漆树酸抑制组蛋白乙酰化修饰对高盐诱导大鼠主动脉血管平滑肌细胞表型转化的影响

The effect of inhibition of histone acetylation modification by anacardic acid on the phenotypic transformation of rat aortic vascular smooth muscle cells induced by high salt

    摘要
  • 摘要: 目的 研究漆树酸抑制组蛋白乙酰化修饰对高盐诱导大鼠主动脉血管平滑肌细胞（VSMC）表型转化的影响。方法 组织贴壁法原代培养SD大鼠主动脉VSMC,免疫荧光鉴定，细胞计数试剂盒-8（CCK-8）法检测不同浓度漆树酸对高盐诱导VSMC增殖的影响，确定漆树酸干预浓度。分为对照组（Na+ 139 mmol/L）、高盐组（Na+ 159 mmol/L）、高盐+漆树酸组，采用划痕、酶联免疫吸附试验（ELISA）、Western blot等实验方法，研究漆树酸抑制组蛋白乙酰化修饰对高盐诱导VSMC表型转化的影响。结果 与对照组相比，高盐组VSMC迁移增加（P<0.05）,Ⅰ型胶原蛋白含量增加（P<0.05）,α-平滑肌肌动蛋白（α-SMA）、平滑肌22α（SM22α）、调宁蛋白表达减少（均P<0.05）,Ⅰ型胶原、纤维连接蛋白（FN）、骨桥蛋白、组蛋白乙酰化酶P300/CBP相关因子（PCAF）、组蛋白H3第9位赖氨酸乙酰化蛋白（H3K9ac）表达增加（均P<0.05）。漆树酸可抑制高盐诱导的VSMC增殖，Pearson相关分析显示，漆树酸浓度与细胞抑制率呈正相关（r=0.984,P<0.05）,即呈剂量依赖性，50%抑制率浓度(IC50)为49.09（95%CI 46.60～51.71）μmol/L,选择50μmol/L漆树酸干预VSMC。与高盐组相比，高盐+漆树酸组VSMC迁移减少（P<0.05）,Ⅰ型胶原蛋白表达减少（P<0.05）,α-SMA、SM22α、调宁蛋白表达增加（均P<0.05）,Ⅰ型胶原、FN、骨桥蛋白、PCAF、H3K9ac表达减少（均P<0.05）。结论 组蛋白乙酰化修饰可能参与高盐诱导的SD大鼠主动脉VSMC表型转化，组蛋白乙酰化酶抑制剂漆树酸可能通过抑制PCAF,降低H3K9ac表达，从而抑制高盐诱导的SD大鼠主动脉VSMC表型转化。

     

    Abstract: Objective To study the effect of anacardic acid（AA） inhibiting histone acetylation modification on the phenotypic transformation of rat aortic vascular smooth muscle cells（VSMC） induced by high salt. Methods SD rat aorta VSMCs were primary cultured by tissue adherence method and identified by immunofluorescence method. Cell counting kit-8（CCK-8）was used to detect the effect of different concentrations of AA on the proliferation of VSMC induced by high salt and to determine the intervention concentration of AA. The cells were treated with normal salt（NS, Na+ 139 mmol/L）, high salt（HS group, Na+ 159 mmol/L） and high salt+anacardic acid（HS+AA group）. Scratch, enzyme linked immunosorbent assay（ELISA） and Western blot were used to study the effect of AA inhibiting histone acetylation modification on the phenotypic transformation of VSMC induced by high salt. Results Compared with the NS group, the migration of VSMC increased（P＜0.05）, the expression of collagen Ⅰ（Col Ⅰ） secreted protein increased（P＜0.05）, the expression of α-smooth muscle actin（α-SMA）, smooth muscle 22α（SM22α） and calponin all decreased（P＜0.05）, while the expression of Col Ⅰ, fibronectin（FN）, osteopontin（OPN）, P300/CBP associated factor（PCAF） and histone 3 acetylation K9（H3 K9 ac） all increased in the HS group（P＜0.05）. AA could inhibit the proliferation of VSMC induced by high salt. Pearson correlation analysis showed that the concentration of AA was positively correlated with the cell inhibition rate（r=0.984, P＜0.05）. The 50% inhibition rate concentration(IC50) was 49.09（95% CI 46.60-51.71） μmol/L, therefore, 50 μmol/L was selected as the intervetion concentration. Compared with the HS group, the migration of VSMC decreased（P＜0.05）, the expression of Col Ⅰ secreted protein decreased（P＜0.05）, the expression of α-SMA, SM22α and calponin all increased（P＜0.05）, while the expression of Col Ⅰ, FN, OPN, PCAF and H3 K9 ac all decreased in the HS+AA group（P＜0.05）. Conclusions Histone acetylation modification is involved in the phenotypic transformation of SD rat aorta VSMC induced by high salt. Histone acetylase inhibitor AA can inhibit PCAF and reduce the expression of H3 K9 ac, thereby inhibits phenotypic transformation of SD rat aorta VSMC induced by high salt.

     

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