A Novel Inhibitor of Poly(ADP-Ribose) Polymerase-1 Inhibits Proliferation of a BRCA-Deficient Breast Cancer Cell Line via the DNA Damage-Activated cGAS-STING Pathway.

Loss-of-function mutations in the Breast Cancer Susceptibility Gene (BRCA1 and BRCA2) are often detected in patients with breast cancer. Poly(ADP-ribose) polymerase-1 (PARP1) plays a key role in the repair of DNA strand breaks, and PARP inhibitors have been shown to induce highly selective killing of BRCA1/2-deficient tumor cells, a mechanism termed synthetic lethality. In our previous study, a novel PARP1 inhibitor─(E)-2-(2,3-dibromo-4,5-dimethoxybenzylidene)-N-(4-fluorophenyl) hydrazine-1-carbothioamide (4F-DDC)─was synthesized, which significantly inhibited PARP1 activity with an IC50 value of 82 ± 9 nM. The current study aimed to explore the mechanism(s) underlying the antitumor activity of 4F-DDC under in vivo and in vitro conditions. 4F-DDC was found to selectively inhibit the proliferation of BRCA mutant cells, with highly potent effects on HCC-1937 (BRCA1-/-) cells. Furthermore, 4F-DDC was found to induce apoptosis and G2/M cell cycle arrest in HCC-1937 cells. Interestingly, immunofluorescence and Western blot results showed that 4F-DDC induced DNA double strand breaks and further activated the cGAS-STING pathway in HCC-1937 cells. In vivo analysis results revealed that 4F-DDC inhibited the growth of HCC-1937-derived tumor xenografts, possibly via the induction of DNA damage and activation of the cGAS-STING pathway. In summary, the current study provides a new perspective on the antitumor mechanism of PARP inhibitors and showcases the therapeutic potential of 4F-DDC in the treatment of breast cancer.

Therapeutic Potential Evaluation of Silk Sericin Stabilized Fisetin to Ulcerative Colitis.

Ulcerative colitis is a chronic inflammatory bowel disease with a high recurrence rate. Natural phytochemical compounds are increasingly being considered as preventative and supportive treatments for this condition. However, the poor water solubility and stability of many of these compounds limit their effectiveness in vivo. To address this issue, fisetin (FT), a natural phytochemical with poor solubility, is stabilized using silk sericin (SS) to create a composite (SS/FT). The therapeutic potential of the SS/FT on ulcerative colitis is extensively investigated, and the results showed that it effectively alleviated the body weight loss and colon length shortening induced by dextran sulfate sodium. Notably, SS/FT downregulated the immune response, decreased colonic histopathological lesions, and reduced the cGAS/STING signal activation. This suggests that SS/FT may offer a promising therapy for treating ulcerative colitis.

Polyvinylpyrrolidone-Polydatin nanoparticles protect against oxaliplatin induced intestinal toxicity in vitro and in vivo.

Oxaliplatin (OXL) is a first-line drug for the treatment of colon cancer, with excellent efficacy. Intestinal toxicity is a common side effect of OXL, with unclear pathogenesis and a lack of effective treatment strategies. Polydatin (PD) has anti-inflammatory and antioxidant activities and is a potential drug for treating intestinal diseases, but its poor water solubility limits its application. In this study, polyvinylpyrrolidone (PVP) was used as a carrier to prepare nanoparticles loaded with PD (PVP-PD), with a particle size of 92.42 nm and exhibiting sustained release properties. In vitro results showed that PVP-PD protected NCM460 cells from OXL induced injury, mitochondrial membrane potential (MMP) disruption, and accumulation of reactive oxygen species (ROS). The in vivo results demonstrated the protective effect of PVP-PD on intestinal toxicity induced by OXL, such as alleviating weight loss and colon length reduction induced by OXL. Both in vivo and in vitro mechanisms indicated that OXL induced DNA damage and activated the cGAS-STING pathway, further inducing the expression of inflammatory factors such as IL-1ß and TNF-α. PVP-PD alleviated the aforementioned changes induced by OXL by inhibiting the DNA damage-cGAS-STING pathway. In summary, our study demonstrated that the DNA damage-cGAS-STING pathway was involved in OXL induced intestinal toxicity, and PVP-PD provided a potential strategy for treating OXL induced intestinal toxicity.

Design, synthesis and SAR of novel 7-azaindole derivatives as potential Erk5 kinase inhibitor with anticancer activity.

The extracellular signal-regulated kinase 5 (Erk5) signaling plays a crucial role in cancer, and regulating its activity may have potential in cancer chemotherapy. In this study, a series of novel 7-azaindole derivatives (4a-5o) were designed and synthesized. Their antitumor activities on human lung cancer A549 cells was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, 4',6-diamidino-2-phenylindole (DAPI) staining and colony formation assay. Among them, compounds 4a, 4 h, 5d and 5j exhibited good anti-proliferative activity with the IC50 values of 6.23 µg/mL, 8.52 µg/mL, 7.33 µg/mL and 4.56 µg/mL, respectively, equivalent to Erk5 positive control XMD8-92 (IC50 = 5.36 µg/mL). The results of structure-activity relationships (SAR) showed that double bond on the piperidine ring and N atoms at the N7 position of 7-azaindole was essential for their antiproliferative activity. Furthermore, compounds 4a and 5j exhibited good inhibition on Erk5 kinase through Western blot analysis and possible action site of compounds with Erk5 kinase was elucidated by molecular docking.

Regulation of Eukaryotic Translation Initiation Factor 4E as a Potential Anticancer Strategy.

Eukaryotic translation initiation factors (eIFs) are highly expressed in cancer cells, especially eIF4E, the central regulatory node driving cancer cell growth and a potential target for anticancer drugs. eIF4E-targeting strategies primarily focus on inhibiting eIF4E synthesis, interfering with eIF4E/eIF4G interactions, and targeting eIF4E phosphorylation and peptide inhibitors. Although some small-molecule inhibitors are in clinical trials, no eIF4E inhibitors are available for clinical use. We provide an overview of the regulatory mechanisms of eIF4E and summarize the progress in developing and discovering eIF4E inhibitor strategies. We propose that interference with eIF4E/eIF4G interactions will provide a new perspective for the design of eIF4E inhibitors and may be a preferred strategy.

Fucoidan-proanthocyanidins nanoparticles protect against cisplatin-induced acute kidney injury by activating mitophagy and inhibiting mtDNA-cGAS/STING signaling pathway.

Fucoidan (FU) is a natural polymer from marine organisms, which has been widely studied and applied in drug delivery. In this study, FU nanoparticles loaded with proanthocyanidins (PCs) (FU/PCs NPs) were prepared and their effect and mechanism in protecting cisplatin-induced acute kidney injury (AKI) were studied. The in vitro studies confirmed that FU/PCs NPs increased the antioxidant activity of free PCs and protected the death of human kidney proximal tubule (HK-2) cells induced by cisplatin. Further mechanism studies showed that FU/PCs NPs protected the mitochondrial damage induced by cisplatin, activated mitophagy, inhibited the release of mitochondrial DNA (mtDNA), and inhibited the cGAS/STING signal pathway. The in vivo results also indicated that FU/PCs NPs protected cisplatin-induced AKI, including inhibiting the increase of blood urea nitrogen (BUN) and serum creatinine (SCr) levels induced by cisplatin. The mechanism studies confirmed that cisplatin induced an increase in the expression of mitophagy-related protein Pink/Pakrin, mitochondrial mtDNA release and cGAS/STING expression in mice kidney tissues. Pre-administration of FU/PCs NPs further activated mitophagy, as well as inhibiting mtDNA release and cGAS/STING expression. In conclusion, our research proved the role of mitophagy-mtDNA-cGAS/STING signal was involved in cisplatin-induced AKI.

Silk fibroin peptide self-assembled nanofibers delivered naringenin to alleviate cisplatin-induced acute kidney injury by inhibiting mtDNA-cGAS-STING pathway.

Silk fibroin (SF) has excellent biocompatibility and biodegradability as a biomaterial. The purity and molecular weight distribution of silk fibroin peptide (SFP) make it more suitable for medical application. In this study, SFP nanofibers (molecular weight ∼30kD) were prepared through CaCl2/H2O/C2H5OH solution decomposition and dialysis, and adsorbed naringenin (NGN) to obtain SFP/NGN NFs. In vitro results showed that SFP/NGN NFs increased the antioxidant activity of NGN and protected HK-2 cells from cisplatin-induced damage. In vivo results also showed that SFP/NGN NFs protected mice from cisplatin-induced acute kidney injury (AKI). The mechanism results showed that cisplatin induced mitochondrial damage, as well as increased mitophagy and mtDNA release, which activated the cGAS-STING pathway and induced the expression of inflammatory factors such as IL-6 and TNF-α. Interestingly, SFP/NGN NFs further activated mitophagy and inhibited mtDNA release and cGAS-STING pathway. Demonstrated that mitophagy-mtDNA-cGAS-STING signal axis was involved in the kidney protection mechanism of SFP/NGN NFs. In conclusion, our study confirmed that SFP/NGN NFs are candidates for protection of cisplatin-induced AKI, which is worthy of further study.

Pterostilbene-Loaded Soluplus/Poloxamer 188 Mixed Micelles for Protection against Acetaminophen-Induced Acute Liver Injury.

Excessive acetaminophen (APAP) induces excess reactive oxygen species (ROS), leading to liver damage. Pterostilbene (PTE) has excellent antioxidant and anti-inflammatory activities, but poor solubility limits its biological activity. In this study, we prepared PTE-loaded Soluplus/poloxamer 188 mixed micelles (PTE-MMs), and the protective mechanism against APAP-induced liver injury was investigated. In vitro results showed that PTE-MMs protected H2O2-induced HepG2 cell proliferation inhibition, ROS accumulation, and mitochondrial membrane potential destruction. Immunofluorescence results indicated that PTE-MMs significantly inhibited H2O2-induced DNA damage and cGAS-STING pathway activation. For in vivo protection studies, PTE-MMs (25 and 50 mg/kg) were administered orally for 5 days, followed by APAP (300 mg/kg). The results showed that APAP significantly induced injury in liver histopathology as well as an increase in serum aspartate aminotransferase and alanine aminotransferase levels. Moreover, the above characteristics of APAP-induced acute liver injury were inhibited by PTE-MMs. In addition, APAP-induced changes in the activities of antioxidant enzymes such as SOD and GSH in liver tissue were also inhibited by PTE-MMs. Immunohistochemical results showed that PTE-MMs inhibited APAP-induced DNA damage and cGAS-STING pathway activation in liver tissues. For in vivo therapeutic effect study, mice were first given APAP (300 mg/kg), followed by oral administration of PTE-MMs (50 mg/kg) for 3 days. The results showed that PTE-MMs exhibited promising therapeutic effects on APAP-induced acute liver injury. In conclusion, our study shows that the Soluplus/poloxamer 188 MM system has the potential to enhance the biological activity of PTE in the protection and therapeutic of liver injury.

Myricetin-Loaded Nanomicelles Protect against Cisplatin-Induced Acute Kidney Injury by Inhibiting the DNA Damage-cGAS-STING Signaling Pathway.

Acute kidney injury (AKI) is the most common side effect of the anti-cancer drug cisplatin, and currently, no effective preventive measures are available in clinical practice. Oxidative stress and DNA damage mechanisms may be involved in cisplatin-induced AKI. In this study, we prepared Kolliphor HS15-based myricetin-loaded (HS15-Myr) nanomicelles and explored the mechanism of protection against cisplatin-induced AKI. In vitro results showed that the HS15-Myr nanomicelles enhanced the antioxidant activity of myricetin (Myr) and inhibited cisplatin-induced proliferation inhibition of HK-2 cells. Moreover, the HS15-Myr nanomicelles inhibited cisplatin-induced reactive oxygen species accumulation, mitochondrial membrane potential reduction, and DNA damage, which might be related to the inhibition of the cyclic GMP-AMP synthase (cGAS)─stimulating interferon gene (STING) signaling pathway. In vivo results in mice showed that the significant reductions in body weight and renal indices and the increased blood urea nitrogen and serum creatinine levels induced by cisplatin could be significantly reversed by pretreating with the HS15-Myr nanomicelles. Furthermore, nanomicelle pretreatment significantly altered the activities of antioxidant enzymes (e.g., GSH, MDA, and SOD) induced by cisplatin. In addition, cisplatin-induced inflammatory responses in mouse kidney tissue were found to be inhibited by pretreatment with HS15-Myr nanomicelles, such as IL-1ß and TNF-α expression. The nanomicelles also significantly inhibited cisplatin-induced activation of the DNA damage-cGAS-STING pathway in kidney tissues. Together, our findings suggest that Myr-loaded nanomicelles are potential nephroprotective drugs.

The Complex of Phycobiliproteins, Fucoxanthin, and Krill Oil Ameliorates Obesity through Modulation of Lipid Metabolism and Antioxidants in Obese Rats.

Phycobiliproteins, fucoxanthin, and krill oil are natural marine products with excellent activities. In the study, we prepared the complex of phycobiliproteins, fucoxanthin, and krill oil (PFK) and assessed the anti-obesity, lipid-lowering, and antioxidant activities in high-fat diet rats. The results showed that the rats significantly and safely reduced body weight gain and regulated serum biochemical parameters at 50 mg/kg phycobiliproteins, 10 mg/kg fucoxanthin, and 100 mg/kg krill oil. Furthermore, the molecular mechanism study suggested that the complex of PFK confined the enzyme activities of lipid synthesis and enhanced antioxidant activity to improve obesity indirectly. The conclusions demonstrated that the complex of PFK has potent anti-obesity and hypolipidemic effects which have potential use as a natural and healthy food and medicine for anti-obesity and lowering blood lipids in the future.

Fucoidan-ferulic acid nanoparticles alleviate cisplatin-induced acute kidney injury by inhibiting the cGAS-STING pathway.

Fucoidan (FU) is a natural sulfated polysaccharide with certain biological activity and has been shown to be an excellent nano-delivery material. In this study, ferulic acid (FA)-loaded FU nanoparticles (FA/FU NPs) were prepared and their nephroprotective mechanism was investigated. With a particle size of 158.6 ± 4.5 nm, FA/FU NPs increased the antioxidant activity of FA in vitro, possibly related to the increased dispersity of FA. In vitro results demonstrated that FA/FU NPs significantly protected human renal proximal tubule (HK-2) cells from cisplatin-induced damage, possibly by suppressing cisplatin-induced DNA damage and activating the cGAS-STING pathway. Furthermore, in vivo experiments confirmed that FA/FU NPs protected mice from cisplatin-induced acute kidney injury (AKI). Mechanistic studies confirmed that FA/FU NPs exerted nephroprotective effects by reducing MDA activity and increasing GSH and SOD activity. Our results demonstrated the potential of FU for delivering poorly soluble drug FA and protecting against cisplatin-induced AKI.

Aramid Nanofibers-Reinforced Rhein Fibrous Hydrogels as Antibacterial and Anti-Inflammatory Burn Wound Dressings.

Burn injuries are one of the most devastating traumas. The development of polymer-based hydrogel dressings to prevent bacterial infection and accelerate burn wound healing is continuously desired. Mechanical strong hydrogels that encapsulated antibacterial drugs have gained increasing attention. Herein, aramid nanofibers (ANFs)-reinforced rhein fibrous hydrogels (ANFs/Rhein) were fabricated through a one-pot procedure to serve as a possible treatment for the Staphylococcus aureus-infected burn wound. ANFs preserved the highly aligned backbones and the mechanical properties of Kevlar, and its combination with an antibacterial drug rhein produced a composite hydrogel that possesses favorable physicochemical properties including appropriate mechanical strength, high water holding capacity, satisfactory antibacterial efficiency, and excellent biocompatibility. As wound dressings, ANFs/Rhein hydrogels provided a moist environment for the wound site and released antibacterial drugs continuously to improve the wound healing rate by efficiently restraining bacterial infection, reducing inflammation, enhancing collagen deposition, and promoting the formation of blood vessels, in this way to offer a potential treatment strategy for bacteria-associated burn wound healing.

Collagen­binding vascular endothelial growth factor (CBD­VEGF) promotes liver regeneration in murine partial hepatectomy.

Liver regeneration is a complex process that needs orchestration of multiple nonparenchymal cells including sinusoid endothelial cells. Vascular endothelial growth factor (VEGF) serves a crucial role in angiogenesis and liver regeneration. However, the lack of an high­efficiency delivery system target to the injured site reduces the local therapeutic efficacy of VEGF. In our previous study, collagen binding VEGF (CBD­VEGF) was established by fusing collagen binding domain (CBD) into the N­terminal of native VEGF and improved cardiac function after myocardial infraction. The present study investigated the therapeutic effect of CBD­VEGF on liver regeneration by a mouse model of partial hepatectomy. After injection through portal vein following 2/3 hepatectomy, CBD­VEGF was largely retained in the hepatic extracellular matrix for 48 h. Furthermore, CBD­VEGF application significantly promoted sinusoidal regeneration and remodeling in remanent liver tissue 48 h after hepatectomy. In addition, CBD­VEGF treatment significantly enhanced the proliferation of hepatocytes at 2 and 3 days post­surgery compared with native VEGF, concomitant with attenuated liver injury. In conclusion, these results demonstrated that CBD­VEGF could be a promising therapeutic strategy for liver regeneration.

Hypoxic bone marrow mesenchymal stromal cells-derived exosomal miR-182-5p promotes liver regeneration via FOXO1-mediated macrophage polarization.

Mesenchymal stromal cells (MSCs) are attractive candidates for treating hepatic disorders given their potential to enhance liver regeneration and function. The paracrine paradigm may be involved in the mechanism of MSC-based therapy, and exosomes (Exo) play an important role in this paracrine activity. Hypoxia significantly improves the effectiveness of MSC transplantation. However, whether hypoxia preconditioned MSCs (Hp-MSCs) can enhance liver regeneration, and whether this enhancement is mediated by Exo, are unknown. In this study, mouse bone marrow-derived MSCs (BM-MSCs) and secreted Exo were injected through the tail vein. We report that Hp-MSCs promote liver regeneration after partial hepatectomy in mice through their secreted exosomes. Interestingly, MSC-Exo were concentrated in liver 6 h after administration and mainly taken up by macrophages, but not hepatocytes. Compared with normoxic MSC-Exo (N-Exo), hypoxic MSC-Exo (Hp-Exo) enhanced M2 macrophage polarization both in vivo and in vitro. Microarray analysis revealed significant enrichment of microRNA (miR)-182-5p in Hp-Exo compared with that in N-Exo. In addition, miR-182-5p knockdown partially abolished the beneficial effect of Hp-Exo. Finally, Hp-MSC-derived exosomal miR-182-5p inhibited theprotein expression of forkhead box transcription factor 1 (FOXO1) in macrophages, which inhibited toll-like receptor 4 (TLR4) expression and subsequently induced an anti-inflammatory response. These results highlight the therapeutic potential of Hp-Exo in liver regeneration and suggest that miR-182-5p from Hp-Exo facilitates macrophage polarization during liver regeneration by modulating the FOXO1/TLR4 signaling pathway.

Baicalein-loaded silk fibroin peptide nanofibers protect against cisplatin-induced acute kidney injury: Fabrication, characterization and mechanism.

Silk fibroin (SF) is a natural polymeric biomaterial widely used in the preparation of drug delivery systems. Herein, silk fibroin peptide (SFP) was self-assembled into nanofibers, encapsulated a poorly water-soluble drug baicalein (SFP/BA NFs), and then used to protect against cisplatin-induced acute kidney injury (AKI). Specifically, the SFP/BA NFs significantly enhanced the aqueous dispersity, storage stability, and in vitro antioxidant activity of BA. SFP/BA NFs increased the drug uptake and localization to mitochondria. In vitro results demonstrated that SFP/BA NFs can relieve the cisplatin-induced HK-2 cell damage, and inhibit the cisplatin-induced accumulation of reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) disruption. Mechanism studies demonstrated that SFP/BA NFs may exert nephroprotective effects by inhibiting both the cisplatin-induced DNA damage and the cGAS/STING pathway activation. In vivo results showed that cisplatin treatment resulted in decreased body weight, increased serum creatinine (SCr), and increased blood urea nitrogen (BUN) levels, while SFP/BA NFs reversed the above symptoms. Furthermore, SFP/BA NFs reversed the cisplatin-induced abnormal changes of antioxidant enzymes (e.g., SOD and GSH), and inhibited the cisplatin-induced DNA damage as well as the activation of cGAS/TING. Above all, our results revealed the potential of SFP/BA NFs to protect against cisplatin-induced AKI.

Collagen-binding fibroblast growth factor ameliorates liver fibrosis in murine bile duct ligation injury.

Cholestatic liver injury, characterized by liver fibrosis, has increasingly become a global health problem, with no effective treatment available. Hepatic stellate cells (HSCs) differentiate into myofibroblasts, leading to excessive deposition of the extracellular matrix (ECM), which is a feature of liver fibrosis. Basic fibroblast growth factor (bFGF) has proven antifibrotic effects in chronic liver disease; however, the lack of an effective delivery system to the injury site reduces its therapeutic efficacy. The aim of this study was to assess the therapeutic effect of collagen-binding bFGF (CBD-bFGF) for the treatment of liver fibrosis in a murine bile duct ligation (BDL) model. We found that CBD-bFGF treatment significantly alleviated liver injury in the early phase of BDL injury, and was associated with decreased necroptotic cell death and inflammatory response. Moreover, CBD-bFGF had enhanced therapeutic effects for liver fibrosis on day 7 after surgery compared to those obtained with native bFGF treatment. In vitro, CBD-bFGF treatment notably inhibited TGF-ß1-induced LX-2 cell activation, migration, and contraction compared with native bFGF. In conclusion, CBD-bFGF may be a promising treatment for hepatic fibrosis.

Silk sericin stabilized proanthocyanidins for synergetic alleviation of ulcerative colitis.

Silk sericin (SS) has become a noticeable drug nanocarrier due to its excellent biocompatibility and bioactivity. To further extend the application of SS, a facile one-step process was constructed to fabricate SS-stabilized-drug composites. Various insoluble drugs can be encapsulated into SS with high loading amount, and showed good dispersity in aqueous solution. For example, proanthocyanidins (PAC), a natural polyphenol with initial antioxidant and anti-inflammatory effects, can be loaded on SS to form SS/PAC composites. The SS/PAC can disperse uniformly in aqueous solution with an average particle diameter of ~136 nm, and showed high drug loading amount of 1767 mg/g. The SS/PAC exhibited high antioxidant efficiency and excellent biocompatibility (non-irritant, non-hemolysis, and non-cytotoxicity), could remarkably alleviate the symptoms of dextran sulfate sodium-induced ulcerative colitis by decreasing the disease activity index scores, inhibiting the shortening of colon length, regulating oxidative stress, suppressing inflammation, and reversing the histopathological injuries. This work provides a simple method to fabricate SS-stabilized-drug composites, promises high potential in therapeutic and pharmaceutical applications.

The NLRP3 Activation in Infiltrating Macrophages Contributes to Corneal Fibrosis by Inducing TGF-ß1 Expression in the Corneal Epithelium.

Purpose: To explore the effect and mechanism of NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3) inflammasomes on corneal fibrosis. Methods: The wild-type, NLRP3 knockout (KO), and myeloid cell-specific NLRP3 KO (NLRP3 Lyz-KO) C57 mice were used to establish a corneal scarring model. NLRP3 inhibitor, IL-1ß neutralizing antibody, and an IL-1R antagonist were used to investigate the role of NLRP3 and IL-1ß in corneal fibrosis. The expression of the NLRP3 signaling pathway related proteins, alpha-smooth muscle actin, TGF-ß was determined by quantitative real-time polymerase chain reaction, Western blotting, and immunofluorescence staining. Flow cytometry was used to detect the infiltration of macrophages during corneal fibrosis. Results: The components of the NLRP3 inflammasomes were elevated and activated during corneal scarring. Additionally, genetic or chemical-mediated blocking of NLRP3 as well as IL-1ß significantly alleviated corneal fibrosis. Moreover, neutrophil (CD45+Ly6G+) and macrophage (CD45+ F4/80+) accumulation increased in the cornea during the progression of corneal fibrosis. Intriguingly, the increased concentrations of NLRP3 and IL-1ß were prominently colocalized with the infiltrating F4/80+ macrophages. Expectedly, NLRP3 Lyz-KO mice exhibited a marked decrease in their corneal fibrosis symptoms. Mechanistically, the activation of IL-1ß or macrophage NLRP3 stimulated the expression of TGF-ß1 in the corneal epithelial cells, whereas an NLRP3 deficiency decreased its expression in the corneal epithelium. Conclusions: These observations revealed that the NLRP3 inflammasome activation in infiltrating macrophages contributes to corneal fibrosis by regulating corneal epithelial TGF-ß1 expression. Targeting the NLRP3 inflammasome might be a promising strategy for the treatment of corneal scarring.

Corneal Epithelium-Derived Netrin-1 Alleviates Dry Eye Disease via Regulating Dendritic Cell Activation.

Purpose: To investigate the expression of corneal epithelium-derived netrin-1 (NTN-1) and its immunoregulatory function in dry eye disease (DED) using a DED mouse model. Methods: We generated DED mouse models with desiccating stress under scopolamine treatment. RNA sequencing was performed to identify differentially expressed genes (DEGs) in the corneal epithelium of DED mice. NTN-1 expression was analyzed via real-time PCR, immunofluorescence staining, and immunoblotting. The DED mice were then treated with recombinant NTN-1 or neutralizing antibodies to investigate the severity of the disease, dendritic cell (DC) activation, and inflammatory cytokine expression. Results: A total of 347 DEGs (292 upregulated and 55 downregulated) were identified in the corneal epithelium of DED mice: corneal epithelium-derived NTN-1 expression was significantly decreased in DED mice compared to that in control mice. Topical recombinant NTN-1 application alleviated the severity of the disease, accompanied by restoration of tear secretion and goblet cell density. In addition, NTN-1 decreased the number of DCs, inhibited the activation of the DCs and Th17 cells, and reduced the expression of inflammatory factors in DED mice. In contrast, blocking endogenous NTN-1 activity with an anti-NTN-1 antibody aggravated the disease, enhanced DC activation, and upregulated the inflammatory factors in the conjunctivae of DED mice. Conclusions: We identified decreased NTN-1 expression in the corneal epithelium of DED mice. Our findings elucidate the role of NTN-1 in alleviating DED and impeding DC activation, thereby indicating its therapeutic potential in suppressing ocular inflammation in DED.

Copolymer Micelle-administered Melatonin Ameliorates Hyperosmolarity-induced Ocular Surface Damage through Regulating PINK1-mediated Mitophagy.

PURPOSE: To investigate the role and mechanism of melatonin-loaded polymer polyvinyl caprolactam-polyvinyl acetate-polyethyleneglycol graft copolymer micelles (Mel-Mic) in dry eye disease (DED). METHODS: In vitro, the apoptosis and reactive oxygen species (ROS) generation in human corneal epithelial cells (HCECs) were analyzed by immunostaining and flow cytometry. The effect of Mel-Mic on autophagy and mitophagy was evaluated by immunostaining and western blots. PINK1 knockdown was analyzed by small interfering RNA. In vivo, sodium fluorescein staining, tear secretion test, and periodic acid-Schiff staining were used to determine whether Mel-Mic can alleviate the severity of DED. Small molecule antagonists were pretreated to investigate whether melatonin type 1 and/or 2 receptors (MT1/MT2) mediate the effects of Mel-Mic. RESULTS: Mel-Mic improved the solubility and biological activities of Mel in aqueous solutions. Treatment with Mel-Mic decreased the apoptosis of HCECs exposed to hyperosmotic medium, accompanied by downregulation of cleaved Caspase-3 and upregulation of Bcl-2. In addition, Mel-Mic application suppressed ROS overproduction, rescued mitochondrial function, and decreased the level of oxidative stress associated biomarkers (COX-2 and 4-HNE) in HCECs. Interestingly, HCECs treated with Mel-Mic exhibited increased levels of mitophagy markers (PINK1, PARKIN, Beclin 1, and LC3B) and restored impaired mitophagic flux under hyperosmolarity. While PINK1 knockdown largely abolished its protective effects. In vivo, compared to vehicle group, topical Mel-Mic-solution-treated mice showed significantly improved clinical parameters, increased tear production, and decreased goblet cells loss in a dose-dependent manner. Also, transmission electron microscopic assay revealed increased autophagosome number in the corneal epithelium of Mel-Mic group. Moreover, luzindole, a nonselective MT1/MT2 antagonist, but not 4-P-PDOT, a selective MT2 antagonist, blocked the protective effect of Mel-Mic. CONCLUSIONS: Our findings demonstrated that Mel-Mic ameliorates hyperosmolarity-induced ocular surface damage via PINK1-mediated mitophagy and may represent an effective treatment for DED possibly through acting MT1 receptor.