MiR221/222 in the conditioned medium of adipose-derived stem cells attenuates particulate matter and high-fat diet-induced cardiac apoptosis

Abstract

Background: Air pollution and obesity are crucial risk factors for cardiovascular disease (CVD), with epidemiological evidence indicating that air pollution exacerbates obesity-induced cardiac damage. Treatment with adipose-derived stem cells (ADSCs) attenuates cardiac damage by releasing paracrine factors. However, the effects of ADSCs on air pollution- and obesity-induced cardiomyocyte apoptosis and the related mechanisms are still unclear.

Methods: Palmitic acid (PA) and a high-fat diet (HFD) were used to cause obesity, and particulate matter (PM) was used to simulate air pollution in the study. We studied the impact of conditioned medium from adipose-derived stem cells (ADSC-CM) on the apoptosis of PA + PM-treated H9c2 cells and HFD + PM-treated mouse cardiomyocytes and the underlying mechanisms involved.

Results: The levels of apoptosis-related proteins (PUMA and cleaved caspase-3) were significantly increased in PA + PM-treated H9c2 cells and HFD + PM-treated mouse cardiomyocytes, whereas the antiapoptotic protein Bcl-2 expression was reduced. However, ADSC-CM treatment effectively reduced the PUMA and cleaved caspase-3 expression but increased the Bcl-2 expression. ADSC-CM significantly reduced PA + PM- and HFD + PM-induced cardiomyocyte apoptosis, as detected by the TUNEL assay. RT-qPCR revealed that PA + PM and HFD + PM significantly reduced miR221/222 levels, whereas ADSC-CM treatment increased miR221/222 levels. Furthermore, knockout (KO) and transgenic (TG) mice were used to demonstrate that miR221/222 in ADSC-CM ameliorated cardiac apoptosis that was induced by HFD + PM treatment. Furthermore, PA + PM treatment increased the reactive oxygen species (ROS) production, which triggered mitochondrial fission and contributed to apoptosis. However, ADSC-CM effectively reduced ROS levels and regulated mitochondrial fission, alleviating cellular apoptosis.

Conclusions: Our findings demonstrated that ADSC-CM attenuated PA + PM-induced cardiomyocyte apoptosis by modulating miR221/222 levels and suppressing ROS production.

Keywords: miR221/222; Conditioned medium from cultured adipose-derived stem cell (ADSC-CM); High-fat diet (HFD); Mitochondrial fission; Palmitic acid (PA); Particulate matter (PM); Reactive oxygen species (ROS).

Fig. 1

Effects of ADSC-CM on PA + PM-induced H9c2 cell apoptosis. A H9c2 cells were treated with PA at various concentration (0, 50, 100, and 200 µM) for 24 h. An MTT method measured cell viability. B H9c2 cells were incubated with various doses (0, 10, 20, and 40 µg/mL) of PM for 24 h. An MTT method measured cell viability. C H9c2 cells were co-treated with the defined concentratin of PA and PM for 24 h. The expression levels of PUMA, Bcl-2, and cCASP3 were checked by Western blot. D H9c2 cells were incubated with PA (50 µM), PM (20 µg/mL), or PA (50 µM) + PM (20 µg/mL) for 24 h. An MTT method measured cell viability. E H9c2 cells were treated with PA (50 µM), PM (20 µg/mL), or PA (50 µM) + PM (20 µg/mL) for 24 h. F H9c2 cells were treated with 50 µM PA, 20 µg/mL PM, and ADSC-CM for 24 h. An MTT method measured cell viability. G H9c2 cells were treated with 50 µM PA, 20 µg/mL PM, and ADSC-CM for 24 h and then examined for apoptosis via a TUNEL method (TUNEL-positive cells: green; nuclei: blue). Scale bar = 100 μm. H The bar graph depicts the quantitative analysis results of counting the number of TUNEL-positive cells. I H9c2 cells were incubated with 50 µM PA, 20 µg/mL PM, and ADSC-CM for 24 h. Cell death was investigated by detecting annexin V-FITC and propidium iodide (PI) staining and analyzing by flow cytometry. J H9c2 cells were incubated with 50 µM PA, 20 µg/mL PM, and ADSC-CM for 24 h. Western blot examined the PUMA, Bcl-2, and cCASP3 expression levels. Experiments were conducted at least five times, and the quantitative data were presented as mean ± SEM. *P < 0.05 is defined as statistically significant. PA: palmitic acid; PM: particulate matter; CM: ADSC-CM; cCASP3: cleaved caspase-3

Fig. 2

Effects of ADSC-CM on PA + PM-induced H9c2 cell apoptosis through miR221/222. A MiR221/222 levels were determined in DMEM, H9c2-CM, and ADSC-CM by qPCR. B H9c2 cells were incubated with 50 µM PA, 20 µg/mL PM, and ADSC-CM for 24 h. The expression levels of miR221/222 were evaluated by qPCR. C, D H9c2 cells were transfected with the corresponding negative control (NC) or miR221/222 mimics for 24 h and then incubated with 50 µM PA and 20 µg/mL PM for 24 h. MiR221/222 expression levels were examined by qPCR (C), and PUMA, Bcl-2, and cCASP3 protein expression levels were investigated by Western blot (D). E, F H9c2 cells were transfected with a scramble sequence or miR221/222 inhibitors for 24 h and then treated with 50 µM PA, 20 µg/mL PM, and ADSC-CM for 24 h. The miR221/222 expression levels were assessed by qPCR (E). PUMA, Bcl-2, and cCASP3 expression levels were investigated by Western blot (F). G ADSCs were transfected with the corresponding negative control (NC) or miR221/222 inhibitors for 24 h. These cells were then incubated in a standard culture medium for another 24 h, and NCKD-CM and miRKD-CM were collected, respectively. The levels of miR221/222 were measured by qPCR. H Apoptosis was assessed via a TUNEL method (TUNEL-positive cells: green; nuclei: blue). Scale bar = 100 μm. I Quantification data of TUNEL-positive cells is shown in the bar graphs. J Western blot measures the PUMA, Bcl-2, and cCASP3 expression levels. Experiments were conducted at least five times, and the quantitative data were presented as mean ± SEM. *P < 0.05 is defined as statistically significant. NC mimic: negative control mimic; miR mimic: miR221/222 mimic; NC inhibitor: negative control inhibitor; miR inhibitor: miR221/222 inhibitor; NCKD-CM: CM obtained after the knockdown of the negative control in ADSCs; miRKD-CM: CM obtained after the knockdown of miR221/222 in ADSCs

Fig. 3

Effects of ADSC-CM on high-fat diet (HFD) + PM-induced apoptosis in B6 and KO mice via miR221/222. A Determination of miR221/222 levels in heart tissues from B6 and KO mice by qPCR. B Mice were fed a HFD for 4 weeks and were injected with or without PM and CM at the third and fourth weeks. The levels of miR221/222 in heart tissues were determined by qPCR. C Mice were weighed before the start of the experiment and weekly thereafter. D Representative photographs of transthoracic echocardiographic morphology in M mode. E–H Measurements of the ejection fraction (EF), fractional shortening (FS), diastolic left ventricular posterior wall thickness (LVPWd), and systolic left ventricular posterior wall thickness (LVPWs). I Representative images of mitral valve waveforms from transthoracic echocardiography. J–M Measurements of the left ventricular isovolumetric contraction time (IVCT), left ventricular isovolumetric relaxation time (IVRT), mitral valve E/A ratio, and stroke volume (SV). N Representative images of HE-stained cardiac tissue (scale bar = 2 mm). O Apoptosis was assessed in cardiac tissue using a TUNEL assay. The positive reaction product is brown. The nuclei were counterstained with hematoxylin solution. P The location of PUMA expression in cardiac tissue was investigated by immunohistochemical staining. The positive reaction product is brown. The nuclei were counterstained with hematoxylin solution. Q PUMA, Bcl-2, and cCASP3 expression levels were examined by Western blot. Experiments were conducted at least six times, and the quantitative data were presented as mean ± SEM. *P < 0.05 is defined as statistically significant. B6: C57B/L6 mice; KO: miR221/222 knockout mice; HFD: high-fat diet

Fig. 4

Effects of miR221/222 on apoptosis of high-fat diet (HFD) + PM-treated TG mice. A Mice were fed an HFD for 4 weeks and injected with or without PM and CM at the third and fourth weeks. The levels of miR221/222 were determined by qPCR. B Mice were weighed before the start of the experiment and weekly thereafter. C Representative M-mode images of transthoracic echocardiography. D–G Measurements of the EF, FS, LVPWd, and LVPWs. H Representative images of mitral valve waveforms from transthoracic echocardiography. I–L Measurements of the IVCT, IVRT, mitral E/A ratio, and SV. M Representative images of HE-stained cardiac tissues (scale bar = 2 mm). N Apoptosis was examined in cardiac tissues using a TUNEL assay. The positive reaction product is brown. The nucleus was counterstained with hematoxylin solution. Scale bar = 100 µm. O Immunohistochemistry was used to detect the location of PUMA expression in cardiac tissue. The positive reaction product is brown. The nuclei were counterstained with hematoxylin solution. P Western blot examined the PUMA, Bcl-2, and cCASP3 expression levels. These experiments were conducted at least six times, and the quantitative data were presented as mean ± SEM. *P < 0.05 is defined as statistically significant. TG: miR221/222 transgenic mice

Fig. 5

Effects of ADSC-CM on ROS production in PA- and PM-treated H9c2 cells. A–E H9c2 cells were incubated with 50 µM PA, 20 µg/mL PM, and 100 nM MitoQ for 24 h. MitoSOX Red staining measures mitochondrial ROS levels using fluorescent microscopy (A) and flow cytometry (B). Apoptosis was detected using a TUNEL method (TUNEL-positive cells: green; nuclei: blue) (C). The statistical graph depicts the quantification of TUNEL-positive cell count (D). The PUMA, Bcl-2, and cCASP3 expression levels were investigated by Western blot (E). F, G H9c2 cells were treated with 50 µM PA, 20 µg/mL PM, and CM for 24 h. They were then treated with MitoSOX Red and detected by fluorescent microscopy (F) and flow cytometry (G). H, I H9c2 cells were incubated with the corresponding negative control (NC) or miR221/222 mimics for 24 h and then incubated with 50 µM PA and 20 µg/mL PM for 24 h. They were treated with MitoSOX Red and checked via fluorescence microscopy (H) or flow cytometry (I). J, K ADSCs were transfected with miR221/222 inhibitors and then miRKD-CMs were collected. H9c2 cells were co-treated with 50 µM PA, 20 µg/mL PM, and DMEM or CM from H9c2, ADSCs, and miRKD (H9c2-CM, ADSC-CM, and miRKD-CM) for 24 h. The cells were then treated with MitoSOX Red and detected by fluorescent microscopy (J) and flow cytometry (K). L The SOD1, SOD2, and SOD3 expression levels were analyzed using Western Blot. The experiments were conducted at least five times, and the quantitative data were presented as mean ± SEM. *P < 0.05 is defined as statistically significant. Scale bar = 100 µm

Fig. 6

Effects of ADSC-CM on PA- and PM-induced mitochondrial fission in H9c2 cells and the relationship between mitochondrial fission and ROS production. A, B H9c2 cells were treated with 50 µM PA, 20 µg/mL PM, and CM for 24 h. ΔΨm was assessed by JC-1 staining via fluorescence microscopy (A; scale bar = 100 µm) and flow cytometry (B). C ATP assay evaluated mitochondrial function. D Mitochondrial morphology was examined using MitoTracker. Scale bars = 20 µm (upper), 10 µm (lower). E Western blot analyzed p-DRP1 and FIS1 expression. F, G TUNEL assay (F; green: TUNEL-positive; blue: nuclei) and quantification (G) assessed Mdivi-1 (10 µM) pretreatment effects on apoptosis. H Western blot examined p-DRP1, PUMA, and cCASP3 expression. I–K Effects of MitoQ on ΔΨm and ATP levels were assessed by JC-1 staining (I; scale bar = 100 µm), flow cytometry (J), and ATP assay (K). L, M MitoTracker staining and Western blot assessed the effects of MitoQ (100 nM) on mitochondrial morphology. Scale bars = 20 µm (upper), 10 µm (lower) (L) and protein expression (M) in H9c2 cells treated with PA and PM. N–R H9c2 cells were treated with H₂O₂ (100 µM) and CM for 24 h. The changes of ΔΨm (N, O), ATP levels (P), mitochondrial morphology (Q), and protein expression (R) were analyzed. S, T mROS levels were assessed by MitoSOX Red staining (S; scale bar = 100 µm) and flow cytometry (T) after Mdivi-1 and PA + PM treatment. U–W Cells transfected with negative control (NC) or miR221/222 mimics for 24 h and then treated with PA and PM for 24 h. ΔΨm was examined by JC-1 (U; scale bar = 100 µm) and flow cytometry (V); ATP levels were measured (W). X Western blot assessed expression of p-DRP1, FIS1, and SOD2. The experiments were conducted at least five times, and the quantitative data were presented as mean ± SEM. *P < 0.05 is defined as statistically significant

Fig. 7

A graphical abstract summarizing the study findings. This schematic illustration summarizes the effects of obesity and air pollution on cardiomyocyte apoptosis and the potential protective role of ADSC-CM. In vitro, PA treatment and PM exposure simulated obesity and air pollution, respectively. In vivo, an HFD and PM were combined to replicate these conditions. This study revealed that ADSC-CM could reduce PA/HFD + PM-induced cardiomyocyte apoptosis in vitro and in vivo studies