OBJECTIVE: To investigate the effects of mTOR inhibitors everolimus (EVE) and gemcitabine (GEM) on the proliferation, apoptosis and cell cycle of diffuse large B-cell lymphoma (DLBCL) cell line U2932, and further explore the molecular mechanisms, so as to provide new ideas and experimental basis for the clinical treatment of DLBCL. METHODS: The effect of EVE and GEM on the proliferation of U2932 cells was detected by CCK-8 assay, the IC₅₀ of the two drugs was calculated, and the combination index (CI=) of the two drugs was calculated by CompuSyn software. The effect of EVE and GEM on apoptosis of U2932 cells was detected by flow cytometry with AnnexinV-FITC/PI staining. Flow cytometry with propidium iodide (PI) staining was used to detect the effect of EVE and GEM on the cell cycle of U2932 cells. Western blot assay was used to detect the effects of EVE and GEM on the channel proteins p-mTOR and p-4EBP1, the anti-apoptotic proteins MCL-1 and Survivin, and the cell cycle protein Cyclin D1. RESULTS: Both EVE and GEM could significantly inhitbit the proliferation of U2932 cells in a time- and dose-dependent manner (r=0.465, 0.848; 0.555, 0.796). According to the calculation of CompuSyn software, EVE combined with GEM inhibited the proliferation of U2932 cells at 24, 48 and 72 h with CI=<1, which had a synergistic effect. After treated U2932 cells with 10 nmol/L EVE, 250 nmol/L GEM alone and in combination for 48 h, both EVE and GEM induced apoptosis, and the difference was statistically significant compared with the control group (P<0.05). The apoptosis rate was significantly enhanced after EVE in combination with GEM compared with single-agent (P<0.05). Both EVE and GEM alone and in combination significantly increased the proportion of cells in G₁ phase compared with the control group (P<0.05). The proportion of cells in G₁ phase was significantly increased when the two drugs were combined (P<0.05). The expression of p-mTOR and effector protein p-4EBP1 was significantly downregulated in the EVE combined with GEM group, the expression of anti-apoptotic proteins MCL-1, Survivin and cell cycle protein cyclin D1 was downregulated too (P<0.05). CONCLUSION EVE combined with GEM can synergistically inhibit the proliferation of U2932 cells, and the mechanism may be that they can synergistically induce apoptosis by downregulating the expression of MCL-1 and Survivin proteins and block the cell cycle progression by downregulating the expression of Cyclin D1.
Humans ; Gemcitabine ; Everolimus/pharmacology* ; Survivin/pharmacology* ; Cyclin D1/pharmacology* ; Myeloid Cell Leukemia Sequence 1 Protein ; Cell Line, Tumor ; Cell Proliferation ; TOR Serine-Threonine Kinases ; Apoptosis ; Apoptosis Regulatory Proteins ; Cell Cycle Proteins ; Lymphoma, Large B-Cell, Diffuse

Aim To study the nephrotoxicity effects of the main monomers in Zuojin Pills. Methods CCK-8 and high-content toxicity screening were used to preliminarily screen the main alkaloids in Zuojin Pills that may cause renal cell damage. Further, by confirmation of cell morphology, release rate of lactate dehydrogenase and cytochrome C, and expression of apoptosis-related proteins, the alkaloids causing cell damage were preliminarily identified, providing in vitro toxicological evidence for the compatibility of components of traditional Chinese medicine and compatibility attenuation. Results Preliminary screening using CCK-8 method and high-content technology showed that evodiamine (EVO) could significantly reduce cell number, increase cell membrane permeability, and reduce mitochondrial membrane potential. In addition, cell morphology, apoptosis and cytochrome C expression were consistent with the results of high-content screening. Western blot experiments indicate that EVO could induce apoptosis and cause renal cell damage. Conclusions EVO can obviously cause renal cell damage, and may induce apoptosis by affecting mitochondria, cytochrome C and cell membrane permeability.

This study investigated the intervention effect and possible mechanism of ophiopogonin D (OPD) in protecting cardiomyocytes against ophiopogonin D' (OPD')-induced injury, and provided relevant experimental data for the clinical use of Ophiopogon japonicas. Cell counting kit-8 (CCK-8) assay was used to evaluate the effect of OPD and OPD' on H9c2 cell viability. The content of reaction oxygen species (ROS) in cells were detected by flow cytometry. The contents of Fe²⁺ in cells were detected by FerroOrange's fluorescence imaging. The content of glutathione (GSH) and glutathione peroxidase (GSH-Px) were detected by kits. The expression of transferrin receptor 1 (TFR1), cyclooxygenase 2 (COX2), NADPH oxidase 1 (NOX1), long-chain acyl-CoA synthetase 4 (ACSL4), cationic amino acid transporter 11 (SLC7A11), glutathione peroxidase 4 (GPX4), and ferritin heavy chain 1 (FTH1) was detected by Western blot. Results showed that OPD' (1 μmol·L^-1) significantly induced the expression of ferroptosis-related proteins, the contents of Fe²⁺, ROS, and GSH-Px were increased, and the content of GSH were decreased. In addition, different concentrations of OPD (0.5, 1, and 2 μmol·L^-1) could partially reverse the myocardial cell injury caused by OPD', and the best effect was obtained when the dose range was 1-2 μmol·L^-1. The experimental results show that OPD can interfere with the ferroptosis caused by OPD', and then have a protective effect on H9c2 cells.

Objective:Through phosphatidylinositol 3-kinase（PI3K）/protein kinase B （Akt）/mammalian rapamycin target protein （mTOR） signaling pathway， explore the effect of Zhigancao Tang on myocardial ischemia-reperfusion injury（MIRI）The role and mechanism of arrhythmia（ventricular tachycardia and ventricular fibrillation）. Method:The 72 SD rats were randomly divided into sham operation group，model group， Zhigancao Tang low，medium and high dose group（11.43，22.86，45.72 g·kg^-1），Wenxin granule group（2.43 g·kg^-1），continuous drug intervention for 10 days. Two hours after the last administration，the MIRI model of rat was prepared by ligating the left anterior descending coronary artery，and the changes of electrocardiogram were recorded. After successful modeling，blood and heart tissue were collected to detect the content of creatine creatine（CK），lactate dehydrogenase（LDH）and aspartate aminotransferase（AST）in the serum, the enzyme-linked immunoassay（ELISA） method was used to detect cardiac troponin（CtnI）content, immunohistochemical detection of myocardial PI3K，Akt，mTOR expression. Western blot was used to detect the myocardial autophagy-related protein microtubule-associated protein 1 light chain 3（LC-3），autophagy markers Beclin1 and PI3K/Akt/mTOR signaling pathway related protein expression and phosphorylated p-PI3K，p-Akt，p-mTOR levels. Result:In model group， 100% of ventricular tachycardia and 91.67% of ventricular fibrillation occurred. Compared with sham operation group， the serum levels of CK，LDH，AST，and CtnI in the model group were significantly increased（P<0.01），PI3K，Akt，mTOR AOD values in myocardial tissue were significantly increased （P<0.01），the relative expression of the ratio of LC3-Ⅱ/LC3-Ⅰ and Beclin1 was significantly increased（P<0.01），p-PI3K/PI3K，p-Akt/Akt，and p-mTOR/mTOR were significantly reduced （P<0.01）. Compared with model group， the incidence of ventricular tachycardia and ventricular fibrillation in the high-dose Zhigancao Tang group was significantly reduced （P<0.01），and the duration was the shortest compared with other administration groups（P<0.01）， CK，LDH，AST level and CtnI content were significantly reduced（P<0.01）， the expression of PI3K，Akt and mTOR of Zhigancao Tang group was significantly decreased with increasing dose（P<0.01）， the expression of LC-3 and Beclin1 was accompanied by Zhigancao Tang increase of each dose group of soup had different degrees of decrease （P<0.01），while the expression ratio of PI3K/Akt/mTOR-related protein was significantly increased （P<0.05，P<0.01）. Conclusion:Pretreatment of Zhigancao Tang can reduce the abusually elevated cardiac enzymes CK, LDH, AST and CtnI, inhibit excessive autophagy of cells, and up-regulate the expression of PI3K, Akt and mTOR, indicating that the anti-MIRI arrhythmia effect of Zhigancao Tang may be related to the PI3K/Akt/mTOR signaling pathway.