Study on the effect and mechanism of bone marrow mesenchymal stem cells on apoptosis of peritoneal mesothelial cells

doi:10.11958/20231064

Abstract

Abstract:

Objective To observe the effect of rat bone marrow mesenchymal stem cells (BMSCs) on the apoptosis of rat peritoneal mesothelium cells (PMCs) induced by high glucose peritoneal dialysis fluid (PDF), and to explore its possible molecular mechanism. Methods The primary BMSCs and PMCs were extracted and identified. Apoptosis of PMCs was induced by high glucose PDF. Cell supernatant from BMSCs after 24 h of culture was collected as the conditioned medium (BMSCs-CM). PMCs were co-cultured with BMSCs by conditioned media or Transwell chambers. PMCs were randomly divided into the control group, the PDF group and the PDF+BMSCs-CM group. The viability of PMCs was measured by CCK-8 in each group. The depolarization of mitochondrial membrane potential was measured by JC-1 method. TUNEL staining was used to detect cell apoptosis. Western blot assay was used to detect the expression levels of apoptosis related proteins B-cell lymphoma-2 (Bcl-2), Bcl-2 associated X protein (Bax), Cleaved cysteine aspartase-3 (Cleaved Caspase-3) and pathway related protein serine/threonine protein kinase (Raf), mitogen-activated extracellular signal-regulated kinase (MEK), extracellular-signal regulated protein kinase (ERK) and their phosphorylated proteins in each group. Results Compared with the control group, the proliferative activity and mitochondrial membrane potential of PMCs were decreased in the PDF group, while the apoptosis rate and the ratio of Bax/Bcl-2, Cleaved Caspase-3/Caspase-3, p-Raf/Raf, p-MEK/MEK and p-ERK/ERK were increased (P<0.05). Compared with the PDF group, the proliferative activity and mitochondrial membrane potential of PMCs were increased in the PDF+BMSCs-CM group, while the apoptosis rate and the ratio of Bax/Bcl-2, Cleaved Caspase-3/Caspase-3, p-Raf/Raf, p-MEK/MEK and p-ERK/ERK were decreased (P<0.05). Conclusion BMSCs can reduce the apoptosis of PMCs induced by high glucose PDF, and its mechanism maybe related to inhibiting the activation of Raf/MEK/ERK signaling pathway.

Key words: peritoneal dialysis, apoptosis, bone marrow mesenchymal stem cells, peritoneal mesothelial cells

CLC Number:

R285.5

Figures/Tables 8

Fig.1 Identification of primary rat BMSCs and PMCs

Tab.1 Comparison of relative proliferative activity, mitochondrial membrane potential and apoptosis rate of PMCs between the three groups

组别	增殖活力/%	线粒体膜电位水平	凋亡率/%
CON组	100.00±3.04	3.05±0.22	7.31±1.75
PDF组	62.00±1.36^a	1.03±0.06^a	54.42±0.88^a
PDF+BMSCs-CM组	78.24±2.15^b	1.74±0.16^b	27.36±1.14^b
F	207.551^＊＊	122.875^＊＊	894.079^＊＊

Fig.2 Results of mitochondrial membrane potential in each group detected by JC-1 fluorescence probe (×200)

Fig.3 Apoptosis in each group of cells detected by TUNEL staining (×200)

Fig.4 The expression of apoptosis-related proteins detected by Western blot assay

Tab.2 Comparison of expression levels of apoptosis-related proteins between the three groups

组别	Bax/Bcl-2	Cleaved Caspase-3/Caspase-3
CON组	0.35±0.01	0.18±0.02
PDF组	0.82±0.08^a	0.30±0.02^a
PDF+BMSCs-CM组	0.44±0.06^b	0.20±0.03^b
F	62.238^＊＊	19.303^＊＊

Fig.5 The expression of Raf/MEK/ERK signal pathway related proteins detected by Western blot assay

Tab.3 Comparison of expression levels of Raf/MEK/ERK signal pathway related proteins between the three groups

组别	p-Raf/Raf	p-MEK/MEK	p-ERK/ERK
CON组	0.18±0.00	0.17±0.01	0.18±0.01
PDF组	0.34±0.03^a	0.32±0.04^a	0.29±0.01^a
PDF+BMSCs-CM组	0.22±0.02^b	0.22±0.02^b	0.21±0.01^b
F	57.076^＊＊	28.990^＊＊	81.448^＊＊

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