Effects And Mechanism Of Asperosaponin Ⅵ On Nonalcoholic Steatohepatitis

Objective: To investigate effects and mechanism of Asperosaponin VI onnonalcoholic steatohepatitis.Methods: Lacking of leptin receptors male mice (ob/ob) was used of the model ofNonalcoholic steatohepatitis (NASH), and nine male C57/BL mice were used as control.Thirty-six ob/ob mice were randomly divided into four groups: model group (n=9) fedhigh-fat diet and the Asperosaponin VI low-dose (30mg/kg) group (n=9), theAsperosaponin VI middle-dose (60mg/kg) group (n=9), and the the Asperosaponin VIhigh-dose (1200mg/kg) group (n=9) fed high-fat diet for4weeks. After4weeks oftreatment, all mice were sacrificed, and the liver tissues were collected to evaluate the liverweight, liver index, blood lipids and liver function. We determined the liver mitochondrialmembrane potential and ROS levels in liver homogenate. The liver histological and ultrastructural changes were each observed by hematoxylin and eosin (H&E) staining andelectron microscopy. To observe the changes of liver tissue with, thiobarbituric acid (TBA)assay was used to detect liver tissue malondialdehyde (MDA) content, colorimetricdetermination of liver tissue glutathione peroxides (GSH-PX) content and super oxidedisputes (SOD) activity. P-JNK, Bax, Bcl-2and Cytochrome C protein expression wasdetected by western blot. Establishment of the vitro NASH model of rat BRL cell inducedby Palmitic acid (PA), based on the experimental requirement set five groups: controlgroup, PA-induced group and Asperosaponin VI low, medium and high concentrationpre-protection groups. MTT assay was used for cell viability, bodipy493/503staining wasused to detect BRL cells lipid deposition, Annexin V-FITC flow cytometry was used todetect the apoptosis rate. P-JNK, Bax, Bcl-2and Cytochrome C protein expression was detected by western blot.Results:1. The results of animal experiments:(1) Compared with the control group,model group were significantly increased body weight and liver index (P <0.05), comparedwith the model group, body weight and liver index were decreased in Asperosaponin VItreat group and the high dose had significant difference (P <0.05).(2) Steatosis, ballooning,mixed acute and chronic lobular inflammation and focal necrosis were presented in modelgroup rats accompanied by elevated serum ALT, AST levels. Mice in model group hadhigher levels of serum TG, TC, LDL-C and liver content of MDA, as well as the serumHDL-C, the in liver contents of GSH-PX and the activity of SOD were significantlydecreased (p<0.05). Compared with model group, a remarkable reduction (P<0.05) wereobserved in serum ALT, AST, TC, TG, LDL-C and liver content of MDA in AsperosaponinVI high dose group, the serum HDL-C, the in liver contents of GSH-PX and the activity ofSOD were significantly increased (p<0.05). Compared with normal group, the livermitochondrial membrane potential has a significantly decreased and the ROS level has asignificantly increased in model group (p<0.05). Compared with the model group theAsperosaponin VI-treated group significantly increased mitochondrial membrane potentialand significantly decreased the ROS level in a dose-dependent manner (p<0.05).Compared with mice in normal group, the P-JNK, Bax and Cytochrome C proteinexpression were significantly increased as well as the Bcl-2protein levels weresignificantly decreased (P <0.05). Compared with the model group the P-JNK, Bax andCytochrome C protein expression were significantly decreased and the Bcl-2protein levelswere significantly increased in Asperosaponin VI dose groups (P <0.05).2. The results ofcell experiments:(1) MTT assay data showed that the growth of cells was inhibited morethan50%at48h when PA concentration was exceed150mol/L. Thus, we adopted thelower cytotoxic concentration of150μmol/L PA for the following experiments.(2)Bodipy493/503staining and lipids quantification revealed cells in the PA-treated group hasa prominent lipids accumulation and the high dose Asperosaponin VI can effectivelyinhibition of lipids deposition(P <0.05).(3) PA caused a significant increase in the number of apoptotic cells of BRL, and the cells displayed with nuclear fragmentation, chromatincondensation compared to control, but the Asperosaponin VI treated can inhibited liver cellapoptosis efficiently.(4) Compared with control, the P-JNK and Bax protein expression inPA-treated group were significantly increased, as well as the Bcl-2protein levels weresignificantly decreased (P <0.05). Compared with the PA-treated group the P-JNK and Baxprotein expression were significantly decreased and the Bcl-2protein levels weresignificantly increased in Asperosaponin VI medium and high dose groups (P <0.05)Conclusions:1. We have successfully established experimental vivo and vitro NASHmodel whose pathological characteristic was very similar to the human nonalcoholicsteatohepatitis, which provides a rational experimental model for the exploration ofpathogenesis and novel treatment of NASH.2. Asperosaponin VI may treat NASH via inhibit oxidative stress and hepatocyteapoptosis of ob/ob mice.3. Asperosaponin VI played protective role in BRL cell by inhibit PA induced lipidsdeposition and apoptosis.4. Asperosaponin VI has anti-therapeutic effects for NASH. Its mechanisms may bepartly through inhibit P-JNK, Bax protein express, promote Bcl-2protein express andinhibit Cytochrome C release, then blocking the mitochondrial apoptosis pathway. But theexact mechanism still needs further study.