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.

Prospective Study of Low- and Standard-dose Chest CT for Pulmonary Nodule Detection: A Comparison of Image Quality, Size Measurements and Radiation Exposure.

OBJECTIVE: To comprehensively and accurately analyze the out-performance of low-dose chest CT (LDCT) vs. standard-dose CT (SDCT). METHODS: The image quality, size measurements and radiation exposure for LDCT and SDCT protocols were evaluated. A total of 117 patients with extra-thoracic malignancies were prospectively enrolled for non-enhanced CT scanning using LDCT and SDCT protocols. Three experienced radiologists evaluated subjective image quality independently using a 5-point score system. Nodule detection efficiency was compared between LDCT and SDCT based on nodule characteristics (size and volume). Radiation metrics and organ doses were analyzed using Radimetrics. RESULTS: The images acquired with the LDCT protocol yielded comparable quality to those acquired with the SDCT protocol. The sensitivity of LDCT for the detection of pulmonary nodules (n=650) was lower than that of SDCT (n=660). There was no significant difference in the diameter and volume of pulmonary nodules between LDCT and SDCT (for BMI <22 kg/m2, 4.37 vs. 4.46 mm, and 43.66 vs. 46.36 mm3; for BMI ≥22 kg/m2, 4.3 vs. 4.41 mm, and 41.66 vs. 44.86 mm3) (P>0.05). The individualized volume CT dose index (CTDIvol), the size specific dose estimate and effective dose were significantly reduced in the LDCT group compared with the SDCT group (all P<0.0001). This was especially true for dose-sensitive organs such as the lung (for BMI <22 kg/m2, 2.62 vs. 12.54 mSV, and for BMI ≥22 kg/m2, 1.62 vs. 9.79 mSV) and the breast (for BMI <22 kg/m2, 2.52 vs. 10.93 mSV, and for BMI ≥22 kg/m2, 1.53 vs. 9.01 mSV) (P<0.0001). CONCLUSION: These results suggest that with the increases in image noise, LDCT and SDCT exhibited a comparable image quality and sensitivity. The LDCT protocol for chest scans may reduce radiation exposure by about 80% compared to the SDCT protocol.

Circular RNA circANKRD36 regulates Casz1 by targeting miR-599 to prevent osteoarthritis chondrocyte apoptosis and inflammation.

Osteoarthritis (OA) is an ageing-related disease characterized by articular cartilage degradation and joint inflammation. circRNA has been known to involve in the regulation of multiple inflammatory diseases including OA. However, the mechanism underlying how circRNA regulates OA remains to be elucidated. Here, we report circANKRD36 prevents OA chondrocyte apoptosis and inflammation by targeting miR-599, which specifically degrades Casz1. We performed circRNA sequencing in normal and OA tissues and found the expression of circANKRD36 is decreased in OA tissues. circANKRD36 is also reduced in IL-1ß-treated human chondrocytes. FACS analysis and Western blot showed that the knockdown of circANKRD36 promotes the apoptosis and inflammation of chondrocytes in IL-1ß stress. We then found miR-599 to be the target of circANKRD36 and correlate well with circANKRD36 both in vitro and in vivo. By database analysis and luciferase assay, Casz1 was found to be the direct target of miR-599. Casz1 helps to prevent apoptosis and inflammation of chondrocytes in response to IL-1ß. In conclusion, our results proved circANKRD36 sponge miR-599 to up-regulate the expression of Casz1 and thus prevent apoptosis and inflammation in OA.

PKCa Agonists Enhance the Protective Effect of Hyaluronic Acid on Nitric Oxide-Induced Apoptosis of Articular Chondrocytes in Vitro.

OBJECTIVE(S): Protein kinase C (PKCα) is involved in modulating articular chondrocytes apoptosis induced by nitric oxide (NO). Hyaluronic acid (HA) inhibits nitric oxide-induced apoptosis of articular chondrocytes by protecting PKCα, but the mechanism remains unclear. The present study was performed to investigate the effects and mechanisms of PKCα regulate protective effect of hyaluronic acid. Materials and Methods The ratio of apoptotic cell and cell viability was surveyed by PCNA and MTT assay. The expression of caspase-3 was determined by real-time PCR and western blot. RESULTS: It was showed that HA was able to reduce the nuclei fragment and PCNA expression, and NO-induced articular apoptosis blocked by HA, pretreated chondrocytes with PMA, HA significantly inhibits the activation of caspase-3 induced by NO, but pretrement with CHR, HA significantly incresed the expression of caspase-3. CONCLUSION: The results may be showed that PKCa regulate the expresion of caspase-3, which contribute to the apoptosis of chondrocytes induced by NO. PKC α agonists enhance the protective effect of hyaluronic acid on nitric oxide-induced articular chondrocytes apoptosis.

Hyaluronic acid inhibits nitric oxide-induced apoptosis and dedifferentiation of articular chondrocytes in vitro.

OBJECTIVE: Nitric oxide is an important mediator in Osteoarthritis (OA), and causes apoptosis and dedifferentiation in articular chondrocytes. Protein kinase Calpha is involved in modulating apoptosis and dedifferentiation of articular chondrocytes induced by nitric oxide. Hyaluronic acid is widely used in the treatment of osteoarthritis and exerts significant chondroprotective effects. The exact mechanisms of its chondroprotective action are not yet fully elucidated. The present study was performed to investigate the effects and mechanisms of hyaluronic acid in NO-induced apoptosis and dedifferentiation of chondrocytes. METHODS: The ratio of apoptotic cell and cell viability was surveyed by TUNEL, MTT assay and flow cytometry. The expression of aggrecan, type II collagen, and PKCalpha were determined by real-time PCR and Western blot. The expression changes of caspase-3 and bcl-2 was detected by Western blot. The mitochondrial membrane potential (DeltaPsim) was evaluated by Rhodamine-123 fluorescence. RESULTS: HA reduces the TUNEL positive cell, nuclei fragment and the impairment of DeltaPsim. NO-induced chondrocyte dedifferentiation was blocked by HA, which restores expression of aggrecan and type II collagen of chondrocytes and cell viability. HA can block inhibition of PKC-alpha by NO. CONCLUSION: Our results show that HA blocks NO-induced apoptosis and dedifferentiation of articular chondrocytes by modulation of DeltaPsim and PKCalpha.

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.

Altered surfactant protein A gene expression and protein homeostasis in rats with emphysematous changes.

BACKGROUND: The decrease of surfactant protein (SP) secreted by the alveolar type II cell is one of the important causes of limiting air of pulmonary emphysema. However, the SP-A gene and protein changes in this disease are rarely studied. This study was undertaken to investigate alterations in SP-A gene activity and protein, and to explore their roles in the pathogenesis of emphysematous changes. METHODS: Twenty Wistar rats were divided randomly into a normal control group (n = 10) and a cigarette smoking (CS) + lipopolysaccharide (LPS) group (n = 10). Ultra-structural changes were observed under an electron microscope. The number of cells positive for SP-A was measured by immunohistochemistry. The mRNA expression and protein level of SP-A in the lung tissues were determined by quantitative polymerase chain reaction (qPCR) and Western blot separately. The protein level of SP-A in lavage fluid was determined by Western blot. RESULTS: The number of cells positive for SP-A of the CS + LPS group (0.35 +/- 0.03) was lower than that of the blank control group (0.72 +/- 0.06, P < 0.05). The level of SP-A in the lung tissues of rats in the CS + LPS group (0.2765 +/- 0.0890) was lower than that in the blank control group (0.6875 +/- 0.1578, P < 0.05). The level of SP-A in the lavage fluid of rats in the CS + LPS group (0.8567 +/- 0.1458) was lower than that in the blank control group (1.3541 +/- 0.2475, P < 0.05). The lung tissues of rats in the CS + LPS group showed an approximate increase (0.4-fold) in SP-A mRNA levels relative to beta-actin mRNA (P < 0.05). CONCLUSIONS: The changes of SP-A may be related to emphysematous changes in the lung. And cigarette smoke and LPS alter lung SP-A gene activity and protein homeostasis.