Int J Stem Cells.  2024 Aug;17(3):309-318. 10.15283/ijsc22200.

Human Endometrium Derived Mesenchymal Stem Cells with Aberrant NOD1 Expression Are Associated with Ectopic Endometrial Lesion Formation

• Li C
• Luo S
• Guo A
• Su Y
• Zhang Y
• Song Y
• Liu M
• Wang L
• Zhang Y

• KMID: 2558608
• DOI: http://doi.org/10.15283/ijsc22200

Abstract

Nucleotide-binding oligomerization domain 1 (NOD1), a cytosolic pattern recognition receptor protein, plays a crucial role in innate immune responses. However, the functional expression of NOD1 in mesenchymal stem cells (MSCs) derived from endometriosis remains unclear. The aim of this study was to explore the functions of NOD1 in ectopic endometrial lesions. Tissues and MSCs were isolated from both normal endometrium and endometriosis. Immunohistochemistry and real time quantitative polymerase chain reaction (RT-qPCR) were used to determine the expression of NOD1 in the tissues/MSCs. Quantification of various cytokines was performed using RT-qPCR and enzyme-linked immunosorbent assay. To confirm the proliferation, invasion/migration, and apoptotic viabilities of the samples, Cell Counting Kit-8, clonogenic formation, transwell assays, and apoptotic experiments were conducted. Higher levels of NOD1 expression were detected in the ectopic-MSCs obtained from endometriosis compared to those from the endometrium. The expression of interleukin-8 was higher in the ectopic-MSCs than in the eutopic-MSCs. Pretreatment with NOD1 agonist significantly enhanced the proliferation and invasion/migration of eutopic-MSCs. Additionally, the NOD1 inhibitor ML-130 significantly reduced the proliferation, clone formation, invasion, and migration abilities of the ectopic-MSCs, having no effect on their apoptosis capacity. Our findings suggest that the expression of NOD1 in ectopic-MSCs may contribute to the progression of ectopic endometrial lesions.

Keyword

Endometrium; Endometriosis; Nucleotide-binding oligomerization domain 1; Mesenchymal stem cells

Figure

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  Fig. 1 Expression of nucleotide-binding oligomerization domain 1 (NOD1) in endometrium (EN) and endometriosis. HE staining identified eutopic (A, 40×) and ectopic (C, 40×) endometrium morphology. Both eutopic (n=5) and ectopic (n=6) endometrium tissues expressed NOD1 (B and D, 40×). NOD1 mRNA expression level in endometrium from healthy donor (EN) and endometriosis (Fig. 1 EN and endometriosis); ectopic-endometrium (ect-EM) and eutopic-endometrium (eut-EM) were compared to the tissues from patients with endometriosis. ect-EM represents endometriosis and eut-EM represents the normal EN from the same patient. EN, n=16; endometriosis, n=41; eut-EM and paired ect-EM, n=11.

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  Fig. 2 Expression of nucleotide-binding oligomerization domain 1 (NOD1) in endometrium (EN) and endometriosis. HE staining identified eutopic (A, 40×) and ectopic (C, 40×) endometrium morphology. Both eutopic (n=5) and ectopic (n=6) endometrium tissues expressed NOD1 (B and D, 40×). NOD1 mRNA expression level in endometrium from healthy donor (EN) and endometriosis (Fig. 1 EN and endometriosis); ectopic-endometrium (ect-EM) and eutopic-endometrium (eut-EM) were compared to the tissues from patients with endometriosis. ect-EM represents endometriosis and eut-EM represents the normal EN from the same patient. EN, n=16; endometriosis, n=41; eut-EM and paired ect-EM, n=11.

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  Fig. 3 Nucleotide-binding oligomerization domain 1 (NOD1)high expression up-regulated the proliferation of eutopic mesenchymal stem cells (eut-MSCs). (A) colony-forming units (CFUs) of ectopic MSCs (ect-MSCs) following pretreatments by various Tri-DAP concentration gradient (0, 1, 10, and 100 ng/ml) (n=3). (B, C) Higher expression of NOD1 induced proliferation of eut-MSCs (one representative picture from three patients). The stimulation of Tri-DAP concentration gradient was 0, 1, 10, 100 ng/ml, and 1 μg/m (reproducible for 3 times). (B) A commercial cell enumeration kit (Cell Counting Kit-8, CCK-8) was used to analyze eut-MSCs growth, OD.450 value presented the activity of cells proliferative capacity. (C) Nucleus of cells stained with 4’,6-diamidino-2-phenylindole (blue color) and while proliferation cells were valued by Ki67 staining (green fluorescence). (D) The transcription levels of interleukin (IL)-8 in eut-MSCs (n=5) and ect-MSCs group (n=4).

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  Fig. 4 ML-130 affected the bio-habits of ectopic mesenchymal stem cells (ect-MSCs). ect-MSCs were stimulated by different concentrations of ML-130 (0, 0.28, and 0.56 μM) at different time points. (A) Colony-forming units (CFUs) decreased with the increase of ML-130 concentration at 48 hours (left panels). The proliferative effects of various concentration gradient of ML-130 on ect-MSCs (n=3) at 48 hours (right panel). (B) The invasion and migration capacities of ect-MSCs were significantly inhibited by the ML-130 at 0.28 and 0.56 μM (n=4) (left panels 200×, the number of invasion and migration cells per field); The transcription (C) and secretion levels (D) of interleukin (IL)-8 of ect-MSCs pretreated with ML-130 (n=4 in each group). **p<0.01.

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  Fig. 5 Apoptotic percentage of ectopic mesenchymal stem cells (ect-MSCs) with nucleotide-binding oligomerization domain 1 (NOD1) down-regulation. After pretreated with various concentration of ML-130 (0, 0.28, and 0.56 μM) for 48 hours, respectively, the apoptotic ratio was measured by flow cytometry (A); (B-E) The percentage of sub-population of cells. The apoptotic ratio was measured by flow cytometry (propidium iodide [PI]-phycoerythrin [PE] and Annexin V-FITC). PI(＋) & Annexin(–) presents necrotic cells debris (B), PI(＋) & Annexin(＋) presents late apoptotic cells (C), PI(−) & Annexin(＋) presents early apoptotic cells (D), PI(−) & Annexin(−) presents live cells/normal cells (E). Representative results of ectopic mesenchymal stem cells from 5 patients.

Reference

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