Intervention effect of Cigu Xiaozhi prescription on ceramide lipoapoptosis in non-alcoholic fatty liver disease.

OBJECTIVE: To explore the mechanism of the Chinese medicine Cigu Xiaozhi prescription (, CGXZ) in the treatment of the non-alcoholic fatty liver disease (NAFLD) by detoxification and phlegm-reducing, the effect of CGXZ prescription on ceramide-mediated lipid apoptosis in Hep G2 cells with NAFLD. METHODS: The experiment was randomly divided into 6 groups: normal control group, model group, CGXZ prescription medicated serum high, medium, and low dose groups, and pioglitazone positive control group. Using 500 µmol/L free fatty acid (FFA) mixture to induce Hep G2 cells to establish NAFLD cell model, respectively, with 2%, 4%, and 6% concentration of CGXZ prescription medicated serum intervention for 24 h. The changes in organelles and lipid droplet accumulation were observed under the electron microscope. Furthermore, TdT-mediated dUTP Nick-End Labeling method was used to assay hepatocyte apoptosis; Biochemical determination of glutamic-pyruvic transaminase, glutamic oxalacetic transaminase, triglycerides, and FFA levels in Hep G2 cells; the content of ceramide was determined by high-performance thin-layer chromatography. Finally, Western Blot and quantitative real-time polymerase chain reaction (qRT-PCR) were used to determine the protein and gene expression levels, such as inducible nitric oxide synthase (iNOS), nuclear factor κB (NF-κB), B cell lymphoma 2 (Bcl-2) and Bcl-2-associated X (Bax). RESULTS: Under the electron microscope, the cells in the model group showed moderate-to-severe steatosis, and apoptotic bodies could be seen. The model group had greater improvements in the apoptosis rate (P < 0.01), and the levels of ceramide C2 and FFA in the cytoplasm (P < 0.01) than the normal control group. The protein expressions of NF-κB, iNOS, and Bax were significantly up-regulated (P < 0.05), while the Bcl-2 had no significant change (P > 0.05). Compared with the model group, the levels of ceramide C2 and FFA (P < 0.01), the protein expressions of NF-κB, iNOS, and Bax (P < 0.05) in the CGXZ prescription treatment group and pioglitazone positive control group were significantly decreased; Only the Bcl-2 protein was significantly up-regulated in the high-dose Chinese medicine group (P < 0.05). The down-regulation of Bax mRNA expression in each Chinese medicine treatment group was significantly better than in the pioglitazone positive control group (P < 0.01). CONCLUSIONS: The CGXZ prescription, formulated with the method of detoxification and phlegm, can inhibit lipoapoptosis in the NAFLD cell model by down-regulating the levels of ceramide C2 and FFA, which may be achieved by regulating ceramide/iNOS/NF-κB signaling pathway.

Effect of artesunate on rat liver fibrogenesis in vitro and in vivo / 中国药理学通报

Aim To study the effect of artesunate on immuno-injured hepatic fibrosis induced by bovine ser-um albumin in rat model and the effect of artesunate on hepatic stellate cells ( HSCs ) proliferation, so as to provide experimental evidence for clinical application of artesunate and the treatment of hepatic fibrosis. Methods The model of immuno-injured hepatic fibro-sis induced by bovine serum albumin was established in Wistar rats. Rats were randomly divided into 5 groups:normal group, model group, low dose of arte-sunate, middle dose of artesunate and high dose of ar-tesunate. Drugs were given to the corresponding thera-peutic groups, and then were continued once a day for two months. Distilled water was given to the rats of normal and model groups according to the same meth-od. Liver tissues were used for measuring the content of collagen, the rat serum activities of albumin( Alb) , alanine aminotransferase ( ALT ) and aspartate amin-otransferase(AST). Liver tissue’ s pathological chan-ges were observed by HE and collagen staining. Isola-ted and cultured rat primary HSCs in the flask for 10 days to make cells activated, MTT assay was used to detect rate of cellular proliferation; concentration of hydroxyproline in supernatant was detected by digestive method; the expression of p53 was investigated by Western blot and RT-PCR. Results Serum levels of Alb in model group were significantly lower ( P <0. 05 ) , and levels of ALT and AST in model group were significantly higher ( P <0. 05 ) compared with normal group. Levels of AST in low, middle and high dose groups(3. 2, 9. 6, 28. 8 mg·kg-1 ) were signifi-cantly lower(P <0. 05) compared with model group, and levels of ALT in high dose groups were significant-ly lower(P<0. 01) compared with model group. The contents of collagen in model groups were significantly higher(P<0. 01) compared with normal group, while the contents of collagen in therapy groups significantly decreased ( P < 0. 05 ) compared with model group. Activated HSCs treated with various concentrations of artesunate (150, 175, 200 μmol·L-1 ) were inhibi-ted on dose and time-effect relationships. Production/secretion of hydroxyproline decreased after HSCs was treated by artesunate for 24 h; the expression of p53 was up-regulated showed by Western blot and RT-PCR in artesunate treated cells. Conclusion Artesunate brings about anti-fibrosis in vitro and in vivo by increas-ing the expression of p53 .

Expression of p53 in the effects of artesunate on induction of apoptosis and inhibition of proliferation in rat primary hepatic stellate cells.

BACKGROUND: Activation of hepatic stellate cells (HSCs) plays an important role in the development of cirrhosis through the increased production of collagen. p53, the "guardian of the genome", is a transcription factor that can bind to promoter regions of hundreds of genes where it either activates or suppresses gene expression. Thereby, p53 serves as a tumor suppressor by inducing cell cycle arrest, apoptosis, senescence and DNA repair. Artesunate is a derivative of Artemisinin, Scholars had found it had more extensive pharmacological effects past 10 years. However, little is known about the expression of p53 in the effects of Artesunate on induction of apoptosis and inhibition of proliferation in rat HSCs. METHODOLOGY/PRINCIPAL FINDINGS: Isolated and cultured rat primary HSCs in the flask for 10 days to make cells activated. HSCs were divided into two groups: experimental groups and control groups, experimental groups included with various concentrations of Artesunate (125, 150, 175, 200, 225 µmol/L) for 24, 48 and 72 hours. Analysis of MTT revealed that activated HSCs treated with various concentrations of Artesunate (150-225 µmol/L) were inhibited on dose and time-effect relationships; Concentration of hydroxyproline in supernatant was detected by digestive method; Analysis of flow cytometry demonstrated that Artesunate could arrest cell cycle in G1 and induce apoptosis; The nuclear morphological changes in apoptotic cells were evaluated with DNA staining by Hoechst 33258 dye; The expression of p53 were up-regulated showed by western blotting and RT-PCR. CONCLUSION: Artesunate could inhibit HSCs proliferation in dose-dependent and time-dependent manners in vitro through increase the expression of p53.