Effects of environmental chemicals on the proliferation and differentiation of neural stem cells.

The objective of the present study was to explore the effects of environmental chemicals, such as methyl mercury, paraquat, and bisphenol A, on cell proliferation and apoptosis, as well as the expression levels of neuronal differentiation-related genes in neural stem cells (NSCs). NSCs originated from human umbilical cord blood (HUCB-NSCs) were used as cell models in the current study. CCK-8 and flow cytometry experiments were performed to assess the effects of methyl mercury, paraquat, and bisphenol A on the proliferation and apoptosis of HUCB-NSCs at different processes, including proliferation and differentiation stages. The expressions of neuronal differentiation-related genes were determined by reverse transcription-polymerase chain reaction and western blot analysis. The results showed that methyl mercury, paraquat, and bisphenol A treatments significantly inhibited cell proliferation and induced cell apoptosis in HUCB-NSCs, as well as decreased the expressions of Oct4, Gdf3, and Sox1, whereas increased Pax6 and Ngn1 expressions at both mRNA and protein levels. In conclusion, this study demonstrates that environmental chemicals can impair the proliferation and differentiation of NSCs, which may cause abnormal development of the nervous system.

[Association of single nucleotide polymorphisms (SNPs) in leptin receptor gene with knee osteoarthritis in the Ningxia Hui population].

To investigate the association between primary knee osteoarthritis (OA) and single nucleotide polymorphism (SNP) (A668G) of leptin receptor gene (LEPR) in the Ningxia Hui population. A case-control association study has been adopted in this thesis. The polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) analysis were performed to investigate the SNP of A668G site within LEPR from 148 patients with knee OA and 155 controls (asymptomatic and radiographically negative) with matched age and gender among Ningxia Hui population. In addition, genotypes of LEPR were verified by direct sequence analysis on PCR products. The result indicates that allele and genotype frequencies (P=0.024 and 0.008, respectively) in LEPR SNP A668G were significantly different in the knee OA patients group and control group, and in the knee OA patients group, the serum levels of leptin decreased significantly (P<0.001) and the serum levels of soluble leptin receptor increased significantly (P<0.001) compared with control group. Therefore, LEPR SNP A668G is associated with susceptibility to knee OA, which would be used as the genetic marker in predicting the risk of knee OA and would be one of the candidate genes in early prevention and control.

Xuebijing injection protects against sepsis-induced myocardial injury by regulating apoptosis and autophagy via mediation of PI3K/AKT/mTOR signaling pathway in rats.

OBJECTIVE: Apoptosis and autophagy are significant factors of sepsis induced myocardial injury (SIMI). XBJ improves SIMI by PI3K/AKT/mTOR pathway. Present study is devised to explore the protective mechanism of XBJ in continuous treatment of SIMI caused by CLP. METHODS: Rat survival was first recorded within 7 days. Rats were randomly assigned to three groups: Sham group, CLP group, and XBJ group. The animals in each group were divided into 12 h group, 1 d, 2 d, 3 d and 5 d according to the administration time of 12 hours, 1 day, 2 days, 3 days or 5 days, respectively. Echocardiography, myocardial injury markers and H&E staining were used to detect cardiac function and injury. IL-1ß, IL-6 and TNF-α in serum were measured using ELISA kits. Cardiomyocyte apoptosis was assayed by TUNEL staining. Apoptosis and autophagy related proteins regulated by the PI3K/AKT/mTOR signaling pathway were tested using western blot. RESULTS: XBJ increased the survival rate in CLP-induced septic Rat. First of all, the results of echocardiography, H&E staining and myocardial injury markers (cTnI, CK, and LDH levels) showed that XBJ could effectively improve the myocardial injury caused by CLP with the increase of treatment time. Moreover, XBJ significantly decreased the levels of serum inflammatory cytokines IL-1ß, IL-6 and TNF-α in SIMI rats. Meanwhile, XBJ downregulated the expression of apoptosis-related proteins Bax, Cleaved-Caspase 3, Cleaved-Caspase 9, Cytochrome C and Cleaved-PARP, while upregulated the protein levels of Bcl-2 in SIMI rats. And, XBJ upregulated the expression of autophagy related protein Beclin-1 and LC3-II/LC3-I ratio in SIMI rats, whereas downregulated the expression of P62. Finally, XBJ administration downregulated the phosphorylation levels of proteins PI3K, AKT and mTOR in SIMI rats. CONCLUSIONS: Our results showed that XBJ has a good protective effect on SIMI after continuous treatment, and it was speculated that it might be through inhibiting apoptosis and promoting autophagy via, at least partially, activating PI3K/AKT/mTOR pathway in the early stage of sepsis, as well as promoting apoptosis and inhibiting autophagy via suppressing PI3K/AKT/mTOR pathway in the late stage of sepsis.

Association between the +104T/C polymorphism in the 5'UTR of GDF5 and susceptibility to knee osteoarthritis: a meta-analysis.

Although the +104T/C polymorphism in the 5' untranslated region (UTR) of growth differentiation factor 5 (GDF5) plays a role in the pathogenesis of knee osteoarthritis, the results have been inconsistent. In this study, we performed a meta-analysis to assess the association of +104T/C polymorphism with knee osteoarthritis. Published literature from PubMed, Google Scholar and China National Knowledge Infrastructure data was retrieved. Pooled odds ratios (ORs) with 95% confidence intervals (CIs) were calculated using fixed- or random-effects models. A total of 6 case-control studies containing 2,744 patients and 4,518 controls were enrolled in this meta-analysis. Overall, a statistically significant association was found between the +104T/C polymorphism and risk of knee osteoarthritis (TT vs. CC: OR 1.68, 95% CI=1.41-2.01; TT vs. TC: OR 1.18, 95% CI=1.01­1.38; dominant model: OR 0.72, 95% CI=0.61-0.86). Taking into account the effect of ethnicity, further stratified analyses were performed. In the subgroup analysis, the same association was identified in Caucasian (TT vs. CC: OR 1.45, 95% CI=1.13-1.85) and Asian (TT vs. CC: OR 1.99, 95% CI=1.53­2.60; TT vs. TC: OR 1.33, 95% CI=1.16-1.52; dominant model: OR 0.64, 95% CI=0.56-0.72; recessive model: OR 1.77, 95% CI=1.37-2.29) populations. The meta-analysis results demonstrated that the +104T/C polymorphism in the 5'-UTR of GDF5 is associated with risk of knee osteoarthritis.

Adenovirus-mediated small interfering RNA targeting tumor necrosis factor-α inhibits titanium particle-induced osteoclastogenesis and bone resorption.

Wear particles are phagocytosed by macrophages, resulting in cellular activation and the release of pro-inflammatory factors, which cause periprosthetic osteolysis and subsequent aseptic loosening, the most common causes of total joint arthroplasty (TJA) failure. During this pathological process, tumor necrosis factor (TNF)-α plays an important role in wear particle-induced osteolysis. Therefore, in this study, we used adenovirus-mediated small interfering RNA (siRNA) targeting TNF-α to suppress the TNF-α release from activated macrophages in response to titanium particles. Our results showed that recombinant adenovirus (Ad-TNF-α-siRNA) suppressed the TNF-α release from activated macrophages in response to titanium particles, and reduced titanium particle-induced osteoclastogenesis and bone resorption in the presence of receptor activator of nuclear factor-κB ligand (RANKL). In addition, the conditioned medium of macrophages challenged with titanium particles (Ti CM) stimulated osteoprogenitor RANKL expression. The conditioned medium of macrophages challenged with titanium particles and Ad-TNF-α-siRNA (Ti-Ad CM) reduced the mRNA expression in MC3T3-E1 cells compared to Ti CM. Based on these data, TNF-α strongly synergizes with RANKL to promote osteoclast differentiation. Furthermore, TNF-α promoted osteoclast differentiation by stimulating osteoprogenitor RANKL expression. Ad-TNF-α-siRNA effectively suppressed osteoclast differentiation and bone resorption following exposure to titanium particles in the presence of RANKL. In addition, recombinant adenovirus (Ad-TNF-α-siRNA) does not have a toxic effect on the murine macrophage cell line, RAW264.7. Consequently, it can be concluded that recombinant adenovirus-mediated siRNA targeting TNF-α (Ad-TNF-α-siRNA) may provide a novel therapeutic approach for the treatment of periprosthetic osteolysis.

[Experimental study on small interfering RNA silencing expression of tumor necrosis factor alpha and inhibiting osteolysis].

OBJECTIVE: To investigate the possibility of gene therapy of osteolysis around artificial joint prosthesis by constructing the recombinant adenovirus which can silence tumor necrosis factor alpha (TNF-alpha). METHODS: The primer of small interfering RNA (siRNA) coding sequence of silent TNF-alpha was designed and amplified, and then RAPAD adenovirus packaging system was used to load the sequence to adenovirus, and the recombinant adenovirus Ad5-TNF-a-siRNA-CMVeGFP which lacked both E1 and E3 regions was constructed. Then 64 female BABL/C mice (weighing, 20-25 g) were randomly divided into 4 groups (n=16): blank control (group A), positive control (group B), simple adenovirus (group C), and treatment group (group D). The prosthetic-model was established in group A, and the prosthetic-loosening-model in groups B, C, and D. At 2 weeks after modeling, PBS solution was injected first, and then the same solution was injected 24 hours later in group A; titanium particle solution was injected, and then PBS solution, Ad5 E1-CMVeGFP (1 x 10(9) PFU/mL), and Ad5-TNF-alpha-siRNA-CMVeGFP (1 x 10(9) PFU/mL) were injected, respectively in groups B, C, and D 24 hours later, every 2 weeks over a 10-week period. The general condition of mice was observed after operation. The tissues were harvested for histological observation, and the expression of TNF-a was detected by Western blot at 12 weeks after operation. RESULTS: The positive clones were achieved by enzyme digestion and confirmed by DNA sequencing after loading the target genes into adenovirus vector, and then HEK293 cells were successfully transfected by recombinant adenovirus Ad5-TNF-alpha-siRNA-CMVeGFP. All mice survived to the completion of the experiment. Histological observation showed that there were few inflammatory cells and osteoclasts in group A, with a good bone formation; there were a large number of inflammatory cells and osteoclasts in groups B and C, with obvious bone destruction; inflammatory cells and osteoclasts in group D was less than those in groups B and C, with no obvious bone destruction. Significant difference was found in the limiting membrane thickness and the number of osteoclasts (group A < group D < group B < group C, P < 0.05). Western blot showed that the TNF-a expression levels were 0.235 +/- 0.022, 0.561 +/- 0.031, 0.731 +/- 0.037, and 0.329 +/- 0.025 in groups A, B, C, and D respectively, showing significant difference among 4 groups (P < 0.05). CONCLUSION: The recombinant adenovirus for silencing TNF-alpha is successfully constructed, which can effectively inhibit osteolysis by silencing TNF-alpha expression in the tissues around prosthesis in mice.