Ethyl Lithospermate Reduces Lipopolysaccharide-Induced Inflammation through Inhibiting NF-κB and STAT3 Pathways in RAW 264.7 Cells and Zebrafish / 中国结合医学杂志

OBJECTIVE@#To explore the anti-inflammatory effects of ethyl lithospermate in lipopolysaccharide (LPS)-stimulated RAW 264.7 murine-derived macrophages and zebrafish, and its underlying mechanisms.@*METHODS@#3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazoliumbromide (MTT) assays were performed to investigate the toxicity of ethyl lithospermate at different concentrations (12.5-100 µ mol/L) in RAW 264.7 cells. The cells were stimulated with LPS (100 ng/mL) for 12 h to establish an inflammation model in vitro, the production of pro-inflammatory cytokines interleukin (IL)-6 and tumor necrosis factor α (TNF-α) were assessed by enzyme linked immunosorbent assay (ELISA). Western blot was used to ascertain the protein expressions of signal transducer and activator of transcription 3 (STAT3), nuclear factor kappa B (NF-κB) p65, phospho-STAT3 (p-STAT3, Tyr705), inhibitor of NF-κB (IκB) α, and phospho-I κB α (p-IκB α, Ser32), and confocal imaging was used to identify the nuclear translocation of NF-κB p65 and p-STAT3 (Tyr705). Additionally, the yolk sacs of zebrafish (3 days post fertilization) were injected with 2 nL LPS (0.5 mg/mL) to induce an inflammation model in vivo. Survival analysis, hematoxylin-eosin (HE) staining, observation of neutrophil migration, and quantitative real-time polymerase chain reaction (qRT-PCR) were used to further study the anti-inflammatory effects of ethyl lithospermate and its probable mechanisms in vivo.@*RESULTS@#The non-toxic concentrations of ethyl lithospermate have been found to range from 12.5 to 100 µ mol/L. Ethyl lithospermate inhibited the release of IL-6 and TNF-α(P<0.05 or P<0.01), decreased IκBα degradation and phosphorylation (P<0.05) as well as the nuclear translocation of NF-κB p65 and p-STAT3 (Tyr705) in LPS-induced RAW 264.7 cells (P<0.01). Ethyl lithospermate also decreased inflammatory cells infiltration and neutrophil migration while increasing the survival rate of LPS-stimulated zebrafish (P<0.05 or P<0.01). In addition, ethyl lithospermate also inhibited the mRNA expression levels of of IL-6, TNF-α, IκBα, STAT3, and NF-κB in LPS-stimulated zebrafish (P<0.01).@*CONCLUSION@#Ethyl lithospermate exerts anti-Inflammatory effected by inhibiting the NF-κB and STAT3 signal pathways in RAW 264.7 macrophages and zebrafish.

Heat-Clearing Chinese Medicines in Lipopolysaccharide-Induced Inflammation / 中国结合医学杂志

Lipopolysaccharide (LPS)-induced inflammation causes massive threatening diseases, such as sepsis, acute lung injury and multiple organ dysfunction syndrome. Efficient treatment to prevent inflammation is crucial in LPS-induced inflammatory diseases. Heat-clearing Chinese medicines (CMs) have been used to ameliorate LPS-induced inflammation in China for centuries. Heat-clearing CMs regulate inflammatory pathways, thereby inhibiting the release of inflammatory factors. This review aimed to introduce promising heat-clearing CMs countering LPS-induced inflammation in the last 5 years, exploring the underlying molecular mechanisms.

Effects of MiR-221-Mediated Wnt/β- Catenin Signaling Pathway on Biological Activity of Childhood Acute Lymphoblastic Leukemia Cells / 中国实验血液学杂志

OBJECTIVE@#To study the effects of miR-221 on the biological activity of childhood acute lymphoblastic leukemia cells and its mechanism.@*METHODS@#Bone marrow mononuclear cells (BMNC) were isolated from bone marrow samples of ALL children diagnosed in our hospital from May 2018 to November 2018. The cells were divided into control group, miR-221-NC group and miR-221 group. After transfection according to the instructions of Lipofectamine 2000 kit, the levels of miR-221 in each group were detected by RT-PCR. Flow cytometry was used to detect the effects of miR-221 on cell cycle and apoptosis. Transwell assay was used to detect cell migration and invasion. Western blot was used to detect the effects of miR-221 on proliferating cell nuclear antigen (PCNA), Caspase 3, Cyclin D1 and MMP-9 proteins in BMNC. Luciferase reporter gene assay was used to detect the targeting relationship between miR-221 and Wnt gene.@*RESULT@#The expression level of miR-221 in the miR-221 group was significantly higher than that in the control group and the miR-221-NC group (P＜0.05). MTT assay showed that, after transfection for 2, 3, 4 and 5 days, the cell proliferation level in miR-221 group was significantly lower than that in the control group and the miR-221-NC group (P＜0.05). The cell ratio of G/G phase was (73.25±8.1)% in the miR-221 group, which was significantly higher than that in the control group and the miR-221-NC group (P＜0.05); moreover, the cell ratio of S phase in the miR-221 group was (12.37±1.6)%，which was significantly lower than that in the control group and the miR-221-NC group (P＜0.05). The percentage of apoptotic cells in the miR-221 group was (24.68±3.87)%, which was significantly higher than that in the control group and the miR-221-NC group (P＜0.05). Transwell cell invasion experiment showed that the number of invasive cells in the miR-221 group was 23.42±3.62, which was significantly lower than that in the control group and the miR-221-NC group (P＜0.05). Transwell cell migration assay showed that the number of migrating cells in the miR-221 group was 34.86±5.32, which was significantly lower than that in the control group and the miR-221-NC group (P＜0.05). The relative level of PCNA, Cyclin D1 and MMP-9 in the miR-221 group was 0.26±0.03, 0.17±3.61 and 0.14±0.02, respectively, which was significantly lower than those in the control group and the miR-221-NC group (P＜0.05), while the relative level of Caspase-3 in the miR-221 group was 0.37±0.05, which was significantly higher than that in the control group and the miR-221-NC group (P＜0.05). Luciferase reporter assay showed that the activity of luciferase in Wnt wild type plasmid was significantly inhibited by miR-221 (P＜0.05).@*CONCLUSION@#miR-221 can inhibit the proliferation, migration and invasion of BMNC, moreover can promote cell apoptosis, which may be related with the inhibition of Wnt/β- catenin signaling pathway.

Inhibitory effect of giganteaside D on hepatocellular carcinoma Hep 3b cells and the underlying mechanisms / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the inhibitory effect of giganteaside D (GD) on hepatocellular carcinoma and its molecular mechanisms.</p><p><b>METHODS</b>The inhibitory effects of GD on Hep 3b cells were determined using MTT assay and colony formation assay. The morphological changes of Hep 3b cells after GD treatment were observed by electron microscopy, and the cell cycle changes was analyzed using flow cytometry. The cell apoptosis and mitochondrial potential collapse in the treated cells were tested with Hoechst staining assay and flow cytometry. The expression levels of Bcl-2, PARP and key proteins in MAPK pathway were detected using Western blotting.</p><p><b>RESULTS</b>GD showed a significant inhibitory effect on Hep 3b cells with an ICvalue of 16.08 µmol/L at 72 h. Flow cytometric analysis demonstrated that the phases of cell cycle remained unchanged and a sub-G1 peak (from 3.3% to 33.6%) appeared as GD concentration increased. GD-induced apoptosis was further conformed by Hoechst staining assay, and flow cytometry showed increased mitochondrial potential collapse in the cells. Western blotting demonstrated the cleavage of PARP, decrease of Bcl-2 and p-Erk1/2 (Thr202/Tyr204), and activation of p-p38 (Thr180/Tyr182) and p-JNK (Thr183/Tyr185) in GD-treated cells.</p><p><b>CONCLUSIONS</b>GD has significant inhibitory effect against hepatocellular carcinoma cells in vitro by inducing apoptosis possibly in association with the MAPK signaling pathway.</p>

Anti-angiogenetic effect of arenobufagin in vitro and in vivo / 药学学报

This study is to investigate the anti-angiogenetic effect of arenobufagin in vitro and in vivo. The anti-proliferation effect of arenobufagin on CNE-2, Hep2, SH-SY5Y, LOVO, PC-3 and DU145 cells as well as human umbilical vein endothelial cells (HUVECs) was determined by MTT assay. Cell morphological changes of LOVO and HUVECs after arenobufagin treatment were observed by microscopy. Arenobufagin inhibited the proliferation of CNE-2, Hep2, SH-SY5Y, LOVO, PC-3, DU145 and HUVECs in a dose-dependent manner. Furthermore, it was obviously observed that the subcytotoxic concentration of arenobufagin in human carcinoma cells induced a marked decrease in the viability of HUVECs. Chick embryo chorioallantoic membrane (CAM) model was used to detect the anti-angiogenetic effect of arenobufagin in vivo. Arenobufagin significantly suppressed the angiogenesis of CAM. Cell cycle analysis demonstrated that G2/M phase was arrested and the sub-G1 peak appeared with the increase of arenobufagin concentration. PI/Annexin V double staining assay further demonstrated that arenobufagin could induce apoptosis in a dose- and time-dependent manner. Mitochondrial potential collapse detected by flow cytometric analysis was increased after arenobufagin treatment. It also observed that PARP was cleaved to p85 active form by Western blotting. Taken together, arenobufagin has significant anti-angiogenetic effect in vitro and in vivo, and the action mechanisms behind its anti-angiogenesis may be associated with cell cycle arrest and apoptosis of vein endothelial cells.