Human Amnion-Derived Mesenchymal Stem Cells Protect Human Bone Marrow Mesenchymal Stem Cells against Oxidative Stress-Mediated Dysfunction via ERK1/2 MAPK Signaling원문보기
Wang, Yuli
(Jiangsu Key Laboratory of Oral Diseases, Nanjing Medical University)
,
Ma, Junchi
(Jiangsu Key Laboratory of Oral Diseases, Nanjing Medical University)
,
Du, Yifei
(Jiangsu Key Laboratory of Oral Diseases, Nanjing Medical University)
,
Miao, Jing
(Jiangsu Key Laboratory of Oral Diseases, Nanjing Medical University)
,
Chen, Ning
(Jiangsu Key Laboratory of Oral Diseases, Nanjing Medical University)
Epidemiological evidence suggests that bone is especially sensitive to oxidative stress, causing bone loss in the elderly. Previous studies indicated that human amnion-derived mesenchymal stem cells (HAMSCs), obtained from human amniotic membranes, exerted osteoprotective effects in vivo. However, t...
Epidemiological evidence suggests that bone is especially sensitive to oxidative stress, causing bone loss in the elderly. Previous studies indicated that human amnion-derived mesenchymal stem cells (HAMSCs), obtained from human amniotic membranes, exerted osteoprotective effects in vivo. However, the potential of HAMSCs as seed cells against oxidative stress-mediated dysfunction is unknown. In this study, we systemically investigated their antioxidative and osteogenic effects in vitro. Here, we demonstrated that HAMSCs significantly promoted the proliferation and osteoblastic differentiation of $H_2O_2$-induced human bone marrow mesenchymal stem cells (HBMSCs), and down-regulated the reactive oxygen species (ROS) level. Further, our results suggest that activation of the ERK1/2 MAPK signal transduction pathway is essential for both HAMSCs-mediated osteogenic and protective effects against oxidative stress-induced dysfunction in HBMSCs. U0126, a highly selective inhibitor of extracellular ERK1/2 MAPK signaling, significantly suppressed the antioxidative and osteogenic effects in HAMSCs. In conclusion, by modulating HBMSCs, HAMSCs show a strong potential in treating oxidative stress- mediated bone deficiency.
Epidemiological evidence suggests that bone is especially sensitive to oxidative stress, causing bone loss in the elderly. Previous studies indicated that human amnion-derived mesenchymal stem cells (HAMSCs), obtained from human amniotic membranes, exerted osteoprotective effects in vivo. However, the potential of HAMSCs as seed cells against oxidative stress-mediated dysfunction is unknown. In this study, we systemically investigated their antioxidative and osteogenic effects in vitro. Here, we demonstrated that HAMSCs significantly promoted the proliferation and osteoblastic differentiation of $H_2O_2$-induced human bone marrow mesenchymal stem cells (HBMSCs), and down-regulated the reactive oxygen species (ROS) level. Further, our results suggest that activation of the ERK1/2 MAPK signal transduction pathway is essential for both HAMSCs-mediated osteogenic and protective effects against oxidative stress-induced dysfunction in HBMSCs. U0126, a highly selective inhibitor of extracellular ERK1/2 MAPK signaling, significantly suppressed the antioxidative and osteogenic effects in HAMSCs. In conclusion, by modulating HBMSCs, HAMSCs show a strong potential in treating oxidative stress- mediated bone deficiency.
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문제 정의
The core purpose of our present study was to investigate the potential molecular signaling pathways engaged by HAMSCs. Signaling pathways involved in oxidative stress-induced inhibition of osteogenic differentiation consist of MAPK, Akt/mTOR/ 4EBP1, p53 and NF-κB (Martindale and Holbrook, 2002; Zeng et al.
가설 설정
, 2014). We, therefore, hypothesized that HAMSCs play a role in oxidative stressinduced bone deficiency. In this study, we used a Transwell coculture system to investigate the in vitro effects of HAMSCs on osteogenic differentiation in H2O2-induced HBMSCs.
제안 방법
A Cell-Light™ EdU Apollo®488 In Vitro Imaging Kit was used to investigate the EdU levels in each group at 5 days.
5%) for 15 min, staining solution was added to each well. After incubating for 30 min, EdU levels were examined by immunofluorescence staining and the area of stained EdU-positive nodules relative to the total culture surface was measured by Image-pro Plus analysis (IPP). DAPI was used to stain the cell nuclei.
All data were expressed as mean ± S.D. from at least three independent experiments, each performed in quintuplicate.
Transwells containing HAMSCs were moved into the corresponding wells of the 6-well culture plate containing HBMSCs in routine culture media supplemented with 10% FBS. At 3, 5 and 7 days, Transwells containing HAMSCs were removed and HBMSCs were harvested. After fixing with 75% ice-cold ethanol at 4℃ in the dark (Wang et al.
-induced HBMSCs seeded in the transwell co-culture system. Cell cycle fractions (G0, G1, S, and G2M phases) were determined by flow cytometry at 3, 5 and 7 days. At 3 and 7 days, the S-phase showed no significant difference between each groups.
In this study, an H2O2-induced oxidative cell injury model was built using HBMSCs and 200 μM H2O2 management for 12 h.
We, therefore, hypothesized that HAMSCs play a role in oxidative stressinduced bone deficiency. In this study, we used a Transwell coculture system to investigate the in vitro effects of HAMSCs on osteogenic differentiation in H2O2-induced HBMSCs. Interestingly, we found that HAMSCs promoted proliferation, activated alkaline phosphatase activity (ALP), enhanced the expression of osteogenic marker proteins and stimulated mineralized matrix deposition.
To elucidate whether the beneficial effects of HAMSCs were linked to their antioxidant properties, the ROS generation in H2O2-induced HBMSCs was measured. Subsequently, macrographs of DCFDA fluorescence were analyzed qualitatively using fluorescence microscopy and the area of stained ROSpositive
대상 데이터
The HBMSCs were obtained from the American Type Culture Collection (ATCC, USA). HAMSCs were collected from abandoned amniotic membrane samples obtained within 24 h using the pancreatin/collagenase digestion method as previously described (Zhang et al.
The Cell-Light™ EdU Apollo®488 In Vitro Imaging Kit was purchased from RiboBio (China).The goat anti-rabbit IgG, phospho-p44/42 MAPK rabbit mAb(p-ERK1/2), p44/42 MAPK (ERK1/2) rabbit mAb, and Runx2 (D1L7F) rabbit mAb were purchased from Cell Signaling Technology (USA).The protein assay kit, RIPA buffer, and DAPI were purchased from Beyotime (China).
U0126 (ERK1/2 inhibitor), 2,7-dichlorodihydrofluoresceindiacetate (DCFH-DA), β-glycerophosphate, ascorbic acid, and dexamethasone were purchased from Sigma–Aldrich (USA).
데이터처리
from at least three independent experiments, each performed in quintuplicate. Data were analyzed using one-way analysis of variance (ANOVA). P-values < 0.
성능/효과
Based on the results that ERK1/2 and Runx2 phosphorylation was activated in H2O2-induced HBMSCs cultured with HAMSCs, we used U0126, a highly selective inhibitor of ERK1/2, to clarify whether the effect of HAMSCs was ERK1/2-dependent. An appropriate HBMSCs: HAMSCs ratio was designed based on previous results.
1A). Further, immunofluorescence staining of EdU, a cell proliferation marker, revealed a statistically significant increase of EdU-positive HBMSCs nodules with the HBMSCs: HAMSCs ratio in co-culture groups compared with the single-culture groups at 5 days (Figs. 1B and 1C). Previous study had shown that co-culturing with HAMSCs promoted HBMSCs proliferation in the absence of hydrogen peroxide (Wang et al.
6D and 6E).The results suggested that by inhibiting the ERK1/2 MAPK signaling, HAMSCs failed to reverse the oxidant injury induced by H2O2.
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