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. 2018 Nov 30;38(11):1277-1287.
doi: 10.12122/j.issn.1673-4254.2018.11.01.

[Effects of sera of rats fed with Huganqingzhi tablets on endoplasmic reticulum stress in a HepG2 cell model of nonalcoholic fatty liver disease]

[Article in Chinese]
Miaoting Yang  1 , Zhijuan Chen  1 , Chunxin Xiao  2 , Waijiao Tang  1 , Beijie Zhou  1   3
Affiliations

[Effects of sera of rats fed with Huganqingzhi tablets on endoplasmic reticulum stress in a HepG2 cell model of nonalcoholic fatty liver disease]

[Article in Chinese]
Miaoting Yang et al. Nan Fang Yi Ke Da Xue Xue Bao. .

Abstract
in English, Chinese

Objective: To investigate the effects of sera from rats fed with Huganqingzhi tablets (HGT) on endoplasmic reticulum (ER) stress in a steatotic hepatocyte model of free fatty acids (FFAs)-induced nonalcoholic fatty liver disease (NAFLD) and explore the possible mechanism.

Methods: FFAs prepared by mixing oleic acid and palmitic acid at the ratio of 2:1. HepG2 cells were treated with the sera from rats fed with low-, moderate-or high-dose HGT (HGT sera) or sera of rats fed with fenofibrate (fenofibrate sera), followed by treatment with 1 mmol/L FFAs for 24 h to induce hepatic steatosis. Oil red O staining was used to observe the distribution of lipid droplets in the cells. The biochemical parameters including triglyceride (TG), lactated hydrogenase (LDH), aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were measured using a commercial kit. The morphological changes of the ER in the cells were observed using transmission electron microscopy. The protein/mRNA expressions of ER stress-related signal molecules including GRP78, PERK, p-PERK, ATF6, ATF4, CASPASE-12, CHOP, XBP-1, PKC, and p-PKC-δ were detected using Western blotting and/or quantitative real-time PCR (qRT-PCR). The changes in the protein expressions of GRP78, p-PERK, CASPASE-12 and CHOP were also detected in cells with transient transfection of PKC-δ siRNA for PKC-δ knockdown.

Results: Compared with the control cells, the cells treated with FFAs showed significantly increased levels of TG, AST, and ALT (P < 0.05). Compared with FFAs-treated cells, the cells pretreated with HGT sera or fenofibrate sera all showed significantly decreased TG, AST and ALT levels (P < 0.05), reduced accumulation of the lipid droplets (P < 0.05), and lowered protein or mRNA expression levels of GRP78, p-PERK, ATF6, ATF4, CHOP, CASPASE-12, XBP-1 and p-PKC-δ (P < 0.05). PKC-δ knockdown caused significantly reduced protein expressions of GRP78, p-PERK, CASPASE-12 and CHOP in the cells with FFA-induced hepatic steatosis (P < 0.001); treatment with high-dose HGT serum more significantly reduced the expressions of GRP78 (P < 0.001) and P-PERK (P < 0.01) in FFAs-induced cells with PKC-δ knockdown.

Conclusions: HGT serum can effectively prevent FFAs-induced steatosis in HepG2 cells by alleviating ER stress, in which PKC-δ may act as an important target.

目的: 探讨护肝清脂片药物血清对混合脂肪酸诱导的非酒精性脂肪肝HepG2细胞模型内质网应激（ER stress）的影响及其可能机制。

方法: 油酸和棕榈酸以2: 1比例混合制备成1 mmol/L游离脂肪酸（FFA）混合液，诱导HepG2细胞24 h发生脂肪变性，建立NAFLD体外模型，将HepG2细胞分为对照组（CON）、FFA组、护肝清脂片低剂量组（HG-L）、护肝清脂片中剂量组（HGM）、护肝清脂片高剂量组（HG-H）及非诺贝特阳性对照组（FF），在加入1 mmol/L FFA前分别加入各组对应的药物血清作用24 h，CON组及FFA组则加入大鼠空白血清作为对照。油红O染色观察细胞内脂滴分布；试剂盒检测细胞内甘油三酯（TG）含量及培养上清液谷草转氨酶（AST/GOT）、谷丙转氨酶（ALT/GPT）的水平；透射电镜观察细胞内质网形态结构的改变；Western blot、qRT-PCR技术检测ERS相关信号分子GRP78、PERK、p-PERK、ATF6、ATF4、XBP-1、CASPASE-12、CHOP、PKC-δ、p-PKC-δ蛋白及（或）mRNA的表达情况。利用siRNA瞬时转染技术沉默PKC-δ在HepG2细胞中的表达后观察GRP78、PERK、p-PERK、CASPASE-12和CHOP蛋白表达变化。

结果: 与CON组相比，FF组HepG2细胞油红染色可见红色脂滴密布细胞质，TG、AST、ALT含量升高（P < 0.001）；与FF组相比，HG-L、HG-M、HG-H及FF均在不同程度上降低TG、AST、ALT含量，减少细胞内脂滴堆积，差异有统计学意义（P < 0.05），并能够在蛋白及mRNA水平上有效地下调ERS相关信号分子GRP78、p-PERK、ATF6、ATF4、CHOP、CASPASE-12、XBP-1、PKC-δ、p-PKC-δ的表达，差异有统计学意义（P < 0.05）。在1 mmol/L FFA共同作用下，PKC-δ siRNA转染组GRP78、p-PERK、CHOP、CASPASE-12蛋白表达均较对照组明显下调，差异具有统计学意义（P < 0.001）。然而，与PKC-δ siRNA +FFA组相比，PKC-δ siRNA+HG-H组GRP78和P-PERK表达下调更显著（P < 0.001，0.01），CHOP和CASPASE-12则无统计学差异（P>0.05）。

结论: 护肝清脂片药物血清可以有效地防治混合脂肪酸诱导的NAFLD细胞模型脂肪变性，改善HepG2功能状态，有效地缓解1 mmol/L游离脂肪酸所致的内质网应激，其作用机制在一定程度上与下调PKC-δ表达有关。

Keywords: Huganqingzhi tablets; endoplasmic reticulum stress; nonalcoholic fatty liver disease; protein kinase C-δ; steatotic hepatocytes.

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Figures

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不同浓度FFA对HepG2细胞生长的影响 Effects of Free fatty acids at different concentrations on viability of HepG2 hepatocytes (Mean±SD). nsP>0.05, ***P < 0.001 vs control group.
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FFA诱导的肝脂肪变性HepG2细胞上清LDH含量的测定 LDH release assay of the cells with different treatments (Mean±SD). A: Cells treated with normal rat serum at different concentrations; B: Cells treated with 10% medicated serum. nsP> 0.05, ***P < 0.001 vs control group.
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护肝清脂片药物血清对FFA诱导的肝脂肪变性HepG2细胞的油红O染色 Effects of Huganqingzhi (HGT)-medicated serum on FFA-stimulated HepG2 hepatocytes (Oil Red O staining, original magnification: ×200).A: CON; B: FFA; C: FFA+10% HG-L; D: FFA+10% HG-M; E: FFA+10% HG-H; F: FFA+10% FF.
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油红O染色法定量测定细胞内脂质含量 Lipid content in the cells determined by Oil red-based colorimetric assay. ***P < 0.001 vs CON; nsP>0.05, #P < 0.05, ##P < 0.01, ###P < 0.001 vs FFA(Mean±SD).
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护肝清脂片药物血清对FFA诱导的肝脂肪变性HepG2细胞TG、AST、ALT含量的影响 Effects of Huganqingzhi (HGT)-medicated serum on TG content (A) and AST/ALT (B) level in FFAs-stimulated HepG2 hepatocytes (Mean ± SD). ***P < 0.001 vs CON; nsP > 0.05, #P < 0.05, ##P < 0.01, ###P < 0.01 vs FFA.
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透射电镜观察各组HepG2细胞内质网结构变化 Transmission electron microscopy of the ER in each group of HepG2 hepatocytes (×1700, ×5000). A-C: CON; D-F: FFA; G-I: FFA+10% HG-H.
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护肝清脂片药物血清对FFA诱导24 h后的GRP78、PERK、p-PERK、ATF6、ATF4、CASPASE-12、CHOP的表达情况的影响 Effects of Huganqingzhi (HGT)-medicated serum on the expressions of GRP78 (A, B), p-PERK/PERK (A, C), ATF6 (A, D), ATF4 (A, E), CASPASE-12 (A, F), and CHOP (A, G) in FFAs-stimulated HepG2 hepatocytes. ***P < 0.001 vs CON; nsP>0.05, #P < 0.05, ##P < 0.01, ###P < 0.01 vs FFA.
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护肝清脂片药物血清对FFA诱导24 h后的GRP78、PERK、ATF6、XBP-1、CASPASE-12、CHOP基因的表达情况的影响 Effects of Huganqingzhi (HGT)-medicated serum on expressions of GRP78 (A), PERK (B), ATF6 (C), XBP-1 (D), CASPASE-12 (E), and CHOP (F) mRNAs in FFAs-stimulated HepG2 hepatocytes. ***P < 0.001 vs CON; nsP>0.05, #P < 0.05, ##P < 0.01, ###P < 0.01 vs FFA.
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护肝清脂片药物血清对FFA诱导24 h的脂肪变性HepG2细胞PKC-δ、p-PKC-δ蛋白及PKC-δ mRNA表达情况的影响 Effects of Huganqingzhi (HGT)-medicated serum on expressions of PKC-δ protein, p-PKC-δ protein, p-PKC-δ/PKC-δ (A and B), and PKC-δ mRNA (C) in FFAs-stimulated HepG2 hepatocytes. ***P < 0.001 vs CON; nsP>0.05, #P < 0.05, ##P < 0.01, ###P < 0.01 vs FFA.
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沉默PKC-δ后，HepG2细胞PKC-δ、p-PKC-δ蛋白表达情况 Expression of total and phosphorylated PKC-δ after treatment with PKC-δ siRNA. ***P < 0.001 vs si-control group.
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沉默PKC-δ后，HepG2细胞GRP78、PERK、p-PERK、CASPASE-12、CHOP蛋白表达情况 Expression of GRP78, PERK, p-PERK, CASPASE-12 and CHOP after treatment with PKC-δ siRNA. ***P < 0.001 vs si-Control group; #P < 0.05, ##P < 0.01, ###P < 0.001 vs si-Control+FFA group; &&P < 0.01, &&&P < 0.001 vs si-PKC-δ group; nsP>0.05,&*P < 0.01, &&*P < 0.001 vs si-PKC-δ+FFA vs si-PKC-δ+FFAgroup.
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