Role of inhibiting lncRNA TUG1 to down⁃regulate nucleotide binding oligomerization domain like receptor protein 1 inflammasome in delaying the progression of Alzheimer’s disease / 解剖学报

Objective To investigate the relieving effects of knockdown of long non-coding RNA(lncRNA)taurine up-regulated gene 1 (TUG1) on inhibiting nucleotide binding oligomerization domain like receptor protein 1 (NLRP1) inflammasome and the progression of Alzheimer’ s disease. Methods Wild-type (WT group, 10 mice) or amyloid precursor protein (APP) / presenilin-1 (PS1) transgenic mice (30 mice) with a genetic background of C57 / BL6 aged 9-10 weeks were used in this study. APP / PS1 transgenic mice were randomly divided into model group, model+lncRNA TUG1 short hairpin RNA (shRNA) group and model + shRNA non target (NT) group (n = 10) . Blood samples, cerebral cortex tissues, primary microglial cells and primary astrocytes were collected from mice 12 weeks of age on day 1 (3-month-old) and 32 weeks of age on day 1 (8-month-old), with 5 mice per group at each time point. Real-time PCR analysis was used to detect the expression levels of lncRNA TUG1 and macrophage migration inhibitory factor (MIF) mRNA in cerebral cortex tissues and primary microglial cells, and C1r and C1s mRNA levels in primary astrocytes of 3-month-old and 8-month-old mice in the above 4 groups, respectively. ELISA was used to determine the MIF in plasma samples of the above 4 groups of mice. Primary microglia and astrocytes from the cerebral cortex of 3-month-old and 8-month-old mice were co-cultured. CCK-8 method was used to determine the proliferation ability of the above cells. Western blotting was used to determine the expression levels of MIF, pro interleukin-1β (pro-IL-1β), apoptosis associated speck-like protein containing a caspase recrult domain(ASC), Caspase-1 (p20), Caspase-1 (full), NLRP1 and NLRP3 in cerebral cortex tissues of 3-month-old and 8-month-old mice. Immunofluorescent staining was used to determine amyloid beta(Aβ) in cerebral cortex of 8-month-old mice. Results At the age of 3-month-old and 8-month-old, compared with the WT group, the relative expression level of lncRNA TUG1 and MIF in cerebral cortex tissues and primary microglia of model group mice was significantly up-regulated, with primary microglial cells and astrocytes proliferation ability enhanced (P0. 05) . There was no significant difference between the model group and the model+shRNA NT group mice of all the above factors (P>0. 05) . Conclusion In APP / PS1 transgenic mice, up-regulation of lncRNA TUG1 and MIF are positively associated with the activation of NLRP1 inflammasome in mice cerebral cortex tissues and primary microglia. Knock-down of lncRNA TUG1 can ameliorate the progression of Alzheimer’ s disease.

The mechanism of tilianin against ischemia-reperfusion injury of H9c2 cells / 药学学报

Z-VAD-FMK was combined with hypoxia-reoxygenation (H/R) injury to establish a necroptosis model of H9c2 cells to mimic the pathological changes of myocardial ischemia reperfusion injury (MIRI) in vitro and to study the effect and mechanism of tilianin against myocardial ischemia-reperfusion injury. A cell counting kit-8 (CCK-8) was used to detect cell viability, and commercial kits were used to detect lactate dehydrogenase (LDH) and superoxide dismutase (SOD) in the cell culture supernatant. Hoechst 33342/PI immunofluorescence staining was used to detect cell death. DCFH-DA, BBcellProbeTMM61, and JC-1 probes were used to detect reactive oxygen species (ROS), mitochondrial permeability transition pore (mPTP), and mitochondrial membrane potential (MMP), respectively. An enzyme-linked immunosorbent assay (ELISA) method was used to detect the release of tumor necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1β), and interleukin-6 (IL-6). The results show that the cell viability, SOD activity, and MMP of the model group induced by H/R injury decreased, as compared with control group, but the necroptosis rate, LDH level, and ROS release increased significantly. Furthermore, mPTP of the model group cells opened, and TNF-α, IL-1β, and IL-6 levels were significantly higher. Molecular docking modeling showed that tilianin can bind to calmodulin-dependent protein kinase II (CaMKII), and Western blot results showed that compared with control group, the expression levels of p-CaMKII and phospho-mixed lineage kinase domain-like protein increased in the model group, and tilianin could decrease the expression level of these proteins. The above results indicate that tilianin can protect H9c2 cells by inhibiting the phosphorylation of CaMKⅡ at threonine 287, protecting mitochondrial function, and inhibiting the opening of mPTP to prevent necroptosis. This study has value for research on new methods to treat H/R injury.

Protective effect of water soluble CoQ10 on rotenone-induced apoptosis in PC12 cells / 西安交通大学学报(医学版)

Objective To investigate the protective effect and the underlying mechanism of water soluble coenzyme Q10 (CoQ10)against rotenone induced injury on PC12 cells model.Methods PC12 cells were cultured with rotenone,water-soluble CoQ1 0 was added to the culture media 3 hours prior to the rotenone incubation.We determined cell viability by CCK8;reactive oxygen species (ROS)was detected by spectrophotometer;and Bcl-2, Bax,active Caspase-3,Caspase-9 and apoptosis-inducing factor (AIF)were measured by Western blotting after 24-hour rotenone incubation.Results After the treatment by rotenone,cell viability decreased significantly (P＜0.01)and ROS level increased (P＜0.01).CoQ10 could improve PC12 cell viability (P＜0.01)and reduce the level of ROS (P＜0.01).Western blotting experiments showed that CoQ10 could reduce rotenone-induced Caspase-9 (P＜0.05),active Caspase-3 (P＜0.05)and Bax (P＜0.01)expressions,increase the expression of Bcl-2 (P＜0.01),and prevent nuclear translocation of AIF (P＜0.05).Conclusion CoQ10 has a protective effect on rotenone-induced apoptosis in PC12 cells,the mechanism of which may be through scavenging ROS in cells;decreasing caspase-9 ,active caspase-3 and Bax expressions;and increasing the expression of Bcl-2 ;and preventing AIF nuclear translocation.

Change Trend of Viablity, Apoptosis and ROS Production of Neutrophil in vitro Culture / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To explore the change trend of cell viability, apoptosis and reactive oxygen species (ROS) production of peripheral blood neutrophils in in vitro culture.</p><p><b>METHODS</b>Neutrophils were isolated by density gradient centrifugation and cultured in medium containing 10% fetal bovine serum for diffentrent times in a fall humidified atmosphere with 5% COat 37 °C. Cell viability, spontaneous apoptosis and ROS level were detected by flow cytometry at 0, 24, 48, 72 and 96 hours.</p><p><b>RESULTS</b>The cell viability of neutrophils could be maintained above 90% at 24 h in culture in vitro, and there was no significant difference between culture in vitro at 0 h and 24 h (P>0.05), but after culture in vitro at 72 h, the cell viability of neutrophils obviously droped, the count of apoptotic cells increased, and after culture in vitro at 96 h the neutrophils almostly all died and the production of ROS at 12 h significantly decreased as compared with that at 0 h.</p><p><b>CONCLUSION</b>The cell viability of neutrophils at 24 h cultures in vitro can be maintained relative stable with cell viability above 90% and unobvious change of count of apoptotic cells, but the neutrophil function test of ROS should be carried out immediately after cell separation.</p>