ABSTRACT
Objective:To study the protective effect and mechanism of Ranae Oviductus protein hydrolysate （ROPH） on the expression of pathway-related proteins in ethanol-induced L-02 cell injury. Method:The ROPH was prepared by compound enzymatic hydrolysis. L-02 cell injury model was induced with 400 mmol·L<sup>-1 </sup>ethanol. Cell viability was detected by cell counting kit-8 （CCK-8） assay. Cell cycle and apoptosis were examined by flow cytometry. JC-1/Hochest staining was employed for qualitative investigation. The expression of related proteins in apoptosis， mitogen-activated protein kinase （MAPK） signaling pathway， and pyroptosis in L-02 cells was detected by Western blot. Result:The results of the CCK-8 assay showed that 400 mmol·L<sup>-1 </sup>ethanol could induce L-02 cell injury within 12 hours. Compared with the blank group， the model group showed decreased viability of L-02 cells （<italic>P</italic><0.01）， elevated percentage of the cell cycle in the G<sub>0</sub>/G<sub>1</sub> phase （<italic>P</italic><0.01）， increased total cell apoptosis rate （<italic>P</italic><0.01）， reduced mitochondrial membrane potential （<italic>P</italic><0.01）， up-regulated expression of apoptosis-related proteins ［B-cell lymphoma-2 （Bcl-2）-associated X protein （Bax）， Cytochrome C （Cyt C）， and cysteine-dependent aspartate specific protease-3 （Caspase-3）］ （<italic>P</italic><0.05， <italic>P</italic><0.01） and MAPK signaling pathway-related proteins ［C-Jun amino-terminal kinase （JNK） and p38 MAPK］ （<italic>P</italic><0.05， <italic>P</italic><0.01）， and potentiated expression of pyrolysis-related proteins Caspase-1 and interleukin-1<italic>β </italic>（IL-1<italic>β</italic>） （<italic>P</italic><0.05）. Compared with the model group， the ROPH treatment group exhibited improved cell cycle arrest （<italic>P</italic><0.05， <italic>P</italic><0.01）， diminished total cell apoptosis rate （<italic>P</italic><0.01）， elevated mitochondrial membrane potential in a dose-dependent manner， down-regulated expression of Bax， Cyt C， and Caspase-3 proteins （<italic>P</italic><0.05， <italic>P</italic><0.01）， up-regulated expression of Bcl-2 protein （<italic>P</italic><0.05， <italic>P</italic><0.01）， and a downward trend in expression of proteins related to MAPK signaling pathway and pyrolysis （<italic>P</italic><0.05， <italic>P</italic><0.01）. Conclusion:ROPH could inhibit oxidative stress-triggered liver injury in ethanol-induced cells by improving mitochondrial membrane potential， reducing the expression of proteins in the mitochondria-mediated apoptosis pathway， and inhibiting the expression of proteins related to the MAPK signaling pathway and pyrolysis pathway to reduce the mitochondrial dysfunction and inflammatory response in ethanol-induced L-02 liver cells and inhibit oxidative stress， thereby exerting a therapeutic role in alcoholic liver injury.
ABSTRACT
Objective::To investigate the protective effect of salvianolic acid B on HepaRG hepatocyte injury induced by arsenic trioxide (As2O3 ) and its mechanism. Method::HepaRG cells were incubated with 5μmol·L-1 As2O3 for 24 h to induce hepatocyte injury. The cells were divided into control group, model group, salvianolic acid B 10 μmol·L-1 group, salvianolic acid B 10 μmol·L-1+ As2O3 group, salvianolic acid B 5 μmol·L-1+ As2O3 group, and salvianolic acid B 2.5 μmol·L-1+ As2O3 group. HepaRG cells were preincubated with salvianolic acid B for 2 h and then incubated with As2O3 for 24 h. At the end of the incubation, cell viability was detected by thiazolyl blue tetrazolium bromide assay, apoptosis was observed by Hoechst33342 fluorescence staining, apoptosis rate was detected by annexin V-FITC/propidium iodide double staining flow cytometry, and mitochondrial membrane was observed by JC-1 fluorescence staining. Western blot was used to detect the protective effect of expressions of relevant proteins Bcl-2, Bax, Akt, p-Akt on salvianolic acid B in the liver. Result::As2O3 concentration-dependently reduced the survival rate of HepaRG cells(P<0.01), salvianolic acid B had no effect on normal cell viability for 2 h, pre-incubation with salvianolic acid B(5, 10 μmol·L-1) for 2 h significantly increased the decreased cell survival rate caused by As2O3 (P<0.01). As2O3 significantly increased hepatocytes apoptosis rate(P<0.01), while pre-incubation with salvianolic acid B(10 μmol·L-1) deceased apoptosis rate(P<0.01). Incubation with As2O3 for 24 h caused decrease of mitochondrial membrane potential, pre-incubation with salvianolic acid B maintained mitochondrial membrane potential, indicating that the anti-apoptotic effect of salvianolic acid B were related to the mitochondrial pathway modulation. Western blot analysis showed that salvianolic acid B promoted the ratio of Bcl-2/Bax and promoted p-Akt/Akt compared with As2O3 group(P<0.01). Conclusion::Salvianolic acid B has a protective effect on hepatocyte injury induced by As2O3, and its mechanism is related to maintenance of mitochondrial function and inhibition of hepatocyte apoptosis.
ABSTRACT
<p><b>OBJECTIVE</b>To observe the effect of crocetin on autophagy in rat hepatocytes exposed to lipopolysaccharide (LPS) and D-galactosamine (D-gal) and explore the mechanism.</p><p><b>METHODS</b>Cultured rat hepatocytes were exposed to LPS (1 mg/L) and Dgal (60 mg/L) to induce cell injury and treated with crocetin, 3MA, or crocetin+3MA. Twelve hours after the treatments, the cells were examined for levels of ALT, AST and LDH in the supernatant using ELISA. LC3 fluorescence in the cells following immunofluorescence staining was observed using fluorescence microscopy. Autophagosomes in the cells were observed by transmission electron microscopy, and the cellular expressions of LC3, p62 and SIRT1 were detected using Western blotting.</p><p><b>RESULTS</b>The levels of ALT, AST and LDH in the hepatocytes were elevated after LPS- and D-gal-induced injury, reached the highest levels after 3MA treatment, but were decreased significantly by crocetin treatment. LC3 fluorescence increased obviously in the injured hepatoctyes, and the increment was the most obvious in crocetin-treated cells; LC3 fluorescence was decreased significantly after 3MA treatment. Cell injury induced obvious increase in autophagy in the hepatocytes, and the number of autophagosomes increased significantly after crocetin treatment but was reduced significantly after 3MA treatment. The cell injury caused an obvious up-regulation of LC3 and SIRT1 expression and down-regulated p62 expression. LC3 and SIRT1 expression levels were the highest and the expression of p62 was the lowest in cells with crocetin treatment. 3MA treatment significantly reduced the expression of LC3 and SIRT1 and increased the expression of p62 in the injured cells.</p><p><b>CONCLUSIONS</b>Autophagy is increased in injured rat hepatocytes, and crocetin can promote autophagy in the injured cells to reduce further cell injury.</p>