The Effect of Ferroptosis on the Activation of Human Hepatic Stellate Cells Induced by Inorganic Arsenic / 医学研究杂志

Objective To investigate the effect of NaAsO2 on ferroptosis in human hepatic stellate cells(LX-2).Methods LX-2 cells were cultured in vitro,and different concentrations of NaAsO2(5μmol/L,10μmol/L,15μmol/L)were infected with LX-2 cells for 24h in a group design to construct the activation model of LX-2 induced by NaAsO2 in vitro,and a control group was set up.Mitochon-drial structure of LX-2 cells treated with NaAsO2 was observed by transmission electron microscopy(TEM).Fe2+levels were detected by fluorescence microscope and fluorescent enzyme label.The content of malondialdehyde(MDA)was determined by the colorimetric meth-od.The protein expression levels of SLC7A11,GPX4,and α-smooth muscle actin(α-SMA)were detected by Western blot.Results TEM showed that mitochondrial membrane integrity was damaged and mitochondrial ridges were reduced and disappeared in the NaAsO2group.In addition,compared with the control group,Fe2+levels in NaAsO2 treatment groups were increased(P＜0.05).There were statistically significant differences in MDA content among different doses of NaAsO2groups(F=7.18,P＜0.05).Compared with the control group,MDA content of LX-2 cells in 5 and 15μmol/L NaAsO2groups was higher than that in the control group(P＜0.05).At the translation level,the expression of fibrosis index α-SMA protein level was up-regulated with the increase of NaAsO2dose(P＜0.05),the protein expression levels of ferroptosis index SLC7A11 and GPX4decreased in a dose-dependent manner with the increase of the dose of NaAsO2(P＜0.05).Conclusion Ferroptosis is involved in the activation of LX-2 cells induced by NaAsO2.

Screening of DNA methylation sites and construction of differential methylation profiles in human hepatic stellate cells fibrosis and autophagy induced by sodium arsenite / 中华地方病学杂志

Objective:To analyze DNA methylation sites related to fibrosis and autophagy in human hepatic stellate cells (LX-2 cells) induced by sodium arsenite (NaAsO 2), and to screen specific methylation genes related to fibrosis and autophagy. Methods:Genome-wide DNA detection was performed using Illumina Infinium Methylation EPIC BeadChips (850K methylation chip) to derive differential methylation sites in LX-2 cells (control group) and the fibrosis and autophagy models of LX-2 cells induced by NaAsO 2(low, medium and high dose groups: the final concentrations were 5, 10, 15 μmol/L NaAsO 2, respectively, after 48 h intervention). Gene ontology (GO) function enrichment analysis and Kyoto encyclopedia of genes and genomes (KEGG) signaling pathway enrichment analysis were used to explore gene function. Results:The model of cell fibrosis and autophagy was established successfully in high dose group. The results of 850K methylation chip detection showed that there were 25 817 significant different methylation sites between the high dose group and the control group, including 12 083 hypermethylation sites and 13 734 hypomethylation sites. GO function enrichment analysis showed that the molecular functions of differentially methylated genes mainly included protein binding, ion binding, catalytic activity, enzyme binding. KEGG signaling pathway enrichment analysis showed that the pathways involved in differentially methylated genes mainly included metabolic pathway, cancer pathway, phosphatidylinositol-3-kinase-protein kinase B (PI3K-Akt) signaling pathway, endocytosis, and mitogen activated protein kinase (MAPK) signaling pathway. In the promoter region, 11 and 29 differentially methylated genes related to fibrosis and autophagy were screened, respectively.Conclusions:A large number of differential methylation sites exist in the process of NaAsO 2 induced fibrosis and autophagy of LX-2 cells. Specific methylation genes related to fibrosis and autophagy are screened out.

Mechanism of total C-21 steroidal glucosides from root of Cynanchum auriculatum in activation of human hepatic stellate cells via TGF-β / 中国药理学通报

Aim To investigate the inhibitory effect of total C-21 steroidal glucosides (TCSG) from the root of Cynanchum auriculatum on activation of human hepatic stellate cells and the underlying mechanism. Methods The fibrosis model in vitro was established by treating LX-2 cells with TGF-β

Study on the Effect and Its Mechanism of Carvedilol on Leptin-induced Activation and Proliferation of Hu-man Hepatic Stellate Cells / 中国药房

OBJECTIVE:To study the effect and its mechanism of carvedilol on leptin-induced activation and proliferation of LX2 human hepatic stellate cells(HSC-LX2). METHODS:HSC-LX2 with logarithmic growth periods were divided into blank con-trol group,leptin-stimulated group and carvedilol low-concentration,medium-concentration,high-concentration groups(5,10,20μmol/L). Except for the blank control group,other groups were added 0.1 g/L leptin and corresponding concentration of carvedilol. After 24 h,MTT method was used to detect the optical density(OD)value of cells and calculate the proliferation rate. Flow cytom-etry was used to detect the cell cycle and apoptosis. Real-time fluorescence quantitative polymerase chain reaction method was used to detect the α-smooth muscle actin (α-SMA),matrix metalloproteinase inhibition factor 1 (TIMP-1),leptin,leptin receptor mRNA expressions. Western blot method was used to detect phosphorylated Janus kinase 2(p-JAK2),phosphorylated signal trans-duction and transcriptional activator 3 (p-STAT3) protein expressions. RESULTS:Compared with blank control group,OD value of cell was increased in leptin-stimulated group;apoptotic rate was decreased;cells of G0/G1 were decreased;α-SMA,TIMP-1, leptin,leptin receptor mRNA expressions and p-JAK2,p-STAT3 protein expressions were increased (P<0.05). Compared with leptin-stimulated group,OD values of cells were decreased in carvedilol concentration groups;apoptotic rate was increased,and the cells were mainly blocked in G0/G1 phase;α-SMA,TIMP-1,leptin,leptin receptor mRNA expressions and p-JAK2,p-STAT3 protein expressions were decreased(P<0.05)and was concentration-depended(P<0.05). CONCLUSIONS:Carvedilol can inhibit the activation and proliferation of leptin-induced HSC-LX2,promote its apoptosis. The mechanism may associate with down-regulat-ing leptin,leptin receptor gene expression and blocking JAK2/STAT3 signal pathway activation by leptin in cells.

Influence of Chaihu Jiedu Decoction on LX-2 Cell Proliferation and Apoptosis / 现代生物医学进展

Objective:To observe the effects of Chaihu Jiedu decoction on human hepatic stellate LX-2 cells,and to explore the potential molecular mechanisms.Methods:The wistar rats were divided into the experimental group and the control group,respectively with Chaihu Jiedudecoction and saline lavage,then centrifugal and get the drug-containing serum and control serum.At the same time,trsuscitate cells.When we got the expected number of cells,we divided into the experimental group and the control group.The human hepatic stellate cells (LX-2) were with the drug serum for 24 h,36 h,48 h,72 h.Then the cell proliferation inhibition rate was measured by CCK-8,the apoptosis were detected by flow cytometry (Annexin V-FITC,PI staining method).Results:ChaihuJiedu decoction could inhibit proliferation of LX-2 cells 24 hours after dosing.The in hibition rate in 36 h,48 h,72 h were 0.37 ％,0.46 ％,0.44 ％ respectively.It could prevent LX-2 cells into proliferation and induce the apoptosis of LX-2 cells.The apoptosis rates in 48 h,72 h were (9.80±0.95)％,(36.40± 5.09)％ respectively,and there are difference of statistical significance(P＜0.05).Conclusion:Chaihu Jiedu decoction can inhibit the proliferation of LX-2 cells proliferation,and induce apoptosis,so as to interfere with course of the liver fibrosis.

Effect of Artesunate on Akt/GSK-3β/β-catenin Signal Pathway in Human Hepatic Stellate Cells / 中国药师

Objective: To explore the effect of artesunate (Art) on Akt/GSK-3β/β-catenin signal pathway.Methods: Art at different concentrations (0, 12.5, 25, 50 μg·ml-1) was used to treat human hepatic stellate cells (LX-2), and CCK-8 assay was used to detect the cell proliferation to determine the optimal concentration.Art inhibitor (MK-2206) at different concentrations (0~8 μmol·L-1) was given to LX-2 cells, and a Western Blot method was applied to determine the optimal inhibition concentration.Art, MK-2206 and MK-2206+Art were respectively given to LX-2 cells, and a Western Blot method was used to detect the levels of Akt, p-Akt, GSK-3β, p-GSK-3β and β-catenin proteins.Results: CCK-8 assay was used to detect the cell survival rate, and the survival rate was 80% after the 24-hour treatment with 25 μg·ml-1 Art.The results of Western Blot showed that MK-2206 at 6 μmol·L-1 could effectively inhibit the expression of p-Akt.Compared with those of the control group, the levels of Akt, p-Akt, p-GSK-3β and β-catenin protein were significantly different (P＜0.05) in Art (25 μg·ml-1) group, MK-2206 (6 μmol·L-1) group and MK-2206 (6 μmol·L-1) + Art (25 μg·ml-1) group.The expression of GSK-3β and Akt in MK-2206+Art group had no significant difference when compared with that in Art group and MK-2206 group (P＞0.05), while the levels of p-Akt, p-GSK-3β and β-catenin were significantly reduced (P＜0.01).Conclusion: Art exhibits the influence on the relative factors in Wnt/β-catenin signal pathway by Akt/β-catenin, subsequently inhibits the cell proliferation and alleviates the liver fibrosis process.

Ursolic Acid Inhibition on Human Hepatic Stellate Cells Proliferation through PDGF-ERK Signaling Pathway / 世界科学技术-中医药现代化

This study was aimed to investigate the effects of ursolic acid on human hepatic stellate cells (HSC-LX-2) proliferation and its mechanism.Different doses of ursolic acid were incubated with HSC-LX-2 cellin vitrof or 48 h.MTT was used for the detection of HSC-LX-2 cell proliferation.The expressions of PDGF-ERK signaling pathway associated proteins were measured by western blot.The results showed that the proliferation of HSC- LX-2 cells was inhibited by ursolic acid in a dose-dependent manner.The inhibition rate of 20,30 and 40μmol·L-1 of ursolic acid was 9.1%,42.3% and 62.6%,respectively.The IC50 was 35.2μmol·L-1.After incubated with ursolic acid for 48 h,protein levels of PDGF-R and p-ERK in 30 and 40μmol·L-1 group were significantly decreased when compared with the normal group (P<0.05 orP<0.01),except the ERK protein.It was concluded that ursolic acid can inhibit HSC-LX-2 cell proliferation.Its mechanism may be related to the blockage of PDGF-ERK signaling pathway.