白桦脂酸对脂肪变性细胞RAGE/NF-κB/iNOS信号通路表达的影响

Effect of Betulic Acid on RAGE/NF-κB/iNOS Signaling Pathway Expression in Steatosis Cell

目的:探讨白桦脂酸(BA)对脂肪变性的LO2细胞的作用。方法:以梯度浓度(0,10,20,40,80,160,250μmol·L^-1)的BA干预LO2细胞24 h,应用噻唑蓝(MTT)比色法观察各组细胞的活力,确定BA干预终浓度。设置空白组,模型组,二甲基亚砜组(DMSO组)和BA干预组(BA组);BA组依据干预终浓度又分为低、中、高浓度3个亚组。首先以含10%Lipid Mix 1培养基培养模型组,DMSO组及BA组的LO2细胞24 h建立非酒精性脂肪肝(NAFLD)离体模型,空白组以不含药培养基培养24 h。然后分别以含0.1%DMSO,20,40,80μmol·L^-1BA培养基干预DMSO组及BA低、中、高浓度组细胞24 h,正常组和模型组予不含药培养基继续培养24 h。干预结束后以油红O染色及尼罗红染色法观察各组细胞内脂滴沉积变化;免疫荧光法检测细胞内诱导型一氧化氮合酶(i NOS)蛋白表达;蛋白免疫印迹法(Western blot)检测细胞氧化应激相关蛋白糖基化终产物受体(RAGE),核转录因子-κB(NF-κB p65)和i NOS的表达。结果:80μmol·L^-1浓度以内的BA干预LO2细胞24 h对细胞无明显毒性作用。与空白组比较,模型组细胞内脂滴沉积明显增多,氧化应激相关蛋白RAGE,NF-κB p65和i NOS表达均明显增加(P<0.05)。与模型组比较,DMSO组细胞内脂滴沉积与模型组相似,两组间各蛋白表达水平无明显差异;而BA组各组细胞内脂滴沉积则均明显减少,高浓度组细胞内RAGE,NF-κB p65及i NOS蛋白表达均明显减少(P<0.05,P<0.01)。结论:BA能显著改善LO2细胞内脂滴沉积,其机制可能通过抑制LO2细胞内氧化应激相关蛋白RAGE,NF-κB p65和i NOS的表达有关。

Objective:To investigate the effect of betulic acid(BA)on steatosis LO2 cells.Method:LO2 cells were intervened with BA at different gradient concentrations(0,10,20,40,80,160,250μmol·L^^-1)for 24 hours.methyl thiazolyl tetrazolium(MTT)staining was used to observed cell viability to determine the final concentration of BA.The cells were divided into control,model,dimethylsulfoxide(DMSO)and BA groups,as well as BA groups intervened with low,middle and high concentrations.First,model,DMSO and BA group’s cells were cultured in 10%Lipid Mix 1 medium for 24 hours to establish a nonalcoholic fatty liver model.Then,DMSO group and low,medium and high-concentration groups were separately cultured with 0.1%DMSO medium and 20,40,80μmol·L^^-1BA medium for 24 hours.And control and model groups were cultured in drug-free medium for24 hours.Oil red O staining and Nile red staining were used to observe the intracellular lipid droplets.Immunofluorescence was used to detect the protein expression of inducible nitric oxide synthase(i NOS).Western blot was used to detect the protein expression levels of receptor for advanced glycation end-products(RAGE),nuclear factorκB p65(NF-κB p55)and i NOS.Result:BA within the concentration of 80μmol·L^^-1had no significant toxicity on LO2 cells.Compared with control group,the intracellular lipid droplets were significantly increased in the model group,and the expressions of oxidative stress-related proteins RAGE,NF-κB p65 and i NOS also increased significantly(P<0.05).Compared with model group,the intracellular lipid droplets in DMSO group were similar to those in model group,with no significant difference in the three protein expressions between the two groups.However,the intracellular lipid droplets deposition in the BA group was significantly decreased.And the expressions of RAGE,NF-κB p65 and i NOS proteins in high-concentration BA group were significantly decreased(P<0.05,P<0.01).Conclusion:BA can significantly improve the intracellular fat deposition in LO2 cells,which was probably related to the inhibition of the expressions of oxidative stress-related proteins RAGE,NF-κB p65 and i NOS.