Human umbilical cord mesenchymal stem cells protect MC3T3-E1 osteoblasts from dexamethasone-induced apoptosis via induction of the Nrf2-ARE signaling pathway.

Objective: To investigate the protective effect human umbilical cord mesenchymal stem cells (hUC-MSCs) have on Dexamethasone (Dex)-induced apoptosis in osteogenesis via the Nrf2-ARE signaling pathway. Methods: Glucocorticoid-induced osteonecrosis of the femoral head (GC-ONFH) was developed in rats through the administration of lipopolysaccharide and methylprednisolone. The incidence of femoral head necrosis, cavity notch, apoptosis of osteoblasts, and bone density were observed by HE staining, TUNEL staining, and Micro-CT. HUC-MSCs were co-cultured with mouse pre-osteoblast MC3T3-E1. The survival rate of osteoblasts was determined by CCK8, and apoptosis and ROS levels of osteoblasts were determined by flow cytometer. The viability of antioxidant enzymes SOD, GSH-Px, and CAT was analyzed by biochemistry. Nrf2 expression levels and those of its downstream proteins and apoptosis-related proteins were analyzed by Western blotting. Results: In rats, hUC-MSCs can reduce the rates of empty bone lacuna and osteoblast apoptosis that are induced by glucocorticoids (GCs), while reducing the incidence of GC-ONFH. hUC-MSCs can significantly improve the survival rate and antioxidant SOD, GSH-Px, and CAT activity of MC3T3-E1 cells caused by Dex, and inhibit apoptosis and oxidative stress levels. In addition, hUC-MSCs can up-regulate the expression of osteoblast antioxidant protein Nrf2 and its downstream protein HO-1, NQO-1, GCLC, GCLM, and apoptosis-related protein bcl-2, while also down-regulating the expression of apoptosis-related protein bax, cleaved caspase-3, cleaved caspase-9, and cytochrome C in MC3T3-E1 cells. hUC-MSCs improve the ability of MC3T3-E1 cells to mineralize to osteogenesis. However, the promoting effects of hUC-MSCs were abolished following the blocking of the Nrf2-ARE signaling pathway for osteoblasts. Conclusion: The results reveal that hUC-MSCs can reduce Dex-induced apoptosis in osteoblasts via the Nrf2-ARE signaling pathway.

Extracellular vesicles from human umbilical cord mesenchymal stem cells prevent steroid-induced avascular necrosis of the femoral head via the PI3K/AKT pathway.

Extracellular vesicles (EVs) secreted by human umbilical cord mesenchymal stem cells (hucMSC) have excellent therapeutic potential for many diseases. The aim of this study was to define the role of hucMSC-EVs in the prevention and treatment of steroid-induced avascular necrosis of the femoral head (SANFH). After establishing the SANFH rat model, the effects of hucMSC-EVs were assessed by measuring the microstructure of the femoral head using HE staining, micro-computed tomography (micro-CT), and TUNEL staining. The administration of hucMSC-EVs caused a significant reduction to glucocorticoids (GCs)-induced osteoblast apoptosis and empty lacuna of the femoral head, while effectively improving the microstructure. HucMSC-EVs rescued the deactivation of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway induced by GCs, and reversed the proliferation and migration of osteoblasts inhibited by GCs. In addition, hucMSC-EVs attenuated the inhibitory effects of GCs on rat osteoblast osteogenesis, angiogenesis of endothelial cells, and prevented osteoblast apoptosis. However, the promoting effects of hucMSC-EVs were abolished following the blockade of PI3K/AKT on osteoblasts. hucMSC-EVs were found to prevent glucocorticoid-induced femoral head necrosis in rats through the PI3K/AKT pathway.

Astaxanthin attenuates osteoarthritis progression via inhibiting ferroptosis and regulating mitochondrial function in chondrocytes.

Ferroptosis is a novel form of regulated cell death that has a close association with mitochondrial dysfunction and is characterized by iron overload, the accumulation of reactive oxygen species (ROS), and lipid ROS. Chondrocytes ferroptosis accelerates the progression of osteoarthritis (OA). Astaxanthin (ATX) is a xanthophyll carotenoid that possesses anti-inflammatory and antioxidant properties and has been explored in research studies for the treatment of diabetes and cardiovascular disease. However, the role it plays in OA, particularly in chondrocyte ferroptosis, has not yet been reported. In this study, ferroptosis-related events were analyzed in rat chondrocytes in vitro. A surgical destabilized medial meniscus was performed for the establishment of in vivo OA model. The results showed that interleukin-1ß (IL-1ß) induced inflammatory injury in chondrocytes through the promotion of the expressions of inflammatory factors including inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX2). IL-1ß triggered chondrocyte ferroptosis by increasing the levels of intracellular ROS, lipid ROS, iron, and mitochondrial iron and inhibiting the expressions of SLC7A11/glutathione peroxidase 4 (GPX4) and Ferritin. The above indices were ameliorated by ferrostatin-1 (Fer-1, a classic ferroptosis inhibitor) and ATX. Furthermore, Fer-1 and ATX rescued the IL-1ß-induced down-regulating collagen type II (collagen Ⅱ) and up-regulating matrix metalloproteinase 13 (MMP13). Following treatment with IL-1ß, mitochondrial membrane potential decreased and the mitochondrial membrane was broken. At the same time, the mitochondrion shrank, becoming deformed as the mitochondrial cristae reduced and became disrupted. These changes were entirely consistent with ferroptosis features. All the aforementioned phenomena were reversed by Fer-1 and ATX. In addition, intra-articular injection of Fer-1 and ATX delayed articular cartilage degradation and OA progression. This study demonstrated that IL-1ß can induce inflammatory damage and ferroptosis in chondrocytes. Both Fer-1 and ATX have the ability to mitigate chondrocyte injury and osteoarthritis progression by inhibiting ferroptosis and the regulation of mitochondrial function. Targeting ferroptosis has the potential to be a promising future treatment method for OA.

Glucocorticoids induce femoral head necrosis in rats through the ROS/JNK/c-Jun pathway.

Osteonecrosis of the femoral head (ONFH) is a common clinical disease with a high disability rate. Apoptosis of osteoblasts caused by high-dose short-term or low-dose long-term glucocorticoid (GC) administration is the biological basis of steroid-induced avascular necrosis of the femoral head (SANFH). The pathogenesis of SANFH has not yet been fully elucidated, and there is currently a lack of effective clinical treatments. Here, we investigated the role of the reactive oxygen species (ROS)/JNK/c-Jun signaling pathway in SANFH. Dexamethasone (Dex) was used to induce apoptosis in osteoblasts, and this resulted in a significant increase in levels of p-JNK, p-c-Jun, Bax, caspase-3, caspase-9, cytochrome C, Beclin-1, and LC3, and a decrease in levels of P62 and Bcl-2. In addition, intracellular ROS levels were increased and mitochondrial membrane potential was decreased. Administration of 3-MA, an autophagy inhibitor, attenuated Dex-mediated changes in autophagy and apoptosis. A rat model of ONFH exhibited severe bone trabecular hollow bone pits along with a significant increase in femoral head cell apoptosis compared with the control group. Additionally, micro-CT analysis showed that both bone tissue content and femoral head integrity were significantly reduced in the ONFH group. Furthermore, 3-MA treatment decreased the effect of Dex on GC-induced ONFH and osteoblast apoptosis in rats and could counteract microstructure destruction due to femoral head necrosis. In summary, our data suggest that GC can induce osteoblast apoptosis and autophagy through the ROS/JNK/c-Jun signaling pathway, which contributes to ONFH.

Anti-inflammatory and anti-apoptosis activity of taraxasterol in ulcerative colitis in vitro and in vivo.

Ulcerative colitis is closely associated with colorectal cancer, the long-standing chronic inflammation being the key etiology of ulcerative colitis. The aim of the present study was to identify the anti-inflammatory and anti-apoptosis activity of taraxasterol in ulcerative colitis. MTT assay was used to obtain the optimal concentrations of lipopolysaccharide (LPS) and taraxasterol for cell treatments in vitro. A mouse model of colitis was established via dextran sodium sulphate (DSS) administration. Levels of IL-6 and TNF-α were detected through ELISA. Flow cytometry and western blotting were used to detect apoptosis and related protein expression levels, respectively. Hematoxylin and eosin staining was performed to detect the pathological damage. The results from the MTT assay identified the optimal concentration of LPS and taraxasterol, and ELISA results demonstrated that taraxasterol treatment decreased the expression levels of IL-6 and TNF-α in vitro and in vivo, in a dose-dependent manner. Taraxasterol treatment inhibited apoptosis, and reduced the protein levels of p53, Bcl-2 associated X (BAX) and caspase-3. Finally, pathological damages were reduced in colonic tissues of mice treated with taraxasterol. Taken together, taraxasterol treatment markedly inhibited inflammation and apoptosis in ulcerative colitis. Therefore, taraxasterol may be a promising agent for decreasing the inflammatory response in ulcerative colitis and other inflammation-related diseases.

Decrease of GSK3ß Ser-9 Phosphorylation Induced Osteoblast Apoptosis in Rat Osteoarthritis Model.

Nowadays, the cumulative intake of glucocorticoids has become the most common pathogenic factor for non-traumatic osteonecrosis of the femoral head (ONFH). Apoptosis of osteoblasts is considered as the main reason of ONFH at the molecular level. Glycogen synthase kinase 3ß (GSK3ß) is an important regulator of cellular differentiation and apoptosis pathway, which can modulate the balance between osteoblasts and osteoclasts. Several studies have reported about its function in osteoporosis, but little is known about it in osteonecrosis. In our study, lipopolysaccharide and methylprednisolone were utilized to establish a rat ONFH model. The phosphorylation of GSK3ß Ser-9 was decreased in the model. Western blotting examination of ß-catenin, Bcl-2, Bax and caspase-3 revealed that the osteoblasts were apoptotic. In dexamethasone (Dex)-incubated primary osteoblasts, the expression profile of GSK3ß phosphorylation and apoptotic factors were consistent with those in the rat ONFH model. To further investigate the regulation of osteonecrosis caused by GSK3ß, the expression and function of GSK3ß were inhibited in Dex-incubated primary osteoblasts. The knockdown of GSK3ß by siRNA decreased the expression of Bax and cleaved caspase-3, but increased Bcl-2 and ß-catenin. On the other hand, selective inhibition of GSK3ß function by LiCl counteracted the activation of caspase-3 induced by Dex. Our work is the first study about the GSK3ß phosphorylation in ONFH, and provides evidence for further therapeutic methods.

Gene expression profiling of osteoblasts subjected to dexamethasone-induced apoptosis with/without GSK3ß-shRNA.

OBJECTIVE: Glucocorticoids (GCs)-induced osteoblast apoptosis has been identified as an important cause of GCs related osteonecrosis of the femoral head (ONFH). Glycogen synthase kinase 3ß (GSK3ß) has been proved to mediate dexamethasone (Dex)-induced osteoblast apoptosis. This study aimed to investigate the underlying mechanism of GSK3ß in Dex-induced osteoblast apoptosis. METHODS: Osteoblast cells were transfected with lentivirus expressing GSK3ß-shRNA, and a DNA microarray was performed to analyze gene expression after Dex treatment with or without GSK3ß-shRNA. Some differentially expressed genes were further validated by quantitative real-time-PCR (qRT-PCR). RESULTS: 460 genes were up-regulated (at least 2-fold) with Dex treatment but down-regulated (at least 2-fold) with GSK3ß-shRNA treatment. In addition, 315 genes were down-regulated (at least 2-fold) with Dex treatment but up-regulated (at least 2-fold) with GSK3ß-shRNA treatment. Among these genes, the apoptosis-related genes Hoxb8, Kif18a, Dock8, Dlk1, Tnfsf14, Casq2, Bcl2l14 and mechanosensation-related gene Piezo2 were selected for further qRT-PCR analysis. 7 of 8 genes (Piezo2, Hoxb8, Kif18a, Dlk1, Tnfsf14, Casq2, Bcl2l14) showed the same tendency between gene chip results and qRT-PCR results. The microarray data also showed that apoptotic pathway, MAPK pathway, TGFß pathway and Wnt pathway might be related to the mechanism of GSK3ß in Dex-induced osteoblast apoptosis. CONCLUSION: Our findings indicate that GSK3ß-shRNA treatment can alter various genes expression levels and change diverse signaling pathways involved in Dex-induced osteoblast apoptosis. Furthermore, Piezo2, Hoxb8, Kif18a, Dlk1, Tnfsf14, Casq2 and Bcl2l14 genes may play an important role in the GSK3ß-mediated osteoblast apoptosis process.

On the significance of Tim-3 expression in pancreatic cancer.

OBJECTIVE: We aim to explore the connection between Tim-3 expression in both cancerous pancreatic and pericarcinous tissues and the clinicopathological features of pancreatic cancer. We will also preliminarily assess the role and significance of Tim-3 in the diagnosis, treatment, and prognosis of pancreatic cancer. METHODS: Cancerous pancreatic and pericarcinous tissues from 50 patients with pancreatic cancer and six healthy pancreatic tissues were collected from the pathological specimens of traumatic patients to distinguish Tim-3 expression using immunohistochemistry. Tim-3 expression was observed to be correlated with cell invasion, metastasis, and recurrence of pancreatic cancer. RESULTS: 1. For the immunohistochemical method, Tim-3 expression in pancreatic cancer tissues was observed to be elevated and statistically significant (P < .01) compared to pericarcinous and normal pancreatic tissues. No statistically significant difference (P > .05) was observed between Tim-3 expression in pericarcinous and normal pancreatic tissues. 2. While Tim-3 expression was observed to be closely related to the history of smoking, fasting blood glucose, tumor size, TNM stage, it was not observed to be related to gender, age, tumor location, pathological type, and degree of tumor differentiation. CONCLUSION: 1. Tim-3 expression in pancreatic cancer tissues was high. 2. The high Tim-3 expression in pancreatic cancer tissues may be closely related to cell invasion, metastasis, and the recurrence of pancreatic cancer.