO1) and heme oxygenase 1( HO-1) were detected by Western blot. The results showed that 50 μg·L-1 TNF-α significantly damaged ECV304 cells,induced the impairment of cell viability( P<0. 01),the increase of ROS production,the decrease of SOD activity,and the increase of MDA,NO,ET-1 and IL-1β( P<0. 01),meanwhile,it caused the up-regulation of Keap1,caspase-9 and Bax protein expression,and down-regulation of NQO1 and Bcl-2 protein expression( P<0. 05) compared with the control group.Compared with the model group,SL extract reduced the damage of ECV304 cells induced by TNF-α,improved cell viability,reduced ROS production,increased SOD activity and decreased MDA,NO,ET-1,IL-1β content( P<0. 01 or P<0. 05). In addition,SL extract also down-regulated the protein expression levels of Keap1,caspase-3,caspase-9 and Bax,and increased the protein expressions of Nrf2,NQO1,HO-1 and Bcl-2( P<0. 01 or P<0. 05). The above results indicate that SL extract can provide protective effect on ECV304 cells injury induced by TNF-α,alleviate oxidative stress injury,inflammation and apoptosis,and its mechanism may be related to regulating Nrf2/Keap1 signaling pathway.
Subject(s)
Apoptosis , Kelch-Like ECH-Associated Protein 1/metabolism , NF-E2-Related Factor 2/metabolism , Oxidative Stress , Plant Extracts , Signal Transduction , Tumor Necrosis Factor-alpha/geneticsABSTRACT
OBJECTIVE To explore the potential molecular mechanism of Shenlian (SL) on myocardial ischemia (MI) on the basis of network pharmacology. METHODS Firstly, the main active ingredients of SL were screened in the Traditional Chinese Medicine Integrated Database, and the MI-associated targets were collected from the DisGeNET database. Then, we used compound-target and target-pathway networks to uncover the therapeutic mechanisms of SL. On the basis of network pharmacology analysis results, we assessed the effects of SL in MI rat model and oxygen glu?cose deprivation model of H9c2 cells and validated the possible molecular mechanisms of SL on myocardial injury in vivo and in vitro. RESULTS The network pharmacology results showed that 37 potential targets were recognized, including TNF-α, Bcl-2, STAT3, PI3K, and MMP2. The pathways revealed that the possible targets of SL were involved in the reg?ulation of inflammation and apoptosis signaling pathway. Then, in vivo experiments indicated that SL significantly reduced the myocardial infarction size of MI rats. Serum CK-MB, cTnT, CK, LDH, and AST levels were significantly decreased by SL (P<0.05 or P<0.01). In vitro, SL significantly increased H9c2 cell viability. The levels of inflammation factors including TNF-α and MMP2 were significantly decreased by SL (P<0.05 or P<0.01). TUNEL and Annexin V/propidium iodide assays indicated that SL could significantly decrease the cell apoptotic rate in vivo and in vitro (P<0.05 or P<0.01). The remarkable upregulation of anti-apoptotic Bcl-2 and downregulation of pro-apoptotic Bax protein level further confirmed this result. Kyoto Encyclopedia of Genes and Genomes pathway analysis showed that the PI3K-Akt and JAK2-STAT3 pathways were significantly enriched in SL. Compared with the model group, SL treatment significantly activated the PI3K-Akt and JAK2-STAT3 pathways in vivo and in vitro according to Western blotting analyses. CONCLU?SION SL could protect the myocardium from MI injury. The underlying mechanism may be related to the reduction of inflammation and apoptosis by activating the PI3K/Akt and JAK2/STAT3 pathways.
ABSTRACT
Objective:To study the effect of Shenlian extract (SL extract) on macrophage function and inflammatory resolution in lipid peroxidation inflammatory injury models. Method:The effects of different concentrations of SL extract (2.5, 5.0, 10.0, 20.0 mg·L-1) on the polarization type, foam formation and chemotactic function of macrophages were detected with RAW264.7 cells induced by oxidized low-density lipoprotein(ox-LDL). Western blot was used to detect pro-inflammatory resolution factor arachidonate 5-lipoxygenase(ALOX5) and inducible inducible nitric oxide synthase(iNOS), and phosphorylated p65 (p-p65) and phosphorylated IκK (p-IκK) in nuclear factor(NF)-κB related signaling pathways. Result:Compared with the control group, ox-LDL enhanced the expressions of M1 macrophage markers TNF-α, IL-1β, iNOS (PPPα, IL-1β, and iNOS (PPPPκK was inhibited significantly (PConclusion:In the inflammatory damage model of lipid peroxidation, SL extract can regulate the polarization of macrophage, inhibit the chemotaxis and foaming of ox-LDL, increase the inflammatory resolution molecular expression, and improve the state of lipid peroxidation, which may be related to the inhibition of NF-κB signaling pathway.