Photosensitizer-loaded hydrogels: A new antibacterial dressing.

Bacterial wound infection has emerged as a pivotal threat to human health worldwide, and the situation has worsened owing to the gradual increase in antibiotic-resistant bacteria caused by the improper use of antibiotics. To reduce the use of antibiotics and avoid the increase in antibiotic-resistant bacteria, researchers are increasingly paying attention to  photodynamic therapy, which uses light to produce reactive oxygen species to kill bacteria. Treating bacteria-infected wounds by photodynamic therapy requires fixing the photosensitizer (PS) at the wound site and maintaining a certain level of wound humidity. Hydrogels are materials with a high water content and are well suited for fixing PSs at wound sites for antibacterial photodynamic therapy. Therefore, hydrogels are often loaded with PSs for treating bacteria-infected wounds via antibacterial photodynamic therapy. In this review, we systematically summarised the antibacterial mechanisms and applications of PS-loaded hydrogels for treating bacteria-infected wounds via photodynamic therapy. In addition, the recent  studies and the research status progresses of novel antibacterial hydrogels are discussed. Finally, the challenges and future prospects of PS-loaded hydrogels are reviewed.

Mitophagy associated self-degradation of phosphorylated MAP4 guarantees the migration and proliferation responses of keratinocytes to hypoxia.

Our previous study has announced that phosphorylated microtubule-associated protein 4 (p-MAP4) accelerated keratinocytes migration and proliferation under hypoxia through depolymerizing microtubules. However, p-MAP4 should exhibit inhibitory effects on wound healing, for it also impaired mitochondria. Thus, figuring out the outcome of p-MAP4 after it impaired mitochondria and how the outcome influenced wound healing were far-reaching significance. Herein, the results revealed that p-MAP4 might undergo self-degradation through autophagy in hypoxic keratinocytes. Next, p-MAP4 activated mitophagy which was unobstructed and was also the principal pathway of its self-degradation triggered by hypoxia. Moreover, both Bcl-2 homology 3 (BH3) and LC3 interacting region (LIR) domains had been verified in MAP4, and they endowed MAP4 with the capability to synchronously function as a mitophagy initiator and a mitophagy substrate receptor. And, mutating any one of them ruined hypoxia-induced self-degradation of p-MAP4, resulting in destroyed proliferation and migration responses of keratinocytes to hypoxia. Our findings unviewed that p-MAP4 experienced mitophagy-associated self-degradation through utilizing its BH3 and LIR domains under hypoxia. As a result, the mitophagy-associated self-degradation of p-MAP4 guaranteed the migration and proliferation responses of keratinocytes to hypoxia. Together, this research provided a bran-new pattern of proteins in regulating wound healing, and offered a new direction for intervening wound healing.

High glucose promotes podocyte movement: From the perspective of single cell motility assay.

Podocytes are highly specialized glomerular epithelial cells that play a crucial role in maintaining the glomerular filtration barrier, impairment of which usually leads to proteinuria. The phenotypic alterations of podocytes are described to be one of the critical mechnisms underlying podocyte detachment from the glomerular basement membrane. High glucose is the major factor mediating the renal damages and podocyte injuries in the process of diabetic nephropathy. It was revealed that high glucose stimulated the epithelial-to-mesenchymal transition of podocyte, thus contributing to proteinuria. When the podocytes converse from epithelial phenotype to mesenchymal phenotype, their migratory capacity significantly increases. Previously, cell migration is conventionally detected by the wound healing assay and the transwell assay. In this study, we investigated and comfirmed the possibility of using single cell motility assay for the anaysis of podocyte motility under high glucose condtition.

Microtubule associated protein 4 phosphorylation-induced epithelial-to-mesenchymal transition of podocyte leads to proteinuria in diabetic nephropathy.

BACKGROUND: Diabetic nephropathy (DN) involves various structural and functional changes because of chronic glycemic assault and kidney failure. Proteinuria is an early clinical manifestation of DN, but the associated pathogenesis remains elusive. This study aimed to investigate the role of microtubule associated protein 4 (MAP4) phosphorylation (p-MAP4) in proteinuria in DN and its possible mechanisms. METHODS: In this study, the urine samples of diabetic patients and kidney tissues of streptozotocin (STZ)-induced diabetic mice were obtained to detect changes of p-MAP4. A murine model of hyperphosphorylated MAP4 was established to examine the effect of MAP4 phosphorylation in DN. Podocyte was applied to explore changes of kidney phenotypes and potential mechanisms with multiple methods. RESULTS: Our results demonstrated elevated content of p-MAP4 in diabetic patients' urine samples, and increased kidney p-MAP4 in streptozocin (STZ)-induced diabetic mice. Moreover, p-MAP4 triggered proteinuria with aging in mice, and induced epithelial-to-mesenchymal transition (EMT) and apoptosis in podocytes. Additionally, p-MAP4 mice were much more susceptible to STZ treatment and showed robust DN pathology as compared to wild-type mice. In vitro study revealed high glucose (HG) triggered elevation of p-MAP4, rearrangement of microtubules and F-actin filaments with enhanced cell permeability, accompanied with dedifferentiation and apoptosis of podocytes. These effects were significantly reinforced by MAP4 hyperphosphorylation, and were rectified by MAP4 dephosphorylation. Notably, pretreatment of p38/MAPK inhibitor SB203580 reinstated all HG-induced pathological alterations. CONCLUSIONS: The findings indicated a novel role for p-MAP4 in causing proteinuria in DN. Our results indicated the therapeutic potential of MAP4 in protecting against proteinuria and related diseases. Video Abstract.

Autophagy-Related LC3 Accumulation Interacted Directly With LIR Containing RIPK1 and RIPK3, Stimulating Necroptosis in Hypoxic Cardiomyocytes.

The exact relationships and detailed mechanisms between autophagy and necroptosis remain obscure. Here, we demonstrated the link between accumulated autophagosome and necroptosis by intervening with autophagic flux. We first confirmed that the LC3 interacting region (LIR) domain is present in the protein sequences of RIPK1 and RIPK3. Mutual effects among LC3, RIPK1, and RIPK3 have been identified in myocardium and cardiomyocytes. Direct LC3-RIPK1 and LC3-RIPK3 interactions were confirmed by pull-down assays, and their interactions were deleted after LIR domain mutation. Moreover, after disrupting autophagic flux under normoxia with bafilomycin A1 treatment, or with LC3 or ATG5 overexpression adenovirus, RIPK1, RIPK3, p-RIPK3, and p-MLKL levels increased, suggesting necroptosis activation. Severe disruptions in autophagic flux were observed under hypoxia and bafilomycin A1 co-treated cardiomyocytes and myocardium and led to more significant activation of necroptosis. Conversely, after alleviating hypoxia-induced autophagic flux impairment with LC3 or ATG5 knockdown adenovirus, the effects of hypoxia on RIPK1 and RIPK3 levels were reduced, which resulted in decreased p-RIPK3 and p-MLKL. Furthermore, necroptosis was inhibited by siRNAs against RIPK1 and RIPK3 under hypoxia or normoxia. Based on our results, LIR domain mediated LC3-RIPK1 and LC3-RIPK3 interaction. Besides, autophagosome accumulation under hypoxia lead to necrosome formation and, in turn, necroptosis, while when autophagic flux was uninterrupted, RIPK1 and RIPK3 were cleared through an autophagy-related pathway which inhibited necroptosis. These findings provide novel insights for the role of LC3 in regulating cardiomyocyte necroptosis, indicating its therapeutic potential in the prevention and treatment of hypoxic myocardial injury and other hypoxia-related diseases.

Microtubule associated protein 4 (MAP4) phosphorylation reduces cardiac microvascular density through NLRP3-related pyroptosis.

Phosphorylation of MAP4 (p-MAP4) causes cardiac remodeling, with the cardiac microvascular endothelium being considered a vital mediator of this process. In the current study, we investigated the mechanism underlying p-MAP4 influences on cardiac microvascular density. We firstly confirmed elevated MAP4 phosphorylation in the myocardium of MAP4 knock-in (KI) mice. When compared with the corresponding control group, we detected the decreased expression of CD31, CD34, VEGFA, VEGFR2, ANG2, and TIE2 in the myocardium of MAP4 KI mice, accompanied by a reduced plasma concentration of VEGF. Moreover, we observed apoptosis and mitochondrial disruption in the cardiac microvascular endothelium of MAP4 KI animals. Consistently, we noted a decreased cardiac microvascular density, measured by CD31 and lectin staining, in MAP4 KI mice. To explore the underlying mechanism, we targeted the NLRP3-related pyroptosis and found increased expression of the corresponding proteins, including NLRP3, ASC, mature IL-1ß, IL-18, and GSDMD-N in the myocardium of MAP4 KI mice. Furthermore, we utilized a MAP4 (Glu) adenovirus to mimic cellular p-MAP4. After incubating HUVECs with MAP4 (Glu) adenovirus, the angiogenic ability was inhibited, and NLRP3-related pyroptosis were significantly activated. Moreover, both cytotoxicity and PI signal were upregulated by the MAP4 (Glu) adenovirus. Finally, NLRP3 inflammasome blockage alleviated the inhibited angiogenic ability induced by MAP4 (Glu) adenovirus. These results demonstrated that p-MAP4 reduced cardiac microvascular density by activating NLRP3-related pyroptosis in both young and aged mice. We thus managed to provide clues explaining MAP4 phosphorylation-induced cardiac remodeling and enriched current knowledge regarding the role of MAP4.

Inwardly rectifying potassium channel 5.1: Structure, function, and possible roles in diseases.

Inwardly rectifying potassium (Kir) channels make it easier for K+ to enter into a cell and subsequently regulate cellular biological functions. Kir5.1 (encoded by KCNJ16) alone can form a homotetramer and can form heterotetramers with Kir4.1 (encoded by KCNJ10) or Kir4.2 (encoded by KCNJ15). In most cases, homomeric Kir5.1 is non-functional, while heteromeric Kir5.1 on the cell membrane contributes to the inward flow of K+ ions, which can be regulated by intracellular pH and a variety of signaling mechanisms. In the form of a heterotetramer, Kir5.1 regulates Kir4.1/4.2 activity and is involved in the maintenance of nephron function. Actually, homomeric Kir5.1 may also play a very important role in diseases, including in the ventilatory response to hypoxia and hypercapnia, hearing impairment, cardiovascular disease and cancer. With an increase in the number of studies into the roles of Kir channels, researchers are paying more attention to the pathophysiological functions of Kir5.1. This minireview provides an overview regarding these Kir5.1 roles.

Severe burn injury alters intestinal microbiota composition and impairs intestinal barrier in mice.

BACKGROUND: The intestinal barrier integrity is crucial for maintaining intestinal homeostasis, and the mechanisms of intestinal barrier disruption induced by burn injury remain obscure. This study was aimed to investigate the changes of intestinal microbiota and barrier function in burned mice to further comprehend the mechanisms of burn-induced intestinal barrier dysfunction. METHODS: Samples were from mice inflicted with 30% total body surface area (TBSA) full-thickness burns. The intestinal permeability, tight junction proteins expressions, zonula occludens-1 (ZO-1) localization, inflammatory cytokines expressions, and short-chain fatty acids (SCFAs) contents were determined. The microbial community was assessed via 16S rDNA Illumina sequencing. RESULTS: The intestinal permeability was increased after severe burn injury, peaking at 6 h post-burn, with approximately 20-folds of the control (p < 0.001). The expression of tight junction proteins (ZO-1, occludin, claudin-1, and claudin-2) was significantly altered (p < 0.05). The ZO-1 morphology was dramatically changed following burn injury. The fecal SCFAs' contents (acetate, propionate, butyrate, isobutyrate, and isovalerate) were noticeably declined after burn injury (p < 0.05). The expressions of pro-inflammatory cytokines (interleukin (IL)-1ß and IL-6) in ileal mucosa were increased, whereas the expressions of anti-inflammatory cytokines (IL-4 and IL-13) were decreased following burn injury (p < 0.05). In addition, burned mice showed an alteration of intestinal microbial community, such as decreased diversity, reduced Bacteroidetes abundance, and increased Firmicutes abundance. CONCLUSIONS: The severe burn-induced intestinal barrier dysfunction is along with the alterations of microbial community.

Antibiotics induced intestinal tight junction barrier dysfunction is associated with microbiota dysbiosis, activated NLRP3 inflammasome and autophagy.

Tight junction barrier is critical to intestinal homeostasis. Applying antibiotics to treat infections is common in clinical practice, which may affect intestinal microbiota. Intestinal microbiota dysbiosis is involved in the occurrence of some gastrointestinal diseases. Therefore, this study was aimed to investigate the influence of antibiotics on intestinal tight junction barrier and the possible underlying mechanisms. Healthy adult female C57BL/6 mice were treated with a broad-spectrum antibiotic cocktail for 14 days. 16S rDNA Illumina sequencing and headspace gas chromatography-mass spectrometry (HS-GC/MS) were respectively used to analyze microbial community and to detect short-chain fatty acids (SCFAs) contents. In vivo intestinal paracellular permeability to fluorescein isothiocyanate-dextran (FITC-dextran) was measured. Protein expression was determined by immunoblotting. Immunofluoresence was applied to observe the distributions of ZO-1, LC3B and ASC. Antibiotics remarkably altered intestinal microbiota composition in healthy mice, accompanying reduced SCFAs' concentrations. In addition, the intestinal tight junction barrier was disrupted by antibiotic treatment, as evidenced by increased intestinal paracellular permeability to FITC-dextran, decreased tight junction protein expressions, and disrupted ZO-1 morphology. Furthermore, NLRP3 inflammasome and autophagy were activated by antibiotic treatment. In conclusion, intestinal epithelial tight junction barrier dysfunction induced by antibiotics is associated with intestinal microbiota dysbiosis, activated NLRP3 inflammasome and autophagy in mice.

Role of Endoplasmic Reticulum Stress-Autophagy Axis in Severe Burn-Induced Intestinal Tight Junction Barrier Dysfunction in Mice.

Severe burn injury induces intestinal barrier dysfunction; however, the underlying mechanisms remain elusive. Our previous studies have shown that the intestinal epithelial tight junction (TJ) barrier dysfunction is associated with both endoplasmic reticulum (ER) stress and autophagy in severely burned mice, but the precise role of ER stress and autophagy in the burn-induced intestinal TJ barrier dysfunction needs to be determined. In this study, female C57/BL6 mice were assigned randomly to either sham burn or 30% total body surface area (TBSA) full-thickness burn. The effects of ER stress and autophagy on the intestinal epithelial TJ barrier were validated by inducing or inhibiting both ER stress and autophagy in mice treated with sham burn or burn injury. The intestinal permeability, expression, and localization of TJ proteins, ER stress, and autophagy were assessed by physiological, morphological, and biochemical analyses. The results showed that inducing ER stress with tunicamycin or thapsigargin caused the activation of autophagy, the increase of intestinal permeability, as well as the reduction and reorganization of TJ proteins in the sham-burned mice, and aggravated the burn-induced activation of autophagy, increase of intestinal permeability, as well as the reduction and reorganization of TJ proteins. In contrast, inhibiting ER stress with 4-phenylbutyrate alleviated the burn-induced activation of autophagy, increase of intestinal permeability, as well as the reduction and reorganization of TJ proteins. In addition, inducing autophagy with rapamycin resulted in the increase of intestinal permeability, as well as the reduction and reorganization of TJ proteins in the sham-burned mice, and aggravated the burn-induced increase of intestinal permeability as well as the reduction and reorganization of TJ proteins. However, inhibiting autophagy with 3-methyladenine attenuated the burn-induced increase of intestinal permeability, as well as the reduction and reorganization TJ proteins. It is suggested that the ER stress-autophagy axis contributes to the intestinal epithelial TJ barrier dysfunction after severe burn injury.

Short-Chain Fatty Acids Manifest Stimulative and Protective Effects on Intestinal Barrier Function Through the Inhibition of NLRP3 Inflammasome and Autophagy.

BACKGROUND/AIMS: Short-chain fatty acids (SCFAs) are the major energy resources of intestinal epithelial cells. It has been reported that SCFAs can repair the dysfunction of intestinal barrier, however, the underlying mechanisms are still not fully understood. Here, we investigated the stimulative and protective effects of SCFAs on intestinal barrier function and the possible mechanisms. METHODS: To investigate the effects of SCFAs on intestinal barrier function, the Caco-2 monolayers were exposed to acetate, propionate, butyrate respectively or simultaneously without or with lipopolysaccharide (LPS). Next, Caco-2 cells were treated with trichostatin A and etomoxir to identify whether SCFAs act as HDAC inhibitors or energy substances. To activate NLRP3 inflammasome and autophagy, Caco-2 cells were treated with LPS+ATP and rapamycin respectively without or with SCFAs. The transepithelial electrical resistance (TER) and paracellular permeability were respectively detected with a Millicell-ERS voltohmmeter and fluorescein isothiocyanate-labeled dextran. Immunoblotting and immunofluorescence were applied to analyze the expression and distribution of tight junction proteins, and the activation of NLRP3 inflammasome and autophagy. RESULTS: Acetate (0.5mM), propionate(0.01mM) and butyrate (0.01mM) alone or in combination significantly increased TER, and stimulated the formation of tight junction. SCFAs also dramatically attenuated the LPS-induced TER reduction and paracellular permeability increase, accompanying significantly alleviated morphological disruption of ZO-1 and occludin. Meanwhile, the activation of NLRP3 inflammasome and autophagy induced by LPS were significantly inhibited by SCFAs. Trichostatin A imitated the inhibiting action of SCFAs on NLRP3 inflammasome, whereas etomoxir blocked the action of SCFAs on protecting intestinal barrier and inhibiting autophagy. In addition, the activation of autophagy and NLRP3 inflammasome by rapamycin and LPS+ATP resulted in TER reduction, paracellular permeability increase and morphological disruption of both ZO-1 and occludin, which was alleviated by SCFAs. CONCLUSION: It is suggested that SCFAs stimulate the formation of intestinal barrier, and protect the intestinal barrier from the disruption of LPS through inhibiting NLRP3 inflammasome and autophagy. In addition, SCFAs act as energy substances to protect intestinal barrier and inhibit autophagy, but act as HDAC inhibitors to suppress NLRP3 inflammasome. Furthermore, the mutual promoting action between NLRP3 inflammasome and autophagy would destroy intestinal barrier function, which could be alleviated by SCFAs.

Severe Burn-Induced Intestinal Epithelial Barrier Dysfunction Is Associated With Endoplasmic Reticulum Stress and Autophagy in Mice.

The disruption of intestinal barrier plays a vital role in the pathophysiological changes after severe burn injury, however, the underlying mechanisms are poorly understood. Severe burn causes the disruption of intestinal tight junction (TJ) barrier. Previous studies have shown that endoplasmic reticulum (ER) stress and autophagy are closely associated with the impairment of intestinal mucosa. Thus, we hypothesize that ER stress and autophagy are likely involved in burn injury-induced intestinal epithelial barrier dysfunction. Mice received a 30% total body surface area (TBSA) full-thickness burn, and were sacrificed at 0, 1, 2, 6, 12 and 24 h postburn. The results showed that intestinal permeability was increased significantly after burn injury, accompanied by the damage of mucosa and the alteration of TJ proteins. Severe burn induced ER stress, as indicated by increased intraluminal chaperone binding protein (BIP), CCAAT/enhancer-binding protein homologous protein (CHOP) and inositol-requiring enzyme 1(IRE1)/X-box binding protein 1 splicing (XBP1). Autophagy was activated after burn injury, as evidenced by the increase of autophagy related protein 5 (ATG5), Beclin 1 and LC3II/LC3I ratio and the decrease of p62. Besides, the number of autophagosomes was also increased after burn injury. The levels of p-PI3K(Ser191), p-PI3K(Ser262), p-AKT(Ser473), and p-mTOR were decreased postburn, suggesting that autophagy-related PI3K/AKT/mTOR pathway is involved in the intestinal epithelial barrier dysfunction following severe burn. In summary, severe burn injury induces the ER stress and autophagy in intestinal epithelia, leading to the disruption of intestinal barrier.