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.

Cigu Xiaozhi pills's influence on lipid peroxidation and TNF-alpha expression in liver tissues of rats with nonalcoholic steatohepatitis / 中国中药杂志

<p><b>OBJECTIVE</b>To investigate the effect of Cigu Xiaozhi pills on expression of tumor necrosis factor alpha (TNF-alpha) in rat with nonalcoholic steatoheptatitis (NASH).</p><p><b>METHOD</b>Fifty male SD rats were divided randomly into five groups: the normal group (n = 10), the model group (n = 10), the high dosage group (n = 10) and the little dosage group of the treatment (n = 10), Dongbao Gantai group (n = 10). Rats in normal control group were fed with standard diet. Rats in other four groups were established models of nonalcoholic steatohepatitis and were treated simultaneously with traditional Chinese medicine and positive medicine. Histopathological changes in the liver were observed. The serum ALT, AST, TG, TC and hepatic MDA, SOD, GPX were detected histologically. The expression of TNF-alpha in the liver was determined using the immunohistochemical technique and RT-PCR.</p><p><b>RESULT</b>In model group, extensive adipose degeneration and inflammatory cell infiltration were found in the liver. In treatment groups steatohepatitis were markedly alleviated. Compared with model group, TG, TC, ALT, AST of the serum and MDA of liver tissue were reduced significantly, and SOD, GPX were increased significantly in treatment groups and positive control group (P < 0.05). TNF-alpha was not expressed almost in normal rat liver and was expressed highly in model rat liver. Compared with model group, the TNF-alpha mRNA and protein expression were significantly lower in liver of treatment groups and positive control group (P < 0.05). And the effects of high dosage group surpass than those of Dongbao Gantai group (P < 0.05).</p><p><b>CONCLUSION</b>TNF-alpha plays an important role in NASH pathogenesis. Cigu Xiaozhi pills can effectively treat experimental nonalcoholic steatohepatitis in rats, and its mechanism may be associated with ameliorating hepatocellular steatosis, removing the free radicals and enhancing the capability of anti-oxidation and anti-inflammatory.</p>