[Effects and mechanism of allogeneic platelet rich plasma on collagen synthesis in wound healing].

Objective: To investigate the effects and mechanism of allogeneic platelet rich plasma (PRP) on collagen in wound surface at different time. Methods: A total of 50 clean 7-week rats were selected for this study, including 10 rats for platelet-rich blood plasma preparation, 20 rats for PRP group and 20 rats for control group, 0.1 ml allogenic PRP and 0.1 ml saline were smeared respectively on wound surfaces of PRP and control group, wound regeneration and healing were examined. Cellular and histological morphology alteration was observed via Masson staining, type Ⅰ and type Ⅲ collagen protein and mRNA expression level were detected by Western blot and real-time PCR. T test was applied for comparison between two samples and one-way ANOVA was utilized for comparison between two groups. Results: The wound healing rate of PRP group was higher than that of control group on 3(rd,) 6(th,) 10(th) and 15(th) day (30.33±3.35 vs.18.35±2.04, 55.51±2.74 vs.36.83±2.34, 79.64±1.40 vs.56.92±1.44, 86.88±2.12 vs.65.80±1.76) after wound surface formation, there were statistic differences (t=13.66-50.48, all P<0.05). The wound collagen of PRP group form faster and coarser, and the fibers arrayed more densely in Masson staining. The protein expression of type Ⅰ collagen(1.92±0.09 vs.1.18±0.11) and type Ⅲ collagen(1.16±0.05 vs.0.74±0.11) of PRP group were higher than that of control group (t=22.99, P<0.01; t=17.62, P<0.05); the mRNA expression of type Ⅰ collagen(5.17±0.11 vs.1.79±0.18, 6.97±0.09 vs.1.96±0.08, 6.00±0.26 vs.2.10±0.05, 4.95±0.11 vs.3.58±0.09)and type Ⅲ collagen(2.35±0.08 vs.1.44±0.05, 3.08±0.05 vs.1.84±0.06, 3.48±0.07 vs.2.36±0.09, 4.42±0.07 vs.2.77±0.10) were higher than that of control group on 3(rd,) 6(th,) 10(th) and 15(th) day after wound surface formation, there were significant differences (t=43.37-188.37, all P<0.05). Conclusion: The allogeneic platelet rich plasma may promote fibroblasts secreted collagen by activated and releasing all kinds of growth factors, especially type Ⅰ and type Ⅲ collagen to accelerate the wound healing.

Hsp90 modulates the stability of MLKL and is required for TNF-induced necroptosis.

The pseudokinase mixed lineage kinase domain-like protein (MLKL) is a key component of tumor necrosis factor (TNF)-induced necroptosis and plays a crucial role in necroptosis execution. However, the mechanisms that control MLKL activity are not completely understood. Here, we identify the molecular chaperone Hsp90 as a novel MLKL-interacting protein. We show that Hsp90 associates with MLKL and is required for MLKL stability. Moreover, we find that Hsp90 also regulates the stability of the upstream RIP3 kinase. Interference with Hsp90 function with the 17AAG inhibitor destabilizes MLKL and RIP3, resulting in their degradation by the proteasome pathway. Furthermore, we find that Hsp90 is required for TNF-stimulated necrosome assembly. Disruption of Hsp90 function prevents necrosome formation and strongly reduces MLKL phosphorylation and inhibits TNF-induced necroptosis. Consistent with a positive role of Hsp90 in necroptosis, coexpression of Hsp90 increases MLKL oligomerization and plasma membrane translocation and enhances MLKL-mediated necroptosis. Our findings demonstrate that an efficient necrotic response requires a functional Hsp90.

Expression and regulation of macrophage inflammatory protein-2 gene by vanadium in mouse macrophages.

Environmental and occupational exposure to vanadium (V) dusts results in inflammation mainly confined to the respiratory tract. Macrophages apparently play an important role in mediating the inflammation via the production of many chemokines. In the current study, we investigated whether vanadium can regulate the gene expression of a CXC chemokine macrophage inflammatory protein-2 (MIP-2), and to determine the molecular mechanisms controlling MIP-2 gene expression. A mouse macrophage cell line RAW 264.7 was treated with sodium metavanadate (NaVO3) at the dose of 0.5, 5, or 10 microg/mi V. Northern blot analysis showed that induction of MIP-2 mRNA expression was in a dose-dependent manner. To define the time course of the inflammatory response, RAW 264.7 cells were exposed to 5 microg/ml V, MIP-2 mRNA in macrophages increased markedly as early as 1 h after treatment, maximally induced at 4 h and reduced to 2-fold above control levels by 6 and 8 h. The protein levels of MIP-2 in conditioned media, measured by enzyme-linked immunosorbent assay (ELISA), was well correlated with the levels of MIP-2 mRNA following all of the treatments in the study. In addition, the increase in MIP-2 mRNA expression by vanadium was attenuated by co-treatment with the antioxidant N-acetylcysteine (NAC), at the doses of 10 and 20 mM, suggesting that the induction of MIP-2 mRNA is mediated via the generation of reactive oxygen species (ROS). To further investigate transcriptional regulation of the MIP-2 gene expression by vanadium, we performed RNA decay assay by measuring the half-life of MIP-2 mRNA. Co-treatment of macrophages with the transcriptional inhibitor actinomycin D at 5 microg/ml following exposure to 5 microg/ml V for 4 h revealed complete stabilization of vanadium-induced MIP-2 mRNA and no sign of mRNA degradation, at least, for 6 h, in comparison to the half-life of MIP-2 mRNA was approximately 2.5 h by bacterial lipopolysaccharide (LPS) treatment, supporting post-transcriptional stabilization as the predominant role of MIP-2 gene expression. In conclusion, these observations demonstrate that in vitro vanadium can induce MIP-2 mRNA expression, mediating, at least in part, via the production of ROS. In addition, the increase in MIP-2 mRNA level involves, most likely, post-transcriptional control via increased mRNA stability.