Optimization of boronic ester-based amphiphilic copolymers for ROS-responsive drug delivery.

This study introduces boronic ester-based ROS-responsive amphiphilic copolymers for antioxidant drug delivery. Tuning the hydrophobic/hydrophilic balance optimized the size, curcumin encapsulation, ROS-triggered release, cellular uptake, and intracellular ROS scavenging. The lead P1b formulation self-assembled into stable 10 nm micelles enabling rapid ROS-triggered curcumin release and preferential cellular internalization. P1b eliminated over 90% of pathogenic intracellular ROS within 10 minutes, demonstrating a rapid antioxidant therapy.

Enhanced Ocular Delivery of Beva via Ultra-Small Polymeric Micelles for Noninvasive Anti-VEGF Therapy.

Pathological ocular neovascularization resulting from retinal ischemia constitutes a major cause of vision loss. Current anti-VEGF therapies rely on burdensome intravitreal injections of Bevacizumab (Beva). Herein ultrasmall polymeric micelles encapsulating Beva (P@Beva) are developed for noninvasive topical delivery to posterior eye tissues. Beva is efficiently loaded into 11 nm micelles fabricated via self-assembly of hyperbranched amphiphilic copolymers. The neutral, brush-like micelles demonstrate excellent drug encapsulation and colloidal stability. In vitro, P@Beva enhances intracellular delivery of Beva in ocular cells versus free drug. Ex vivo corneal and conjunctival-sclera-choroidal tissues transport after eye drops are improved 23-fold and 7.9-fold, respectively. Anti-angiogenic bioactivity is retained with P@Beva eliciting greater inhibition of endothelial tube formation and choroid sprouting over Beva alone. Remarkably, in an oxygen-induced retinopathy (OIR) model, topical P@Beva matching efficacy of intravitreal Beva injection, is the clinical standard. Comprehensive biocompatibility verifies safety. Overall, this pioneering protein delivery platform holds promise to shift paradigms from invasive intravitreal injections toward simplified, noninvasive administration of biotherapeutics targeting posterior eye diseases.

Self-Report Amphiphilic Polymer-Based Drug Delivery System with ROS-Triggered Drug Release for Osteoarthritis Therapy.

The development of drug delivery systems with real-time cargo release monitoring capabilities is imperative for optimizing nanomedicine performance. Herein, we report an innovative self-reporting drug delivery platform based on a ROS-responsive random copolymer (P1) capable of visualizing cargo release kinetics via the activation of an integrated fluorophore. P1 was synthesized by copolymerization of pinacol boronate, PEG, and naphthalimide monomers to impart ROS-sensitivity, hydrophilicity, and fluorescence signaling, respectively. Detailed characterization verified that P1 self-assembles into 11 nm micelles with 10 µg mL-1 CMC and can encapsulate hydrophobic curcumin with 79% efficiency. Fluorescence assays demonstrated H2O2-triggered disassembly and curcumin release with concurrent polymer fluorescence turn-on. Both in vitro and in vivo studies validated the real-time visualization of drug release and ROS scavenging, as well as the therapeutic effect on osteoarthritis (OA). Overall, this nanotheranostic polymeric micelle system enables quantitative monitoring of drug release kinetics for enhanced treatment optimization across oxidative stress-related diseases.

Tespa1 deficiency reduces the antitumour immune response by decreasing CD8+T cell activity in a mouse Lewis lung cancer model.

Thymocyte-expressed, positive selection-associated 1 (Tespa1) is a key molecule in T-cell development and has been linked to immune diseases. However, its role in antitumour CD8+T cell immunity remains unclear. Here, we demonstrated that Tespa1 plays an important role in antitumour CD8+T cell immunity. First, compared with wild-type (WT) mice, Lewis lung cancer cells grew faster in Tespa1 knockout (Tespa1-/-) mice, with reduced apoptosis, and decreased CD8+T cells in peripheral blood and tumor tissues. Second, the proportion of CD8+T and Th1 cells in the splenocytes of Tespa1-/- mice was lower than that in WT mice. Third, Tespa1-/- CD8+ tumor-infiltrating lymphocytes (TILs) showed weakened proliferation, invasion, cytotoxicity, and protein expression of IL-2 signalling pathway components compared to WT CD8+TILs. Furthermore, PD-1 expression in CD8+TILs was higher in Tespa1-/- than in WT mice. Lastly, CD8+TILs in WT mice improved the antitumour ability of Tespa1-/- mice. In conclusion, these findings suggest that Tespa1 plays a critical role in the tumor immune system by regulating CD8+T cells.

pH-Responsive Hexa-Histidine Metal Assembly (HmA) with Enhanced Biocatalytic Cascades as the Vehicle for Glucose-Mediated Long-Acting Insulin Delivery.

Diabetes has been listed as one of the three major diseases that endanger human health. Accurately injecting insulin (Ins) depending on the level of blood glucose (LBG) is the standard treatment, especially controlling LBG in the long-term by a single injection. Herein, the pH-responsive hexa-histidine metal assembly (HmA) encapsulated with enzymes (GOx and CAT) and Ins (HmA@GCI) is engineered as the vehicle for glucose-mediated insulin delivery. HmA not only shows high proteins loading efficiency, but also well retained proteins activity and protect proteins from protease damage. Within HmA, the biocatalytic activities of enzymes and the efficiency of the cascade reaction between GOx and CAT are enhanced, leading to a super response to the change of LBG with insulin release and efficient clearance of harmful byproducts of GOx (H2 O2 ). In the treatment of diabetic mice, HmA@GCI reduces LBG to normal in half an hour and maintains for more than 5 days by a single subcutaneous injection, and nearly 24 days with four consecutive injections. During the test period, no symptoms of hypoglycemia and toxicity to tissues and organs are observed. These results indicate that HmA@GCI is a safe and long-acting hypoglycemic agent with prospective clinical application.

Hydrazone-Based Amphiphilic Brush Polymer for Fast Endocytosis and ROS-Active Drug Release.

Due to the high reactivity of reactive oxygen species (ROS), it is essential to sweep them away in time. In this study, ClO--responsible amphiphilic brush polymers were prepared by free radical polymerization using two monomers consisting of polyethylene glycol as the hydrophilic part, and an alkyl chain connected by hydrazone as the hydrophobic part. The macromolecules assemble into particles with nanoscaled dimensions in a neutral buffer, which ensures quick cellular internalization. The polymer has a low critical micellization concentration and can encapsulate hydrophobic drug molecules up to 19% wt. The micelles formed by the polymer disassemble in a ClO--rich environment and release 80% of their cargo within 2 h, which possesses a faster release rate compared to the previous systems. The relatively small size and the quick response of hydrazone toward ClO- ensure a quick uptake and elimination of ROS in vitro and in vivo.

Combined delivery of small molecule and protein drugs as synergistic therapeutics for treating corneal neovascularization by a one-pot coassembly strategy.

Combined drug administration is a potential strategy to increase efficacy through therapeutic synergy. Small molecule drugs and protein drugs are the two most popular kinds of drugs in medicine. However, efficiently encapsulating these two drugs still have key challenges due to their distinct properties (molecular weight, hydrophilicity, chemical groups, etc.), weak ability to penetrate through various biobarriers (cell membrane, endosome escape, tissue barriers dependent on the method of administration, etc.) and the easy deactivation of protein drugs during the construction of carrier and delivery process. Here, we utilize the hexahistidine-metal assembly (HmA), which can encapsulate a wide spectrum of drugs with high loading efficiency, to coencapsulate Dexp (a small molecule drug) and BVZ (protein drug) by a one-pot coassembly strategy. Our data demonstrated that Dexp and BVZ were coloaded into Dexp&BVZ@HmA with high efficiency, while the bioactivity of BVZ was well-maintained. Most importantly, when evaluating the therapeutic outcomes of drugs@HmA in a corneal neovascularization (CNV) model in vitro and in vivo, the combination group presented overwhelming efficacy compared to the monotherapy group. This strategy offers a platform to codeliver protein and small drugs and has the potential for treating anterior segment diseases as well as other diseases that need combination therapy.

Decorating hexahistidine-metal assemblies with tyrosine enhances the ability of proteins to pass through corneal biobarriers.

In recent decades, the use of protein drugs has increased dramatically for almost every clinical indication, including autoimmunity and cancer infection, given their high specificity and limited side effects. However, their easy deactivation by the surrounding microenvironment and limited ability to pass through biological barriers pose large challenges to the use of these agents for therapeutic effects; these deficits could be greatly improved by nanodelivery using platforms with suitable physicochemical properties. Here, to assess the effect of the hydrophobicity of nanoparticles on their ability to penetrate biological barriers, the hydrophobic amino acid tyrosine (Y) was decorated onto hexahistidine peptide, and two nanosized YHmA and HmA particles were generated, in which Avastin (Ava, a protein drug) was encapsulated by a coassembly strategy. In vitro and in vivo tests demonstrated that these nanoparticles effectively retained the bioactivity of Ava and protected Ava from proteinase K hydrolysis. Importantly, YHmA displayed a considerably higher affinity to the ocular surface than HmA, and YHmA also exhibited the ability to transfer proteins across the barriers of the anterior segment, which greatly improved the bioavailability of the encapsulated Ava and produced surprisingly good therapeutic outcomes in a model of corneal neovascularization. STATEMENT OF SIGNIFICANCE: Improving the ability to penetrate tissue barriers and averting inactivation caused by surrounding environments, are the keys to broaden the application of protein drugs. By decorating hydrophobic amino acid, tyrosine (Y), on hexahistidine peptide, YHmA encapsulated protein drug Ava with high efficiency by co-assembly strategy. YHmA displayed promising ability to maintain bioactivity of Ava during encapsulation and delivery, and protected Ava from proteinase K hydrolysis. Importantly, YHmA transferred Ava across the corneal epithelial barrier and greatly improved its bioavailability, producing surprisingly good therapeutic outcomes in a model of corneal neovascularization. Our results contributed to not only the strategy to overcome shortcomings of protein drugs, but also suggestion on hydrophobicity as a nonnegligible factor in nanodrug penetration through biobarriers.

Lumiflavin Reduces Cisplatin Resistance in Cancer Stem-Like Cells of OVCAR-3 Cell Line by Inducing Differentiation.

Ovarian cancer stem-like cells (CSCs) play a vital role in drug resistance and recurrence of ovarian cancer. Inducing phenotypic differentiation is an important strategy to enhance the effects of chemotherapy and reduce the drug resistance of CSCs. This study found that lumiflavin, a riboflavin decomposition product, reduced the development of CSC resistance and enhanced the chemotherapy effect of cisplatin (DDP) on CSCs in DDP-resistant ovarian cancer OVCAR-3 cell line (CSCs/DDP) and was related to the induction of CSC phenotypic differentiation. Results showed that the development of DDP-resistant OVCAR-3 cells was related to the increase in the proportion of CSCs/DDP, and the treatment with lumiflavin reduced the DDP-resistance levels of OVCAR-3 cells and proportion of CSCs/DDP. Further investigation found that lumiflavin synergistic with DDP increased apoptosis, decreased mitochondrial membrane potential, and inhibited the clonal formation of CSCs/DDP. Meanwhile, in vivo experiments showed that lumiflavin dose-dependently enhanced the chemotherapy effect of DDP on tumor-bearing nude mice inoculated by CSCs/DDP. Lumiflavin treatment also reduced the ratio of CD133+/CD177+ to CD44+/CD24 cells, which is the identification of CSCs, in CSCs/DDP. In addition, transcriptome sequencing results suggested that the role of lumiflavin was related to the notch and stem cell pathway, and Western blot analysis showed that lumiflavin inhibited the protein expression of notch signaling pathway in CSCs/DDP. In conclusion, lumiflavin reduces the development of the drug resistance of OVCAR-3 cell and increases the sensitivity of CSCs/DDP to DDP by inducing phenotypic differentiation, which may have a potential role in the chemotherapy treatment of ovarian cancer.

Panax notoginseng protects the rat brain function from traumatic brain injury by inhibiting autophagy via mammalian targeting of rapamycin.

Traumatic brain injury (TBI) remains one of the leading causes of death and disability worldwide. Our previous studies have found that traditional Chinese medicine, Panax notoginseng (P. notoginseng) can reduce cerebral hemorrhage in rats with TBI. Yet, the exact mechanism still remains unclear. According to the random number table, 36 SD rats were randomly divided into six groups: Sham group (negative control group), Model group, PIK inhibitor group (positive group), P. notoginseng group (experimental group), Rapamycin group, and Panax notoginseng+Rapamycin group (experimental group). In the Model group (M group, the group showing signs of TBI without any treatment), the neural function defect score was significantly decreased, while sequestosome 1 (P62), Beclin 1, and microtubule-associated protein 1 light chain 3 (LC3-II) were significantly increased. The brain tissue was significantly damaged, and many autophagosomes were observed in the cytoplasm. Compared with the Model group and the Rapamycin group (M+Rapa group, the group showing signs of TBI with Rapamycin treatment), P62, Beclin 1, and LC3-II were significantly decreased, the score of neural function defect was significantly improved, and the brain tissue damage was significantly reduced in the PIK (phosphatidylinositol 3-kinase) inhibitor group (M+LY group, the group showing signs of TBI with PIK inhibitor treatment). Compared with the Model group, mTOR was decreased and LC3-II was increased; however, there were no significant changes in neural function defect score, HE staining, Nissl staining, and transmission electron microscopy in the Rapamycin group. Compared with the Model group, the neural function defect score at 72h was significantly improved, mTOR was significantly increased, P62, Beclin 1, and LC3-II significantly decreased, brain tissue damage was reduced in HE staining and Nissl staining, autophagosomes were reduced in cytoplasm by transmission electron microscopy in the P. notoginseng group (M+PN group, the group showing signs of TBI with P. notoginseng treatment). Also, there was no significant difference between P. notoginseng group and P. notoginseng+Rapamycin group (M+PN+Rapa group, the group showing signs of TBI with P. notoginseng+Rapamycin treatment). P. notoginseng protects the rat brain function from TBI by inhibiting autophagy through the mTOR signaling pathway and other autophagy pathways.

Lumiflavin Enhances the Effects of Ionising Radiation on Ovarian Cancer Stem-Like Cells by Inhibiting Autophagy.

BACKGROUND: The development of Cancer Stem-like Cells (CSCs) is one of the main causes of ovarian cancer tolerance to radiotherapy. Autophagy is an adaptive process by which cells damage due to radiation. As a metabolite of riboflavin, lumiflavin can enhance the chemotherapeutic effects of cisplatin on ovarian cancer CSCs. OBJECTIVE: This study aimed to investigate the synergistic effects of lumiflavin and ionising radiation on ovarian cancer CSCs and explore the association of this metabolite with autophagy. METHODS: CSCs of human ovarian cancer cell lines HO8910 were treated with lumiflavin and rapamycin and then subjected to irradiation at a cumulative dose of 8 Gy. Cell proliferation ability, clonal formation ability, apoptosis rate, autophagy changes and autophagy-related protein changes were detected. RESULTS: Lumiflavin and ionising radiation synergistically reduced cell vitality and clone formation and increased the apoptosis of CSCs compared with irradiation alone. In addition, ionising radiation increased autophagy and the expression of associated proteins, whereas lumiflavin reduced those changes in autophagy progression. Moreover, rapamycin, an autophagy inhibitor, was observed to block the synergistic effects of lumiflavin and ionising radiation on CSC apoptosis. CONCLUSION: Lumiflavin can enhance the effects of ionising radiation on ovarian cancer CSCs. The mechanism by which these effects are exerted is related to blocking the autophagy pathway.

Tespa1 plays a role in the modulation of airway hyperreactivity through the IL-4/STAT6 pathway.

BACKGROUND: Thymocyte-expressed, positive selection-associated 1 (Tespa1) is a critical signaling molecule in thymocyte development. This study aimed to investigate the regulatory effect of Tespa1 on mast cells in the pathogenesis of asthma and its relationship with the interleukin (IL)-4/signal transducers and activators of transcription 6 (STAT6) signaling pathway. METHODS: Tespa1 mRNA expression analysis and IgE levels were carried out using the induced sputum of 33 adults with stable asthma and 36 healthy controls. Tespa1-knockout mice (Tespa1-/-, KO) and C57BL/6 background (wild-type, WT) mice were sensitized and treated with ovalbumin (OVA) to establish an asthma model. Pathological changes, number and activity of mast cells, and changes in activation of the IL-4/STAT6 pathway in lung tissue were detected. The changes of tryptase expression and STAT6 activation after mast cell gene knockout were analyzed in vitro. The changes of enzyme expression and STAT6 activation after mast cell gene knockout were analyzed in vitro. The association between the Tespa1 and p-STAT6 was analyzed by co-immunoprecipitation method. RESULTS: Compared with the healthy controls, Tespa1 expression was decreased, and IgE levels were elevated in the sputum of asthmatic patients. Animal experiments showed that Tespa1-/- mice exhibited more severe inflammation, higher quantity of goblet cells and mast cells in the bronchium, and greater expression of mast cell tryptase, which is induced by ovalbumin, than WT mice. And IL-4, IL-13, phospho-Janus kinase 1, and p-STAT6 expressions presented a higher increase in the Tespa1-/- mouse model than in the WT mouse model. Further in vitro studies confirmed that IL-4 could more significantly promote tryptase and p-STAT6 activities in Tespa1-/- mast cells than their WT counterparts. Correlation analysis results showed a negative correlation between Tespa1 and p-STAT6. Co-immunoprecipitation results demonstrated an association between Tespa1 and p-STAT6. CONCLUSIONS: Altogether, our results indicate that Tespa1 can negatively regulate mast cell activity, and this event is related to the mast cell IL-4/STAT6 signaling pathway and could be therapeutically exploited to treat asthma attacks.

Phenytoin sodium-ameliorated gingival fibroblast aging is associated with autophagy.

BACKGROUND AND OBJECTIVE: Human gingival fibrolasts aging is an important cause of periodontal disease. Phenytoin sodium (phenytoin) has a side effect of gingival hyperplasia and an effect on the autophagy progress. This study investigated whether the effect of phenytoin on aging gingival fibroblast is related to the autophagy pathway. MATERIAL AND METHODS: The aging model of gingival fibroblast cell line HGF-1 was induced by hydrogen peroxide (H2 O2 ), and the treatment of phenytoin and 3-methyladenine (3-MA) was performed simultaneously. Cell viability, cell cycle, and intracellular calcium ion were measured by flow cytometry. Changes in expression of basic fibroblast growth factor (bFGF), P16INK4A , P21cip1 , and bFGF, P16INK4A , P21cip1 , LC3II, p62, and Beclin were tested by using reverse transcription polymerase chain reaction, western blot, and immunofluorescence staining. RESULTS: The results showed that aging HGF-1 proliferation was inhibited by H2 O2 , gene, protein expression of bFGF, P16INK4A , and P21cip1 were decreased, autophagy-related proteins LC3II, p62, and Becline were decreased, and the proportion of G0/G1 phase and intracellular calcium ion of cell cycle was increased. Phenytoin treatment could recovery above changes, but the effect of phenytoin could be blocked by 3-MA. CONCLUSION: We propose that phenytoin alleviates the aging of gingival fibroblasts induced by H2 O2 . This condition is related to the enhancement of autophagy pathway.

Healing characteristics of acellular porcine corneal stroma following therapeutic keratoplasty.

BACKGROUND: Acellular porcine corneal stroma (APCS) has proven to be a promising alternative to traditional corneal grafts. This prospective case series was conducted to further investigate the healing characteristics of APCS following keratoplasty. METHODS: Twenty-seven patients undergoing APCS implantation to treat infectious keratitis were included. The patients were followed up for 12 months after surgery. The main outcome measures included visual acuity, corneal transparency, graft thickness, and cellular and nerve regeneration. RESULTS: In the operated eyes, the best-corrected visual acuity (BCVA, in logarithm of the minimal angle of resolution [logMAR] units) increased from 1.23 ± 0.95 logMAR before surgery to 0.23 ± 0.18 logMAR at 12 months after surgery (P < .001). The contrast sensitivity was still evidently reduced, especially at higher spatial frequencies. Gradual transparency improvement was observed in APCS grafts post-operatively. After implantation, the APCS graft thickness initially increased (day 1 = 592.41 ± 52.69 µm) but then continuously decreased until 3 months after surgery (1 month = 449.26 ± 50.38 µm; 3 months = 359.63 ± 34.14 µm, P < .001). Graft reepithelialization was completed within 1 week. In the in vivo confocal microscopy scans, host keratocytes began to repopulate the APCS grafts between 3 and 6 months post-operatively; subbasal nerve regeneration was only noted in 18.52% (5/27) of the eyes by 12 months after surgery. CONCLUSIONS: Acellular porcine corneal stroma functions as an effective alternative to human corneal tissue in lamellar keratoplasty. However, APCS is somewhat different from fresh human cornea in term of the post-operative healing process, which warrants the attention of both clinicians and patients.

Lumiflavin increases the sensitivity of ovarian cancer stem-like cells to cisplatin by interfering with riboflavin.

Here, we used lumiflavin, an inhibitor of riboflavin, as a new potential therapeutic chemosensitizer to ovarian cancer stem-like cells (CSCs). This study demonstrates that the enrichment of riboflavin in CSCs is an important cause of its resistance to chemotherapy. Lumiflavin can effectively reduce the riboflavin enrichment in CSCs and sensitize the effect of cisplatin Diamminedichloroplatinum (DDP) on CSCs. In this study, CSCs of human ovarian cancer cell lines HO8910 were separated using a magnetic bead (CD133+). We also show the overexpression of the mRNA and protein of riboflavin transporter 2 and the high content of riboflavin in CSCs compared to non-CSCs (NON-CSCs). Moreover, CSCs were less sensitive to DDP than NON-CSCs, whereas, the synergistic effect of lumiflavin and DDP on CSCs was more sensitive than NON-CSCs. Further research showed that lumiflavin had synergistic effects with DDP on CSCs in increasing mitochondrial function damage and apoptosis rates and decreasing clonic function. In addition, we found that the combination of DDP and lumiflavin therapy in vivo has a synergistic cytotoxic effect on an ovarian cancer nude mice model by enhancing the DNA-damage response and increasing the apoptotic protein expression. Notably, the effect of lumiflavin is associated with reduced riboflavin concentration, and riboflavin could reverse the effect of DDP in vitro and in vivo. Accordingly, we conclude that lumiflavin interfered with the riboflavin metabolic pathways, resulting in a significant increase in tumour sensitivity to DDP therapy. Our study suggests that lumiflavin may be a novel treatment alternative for ovarian cancer and its recurrence.

Probucol ameliorates hepatic stellate cell activation and autophagy is associated with farnesoid X receptor.

Probucol has antioxidant effects and inhibits inflammation. Farnesoid X receptor (FXR) is a nuclear receptor that regulates autophagy, which is regarded as the key cause of the activation of hepatic stellate cell (HSC). In this study, the effects of probucol on HSC activation and autophagy in vitro and vivo and the role of FXR in this progress were investigated. Results showed that probucol ameliorated hepatic fibrosis and autophagy, and increased the expression of FXR in liver in a mouse model of fibrosis induced by CCl4. And probucol could alleviate lipopolysaccharide-induced autophagy and HSC activation in vitro. In addition, probucol increased FXR expression, and the Z-guggulsterone, an antagonist of FXR, could block the effects of probucol on HSC activation and autophagy. Additionally, agonists of FXR could suppress LPS-induced autophagy and activation. These results suggest that probucol could ameliorate hepatic fibrosis, and inhibit HSC autophagy and activation, and these effects are associated with FXR.

Toll-like receptor 4 contributes to a myofibroblast phenotype in cardiac fibroblasts and is associated with autophagy after myocardial infarction in a mouse model.

BACKGROUND AND AIMS: Cardiac fibrosis after myocardial infarction (MI) is involved in fibroblast transforming and differentiating into myofibroblast phenoconversion, however, the underlying mechanisms are poorly understood. Toll-like receptor 4 (TLR4)-mediated pathogen-associated molecular patterns are key factors that deteriorate cardiac remodelling after MI. Moreover, autophagy has dual roles in cell survival in myocardial tissues after MI. We evaluated the relationship between TLR4 signalling and cardiac myofibroblast transformation-differentiation after MI in vivo and in vitro and analysed the role of autophagy. METHODS: We reproduced a model of MI by the permanent ligation of the left anterior descending coronary artery of Tlr4-knockout (Tlr4-/-) and wild-type (WT) male mice. We evaluated scar formation, myofibroblast phenoconversion, LC3 dot formation, autophagy related proteins and α-smooth muscle actin (SMA) in cardiac tissues, 7, 14, and 28 days after myocardial infarction. Cardiac fibroblasts were cultured from Tlr4-/- or WT mice. Vimentin, α-SMA, bilayer membrane vesicle structures of autophagosomes, and autophagy related proteins were observed after treatment with lipopolysaccharide (LPS) or 3-methyladenine (3-MA) at 24 h. RESULTS: After MI on 7, 14, and 28 days, Tlr4-/- mice showed that heart tissue fibrosis and expression of α-SMA, a marker of myofibroblasts, were decreased compared to WT mice. Additionally, levels of LC3II, Atg5, Atg7, and Beclin-1, which are involved in autophagy, were lower than those in WT mice. Further, p62 expression, which is negatively correlated with autophagy levels, was higher in Tlr4-/- mice. Moreover, LC3-labelled autophagosomes in cardiac tissues were reduced in these animals. In vitro, LPS, a ligand of TLR4, stimulated α-SMA expression in cardiac fibroblasts, enhanced autophagic flux, and increased autophagosome numbers. In contrast, these effects were not obvious in Tlr4-/- cardiac fibroblasts. LC3II, Atg5, Atg7, and Beclin-1 were upregulated, and p62 was downregulated in cardiac fibroblasts of WT mice stimulated with LPS. However, these effects were blocked by 3-methyladenine, an inhibitor of autophagy. CONCLUSIONS: These results suggest that TLR4 signalling executes the development of a myofibroblast phenotype after MI via autophagy and could be therapeutically exploited to improve outcome after myocardial injury.

Tacrolimus interacts with voriconazole to reduce the severity of fungal keratitis by suppressing IFN-related inflammatory responses and concomitant FK506 and voriconazole treatment suppresses fungal keratitis.

Purpose: To investigate the expression and roles of type I and II interferons (IFNs) in fungal keratitis, as well as the therapeutic effects of tacrolimus (FK506) and voriconazole on this condition. Methods: The mRNA and protein expression levels of type I (IFN-α/ß) and II (IFN-γ) IFNs, as well as of related downstream inflammatory cytokines (interleukin (IL)-1α, IL-6, IL-12, and IL-17), were detected in macrophages, neutrophils, lymphocytes, and corneal epithelial cells (A6(1) cells) stimulated with zymosan (10 mg/ml) for 8 or 24 h. A fungal keratitis mouse model was generated through intrastromal injection of Aspergillus fumigatus, and the mice were then divided into four groups: group I, the PBS group; group II, the voriconazole group; group III, the FK506 group; and group IV, the voriconazole plus 0.05% FK506 group. Corneal damage was evaluated with clinical scoring and histological examination. In addition, the mRNA and protein expression levels of type I (IFN-α/ß) and type II (IFN-γ) IFNs, as well as related inflammatory cytokines, were determined at different time points using quantitative real-time PCR (qRT-PCR) and western blotting. Results: After zymosan stimulation of mouse neutrophils, lymphocytes, macrophages, and A6(1) cells, the IFN mRNA and protein expression levels were markedly increased until 24 h, peaking at 8 h (p<0.001). The mRNA and protein expression levels of inflammatory cytokines (IL-1α, IL-6, IL-12, and IL-17) were also upregulated after zymosan stimulation. Moreover, type I (IFN-α/ß) and type II (IFN-γ) IFN expression levels were increased and positively correlated with the progression of fungal keratitis in vivo. FK506 administered with voriconazole reduced the pathological infiltration of inflammatory cells into the cornea and downregulated the expression levels of IFNs and related inflammatory cytokines. Conclusions: In conclusion, this study demonstrated that type I and II IFN levels were markedly increased in fungal keratitis and that FK506 combined with voriconazole decreased the severity of fungal keratitis by suppressing type I and II IFNs and their related inflammatory responses.

Aspirin modulates the inflammatory response in a thrombus­stimulated LMVEC model.

The purpose of the present study was to examine whether aspirin interferes with the inflammatory response in a thrombus­stimulated lung microvascular endothelial cell (LMVEC) model. The LMVECs were randomly divided into eight groups: Normal group (group N), model group (group M), model + ASP group (group M+A), model+CX3CL1­short hairpin (sh)RNA group (group M+SH), model + CX3CL1­overexpression vector group (group M+CX3), model + ASP + shRNA group (group M+A+SH), model + ASP + CX3CL1­overexpression vector group (group M+A+CX3), and normal + virus control group (group N+V). The endothelial cells were cultured, and a thrombus was added to the cells. Briefly, 12 h following the precipitation of the thrombus, data from ELISA, reverse transcription­quantitative polymerase chain reaction analysis and confocal microscopy revealed that the levels of tumor necrosis factor (TNF)­α, interleukin (IL)­6, CX3C chemokine ligand 1 (CX3CL1), CX3C chemokine receptor 1 (CX3CR1) and nuclear factor­κB (NF­κB) in group M were increased, compared with those in group N (P<0.01). These levels, with the exception of TNF­α, were significantly lower in group M+SH, compared with those in group M (P<0.01). Furthermore, the levels of IL­6 in groups M+A, M+CX3 and M+A+CX3 were decreased, compared with those in group M (P<0.01); the level of TNF­α in group M+A+SH was decreased, compared with that in group M (P<0.01); the level of CX3CR1 waslower in groups M+A and M+A+SH, compared with that in group M (P<0.01), and the level of NF­κB in group M+SH was decreased, compared with the level in group M and group M+A (P<0.05). In conclusion, the thrombus­stimulated LMVEC model exhibited induced production of TNF­α, IL­6, CX3CL, CX3CR1, NF­κB and intercellular adhesion molecule­1. Furthermore, it was confirmed that the signaling pathways involving CX3CL1­NF­κB, IL­6 and TNF­α were partly inhibited by aspirin.

Microvascular abnormalities in dry eye patients.

This study was conducted to evaluate conjunctival blood flow velocities and microvascular network density in patients with dry eye disease (DED). Twenty-five patients with DED and 25 healthy controls were recruited. The microvasculature and microcirculation of the temporal bulbar conjunctiva of the right eyes were assessed using a functional slit-lamp biomicroscope. Vascular variables included blood flow velocity (BFV), blood flow rate (BFR), microvascular network density and vessel diameter. A fractal analysis was performed using the box counting method to measure the fractal dimension (Dbox) representing the vessel density. The bulbar BFV was 0.59 ±â€¯0.09 mm/s in the DED group and 0.47 ±â€¯0.12 in the control group (P < 0.001). BFR was 169.5 ±â€¯1.8 in the DED group compared to the control group (107.2 ±â€¯49.6) (P < 0.001). Dbox was higher in DED patients (1.65 ±â€¯0.04) than controls (1.60 ±â€¯0.07, P < 0.05). Moreover, the vessel diameter was larger in the DED group (21.8 ±â€¯1.8 µm) compared with controls (17.9 ±â€¯2.2 µm, P < 0.001). Dbox was positively related with ocular surface disease index (OSDI) in patients with DED (r = 0.54, P = 0.008). Microvascular alterations were found in the bulbar conjunctiva of DED patients, including increased blood flow velocity, higher vessel density and larger vessel diameter.