ABSTRACT
Objective:To observe the effect of rhGLP-1 (7-36) on Akt/GSK3 signaling pathway in hepatocytes.Methods:Human HL7702 cell line was cultured to the logarithmic growth stage and divided into experimental group and blank control group. The cultures were incubated with 100nM medium containing rhglp-1 (7-36) and without rhglp-1 (7-36) for 90min. The levels of Akt, Glycogen synthase Kinase 3 (GSK3) and Glycogen synthase (GS) in the two groups were detected by Western Blot.Results:Compared with blank control group, the protein expression of p-Akt (Thr308) in experimental group (1.81±0.28) was significantly increased ( P=0.01), but the protein expression of Akt and p-Akt (Ser473) was not significantly changed. The protein expression levels of p-GSK3α (Ser21) (1.27±0.09) and p-GSK3β (Ser9) (1.24±0.09) in the experimental group were significantly increased ( P=0.003, 0.002), while the protein expression levels of GSK3α and GSK3β were not significantly changed. The protein expression level of p-GS (Ser641) (0.70±0.16) was decreased in the experimental group ( P=0.03), but the protein expression level of GS did not change significantly. Conclusion:Glp-1 can inhibit GSK3/GS signaling pathway, activate GS activity and promote glycogen synthesis.
ABSTRACT
Objective: Sweet Tea (ST), derived from the leaves of Lithocarpus polystachyus, is a Chinese folk medicine with wide pharmacological activities. However, the promotive effects of ST water extract on hepatocytes proliferation and its underlying mechanism remains still unknown. In the present study, the beneficial effects of ST water extract on human hepatocytes and its possible mechanism were investigated. Methods: MTT assay was used to detect the safety range of ST; HL7702 cells were divided into four groups: control group, ST low- (50 μg/mL), medium- (200 μg/mL) and high-concentration (800 μg/mL) groups; BrdU ELISA and EDU staining were used to observe DNA content and cell proliferation; Moreover, flow cytometry was applied to analyze the distribution of cell cycle. Furthermore, the expression of cyclin D1, CDK4, HGF/c-Met, Akt, Erk1/2 were detected by Western blot. Results: It was found that ST water extract concentration-dependent promoted human hepatocytes HL7702 cell proliferation within 72 h through accumulating the cells in S phase and G2/M phase. Furthermore, ST water extract up-regulated expression of Cyclin D1 and CDK4 proteins. Moreover, ST water extract not only increased HGF expression and phosphorylation of c-Met level, but also activated the phosphorylation levels of AKT, ERK1/2. Interestingly, both of AKT inhibitor A6730 and ERK1/2 inhibitor U0126 reversed the promotive effects of ST water extract, which further confirmed that activation of AKT and ERK1/2 were involved. Conclusion: The findings reveal that ST water extract promoted HL7702 cells proliferation through the stimulation of cell cycle mediated by activating the AKT- and ERK1/2-related pathway.
ABSTRACT
OBJECTIVE：To stu dy in vitro lipid-lowering effect of ganoderic acid C 2（GAC2），and to investigate its potential mechanism on the basis of S 6K/SREBPs signaling pathway. METHODS ：Using human liver cells HL- 7702 as objects ，MTT assay was used to test relative cell viability after treated with low ，medium and high doses （5，10，20 μmol/L，hereinafter）of GAC 2. Using lovastatin as positive control ，ELISA method was used to detect the contents of TC and TG in cells after treated with low ， medium and high doses of GAC 2. Nile red staining was used to observe the accumulation of lipids in cells. After transfected SREBPs report gene plasmid ，using 25-HC as positive control ，relative viability of SREBPs luciferase in cells were determined by luciferase assay after treated with low ，medium and high doses of GAC 2. Using 25-HC as positive control ，real-time fluorescent quantitative PCR was used to measure the mRNA expression of SREBPs and their downstream genes in cells after treated with medium and high doses of GAC 2. Using SREBPs inhibitor （25-HC）and S 6K inhibitor （rapamycin）as control ，Western blotting assay was adopted to determine the expression of SREBP- 1 and SREBP- 2（in the case of n-SREBPs ），relative expression ratio of phosphorylated S 6K to S 6K（p-S6K/S6K ratio ）. AutoDock 4.0 and other softwares were used for molecular docking of S 6K and GAC2. RESULTS ：There was no significant effect of low ， 0.05）. Compared with blank control group ，the content of TC qq.com in lovastatin group and GAC 2 high-dose group as well as thecontent of TG in lovastatin group ， GAC2 medium- and 床应用。电话：0371-65962746。E-mail：whui3697@126.com high-dose groups were decreased significantly （P＜0.05 or P＜ 0.01）；the number of lipid droplets in the cells of all medication groups decreased. Compared with blank control group ，relative viability of SREBPs luciferase in 25-HC group ，GAC2 low-，medium- and high-dose groups were decreased significantly ；mRNA expression of HMGCS1，MVK，SCD，HMGCR gene in 25-HC group and GAC 2 medium-，high-dose groups ，mRNA expression of DHCR7 gene in 25-HC group ，mRNA expression of SREBP-2 gene in GAC- 2 high-dose group as well as mRNA expression of DHCR24 and MSMO2 gene in 25-HC group and GAC 2 high-dose group were all decreased significantly ；relative protein expression of n-SREBP- 1 in 25-HC group ，GAC2 low-，medium- and high-dose groups ，relative protein expression of n-SREBP- 2 in 25-HC group and GAC 2 high-dose group as well as p-S 6K/S6K ratio in rapamycin group and GAC 2 groups were decreased significantly （P＜0.05 or P＜0.01）. The molecular docking results showed that GAC 2 could bound to amino acid residues Arg 335，Arg330 and Ala332 of S 6K via hydrogen bond. CONCLUSIONS ：GAC2 can reduce the lipid level of HL- 7702 cells，which may be associated with inhibiting the expression of S 6K/SREBPs signaling pathway.
ABSTRACT
OBJECTIVE: To investigate the toxicity of N-succinyl-chitosan (NSCS) to bovine hemoglobin (BHb) and human liver cells (HL-7702). METHODS: BHb was used as a research object and the toxic effect of NSCS was investigated by UV-Vis absorption spectroscopy, fluorescence spectroscopy and synchrotron spectroscopy under the simulative human physiological condition. At the same time, human HL-7702 cells was used as a research object and methyl thiazolyl tetrazolium (MTT) assay was employed to examine the cytotoxicity of NSCS. RESULTS: The results of UV-Vis absorption spectroscopy and MTT showed that the toxicity of NSCS was weak, and substitution degree had little effect on it. The result of fluorescence spectroscopy demonstrated that the intrinsic fluorescence of BHb was quenched by NSCS and the quenching effect slightly increased with the increase of substitution degree. The quenching mechanism was mainly dynamic quenching, and the major driving forces were hydrophobic and electrostatic force. CONCLUSION: The result of synchronous fluorescence spectroscopy reveals that NSCS has almost no influence on the conformation of BHb. The toxicity of NSCS to BHb and HL-7702 is weak.
ABSTRACT
Aim To study the inhibitory effect of gast-rodin (GSTD) on oleic acid (OA)-induced fat accu-mulation in HL-7702 cells and explore possible cellular signaling pathways. Methods The MTT method was used to study the impact of GSTD on cell viability in HL-7702 cells. Cellular steatosis was induced by 1 mmol·L-1 of OA administration for 24 h, and differ-ent concentrations of GSTD were added at the same time. Oil red O ( ORO) staining was used to determine fat accumulation in cells, and intracellular triglyceride ( TG) contents were assayed. Western blot was used to determine the phosphorylation levels of AMPKα and ACC in cells after GSTD administration. Compound C was used to treat the cells in order to study its influ-ence on the efficacies of GSTD. Results GSTD had no obvious toxicity in HL-7702 cells when its concen-tration was≤3 386. 5 μmol · L-1 . After 24 h of OA administration, there were large amounts of lipid drop-lets accumulated in HL-7702 cells, and intracellular TG contents greatly increased as well. However, when 169. 3 or 338. 7 μmol · L-1 of GSTD was added to-gether with OA, fat accumulation in cells was greatly inhibited, and intracellular TG contents were reduced averagely by 35% and 43 . 6%, respectively ( P<0. 01 vs OA alone ) . After administration, GSTD could in-crease the levels of p-AMPKα and p-ACC in HL-7702 cells time and dose dependently. Compound C could completely abolish the stimulating activity of GSTD on AMPK pathway and block its reducing effect on hepatic TG accumulation. Conclusions GSTD greatly inhibits OA-induced fat accumulation and reduces intracellular TG contents in HL-7702 cells;the efficacy of GSTD is dependent on the activation of cellular AMPK pathway.