Intervention effect of Cigu Xiaozhi prescription(慈菇消脂方)on ceramide lipoapoptosis in non-alcoholic fatty liver disease

OBJECTIVE:To explore the mechanism of the Chinese medicine Cigu Xiaozhi prescription(慈菇消脂方,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 6groups:normal control group,model group,CGXZ prescription medicated serum high,medium,and low dose groups,and pioglitazone positive control group.Using 500μmol/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,Td T-mediated d UTP 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 G2cells;the content of ceramide was determined by highperformance thin-layer chromatography.Finally,Western Blot and quantitative real-time polymerase chain reaction(q RT-PCR)were used to determine the protein and gene expression levels,such as inducible nitric oxide synthase(i NOS),nuclear factorκB(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,i NOS,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,i NOS,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 downregulation of Bax m RNA 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 downregulating the levels of ceramide C2 and FFA,which may be achieved by regulating ceramide/i NOS/NF-κB signaling pathway.