Effect of puerarin on cholesterol influx and efflux in RAW264.7-treated foam cells

AIM: To study the protective effect of puerarin on the atherosclerosis of RAW264.7-derived foam cells. METHODS: The model of foam cells was established by incubating the RAW264.7 cells with ox-LDL. The cholesterol uptake was evaluated by a DiI-ox-LDL binding assay. The ability of cholesterol efflux of the RAW264.7-derived foam cells was detected by cholesterol efflux assay. The protein levels of LC3Ⅱ, P62, CD36, ABCA1, LAL and p-AMPK were determined by Western blot. RESULTS: Puerarin treatment reduced the cholesterol uptake capacity and enhanced the cholesterol efflux rate. The protein levels of LC3Ⅱ, ABCA1 and LAL in puerarin group were higher than that in ox-LDL group, while the protein levels of P62 and CD36 were obviously decreased, and those in rapamycin treatment group had the same change as puerarin group. The protein levels of LC3Ⅱ, ABCA1 and LAL were obviously decreased and the protein level of p-AMPK was increased after co-treated with 3-MA. CONCLUSION: Puerarin promotes LAL and ABCA1-mediated cholesterol efflux in ox-LDL-treated RAW264.7 macrophages, which might enhance autophagy through AMPK-dependent pathway for cholesterol efflux regulation, and reduce the uptake of lipids by CD36 negative regulation.     

Notch3 pathway mediates SAHA-induced apoptosis in human small-cell lung cancer H446 cells

AIM: To investigate the effect of suberoylanilide hydroxamic acid (SAHA) on the apoptosis of human small-cell lung cancer H446 cells and its possible mechanism. METHODS: H446 cells were incubated in the medium containing SAHA. CCK-8 assay was used to detect the anti-tumor effect of SAHA on the H446 cells, and IC₅₀ values of SAHA were calculated. Flow cytometry was used to analyze the apoptosis. After Notch3 gene was silenced, the pro-apopto-tic effect of SAHA on the H446 cells was inhibited (P<0.05). Eukaryotic expression plasmid containing N3ICD was transfected into the H446 cells, so that N3ICD was expressed in the H446 cells. The mRNA expression of Notch3 was measured by RT-PCR. The protein levels of Notch3, N3ICD, Puma and cleaved caspase-3 were determined by Western blot. RESULTS: SAHA remarkably reduced the cell viability in a dose-dependent manner (P<0.05), and the IC₅₀ value of SAHA was 1.91 μmol/L. SAHA induced apoptosis in a dose-dependent manner (P<0.05). The expression of Notch3 gene was negative in the H446 cells, SAHA reactivated Notch3 gene and Notch3 pathway in a dose-dependent manner (P<0.05).Notch3 knockdown inhibited apoptosis induced by SAHA (P<0.05). Over-expression of N3ICD up-regulated the protein levels of Puma and cleaved caspase-3.CONCLUSION: SAHA induces apoptosis in human small-cell lung cancer H446 cells by activating Notch3 pathway and up-regulating the protein level of Puma.     

Protective effect of ecdysterone on H9c2 cells against oxidative stress

AIM: To investigate the effect of ecdysterone (EDS) on H9c2 cardiomyocytes after oxidative stress. METHODS: H9c2 cells were cultured in vitro and divided into control group, high dose (2 μmol/L) of EDS group, middle dose (1.5 μmol/L) of EDS group, low dose (1 μmol/L) of EDS group, and H₂O₂ group. H9c2 cardiomyocytes in H₂O₂ group and high, middle and low doses of EDS groups were exposed to H₂O₂ for 6 h to establish the model of oxidative stress. The viability of the H9c2 cells was detected by CCK-8 assay. The apoptosis of H9c2 cells was analyzed by flow cytometry. The levels of lactate dehydogenase (LDH) and creatine kinase-MB (CK-MB) in the culture medium, and the levels of superoxide dismutase (SOD) and malondialdehyde (MDA) in the H9c2 cells were measured by colorimetry. The generation of reactive oxygen species (ROS) and the mitochondrial membrane potential were evaluated by flow cytometry and confocal laser scanning microscopy. The protein levels of Bax, Bcl-2 and cleaved caspase-3 in the H9c2 cells were determined by Western blot. RESULTS: Ecdysterone at the selected concentrations had no effect on the viability of H9c2 cells. Compared with control group, the levels of LDH, CK-MB, ROS and MDA, and the apoptotic rates of the H9c2 cells were significantly increased after treated with H₂O₂, but were decreased by EDS treatment in a dose-dependent manner. The levels of SOD and mitochondrial membrane potential of the H9c2 cells in H₂O₂ group were reduced significantly compared with control group, but high, middle and low doses of EDS treatments up-regulated the levels of SOD and mitochondrial membrane potential in H₂O₂-treated H9c2 cells. The protein levels of Bax and cleaved caspase-3 in the H9c2 cells in H₂O₂ group showed significant elevation in comparison with control group, and the protein expression of Bcl-2 declined in H₂O₂ group compared with control group, but high, middle and low doses of ecdysterone treatments down-regulated the protein levels of Bax, cleaved caspase-3 and up-regulated the expression of Bcl-2 in H₂O₂-treated H9c2 cells. CONCLUSION: Ecdysterone attenuates the effect of H₂O₂-induced oxidative stress on H9c2 cardiomyocytes. The mechanism may be involved in scavenging oxidative stress products, increasing antioxidant enzyme activity and improving mitochondrial function.     

MEK/ERK pathway activated by changing insulin receptor isoform is associated with abnormal proliferation of intestinal epithelial cells in diabe-tic mice

AIM: To investigate the role of insulin receptor (IR)-A/IR-B ratio and the downstream pathway in abnormal proliferation of intestinal epithelial cells (IECs) in diabetic mice. METHODS: Diabetes mouse models were induced by intraperitoneal streptozocin injection. The proliferating cell nuclear antigen (PCNA) proliferation rates in the small intestine tissue were evaluated by immunohistochemical methods. The expression of IR isoforms was detected by RT-PCR. To ensure that the downstream pathways of IR are involved, real-time PCR and Western blot were performed to detect the expression of MEK1/2, ERK1/2, PI3K and Akt. RESULTS: In diabetic mice, the PCNA proliferation rates were higher than those in control group (P<0.05), and a high ratio of IR-A/IR-B was detected in the IECs (P<0.05). The mRNA expression of MEK1, MEK2, ERK1/2 and their phosphorylated protein levels in the diabetic mice were significantly higher than those in control group (P<0.05). CONCLUSION: The over-proliferation of IECs in the diabetic mice is associated with high IR-A/IR-B ratio and up-regulation of IR/MEK/ERK pathway.     

Effect of Sonic Hedgehog signaling blockade on growth of hepatocarcinoma cells

AIM: To investigate the effect of Sonic Hedgehog (Shh) signaling blockade on the growth of hematocarcinoma cells and underlying mechanisms. METHODS: The expression of Shh signaling molecules in hematocarcinoma cell lines BEL-7402, Huh7 and HepG2 was detected by RT-PCR. The cell viability was detected by MTT assay. The cell cycle and apoptosis were analyzed by flow cytometry. The expression of apoptosis-related proteins was determined by Western blot. RESULTS: Shh signaling molecules were all expressed in BEL-7402, Huh7 and HepG2 cells. The mRNA expression of Patched (Ptch), Gli1 and Gli2 was down-regulated by anti-Shh antibody. Blockade of Shh signaling pathway inhibited the proliferation of hepatocarcinoma cells with increasing cells in G₀/G₁ phase and induced the apoptosis of hepatocarcinoma cells. Treatment with anti-Shh antibody down-regulated the protein expression of pro-caspase-3, pro-caspase-8 and pro-caspase-9, while up-regulated the protein levels of cleaved caspase-3, cleaved caspase-8 and cleaved caspase-9 in BEL-7402 cells. CONCLUSION: Blockade of Shh signaling pathway inhibits the growth of hepatocarcinoma at different levels by cell cycle arrest and inducing apoptosis of hematocarcinoma cells.     

Effects of Chinese yellow wine on homocysteine-induced dysfunction in rat endothelial progenitor cells

AIM: To investigate whether Chinese yellow wine has influences on homocysteine (Hcy)-induced dysfunction in rat endothelial progenitor cells (EPCs). METHODS: Rat bone marrow was extracted to harvest mononuclear cells (MNCs) by density gradient centrifugation. The MNCs were plated on fibronectin-coated culture dishes, and were induced into EPCs by EGM-2 complete medium supplemented with cell growth factor. The adherent cells were collected 7 d later for all studies. EPCs were characterized as adherent cells double positive for DiI-ac-LDL uptaking and lectin binding by direct fluorescent staining under a laser scanning confocal microscope. The viability, migration, apoptosis and in vitro vasculogenic activity of the EPCs were determined by MTT assay, Transwell chamber assay, apoptosis kit and in vitro vasculogenesis kit, respectively. RESULTS: Compared with control group, the viability, migration and in vitro vasculogenic capacity of the EPCs in Hcy group were significantly decreased (P<0.01). Compared with Hcy group, yellow wine group and red wine group both significantly improved the viability, migration and in vitro vasculogenic capacity of Hcy-induced EPCs (P<0.01). Compared with control group, yellow wine group and red wine group both significantly improved the above-mentioned functions of EPCs (P<0.05). However, no significant difference of apoptosis in all groups was observed. CONCLUSION: Hcy may result in dysfuction of EPCs. Treatment with yellow wine improves Hcy-induced EPC functions.     

Construction of Eukaryotic Expression Vector of Sox2 Gene and Establishment of Sheep Bone Marrow Mesenchymal Stem Cell Lines Transfected with Sox2 Gene

Sox2 is one important marker of pluripotent stem cells, a study found that neural stem cells with high expression of Sox2 as donor cells showed higher reprogramming ability in nuclear transplantation.In this study, through enhancing exogenous Sox 2 gene expression of sheep bone marrow mesenchymal stem cells, in order to raise their reprogramming ability, and improve the efficiency of somatic cell cloning in animal.Total RNA was extracted from sheep testicular tissue, and with this template, Sox2 cDNA sequence was amplified and inserted into the eukaryotic expression vector pEGFP-N1 to build a recombinant vector pEGFP-N1-Sox2.The vector was transfected into the sheep bone marrow mesenchymal stem cells by liposome method, and through G418 and fluorescence screening to obtain and amplify monoclone.DNA sequencing showed that sheep Sox 2 gene CDS sequence was obtained, and recombinant plasmid was successfully constructed.Identification of fluorescence confirmed that stable sheep bone marrow mesenchymal stem cell lines transfected with Sox2 were established.This study obtained the sheep bone marrow mesenchymal stem cell lines with high expression of Sox2, and provided a new idea for raising reprogramming efficiency in the process of somatic cell cloning.     

Study on Differentiation of Sheep Umbilical Cord Mesenchymal Stem Cells into Muscle Cells Induced by Mouse MyoD Gene

In order to investigate the differentiation of sheep umbilical cord mesenchymal stem cells (UCMSCs) into muscle cells induced by mouse MyoD gene. This study based on the previous work constructed the eukaryotic expression vector of MyoD-pcDNA3.1 in mice, and the vector was transfected into sheep UCMSCs. The morphological changes of cells were observed by fluorescent microsco, the expression of MyoD, Desmin and MyoG genes were detected by immunofluorescence, the percentage of cells expressing the cell specific factor (MyoD, Desmin and MyoG) was analyzed by flow cytometry and Real-time quantitative PCR to detect the relative expression of mRNA relative to muscle cell specific factor. Compared with the control group (no transfection), the vector was transfected into sheep UCMSCs, it was found that the cells were transformed into a long, slender, muscular cell state, and the cell spiral gradually disappeared at 21th day. It was found that MyoD and Desmin showed positive expression by immunofluorescence assay at 8th day, the expression of MyoG was also found after 16 d of induction, and the expression of MyoD decreased, the amount of Desmin expression was no change;By flow cytometry, the percentages of the expression of MyoD, MyoG and Desmin were 93.5%, 97.4% and 99.5%,respectively;Real-time quantitative PCR results showed that the relative expression of MyoD, MyoG and Desmin were increased and compared with the control group (non transfected cells), the cells were increased by 2.046, 2.389 and 5.489 times, respectively. The results showed that mouce MyoD gene could induce the differentiation of sheep UCMSCs into muscle cells.