Chinese Medical E-ournals Database

Chinese Journal of Obstetrics & Gynecology and Pediatrics(Electronic Edition) ›› 2024, Vol. 20 ›› Issue (05): 525 -533. doi: 10.3877/cma.j.issn.1673-5250.2024.05.008

Special Column of Frontiers in Obstetrics

Inosine inhibits opening of mitochondrial permeability transition pore to alleviate hypoxic/reoxygenationinduced apoptosis of human chorionic trophoblast cells

Yawen Zhong1, Yu Wang1, Haizhen Wang2, Liping Huang1,()   

  1. 1.Department of Obstetrics and Gynecology,Nanfang Hospital,Southern Medical University,Guangzhou 510000,Guangdong Province,China
    2.Department of Obstetrics and Gynecology,The Fifth Affiliated Hospital,Southern Medical University,Guangzhou 510000,Guangdong Province,China
  • Received:2024-08-31 Revised:2024-09-03 Published:2024-10-01
  • Corresponding author: Liping Huang

Objective

To investigate the effect and mechanism of inosine inhibiting the opening of mitochondrial permeability transition pore (mPTP)on hypoxic/reoxygenation (H/R)-induced apoptosis of human chorionic trophoblast cells (HTR-8/SVneo cell line).

Methods

Human chorionic trophoblast cells (HTR-8/Svneo cell line)was used as research material,and H/R-induced HTR-8/SVneo cell model was established invitro.According to the cell culture mode,HTR-8/SVneo cells were divided into 4 groups:H/R group(n=3,only H/R-induced),H/R+inosine group(n=3,H/R+inosine co-coculture treatment for 24 h),H/R+cyclosporine A(Cs A)group(n=3,H/R+Cs A co-culture treatment for 24 h),negative control(NC)group(n=3,conventional cell culture).Calcein/PI staining was used to detect the activity of HTR-8/SVneo cells (relative positive rate of PI staining).DHE probe was used to detect the content of active oxygen species (relative fluorescence intensity of active oxygen species).JC-1 probe was used to detect mitochondrial membrane potential(ratio of green to red fluorescence intensity).Fluo-4AM probe was used to detect Ca2+content in cells (Fluo-4AM relative fluorescence intensity).mPTP assay kit was used to detect mPTP openness(mPTP relative fluorescence intensity).The apoptosis rate was detected by flow cytometry.Real-time fluorescence quantitative enzyme-linked immunosorbent assay (RT-qPCR)was used to detect the m RNA expression levels of BAX and BCL2.Western blotting detected cleaved Caspase3 protein relative expression level and the ratio of BAX to BCL2(BAX/BCL2).The above indexes were statistically compared between H/R group and the other three groups,respectively by independent-samples t test.

Results

PI staining relative positive rate,reactive oxygen species relative fluorescence intensity,ratio of green to red fluorescence intensity detected by JC-1 probe,Fluo-4AM relative fluorescence intensity,apoptosis rate,BAX mRNA level,cleaved Caspase3 relative expression and BAX/BCL2 in H/R group was respectively higher than those in NC group,H/R+inosine group and H/R+Cs A group,while the mPTP relative fluorescence intensity and BCL2 mRNA level in H/R group was respectively lower than those in the other three group,and the differences were statistically significant(all with P <0.05).

Conclusions

Inosine can alleviate mitochondrial dysfunction by inhibiting the mPTP opening of HTR-8/SVneo cells,hence suppressing H/R-induced apoptosis of HTR-8/SVneo cells.

图1 4组HTR-8/SVneo细胞活性检测荧光图(calcein/PI染色,高倍)及PI染色相对阳性率比较 注:NC为阴性对照,H/R 为缺氧/复氧,CsA 为环孢素A。 a表示P<0.05, b表示P<0.01
图2 4组HTR-8/SVneo细胞DHE探针检测细胞活性氧水平荧光图(高倍)及活性氧相对荧光强度比较 注:NC为阴性对照,H/R 为缺氧/复氧,CsA 为环孢素A,DHE为二氢乙锭,DAPI为4',6-二咪基-2-苯基吲哚。 a表示P<0.001
图3 4组HTR-8/SVneo细胞JC-1探针检测细胞线粒体膜电位荧光图(高倍)及绿色与红色荧光强度比值比较 注:NC为阴性对照,H/R 为缺氧/复氧,Cs A 为环孢素A。 a表示P<0.001
图4 4组HTR-8/SVneo细胞Fluo-4AM 探针检测细胞Ca2+含量荧光图(高倍)及Fluo-4AM 相对荧光强度比较 注:NC为阴性对照,H/R 为缺氧/复氧,Cs A 为环孢素A。 a表示P<0.01,b 表示P<0.001
图5 4组HTR-8/SVneo细胞mPTP开放程度荧光图(高倍)及mPTP相对荧光强度比较 注:NC为阴性对照,H/R 为缺氧/复氧,CsA 为环孢素A,mPTP为线粒体通透性转换孔。 a表示P<0.05,b 表示P<0.01
图6 4组HTR-8/Svneo细胞凋亡的流式细胞术图及细胞凋亡率比较 注:NC为阴性对照,H/R 为缺氧/复氧,Cs A 为环孢素A。 a表示P<0.001
图7 4组HTR-8/SVneo细胞BAX、BCL2 mRNA 表达水平比较 注:NC为阴性对照,H/R 为缺氧/复氧,Cs A 为环孢素A。 a表示P<0.01,b 表示P<0.001
图8 4组HTR-8/SVneo细胞cleaved-Caspase3、BAX、BCL2蛋白电泳图及表达水平比较 注:NC为阴性对照,H/R 为缺氧/复氧,CsA 为环孢素A。 a表示P<0.05
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