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AIM: To evaluate the effect of GYY4137, a novel hydrogen sulfide (H2S) donor, on cytosolic lipid decomposition in mouse primary steatosis hepatocytes.METHODS: Oleic acid (OA) was used to induce hepatic steatosis model in vitro.The C57BL/6 mouse primary hepatocytes isolated and cultured by 2-step in situ perfusion were divided into 4 groups: the cells in control group were incubated with normal medium for 54 h;the cells in model group were incubated with OA at 1.2 mmol/L for 48 h followed by serum-free phenol red-free RPMI-1640 for 6 h;the cells in H2S group or DL-propar-gylglycine (PAG;an inhibitor of cystathione γ-lysase, inhibiting H2S synthesis) group were incubated with OA at 1.2 mmol/L for 48 h followed by serum-free phenol red-free RPMI-1640 which contained 1 mmol/L GYY4137 or 200 μmol/L PAG for 6 h.The glycerin release and the protein expression of hormone-sensitive lipase (HSL) in the cells were mea-sured.RESULTS: Compared with model group, the glycerin release and the protein expression of phosphorylated HSL (p-HSL) in H2S group decreased significantly, while those increased significantly in PAG group.CONCLUSION: In steatosis hepatocytes, exogenous H2S possibly decreases cytosolic lipid decomposition by decreasing the protein level of p-HSL.
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Nitrites play multiple characteristic functions in invasion and metastasis of hepatic cancer cells, but the exact mechanism is not yet known. Cancer cells can maintain the malignant characteristics via clearance of excess mitochondria by mitophagy. The purpose of this article was to determine the roles of nitrite, reactive oxygen species (ROS) and hypoxia inducing factor 1 alpha (HIF-1 α) in mitophagy of hepatic cancer cells. After exposure of human hepatocellular carcinoma SMMC-7721 cells to a serial concentrations of sodium nitrite for 24 h under normal oxygen, the maximal cell vitality was increased by 16 mg x (-1) sodium nitrite. In addition, the potentials of migration and invasion for SMMC-7721 cells were increased significantly at the same time. Furthermore, sodium nitrite exposure displayed an increase of stress fibers, lamellipodum and perinuclear mitochondrial distribution by cell staining with Actin-Tracker Green and Mito-Tracker Red, which was reversed by N-acetylcysteine (NAC, a reactive oxygen scavenger). DCFH-DA staining with fluorescent microscopy showed that the intracellular level of ROS concentration was increased by the sodium nitrite treatment. LC3 immunostaining and Western blot results showed that sodium nitrite enhanced cell autophagy flux. Under the transmission electron microscopy (TEM), more autolysosomes formed after sodium nitrite treatment and NAC could prevent autophagosome degradation. RT-PCR results indicated that the expression levels of COX I and COXIV mRNA were decreased significantly after sodium nitrite treatment. Meanwhile, laser scanning confocal microscopy showed that sodium nitrite significantly reduced mitochondrial mass detected by Mito-Tracker Green staining. The expression levels of HIF-1α, Beclin-1 and Bnip3 (mitophagy marker molecular) increased remarkably after sodium nitrite treatment, which were reversed by NAC. Our results demonstrated that sodium nitrite (16 mg x L(-1)) increased the potentials of invasion and migration of hepatic cancer SMMC-7721 cells through induction of ROS and HIF-1α mediated mitophagy.
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Aim To investigate the effect of hydrogen sulfide on hepatic lipid accumulation in obese mice. Methods C57 BL/6 J mice were randomly divided into control group, model group, and NaHS group. The mice of the control group were fed with normal diet. The mice of the model group and the NaHS group were fed with high-fat diet. From the thirteenth week, the mice of NaHS group were injected intraperitoneally with NaHS (H2S donor) in a dose of 50 μmol·kg-1 per day for 4 weeks and the mice of the model group were injected with the same volume of saline. All mice were sacrificed at the end of the 16th week. The tis-sues of liver were homogenized and centrifugated. The supernatants were used for the determination of triglyc-eride and cholesterol in liver. The morphology of liver was tested by H&E staining. Liver lipid accumulation was determined by oil red staining. Total RNA was ex-tracted from frozen tissue of liver. PCR was used to de-tect CPT-1 , FAS gene expression and ELISA method was used to detect CPT-1,FAS activity in mice liver. Results The body weight of the mice from NaHS group and model group was bigger than that of the mice from control group. Compared with the model group, the body weight of the mice from NaHS group was less;the content of triglyceride and cholesterol in liver was lower; the degree of liver tissue pathological changes and lipid accumulation were alleviated; CPT-1 expres-sion and activity were increased; FAS expression and activity were decreased. Conclusions These data in-dicate that hydrogen sulfide can reduce the lipid con-tent of liver tissue in obese mice and alleviate fatty liv-er. The mechanism may be associated with the in-creased expression of CPT-1 and the decreased expres-sion of FAS in liver.
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Recent data have revealed that inhibiting autophagy exacerbates lipid accumulation in hepatocytes and nitrite treatment reduces total triglyceride levels in the high-fat diet mice. Therefore, the present study aimed to determine the effects of nitrite on simple hepatic steatosis and the possible role of autophagy. Firstly, steatotic L-02 cells were induced by incubating L-02 cells with 1.2 mmol · L(-1) oleic acid (OA) for 24 h. Secondly, steatotic L-02 cells were treated with 0.2 mmol · L(-1) sodium nitrite (SN) plus 3-methyladenine (3-MA), or chloroquine (CQ) for 24 h, and then lipid accumulation was measured with oil red O staining and triglyceride quantification. The notable steatosis could be observed in L-02 cells following exposure to 1.2 mmol · L(-1) OA for 24 h. Treatment with 0.2 mmol · L(-1) sodium nitrite reduced lipid accumulation in steatotic L-02 cells. 3-MA weakened the ability of sodium nitrite to ameliorate hepatic steatosis. Additionally, the sodium nitrite increased number of LC3-II immunostaining puncta and LC3-II protein expression was confirmed by immunofluorescence or Western blot analysis, and the effects were enhanced by CQ treatment. The number of increased cytoplasm vacuoles and lysosomes increased was confirmed by phase contrast and fluorescence microscope respectively. The increased autolysosome was detected by electron microscopy, this phenomenon could be reversed by CQ treatment. These data demonstrated that sodium nitrite enhanced the autophagic flux and decomposition of triglycerides in steatotic L-02 cells.
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ObjectiveTo a nalyze the morphologic features of SMMC-7721 cannibalistic cells that induced by triazole Schiff base derivative(LH-37) in vitro. Methods The SMMC-7721 cells (1×10~4/ml)were cultured in the medium containing of 1×10~(-5) mol/L LH-37 for 24h,48h.The character of cells was detected by Papanicolaou and Wright′s Staining. Immunohistochemical method was used to observe the cleaved Caspase-3 positive cells. The ultrastructure of cannibalism cells was observed by JEM 100CX-II transmission electronic microscope. Results Microscopic analysis demonstrated the complete internalization of one cell within another. We noted that some cannibalistic cells in small aggregates appeared to be inside of large vacuoles, suggesting that they were internalized within a neighboring cell. The proportion of cannibalistic cells were increased after SMMC-7721 cells were cultured in the presence of LH-37 for 48 hours. The proportion of the cannibalistic cells in control and LH-37 group was 0.47% and 5.23% respectively . Many internalized cells were positive for cleaved caspase-3 staining . Ultrastructural analysis of engulfed cells from 24 hours exhibited evidence of live-cell internalization consistent with cannibalism, The most common fate for internalized cells was death after treatment with LH-37 for 48 hours, as evidenced by nuclear degradation and the eventual disappearance of some cells within the enveloping cell . Conclusion The data presented indicate that LH-37 can lead to an increase of cannibalism in human hepatocarcinoma cell in vitro.
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AIM: To study the cytoprotective role of NaNO_2 preconditioning against H_2O_2 induced damage in PC12 cells. METHODS: PC12 cells were treated with different concentrations of NaNO_2 for 6 h, 12 h, 24 h and 48 h, respectively. The viability of the cells was measured by MTT method and cell counting. The apoptotic rate of PC12 was determined by Hoechst 33258 staining to calculate the ratio of the cells between concentrated and broken nucleus in the total cell count. PC12 cells were pretreated with NaNO_2 at concentration of 3 mmol/L for 24 h. The cytoprotective role to the toxicity of H_2O_2 at concentration of 1.1 mmol/L for 6 h was observed by MTT. The cell apoptosis was measured by flow cytometry and staining with Hoechst 33258 and PI. The activities of catalase (CAT), superoxide dismutase (SOD), and the changes of glutathione peroxidase (GSH-Px) and malondialdehyde (MDA) were measured by the method of colorimetry. RESULTS: The dose-response results showed that the effect of NaNO_2 on PC12 cell proliferation was a typical β-shape curve. The maximal stimulatory response was at 24 h, and the concentration of the maximum stimulatory response was 1.4 mmol/L. The maximal stimulation of the concentration-responses was 156% above the control. No observable adverse effect level (NOAEL) was 6 mmol/L. IC_(50) was 45 mmol/L. When the cells were pretreated by NaNO_2 at concentration of 3 mmol/L for 24 h, and then exposed to H_2O_2 at concentration of 1.1 mmol/L for 6 h, the proliferation rate was increased as compared to the cells treated with H_2O_2 alone. Under the conditions of treating the cells with NaNO_2 at concentration of 3 mmol/L to induce the adaptive response, then exposing the cells to H_2O_2 at concentration of 1.1 mmol/L, the apoptosis rate in non-preconditioning group was 44.9%, the apoptosis rate in preconditioning group was 19.1%, the difference was significant (P<0.05). The cytoprotective effect of NaNO_2 was inhibited by nitric oxide (NO) scavenger ferrohemoglobin. The activities of SOD, CAT and the level of GSH-Px were markedly increased, the content of MDA decreased in preconditioning group. CONCLUSION: Exposure of NaNO_2 at concentration of <6 mmol/L induces hormesis on PC12 cells. Low dose of nitrite plays an important role in cytoprotection by reducing nitrite to NO, indicating that decrease in lipid peroxidation and increase in endogenous antioxidants may play a key role in cytoprotection induced by preconditioning with low dose of NaNO_2 in PC12 cells.
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Objective To study the effects of matrine on inducing autophagy and autophagic death in BEL-7402 cells. Methods BEL-7402 cells were cultured in RPMI1640 alone or exposed to different concentration of matrine. Cell growth inhibition was assessed by MTT assay. Apoptosis was evaluated by flow cytometry. Autophagosome marker LC3 was examined by immunofluorescence microscopy. The ultrastructure change of cells was analyzed by transmission electron microscope. Results The cell proliferation was inhibited by matrine in a dose dependent manner. Its IC50 value was 1.1 mg/mL at 48 h. Treatment with 0.6-1.6 mg/mL matrine for 12 h induced apoptosis of BEL-7402 cells and the apoptosis rate reached (44.88?0.78)% at concentration of 1.2 mg/mL. The autophagy positive cells were greatly increased after 1.2 mg/mL matrine treated in BEL-7402 cells and the number of cells reached (63.16?0.29)%. Morphological features of typical autophagic death were apparent in the cytoplasm at the ultrastructural level. Conclusion Matrine could induce autophagy and autophagic death of BEL-7402 cells in vitro.
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Aim To study the effect of 3-pyridin-3-yl-4-[(4-hydroxy-3-methoxy-benzylidene)amino]-5-me-thylsulfanyl-4H-[1,2,4] triazole (LH-37) on induction of differentiation and apoptosis of hepatocarcinoma BEL-7402 cells. Methods BEL-7402 cells were cultured in RPMI 1640 and treated by LH-37 at different doses. The proliferation of the cells and the inhibition effect of LH-37 on the cell proliferation were examined by MTT assay. The reverse transcriptase-polymerase chain reaction (RT-PCR) was used to detect the changes of alpha fetoprotein(?-FP)mRNA and albumin(Alb). The concentration of ?-FP in the cells was detected by ELISA assay. Cell morphology was observed by fluorescence microscope techniques. The protein expressions of active caspase-3 in BEL-7402 cells were observed by immunohistochemical staining. Western blot was used to assay caspase-3 and caspase-9. Colorimetric method was used to assay activity of superoxide dismutase (SOD) and catalase (CAT). The apoptotic cells were assayed by flow cytometry (FCM) with annexin V-FITC conjugated and propidium iodide (PI) staining. Results The cell proliferation was inhibited by LH-37 at 10 ?mol?L-1~1 mmol?L-1 in dose dependent manners. After treated with 10 ?mol?L-1 or 1.0 ?mol?L-1 LH-37, the mRNA and protein expression of ?-FP were significantly reduced but mRNA expression of Alb was significantly increased. Treatment of BEL-7402 cells with different LH-37 concentrations for 48 hours increased the percentage of active caspase-3 positive cells and protein expression of caspase-3 and caspase-9. More apoptosis features of cells were observed in LH-37 ( 100 ?mol?L-1) treatment groups than in control group. LH-37 markedly promoted the viability of SOD and decreased CAT in BEL-7402 cells. Counclusion LH-37 might inhibit proliferation and induce differentiation and apoptosis of human hepatocarcinoma BEL-7402 cells, which might be related to the cytotoxicity of the intracellular hydrogen peroxide (H2O2).