SIRT1 modulates microglia phenotypes via inhibiting drp1 phosphorylation reduces neuroinflammation in heatstroke.

BACKGROUND: Brain injury often results in high mortality rates and significant sequelae following severe heatstroke (HS). Neuroinflammation aggravates HS-induced brain injury, yet the involvement of microglia in heat-induced neuroinflammation deserves further investigation. METHODS: Our study investigated activation status, phenotype markers, production of pro-inflammatory cytokine and reactive oxygen species (ROS) of microglia both in vitro and in vivo under HS. Utilizing high-throughput sequencing, we identified SIRT1 as a potential modulator of microglia phenotype, and observed that SIRT1 alleviated severe heatstroke-induced brain injury following intraperitoneal administration of the SIRT1 agonist SRT-1720 and the inhibitor selisistat. Additionally, the effects of SRT-1720 and selisistat on mitochondrial dynamics and microglial phenotype transition were examined in BV2 cells in vitro. RESULTS: Heatstroke promotes microglia activation, as evidenced by the increased production of pro-inflammatory cytokine and reactive oxygen species. High-throughput sequencing revealed elevated expression of SIRT1 in BV2 cells under HS. Upon inhibition of SIRT1 expression, there was a corresponding increase in pro-inflammatory cytokine, iNOS, and ROS expression in BV2 cells. In vivo experiments with the SIRT1 agonist SRT-1720 showed a mitigation of neuron injury under HS, as assessed by Nissl and HE staining. Activation of SIRT1 was associated with a reduction in mitochondrial injury and a decrease in the phosphorylation of mitochondrial fission protein Drp1ser616. Furthermore, the heat-induced activation of microglia was reversed by the Drp1 inhibitor, Mdivi. CONCLUSIONS: Our findings provided evidence that SIRT1 played a crucial role in inhibiting heat stress-induced microglial activation. By suppressing the phosphorylation of mitochondrial fission protein Drp1, SIRT1 contributed to the reduction of neuroinflammation and severity of heatstroke-induced brain injury.

Platelets Facilitate Wound Healing by Mitochondrial Transfer and Reducing Oxidative Stress in Endothelial Cells.

As a critical member in wound healing, vascular endothelial cells (ECs) impaired under high levels of reactive oxygen species (ROS) would hamper neovascularization. Mitochondria transfer can reduce intracellular ROS damage under pathological condition. Meanwhile, platelets can release mitochondria and alleviate oxidative stress. However, the mechanism by which platelets promote cell survival and reduce oxidative stress damage has not been clarified. Here, first, we selected ultrasound as the best method for subsequent experiments by detecting the growth factors and mitochondria released from manipulation platelet concentrates (PCs), as well as the effect of manipulation PCs on the proliferation and migration of HUVECs. Then, we found that sonicate platelet concentrates (SPC) decreased the level of ROS in HUVECs treated with hydrogen peroxide in advance, increased mitochondrial membrane potential, and reduced apoptosis. By transmission electron microscope, we saw that two kinds of mitochondria, free or wrapped in vesicles, were released by activated platelets. In addition, we explored that platelet-derived mitochondria were transferred to HUVECs partly by means of dynamin-dependent clathrin-mediated endocytosis. Consistently, we determined that platelet-derived mitochondria reduced apoptosis of HUVECs caused by oxidative stress. What is more, we screened survivin as the target of platelet-derived mitochondria via high-throughput sequencing. Finally, we demonstrated that platelet-derived mitochondria promoted wound healing in vivo. Overall, these findings revealed that platelets are important donors of mitochondria, and platelet-derived mitochondria can promote wound healing by reducing apoptosis caused by oxidative stress in vascular endothelial cells. And survivin is a potential target. These results further expand the knowledge of the platelet function and provide new insights into the role of platelet-derived mitochondria in wound healing.

Effectiveness of Nano Bioactive Glass Fiber Loaded with Platelet-Rich Plasma on Thermal Wound Healing Process in Rats.

In this study we evaluated the impact of topical application of bioactive glass fibers loaded PRP on a deep seconddegree thermal wound and its healing process sub-streaming molecular pathway of re-epithelialization. Wistar rats were randomly divided into four groups: normal control group, model group (deep second-degree thermal wound), PRP group, and PRP+nanobioactive glass fiber group. After treatment, the changes of wounds were observed daily. H&E staining was used to evaluate the pathological changes and also, qRT-PCR was used to detect the mRNA expression of KGF, IL-1, IL-6, IL-10, TGF-ß, EGF, VEGF, HIF-1α, integrin α3 and integrin ß1 in wound tissues. In the current study, we observed that PRP group and the PRP group basically re-epithelized on the 21st day. The wound healing rates of the PRP+nanobioactive glass fiber group and PRP group at each time point were higher than those in the model group, while there was no significant difference in wound healing rate between the PRP+nanobioactive glass fiber group and PRP group at each time point. H&E staining showed that the pathological scores of skin wound repairing in the PRP+nanobioactive glass fiber group on the 7th, 14th and 21st day were higher than that of in the model group. The qPCR results suggested the mRNA expression of IL-1, IL-6 and IL-10 in the PRP+nanobioactive glass fiber group and the PRP group were lower than those in the untreated group on the 14th day; the expression of VEGF and EGF mRNA were higher on the 3rd day; the mRNA expression of TGF-ß, HIF-1α showed a tendency of increasing first and decreasing then; integrin ß1 mRNA expression increased significantly, which was highest; integrin α3 mRNA expression was higher on day 3rd and 21th, respectively. The PRP+nanobioactive glass fibers and PRP can shorten the wound healing time and improve the healing quality mainly by promoting the wound epithelization through increasing the expression of EGF, VEGF, TGF-ß, HIF-1α, Integrin α3, and meanwhile increasing the release of Integrin ß1 and other mechanisms.

Treatment of Acute Wounds With Recombinant Human-Like Collagen and Recombinant Human-Like Fibronectin in C57BL/6 Mice Individually or in Combination.

Wound repair is accomplished by the interaction between the cells involved in the repair and the extracellular matrix (ECM). Collagen is the main component of ECM, which is involved in transduction of signal, transportation of growth factors and cytokines. Fibronectin (FN) is also an important ECM, which participates in the initiation of fibroblast cell (FC) and promotes adhesion, migration, proliferation and differentiation of target cells. Compared with natural protein, the recombinant protein prepared by artificial method has the advantages of poor immunogenicity, wide range of sources, low cost and high activity. In this study, we used recombinant human-like collagen (RHC) and recombinant human-like fibronectin (rhFN) to treat acute wounds in C57BL/6 mice individually or in combination, and explored their effects on wound healing. Our study confirmed that these two recombinant proteins could effectively promote the proliferation, migration and adhesion of FCs. Meanwhile, it could positively regulate the healing speed and quality of acute wounds, re-epithelialization, collagen deposition, inflammation and angiogenesis. Moreover, we proved that the combination of the two was better than the treatment alone. Consequently, it has a good prospect as a new tissue material in the field of skin repair.

A composite hydrogel containing resveratrol-laden nanoparticles and platelet-derived extracellular vesicles promotes wound healing in diabetic mice.

Diabetic wounds are difficult to heal because of persistent inflammation and limited angiogenesis. Resveratrol (RES) is an anti-inflammatory and antioxidant agent. Platelet-derived extracellular vesicles (PDEVs) are rich in growth factors and cytokines, which promote proliferation and angiogenesis. However, single drug treatment has limited efficacy and delivery efficiency. Bioengineering can improve the limited effect of single drugs by combining drugs and materials to obtain complementary or cooperative bioengineered drugs. In this study, gelatin methacrylate (GelMA) and silk fibroin glycidyl methacrylate (SFMA) were used to synthesize GelMA/SFMA composite hydrogels with suitable mechanical properties, swelling ratio and biodegradability. The composite hydrogel was used as a wound dressing for sustained drug release. RES was loaded into mesoporous silica nanoparticles (MSNs) to synthesize MSN-RES to enhance the release dynamic, and MSN-RES and PDEVs were combined with the composite hydrogels to form GelMA/SFMA/MSN-RES/PDEVs hydrogels. The GelMA/SFMA/MSN-RES/PDEVs had low cytotoxicity and good biocompatibility, inhibited macrophage iNOS expression, and promoted the tube formation by human umbilical vein endothelial cells (HUVECs) in vitro. In a diabetic mouse wound model, the GelMA/SFMA/MSN-RES/PDEVs hydrogels decreased the expression of pro-inflammatory factors TNF-α and iNOS, increased the expression of anti-inflammatory factors TGF-ß1 and Arg-1, promoted angiogenesis, and accelerated wound healing. Interestingly, the GelMA/SFMA/MSN-RES/PDEVs hydrogels promoted the expression of extracellular purinergic signaling pathway-related CD73 and adenosine 2A receptor (A2A-R). Therefore, the GelMA/SFMA/MSN-RES/PDEVs hydrogels could be used as wound dressings to regulate the inflammation and angiogenesis of diabetic wounds and accelerate wound healing. STATEMENT OF SIGNIFICANCE: Drugs often fail to function because of a continuous oxidative stress microenvironment and inflammation. Here, a GelMA/SFMA hydrogel, with enhanced mechanical properties and liquid absorption ability, is proposed for sustained release of drugs. In addition to carrying platelet-derived extracellular vesicles (PDEVs) with pro-angiogenic effects, the hydrogels were also loaded with nanoparticle-encapsulated resveratrol with anti-inflammatory activities, aiming to reduce inflammation and oxidative stress in the wound microenvironment, such that the wound could receive proliferative repair signals to achieve sequential treatment and heal quickly. We also experimentally predicted that the regulatory mechanism of the GelMA/SFMA/MSN-RES/PDEVs in wound healing might be related to the extracellular purinergic signaling pathway.

Customized Design 3D Printed PLGA/Calcium Sulfate Scaffold Enhances Mechanical and Biological Properties for Bone Regeneration.

Polylactic glycolic acid copolymer (PLGA) has been widely used in tissue engineering due to its good biocompatibility and degradation properties. However, the mismatched mechanical and unsatisfactory biological properties of PLGA limit further application in bone tissue engineering. Calcium sulfate (CaSO4) is one of the most promising bone repair materials due to its non-immunogenicity, well biocompatibility, and excellent bone conductivity. In this study, aiming at the shortcomings of activity-lack and low mechanical of PLGA in bone tissue engineering, customized-designed 3D porous PLGA/CaSO4 scaffolds were prepared by 3D printing. We first studied the physical properties of PLGA/CaSO4 scaffolds and the results showed that CaSO4 improved the mechanical properties of PLGA scaffolds. In vitro experiments showed that PLGA/CaSO4 scaffold exhibited good biocompatibility. Moreover, the addition of CaSO4 could significantly improve the migration and osteogenic differentiation of MC3T3-E1 cells in the PLGA/CaSO4 scaffolds, and the PLGA/CaSO4 scaffolds made with 20 wt.% CaSO4 exhibited the best osteogenesis properties. Therefore, calcium sulfate was added to PLGA could lead to customized 3D printed scaffolds for enhanced mechanical properties and biological properties. The customized 3D-printed PLGA/CaSO4 scaffold shows great potential for precisely repairing irregular load-bearing bone defects.