目的 探讨葛根芩连汤（GQD）是否通过P53/SAT1/ALOX15信号通路调控铁死亡改善棕榈酸（PA）诱导的AML-12细胞胰岛素抵抗（IR）。方法 网络药理学筛选GQD干预IR的相关靶点，构建蛋白质蛋白质相互作用网络并进行基因本体论（GO）和京都基因与基因组百科全书（KEGG）富集分析。采用PA联合胰岛素处理AML-12细胞构建IR模型，通过CCK-8法筛选PA最佳造模浓度；模型验证阶段分为对照组、胰岛素组、PA组、模型组，试剂盒法检测细胞内葡萄糖水平，Western blotting法检测蛋白激酶B（Akt）、p-Akt、胰岛素受体底物1（IRS-1）、p-IRS-1蛋白表达水平验证模型成功。将60只大鼠随机分为空白（蒸馏水）组30只、GQD(生药10 g·kg^-1)组20只、盐酸吡格列酮(Pio，阳性药，2.7 mg·kg^-1)组10只，每组大鼠每天给药2次，连续ig 7 d，第8天末次给药后0.5 h腹主动脉取血，制得各组血清。干预实验分为对照组、模型组、Pio（10%含药血清）组和GQD含药血清高、中、低浓度（20%、15%、10%）组，干预后，CCK-8法检测细胞存活率；培养上清液中葡萄糖消耗；进行油红O染色；试剂盒法检测细胞三酰甘油（TG）、总胆固醇（TC）、游离脂肪酸（FFA）、亚铁离子(Fe²⁺)、丙二醛（MDA）、谷胱甘肽（GSH）、谷胱甘肽过氧化物酶（GSH-Px）水平，实时荧光定量PCR（qRT-PCR）法检测GPX4、SLC7A11、FTH1、LPCAT3、TFR1、P53、SAT1、ALOX15、ACSL4基因表达，Western blotting法检测GPX4、SLC7A11、FTH1、P53、SAT1、ALOX15、ACSL4蛋白表达。结果 网络药理学分析显示，GQD与IR的交集靶点主要富集于P53信号、脂质代谢和氧化应激等通路。CCK-8结果表明，200μmol·L^-1 PA为最佳造模浓度，模型验证结果显示造模成功。GQD干预后，与模型组相比，GQD各浓度组及Pio组细胞存活率均显著升高（P<0.05、0.01、0.001）；细胞上清液葡萄糖消耗显著升高（P<0.05、0.01）；油红O染色显示脂质沉积明显减少，TG、TC、FFA水平显著降低（P<0.05、0.01）;Fe²⁺、MDA水平显著降低（P<0.05、0.01）,GSH、GSH-Px水平显著升高（P<0.05、0.01）;qRT-PCR结果显示，GPX4、SLC7A11、FTH1基因表达显著上调（P<0.05、0.01）,TFR1、LPCAT3、P53、SAT1、ALOX15、ACSL4基因表达显著下调（P<0.05、0.01）;Western blotting结果显示，GPX4、SLC7A11、FTH1蛋白表达显著上调（P<0.05、0.01）,P53、SAT1、ALOX15、ACSL4蛋白表达显著下调（P<0.05、0.01）。结论 GQD可改善PA诱导的肝细胞IR，其机制可能与调节脂质代谢、抑制肝细胞铁死亡、调控P53/SAT1/ALOX15信号通路相关基因及蛋白表达有关。

Objective To investigate the effect of Gegen Qinlian Decoction（GQD） on insulin resistance（IR） induced by palmitic acid（PA） in AML-12 cells by regulating ferroptosis through P53/SAT1/ALOX15 signaling pathway. Methods Network pharmacology was used to screen the relevant targets of GQD intervention in IR, and a protein-protein interaction network was constructed. Gene Ontology（GO） and Kyoto Encyclopedia of Genes and Genomes（KEGG） enrichment analyses were performed. A model of IR was established by treating AML-12 cells with PA combined with insulin. The optimal concentration of PA for model establishment was screened by the CCK-8 method. In the model validation stage, the rats were randomly divided into the control group, insulin group, PA group, and model group. The intracellular glucose level was detected by the kit method, and the protein expression levels of Akt, p-Akt, insulin receptor substrate 1（IRS-1）, and p-IRS-1 were detected by Western blotting to verify the success of the model. Sixty rats were randomly divided into a control（distilled water） group（30 rats）, a GQD(10 g·kg^-1 crude drug) group（20 rats）, and a pioglitazone hydrochloride(Pio, positive control, 2.7 mg·kg^-1) group（10 rats）. Each group of rats was administered twice a day by intragastric administration for 7 consecutive days. Blood was collected from the abdominal aorta 0.5 h after the last administration on the 8 th day to obtain the serum of each group. The intervention experiment was divided into the control group, the model group, the Pio（10% drug-containing serum） group, and the high, medium, and low concentration（20%, 15%, and 10%） GQD drug-containing serum groups. After intervention, the cell survival rate was detected by the CCK-8 method; the glucose consumption in the culture supernatant was measured; oil red O staining was performed; the levels of triglycerides（TG）, total cholesterol（TC）, free fatty acids（FFA）, ferrous ions(Fe²⁺), malondialdehyde（MDA）, glutathione（GSH）, and glutathione peroxidase（GSH-Px） in the cells were detected by the kit method; the expression of GPX4, SLC7A11, FTH1, LPCAT3, TFR1, P53, SAT1, ALOX15, and ACSL4 genes was detected by real-time fluorescence quantitative PCR（qRT-PCR）; and the protein expression of GPX4, SLC7A11, FTH1, P53, SAT1, ALOX15, and ACSL4 was detected by Western blotting. Results Network pharmacology analysis showed that the intersection targets of GQD and IR were mainly enriched in the P53 signaling, lipid metabolism, and oxidative stress pathways. The CCK-8 results indicated that 200 μmol·L^-1 PA was the optimal concentration for model establishment. The model validation results showed that the model was successfully established. After GQD intervention, compared with the model group, the cell survival rate in each concentration group of GQD and the Pio group was significantly increased（P < 0.05, 0.01, 0.001）; the glucose consumption in the cell supernatant was significantly increased（P < 0.05, 0.01）; oil red O staining showed that lipid deposition was significantly reduced, and the levels of TG, TC, and FFA were significantly decreased（P < 0.05, 0.01）; the levels of Fe²⁺ and MDA were significantly decreased（P < 0.05, 0.01）, and the levels of GSH and GSH-Px were significantly increased（P < 0.05, 0.01）; qRT-PCR results showed that the expression of GPX4, SLC7A11, and FTH1 genes was significantly upregulated（P < 0.05, 0.01）, and the expression of TFR1, LPCAT3, P53, SAT1, ALOX15, and ACSL4 genes was significantly downregulated（P < 0.05, 0.01）; Western blotting results showed that the protein expression of GPX4, SLC7A11, and FTH1 was significantly upregulated（P < 0.05, 0.01）, and the protein expression of P53, SAT1, ALOX15, and ACSL4 was significantly downregulated（P < 0.05, 0.01）. Conclusion GQD can improve PA-induced IR in hepatocytes, and its mechanism may be related to the regulation of lipid metabolism, inhibition of hepatocyte ferroptosis, and regulation of P53/SAT1/ALOX15 signaling pathway-related genes and protein expression.

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赣鄱俊才-江西省主要学科学术和技术带头人培养项目(领军人才-学术类,20232BCJ22022); 江西省教育厅科技项目(GJJ213111); 江西省中药药理重点实验室(2024SSY07111); 企业委托横向项目(横20250106); 江西中医药大学校级项目(81525180)