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Title: [Effects of celastrol on autophagy and endoplasmic reticulum stress-mediated apoptosis in a mouse model of nonalcoholic fatty liver disease].
Author: Tian T, Liao XC, Zhang M, Wu XM, Guo YT, Tan SY.
Journal: Zhonghua Gan Zang Bing Za Zhi; 2022 Jun 20; 30(6):656-662. PubMed ID: 36038329.
Abstract:
Objective: To investigate the effect of celastrol (CEL) on autophagy and endoplasmic reticulum stress-mediated apoptosis in a mouse model of nonalcoholic fatty liver disease (NAFLD). Methods: Eighteen male C57BL/6J mice were randomly divided into normal control (NC, n=6), high-fat diet (HFD, n=6) and celastrol group (HFD+CEL, n=6). The normal control group was fed with regular diet, and the high-fat diet and celastrol group were fed with high-fat diet for 12 weeks. After successful modeling, celastrol group were injected with 100 μg⋅kg^-1⋅d^-1 celastrol intraperitoneally for 4 weeks, and NC and HFD group were injected intraperitoneally with the same doses of normal saline. Serum concentrations of alanine aminotransferase (ALT), aspartate aminotransferase (AST), triglyceride (TG), total cholesterol (TC), high density lipoprotein cholesterol (HDL-C) and low density lipoprotein cholesterol (LDL-C) were measured in mouse after 4-weeks of intervention. HE and Oil Red O staining were used to observe the pathomorphological changes and lipid droplet deposition in mouse liver, and the findings were scored according to NAFLD activity score (NAS). Western blot was used to detect the expression levels of liver microtubule associated protein 1 light chain 3 (LC3), P62, glucose-regulated protein 78 (GRP78), protein kinase R-like endoplasmic reticulum kinase (PERK), phosphorylated PERK (p-PERK), activated transcription factor 4 (ATF4), C/EBP homologous protein (CHOP), cleaved Caspase-3(cleaved caspase-3), B-cell lymphoma-2 (Bcl-2) and Bcl-2 related X protein (Bax).TUNEL staining was used to observe the apoptosis of hepatocytes. One-way analysis of variance was used for the intergroup comparison. Results: Serum levels of ALT (68.71±8.57) U/L, AST (209.63±28.64) U/L, TG (0.97±0.14) mmol/L, TC (4.12±0.64) mmol/L, and LDL -C (0.40±0.06) mmol/L were lower in celastrol group mouse than HFD group [(110.19±10.79) U/L, (399.72±73.47) U/L, (1.44±0.13) mmol/L, (5.65±0.54) mmol /L, (0.61±0.07) mmol/L] (P<0.05); while the serum HDL-C level (1.29±0.17) mmol/L was higher in celastrol than HFD group (0.72±0.13) mmol/L (P<0.05). HE and Oil Red O staining showed that lipid deposition and intralobular inflammation were apparent in the liver tissue of HFD group mouse, and the NAS score was significantly increased, while the hepatocyte steatosis and intralobular inflammation were alleviated after celastrol intervention, and the NAS score was decreased significantly (P<0.05). Compared with HFD group, the ratio of LC3II/I was significantly increased in the liver of celastrol group mouse, and the P62 was significantly decreased (P<0.05). Meanwhile, the expression level of GRP78, p-PERK/PERK , ATF4, and CHOP was significantly lower in celastrol than HFD group (P<0.05). In addition, the expressions of cleaved caspase-3 and Bax were significantly lower in celastrol than HFD group, and the expression of Bcl-2 was significantly increased (P<0.05). At the same time, the apoptosis rate of hepatocytes was also significantly lower in celastrol than HFD group (P<0.05). Conclusion: Celastrol can effectively alleviate the lipid deposition, protect hepatocytes and delay the progression of non-alcoholic fatty liver disease in mouse liver with non-alcoholic fatty liver disease. In addition, its mechanism of action may be related to the induction of autophagy, inhibition of endoplasmic reticulum stress PERK/ATF4/CHOP pathway and its mediated apoptosis. 目的： 探讨雷公藤红素（CEL）对非酒精性脂肪性肝病（NAFLD）小鼠模型自噬及内质网应激介导的凋亡的影响。 方法： 将18只雄性C57BL/6J小鼠随机分为正常对照组（NC，n=6）、高脂饮食组（HFD，n=6）和CEL组（HFD+CEL，n=6）。正常对照组给予普通饲料喂养，高脂饮食组和CEL组给予高脂饲料喂养12周。造模成功后，CEL组给予100 μg·kg^-1·d^-1的CEL溶液腹腔注射4周，NC组和HFD组给予等剂量的等渗盐水腹腔注射。4周干预结束后，检测小鼠血清丙氨酸转氨酶（ALT）、天冬氨酸转氨酶（AST）、甘油三酯（TG）、总胆固醇（TC）、高密度脂蛋白胆固醇（HDL-C）和低密度脂蛋白胆固醇（LDL-C）的含量。HE染色和油红O染色观察肝脏病理学形态改变及脂滴沉积，并根据NAFLD活动度积分（NAS）评分。蛋白质印迹法检测肝脏微管相关蛋白1轻链3（LC3）、P62、葡萄糖调节蛋白78（GRP78）、蛋白激酶R样内质网激酶（PERK）、磷酸化PERK（p-PERK）、激活转录因子4（ATF4）、C/EBP同源蛋白（CHOP）、剪切的半胱氨酸天冬氨酸蛋白酶3（cleaved caspase-3）、B细胞淋巴瘤-2（Bcl-2）及Bcl-2相关X蛋白（Bax）的表达水平。TUNEL染色观察肝细胞凋亡情况。组间比较采用单因素方差分析。 结果： CEL组小鼠血清ALT（68.71±8.57）U/L、AST（209.63±28.64）U/L、TG（0.97±0.14）mmol/L、TC（4.12±0.64）mmol/L、LDL-C（0.40±0.06）mmol/L水平均低于HFD组［（110.19±10.79）U/L、（399.72±73.47）U/L、（1.44±0.13）mmol/L、（5.65±0.54）mmol/L、（0.61±0.07）mmol/L］（P＜0.05）；CEL组血清HDL-C（1.29±0.17）mmol/L水平高于HFD组的（0.72±0.13）mmol/L（P＜0.05）。苏木精-伊红染色及油红O染色显示HFD组小鼠肝组织内出现脂质沉积及小叶内炎症，NAS评分明显增加，而CEL干预后肝细胞脂肪变及小叶内炎症缓解，NAS评分明显降低（P＜0.05）。CEL组小鼠肝脏内微管相关蛋白1轻链3（LC3）II/I比率较HFD组显著增加，P62显著降低（P＜0.05）；同时，CEL组GRP78、p-PERK/PERK、ATF4、CHOP表达水平较HFD组明显降低（P＜0.05）；此外，CEL组cleaved caspase-3、Bax表达较HFD组明显降低，Bcl-2表达明显增加（P＜0.05）。同时，CEL组肝细胞凋亡率较HFD组也明显降低（P＜0.05）。 结论： CEL可有效减轻NAFLD小鼠肝脏的脂质沉积，保护肝细胞并延缓NAFLD的进展，其作用机制可能与诱导自噬、抑制内质网应激PERK/ATF4/CHOP通路及其介导的细胞凋亡有关。.

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