CircSPI1_005 ameliorates osteoarthritis by sponging miR-370-3p to regulate the expression of MAP3K9.

BACKGROUND: Osteoarthritis (OA), caused by the destruction of joint cartilage, is the most prevalent form of arthritis, causing pain and stiffness in joints among millions of patients worldwide. Increasing evidence suggests that non-coding RNAs, including circular RNAs, play important roles in the pathogenesis of OA, but the precise signaling pathway is still unclear. METHODS: To study OA, we established a mouse model by the destabilized medial meniscus (DMM) surgery and used IL-1ß stimulated human cell line C28/I2 as an in vitro study. To further study the role of circSPI1_005 in regulating cell proliferation and apoptosis, EdU staining and FACS-based (fluorescence-activated cell sorting) apoptosis examination were performed after the manipulation of the expression of circSPI1_005. Also, bioinformatics predictions were conducted to analyze the downstream microRNAs of circSPI1_005 and the protein regulated by circSPI1_005. The luciferase assay and the RNA immunoprecipitation (RIP) assay were used to confirm the binding between circSPI1_005 and the predicted microRNA. To verify the role of circSPI1_005 in regulating OA in vivo, we also over-expressed circSPI1_005 by injecting AAV into previously injured knees to improve the OA symptoms. RESULTS: In this study, we found that circSPI1_005 was significantly down-regulated in IL-1ß treated chondrocyte cell lines and cartilage tissues of the OA mouse model. Overexpression of circSPI1_005 ameliorated OA by increasing proliferation and inhibiting apoptosis, and knockdown of circSPI1_005 in chondrocytes mimicked OA phenotypes. Bioinformatics study showed circSPI1_005 could sponge to miR-370-3p, and mechanistic studies confirmed the functional binding between circSPI1_005 and miR-370-3p. Furthermore, we conducted a TargetScan analysis and found that MAP3K9 (mitogen-activated protein kinase kinase kinase 9) could be the downstream protein effector. The expression level of MAP3K9 was regulated by miR-370-3p and overexpression of MAP3K9 could efficiently ameliorate OA. Also, we over-expressed circSPI1_005 in vivo and found that the cartilage surface in the OA mouse model was improved with overexpression of circSPI1_005. CONCLUSIONS: Collectively, circSPI1_005 could sponge to miR-370-3p to regulate the expression of MAP3K9, ameliorating the progression of osteoarthritis.

Glucocorticoids induce osteonecrosis of the femoral head in rats via PI3K/AKT/FOXO1 signaling pathway.

Background: Steroid-induced osteonecrosis of the femoral head (SONFH) is a disorder that causes severe disability in patients and has a high incidence worldwide. Although glucocorticoid (GC)-induced apoptosis of osteoblasts is an important cytological basis of SONFH, the detailed mechanism underlying SONFH pathogenesis remains elusive. PI3K/AKT signaling pathway was reported to involve in cell survival and apoptosis. Objective: We explored the role of PI3K/AKT/FOXO1 signaling pathway and its downstream targets during glucocorticoid -induced osteonecrosis of the femoral head. Methods: We obtained gene expression profile of osteoblasts subjected to dexamethasone (Dex) treatment from the Gene Expression Omnibus (GEO) database. Differentially expressed genes (DEGs) were screened out and functional enrichment analysis were conducted by bioinformatics analysis. In vitro, we analyzed Dex-induced apoptosis in MC3T3-E1 cells and explored the role of PI3K/AKT/FOXO1 signaling pathway in this phenomenon by employing siRNA-FOXO1 and IGF-1(PI3K/AKT agonist). Finally, we verified our results in a rat model of SONFH. Results: In Dex-treated osteoblasts, DEGs were mainly enriched in the FOXO signaling pathway. Dex inhibited MC3T3-E1 cell viability in a dose-dependent effect and induced apoptosis by increasing the expression levels of FOXO1, Bax, cleaved-Caspase-3, and cleaved-Caspase-9, while reducing the expression of Bcl-2. Notably, these results were reversed by siRNA-FOXO1 treatment. Dex inhibited PI3K/AKT signaling pathway, upregulated FOXO1 expression and increased FOXO1 nuclear translocation, which were reversed by IGF-1. Compared to normal rats, the femoral head of SONFH showed increased expression of FOXO1, increased number of apoptotic cells, and empty osteocytic lacunas, as well as decreased bone tissue content and femoral head integrity. Significantly, the effects of GC-induced SONFH were alleviated following IGF-1 treatment. Conclusion: Dex induces osteoblast apoptosis via the PI3K/AKT/FOXO1 signaling pathway. Our research offers new insights into the underlying molecular mechanisms of glucocorticoid-induced osteonecrosis in SONFH and proposes FOXO1 as a therapeutic target for this disease.

Dexamethasone induces ferroptosis via P53/SLC7A11/GPX4 pathway in glucocorticoid-induced osteonecrosis of the femoral head.

Recently, ferroptosis as new regulatory necrosis has attracted the scientific community. However, the study focused on the effect of ferroptosis on osteocytes in steroid (glucocorticoid)-induced osteonecrosis of the femoral head (SONFH) is still scarce. In this study, we use bioinformatic analysis to screen out differentially expressed genes (DEGs) in osteoblasts that treated by dexamethasone (Dex) in GSE10311 and found these DEGs are enriched in the ferroptosis signaling pathway. The results in vitro experiments show that Dex can induce MC3T3-E1 cells ferroptosis by down-regulating SLC7A11. Specifically, Dex inhibits the expression of SLC7A11/GPX4, decreases the activity of the intracellular antioxidant system such as intracellular glutathione (GSH), while increasing Malondialdehyde (MDA), reactive oxygen species (ROS), and lipid ROS, and reduces the volume of mitochondria, the mitochondrial ridges and a series of obvious ferroptosis features. The overexpression of SLC7A11 and the use of ferroptosis inhibitor (Fer-1) can reverse the Dex-induced MC3T3 ferroptosis. Dex can induce an increase in the expression of p53 and knocking down the expression of p53 by small interfering ribonucleic acid (siRNA) can reverse the suppression of SLC7A11 and GPX4 expression in MC3T3-E1 and MOLY4 cells, thereby reducing the production of ferroptosis. Thus, this study demonstrated that Dex induces MC3T3-E1cells ferroptosis via p53/SLC7A11/GPX4 pathway. The present finding offers novel insight to understand the underlying molecular mechanisms for glucocorticoid-induced osteonecrosis. Moreover, the suppression of ferroptosis may be a novel and promising treatment option for SONFH.

Protective Effect of Human Umbilical Cord Mesenchymal Stem Cells in Glucocorticoid-induced Osteonecrosis of Femoral Head.

OBJECTIVE: To evaluate the effect of human umbilical cord mesenchymal stem cells (hUC-MSCs) on preventing rats from glucocorticoid-induced osteonecrosis of femoral head (GCONFH) in the early stage in vivo and to investigate the possible mechanism of hUC-MSCs in regulating the balance of osteogenesis and adipogenesis. METHODS: All rats were randomly divided into 3 groups: control group (C group), model group (M group), and intervention group (I group). The model of GC-ONFH was developed by a sequential administration of lipopolysaccharide and methylprednisolone. The rats in the I group were treated with caudal vein injection of hUC-MSCs. Six weeks later, the blood samples were obtained to measure the activity of alkaline phosphatase (ALP) and the content of triglyceride (TG) in serum, and the femoral heads were harvested and observed by hematoxylin-eosin staining, Micro-CT, Western blot and real-time quantitative polymerase chain reaction. RESULTS: After intervention of hUC-MSCs, the necrosis rate of femoral head decreased from 83% (10/12) to 33% (4/12), the rate of empty bone lacuna was significantly decreased, the activity of ALP increased significantly, the content of TG decreased significantly, the bone density increased obviously, the expression of RUNX2 and Col I increased significantly and the expression of PPARγ decreased significantly. CONCLUSION: These results revealed that caudal vein injection of hUC-MSCs can effectively reduce the incidence of GC-ONFH in rats by increasing ALP activity and reducing TG content in serum, increasing bone mineral density, promoting the expression of RUNX2 and Col I, and inhibiting the expression of PPARγ.

Human papillomavirus distribution and cervical cancer epidemiological characteristics in rural population of Xinjiang, China.

BACKGROUND: Cervical cancer remains a major public health issue for the Uyghur women and other women living mainly in rural areas of Xinjiang. This study aims to investigate the distribution of human papillomavirus (HPV) infection and cervical cancer in rural areas of Xinjiang, China. METHODS: Cervical cancer screening was performed on rural women aged 35 to 64 years from Xinjiang, China in 2017 through gynecological examination, vaginal discharge smear microscopy, cytology, and HPV testing. If necessary, colposcopy and biopsy were performed on women with suspicious or abnormal screening results. RESULTS: Of the 216,754 women screened, 15,518 received HPV testing. The HPV-positive rate was 6.75% (1047/15,518). Compared with the age 35-44 years group, the odds ratios (ORs) of HPV positivity in the age 45-54 years and 55-64 years groups were 1.18 (95% confidence interval [CI]: 1.02-1.37) and 1.84 (95% CI: 1.53-2.21), respectively. Compared with women with primary or lower education level, the ORs for HPV infection rates of women with high school and college education or above were 1.37 (95% CI: 1.09-1.72) and 1.62 (95% CI: 1.23-2.12), respectively. Uyghur women were less likely to have HPV infection than Han women, with an OR (95% CI) of 0.78 (0.61-0.99). The most prevalent HPV types among Xinjiang women were HPV 16 (24.00%), HPV 33 (12.70%), and HPV 52 (11.80%). The detection rate of cervical intraepithelial neoplasia (CIN)2+ was 0.14% and the early diagnosis rate of cervical cancer was 85.91%. The detection rates of vaginitis and cervicitis were 19.28% and 21.32%, respectively. CONCLUSIONS: The HPV infection rate in Xinjiang is low, but the detection rate of cervical cancer and precancerous lesions is higher than the national average level. Cervical cancer is a prominent public health problem in Xinjiang, especially in southern Xinjiang.

Hsa-let-7g promotes osteosarcoma by reducing HOXB1 to activate NF-kB pathway.

MicroRNA (miRNA) is known to be involved in regulating the proliferation, migration and apoptosis of cancer cells in osteosarcoma. In this study, We aim to explore the expression of hsa-let-7 g and its role in pathogenesis of osteosarcoma. By analyzing clinical data. We found high expression of hsa-let-7 g in patients with osteosarcoma. The patients with higher expression of hsa-let-7 g showed poorer prognosis and lower survival rate. After downregulation of hsa-let-7 g in cell model and animal model, we found that with downregulation of hsa-let-7 g, the proliferation of osteosarcoma cells was significantly reduced, the level of migration and invasion was down-regulated, the cell cycle was inhibited, and cell apoptosis was increased. Through Dual Luciferase Reporter, immunohistochemistry, western blot and other experiments, it was found that hsa-let-7 g down-regulated HOXB1 gene and activated NF-kB pathway to promote the development of osteosarcoma. In conclusion, hsa-let-7 g is highly expressed in osteosarcoma tissues, and high expression of hsa-let-7 g can promote the occurrence of osteosarcoma by down-regulating HOXB1 and activating NF-kB pathway.

Local intra­articular injection of vascular endothelial growth factor accelerates articular cartilage degeneration in rat osteoarthritis model.

In the pathophysiology of osteoarthritis (OA), articular cartilage degeneration exhibits a significant role. Vascular endothelial growth factor (VEGF) is considered to be an effective angiogenic factor and a crucial regulator of articular cartilage degeneration in the development of OA. Therefore, the present study aimed to investigate the underlying influences of exogenous VEGF on articular cartilage degeneration in OA model rat. A total of 24 male Sprague­Dawley rats were randomly allocated into 3 groups. In the normal saline (NS) and VEGF groups, animals received bilateral anterior cruciate ligament (ACL) transection to establish the OA model; at 4 weeks post­surgery, the rats received local intra­articular injections of 100 µl NS or VEGF solution, respectively, every week for 4 weeks. The Control group received neither surgery nor injections. All animals were sacrificed at 12 weeks following surgery. Prominent cartilage degeneration was observed in rats in the NS­ and VEGF­injected groups. The extent and the grade of cartilage damage in the VEGF­injected group were notably more severe compared with those in the NS­treated group. Western blotting results demonstrated that the expression levels of aggrecan and type II collagen were significantly reduced in OA model rats that were treated with VEGF. In addition, the expression levels of matrix metalloproteinase (MMP)­3, MMP­9, MMP­13, a disintegrin and metalloproteinase with thrombospondin motifs (a disintegrin and metalloproteinase; ADAMTS)­4, ­5 and ­12, type III collagen and transforming growth factor­ß1 were significantly increased following VEGF administration. Results from the present study indicated that VEGF may exhibit a promoting role in the development of OA by destroying articular cartilage matrix.

The proteasome activator PA28γ, a negative regulator of p53, is transcriptionally up-regulated by p53.

PA28γ (also called REGγ, 11Sγ or PSME3) negatively regulates p53 activity by promoting its nuclear export and/or degradation. Here, using the RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE) method, we identified the transcription start site of the PA28γ gene. Assessment with the luciferase assay demonstrated that the sequence -193 to +16 is the basal promoter. Three p53 binding sites were found within the PA28γ promoter utilizing a bioinformatics approach and were confirmed by chromatin immunoprecipitation and biotinylated DNA affinity precipitation experiments. The p53 protein promotes PA28γ transcription, and p53-stimulated transcription of PA28γ can be inhibited by PA28γ itself. Our results suggest that PA28γ and p53 form a negative feedback loop, which maintains the balance of p53 and PA28γ in cells.

Pulsed electromagnetic fields protect the balance between adipogenesis and osteogenesis on steroid-induced osteonecrosis of femoral head at the pre-collapse stage in rats.

This study was designed to investigate the effects of pulsed electromagnetic fields (PEMF) on the balance of adipogenesis and osteogenesis on steroid-induced osteonecrosis of the femoral head (OFH) in rats. Forty-two rats were divided into three groups: Steroid group (S, n = 16); Steroid + PEMF group (S + P, n = 16); and Control group (C, n = 10). For groups S and S + P, all rats were first intravenously given 10 µg/kg lipopolysaccharide on day 1, and then intramuscularly injected with 20 mg/kg methylprednisolone acetate on days 2, 3, and 4, with an interval of 24 h. After 4 weeks, the S + P group was treated with PEMF (4.5-ms square pulse, repeated at 15 Hz, with a peak of 1.2 mT) for 4 h a day for the next 8 weeks. Group S was not exposed to PEMF. Group C was chosen as the control group, without steroid use and exposure to PEMF. After 8 weeks of treatment, the histological changes, and mRNA and protein expressions of PPAR-γ2 and Runx2 were measured and analyzed. Compared with the S group, lower incidence of osteonecrosis (31% vs. 69%, P < 0.05) and empty osteocyte lacuna rate (36.16 ± 15.34 vs. 59.55 ± 21.70, P < 0.01) was observed in the S + P group. Furthermore, PEMF suppressed the expressions of PPAR-γ2 and improved the expressions of Runx2 in the femoral head (P < 0.05). All data suggest that PEMF is an effective physiotherapy in the treatment of steroid-induced ONFH, and the possible underlying mechanisms include protecting the balance between adipogenesis and osteogenesis.

The effect of vascular endothelial growth factor on aggrecan and type II collagen expression in rat articular chondrocytes.

The expression of vascular endothelial growth factor (VEGF) directly correlates with the Mankin score and the degree of cartilage destruction. The biological activity of VEGF on articular cartilage remains unknown, so this study was performed to investigate the effect of VEGF on aggrecan and type II collagen expression in vitro. We carried out this study at the Center Laboratory of Renmin Hospital at Wuhan University. Rat articular chondrocytes were cultured in a monolayer. Then, the experiment was divided into 4 groups: group A (control group), without any disposal; group B, treated with 10 ng/ml VEGF; group C, treated with 10 ng/ml IL-1ß; and group D, treated with 10 ng/ml VEGF + 10 ng/ml IL-1ß. After 48 h, messenger RNA (mRNA) expression of aggrecan and type II collagen was evaluated by real-time polymerase chain reaction (real-Time PCR), and protein expression of aggrecan and type II collagen was detected by Western blotting. VEGF was found to significantly inhibit the expression of aggrecan and type II collagen at the gene and protein levels. These findings suggest that VEGF may result in degeneration of articular cartilage by inhibiting the synthesis and expression of aggrecan and type II collagen.

Pulsed electromagnetic fields stimulation prevents steroid-induced osteonecrosis in rats.

BACKGROUND: Pulsed electromagnetic fields (PEMF) stimulation has been used successfully to treat nonunion fractures and femoral head osteonecrosis, but relatively little is known about its effects on preventing steroid-induced osteonecrosis. The purpose of the study was to investigate the effects of PEMF stimulation on the prevention of steroid-induced osteonecrosis in rats and explore the underlying mechanisms. METHODS: Seventy-two male adult Wistar rats were divided into three groups and treated as follows. (1) PEMF stimulation group (PEMF group, n = 24): intravenously injected with lipopolysaccharide (LPS, 10 µg/kg) on day 0 and intramuscularly injected with methylprednisolone acetate (MPSL, 20 mg/kg) on days 1, 2 and 3, then subjected to PEMF stimulation 4 h per day for 1 to 8 weeks. (2) Methylprednisolone-treated group (MPSL group, n = 24): injected the same dose of LPS and MPSL as the PEMF group but without exposure to PEMF. (3) Control group (PS group, n = 24): injected 0.9% saline in the same mode at the same time points. The incidence of osteonecrosis, serum lipid levels and the mRNA and protein expression of transforming growth factor ß1 (TGF-ß1) in the proximal femur were measured 1, 2, 4 and 8 weeks after the last MPSL (or saline) injection. RESULTS: The incidence of osteonecrosis in the PEMF group (29%) was significantly lower than that observed in the MPSL group (75%), while no osteonecrosis was observed in the PS group. The serum lipid levels were significantly lower in the PEMF and PS groups than in the MPSL group. Compared with the MPSL and PS groups, the mRNA expression of TGF-ß1 increased, reaching a peak 1 week after PEMF treatment, and remained high for 4 weeks, then declined at 8 weeks, whereas the protein expression of TGF-ß1 increased, reaching a peak at 2 weeks after PEMF treatment, and remained high for 8 weeks. CONCLUSIONS: PEMF stimulation can prevent steroid-induced osteonecrosis in rats, and the underlying mechanisms involve decreased serum lipid levels and increased expression of TGF-ß1.

Effects of hyaluronan on vascular endothelial growth factor and receptor-2 expression in a rabbit osteoarthritis model.

BACKGROUND: Little is known about the expression of vascular endothelial growth factor (VEGF) and its receptor-2 (VEGFR-2) mRNA in the cartilage of a rabbit osteoarthritis model or the influence of intraarticular injection of hyaluronan (HA) on the expression of VEGF and VEGFR-2 in cartilage during the process of osteoarthritis (OA). The therapeutic mechanism of HA is not completely understood, and we hypothesize that the mechanism is through the effects of VEGF and VEGFR2. In this study, we determined the VEGF and VEGFR-2 mRNA expression in a rabbit OA model and assessed the therapeutic mechanism of HA against OA. METHODS: We carried out this study at the Center Laboratory of Renmin Hospital at Wuhan University and the Key Laboratory of Respiratory Disease of the Ministry of Public Health, Huazhong University of Science and Technology. Between October 2006 and April 2008 a total of 24 mature New Zealand white rabbits were divided into three groups: normal controls, a no-HA group, and an HA group. The no-HA and HA groups underwent unilateral anterior cruciate ligament transection. At 4 weeks after the operation, animals in the HA group received intraarticular injections of 1% sodium hyaluronate (HA) once a week for 5 weeks as per the clinical treatment presently utilized. The no-HA rabbits were not given HA. At death, 11 weeks following surgery, cartilage was harvested and total RNA was extracted. VEGF and VEGFR mRNAs were analyzed using the reverse transcription-polymerase chain reaction (RT-PCR) and real-time PCR for each group. RESULTS: Cartilage damage (both extent and grade) was less severe in the HA group than in the no-HA group. VEGF and VEGFR-2 mRNA expression was enhanced in the cartilage of the OA model. Intraarticular 1% sodium hyaluronate injection inhibited VEGFR-2 expression but had no effect on reducing the VEGF mRNA expression in cartilage. CONCLUSIONS: These results suggested that VEGF and VEGFR-2 may be involved in the progression of OA and in the therapeutic mechanism of HA at least partly through the influence of VEGFR-2 expression during the development of OA.

The protective effect of sodium hyaluronate on the cartilage of rabbit osteoarthritis by inhibiting peroxisome proliferator-activated receptor-gamma messenger RNA expression.

PURPOSE: The purpose of this study was to study the protective effect and influence of sodium hyaluronate (Na-HA) on mRNA expression of peroxisome proliferators-activated receptor gamma (PPAR-gamma) in cartilage of rabbit osteoarthritis (OA) model. MATERIALS AND METHODS: Forty eight white rabbits were randomly divided into A, B, and C groups. Group A was normal control group, B and C groups underwent unilateral anterior cruciate ligament transection (ACLT). The rabbits in group B were injected normal saline after ACLT; and Group C received intra-articular 1% sodium hyaluronate (HA) injection 5 weeks after surgery, 0.3 mL once a week. At 11th week after surgery, all the rabbits were sacrificed. The cartilage changes on the medial femoral condyles were graded separately. Cartilage sections were stained with safranin-O and HE, and messenger RNA (mRNA) expression of PPAR-gamma was detected by using real time polymerase chain reaction (Real Time-PCR). RESULTS: Cartilage degeneration in group B was significantly more severe than in A and C injection group. The grey value of Safranin-O of B group was higher than A and C groups. Expression of PPAR-gamma mRNA in group B was higher than group A and C. CONCLUSION: This study shows that Na-HA has a protective effect on articular cartilage degeneration, and the inhibitory effect on the PPAR-gamma mRNA expression may be one of therapeutic mechanism of Na-HA.

Comparison of therapeutic effect between percutaneous kyphoplasty and pedicle screw system on vertebral compression fracture.

OBJECTIVE: To compare the clinical efficacy of percutaneous kyphoplasty (PKP) with pedicle screw system (PS) in the treatment of vertebral compression fracture(VCF). METHODS: Eighty-six patients with VCF were treated either by PKP (Group A, n equal to 30) or PS (Group B, n equal to 56). The anterior, intermediate, and posterior heights of the vertebrae body, visual analogue pain scale (VAS) before and after operation, the duration of operation, and amount of blood loss between two groups were compared. RESULTS: No statistical difference was noted regarding the vertebral height between two groups. Significant difference was seen in VAS, duration of operation and amount of blood loss between the two groups (P less than 0.01). CONCLUSIONS: Percutaneous kyphoplasty has the similar therapeutic efficacy with pedicle screw system in treatment of VCF with a minimal invasion, less operation time and blood loss. For those with posterior wall destruction, PS is deemed favorable.

[Preparation of mouse KCTD10 antibody and expression analysis of KCTD10 in neuroepithelium of neural tube and dorsal root ganglion].

KCTD10 is a TNF-alpha inducible protein that can interact with the small subunit of DNA polymerase a and PCNA. In order to study the function of KCTD10, we prepared the rabbit anti-mouse KCTD10 polyclonal antibody by using the His-tagged recombinant mouse KCTD10 protein to immune New Zealand white rabbit. Mouse KCTD10 shares significant similarity with PDIP1 (polymerase delta-interacting protein 1) and TNFAIP1 (tumor necrosis factor alpha-induced protein 1) protein,and then KCTD10 polyclonal antiserum possesses cross-reactivity with PDIP1 protein and TNFAIP1 protein. The partially digested fragments of homogeneous proteins PDIP1 and TNFAIP1 were mixed and incubated with anti-KCTD10 antiserum at 4 degrees C for 3 h to deplete unspecific antibodies. Through this method, we removed successfully the cross-reactivity of anti-KCTD10 antibody with PDIP1 and TNFAIP1 and obtained specific anti-KCTD10 antibody. Then, the anti-KCTD10 antibody was used in immunohistochemistry experiments of mouse. The results of immunohistochemistry on whole-mount embryo and paraffin section demonstrated that KCTD10 is highly expressed in neuroepithelium of neural tube and dorsal root ganglion of 12.5 d embryos. These results suggest that KCTD10 may play roles in the development of neuroepithelium of neural tube and dorsal root ganglion.