Abstract：
BACKGROUND: Hepatic fibrosis is a reversible pathological phenomenon caused by the abnormal proliferation of connective tissues in the liver for self-repair after persistent liver injury. Among these tissues, the activation status of hepatic stellate cells (HSCs) is crucial. Glycyrrhizic acid (GA) agents have been proven to have excellent anti-fibrosis effects, but their targets are unclear.
OBJECTIVE: To investigate the anti-hepatic fibrosis effect of GA and its target in activated HSCs.
METHODS: A mouse model of hepatic fibrosis was prepared with 20 % carbon tetrachloride (CCl4) and GA was administered continuously for 4 weeks. Subsequently, the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), type Ⅲ procollagen peptide (P III P), laminin (LN), hyaluronic acid (HA), and type Ⅳ collagen (Col Ⅳ) were measured. Liver tissues were subjected to hematoxylin and eosin (HE), Masson, and Sirius red staining and proteome sequencing analysis. Based on LX-2 cells, activity-based protein profiling (ABPP) was used to investigate the potential targets of GA, which was further validated by the cellular thermal shift assay (CETSA), immunofluorescence co-localization, molecular docking, small interfering RNA (siRNA) and western blot (WB) assays.
RESULTS: In vivo, GA significantly reduced serum ALT, AST, HA, P III P, Col IV, and LN levels. HE, Masson, and Sirius red staining showed that GA significantly ameliorated hepatic inflammatory response and collagen deposition in CCl4-treated mice. Proteome sequencing results showed that GA mainly regulated glutathione S-transferase family members involved in glutathione metabolism. In vitro, GA significantly inhibited LX-2 cell proliferation and reduced reactive oxygen species accumulation. ABPP suggested that aldo-keto reductase family 7 member A2 (AKR7A2) was the major binding protein of GA in LX-2 cells. CETSA, fluorescence co-localization, molecular docking, and surface plasmon resonance further validated GA binding to AKR7A2. The WB results showed that GA up-regulated AKR7A2 expression both in vitro and in vivo and was corroborated by siRNA experiments.
CONCLUSIONS: GA targeted AKR7A2 in LX-2 cells to defend against sustained oxidative stress injury, thereby inhibiting the proliferation of activated HSCs and reversing hepatic fibrosis.
OBJECTIVE: To investigate the anti-hepatic fibrosis effect of GA and its target in activated HSCs.
METHODS: A mouse model of hepatic fibrosis was prepared with 20 % carbon tetrachloride (CCl4) and GA was administered continuously for 4 weeks. Subsequently, the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), type Ⅲ procollagen peptide (P III P), laminin (LN), hyaluronic acid (HA), and type Ⅳ collagen (Col Ⅳ) were measured. Liver tissues were subjected to hematoxylin and eosin (HE), Masson, and Sirius red staining and proteome sequencing analysis. Based on LX-2 cells, activity-based protein profiling (ABPP) was used to investigate the potential targets of GA, which was further validated by the cellular thermal shift assay (CETSA), immunofluorescence co-localization, molecular docking, small interfering RNA (siRNA) and western blot (WB) assays.
RESULTS: In vivo, GA significantly reduced serum ALT, AST, HA, P III P, Col IV, and LN levels. HE, Masson, and Sirius red staining showed that GA significantly ameliorated hepatic inflammatory response and collagen deposition in CCl4-treated mice. Proteome sequencing results showed that GA mainly regulated glutathione S-transferase family members involved in glutathione metabolism. In vitro, GA significantly inhibited LX-2 cell proliferation and reduced reactive oxygen species accumulation. ABPP suggested that aldo-keto reductase family 7 member A2 (AKR7A2) was the major binding protein of GA in LX-2 cells. CETSA, fluorescence co-localization, molecular docking, and surface plasmon resonance further validated GA binding to AKR7A2. The WB results showed that GA up-regulated AKR7A2 expression both in vitro and in vivo and was corroborated by siRNA experiments.
CONCLUSIONS: GA targeted AKR7A2 in LX-2 cells to defend against sustained oxidative stress injury, thereby inhibiting the proliferation of activated HSCs and reversing hepatic fibrosis.
摘要：
背景：肝纤维化是在持续的肝损伤后，由于肝脏中结缔组织的异常增殖进行自我修复而引起的一种可逆性病理现象。在这些组织中，肝星状细胞（HSC）的活化状态至关重要。甘草酸（GA）剂已被证明具有优异的抗纤维化作用，但他们的目标还不清楚.
目的：探讨GA及其靶点对活化的HSCs的抗肝纤维化作用。
方法：用20％四氯化碳（CCl4）制备小鼠肝纤维化模型，连续给药GA4周。随后，丙氨酸氨基转移酶（ALT）的水平，天冬氨酸转氨酶（AST），Ⅲ型前胶原肽(PⅢP),层粘连蛋白(LN),透明质酸（HA），并测定Ⅳ型胶原(ColⅣ)。肝脏组织接受苏木精和伊红（HE），Masson,天狼星红染色和蛋白质组测序分析。基于LX-2细胞，基于活性的蛋白质谱分析（ABPP）用于研究GA的潜在靶标，通过细胞热转移试验（CETSA）进一步验证，免疫荧光共定位，分子对接,小干扰RNA（siRNA）和蛋白质印迹（WB）测定。
结果：体内，GA显著降低血清ALT，AST,HA,PIIIP,ColIV,LN水平。他,Masson,天狼星红染色显示GA显著改善CCl4处理小鼠的肝脏炎症反应和胶原沉积。蛋白质组测序结果表明，GA主要调节参与谷胱甘肽代谢的谷胱甘肽S-转移酶家族成员。体外，GA显著抑制LX-2细胞增殖并减少活性氧积累。ABPP提示醛酮还原酶家族7成员A2（AKR7A2）是LX-2细胞中GA的主要结合蛋白。CETSA,荧光共定位，分子对接,和表面等离子体共振进一步验证了GA与AKR7A2的结合。WB结果显示GA在体外和体内上调AKR7A2表达,并通过siRNA实验得到证实。
结论：GA靶向LX-2细胞中的AKR7A2以防御持续的氧化应激损伤，从而抑制活化的HSCs的增殖并逆转肝纤维化。
目的：探讨GA及其靶点对活化的HSCs的抗肝纤维化作用。
方法：用20％四氯化碳（CCl4）制备小鼠肝纤维化模型，连续给药GA4周。随后，丙氨酸氨基转移酶（ALT）的水平，天冬氨酸转氨酶（AST），Ⅲ型前胶原肽(PⅢP),层粘连蛋白(LN),透明质酸（HA），并测定Ⅳ型胶原(ColⅣ)。肝脏组织接受苏木精和伊红（HE），Masson,天狼星红染色和蛋白质组测序分析。基于LX-2细胞，基于活性的蛋白质谱分析（ABPP）用于研究GA的潜在靶标，通过细胞热转移试验（CETSA）进一步验证，免疫荧光共定位，分子对接,小干扰RNA（siRNA）和蛋白质印迹（WB）测定。
结果：体内，GA显著降低血清ALT，AST,HA,PIIIP,ColIV,LN水平。他,Masson,天狼星红染色显示GA显著改善CCl4处理小鼠的肝脏炎症反应和胶原沉积。蛋白质组测序结果表明，GA主要调节参与谷胱甘肽代谢的谷胱甘肽S-转移酶家族成员。体外，GA显著抑制LX-2细胞增殖并减少活性氧积累。ABPP提示醛酮还原酶家族7成员A2（AKR7A2）是LX-2细胞中GA的主要结合蛋白。CETSA,荧光共定位，分子对接,和表面等离子体共振进一步验证了GA与AKR7A2的结合。WB结果显示GA在体外和体内上调AKR7A2表达,并通过siRNA实验得到证实。
结论：GA靶向LX-2细胞中的AKR7A2以防御持续的氧化应激损伤，从而抑制活化的HSCs的增殖并逆转肝纤维化。
