沉默PKCδ表达对L02肝细胞脂肪变性和内质网应激的影响

PKCδ silencing suppresses endoplasmic reticulum stress and attenuates fatty degeneration in steatotic L02 hepatocytes

目的研究蛋白激酶Cδ亚型(protein kinase Cδ,PKCδ)与肝细胞脂肪变性、内质网应激的关系,探讨其在非酒精性脂肪性肝炎(non-alcoholic steatohepatitis,NASH)发病机制中的作用。方法用混合脂肪酸构建人正常肝细胞L02脂肪变性模型,油红O染色和甘油三酯(TG)试剂盒检测细胞脂变程度,实时荧光定量PCR检测PKCδ、Bip、XBP-1s mRNA表达,Western blot检测其蛋白表达。通过siRNA瞬时转染技术沉默PKCδ在L02细胞中的表达后观察上述指标表达变化。结果油酸和棕榈酸酯2∶1混合比例能够成功诱导肝细胞脂肪变性,与对照组单个细胞脂变面积(14.47±9.28)和TG含量(2.30±0.62)μg/mg相比,脂肪酸16 h组脂变面积(333.06±42.36)和TG含量(30.86±6.24)μg/mg均显著增加(P<0.05)。脂肪酸组肝细胞PKCδ、Bip、XBP-1s mRNA和蛋白表达水平较对照组显著升高(P<0.05),具有时间依赖性。在相同脂肪酸诱导液处理的条件下,PKCδsiRNA转染组细胞脂变程度(30.92±1.29)%较对照组(55.32±6.58)%明显减轻(P<0.05),且PKCδsiRNA转染组Bip、XBP-1s的表达较对照siRNA转染组明显降低。结论 PKCδ在NASH形成发展中发挥重要作用,沉默PKCδ能够通过抑制内质网应激减轻肝细胞脂肪变性程度。

Objective To investigate the relationship of the expression of protein kinase Cδ （PKCS） with fatty degeneration and endoplasmic retieulum stress in the steatotie hepatocytes, and investigate the role of PKCδ in pathogenesis of non-alcoholic steatohepatitis （NASH）. Methods The steatosis model of human LO2 hepatocytes was established by induction with fatty acid mixture. Triglyceride （TG） kit was used to measured lipid accumulation in the hepatocytes with Oil Red O staining. The expression of PKCδ, binding immunoglobulin protein （Bip）, and spliced X-box binding protein 1 （XBP-1） at mRNA and protein levels were analyzed by real-time PCR and Western blotting respectively. After transient transfeetion of PKC8 siRNA was used to knock down PKC8 expression in LO2 cells, the above expression was detected again. Results Fatty acid mixture （ oleate-to-palmitate at 2：1 ） induced hepatic steatosis, the average LD area per cell of fatty acid 16 h group was 333.06 ± 42.36 pixels, which was apparently more than that of the control group （ 14.47±9.28 ）, and TG content was significantly higher than in control group （30.86 ± 6.24 vs 2.30 ± 0.62 μg/mg, P 〈 0.05 ）. Fatty acid mixture up-regnlated the expression of PKCS, Bip and XBP-1 in a time-dependent manner （ P 〈 0.05 ）. Knock-down of PKC8 gene expression led to a reduction in both lipid accumulation [ （30.92 ± 1.29 ）% vs （55.32± 6.58） %, P 〈 0.05 ] and the expression of Bip and XBP-1 during fatty acid mixture-induced hepatic steatosis. Conclusion PKCδ may play an important role in the prognosis of NASH. Silencing the gene expression of PKCδ can reduce the degree of fatty degeneration and endoplasmic reticulum stress.