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目的 基于网络药理学和细胞实验验证探究2，2，6，6-四甲基哌啶-1-氧基-4-氨基-2-氧基乙基(MT)对过氧化氢(H2O2)诱导活化的大鼠肝星状细胞(HSC-T6)细胞凋亡的作用。方法 以网络药理学为研究手段，先预测MT治疗肝纤维化(HF)的候选作用靶点与相关信号通路，并将目标靶点蛋白与MT进行分子对接和分子动力学模拟初步验证，再进一步结合体外细胞实验验证上述结论。将HSC-T6细胞分为6组：对照组、MT对照组(40μmol/L)、H2O2组(50μmol/L)、MT不同浓度组(20、40μmol/L)和姜黄素组(15μmol/L)。CCK-8法检测细胞活力；细胞克隆形成实验检测细胞增殖能力；试剂盒检测细胞中氧化应激水平；流式细胞术检测细胞凋亡情况；免疫荧光染色法检测细胞中I型胶原(COLI)的表达；Westernblot检测细胞中α-平滑肌肌动蛋白(α-SMA)、COLI与凋亡相关蛋白表达。结果 通过在线数据库筛选并获得147个MT与HF相关的交集靶点，其中涉及B淋巴细胞瘤-2(Bcl-2)、半胱天冬酶3(Casp3)等；分子对接和分子动力学模拟结果表明MT与Bcl-2、Casp3结合良好；京都基因与基因组百科全书(KEGG)通路富集分析表明，MT可能通过细胞凋亡途径调控HF进程；体外实验结果显示，MT干预可显著降低HSC-T6细胞的活力与增殖能力，减少丙二醛(MDA)、α-SMA、COLI及Bcl-2水平，同时上调超氧化物歧化酶(SOD)、谷胱甘肽(GSH)、Bcl-2相关X蛋白(Bax)、活化的半胱天冬酶3(Cleaved-Caspase-3)水平，促进HSC-T6细胞凋亡。结论 MT可抑制HSC-T6细胞的增殖和活化，其机制可能与促进HSC-T6细胞凋亡有关。
Abstract：Objective To investigate the effects of 2， 2， 6， 6-Tetramethylpiperidin-1-oxy-4-amino-2-oxoethyl (MT) on apoptosis in hydrogen peroxide (H2O2)-activated HSC-T6 cells through network pharmacology and in vitro cellular experiments. Methods Network pharmacology was employed to predict potential target proteins and signaling pathways of MT in the treatment of hepatic fibrosis (HF). Molecular docking was performed to preliminarily validate the interactions between MT and identified target proteins. These findings were further validated through in vitro cellular experiments. The HSC-T6 cells were divided into 6 groups: the control group， the MT control group (40 μmol/L)， the H2O2 group (50 μmol/L)， the different concentrations of MT groups (20， 40 μmol/L)， and the curcumin group (15 μmol/L). The cell viability was detected using the CCK-8 assay. The cell proliferation ability were detected using the cell cloning formation experiment. The levels of oxidative stress were detected by kits. Apoptosis was detected by flow cytometry. Type I collagen (COL Ⅰ) expression was detected by immunofluorescence staining. Western blot assay was used to measure expression of α-smooth muscle actin (α-SMA)， COL Ⅰ， and apoptosis-related proteins in cells. Results Through the screening of an online database identified 147 intersection targets associated with MT and HF， including B-cell lymphoma-2 (Bcl-2) and Casp3. The results of molecular docking and molecular dynamics simulation indicated that strong binding affinity between MT and Bcl-2/Casp3. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis revealed that MT regulates HF progression by the apoptosis pathway. The results of the in vitro experiments showed that MT intervention could significantly reduce the viability and proliferation ability of HSC-T6 cells， decrease the levels of malondialdehyde (MDA)， α-SMA， COL I and Bcl-2， while up-regulating the levels of superoxide dismutase (SOD)， glutathione (GSH)， Bcl-2 associated X protein (Bax) and activated caspase-3 (Cleaved-Caspase-3)， and promoting the apoptosis of HSC-T6 cells. Conclusion In conclusion， MT can inhibit the proliferation and activation of HSC-T6 cells， and its mechanism may be related to the promotion of HSC-T6 cells apoptosis.
[1] Lee J E， Lee Y J， Yoon J K. Advanced 3D in vitro liver fibrosis models: spheroids， organoids， and liver-on-chips[J]. Biomimetics， 2025， 10(10): 639. doi:10.3390/biomimetics10100639.
[2] 凌 辉， 汪先晨， 尤峻柏， 等. RUNX3对肝星状细胞活化增殖和迁移能力的影响[J]. 安徽医科大学学报， 2026， 61(2): 277-84. doi:10.19405/j.cnki.issn1000-1492.2026.02.013.
[3] Choudhury A， Ratna A， Lim A， et al. Loss of heat shock factor 1 promotes hepatic stellate cell activation and drives liver fibrosis[J]. Hepatol Commun， 2022， 6(10): 2781-97. doi:10.1002/hep4.2058.
[4] Ali Hassan H， Banchi P， Domain G， et al. Mito-Tempo improves acrosome integrity of frozen-thawed epididymal spermatozoa in tomcats[J]. Front Vet Sci， 2023， 10: 1170347. doi:10.3389/fvets.2023.1170347.
[5] Wang P F， Xie K， Cao Y X， et al. Hepatoprotective effect of mitochondria-targeted antioxidant mito-TEMPO against lipopolysaccharide-induced liver injury in mouse[J]. Mediat Inflamm， 2022， 2022(1): 6394199. doi:10.1155/2022/6394199.
[6] Shetty S， Anushree U， Kumar R， et al. Mitochondria-targeted antioxidant， mito-TEMPO mitigates initiation phase of N-Nitrosodiethylamine-induced hepatocarcinogenesis[J]. Mitochondrion， 2021， 58: 123-30. doi:10.1016/j.mito.2021.03.001.
[7] Ali F E， Abd El-Aziz M K， Sharab E I， et al. Therapeutic interventions of acute and chronic liver disorders: a comprehensive review[J]. World J Hepatol， 2023， 15(1): 19-40. doi:10.4254/wjh.v15.i1.19.
[8] Xue X， Zhou H， Gao J， et al. The impact of traditional Chinese medicine and dietary compounds on modulating gut microbiota in hepatic fibrosis: a review[J]. Heliyon， 2024， 10(19): e38339. doi:10.1016/j.heliyon.2024.e38339.
[9] Li K X， Wang Z C， Machuki J O， et al. Benefits of curcumin in the vasculature: a therapeutic candidate for vascular remodeling in arterial hypertension and pulmonary arterial hypertension?[J]. Front Physiol， 2022， 13: 848867. doi:10.3389/fphys.2022.848867.
[10] Yu S， Pu J， Liu K， et al. Curcumin mediates oxidative stress to play an anti-fibrotic role， focusing on liver， renal， myocardial and pulmonary fibrosis[J]. Front Pharmacol， 2025， 16: 1636538. doi:10.3389/fphar.2025.1636538.
[11] Lu J L， Yu C X， Song L J. Programmed cell death in hepatic fibrosis: current and perspectives[J]. Cell Death Discov， 2023， 9(1): 449. doi:10.1038/s41420-023-01749-8.
[12] Shahcheraghi S H， Salemi F， Peirovi N， et al. Nrf2 regulation by curcumin: molecular aspects for therapeutic prospects[J]. Molecules， 2021， 27(1): 167. doi:10.3390/molecules27010167.
[13] Solhjoo S， Liu T， Sidor A， et al. Oxidative stress in the mitochondrial matrix underlies ischemia/reperfusion-induced mitochondrial instability[J]. J Biol Chem， 2023， 299(1): 102780. doi:10.1016/j.jbc.2022.102780.
[14] Du K， Jun J H， Dutta R K， et al. Plasticity， heterogeneity， and multifunctionality of hepatic stellate cells in liver pathophysiology[J]. Hepatol Commun， 2024， 8(5): e0411. doi:10.1097/hc9.0000000000000411.
[15] Huang Z， Zhou Z， Ma Y， et al. Mito-Tempo alleviates ox-LDL-provoked foam cell formation by regulating Nrf2/NLRP3 signaling[J]. Biosci Biotechnol Biochem， 2024， 88(7): 759-67. doi:10.1093/bbb/zbae058.
[16] Duan T， Sun L， Xi Y， et al. Mito-tempo ameliorates tubular injury of diabetic nephropathy via inhibiting mt-dsRNA release and PKR/eIF2α pathway activation[J]. Free Radic Biol Med， 2025， 237: 147-59. doi:10.1016/j.freeradbiomed.2025.05.431.
基本信息:
中图分类号:R965
引用信息:
[1]袁满琴,马利,赵珮伶,等.基于网络药理学与实验验证探究Mito-TEMPO对大鼠肝星状细胞凋亡的影响[J].安徽医科大学学报().
基金信息:
国家自然科学基金（编号：81860118）; 贵州省大学生创新创业训练项目（编号：S2024106601260）; 贵州医科大学慢性疾病标志物研究重点实验室项目（编号：2024FY-004）;贵州医科大学附属医院科研育人与科研反哺教学项目（编号：gyfykj-2025-y05）
2026-08-26
2026-08-26
2026-08-26