基于CXCL12/CXCR4轴探讨琥珀散含药血清促进子宫内膜异位症患者经血源间充质干细胞自噬活性的作用机制

Mechanistic study of Hupo Powder containing serum promoting autophagy of menstrual blood-derived mesenchymal stem cells with endometriosis based on CXCL12/CXCR4 axis

目的 基于CXCL12/CXCR4轴探讨琥珀散对子宫内膜异位症(EMT)经血源间充质干细胞(MenSCs)自噬活性的调控作用。方法 从EMT患者和健康女性志愿者的月经血中分离提取EMT MenSCs(E-MenSCs)和正常MenSCs(HMenSCs)。采用12只7周龄雄性SD大鼠制备空白血清和含药血清(ig给予大鼠琥珀散9.9 g·kg^(-1),临床等效剂量)。将细胞分为3组:对照组(H-MenSCs)、模型组(E-MenSCs)和琥珀散组(E-MenSCs),对照组和模型组使用20%空白血清干预,琥珀散组使用20%含药血清干预,均干预48 h。分别采用CCK-8法、透射电镜、实时荧光定量PCR(qRT-PCR)和Western blotting法检测3组细胞的细胞活力、自噬体和自噬溶酶体表达、CXCL12和CXCR4的mRNA水平和CXCL12/CXCR4及自噬标志分子(Beclin1和LC3Ⅱ)的蛋白表达量。结果 与对照组比较,模型组的细胞活力、CXCL12和CXCR4的mRNA和蛋白表达水平均显著提高(P<0.05、0.01、0.001),自噬体和自噬溶酶体数量、Beclin1和LC3Ⅱ的蛋白表达均显著降低(P<0.05、0.01);与模型组比较,琥珀散组的细胞活力、CXCL12和CXCR4的mRNA及蛋白表达水平均显著下调(P<0.05、0.01、0.001),自噬体和自噬溶酶体数量、Beclin1和LC3Ⅱ的蛋白表达均显著上调(P<0.05、0.01、0.001)。结论 琥珀散含药血清能够上调E-MenSCs自噬活性,其作用机制可能与其抑制CXCL12/CXCR4轴有关。

Objective Based on CXCL12/CXCR4 axis,the regulation of Hupo Powder on the autophagy of menstrual blood-derived mesenchymal stem cells(MenSCs) with endometriosis(EMT) was studied.Methods EMT MenSCs(E-MenSCs) and healthy MenSCs(H-MenSCs) from the menstrual blood of patients with EMT and healthy volunteers were isolated,respectively.Twelve seven-weeks old male SD rats were used to prepare blank serum and drug containing serum(9.9 g·kg^(-1) of amber powder was given to rats by ig,clinical equivalent dose).These cells were divided into three groups:control(H-MenSCs),model(E-MenSCs),and Hupo Powder(E-MenSCs) groups.The control and model groups were intervened in 20% concentration of blank serum for 48hours,while the Hupo Powder group was intervened in 20% concentration of Hupo Powder containing serum for 48 hours.CCK-8assay,transmission electron microscope,qRT-PCR,and Western blotting were used to detect the cell viability,autophagosomes and autolysosomes,the mRNA levels of CXCL12 and CXCR4,and the protein level of CXCL12,CXCR4,and autophagy marker proteins(Beclin1 and LC3Ⅱ),respectively.Results Compared to the control group,cell viability,mRNA and protein expression of CXCL12 and CXCR4 in the model group were increased(P<0.05,0.01,and 0.001),and autophagosomes and autolysosomes,Beclin1 and LC3Ⅱ protein expression were decreased(P<0.05 and 0.01).Compared to the model group,cell viability,mRNA and protein expression of CXCL12 and CXCR4 in the Hupo Powder group were down-regulated(P<0.05,0.01,and 0.001),and autophagosomes and autolysosomes,Beclin1 and LC3 Ⅱ protein expression were upregulated(P<0.05,0.01,and 0.001).Conclusion Hupo Powder containing serum can up-regulate E-MenSCs autophagy,which might relate to the inhibition of CXCL12/CXCR4 from Hupo Powder.