The Effect of Methyl-β-cyclodextrin on Apoptosis, Proliferative Activity, and Oxidative Stress in Adipose-Derived Mesenchymal Stromal Cells of Horses Suffering from Metabolic Syndrome (EMS)

Abstract

Methyl-β-cyclodextrin (MβCD) is a cyclic oligosaccharide, commonly used as a pharmacological agent to deplete membrane cholesterol. In this study, we examined the effect of MβCD on adipose-derived mesenchymal stromal cells (ASCs) isolated form healthy horses (ASC_CTRL) and from horses suffering from metabolic syndrome (ASC_EMS). We investigated the changes in the mRNA levels of the glucose transporter 4 (GLUT4) and found that MβCD application may lead to a significant improvement in glucose transport in ASC_EMS. We also showed that MβCD treatment affected GLUT4 upregulation in an insulin-independent manner via an NO-dependent signaling pathway. Furthermore, the analysis of superoxide dismutase activity (SOD) and reactive oxygen species (ROS) levels showed that MβCD treatment was associated with an increased antioxidant capacity in ASC_EMS. Moreover, we indicated that methyl-β-cyclodextrin treatment did not cause a dysfunction of the endoplasmic reticulum and lysosomes. Thereby, we propose the possibility of improving the functionality of ASC_EMS by increasing their metabolic stability.

Keywords: cyclodextrin; equine metabolic syndrome; mesenchymal stem cells; methyl-β-cyclodextrin; stem cells.

Figure 1

Proliferation of equine ASCs during a five-day culture. The growth of cells (a) was determined using a resazurin-based assay (TOX-8). The population doubling time (PDT) was expressed in hours (b). The proliferation factor (PF) was calculated in comparison with the control group (c). PF values higher than 1 indicated an increased proliferation potential. A hashtag (#) indicates a statistically significant difference between the experimental groups, while an asterisks (*) indicates a statistically significant difference in comparison to the control ASC_CTRL group. The results are expressed as mean ± SD; */# p value < 0.05, **/## p value < 0.01, and *** p value < 0.001. ASC_CTRL—control cells isolated form healthy horses, ASC_EMS - cells isolated from individuals with EMS.

Figure 2

The effect of methyl-β-cyclodextrin on cell morphology. Staining for DAPI, DAPI/phalloidin merged, and MitoRed (a). Enlarged cells with oversized nuclei and improper mitochondria distribution are marked on the photographs. The percentage of the enlarged cell area versus the total cell area is presented in (b). An asterisks (*) indicates a statistically significant difference in comparison to the control ASC_CTRL group. The results expressed as mean ± SD; * p value < 0.05. Magnification: DAPI × 100, scale bar: 200 µm; DAPI/phalloidin × 100, scale bar: 200 µm.

Figure 3

Ultrastructure of EqACSs (a). The number (b) and size of caveolae (c) within the field of view. In TEM images, caveolae are indicated by arrows. Magnification: 30,000× and 100,000×, scale bar: 200 nm for magnification 30,000 and 100 nm for magnification 100,000×. An asterisks (*) indicates a statistically significant difference in comparison to the control ASC_CTRL group. The results expressed as mean ± SD; * p value < 0.05 and ## p value < 0.01.

Figure 4

Analysis of the extracellular levels of reactive oxygen species (ROS) (a); superoxide dismutase (SOD) activity (b); nitric oxide (NO) concentration (c) and nitrite-to-nitrate molecular ratio (d) in EqASCs. Protein kinase RNA-like endoplasmic reticulum kinase (PERK) and lysosome-associated membrane protein 2 (LAMP2) expression in equine ASCs (e,f). An asterisks (*) indicates a statistically significant difference in comparison to the control ASC_CTRL group. The results expressed as mean ± SD; */# p value < 0.05, **/## p value < 0.01, and ***/### p value < 0.001.

Figure 5

The analysis of the apoptotic process in EqASCs. mRNA levels of p53 (a); BAX (b); and the BCL-2/BAX ratio (c) in control and experimental cultures. The level of expression of the indicated genes was calculated in relation to the housekeeping gene GAPDH. An asterisks (*) indicates a statistically significant difference in comparison to the control ASC_CTRL group. The results expressed as mean ± SD; * p value < 0.05, **/## p value < 0.01, and *** p value < 0.001. Results of the TUNEL staining (d); BrdU-positive cells were stained red. DAPI counterstain was used to determine the total cell number. Percentage of the positive stained area versus the total stained area (e). Magnification: ×100, scale bar: 200 µm.

Figure 6

Extracellular levels of insulin (a,b) and adiponectin (c) evaluated by quantitative ELISA. Furthermore, using RT-PCR, the mRNA levels of GLUT4 (d) and leptin (LEP, e) were assessed. A hashtag (#) indicates a statistically significant difference between the experimental groups, while an asterisks (*) indicates a statistically significant difference in comparison to the control ASC_CTRL group. The results expressed as mean ± SD; */# p value < 0.05, ** p value < 0.01.