| Object : Using obesity mice induced by high-fat diet and mice behavior test to Screen the inhibitors of CB1 R to achieve compounds with therapeutic effect and lower side effect and investigate the protective mechanism of reduce adipose, which will provide novel experimental and theoretical basis for novel peripheral CB1 R antagonist in treating obesity.Methods : In vivo: Obese mice induced by high-fat diet were treated with new compounds and the changes of weight loss in treatment group and control group were observed. The depression test were measured after 18-day-treatment by EPM, FST and TST.Fasting blood glucose, insulin tolerance test and glucose tolerance test in mice were determined before sacrifice.The content of total cholesterol(TC), triacylglycerol(TG), high density lipoprotein cholesterol(HDL-C), Glucose, low density lipoprotein cholesterol(LDLC), glutamic-oxalacetic transaminase(AST), glutamic-pyruvic transaminase(ALT) and lactic dehydrogenase(LDH) in serum in mice blood serum were tested.The genital peripheral adipose tissue and liver were weighted and fixed by 4% formaldehyde to cut into paraffin section. The morphology of tissue and liver were examined by HE staining.In vitro: Hep G-2 cells were treated PA-Na(0.5 m M) for 24 h to mimic the fatty liver in cell model, and different concentrations of ZH-101-S were added for another 24 h. Cell viabilities were measured using MTT. The levels of TG and CHOL were determined by kit.Mito Tracker Red measuring the number of mitochondria, luciferase assay testing ATP contents in cell and HCFH-DA staining detecting cellular ROS were used to test mitochondrial associated function. The expression of CB1 R in membrane of mitochondria, pAMPK, and PGC1 were detected using Western blot.Results: Compared with high fat group, treatment with ZH-101-S can decrease weight significantly. The results from EPM showed the frequency, distance, duration of open arm were significant decreased by peripheral CB1 R antagonist ZH-101-S treatment compared to rimonabant group. Additionally, the immobility time of FST, TST were attenuated by ZH-101-S treatment compared to rimonabant group. ZH-101-S improved insulin resistance and glucose tolerance and decreased the level of CHOL, TG, Glu, LDL, AST, ALT, HDL as well as increase HDL-C. The diameter of fat cell was reduced in ZH-101 group combined with improved morphology and metabolism of liver tissue.MTT results 0.5 m M PA-Na and 3, 10, 30 μM ZH-101-S have no cytotoxicity in Hep G-2.Oil red analysis showed that compare with PA-Na treatment, the content of lipid droplet wasdecreased 30.0% after 30 μM ZH-101-S treatment for 24 h. Compare with PA-Na treatment,the content of TG was decreased 63.6%, the rate was increased in ZH-101-S treatment in a dose-dependment manner, moreover, the content of CHOL was decreased 30.0% after 30 μM ZH-101-S treatment. 30 μM ZH-101-S can decrease CHOL, TG significantly in oil red staining. ZH-101-S had protective effect on increase mitochondrial number, ATP level, and decrease ROS content. Western blot result verified Cannabinoid CB1 receptor is expressed on the outer mitochondrial membrane(mt CB1R), ZH-101-S promoted the expression of pAMPK and PGC-1α.Conclusions :ZH-101-S can lose weight in fat mice significantly and improve fatty liver. What’s more, it avoids depression-like behavior at the same time of decrease weight. The potential weight loss and liver protection mechanism of ZH-101-S might be associated with mitochondrial function to enhance energy metabolism via CB1 R located in outer membrane of mitochondria. |
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