Nobiletin attenuates palmic acid-induced lipidosis in hepatocytes and regulatory mechanism of lncLSTR

AIM: To observe the effects of nobiletin on palmic acid (PA)-induced lipidosis in hepatocytes and to discuss the regulatory mechanism of lncLSTR. METHODS: AML12 cells were cultured in vitro. The control group, PA group (0.2 mmol/L) and protection group (exposure to nobiletin at 1 mg/L, 5 mg/L, 15 mg/L or 50 mg/L for 2 h, followed by treatment with 0.2 mmol/L PA) were established according to the experimental requirements. The lipid accumulation was morphologically observed by Oil red O staining in the cells. The qPCR was applied to detect mRNA expression, and the protein expression was determined by and Western blot. RESULTS: PA treatment (0.2 mmol/L) induced lipidosis, while 50 mg/L nobiletin pretreatment suppressed the lipidosis. Compared with control group, the mRNA expression of Apoc2 in PA group was significantly down-regulated (P<0.05), but increased by nobiletin pretreatment compared with PA group (P<0.05). The protein expression of Apoc2 in PA group was significantly down-regulated (P<0.05), but increased by nobiletin pretreatment (P<0.05). The expression of lncLSTR in PA group was significantly increased (P<0.05) and that was inhibited by nobiletin pretreatment (P<0.05). A negative correlation between Apoc2 protein and lncLSTR expression in PA group (R²=0.717 9, P<0.01) was observed. A negative correlation between Apoc2 protein and lncLSTR expression in protection group (R²=0.525 3, P<0.05) was also found. CONCLUSION: Nobiletin has anti-lipidosis effect on hepatocytes. The mechanism is partially related to the inhibition of up-regulation of lncLSTR, thus down-regulating Apoc2 expression.     

Role of TRIM8 in mouse cardiomyocyte apoptosis induced by high glucose and high free fatty acid

AIM: To investigate the effects of tripartite motif-containing protein 8 (TRIM8) on the apoptosis of mouse cardiomyocytes (MCMs) induced by high glucose and high free fatty acid (HGHF) and the underlying mechanism. METHODS: The MCMs were divided into normal glucose (NG) group (glucose at 5.5 mmol/L), high glucose (HG) group (glucose at 33 mmol/L), high free fatty acid (HF) group (sodium palmitate at 300 μmol/L) and HGHF group (glucose at 33 mmol/L and sodium palmitate at 300 μmol/L). The expression of TRIM8 in the MCMs was knocked down by siRNA, and the MCMs was further divided into control group, scrambled siRNA (Scra-siRNA)/PBS group, TRIM8-siRNA/PBS group, Scra-siRNA/HGHF group and TRIM8-siRNA/HGHF group. To further confirm the specific mechanism of TRIM8 in the MCM injury induced by HGHF, the MCMs were subgrouped into HGHF/DMSO group, HGHF+TRIM8-siRNA+DMSO (HGHF+Ts/DMSO) group, HGHF/ML385 group and HGHF+Ts/ML385 group. Accordingly, apoptosis was analyzed by flow cytometry, and the levels of reactive oxygen species (ROS) were measured by flow cytometry and DHE staining. The expression of TRIM8, nuclear factor E2-related factor 2 (Nrf2), glutamate-cysteine ligase catalytic subunit (GCLC), heme oxygenase-1 (HO-1) and NAD(P)H:quinone oxidoreductase 1 (NQO-1) at mRNA and protein levels was determined by qPCR and Western blot. RESULTS: HGHF increased the expression of TRIM8, and suppressed the expression of Nrf2, GCLC, HO-1 and NQO-1 in the MCMs (P < 0.05). Compared with Scra-siRNA/HGHF group, the intracellular ROS content and apoptotic rate were decreased in TRIM8-siRNA/HGHF group (P < 0.05). Correspondingly, the expression of the antioxidant molecule Nrf2 and its downstream genes GCLC, HO-1 and NQO-1 was increased (P < 0.05). In contrast, the addition of Nrf2 inhibitor ML385 partially reversed the inhibitory effect of TRIM8 expression knock-down on HGHF-induced apoptosis of MCMs. CONCLUSION: TRIM8 exacerbates the HGHF-induced cardiomyocyte apoptosis by modulating Nrf2 antioxidative pathway.     

Extracellular cysteine/cystine redox potential affects mitochondrial function in NAFLD LO2 cells

AIM: To investigate the effects of extracellular cysteine/cystine redox potential (E_hCys/CySS) on the mitochondrial function of nonalcoholic fatty liver disease (NAFLD) hepatocytes. METHODS: LO2 cells were incubated with E_hCys/CySS of the oxidized (0 mV), the normal (-80 mV), or the reduced (-150 mV) status medium, then treated with oleic acid to establish NAFLD model in vitro. DCFH-DA and MitoSOX were used as the fluorescent probes for determining reactive oxygen species (ROS). Apocynin (NADPH oxidase inhibitor), MitoQ₁₀ (mitochondria-targeted antioxidant), rotenone (mitochondrial respiratory chain complex I inhibitor) and antimycin A (mitochondrial respiratory chain complex III inhibitor) were used to investigate the sources of ROS. RESULTS: An increase in ROS in LO2 cells by oleic acid was aggravated by the oxidized extracellular E_hCys/CySS (0 mV), which was removed by the reduced E_hCys/CySS (-150 mV). ROS generation by 0 mV was significantly eliminated by MitoQ₁₀. ROS levels were dependent on extracellular E_hCys/CySS in rotenone treated LO2 cells. A decline of mitochondrial membrane potential in the cells with NAFLD was aggravated by 0 mV and reversed by -150 mV. CONCLUSION: The oxidized extracellular E_hCys/CySS via inhibitiing of complex I intensifies ROS generation and reducing the mitochondrial membrane potential in the NAFLD hepatocytes, which were reversed by reduced E_hCys/CySS.     

Role of calreticulin-induced mitochondrial damage in high glucose-induced apoptosis of myocardial cells

AIM: To observe the effect of high glucose on the protein expression of calreticulin (CRT) and its association with cell apoptosis and mitochondrial dysfunction in the cardiomyocytes. METHODS: AC-16 cardiomyocytes were randomly divided into normal glucose group, high glucose group, high glucose+ CRT siRNA group and isotonic control group. The cell apoptotic rate, reactive oxygen species (ROS), mitochondrial membrane potential level, respiratory enzyme activity, and protein expression of CRT were observed. RESULTS: Compared with the cardiomyocytes in normal glucose group, the apoptotic rate and ROS production of cardiomyocytes increased in high glucose group, accompanying with the decreases in the mitochondrial membrane potential level and enzyme activitiy of the respiratory chain. The protein expression of CRT was significantly increased in high glucose group. However, compared with high glucose group, high glucose+ CRT siRNA decreased the expression of CRT and attenuated the damage of mitochondria, but CRT siRNA did not reduce the ROS level in cardiomyocytes. CONCLUSION: High glucose brings about CRT over-expression to induce mitochondrial injury, thus increasing myocardial apoptosis.     

Effect of betaine on liver injuries in aging db/db mice induced by lipids

AIM: To evaluate the effect of betaine on lipid metabolism disorder in inherited db/db mice with long-term nonalcoholic fatty liver disease (NAFLD).METHODS: Experimental NAFLD models were established by feeding the db/db mice with high-fat diet. Fifty 7-month-old db/db mice were randomly divided into 5 groups: the mice in low, medium and high dose groups were given betaine by intragastric administration at doses of 200 mg/kg, 400 mg/kg and 800 mg/kg for 6 weeks, respectively,while the mice in saline control group and positive drug group were given normal saline and positive control drug,respctively. All the animals were killed, and serum alanine aminotransferase (ALT), triglyceride (TG), total cholesterol (TC), low-density lipoprotein (LDL) and glucose tolerance were detected. The pathological changes of the liver tissues were also observed.RESULTS: Betaine significantly decreased the levels of ALT, TC and LDL (P<0.05 or P<0.01). The pathological changes of the liver tissues indicated that the content of lipid in the hepatocytes of betaine treatment groups was less than that in saline control group.CONCLUSION: Betaine significantly improves the lipid metabolism and the liver function in the aging db/db mice, and reduces the accumulation of lipid in the hepatocytes.     

Schisandra total lignin attenuates apoptosis of endoplasmic reticulum pathway to delay mouse brain aging

AIM:To investigate the role of schisandra total lignin (SCL) in anti-aging of the mouse brain. METHODS:A D-galactose induced mouse aging model was established. The following groups in this study were set up: control group, model group, SCL low dose group ［SCL (L)］, SCL moderate dose group ［SCL (M)］ and SCL high dose group ［SCL (H)］. Learning and memory abilities were measured by mouse jumping experiments. The expression of ubiquitin (Ub), glucose-regulated protein 78 （GRP78）, protein disulfide isomerase (PDI), CCAAT /enhancer-binding protein homologous protein (CHOP), Bcl-2 and Bax proteins was detected by Western blotting. In addition, the protein expression of Bcl-2 and Bax in the aging cerebral cortex was also observed by immunohistochemistry. RESULTS:In learning test, compared with control group, the number of errors within 5 min increased in model group (P<0.05). Compared with model group, the number of errors within 5 min decreased in SCL (L) group, SCL(M) group and SCL(H) group (P<0.05). In memory test, compared with control group, incubation period of the first jumping off the platform was shorter and the number of errors within 5 min increased in model grou(P<0.05). Compared with model group, the incubation period of the first jumping off the platform was longer and the number of errors within 5 min decreased in SCL (L) group , SCL(M) group and SCL(H) group (P<0.05). Compared with control group, the protein expression of Ub, GRP78, PDI, CHOP and Bax was increased (P<0.05), while Bcl-2 protein level and Bcl-2/Bax ratio were decreased in model group(P<0.05). Compared with model group, the protein expression of Ub, GRP78, PDI, CHOP and Bax was decreased in SCL (L) group, SCL(M) group and SCL(H) group (P<0.05), while Bcl-2 protein level and Bcl-2/Bax ratio were increased (P<0.05). In control group, neuronal morphology was normal, the protein expression of Bcl-2 was positive and Bax was negative in the cytoplasm. In model group, the neurons were degeneration, the protein expression of Bcl-2 was negative and Bax was positive in the cytoplasm. In SCL (L) group, SCL (M) group and SCL (H) group, the number of degenerative neurons decreased, the protein expression of Bcl-2 was positive and Bax was negative in the cytoplasm. CONCLUSION: SCL inhibit D-galactose-induced brain aging in mice by attenuating apoptosis of endoplasmic reticulum pathway.     

Preliminary mechanism of Mcl-1 silencing in regulating apoptosis of mouse peritoneal macrophages infected with MTB

AIM: To investigate the mechanism of myeloid cell leukemia-1 (Mcl-1) silencing in regulating the apoptosis of mouse peritoneal macrophages infected with Mycobacterium tuberculosis (MTB) by observing the changes of Bcl-2 and Bax expression. METHODS: The suspensions of MTB strains with different virulence, BCG, H37Ra, H37Rv and XJ-MTB, were prepared to infect BALB/c mice. The transfection of Mcl-1-shRNA plasmid was used to establish a mouse model, and a corresponding control group at the same time was set up. The mice were executed and their peritoneal macrophages were collected 1 d, 3 d, 5 d and 7 d after the treatment. The apoptosis of the macrophages treated with diffe-rent virulence of MTB strains at different time points was analyzed by flow cytometry. The expression of Bcl-2 and Bax at mRNA and protein levels was determined by real-time PCR and Western blot. RESULTS: The apoptotic rate of mouse peritoneal macrophages increased to some extent after transfection with Mcl-1-shRNA plasmid compared with control group. The order of apoptotic rates was BCG > H37Ra > H37Rv≈XJ-MTB (P<0.05). The expression of Bcl-2 at mRNA and protein levels was significantly decreased, while the expression of Bax at mRNA and protein levels was significantly increased. The changes in BCG infection group were the most significant, and the negative correlation between the Bcl-2/Bax ratio at mRNA level and the virulence of the MTB strains was observed (P<0.05). CONCLUSION: Inhibition of Mcl-1 expression significantly promotes the apoptosis of peritoneal macrophages in mice infected with different virulence of MTB strains. The regulatory mechanism may be closely related to the protein expression of Bcl-2 and Bax and the virulence of MTB strains.     

Oxidative stress induces apoptosis in HepG2 cells

AIM: Direct exposure of cells to reactive oxygen species can induce apoptosis. In this study we investigate how oxidative stress induces cell death in HepG2 cells and characterize the molecular events involved.METHODS: Oxidative stress was created by exposing HepG2 cells to 2 mmol/L H2O2. Apoptosis was determined by analysis of DNA fragmentation by agarose gel electorphoresis. The mitochondrial membrane potential was analyzed using DePsipher fluorescent staining and the expression of cytochrome c in the cytosolic fraction was measured by Western blotting analysis. The caspase activity was detected using fluorometric assay kit by a fluorescence microplate reader.RESULTS: When HepG2 cells were treated with 2 mmol/L H2O2, the cells displayed DNA fragmentation, a typical feature of apoptosis, after 12 h. The mitochondrial membrane potential appeared different in two group of cells. H2O2-treated cells appeared green fluorescence as early as 4 h, which represents de-energized mitochondria, the untreated cells appeared red fluorescence, a feature of mitochondria with intact membrane potential. In treated cells, the expression of cytochrome c increased and accumulated in cytosolic fraction with treatment time, caspase-3 activity increased by 6.7-fold (P<0.01) at 8 h and caspase-9 activity increased by 3.6-fold (P<0.01) at 12 h, respectively, however, the activity of caspase-8 remained unchanged.CONCLUSION: These findings suggest that oxidative stress can induce apoptotic cell death in HepG2 cells, and the mechanism is related to mitochondrial pathway, which activates caspase-9 and-3, but not caspase-8.     

Effects of edaravone on high glucose-induced apoptosis in SH-SY5Y cells via regulating microRNA-25

AIM: To observe the effects of edaravone on high glucose-induced apoptosis of SH-SY5Y cells and its potential mechanism. METHODS: The SH-SY5Y cells were cultured in the DMEM medium with 100 mmol/L glucose and 100 μmol/L edaravone for 24 h. The viability of the SH-SY5Y cells was detected by MTT assay. The levels of ROS in the cells were determined by DCFH-DA fluorescent probing. The apoptotic rates of the cells were analyzed by flow cytometry. The protein expression of Bax and Bcl-2 in the cells were detected by Western blot. The expression levels of micro-RNA-25 (miR-25) were determined by real-time PCR. To further clarify the target sites of edaravone on inhibiting apoptosis induced by high glucose, miR-25 inhibitor was applied to the SH-SY5Y cells and the activity of caspase-3 was measured.RESULTS: Compared with control group, the cell viability was decreased significantly in model group, and the ROS level was increased significantly. The protein expression of Bax was up-regulated significantly, while the expression levels of Bcl-2 and miR-25 were significantly down-regulated. Compared with model group, the cell viability was increased significantly in edaravone group. The ROS level was decreased significantly. Meanwhile, the expression of Bax was down-regulated, while the expression of Bcl-2 and miR-25 was up-regulated with statistical significance. The caspase-3 activity of the cells incubated with 100 mmol/L glucose and miR-25 inhibitor was increased. However, no alteration of caspase-3 activity with edaravone added simultaneously was observed. CONCLUSION: Edaravone inhibits the apoptosis of SH-SY5Y cells induced by high glucose with the potential target site of miR-25.     

Preliminary mechanism of senegenin against H/R-induced apoptosis of primary cortical neurons

AIM：To explore the preliminary mechanism of senegenin (Sen) on inhibiting hypoxia/reoxygenation(H/R)-induced apoptosis of primary cortical neurons. METHODS：The cultured cortical neurons were randomly divided into normal group (control group), model group (H/R group), Sen+H/R group and Sen group. Flow cytometry was used to evaluate the effect of Sen on H/R-induced cell apoptosis. The protein levels of JNK, p-JNK, c-Jun, p-c-Jun, Bcl-2 and Bax were assessed by Western blotting. RESULTS：The apoptotic rate in H/R group was obviously higher than that in control group (P<0.05), while the apoptotic rate in Sen+H/R group was obviously lower than that in H/R group (P<0.05), suggesting that the model of apoptosis was established successfully. The results of Western blotting showed that Sen increased the expression of JNK and c-Jun, inhibited the phosphorylation of JNK and c-Jun (P<0.05), increased the protein level of Bcl-2 and inhibited the protein level of Bax in H/R treated primary cortical neurons (P<0.05). CONCLUSION：Sen has a protective effect against H/R-induced neuronal apoptosis by increasing the expression of JNK and c-Jun, inhibiting the phosphorylation of JNK and c-Jun, increasing the protein level of Bcl-2 and decreasing the protein level of Bax.     

MicroRNA expression in hepatocytes with hydrogen peroxide-induced oxidative stress-injury and alleviating effect of mesenchymal stem cell-conditioned medium

AIM: To evaluate the changes of microRNA (miRNA) in hepatocytes during hydrogen peroxide-induced oxidative stress injury, and to observe the alleviating effect of mesenchymal stem cell-conditioned medium (MSC-CM) in this progress. METHODS: The hepatocyte oxidative stress injury model was established using hydrogen peroxide and human normal liver cell line L02. MSC-CM was prepared using centrifugation and filter. The effects of MSC-CM on hepatocyte injury were evaluated by apoptosis analysis, cell viability detection, cell cycle, and mitochondrial membrane potential (MMP). Twenty-one differentially expressed miRNAs were selected by gene chip hybridization, in which miR-143, miR-145, miR-301a and let-7a were confirmed by RT-qPCR. Bioinformatics software was utilized to predict target proteins of these miRNAs, and then the proteins were verified by Western blot.RESULTS: MSC-CM markedly attenuated hydrogen peroxide-induced oxidative stress injury by reducing apoptosis, promoting cell viability and regulating cell cycle. The expression of miR-143, miR-145, miR-301a and let-7a, indentified by RT-qPCR, increased under the condition of oxidative stress injury, while decreased after MSC-CM treatment. The expression of miR-143 predicted target proteins, HK2 and ADRB1, decreased under the hydrogen peroxide-exposure, while increased after MSC-CM treatment, which is consistent with the regulatory trend of miR-143. CONCLUSION: MSC-CM might attenuate hydrogen peroxide induced oxidative stress injury via inhibiting apoptosis and regulating some miRNA expression.     

Effects of HDL from patients with coronary artery disease on lipid deposition and apoptosis in mouse peritoneal macrophages

AIM: To compare the effects of high-density lipoprotein (HDL) from healthy subjects (HDL_heathy) and HDL from the patients with coronary artery disease (HDL_CAD) on the lipid deposition and apoptosis in mouse peritoneal macrophages. METHODS: HDL was isolated from healthy subjects, stable CAD patients (HDL_SCAD) and acute myocar-dial infarction patients (HDL_AMI). The accumulation of intracellular lipids was determined by oil red O staining. The apoptosis of macrophages was measured by fluorescence microscopy with annexin-V/PI staining. DCHF-DA, a redox-sensitive dye, was used to detect intracellular reactive oxygen species (ROS) levels. The protein expression of ATP-binding cassette transporter (ABC) A1, ABCG1, Bcl-2 and Bax was determined by Western blot analysis. RESULTS: Lipid deposition in the macrophages was increased significantly after oxidized low-density lipoprotein (ox-LDL) treatment, and the expression of ABCA1 and ABCG1 was up-regulated (P<0.05). Compared with ox-LDL treatment alone, HDL_healthy decreased lipid deposition in the macrophages and up-regulated the expression of ABCA1 and ABCG1 (P<0.05), while treatment with HDL_SCAD or HDL_AMI further decreased lipid deposition in the macrophages and down-regulated the expression of ABCA1 and ABCG1 (P<0.05). Compared with HDL_SCAD treatment, lipid deposition in the macrophages was further increased after HDL_AMI treatment, and the expression of ABCA1 and ABCG1 was down-regulated (P<0.05). HDL_healthy decreased the levels of intracellular ROS and apoptosis by increasing the level of antiapoptotic protein Bcl-2 and reducing the expression of proapoptotic protein Bax. In contrast, HDL_SCAD and HDL_AMI had opposite effects on the intracellular ROS, the cell apoptosis and the expression of apoptosis-related proteins Bcl-2 and Bax. CONCLUSION: HDL_CAD promotes lipid accumulation in macrophages and induces macrophage apoptosis. These findings provide novel insights into mechanisms leading to altered vascular effects of HDL in CAD.     

Effect of berberine on oxidative damage and the SIRT1/p53 pathway in liver tissues of NAFLD rats

AIM:To investigate the effect of berberine on oxidative damage and silent mating type information regulation 2 homolog 1 (SIRT1)/p53 pathway in the liver tissues of nonalcoholic fatty liver disease (NAFLD) rats and to explore the mechanism of berberine against NAFLD. METHODS:The male SD rats (n=24) were randomly divided into normal group, model group and berberine group (8 rats in each group). The rats in normal group was fed with normal diet, while the rats in model group and berberine group were fed with high-fat diet. The rats in berberine group was intragastrically administered with daily doses (100 mg/kg) of berberine for 16 weeks. The levels of liver total cholesterol (TC), triglyceride (TG), superoxide dismutase (SOD), malondialdehyde (MDA) and total anti-oxidant capatity (T-AOC) were measured. HE staining, oil red O straining and transmission electron microscopy were used to observe the histological changes of the livers. The protein levels of SIRT1, p53 and acetylated p53 (Ac-p53) were determined by Western blot. RESULTS:Compared with model group, the levels of liver TC, TG and MDA in berberine group were significantly reduced (P<0.05 or P<0.01), and the levels of SOD and T-AOC were significantly increased (P<0.01). The results of pathological observation showed that the lipid accumulation in the liver of berberine group was significantly attenuated. Compared with model group, the expression of SIRT1 was significantly increased and the expression of Ac-p53 was obviously reduced in berberine group (P<0.01). CONCLUSION:Berberine reduces hepatic steatosis and oxidative damage in NAFLD rats induce by high-fat diet, and this effect may be associated with regulation of the SIRT1/p53 signal pathway.     

Etoposide induces apoptosis via mitochondrial signaling pathway with cytochrome c release in Jurkat leukemia cells

AIM:To investigate the molecular mechanisms of apoptosis and to elucidate the apoptosis signaling pathway triggered by etoposide in Jurkat human leukemia cells. METHODS:Apoptosis was detected using annexin V-FITC and propidium iodide (PI) staining, respectively, and annexin V-FITC positive cells and hypodiploid cells were analyzed by flow cytometry. Mitochondrial membrane potential (△Ψm) was detected using 3, 3-dihexyloxycarbocyanine iodide ［DiOC6(3)］ staining and △Ψm low cells were analyzed by flow cytometry. Preparation of cytosolic extracts and isolation of mitochondria were completed by centrifugation. Western blotting analysis was used to evaluate the level of cytochrome c, caspase-3, and poly (ADP-ribose) polymerase (PARP) expression. RESULTS:Etoposide induced apoptosis showing phosphatidylserine externalization and DNA fragmentation in a time-dependent manner and the apoptosis could be inhibited by a broad caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD.fmk). Collapse of △Ψm induced by etoposide preceded DNA fragmentation and phosphatidylserine externalization. In contrast, it was not blocked by zVAD.fmk. Etoposide caused cytochrome c release from mitochondria into cytosol, subsequent activation of caspase-3 (32 kD) presented with an intermediate (20 kD) and its active product (17 kD), and cleavage of full-length PARP (116 kD) into the so-called apoptotic 85 kD fragment. CONCLUSION:Etoposide-induced Jurkat cell apoptosis is initiated through mitochondria signaling pathway with cytochrome c release into cytoplasm and caspase is the ultimate executioner of cell apoptosis.     

Effect of rapamycin on apoptosis of mouse astrocytes in vitro

AIM: To observe the effect of rapamycin on the apoptosis of mouse astrocytes in vitro.ME-THODS:The astrocytes from C57BL/6J newborn mouse pups were isolated and primarily cultured. The effect of rapamycin on the viability of astrocytes was assessed by MTT assay. The mean fluorescence intensity of SYTOX^® Green stain in the astrocytes was detected by fluorescence microplate reader in order to analyze the effects of rapamycin on the cell death induced by H₂O₂, ionomycin and/or deferorxamin. DiOC₆(3) staining was used to analyze the mitochondrial membrane potential of the astrocytes induced by H₂O₂. Flow cytometry analysis was used to determine the production of ROS in the astrocytes and mitochondria by staining with H₂DCFDA and MitoSOX^TM Red reagent, respectively.RESULTS: Rapamycin at concentration of 0.5 μmol/L protected the astrocytes against cell death induced by H₂O₂ or deferoxamine plus ionomycin. Rapamycin protected the mitochondrial membrane potential of astrocytes from the injury of H₂O₂. It also reduced the production of ROS in the astrocytes and decreased the level of ROS in the mitochondria.CONCLUSION: Rapamycin reduces the ROS overload in the mitochondria, keeps mitochondrial membrane potential safety and protects the astrocytes against apoptosis in vitro.     

Effects of sinapic acid on proliferation and apoptosis of rat vascular smooth muscle cells induced by high glucose

AIM: To investigate the effects of sinapic acid(SA) on the proliferation and apoptosis of rat vascular smooth muscle cells(VSMCs) induced by high glucose(HG). METHODS: Cultured A7r5 cells were randomly divided and treated as indicated. The cell viability was determined by MTT assay. DNA synthesis was measured by BrdU assay. Cell cycle progression and cell apoptotic rate were determined by flow cytometry analysis. The levels of reactive oxygen species(ROS) were detected by ELISA. The protein levels of cyclin D1, P21, P27, phosphorylated protein kinase C(p-PKC), p-P38 and β-actin were evaluated by Western blot. RESULTS: Compared with control group, the viability of A7r5 cells was significantly enhanced, the DNA synthesis was increased, the cell cycle progression was promoted, the levels of ROS were elevated, the cell apoptotic rate was reduced, the protein expression of P21 and P27 was decreased, and the protein levels of cyclin D1, p-PKC and p-P38 were increased in HG group(all P<0.05). These effects were reversed by SA(0.1, 1 and 10 μmol/L) treatment in a dose-dependent manner(all P<0.05). Both P38 inhibitor SB203580 and PKC inhibitor chelerythrine significantly inhibit HG-induced PKC/P38 activation and cell viability(P<0.05).CONCLUSION: SA inhibits HG-induced VSMCs proliferation and promotes cell apoptosis via reducing PKC/P38 activation.     

Calcium overload and reactive oxygen species mediate high glucose-induced apoptosis of mouse osteoblast MC3T3-E1 cells

AIM: To investigate the role of reactive oxygen species (ROS) and calcium overload in the apoptosis of MC3T3-E1 cells induced by high glucose. METHODS: Cultured mouse skull bone-derived osteoblast cell line MC3T3-E1 was treated with high concentration of D-glucose to induce apoptosis. The proliferation of MC3T3-E1 cells was detected by MTT assay after treated with different concentrations of D-glucose for 24 h and 48 h. The apoptotic rate and the intracellular levels of calcium and ROS were also measured after the cells were treated with high glucose (35 mmol/L) for 24 h. RESULTS: After high glucose treatment, the cell proliferation was inhibited. The early apoptosis and total cell death increased to (24.16?3.53)% and (63.74?4.32)%,respectively. High glucose treatment significantly increased intracellular levels of ROS and Ca²⁺. The increased apoptotic rate was reduced by addition of antioxidant N-acetylcysteine and calcium chelator BAPTA-AM. Inhibition of store-operated Ca²⁺ channels by La³⁺ also decreased the intracellular level of Ca²⁺ and cell apoptosis induced by high glucose. CONCLUSION: High glucose increases intracellular ROS level and the release of Ca²⁺ through the store-operated Ca²⁺ channels, thus resulting in intracellular Ca²⁺ overload and leading to apoptosis of osteoblasts.     

Effects of Anhua dark tea on ApoE-/- mice with non-alcoholic fatty liver induced by high-fat diet

AIM To explore the effects of Anhua dark tea on the prevention and treatment of non-alcoholic fatty liver induced by high-fat diet in apolipoprotein E knockout (ApoE^-/-) mice and the relevant mechanisms. METHODS Male ApoE^-/- mice (n=50, 8 weeks old) were randomly divided into model group, atorvastatin group, and high-, medium- and low-dose Anhua dark tea groups, with 10 mice in each group. In addition, 10 homologous wild-type male C57BL/6J mice were selected as normal control group. The ApoE^-/- and wild-type mice were fed with the same amount of high-fat feed and common feed for 17 weeks, respectively, and intervened by the corresponding drugs and normal saline. At the end of the experiment, HE staining was used to observe the histopathological changes of the liver. The levels of alanine aminotransfease (ALT), aspartate aminotransfease (AST), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and malondialdehyde (MDA) in the liver tissues were detected. The mRNA expression levels of hydroxymethyl glutaryl coenzyme A reductase (HMGCR), peroxisome proliferator-activated receptor-γ (PPAR-γ) and steroyl coenzyme A desaturase-1 （SCD-1） were detected by RT-qPCR to observe the lipid synthesis. RESULTS In model group, increased volume, smooth surface, tight membrane and dull edge of the liver were observed, and microscopic images with HE staining showed the formation of vacuoles with varying sizes, indicating the success of establishing the model. Compared with the model group, the degree of liver steatosis, the levels of ALT, AST and MDA, and the mRNA expression of HMGCR, SCD-1 and PPAR-γ in different doses of Anhua dark tea groups were significantly decreased, while the activity of SOD and GSH-Px was increased (P<0.05). The effect of Anhua dark tea in high- and medium-dose groups was better than that in low-dose group (P<0.05). CONCLUSION Anhua dark tea prevents high-fat diet-induced non-alcoholic fatty liver in ApoE^-/- mice. The mechanism may be related to reducing lipid synthesis by inhibition of HMGCR, SCD-1 and PPAR-γ expression, and protecting liver cells through anti-oxidative activity.     

GLP-1 down-regulates mRNA expression of SOCS-3 and SREBP-1c in rats with nonalcoholic fatty liver disease

AIM: To investigate the effects of glucagon-like peptide-1(GLP-1) on mRNA expression of SOCS-3 and SREBP-1c in the rats with nonalcoholic fatty liver disease. METHODS: Male SD rats were randomly divided into normal control(NC) group, high fat(HF) group and HF+liraglutide(Lira) group. The rats in HF group and HF+Lira group were given high-fat diet for 16 weeks. After 12 weeks of high-fat diet feeding in HF+Lira group, Lira(600 μg·kg^-1·d^-1) was intraperitoneally injected for 4 weeks. At the end of the 16th week, the rats were killed. The pathological changes of the liver were observed under optical microscope. The serum levels of alanine aminotransferase(ALT), aspartate aminotransferase(AST), triglyceride(TG) and total cholesterol(TC) were detected by automatic biochemical analyzer. TG contents of liver were measured by GPO-PAP method. The fasting insulin(FINS) was determined by ELISA, and insulin resistance index was assessed by homeostasis mode assessment(HOMA-IR). The mRNA expression of SOCS-3 and SREBP-1c in the liver tissues was detected by RT-qPCR. RESULTS: Compared with NC group, HOMA-IR, TG of liver, and the serum levels of ALT, AST, TG, TC and FINS in HF group were obviously increased(P<0.01). Compared with HF group, HOMA-IR, TG of liver, and the serum levels of ALT, AST, TG, TC and FINS in HF+Lira group were all obviously decreased(P<0.05 or P<0.01). The mRNA expression of SOCS-3 and SREBP-1c in HF group was significantly higher than that in NC group(P<0.01). The mRNA expression of SOCSV3 and SREBP-1c in HF+Lira group was significantly decreased as compared with HF group(P<0.05). CONCLUSION: Liraglutide may improve the IR and reduce TG of liver through decreasing the mRNA expression of SOCS-3 and SREBP-1c, so as to play a therapeutic role in nonalcoholic fatty liver disease.