Effects of Estrogen on Proliferation and Apoptosis of Osteoblasts through Regulating GPER/AKT Pathway.

This experiment was carried out to study the effects of estrogen on the proliferation and apoptosis of osteoblasts through regulating the G protein-coupled estrogen receptor (GPER)/protein kinase B (AKT) pathway. For this aim, osteoblasts were cultured in vitro and divided into control group, estrogen group and inhibitor group after passage. The osteoblasts in the control group were cultured normally, estrogen intervention was made in the estrogen group and G15 inhibitor intervention was made in the inhibitor group. After intervention for 24 h, osteoblasts were collected for detection. The positive expression of GPER and the double-positive expression of Tom20/Lamp2 were detected via immunofluorescence assay. The protein expressions of GPER, AKT and phosphorylated (p)-AKT were detected via Western blotting. The mRNA expression of GPER was detected via qPCR. Moreover, the autophagosomes were observed under a transmission electron microscope, and the apoptosis and cell proliferation were detected via terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay and cell counting kit-8 (CCK8) assay, respectively. Results of the immunofluorescence assay revealed that the positive expression of GPER in the estrogen group was higher than that in the control group and inhibitor group (p<0.05), while the double-positive expression of Tom20/Lamp2 in the estrogen group was lower than that in control group and inhibitor group (p<0.05). According to the results of Western blotting, the relative protein expression of AKT had no differences among the three groups (p>0.05), while the relative protein expressions of GPER and p-AKT in the estrogen group were higher than those in the control group and inhibitor group (p<0.05). The results of qPCR showed that the relative mRNA expression of GPER in the estrogen group was higher than that in the control group and inhibitor group (p<0.05). There were a small number of autophagosomes in osteoblasts in the control group and inhibitor group, while the number of autophagosomes in osteoblasts was smaller in the estrogen group. Besides, the estrogen group had a remarkably lower apoptosis rate of osteoblasts than the control group and inhibitor group and a remarkably higher proliferation rate than the control group and inhibitor group. Then estrogen can inhibit the mitochondrial autophagy of osteoblasts by regulating the GPER/AKT pathway, thereby inhibiting apoptosis and promoting cell proliferation.

Pactacin is a novel digestive enzyme in teleosts.

Generally, animals extract nutrients from food by degradation using digestive enzymes. Trypsin and chymotrypsin, one of the major digestive enzymes in vertebrates, are pancreatic proenzymes secreted into the intestines. In this investigation, we report the identification of a digestive teleost enzyme, a pancreatic astacin that we termed pactacin. Pactacin, which belongs to the astacin metalloprotease family, emerged during the evolution of teleosts through gene duplication of astacin family enzymes containing six cysteine residues (C6astacin, or C6AST). In this study, we first cloned C6AST genes from pot-bellied seahorse (Hippocampus abdominalis) and analyzed their phylogenetic relationships using over 100 C6AST genes. Nearly all these genes belong to one of three clades: pactacin, nephrosin, and patristacin. Genes of the pactacin clade were further divided into three subclades. To compare the localization and functions of the three pactacin subclades, we studied pactacin enzymes in pot-bellied seahorse and medaka (Oryzias latipes). In situ hybridization revealed that genes of all three subclades were commonly expressed in the pancreas. Western blot analysis indicated storage of pactacin pro-enzyme form in the pancreas, and conversion to the active forms in the intestine. Finally, we partially purified the pactacin from digestive fluid, and found that pactacin is novel digestive enzyme that is specific in teleosts.

circRNA expression pattern and ceRNA network in the pathogenesis of aseptic loosening after total hip arthroplasty.

Increasing evidence has demonstrated that circular RNA (circRNA) exerts important function in the pathogenesis of some diseases. While, the contributions of circRNAs to aseptic loosening after total hip arthroplasty (THA) remain largely unknown. Our research is to explore the differentially expressed circRNAs (DEcircRNAs) and elucidate complex regulated mechanism of circRNAs in aseptic loosening. The DEcircRNAs were identified by RNA sequencing (RNA-seq) analysis. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was adopted to corroborate these DEcircRNAs. The potential function of circRNAs in aseptic loosening tissue was identified by competing endogenous RNA (ceRNA) analysis. Enrichment analysis was performed for target mRNAs and host genes of the DEcircRNAs by Gene Oncology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). 257 DEcircRNAs were obtained from RNA-seq results. Following the RT-qPCR corroboration, 6 circRNAs (hsa_circ_0007482, hsa_circ_0005232, hsa_circ_0000994, hsa_circ_0000690, hsa_circ_0058092 and hsa_circ_0004496) were selected for further analysis. By circRNA-miRNA and miRNA-mRNA prediction, 6 circRNAs, 138 miRNAs and 1667 mRNAs were identified. Then, circRNA-miRNA-mRNA network was established. The result of GO and KEGG enrichment analysis suggested that the circRNAs were related with some biological functions and pathways of aseptic loosening. A novel pathogenesis and treatment strategy about aseptic loosening after THA was revealed from our study of circRNA-miRNA-mRNA network.

17ß-Estradiol Induces Mitophagy Upregulation to Protect Chondrocytes via the SIRT1-Mediated AMPK/mTOR Signaling Pathway.

Increasing evidence reveals that estrogen, especially 17ß-estradiol (17ß-E2), is associated with articular cartilage metabolism disorder and postmenopausal osteoarthritis (OA). SIRT1, AMPK, and mTOR are regarded as critical mitophagy regulators. Recent studies have shown that mitophagy displays a protective effect against OA, but the molecular mechanism is not well known. This study aimed to investigate the effect of 17ß-E2 on Sirtuin-1 (SIRT1) expression and the induction of mitophagy upregulation by 17ß-E2 via the SIRT1-mediated AMP-activated protein kinase (AMPK)/mammalian target of the rapamycin (mTOR) signaling pathway to protect chondrocytes. ATDC5 chondrocytes were treated with different concentrations of 17ß-E2 (0 M, 1 × 10-9 M, 1 × 10-8 M, and 1 × 10-7 M) for 24 h or pretreatment with or without NAM (SIRT1 inhibitor), Compound C (AMPK inhibitor) and S1842 (mTOR inhibitor) for 30 min prior to treatment with 17ß-E2 (1 × 10-7 M) for 24 in each groups. Expression of SIRT1 was evaluated by real-time PCR, Western blotting and confocal immunofluorescence staining. Then, the mitophagosomes in cells were observed under a transmission electron microscopy (TEM), and the AMPK/mTOR signaling pathway was detected by Western blotting. The mitophagy-related proteins, p-AMPK, p-mTOR, p-JNK, and p-p38 were also identified by Western blot analysis. The chondrocytes viability and proliferation were determined by MTT and 5-Bromo-2'-deoxyuridine (BrdU) assay. These experiments were independently repeated 3 times The study found that 17ß-E2 increased the expression level of SIRT1, p-AMPK, and mitophagy-related proteins but decreased p-mTOR expression, and then induced mitophagy upregulation in chondrocytes. More mitochondrial autophagosomes were observed in 17ß-E2-treated chondrocytes under a transmission electron microscope. Also, 17ß-E2 improved cell viability and proliferation with the higher expression of SIRT1 and activation of the AMPK/mTOR signaling pathway. However, SIRT1 inhibitor nicotinamide (NAM) and AMPK inhibitor Compound C blocked the beneficial effect of 17ß-E2. In summary, this study was novel in demonstrating that 17ß-E2 induced mitophagy upregulation to protect chondrocytes via the SIRT1-mediated AMPK/mTOR signaling pathway.

Cryotherapy on postoperative rehabilitation of joint arthroplasty.

PURPOSE: The effectiveness of cryotherapy on joint arthroplasty recovery remains controversial. This systematic review was conducted to assess the effectiveness of cryotherapy in patients after joint arthroplasty. METHODS: Comprehensive literature searches of several databases including Cochrane Library (2013), MEDLINE (1950-2013), and Embase (1980-2013) were performed. We sought randomised controlled trials that compared the experimental group received any form of cryotherapy with any control group after joint arthroplasty. The main outcomes were postoperative blood loss, adverse events, and pain. Analyses were performed with Revman 5.0. Results were shown as mean differences (MD) and standard deviations or as risk difference and 95 % confidence intervals (CIs). RESULTS: Ten trials comprised 660 total knee arthroplastys and three trials comprised 122 total hip arthroplastys (THAs) met the inclusion criteria. Blood loss was significantly decreased by cryotherapy (MD = -109.68; 95 % CI -210.92 to -8.44; P = 0.03). Cryotherapy did not increase the risk of adverse effect (n.s.). Cryotherapy decreased pain at the second day of postoperative (MD = -1.32; 95 % CI -2.37 to -0.27; P = 0.0003), but did not decreased pain at the first and third day of postoperative (n.s.). CONCLUSIONS: Cryotherapy appears effective in these selected patients after joint arthroplasty. The benefits of cryotherapy on blood loss after joint arthroplasty were obvious. However, the subgroup analysis indicated that cryotherapy did not decreased blood loss after THA. Cryotherapy did not increase the risk of adverse effect. Cryotherapy decreased pain at the second day of postoperative, but did not decreased pain at the first and third day of postoperative. LEVEL OF EVIDENCE: II.

Press-fit cementless acetabular fixation with and without screws.

PURPOSE: Cementless acetabular fixation for total hip arthroplasty (THA) is widely used. The question of using screws for a better primary and secondary acetabular fixation has been discussed in the literature in recent years. The aim of this meta-analysis was to compare fixation of acetabular cups with and without screws in total hip arthroplasty. METHODS: Electronic databases Embase, PubMed and Cochrane Library were used to search for randomised controlled trials reported through May 2013 of cementless acetabular fixation for THA with and without screws. Two independent reviewers assessed the trials for eligibility and quality. All related data matching our standards were abstracted for meta-analysis by RevMan 5.0. Evaluation criteria included revisions, migration and osteolysis. RESULTS: A total of 1,130 THAs enrolled into five trials were included in this meta-analysis. All studies compared fixation of acetabular cups with and without screws, and our pooled data showed no statistical significance between the two surgical methods in revision, migration and osteolysis. CONCLUSION: There is no significant difference between cementless acetabular fixation for THA with and without screws in revisions, migration or osteolysis.