Effect Of Procyanidins On Proliferation And Differetiation Of Pig Preadipocytes

With the development of the living standard of people,consumers put forward higher requirements for ratio of carcass lean meat.And the utilization of antibiotics and chemical synthetic drugs contribut to decline of pork flavor.So the development and application of green extract has great significance.There were some studies have reports that lipid production of mice can be inhibit by Grape seed procyanidin extract(GSPE).But the study of GSPE on the proliferation and differentiation of pig preadipocytes have not yet been reported.Therefore,the objective of this study was to investigate the effect of GSPE on proliferation and differentiation and mechanism of pig preadipocytes,and Meishan pig preadipocytes were used as research model in our experiments.These provided theoretical basis to discuss the fat formation mechanism,further to improve fat deposition of pigs by nutritional regulation.The first part:The aim of this study was to establish the preadipocyte in vitro culture system of Meishan pig.Preadipocytes were obtained from subcutaneous adipocyte tissue of neck and back from 3-day Meishan pigs,then primary cultured and sub-cultured.Morphological change,growth curve,Oil red O staining and fat content were studied,and mRNA expressions of PPAR?,CEBP?,LPL,DLK1,FABP4 and ADIPOQ were measured during Meishan pig preadipocyte differentiation by Real-time fluorescent quantitative RT-PCR.The results show that meishan piglet primary preadipocytes began to paste the wall about 6 hours and presented small spindle-shape and irregular triangle.The cells were highly homogeneous,adhered well and presented fibroblast-like morphology after sub-culturing.During 1st-9th day,the cells entered logarithmic growth phase and growth curve showed "S" shape.On 11th day of induced culture,the cells had a high differentiation rate and showed red by Oil red O staining.Fat content was exceeded significantly than the un-induced groups(P<0.01).Thus,the cells presented typical characteristics of preadipocyte.PPARy gene expressed in the early stage of preadipocyte differentiation and highly expressed on 3rd day(P<0.01).CEBPa and LPL gene levels gradually increased with preadipocyte differentiation(P<0.01),while DLK1 gene levels gradually decreased(P<0.01).FABP4 and ADIPOQ gene highly expressed on 6th day(P<0.01).In all,this study established a culture system and induced differentiation model for Meishan pigs successfully,providing a material for further studying body fat metabolic regular in vitro using preadipocytes.The second part:The aim of this research was to investigate the effect of different concentrations of GSPE on morphology and proliferation of pig preadipocytes.Pig preadipocytes were treated by different concentrations(5,10,25,50,100,200 and 300?g/mL)of GSPE,morphological observation and CCK-8 proliferation activity were measured after 24 h.The results show that 5-50 ?g/mL GSPE had no significant effect on the preadipocyte morphology compared with control.Preadipocytes adhered well and presented spindle-shape fibroblast-like morphology,which were highly homogeneous.100?300 ?g/mL GSPE significantly affected preadipocyte morphology.Specifically,preadipocytes adhered unstable and nucleus condensation,irregular triangular cells and dead cells increased in a dose-dependent.In addition,CCK-8 showed that 5 and 10?g/mL had no significant effect on cell proliferation compared with control(P>0.05),while 25-300 ?g/mL GSPE significantly inhibited cell proferation in a dose dependent manner(P<0.01).Half maximal inhibitory concentration(IC50)was 166.89 ?g/mL.In conclusion,morphology and proliferation of pig preadipocytes can be inhibited by certain concentrationa of GSPE.The third part:This research was conducted to study the effect and mechanism of GSPE on cell cycle of pig preadipocytes.Preadipocytes were treated by 100,150 and 200?g/mL GSPE,respectively.Preadipocytes were fluorescently stained by Hoechst 33258 and the apoptosis rate was analyzed by flow cytometry.In addition,preadipocytes were treated by 100 ?g/mL GSPE.Cell cycle percentage was analysised by flow cytometry and cell cycle related gene including p16,p21,CDK2,CDK4,CDK6,Cyclin A and Cyclin D1 mRNA expressions were detected by real-time fluorescent quantitative RT-PCR after 24 h.The results showed that apoptosis can be significantly induced by 150 and 200 ?g/mL GSPE(P<0.01),while no difference was observed by 100 ?g/mL GSPE(P>0.05).100?g/mL GSPE can remarkably induce the cell cycle arrest at GO/G1 phase(P<0.01),which was associated with expressions of CDK2,CDK6 and Cyclin Dl significantly decreased(P<0.01),and expressions of CDK4 and Cyclin A decreased(P<0.05).No significant change was observed for p16 and p21(P>0.05).('onclusions:150 and 200 ?g/mL GSPE induced apoptosis,while 100 ?g/mL GSPE incuced cell cycle arrest,which may be associated with down-regulated expression of CDK2,CDK4,CDK6,Cyclin A and Cyclin D1.The fourth part:This research was conducted to study the effect and mechanism of different concentrations of GSPE on differentiation of pig preadipocytes.Preadipocytes were treated by different concentrations(5,10,25,50,100,200 and 300 ?g/mL)of GSPE for 24 h on 0th day of cell differentiation.Oil red O staining,intracellular TG content were determinated on 11st day.Then preadipocytes were treated by 100 ?g/mL GSPE for 24 h,GPDH activity were determinated on 11st day.In additon,mRNA expressions of key genes during adipocyte differentiation,including PPARy,CEBPa,FABP4 and DLK1 were detected respectively on 1st day,3rd day,6th day and 11st day of cell differentiation.The results showed that lipid contents in adipocytes were reduced by 25-50 ?g/mL GSPE(P<0.05),and significantly reduced by 100?300 ?g/mL GSPE in a dose dependent manner(P<0.01)by Oil red O staining.TG assay showed that 100?300 ?g/mL GSPE could decrease TG content in the cells significantly(P<0.01).Thus,we choose 100 ?g/mL GSPE as the best concentration to inhibite the cell differentiation.GPDH activity was significantly decreased by 100 ?g/mL GSPE treatment(P<0.01).Real-time fluorescent quantitative RT-PCR showed that the expressions of PPARy and FABP4 were reduced(P<0.05),C/EBPa had no significant change(P>0.05),while DLK1 was increased significantly(P<0.01)at all of differetiation.Thus,preadipocyte differentiation can be inhibited by 100 ?g/mL GSPE,which by down-regulating the expressions of PPARy and FABP4 and increasing expressions of DLK1 at the late differentiation stage.