Vaspin对棕榈酸诱导的INS-1细胞自噬水平的影响

Effectofvaspinonthelevelofpalmiticacid-inducedautophagyinINS-1cells

摘要:

目的：研究脂肪细胞因子Vaspin对棕榈酸诱导的INS-1细胞自噬水平的影响。方法：培养INS-1细胞，分为正常组：用普通培养基培养24h；棕榈酸（palmiticacid，PA）组：以0.5mmol/LPA培养24h；PA+Vaspin组：先加入320ng/mLVaspin预培养2h，再以0.5mmol/LPA+320ng/mLVaspin共培养24h；PA+Vaspin+雷帕霉素（rapamycin）组：先以50nmol/LRapamycin预培养2h，再以0.5mmol/LPA+320ng/mLVaspin共培养24h；PA+Vaspin+3-甲基腺嘌呤（3-methyladenine，3-MA）组：先以1mmol/L3-MA预培养2h，再以0.5mmol/LPA+320ng/mLVaspin共培养24h。葡萄糖（3.3mmol/L和16.7mmol/L）刺激胰岛素分泌实验检测各组胰岛素分泌水平，Westernblot检测哺乳动物雷帕霉素靶蛋白（mammaliantargetofrapamycin，mTOR）、p-mTOR、Beclin-1、LC3-Ⅰ、LC3-Ⅱ及P62蛋白水平，mRFP-GFP-LC3检测自噬流水平，流式细胞仪检测细胞凋亡水平。结果：与正常组相比，PA组INS-1细胞上清液中胰岛素分泌水平在3.3mmol/L葡萄糖和16.7mmol/L葡萄糖刺激下均降低[（1.02±0.08）ng/mLvs.（0.54±0.06）ng/mL，P=0.000；（1.15±0.13）ng/mLvs.（0.71±0.05）ng/mL，P=0.000）]，PA+Vaspin组较PA组升高[（0.76±0.03）ng/mLvs.（0.54±0.06）ng/mL，P=0.001；（0.91±0.04）ng/mLvs.（0.71±0.05）ng/mL，P=0.011）]；与正常组相比，PA组INS-1细胞p-mTOR与mTOR蛋白比值低（2.04±0.14vs.1.19±0.09，P=0.000），PA+Vaspin组p-mTOR与mTOR蛋白比值较PA组高（1.51±0.05vs.1.19±0.09，P=0.005）；同时，PA组自噬相关蛋白Beclin-1、LC3Ⅱ/LC3Ⅰ比值与正常组相比明显增加（0.81±0.07vs.0.65±0.04，P=0.005；2.35±0.25vs.1.16±0.11，P=0.000），P62蛋白水平降低（1.04±0.19vs.0.60±0.08，P=0.000）；PA+Vaspin组Beclin-1、LC3Ⅱ/LC3Ⅰ蛋白水平较PA组低（0.58±0.04vs.0.81±0.07，P=0.001；1.95±0.08vs.2.35±0.25，P=0.007），P62蛋白增加（0.82±0.03vs.0.60±0.08，P=0.032）；PA组自噬溶酶体数量较正常组多（8.33±1.53vs.0.33±0.58，P=0.000），Vaspin干预后降低（3.67±1.53vs.8.33±1.53，P=0.001）；同时，PA干预INS-1细胞24h后细胞凋亡明显增加（7.91±0.50vs.22.15±2.05，P=0.000），PA+Vaspin组较PA组细胞凋亡减少（13.23±1.97vs.22.15±2.05，P=0.000）。结论：Vaspin能够改善棕榈酸诱导的INS-1细胞过度自噬和细胞凋亡，改善胰岛β细胞分泌功能。

Abstract:

Objective：Toinvestigatetheeffectoftheadipocytokinevaspinonthelevelofpalmiticacid（PA）-inducedautophagyinINS-1cells.Methods：INS-1cellsweredividedintogroupsandculturedasfollows：normalgroup：culturedinanordinarymediumfor24hours；PAgroup：culturedwith0.5mmol/LPAfor24hours；PA+vaspingroup：pre-culturedwith320ng/mlvaspinfor2hours，andthenco-culturedwith0.5mmol/LPAand320ng/mLvaspinfor24hours；PA+vaspin+rapamycingroup：pre-cul－turedwith50nmol/Lrapamycinfor2hours，andthenco-cul－turedwith0.5mmol/LPAand320ng/mLvaspinfor24hours；PA+vaspin+3-methyladenine（3-MA）group：pre-culturedwith1mmol/L3-MAfor2hours，andthenco-culturedwith0.5mmol/LPAand320ng/mLvaspinfor24hours.Theglucose-stimulatedinsulinsecretion（GSIS）test（withglucoselevelsof3.3and16.7mmol/L）wasperformedtodeterminetheinsulinlevelineachgroup；Westernblotwasusedtodeterminethelevelsofmammaliantargetofrapamycin（mTOR），p-mTOR，Beclin-1，LC3-I，LC3-II，andP62；mRFP-GFP-LC3andflowcytometrywereusedtodeterminetheautophagicflowandcellapoptosis，respectively.Results：Comparedwiththenormalgroup，thePAgrouphadsignificantlyreducedGSISlevelsintheINS-1cellsupernatant[（1.02±0.08）ng/mLvs.（0.54±0.06）ng/mL，P=0.000；（1.15±0.13）ng/mLvs.（0.71±0.05）ng/mL，P=0.000）]，whilethevaspin-treatedgrouphadsignificantlyincreasedGSISlevelsthanthePAgroup[（0.76±0.03）ng/mLvs.（0.54±0.06）ng/mL，P=0.001；（0.91±0.04）ng/mLvs.（0.71±0.05）ng/mL，P=0.011）].Comparedwiththenormalgroup，thePAgrouphadasignificantlyreducedp-mTOR-to-mTORratio（2.04±0.14vs.1.19±0.09，P=0.000），whilethevaspin-treatedgrouphadasignificantlyincreasedratiocomparedwiththePAgroup（1.51±0.05vs.1.19±0.09，P=0.005）.Comparedwiththenormalgroup，thePAgrouphadsignificantincreasesintheleveloftheautophagy-associatedproteinBeclin-1andtheratioofLC3-IItoLC3-I（0.65±0.04vs.0.81±0.07，P=0.005；1.16±0.11vs.2.35±0.25，P=0.000）andasignificantreductioninP62level（1.04±0.19vs.0.60±0.08，P=0.000）；thevaspin-treatedgrouphadsignificantdecreasesintheBeclin-1levelandtheLC3-Ⅱ-to-LC3-Ⅰratio（0.58±0.04vs.0.81±0.07，P=0.001；1.95±0.08vs.2.35±0.25，P=0.007）andasignificantincreaseinP62level（0.82±0.03vs.0.60±0.08，P=0.032）comparedwiththePAgroup.ThePAgrouphadasignificantlyhighernumberofautolysosomesthanthenormalgroup（8.33±1.53vs.0.33±0.58，P=0.000），whilethevaspin-treatedgrouphadasignificantlyreducednumberofautolysosomescom－paredwiththePAgroup（3.67±1.53vs.8.33±1.53，P=0.001）.Aftertreatmentfor24hours，thePAgrouphadasignificantlyincreasedapoptosisrateofINS-1cellscomparedwiththenormalgroup（22.15±2.05vs.7.91±0.50，P=0.000），whilethevaspin-treatedgrouphadasignificantlyreducedapoptosisratecomparedwiththePAgroup（13.23±1.97vs.22.15±2.05，P=0.000）.Conclusion：VaspincaninhibitPA-inducedexcessiveautophagyandapoptosisofINS-1cells，thusimprovingthesecretoryfunctionofisletβcells.