UBE2L3 Promotes Oxidative Stress-regulated Necroptosis to Accelerate Osteosarcoma Progression.

BACKGROUND: Osteosarcoma is a highly invasive bone marrow stromal tumor with limited treatment options. Oxidative stress plays a crucial role in the development and progression of tumors, but the underlying regulatory mechanisms are not fully understood. Recent studies have revealed the significant involvement of UBE2L3 in oxidative stress, but its specific role in osteosarcoma remains poorly investigated. OBJECTIVE: This study aimed to explore the molecular mechanisms by which UBE2L3 promotes oxidative stress-regulated necroptosis to accelerate the progression of osteosarcoma using in vitro cell experiments. METHODS: Human osteoblast hFOB1.19 cells and various human osteosarcoma cell lines (MG-63, U2OS, SJSA-1, HOS, and 143B) were cultured in vitro. Plasmids silencing UBE2L3 and negative control plasmids were transfected into U2OS and HOS cells. The cells were divided into the following groups: U2OS cell group, HOS cell group, si-NC-U2OS cell group, si-UBE2L3-U2OS cell group, si-NC-HOS cell group, and si-UBE2L3-HOS cell group. Cell viability and proliferation capacity were measured using the Tunnel method and clonogenic assay. Cell migration and invasion abilities were assessed by Transwell and scratch assays. Cell apoptosis was analyzed by flow cytometry, and ROS levels were detected using immunofluorescence. The oxidative stress levels in various cell groups and the expression changes of necroptosis-related proteins were assessed by PCR and WB. Through these experiments, we aim to evaluate the impact of oxidative stress on necroptosis and uncover the specific mechanisms by which targeted regulation of oxidative stress promotes tumor cell necroptosis as a potential therapeutic strategy for osteosarcoma. RESULTS: The mRNA expression levels of UBE2L3 in human osteosarcoma cell lines were significantly higher than those in human osteoblast hFOB1.19 cells (p <0.01). UBE2L3 expression was significantly decreased in U2OS and HOS cells transfected with si-UBE2L3, indicating the successful construction of stable cell lines with depleted UBE2L3. Tunnel assay results showed a significant increase in the number of red fluorescent-labeled cells in si-UBE2L3 groups compared to si-NC groups in both cell lines, suggesting a pronounced inhibition of cell viability. Transwell assay demonstrated a significant reduction in invasion and migration capabilities of si-UBE2L3 groups in osteosarcoma cells. The clonogenic assay revealed significant suppression of proliferation and clonogenic ability in both U2OS and HOS cells upon UBE2L3 knockdown. Flow cytometry confirmed that UBE2L3 knockdown significantly enhanced apoptosis in U2OS and HOS cells. Immunofluorescence results showed that UBE2L3 silencing promoted oxidative stress levels in osteosarcoma cells and facilitated tumor cell death. WB analysis indicated a significant increase in phosphorylation levels of necroptosis-related proteins, RIP1, RIP3, and MLKL, in both osteosarcoma cell lines after UBE2L3 knockdown. In addition, the expression of necrosis-associated proteins was inhibited by the addition of the antioxidant N-acetylcysteine (NAC). CONCLUSION: UBE2L3 is upregulated in osteosarcoma cells, and silencing of UBE2L3 promotes oxidative stress in these cells, leading to enhanced necroptosis and delayed progression of osteosarcoma.

Percutaneous kyphoplasty for osteoporotic vertebral compression fractures improves spino-pelvic alignment and global sagittal balance maximally in the thoracolumbar region.

BACKGROUND: Osteoporotic vertebral compression fractures (OVCFs) often cause local kyphosis. Percutaneous kyphoplasty (PKP) is a common method for the treatment of local kyphosis. However, the influence of kyphoplasty on spino-pelvic alignment and global sagittal balance when performed at specific treatment sites in the spine remains unclear. The purpose of the study is to investigate the influence of different fracture sites and PKP treatment on the spino-pelvic alignment and global sagittal balance in patients with OVCFs. METHODS: 90 patients with OVCF who underwent PKP were included in the retrospective study. According to the site of the fractured vertebrae, all the cases were divided into 3 groups: Main thoracic (MT) group (T1 to T9), Thoracolumbar (TL) group (T10 to L2) and Lumbar (LU) group (L3 to L5). 26 healthy elderly volunteers (aged over 59) were enrolled as the control group. Sagittal spino-pelvic parameters were measured on the full-spine radiographs preoperatively and postoperatively. Information of sagittal spino-pelvic parameters and global sagittal balance was gathered. RESULTS: Compared with the Control group, TL group showed significant differences in almost all parameters, except pelvic incidence (PI) and lumbar lordosis (LL). While only local sagittal parameters (Thoracic kyphosis (TK), Thoracolumbar kyphosis (TLK), LL) were significantly different in MT group. There was no significant difference in almost all of the parameters except for PT and TPA in LU group. Correspondingly, the sagittal parameters of TL group improved best after PKP, except for thoracic kyphosis (TK) and sagittal vertical axis (SVA). In MT group, only TLK was significantly decreased, while in LU group, only local kyphosis Cobb angle and SSA were improved. CONCLUSIONS: OVCF mainly occurs in the thoracolumbar region. Compared with MT group and LU group, OVCF occurred in the thoracolumbar region had greater influence on the spino-pelvic alignment and global sagittal balance. When PKP was performed, the improvement of sagittal balance parameters of TL group was the best in the three groups.

Butyrate inhibit collagen-induced arthritis via Treg/IL-10/Th17 axis.

Rheumatoid arthritis (RA) is a chronic autoimmune disorder demanding the development of novel therapeutic strategy. Butyrate is a functional short-chain fatty acid produced by the anaerobic intestinal microbiota. This study aimed to investigate the attenuation of butyrate on RA. The collagen-induced arthritis (CIA) mouse model was established and butyrate was administered in drinking water along with the collagen immunization. The histopathological features, clinical score, paw swelling, as well as the production of pro-inflammatory cytokines including interleukin (IL)-1ß, IL-6 and IL-17A were measured to determine the amelioration of butyrate on arthritis. The differentiation of Treg cells and Th17 cells in the splenic cells was assessed by flow cytometry. The expression of Foxp3, IL-10, Rorγt and IL-17A were detected by RT-PCR and FACS immunostaining. Anti-IL10R antibody was used in the CIA and CD4+ cell cultures to mediate the effects of butyrate. Butyrate significantly inhibited expressions of IL-1ß, IL-6 and IL-17A, but promoted the expression of IL-10. Butyrate also increased systematical Treg cells and reduced Th17 cells. Mechanism study revealed that butyrate directly enhanced the polarization of Treg cells but not Th17 cells. All effects of butyrate on RA were inversed by the co-administered anti-IL10R antibody. This study showed that butyrate administration inhibited arthritis in CIA mice model, suppressed the expression of inflammatory cytokines. The modulation may be mediated the differentiation of CD4 T cells towards Treg cells, which produce anti-inflammatory cytokine IL-10, and thus influenced the function of Th17 cells.

Association of matrix Gla protein polymorphism and knee osteoarthritis in a chinese population.

Several studies have explored the association between matrix Gla protein (MGP) gene polymorphism and knee osteoarthritis (OA) risk; however, they obtained conflicting findings. The present study aims to explore the association of MGP gene polymorphism and OA risk in a Chinese Han population. A total of 256 patients with radiographic knee OA and 327 control subjects were recruited in this case-control study. The genotypes of MGP gene rs1800802 polymorphism was determined by standard PCR and restriction fragment length polymorphism (PCR-RLFP). In this case-control study, we observed that MGP gene rs1800802 polymorphism increased the risk of knee OA. Subgroup analyses also found that rs1800802 polymorphism was related to the elevated risk for knee OA among the female, smoker, drinker, and body mass index (BMI) ≥25 kg/m2 groups. In conclusion, this study shows that MGP gene rs1800802 polymorphism is associated with increased risk for knee OA in Chinese Han population and the rs1800802 polymorphism may be a diagnostic marker of radiographic knee OA.