漆树酸抑制组蛋白H3K9乙酰化对高果糖诱导大鼠主动脉平滑肌细胞表型转化的影响

The effect of anacardic acid inhibiting histone H3K9 acetylation on the phenotypic transformation of rat aortic vascular smooth muscle cells induced by high fructose

    摘要
  • 摘要: 目的 研究漆树酸抑制组蛋白H3K9乙酰化修饰对高果糖诱导血管平滑肌细胞（VSMC）表型转化的影响及其可能的机制。方法 组织块贴壁法培养SD大鼠主动脉VSMC,细胞计数试剂（CCK-8）法检测果糖对VSMC增殖的影响，确定果糖干预浓度，进一步检测不同浓度漆树酸对高果糖致VSMC增殖的影响，确定漆树酸干预浓度。采用Western-blot、免疫荧光等实验方法探究漆树酸抑制组蛋白H3K9乙酰化修饰对高果糖诱导VSMC表型转化标志蛋白和核因子κB（NF-κB）/核苷酸结合寡聚结构域样受体蛋白3（NLRP3）表达的影响。结果 果糖浓度在5～30 mmol/L干预48、72 h可促进VSMC增殖（P<0.05）,其中浓度为10 mmol/L作用72 h可显著促进VSMC增殖；漆树酸以浓度依赖的方式抑制高果糖诱导的VSMC增殖。Spearman相关分析显示漆树酸浓度与细胞抑制率呈正相关（rs=0.98,P<0.05）,其半数抑制浓度(IC50)为65.34（95%CI 60.60～70.45）μmol/L。高果糖致使VSMC的收缩表型标志蛋白α-平滑肌肌动蛋白（α-SMA）、平滑肌22α（SM22α）、调宁蛋白（Calponin）表达减少（P<0.05）,Ⅰ型胶原（ColⅠ）、纤维连接蛋白（FN）、合成表型标志蛋白骨桥蛋白（OPN）、组蛋白乙酰转移酶P300/CBP相关因子（PCAF）、组蛋白H3第9位赖氨酸乙酰化蛋白（H3K9ac）、p-NF-κBp65、NLRP3表达增加（P<0.05）。漆树酸可使α-SMA、SM22α、Calponin表达增加（P<0.05）,ColⅠ、FN、OPN、PCAF、H3K9ac、p-NF-κBp65、NLRP3表达减少（P<0.05）。结论 漆树酸抑制高果糖诱导大鼠主动脉VSMC收缩表型向合成表型转化，其机制可能与漆树酸抑制PCAF介导的H3K9乙酰化修饰和NF-κB/NLRP3表达有关。

     

    Abstract: Objective To study the effect of anacardic acid inhibiting histone H3K9 acetylation modification on the phenotypic transformation of rat aortic vascular smooth muscle cells（VSMC） induced by high fructose and its possible mechanism. Methods The VSMC of SD rat were cultured by tissue adherent method. The effect of different fructose concentrations on the proliferation of VSMC was detected by CCK-8 assay to determine the fructose intervention concentration. Further, the effect of different concentrations of anacardic acid on the proliferation of high fructose-induced VSMC was detected to determine the intervention concentration of anacardic acid. Western blot and immunofluorescence were used to investigate the effect of anacardic acid inhibiting histone H3K9 acetylation modification on the expression of protein of phenotypic transformation and NF-κB/nucleotide binding oligomerization domain-like receptor protein 3（NLRP3） of rat VSMC induced by high fructose. Results Treatment with fructose of 5-30 mmol/L for 48 h and 72 h enhanced the proliferation of VSMC（P＜0.05） and the effect of fructose 10 mmol/L for 72 h was the most significant. Anacardic acid inhibited the proliferation of VSMC induced by high fructose in a concentration-dependent manner. Spearman correlation analysis showed that the concentration of Anacardic acid was positively correlated with cell inhibition rate（rs=0.98, P＜0.05） and the half inhibitory concentration(IC50) was 65.34（95%CI 60.60-70.45） μmol/L. High fructose made the expression of α-smooth muscle actin（α-SMA）, smooth muscle 22α（SM22α） and Calponin which are contractive biomarkers decreased（P＜0.05）, while the expression of collagen Ⅰ（Col Ⅰ）, fibronectin（FN）, osteopontin（OPN） which is synthetic biomarker, P300/CBP related factor（PCAF）, Histone 3 acetylation K9（H3K9ac）, p-NF-κBp65 and NLRP3 increased（P＜0.05）. Anacardic acid made the expression of α-SMA, SM22α, Calponin increased（P＜0.05）, while the expression of Col Ⅰ, FN, OPN, PCAF, H3K9ac, p-NF-κBp65 and NLRP3 decreased（P＜0.05）. Conclusion Anacardic acid inhibits high fructose-induced aortic VSMC phenotypic transformation from a contractile to a synthetic type in rats and the mechanism may be related to the inhibition of PCAF-mediated H3K9 acetylation and NF-κB/NLRP3 expression by anacardic acid.

     

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