目的:研究低氧性大鼠肺动脉平滑肌细胞(PASMC)的增殖、凋亡与丝裂原活化蛋白激酶(MAPK)关系。方法:用组织酶消化法获取肺动脉平滑肌细胞(PASMCs),进行原代培养;采用普通光学显微镜和免疫荧光染色法,分别鉴定PASMCs;选择处于对数生长期的...目的:研究低氧性大鼠肺动脉平滑肌细胞(PASMC)的增殖、凋亡与丝裂原活化蛋白激酶(MAPK)关系。方法:用组织酶消化法获取肺动脉平滑肌细胞(PASMCs),进行原代培养;采用普通光学显微镜和免疫荧光染色法,分别鉴定PASMCs;选择处于对数生长期的4~6代PASMCs,随机分为7组进行造模:常氧对照组(N)、低氧组(H)、DMSO组(D)、U0126组(U)、SB203580组(S)、Anisomycin组(A)、Staurosporine Aglycone组(SA);N组加入10%培养基后置于常氧培养箱中,其它各组分别加入含相应药物的10%培养基后置于低氧培养箱(3%O2,5%CO2,37℃)中,造模时间均为48 h。CCK-8法检测各组PASMCs增殖情况;TUNEL法测定各组PASMCs凋亡情况。结果:与N组相比,H组PASMCs的OD值显著上调(0.990±0.041 vs 1.143±0.033,P<0.01),凋亡指数没有明显变化(4.913±0.451 vs 5.452±0.557,P>0.05);与H组相比,D组PASMCs的OD值和凋亡指数均无显著变化(1.143±0.033 vs 1.142±0.049,5.452±0.557 vs 5.402±0.651,均P>0.05);U组PASMCs的OD值下降,凋亡指数升高(1.143±0.033 vs 0.985±0.078,5.452±0.557 vs 10.145±2.545,均P<0.01);S组PASMCs OD值上调,凋亡指数明显下调(1.143±0.033 vs 1.295±0.039,5.452±0.557 vs 3.093±0.409,均P<0.01);A组PASMCs的OD值下降,凋亡指数升高(1.143±0.033 vs 0.347±0.067,5.452±0.557 vs 25.753±1.262,均P<0.01);SA组PASMCs OD值上调,凋亡指数下调(1.143±0.033 vs 1.685±0.100,5.452±0.557 vs 1.700±0.095,均P<0.01)。结论:低氧对PASMCs增殖和凋亡的调控与MAPK信号通路有关。展开更多
This study delved into the mechanism by which the principal component of Astragali Radix regulated ferroptosis in the context of hypoxia-induced pulmonary hypertension,employing a combination of network pharmacology a...This study delved into the mechanism by which the principal component of Astragali Radix regulated ferroptosis in the context of hypoxia-induced pulmonary hypertension,employing a combination of network pharmacology and experimental validation techniques.Active constituents of Astragali Radix and their corresponding targets were identified using the TCMSP database,while therapeutic targets associated with hypoxia-induced pulmonary hypertension were sourced from the GeneCards database.The Venn online tool facilitated the identification of overlapping targets between the active constituents of Astragali Radix and hypoxia-induced pulmonary hypertension.Interaction network diagrams depicting the relationship between Astragali Radix’s active constituents and their targets were constructed using Cytoscape software,with core targets and sub-networks identified using the CytoHubba plug-in.Gene ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analyses were conducted using the DAVID database.Additionally,the FerrDb database was consulted to analyze genes implicated in regulating ferroptosis.The investigation revealed 18 active constituents selected from Astragali Radix,with quercetin emerging as the key component.A total of 35 potential targets associated with Astragali Radix in regulating ferroptosis and addressing hypoxia-induced pulmonary hypertension were predicted.Experimental validation demonstrated that quercetin could inhibit the MAPK signaling pathway,resulting in reduced Fe2+and lipid peroxide levels,increased GPX4 expression,and the reversal of ferroptosis.In summary,this study elucidated the fundamental constituents and pivotal signaling pathways through which Astragali Radix modulated ferroptosis and mitigated hypoxia-induced pulmonary hypertension.Specifically,quercetin,a core constituent of Astragali Radix,was observed to inhibit ferroptosis in pulmonary arterial smooth muscle cells via the MAPK pathway and alleviate hypoxia-induced pulmonary hypertension.展开更多
文摘目的:研究低氧性大鼠肺动脉平滑肌细胞(PASMC)的增殖、凋亡与丝裂原活化蛋白激酶(MAPK)关系。方法:用组织酶消化法获取肺动脉平滑肌细胞(PASMCs),进行原代培养;采用普通光学显微镜和免疫荧光染色法,分别鉴定PASMCs;选择处于对数生长期的4~6代PASMCs,随机分为7组进行造模:常氧对照组(N)、低氧组(H)、DMSO组(D)、U0126组(U)、SB203580组(S)、Anisomycin组(A)、Staurosporine Aglycone组(SA);N组加入10%培养基后置于常氧培养箱中,其它各组分别加入含相应药物的10%培养基后置于低氧培养箱(3%O2,5%CO2,37℃)中,造模时间均为48 h。CCK-8法检测各组PASMCs增殖情况;TUNEL法测定各组PASMCs凋亡情况。结果:与N组相比,H组PASMCs的OD值显著上调(0.990±0.041 vs 1.143±0.033,P<0.01),凋亡指数没有明显变化(4.913±0.451 vs 5.452±0.557,P>0.05);与H组相比,D组PASMCs的OD值和凋亡指数均无显著变化(1.143±0.033 vs 1.142±0.049,5.452±0.557 vs 5.402±0.651,均P>0.05);U组PASMCs的OD值下降,凋亡指数升高(1.143±0.033 vs 0.985±0.078,5.452±0.557 vs 10.145±2.545,均P<0.01);S组PASMCs OD值上调,凋亡指数明显下调(1.143±0.033 vs 1.295±0.039,5.452±0.557 vs 3.093±0.409,均P<0.01);A组PASMCs的OD值下降,凋亡指数升高(1.143±0.033 vs 0.347±0.067,5.452±0.557 vs 25.753±1.262,均P<0.01);SA组PASMCs OD值上调,凋亡指数下调(1.143±0.033 vs 1.685±0.100,5.452±0.557 vs 1.700±0.095,均P<0.01)。结论:低氧对PASMCs增殖和凋亡的调控与MAPK信号通路有关。
基金National Natural Science Foundation of China(Grant No.82305214)Hunan Province’s Natural Science Fund(Grant No.2023JJ40401)+2 种基金Hunan Administration of Traditional Chinese Medicine(Grant No.B2023024)Hunan Provincial Department of Education Outstanding Youth Project(Grant No.22B0394)State Key Laboratory Project of Chinese Medicine Powder and Innovative Drugs Project(Grant No.21PTKF1002).
文摘This study delved into the mechanism by which the principal component of Astragali Radix regulated ferroptosis in the context of hypoxia-induced pulmonary hypertension,employing a combination of network pharmacology and experimental validation techniques.Active constituents of Astragali Radix and their corresponding targets were identified using the TCMSP database,while therapeutic targets associated with hypoxia-induced pulmonary hypertension were sourced from the GeneCards database.The Venn online tool facilitated the identification of overlapping targets between the active constituents of Astragali Radix and hypoxia-induced pulmonary hypertension.Interaction network diagrams depicting the relationship between Astragali Radix’s active constituents and their targets were constructed using Cytoscape software,with core targets and sub-networks identified using the CytoHubba plug-in.Gene ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analyses were conducted using the DAVID database.Additionally,the FerrDb database was consulted to analyze genes implicated in regulating ferroptosis.The investigation revealed 18 active constituents selected from Astragali Radix,with quercetin emerging as the key component.A total of 35 potential targets associated with Astragali Radix in regulating ferroptosis and addressing hypoxia-induced pulmonary hypertension were predicted.Experimental validation demonstrated that quercetin could inhibit the MAPK signaling pathway,resulting in reduced Fe2+and lipid peroxide levels,increased GPX4 expression,and the reversal of ferroptosis.In summary,this study elucidated the fundamental constituents and pivotal signaling pathways through which Astragali Radix modulated ferroptosis and mitigated hypoxia-induced pulmonary hypertension.Specifically,quercetin,a core constituent of Astragali Radix,was observed to inhibit ferroptosis in pulmonary arterial smooth muscle cells via the MAPK pathway and alleviate hypoxia-induced pulmonary hypertension.
