Preparation and in vivo and ex vivo studies of sirolimus nano-in-situ gel ophthalmic formulation.

Sirolimus (SR) is a macrolide with antifungal and antitumor immunosuppressant properties, classified as a selective inhibitor of mammalian target of rapamycin (mTOR). In this study, an ionic in situ gel of SR (SR-SUS-ISG) was formulated using gellan gum, exhibiting stability regardless of temperature and pH variations, causing minimal irritation. Harnessing the physiological conditions of the eye, SR-SUS-ISG underwent gelation upon contact with ions, increasing drug viscosity and prolonging retention on the ocular surface. Concurrently, SR-SUS-ISG displayed favorable shear dilution properties, reducing viscosity at ambient temperature, enhancing fluidity, and facilitating convenient packaging and transport. Biocompatibility assessments on both human corneal epithelial cells and rabbit eyes demonstrated that SR-SUS-ISG could well be tolerated. Pharmacokinetic investigations in rabbit ocular aqueous humor revealed sustained release, improved corneal penetration, and enhanced bioavailability. Additionally, in a rat corneal alkali burn model, SR-SUS-ISG exhibited inhibitory effects on corneal neovascularization, associated with decreased levels of the inflammatory factors VEGF and MMPs. These findings suggested that SR-SUS-ISG held promise as an effective ocular drug delivery system.

Precise nanodrug delivery systems with cell-specific targeting for ALI/ARDS treatment.

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are common acute and critical diseases in clinics and have no effective treatment to date. With the concept of "precision medicine", research into the precise drug delivery of therapeutic and diagnostic drugs has become a frontier in nanomedicine research and has entered the era of design of precise nanodrug delivery systems (NDDSs) with cell-specific targeting. Owing to the distinctive characteristics of ALI/ARDS, designing NDDSs for specific focal sites is an important strategy for changing drug distribution in the body and specifically increasing drug concentration at target sites while decreasing drug concentration at non-target sites. This strategy enhances drug efficacy, reduces adverse reactions, and ensures accurate nano-targeted treatment. On the basis of the characteristics of pathological ALI/ARDS microenvironments, this paper reviews NDDSs targeting vascular endothelial cells, neutrophils, alveolar macrophages, and alveolar epithelial cells to provide reference for designing accurate NDDSs for ALI/ARDS and novel insights into targeted treatments for ALI/ARDS.

A study on puerarin in situ gel eye drops: Formulation optimization and pharmacokinetics on rabbits by microdialysis.

Puerarin (PUE), an isoflavonoid isolated from Pueraria lobata (Willd) Ohwi root, is a ß-adrenergic receptor inhibitor used in treating glaucoma. The concentration range of gellan gum was determined based on the formulation viscosity and gelling capacity. PVP-K30 and gellan gum were used as variables, with the viscosity of formulation: STF = 40: 21, the 4 h permeation rate of rabbit isolated sclera, and 2 h in vitro release rate as response values. The JMP software was used to optimize the results, presenting that gellan gum was the main factor influencing viscosity. The in vitro release and permeation rate were primarily influenced by PVP-K30. The optimal prescription was 0.45% gellan gum and 6.0% PVP-K30. The in vitro release and permeation characteristics of puerarin in situ gel (PUE-ISG) were investigated using PUE solution as a control. The dialysis bag method results indicated that the release of the solution group leveled off after 4 h, while the PUE-ISG group had been continuously releasing. However, the cumulative release rates of the two were no longer significantly different at 10 h. The cumulative permeation rates of the ISG and solution groups were not significantly different (P > 0.05) in the rabbit isolated sclera. The apparent permeability Papp and steady-state flux Jss of PUE-ISG were 0.950 ± 0.059 cm/h and 9.504 ± 0.587 mg·(cm·h), respectively. A sensitive and stable HPLC-MS/MS analytical method for quantifying aqueous humor concentrations of PUE was validated. A microdialysis technique was successfully used in the aqueous humor pharmacokinetics study to sample aqueous humor from rabbit eye continuously. The results revealed that PUE-ISG significantly increased the drug concentration in the aqueous humor, with Cmax and AUC(0-t) 3.77 and 4.40 times higher than those of the solution group, respectively. Tmax was also significantly prolonged, indicating good prospects for clinical application. The developed PUE-ISG preparation has the characteristics of rapid drug release and sustained permeation, and increase the drug concentration in aqueous humor, with all inactive ingredients remaining within the maximum allowable limits recommended by the FDA guideline.

Research progress of ophthalmic preparations of immunosuppressants.

Immune ophthalmopathy is a collection of autoimmune eye diseases. Immunosuppressants are drugs that can inhibit the body's immune response. Considering drug side effects such as hepatorenal toxicity and the unique structure of the eye, incorporating immunosuppressants into ophthalmic nanodrug delivery systems, such as microparticles, nanoparticles, liposomes, micelles, implants, and in situ gels, has the advantages of improving solubility, increasing bioavailability, high eye-target specificity, and reducing side effects. This study reviews recent research and applications of this aspect to provide a reference for the development of an ophthalmic drug delivery system.

Preparation and Characterization of Ion-Sensitive Brimonidine Tartrate In Situ Gel for Ocular Delivery.

Brimonidine tartrate (BRT) is a highly selective α2 adrenergic receptor agonist as treatment for patients with open angle glaucoma and high intraocular pressure. The objective of this study was to formulate an ophthalmic ion-sensitive in situ gel (ISG) of BRT to increase the retention time of the drug and its bioavailability. The optimum formulation of 2 mg/mL BRT-ISG was obtained with 0.45% gellan gum as the gel matrix. In vitro release results showed that the water-soluble drug bromonidine tartrate in ocular in situ gels exhibited a high burst effect and fast release in solution. The results of dialysis membrane permeation showed that there was a significant difference between the commercially available and BRT-ISG groups after 45 min. The results of the pre-corneal retention study indicated that gellan gum can effectively prolong ocular surface retention. Preliminary stability results showed that it should be stored in a cool and dark place, and the formulation under long-term preservation can be basically stable. The pharmacokinetic study of the BRT-ISG in the anterior chamber of the rabbit eye was studied by microdialysis technique, and microdialysis samples were analyzed by LC-MS/MS. The pharmacokinetic study showed that the BRT-ISG reached Cmax (8.16 mg/L) at 93 min after administration, which was 2.7 times that of the BRT eye drops, and the AUC(0-t) (1397.08 mg·min/L) was 3.4 times that of the BRT eye drops. The optimal prescription can prolong the retention time of BRT in front of the cornea and significantly improve the bioavailability of BRT in the eye. Combined with the results of in vitro release, permeation and pre-corneal retention studies, the improvement of BRT-ISG bioavailability in rabbit eyes was found to be mainly due to the retention effect after the mixture of ISG and tears.

Investigation on Potential Biomarkers and Immune-Related Mechanisms of Ischemic Stroke.

BACKGROUND: This study aimed to reveal the detailed immune-related mechanisms underlying ischemic stroke (IS) and identify new immune-associated biomarkers for clinical management. METHODS: Differentially expressed genes (DEGs) between IS samples and normal controls were identified using the GSE16561 dataset. The feature genes of the immune cells were investigated using the GSE72642 dataset. Weighted correlation network analysis (WGCNA) was performed to reveal module genes, followed by an investigation of common DEGs and a functional enrichment analysis. Potential biomarkers were identified based on hub genes in protein-protein interaction networks and WGCNA. Finally, GSE158312 was used for biomarker verification. RESULTS: In total, 1230 DEGs were identified between the IS samples and normal controls. Seven clinically significant modules were identified using WGCNA. The yellow module genes were positively correlated with polymorphonuclear cells (PMNC), whereas the brown module genes were positively correlated with CD4+ T cells. Eight genes were selected as hub genes. These genes are mainly involved in functions such as the innate immune response. Upregulated TLR2 and ARG1 levels were significantly different between the two groups in the verification dataset. CONCLUSIONS: Our findings suggest ARG1 and TLR2 as novel biomarkers for IS. Upregulated TLR2 might play a role in IS development by participating in the innate immune response function.

FYU-Net: A Cascading Segmentation Network for Kidney Tumor Medical Imaging.

Automated segmentation of renal tumors is essential for the diagnostic evaluation of kidney cancer. However, renal tumor volume is generally small compared with the volume of the kidney and is irregularly distributed; moreover, the location and shape of renal tumors are highly variable, making the segmentation task extremely challenging. To solve the aforementioned problems, a cascaded segmentation model (FYU-Net) for computed tomography (CT) images is proposed in this paper to achieve automatic kidney tumor segmentation. The proposed model involves two main steps. In the first step, a fast scan of the kidney CT data is performed using a localization network to find slices containing tumors, and coarse segmentation is performed simultaneously. In the second step, a segmentation framework embedded with the feature pyramid network module is employed to finely segment kidney tumors. By building a feature pyramid structure, targets of different sizes are distributed to be detected on different feature layers to extract richer feature information. In addition, the top-down structure allows the information of the higher-level feature maps to be transferred to the lower-level feature maps, enhancing the semantic information of the lower-level feature maps. Comparative experiments were conducted on the Kidney PArsing Challenge 2022 public dataset; the average Jaccard coefficient and average Dice coefficient of tumor structure segmentation were more than 70.73% and more than 82.85%, respectively. The results demonstrate the effectiveness of the proposed model for kidney tumor segmentation.

Gut microbiota-mitochondrial inter-talk in non-alcoholic fatty liver disease.

The increasing prevalence of non-alcoholic fatty liver disease (NAFLD), which is a progressive disease, has exerted huge a healthcare burden worldwide. New investigations have suggested that the gut microbiota closely participates in the progression of NAFLD through the gut-liver axis or gut-brain-liver axis. The composition of the microbiota can be altered by multiple factors, primarily dietary style, nutritional supplements, or exercise. Recent evidence has revealed that gut microbiota is involved in mitochondrial biogenesis and energy metabolism in the liver by regulating crucial transcription factors, enzymes, or genes. Moreover, microbiota metabolites can also affect mitochondrial oxidative stress function and swallow formation, subsequently controlling the inflammatory response and regulating the levels of inflammatory cytokines, which are the predominant regulators of NAFLD. This review focuses on the changes in the composition of the gut microbiota and metabolites as well as the cross-talk between gut microbiota and mitochondrial function. We thus aim to comprehensively explore the potential mechanisms of gut microbiota in NAFLD and potential therapeutic strategies targeting NAFLD management.

HIF­1α protects PC12 cells from OGD/R­induced cell injury by regulating autophagy flux through the miR­20a­5p/KIF5A axis.

Hypoxia inducible factor 1α (HIF­1α) has been reported to play a key role in protecting neurons from ischaemic injury. However, the exact molecular mechanisms remain largely unclear. PC12 cells were exposed to oxygen glucose deprivation/reoxygenation (OGD/R) conditions to mimic ischaemic injury in vitro. The expression of the HIF­1α mRNA, miR­20a­5p, and kinesin family member 5A (KIF5A) mRNA was tested using qRT-PCR. Levels of the HIF­1α, LC3I/II, P62, LAMP2, cathepsin B (CTSB) and KIF5A proteins were determined using western blotting. The CCK­8 assay was conducted to assess PC12 cell viability. DQ­Red­BSA and LysoSensor Green DND­189 dyes were employed to measure the proteolytic activity and pH of lysosomes, respectively. The interaction between miR­20a­5p and HIF­1α or KIF5A was verified by performing chromatin immunoprecipitation (ChIP) and/or dual­luciferase reporter assays. TUNEL staining was adopted to assess PC12 cell death. GFP­LC3 and RFP­GFP­LC3 probes were used to examine the autophagy status and autophagy flux of PC12 cells. A rat middle cerebral artery occlusion­reperfusion (MCAO/R) model was established to investigate the role of the HIF­1α/miR­20a­5p/KIF5A axis in ischaemic stroke in vivo. OGD/R exposure initiated PC12 cell autophagy and injury. HIF­1α expression was substantially increased in PC12 cells after OGD/R exposure. Overexpression of HIF­1α reversed the effects of OGD/R on reducing cell viability, blocking autophagy flux and inducing lysosome dysfunction. These rescue effects of HIF­1α depended on KIF5A. HIF­1α negatively regulated miR­20a­5p expression by targeting its promoter region, and miR­20a­5p directly targeted and negatively regulated the KIF5A mRNA. Overexpression of miR­20a­5p abolished the effects of HIF­1α on rescuing OGD/R­induced injury in PC12 cells. The effects of the HIF­1α/miR­20a­5p/KIF5A axis were verified in MCAO/R rats. HIF­1α protects PC12 cells from OGD/R­induced cell injury by regulating autophagy flux through the miR­20a­5p/KIF5A axis.

LncRNA nuclear-enriched abundant transcript 1 aggravates cerebral ischemia/reperfusion injury through activating early growth response-1/RNA binding motif protein 25 axis.

Ischemic stroke is a major global health issue. Ischemia and subsequent reperfusion results in stroke-related brain injury. Previous studies have demonstrated that nuclear-enriched abundant transcript 1 (NEATa and early growth response 1 (EGR1) are involved in ischemia reperfusion (IR) injury). In this study, we aimed to explore the roles of NEAT1/EGR1 axis as well as its downstream effector RNA binding motif protein 25 (RBM25) in cerebral IR injury. Oxygen-glucose deprivation/reperfusion (OGD/R) and middle cerebral artery occlusion (MCAO) were used to establish in vitro and in vivo models of cerebral IR injury, respectively. According to our data, NEAT1, EGR1, and RBM25 levels were elevated in OGD/R-exposed SK-N-SH and SH-SY5Y cells and cerebral cortex of MCAO mice. NEAT1, EGR1, or RBM25 knockdown effectively reduced infarct volumes and apoptosis, and improved neurological function. Mechanistically, NEAT1 directly interacted with EGR1, which restrained WW domain containing E3 ubiquitin protein ligase 1 (WWP1)-mediated ubiquitination of EGR1 and subsequently caused EGR1 accumulation. EGR1 bound to RBM25 promoter and transcriptionally activated RBM25. Rescue experiments indicated that RBM25 overexpression abolished the therapeutic effects of NEAT1 knockdown. In conclusion, this work identified a novel NEAT1/EGR1/RBM25 axis in potentiating brain injury after IR insults, suggesting a potential therapeutic target for ischemic stroke.

LncRNA RMRP accelerates autophagy-mediated neurons apoptosis through miR-3142/TRIB3 signaling axis in alzheimer's disease.

BACKGROUND: Alzheimer's disease (AD) is a major neurodegenerative disorder. The functions of lncRNA RMRP have been characterized mainly in various human cancers. However, the functional network of RMRP in AD progression remains unknown. METHODS: Human serum samples, AD transgenic (Tg) mice as well as SH-SY5Y cells were used in this study. The RNA expression patterns of RMRP, miR-3142 and TRIB3 were assessed by quantitative real-time PCR (qRT-PCR). Levels of apoptosis- or autophagy-associated biomarkers and TRIB3 level were evaluated using immunohistochemistry (IHC), western blotting or immunofluorescence assays, respectively. Bioinformatics methods and luciferase assays were used to predict and validate the interactions among RMRP, miR-3142, and TRIB3. Flow cytometry, TUNEL staining and EdU assays were used to examine the apoptosis and proliferation of neurons, respectively. RESULTS: The elevated RMRP and TRIB3 expressions and activation of autophagy were observed in AD. Knockdown of RMRP restrained neuronal apoptosis and autophagy activation in vitro and in vivo. Interestingly, TRIB3 overexpression reversed the biological effects of RMRP silencing on Aß1-42-induced cell apoptosis and autophagy. Further mechanistic analysis showed RMRP acted as a sponge of miR-3142 to elevate TRIB3 level. CONCLUSION: These data illustrated that knockdown of RMRP inhibited autophagy and apoptosis via regulating miR-3142/TRIB3 axis in AD, suggesting that inhibition of RMRP maybe a therapeutic strategy for AD.

HPLC-MS/MS studies of brimonidine in rabbit aqueous humor by microdialysis.

Aim: The pharmacokinetic study of the brimonidine tartrate in situ gel in the anterior chamber of the rabbit eye was studied by microdialysis technique, and samples were analyzed by HPLC-MS/MS. Materials & methods: It was monitored in ESI mode at transition 291.9→212.0 and 296.0→216.0 for brimonidine and internal standard, respectively. Acetonitrile and 0.1% aqueous formic acid (50:50, v/v) were used as the mobile phase at 0.4 ml/min. Results & conclusion: It showed a good linear correlation between 5 and 5000 ng/ml in microdialysis solution, and the inter- and intra-day precision (relative standard deviation) was less than 4.0%. The pharmacokinetic study showed that the AUC(0-t) of in situ gel was 3.5-times than that of eyedrops, which significantly improve the bioavailability of brimonidine.

Preparation, characterization, and anti-colon cancer activity of oridonin-loaded long-circulating liposomes.

In this study, oridonin-loaded long-circulating liposomes (LC-lipo@ORI) were prepared with the ethanol injection method. Its physicochemical properties and the morphology were characterized, and its stability and release profiles were evaluated. Furthermore, its antitumor effects were studied using two in vitro cell models of colon cancer and two tumor-bearing models in nude mice. The prepared LC-lipo@ORI was quasi-spherical, with a mean particle size of 109.55 ± 2.30 nm. The zeta potential was -1.38 ± 0.21 mV, the encapsulation efficiency was 85.79%±3.25%, and the drug loading was 5.87%±0.21%. In vitro release results showed that the cumulative release rate of LC-lipo@ORI at 12 h was 63.83%. However, ORI dispersion was almost completely released after 12 h. In vitro cytotoxicity results showed that, the inhibiting effects of LC-lipo@ORI on the proliferation of two types of colon cancer cells were apparently higher than those of ORI dispersion, whereas those of the blank carrier were not noticeable. In vivo studies confirmed that, the encapsulation of LC-lipo enhanced the inhibitory effects of ORI on tumor growth. These results indicated that LC-lipo@ORI a promising formulations for colon cancer treatment.

LncRNA MEG3 suppresses PI3K/AKT/mTOR signalling pathway to enhance autophagy and inhibit inflammation in TNF-α-treated keratinocytes and psoriatic mice.

BACKGROUND: Psoriasis is a common chronic inflammatory skin disorder that causes patches of thick red skin and silvery scales and affects 1-3% of the population, which reduces patient's quality of life. Understanding the pathogenesis of psoriasis is crucial for developing novel therapeutic strategies. METHODS: HaCaT and NHEK cells were treated with TNF-α in vitro. A mouse model of psoriasis was established by topical imiquimod application on back skin. LncRNA MEG3 was cloned into the pcDNA3.1 vector and transfected in TNF-α-treated HaCaT and NHEK cells to overexpress its expression. Liposome-encapsulated pcDNA3.1-MEG3 was injected into imiquimod-treated mice via tail vein. RT-qPCR and western blot assays were used to examine the expression of lncRNA MEG3, IL-6, IL-8, IFN-γ, IL-1ß, LC3, Beclin 1, p62, p-p65, p65, NLRP3, p-PI3K, PI3K, p-AKT, AKT, p-mTOR, mTOR respectively. The secretion of IL-6, IL-8, IFN-γ and IL-1ß was determined using ELISA assay. Immunofluorescence and immunohistochemistry methods were performed for analyzing the expression of LC3 and NLRP3 in cells and skin tissues respectively. LY294002 was used to block the PI3K/AKT/mTOR signalling. MTT assay was applied to test the toxicity of LY294002 to HaCaT and NHEK cells. RESULTS: LncRNA MEG3 expression levels were downregulated in TNF-α-treated HaCaT and NHEK cells and skin tissues of psoriatic mice model. TNF-α treatment enhanced inflammation and suppressed autophagy in HaCaT and NHEK cells, which were largely reversed by overexpression of lncRNA MEG3. Autophagy puncta and NLRP3 inflammasome assembly showed the same patterns with the expression of inflammation and autophagy markers in TNF-α-treated HaCaT and NHEK cells with or without lncRNA MEG3 overexpression. TNF-α-induced activation of the PI3K/AKT/mTOR signalling was abolished by lncRNA MEG3 overexpression in HaCaT and NHEK cells. Blocking the PI3K/AKT/mTOR signalling inhibited TNF-α-induced inflammation and restored autophagy level in TNF-α-treated HaCaT and NHEK cells. Overexpression of lncRNA MEG3 suppressed inflammation, promoted autophagy and inhibited the activation of the PI3K/AKT/mTOR signalling in a mouse model of psoriasis. CONCLUSION: LncRNA MEG3 facilitates autophagy and suppresses inflammation in TNF-α-treated keratinocytes and psoriatic mice, which is dependent on the PI3K/AKT/mTOR signalling pathway. Our study enhances the understanding of psoriasis and provides potential therapeutic targets for psoriasis.

pH-sensitive chitosan-deoxycholic acid/alginate nanoparticles for oral insulin delivery.

Oral absorption of peptides/proteins is usually compromised by various gastrointestinal tract barriers. To improve delivery efficiency, chitosan-conjugated deoxycholic acid (CS-DCA) coupled with sodium alginate (ALG) was prepared to load insulin into pH-sensitive nanoparticles. The insulin-loaded chitosan-deoxycholic acid/alginate nanoparticles (CDA NPs) were characterized by size (143.3 ± 10.8 nm), zeta potential (19.5 ± 1.6 mV), entrapment efficiency (61.14 ± 1.67%), and insulin drug loading (3.36 ± 0.09%). The CDA NPs exhibited pH-triggered release characteristics in vitro and protected the wrapped insulin from gastric degradation. Stability of the CDA NPs in enzyme-containing simulated gastrointestinal fluids suggested that the NPs could partially protect the wrapped insulin from enzymatic degradation. Additionally, CS-DCA-modified NPs promoted the permeability of Caco-2 cells and enhanced intracellular absorption of FITC-labeled insulin by 9.4 and 1.2-folds, when compared to insulin solution and unmodified NPs, respectively. The positively charged NPs increased intestinal villi adhesion and enhanced insulin absorption in the intestines of diabetic rat models. Furthermore, the hypoglycemic test showed that CDA NPs prolonged insulin release in vivo and exerted a remarkable hypoglycemic effect on diabetic rats with an oral bioavailability of 15%. In conclusion, CDA NPs is a potential oral insulin delivery system.

[Effects of Biochar and Straw on Soil N2O Emission from a Wheat Maize Rotation System].

The effects of biochar and straw return on soil N2O emissions were studied in the winter wheat-summer maize rotation system of intensively farmed land in North China to provide a theoretical basis for N2O emission reduction and the efficient straw utilization. The experiment included the following four treatments:① Control (CK); ② Biochar application at a rate of 9.0 t·(hm2·a)-1 (C); ③ Straw return (SR); and ④ Straw return plus biochar application at a rate of 9.0 t·(hm2·a)-1 (C+SR). The results showed that in the wheat season, the CK treatment showed a slight decrease in soil N2O emission while the SR and C+SR treatments promoted soil N2O emission by 47.4% and 71.8%, respectively. In the maize-growing season, the CK treatment reduced soil N2O emission by 29.8% while the SR and C+SR treatments increased soil N2O emission by 13.4% and 35.8%, respectively. During the wheat-growing season, the soil water, NH4+-N, and MBN content were the main environmental factors affecting N2O emissions; during the maize-growing season, NO3--N, NH4+-N, and MBC content were the main environmental factors affecting emissions. Based on our results, the application of biochar to cropland is an effective option for mitigating greenhouse gas emissions, whereas direct straw return to fields might not be an effective strategy. More research is now needed to examine the effect of the return of straw of different maturity on N2O emissions.