The Study Of Vaccarin On Inhibition Of Adipogenesis Via Activating Hedgehog Signaling In Vitro And In Vivo

Objective: Recently,obesity is one of the most widespread problematic health conditions,and to be a risk factor for several diseases.An increase in the number of adipose cells(hyperplasia)and an increase in the size of adipose cells(hypertrophy),resulting in obesity,and inhibition of adipogenic differentiation was very important against obesity.VAC has been demonstrated exerts various bioactivities,including anti-inflammatory,anti-apoptosis,anti-hyperlipidemia and promoting angiogenesis.However,the effects of vaccarin on adipogenesis and the possible mechanism of inhibiting lipid accumulation in vitro and in vivo was not elucidated.Therefore,this study was to explore anti-obesity effects of VAC based on 3T3-L1 adipocytes and high-fat diet induced obese mice.Methods:(1)The effect of Hedgehog(Hh)signaling pathway on adipogenesis was investigated using 3T3-L1 preadipocytes.The cells were differentiated by a "cocktail" method and cotreated with Hh pathway activator(SAG)and inhibitor(VIS),respectively.Oil red O staining,qPCR and Western blot were used to investigate the effect of Hh signaling pathway activation and inhibition on lipid accumulation.Gli1 was further overexpressed to explore the key time points of inhibiting adipogenesis during the adipogenic differentiation of 3T3-L1 cells.(2)To investigate the anti-obesity effect of VAC in vitro.The effect of VAC on Gli1 expression was detected in Gli1 stable transfected Shh-light 2 cells by dual luciferase reporting assay.Furthermore,the effect of VAC on lipid accumulation was observed by oil red O staining and Bodipy staining.The m RNA and protein levels of adipogenesis-related transcription factors such as PPARg,C/EBPg,Hh signaling pathway members such as Gli1,Smo,and Ptch were detected in 3T3-L1 cells after treatment different dose of VAC by qPCR and Western blot,respectively.In addition,the key time point of VAC regulating Gli1 was explored during adipogenic differentiation of 3T3-L1 cells.Glycerol release in 3T3-L1 mature adipocytes was detected by kit,and m RNA expression levels of lipolysis-related genes were detected by qPCR.(3)The obesity mice model was established by high-fat diet to investigate the antiadipogenesis effects of VAC in vivo.High fat diet induced mice were received 10 mg/kg(bodyweight)of VAC and received 25 mg/kg(bodyweight)of VAC,respectively.And body weight and food intake of mice were monitored twice a week,glucose tolerance and insulin tolerance tests were performed after 8-week administration.After 12-week administration,blood samples,liver,white adipose tissue and brown adipose tissue of mice were collected and weighed.qPCR and Western blot were used to detect the expression of adipogenesis-related transcription factors and Hh signaling pathway related genes.The adipocyte size of white adipose tissue and lipid accumulation in liver were observed by hematoxylin and eosin(H&E)staining.Results:(1)Lipid accumulation in 3T3-L1 cells was significantly reduced at 1 mM of SAG treatment,while no significantly changed in VIS treatment(5 mM).The protein expression levels of PPARg and C/EBPa,the adipogenesis-related transcription factors were decreased with SAG treatment,there was no significant effect after VIS treatment.In addition,Hh signaling inhibited adipogenesis mainly in the MDI stage of 3T3-L1 cells during adipogenic differentiation.(2)There was no cytotoxicity of VAC in 3T3-L1 cells under 200 mM.The lipid content decreased with the increase of VAC(1,5,10 mM)treated dose in the 3T3-L1 preadipocytes.The Gli1 transcriptional activity was significantly increased in Shh-light 2 cells by VAC treatment.The expression of Hh signaling components Gli1,Smo and Ptch were activated and the expression of adipogenesis related genes PPARg and C/EBPa were suppressed by VAC treatment.In addition,the m RNA expressions of lipolysis genes LPL,ATGL,HSL and MGL were significantly upregulated by VAC in mature 3T3-L1 adipocytes,and the release of glycerol was also significantly increased.(3)The body weight of high-fat diet induced mice were significantly alleviated by high and low doses of VAC.VAC significantly inhibited the increase of total triglyceride,total cholesterol and low-density lipoprotein in serum and up-regulated the content of high-density lipoprotein in serum of high fat induced mice.Pathological section results showed that VAC significantly reduced the degree of steatosis in abdominal fat and the extent of damage in liver.In addition,VAC treatment promoted Gli1 expression in white adipose tissue of high-fat diet induced mice and suppressed the levels of adipogenesis-related genes PPARg,C/EBPa,and the m RNA expressions of lipolysis-related genes LPL,ATGL,HSL and MGL was promoted by VAC.In conclusion,the mechanism of anti-obesity effects of VAC may be related to Hh signaling pathway activation.Conclusion:VAC showed significant inhibitory effects on adipogenesis in 3T3-L1 cells and weight gain in high-fat diet induced obese mice via activating the Hh signaling pathway.These results suggest that VAC may be an effective candidate for anti-obesity drugs.