Shuang Deng1, Guo Dai2, Sen Chen1, Zhigang Nie1, Jianlin Zhou1, Hongsong Fang1, Hao Peng3. 1. Department of Orthopedics, Renmin Hospital of Wuhan University, Jiefang Road 238, Wuhan, Hubei, 430060, China. 2. Department of Spine Surgery, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, 510080, Guangdong, China. 3. Department of Orthopedics, Renmin Hospital of Wuhan University, Jiefang Road 238, Wuhan, Hubei, 430060, China. Electronic address: PengHao0503@163.com.
Abstract
BACKGROUND: Osteoblasts play important roles in the process of osteogenesis and prevention of osteonecrosis. Dexamethasone (Dex), a type of glucocorticoids (GCs), induces apoptosis of osteoblasts and leads to the occurrence of non-traumatic osteonecrosis. This study aimed to explore the effects of phosphatidylinositol 3-kinase/Protein kinase 3 (PI3K/AKT) and glycogen synthase kinase 3β (GSK3β) on Dex-induced osteoblasts apoptosis. METHODS: Viabilities, proliferation, and apoptosis of primary osteoblasts and pre-osteoblast MC3T3-E1 cells after Dex treatment were detected using cell counting kit-8 (CCK-8) assay, 5-bromo-2'-deoxyuridine (BrdU) incorporation assay, FITC-Annexin V/PI staining and western blotting, respectively. 2',7'-Dichlorodihydrofluorescein diacetate (DCFH-DA) staining was performed to measure the intracellular reactive oxygen species (ROS) levels after Dex treatment. N-acetyl-l-cysteine (NAC) was used as ROS scavenger in this research. The expressions of PI3K/AKT and GSK3β in osteoblasts and MC3T3-E1 cells after Dex treatment were analyzed using western blotting and qRT-PCR, respectively. Then the effects of GSK3β knockdown on Dex-induced apoptosis of osteoblasts were explored. Alkaline phosphatase (ALP) activity assay was used to detect the role of Dex in regulating ALP activity. RESULTS: Dex remarkably inhibited proliferation and induced apoptosis of osteoblasts and MC3T3-E1 cells. Dex potentially attenuated the osteoblast differentiation. The intracellular ROS levels were significantly increased after Dex treatment. Dex suppressed the activation of PI3K/AKT pathway in osteoblasts and MC3T3-E1 cells by down-regulating the expressions of p-PI3K and p-AKT. The expressions of GSK3β in osteoblasts and MC3T3-E1 cells were obviously up-regulated after Dex treatment. Knockdown of GSK3β alleviated Dex-induced osteoblast and MC3T3-E1 cell apoptosis by decreasing the expressions of Bax, cleaved-caspase 3, cleaved-caspase 9 and increasing the expression of Bcl-2. CONCLUSION: Our research verified that Dex induced osteoblasts apoptosis by ROS-PI3K/AKT/GSK3β signaling pathway.
BACKGROUND: Osteoblasts play important roles in the process of osteogenesis and prevention of osteonecrosis. Dexamethasone (Dex), a type of glucocorticoids (GCs), induces apoptosis of osteoblasts and leads to the occurrence of non-traumatic osteonecrosis. This study aimed to explore the effects of phosphatidylinositol 3-kinase/Protein kinase 3 (PI3K/AKT) and glycogen synthase kinase 3β (GSK3β) on Dex-induced osteoblasts apoptosis. METHODS: Viabilities, proliferation, and apoptosis of primary osteoblasts and pre-osteoblast MC3T3-E1 cells after Dex treatment were detected using cell counting kit-8 (CCK-8) assay, 5-bromo-2'-deoxyuridine (BrdU) incorporation assay, FITC-Annexin V/PI staining and western blotting, respectively. 2',7'-Dichlorodihydrofluorescein diacetate (DCFH-DA) staining was performed to measure the intracellular reactive oxygen species (ROS) levels after Dex treatment. N-acetyl-l-cysteine (NAC) was used as ROS scavenger in this research. The expressions of PI3K/AKT and GSK3β in osteoblasts and MC3T3-E1 cells after Dex treatment were analyzed using western blotting and qRT-PCR, respectively. Then the effects of GSK3β knockdown on Dex-induced apoptosis of osteoblasts were explored. Alkaline phosphatase (ALP) activity assay was used to detect the role of Dex in regulating ALP activity. RESULTS:Dex remarkably inhibited proliferation and induced apoptosis of osteoblasts and MC3T3-E1 cells. Dex potentially attenuated the osteoblast differentiation. The intracellular ROS levels were significantly increased after Dex treatment. Dex suppressed the activation of PI3K/AKT pathway in osteoblasts and MC3T3-E1 cells by down-regulating the expressions of p-PI3K and p-AKT. The expressions of GSK3β in osteoblasts and MC3T3-E1 cells were obviously up-regulated after Dex treatment. Knockdown of GSK3β alleviated Dex-induced osteoblast and MC3T3-E1 cell apoptosis by decreasing the expressions of Bax, cleaved-caspase 3, cleaved-caspase 9 and increasing the expression of Bcl-2. CONCLUSION: Our research verified that Dex induced osteoblasts apoptosis by ROS-PI3K/AKT/GSK3β signaling pathway.
Authors: Mariam I Gamal El-Din; Nouran M Fahmy; Fulin Wu; Maha M Salem; Omar M Khattab; Hesham R El-Seedi; Michal Korinek; Tsong-Long Hwang; Ahmed K Osman; Mohamed El-Shazly; Shaimaa Fayez Journal: Plants (Basel) Date: 2022-06-27
Authors: Mohd Amir Kamaruzzaman; Kok-Yong Chin; Elvy Suhana Mohd Ramli Journal: Evid Based Complement Alternat Med Date: 2019-09-19 Impact factor: 2.629