Along with initiatives to understand the pathophysiology of stroke in detail and to identify neuroprotective targets, cell-stabilizing elements have gained increasing attention. Although cell culture experiments have indicated that tricellulin, α-catenin and microfibrillar-associated protein 5 (MFAP5) contribute to cellular integrity, these elements have not yet been investigated in the ischemic brain. Applying immunofluorescence labeling, this study explored tricellulin, MFAP5 and α-catenin in non-ischemic and ischemic brain areas of mice  and rats (4 h of ischemia), along with collagen IV and fibronectin as vascular and extracellular matrix constituents and microtubule-associated protein 2 (MAP2) and neurofilament light chain (NF-L) as cytoskeletal elements. Immunosignals of tricellulin and notably MFAP5 partially appeared in a fiber-like pattern, and α-catenin appeared more in a dotted pattern. Regional associations with vascular and extracellular constituents were found for tricellulin and α-catenin, particularly in ischemic areas. Due to ischemia, signals of tricellulin, MFAP5 and α-catenin decreased concomitantly with MAP2 and NF-L, whereby MFAP5 provided the most sensitive reaction. For the first time, this study demonstrated ischemia-related alterations in tricellulin, MFAP5 and α-catenin along with the vasculature, extracellular matrix and cytoskeleton. Confirmatory studies are needed, also exploring their role in cellular integrity and the potential for neuroprotective approaches in stroke. Ischemic stroke is characterized by a critical reduction in blood supply for a brain area caused by acute vessel occlusion with various mechanisms, ultimately leading to tissue damage . TreatmeRegarding the cellular consequences of stroke, the neurovascular unit (NVU) has been established as a construct summarizing the local formation of neuronal, glial, and vascular elements ,7,8. AccA novel cell-stabilizing element critically impacting the NVU’s integrity might be tricellulin, a trans-membrane protein that has been found to realize the contact between three adjoining cells, seen in epithelial and non-epithelial cells ,21,22,23From the variety of extracellular filaments potentially contributing to cellular integrity, the family of microfibrillar-associated proteins (MFAPs) has recently turned into focus. MFAPs represent extracellular matrix glycoproteins involved in processes such as assembling elastic microfibrils, remodeling and tissue homeostasis, which was particularly demonstrated for metabolic and cancer diseases . MFAP5 iHowever, tricellulin and MFAP5 have not yet been investigated in the non-ischemic and the ischemic brain along with elements of the NVU and the extracellular matrix. This study thus aimed to explore tricellulin, MFAP and α-catenin in rodent models of focal cerebral ischemia in conjunction with vascular and extracellular constituents, as well as the cytoskeletal elements MAP2 and NF-L. These insights might help to extend the perspective of the NVU and, especially, the knowledge on cell-stabilizing elements, which might serve as a target for neuroprotective strategies in stroke. In mice subjected to 24 h of focal cerebral ischemia, typical alterations in NVU elements were visible especially in the ischemic border zone of the neocortex, thus qualifying for an explorative analysis of cell-stabilizing elements in naive and ischemia-affected brain regions. In detail, slightly increased immunosignals of collagen IV and fibronectin were observed in the ischemic area, representing markers associated with the vasculature and the extracellular matrix A,B. ThesIn brain tissues from mice subjected to 24 h of focal cerebral ischemia, tricellulin was detectable via immunofluorescence labeling, with a signal appearing diffusely arranged and, at least in part, in a fiber-like configuration in non-ischemic areas , whereas the performed inter-hemispheric comparison of signals covering the neocortical border zone  and the associated ischemic region of the neocortex (both p = 0.054) failed to reach statistical significance after careful consideration of performed multiple comparisons. Nevertheless, comparing the most medial area with the most lateral area covering the least affected and the most ischemic region, a significantly reduced tricellulin signal was detected in the most ischemic area (p = 0.012). An inter-hemispheric comparison of signals visualizing MAP2 and NF-L revealed a non-significantly increased signal for NF-L close to the border zone, followed by a significant decrease in both signals along the neocortex starting in the border zone and maintaining in the ischemic region (each p = 0.014). Further, significantly decreased signals of MAP2 and NF-L were found in the ischemic subcortex (each p = 0.014). Regarding the regional arrangement of tricellulin, MAP2 and NF-L, the merged immunofluorescence labeling indicated partial overlapping of fiber-like structures seen in both the non- and the ischemia-affected area  with an even more apparent reduction toward the region affected by ischemia (each p = 0.014). A significantly decreased MFAP5 signal was consistently found when comparing the most naive and most ischemia-affected areas (p = 0.012). Further, a significantly decreased MFAP5 signal was observed in the ischemic subcortex compared to the contralateral hemisphere (p = 0.014). Accompanied by the decreasing immunosignal of MFAP5 along the ischemia-affected neocortex, signals of MAP2 and NF-L decreased significantly , even though in this subset of analyses NF-L was found to decrease to a lesser degree. This observation was also evident for MAP2 and NF-L the ischemic subcortex . Concerning regional arrangements, MFAP5 seemed to overlap with a relevant part of fiber-like structures visualized by MAP2 und NF-L  and maintaining toward the neocortical area affected by ischemia (each p = 0.014). In addition, the ischemic subcortex showed a significantly lowered α-catenin signal (p = 0.014). When comparing the most medial region with the most lateral region analyzed, a significantly decreased α-catenin signal was found in the most ischemic area (p = 0.012). Regarding MAP2 and NF-L, a non-significant increase in the NF-L signal was seen at the medial part of the border zone, whereas toward the ischemia-affected region, a reduction in both signals was observed , which was also evident for the ischemic subcortex . The regional arrangement of α-catenin with MAP2 and NF-L primarily appeared in a complementary manner, as α-catenin with its dotted appearance in the ischemia-affected area did not provide certain evidence of overlaps with both cytoskeletal elements . ,19. 14,1Collectively, this is the first study showing tricellulin, MFAP5 and α-catenin along with vascular, extracellular and cytoskeletal elements in non-ischemic and ischemic areas of the rodent brain. Whereas the immunosignals of tricellulin, MFAP5 and α-catenin mainly appeared with a diffusely arranged pattern, the tricellulin and notably MFAP5 signals partially appeared in a fiber-like pattern, and that of α-catenin appeared in a more dotted pattern. Regional associations with vascular and extracellular elements were partly seen for tricellulin and α-catenin, particularly in regions of focal cerebral ischemia. Along the ischemic neocortex, immunosignals of tricellulin, MFAP5 and α-catenin gradually decreased toward the most affected area concomitantly with altered signals of cytoskeletal elements. Compared with MAP2 and NF-L, MFAP5 was identified as most sensitive, as its signal changed very early with reference to the ischemic lesion. Although these findings need to be confirmed by further studies, the present observations suggest that tricellulin, α-catenin and notably MFAP5 may play a role as cell-stabilizing elements in the brain and may thus represent a target for neuroprotective approaches in stroke.n = 4) or 24 h (n = 6) of ischemia, and rats (n = 4) were sacrificed after 4 h, using isoflurane .Applying an exploratory approach, this study used brains from mice and rats subjected to focal cerebral ischemia to address changes in different vascular, extracellular and cytoskeletal elements as visualized by immunohistochemical techniques. Analyses were based on 10 male C57/BL/6J mice with a mean body weight of 25.4 g, bred by Charles River Laboratories , and 4 male Wister rats with a mean body weight of 289.6 g, also bred by Charles River Laboratories (Sulzfeld). Focal cerebral ischemia was induced as described below. For histochemical investigations, mice were sacrificed after 4 . For the prevention of anesthesia-associated cooling, a thermostatically controlled warming pad with a rectal probe  was used during surgical procedures, and 37 °C was adjusted as the target temperature. Mice and rats received a complex pain medication including lidocaine , meloxicam  and metamizole , respectively. Surgical procedures were generally conducted under anesthesia using about 2–2.5% isoflurane  in a mixture of 70% 493 NThe sufficient induction of focal cerebral ischemia was verified clinically by at least a score of two using Menzies scoring  during tAfter transcardial perfusion with saline and 4% phosphate-buffered paraformaldehyde (PFA), fixed brains from mice and rats were equilibrated with 30% phosphate-buffered sucrose. Forebrains were cut with a freezing microtome , and 10 series of 30 µm-thick sections were collected. Until further processing, brain sections were stored at 4 °C in 0.1 M Tris-buffered saline, with a pH of 7.4 (TBS), containing sodium azide. Immunofluorescence labeling started with rinsing sections in TBS followed by a one-hour antigen retrieval treatment with 60% methanol in water. For blocking non-specific binding sites, 5% normal donkey serum in TBS containing 0.3% Triton X-100 was applied for one hour. Next, sections were processed with different primary and secondary antibodies, as described in In control experiments addressing the validity of visualized structures via immunofluorescence labeling, the primary antibodies were omitted, which resulted in the absence of specific signals.Brain sections, immunochemically stained with Cyanine-labeled secondary antibodies, were screened with an AxioScan.Z1 microscope  equipped with a Colibri7 light source. Images of selected brain regions were taken with the Axiocam 506 (Carl Zeiss) and a Plan-Apochromat objective  with an exposure time of 7 ms for Cy2, 10 ms for Cy3, 5 ms for Cy5 and 2 ms for Hoechst 33342. Images were digitized using the ZEN 2.6 software (Carl Zeiss), and quantifications on immunosignals of tricelluin, MFAP5 and α-catenin were performed with ZEN 3.7 Intellesis software (Carl Zeiss) by measuring fluorescence intensities within defined regions of interests (ROIs). Therefore, 6 ROIs were arranged along the hemisphere that was affected by focal cerebral ischemia, and the third and fourth ROIs were located directly within the ischemic border zone. These 6 ROIs were mirrored to the contralateral hemisphere, which was naturally not affected by ischemia. In addition, a single ROI was placed in the ischemic subcortex and mirrored to the contralateral hemisphere. Each ROI had dimensions of 150 × 600 µm, and the squares were placed with a 50 µm gap within the neocortex. Quantifications were based on three brain sections per animal, and mean values of fluorescence intensities from each ROI were calculated across the three sections and used for intra- and interhemispheric comparison between ROIs. In case a single ROI failed to provide a valid fluorescence signal, e.g., due to mechanical alterations in the tissue, this ROI was not included in further processing.p-value of <0.05 was considered statistically significant. Based on immunosignals of tricellulin, MFAP5 and α-catenin, descriptive analyses and tests concerning statistical significance between regions were performed with the statistics software SigmaPlot 14.0 . Due to the relatively small sample size, a non-parametric test, i.e., the Wilcoxon Rank-Sum test, was applied. The Bonferroni–Holm correction was used to consider multiple comparisons and, thus, to reduce the risk of false-positive results . GeneralFigures comprising selected micrographs and a summary of quantifications were arranged with CorelDraw 24  and Microsoft PowerPoint , and the brightness and contrast of micrographs were slightly adjusted if necessary but without deleting or creating signals.


Lysyl oxidase-like 2 (LOXL2) is an extracellular copper-dependent enzyme that plays a central role in fibrosis by catalyzing the crosslinking and deposition of collagen. Therapeutic LOXL2 inhibition has been shown to suppress liver fibrosis progression and promote its reversal. This study investigates the efficacy and underlying mechanisms of human umbilical cord-derived exosomes (MSC-ex) in LOXL2 inhibition of liver fibrosis. MSC-ex, nonselective LOX inhibitor β-aminopropionitrile (BAPN), or PBS were administered into carbon tetrachloride (CCl4)-induced fibrotic livers. Serum LOXL2 and collagen crosslinking were assessed histologically and biochemically. MSC-ex’s mechanisms on LOXL2 regulation were investigated in human hepatic stellate cell line LX-2. We found that systemic administration of MSC-ex significantly reduced LOXL2 expression and collagen crosslinking, delaying the progression of CCl4-induced liver fibrosis. Mechanically, RNA-sequencing and fluorescence in situ hybridization (FISH) indicated that miR-27b-3p was enriched in MSC-ex and exosomal miR-27b-3p repressed Yes-associated protein (YAP) expression by targeting its 3ʹ untranslated region in LX-2. LOXL2 was identified as a novel downstream target gene of YAP, and YAP bound to the LOXL2 promoter to positively regulate transcription. Additionally, the miR-27b-3p inhibitor abrogated the anti-LOXL2 abilities of MSC-ex and diminished the antifibrotic efficacy. miR-27b-3p overexpression promoted MSC-ex mediated YAP/LOXL2 inhibition. Thus, MSC-ex may suppress LOXL2 expression through exosomal miR-27b-3p mediated YAP down-regulation. The findings here may improve our understanding of MSC-ex in liver fibrosis alleviation and provide new opportunities for clinical treatment.The online version contains supplementary material available at 10.1186/s12951-023-01942-y. Liver fibrosis develops in response to various chronic injuries such as viral infection, nonalcoholic steatohepatitis (NASH), nonalcoholic fatty liver disease (NAFLD), alcohol-related fatty liver diseases (AFLD), and autoimmune hepatitis. It can give rise to cirrhosis or hepatocellular carcinoma (HCC) and has become one of the leading causes of death, accounting for approximately 2 million deaths per year worldwide , 2. HoweThe Lysyl oxidase (LOX) family (LOX and LOXL1–4) are essential enzymes that promote crosslinking of collagens by oxidatively deaminating lysine residues. Like all LOX members, LOXL2 is secreted to the extracellular matrix (ECM), where they participate in crosslinking of fibrillar collagen, a significant component of fibrosis. The distinct role of LOXL2 in a wide range of diseases, including liver fibrosis, has been elaborated . Recent Recently, MSC transplantation has emerged as an attractive therapeutic for treating liver fibrosis and cirrhosis . MSC-basIn this study, we investigated the effects and molecular mechanisms of MSC-ex on LOXL2 modulation in carbon tetrachloride-induced liver fibrosis mouse models and in vitro cell culture systems. Hepatic stellate cells (HSC) are the primary cellular sources of LOXL2 in fibrotic livers. MSC-ex efficiently inhibits LOXL2 secretion of activated HSC and collagen crosslinking and suppresses liver fibrosis progression. We also demonstrate that LOXL2 can be transcriptionally regulated by Yes-associated protein (YAP), which was overexpressed in TGFβ activated HSC. In addition, MSC-ex-derived miR-27b-3p promoted YAP down-regulation, reducing LOXL2 expression, and inhibited collagen crosslinking and fibrosis progression in vivo. These findings provide a better understanding of using MSC-ex for LOXL2 inhibition and liver fibrosis therapy.2 and tested for mycoplasma contamination.The human umbilical cord was gained from informed, healthy parturients, and MSC was isolated from the human umbilical cord and identified as described previously . All clig for 16 h at 4 °C. 500 mL condition medium from MSC at passages 3 to 6 was collected, and centrifuged at 2000×g for 20 min to remove cell debris. Then, Supernatants were concentrated using a 100 KDa molecular weight cut-off (MWCO) ultrafiltration filter per the manufacturer’s instructions . After filtration with 0.22 μM filter membrane, the exosomes-enriched fraction was transferred to a 15 mL sterile centrifuge tube. MSC-ex was precipitated from the concentrates using ExoQuick-TC extracellular vesicle (EV) isolation Kit following the protocol . The protein concentration of the extracted exosomes was quantified by a BCA protein assay kit . The morphology of MSC-ex was observed by transmission electron microscopy . The amount and size distribution of MSC-ex was measured by NanoSight tracking analysis (NTA) with NTA 3.1 Software .MSC-ex was isolated and purified as our previously established method . MSC wasn = 6); 3-aminopropionitrile fumarate salt (BAPN) group, mice treated with BAPN  once per day; and MSC-ex 12.5 mg/kg body weight (n = 6), 25 mg/kg body weight (n = 6) groups, mice treated with MSC-ex twice a week. PBS, BAPN, and MSC-ex were administered by tail vein. At 4 weeks post MSC-ex injection, mice were sacrificed to collect blood and liver samples for further analysis.All experiments involving animals were conducted according to the ethical policies and procedures approved by the Jiangsu University ethics committee . BALB/c female mice, 4–5 weeks old, were treated with carbon tetrachloride (CCl4) (10%) for 6 weeks to induce liver fibrosis as described. To analyze the effect of MSC-ex on LOXL2 expression and liver fibrosis, mice were randomized into four groups: PBS group, mice injected with 1 mL PBS . In vivo, spectral imaging from 690–850 nm was performed using an exposure time of 150 ms per image frame. For the distribution of MSC-ex in LX-2, PKH67 labeled MSC-ex (PKH67-ex) was incubated with LX-2 cells at 37 °C for 24 h and observed with confocal microscopy.MSC-ex was incubated with DiR (DiIC 18(7); 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindotricarbocyanine iodide)  or PKH67  for 30 min at 37 °C according to the manufacturer’s instructions. After washing with PBS, DiR or PKH67 labeled MSC-ex were concentrated with a 100 KDa molecular weight cut-off (MWCO) ultrafiltration filter at 1000×Whole-cell or MSC-ex lysates were prepared in RIPA lysis buffer . Protein concentration was determined using the BCA assay kit . Equal amounts of lysates were loaded and separated on a 10% or 12% SDS-PAGE gel. Standard Western blot used primary antibodies and a peroxidase-linked, species-specific, anti-mouse, anti-rat, or anti-rabbit IgG . The following primary antibodies were used: CD9 , CD63 , Calnexin , TSG101 , α-SMA , LOXL2 , YAP , β-actin . and GAPDH . Proteins were detected with an ECL detection system . Western blot results were quantitated using ImageJ software; protein expression was normalized to GAPDH.−ΔΔCT method and normalized to the β-actin gene. The PCR primers are listed in Table −ΔΔCt method. There were three or six replicates per group.The total RNA of LX-2 cells and mouse livers were extracted with Trizol according to the manufacturer’s instructions (Invitrogen). 1 µg of total RNA was used for the reverse transcription of RNA into cDNA in a reaction using the SuperScriptTM II RT kit according to the manufacturer’s instructions (Invitrogen). SYBR-Green I-based Real-Time PCR kit  was used, and relative gene expression quantitation was determined using the 2Serum LOXL2 levels in fibrotic mice treated with MSC-ex or BAPN were determined using an ELISA (Enzyme-linked Immunosorbent Assay) kit  according to the manufacturer’s instructions. Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), and UREA were measured with an automated biochemical analyzer.LOXL2, YAP, and α-SMA protein expressions were analyzed using MSC-ex or BAPN-treated mouse tissue samples. Mouse tissue sections (4 µm thick) of formalin-fixed, paraffin-embedded liver specimens were deparaffinized in xylene and rehydrated in graded alcohol. Standard immunohistochemical procedures were performed on liver tissue sections using anti-LOXL2 , anti-YAP , or anti-α-SMA antibody . Signals were visualized using 3, 3′-Diamino-benzidine tetrahydrochloride . A brown membrane, cytoplasmic, and nuclear staining indicated a positive reaction according to different markers. The staining result of LOXL2, YAP, and α-SMA expression was determined by the percentage of positive cells by two investigators blinded to the data. Immunofluorescence staining was performed using anti-CD9 , anti-LOXL2 (1:50), anti-YAP (1:50), or anti-α-SMA antibody (1:50). Negative controls with isotype IgG were run in parallel. Images were acquired using a laser scanning confocal microscope .Formalin-fixed paraffin-embedded cardiac, liver, spleen, lung, and kidney tissues sections were stained with Masson Trichrome (MT)  and Sirius Red  according to the instruction of the manufacturer. To analyze hepatic collagen distribution, 10 fibrotic septa randomly selected from the right and left liver lobes of 6 individual mice/groups were assessed. Collagen extent was expressed as a percentage of stained area in each liver section.LX-2 was cultured in a 6-well plate until it reached approximately 50–60% confluence. Then cells were randomized into four groups: PBS, LX-2 treated with PBS, TGFβ: LX-2 treated with 10 ng/mL TGFβ, TGFβ/MSC-ex, LX-2 treated with 10 ng/mL TGFβ and 100 or 200 μg/mL MSC-ex, TGFβ/BAPN: LX-2 treated with 10 ng/mL TGFβ and 1.0 mg/mL BAPN. These cells were treated for 48 h for further investigation.LX-2 cells were fixed in 4% paraformaldehyde for 10 min and permeabilized with 0.1% Triton X-100 for 10 min at room temperature. For examining LOXL2, YAP, and α-SMA protein levels, LX-2 cells were blocked with 5% BSA and incubated with anti-LOXL2, anti-YAP, or anti-α-SMA primary antibody at 4 °C overnight; after incubation and washing, fluorescent-labeled secondary antibody was added with the necessary incubation and washing. Then the slides were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) for nuclear staining and examined under a laser scanning confocal microscope .Adenoviruses expressing full-length human YAP (Ad-YAP), GFP alone (Ad-GFP), YAP shRNA (sh-YAP), and control shRNA (sh-Ctr) were constructed. Full-length human YAP cDNA was inserted into pAV(Exp)-CMV > YAP/HA-IRES-Egfp adenoviral vectors to generate Ad-YAP expression vectors. The adenoviral YAP shRNA vector was packaged by the vector ADV1(U6/CMV-GFP) with YAP shRNA oligonucleotides. The shRNA oligonucleotide sequences are listed in Table The LOXL2 promoter constructs pcDNA3.1-LOXL2-907 (pLOXL2-907), pcDNA3.1-LOXL2-826 (pLOXL2-826) and pcDNA3.1-LOXL2-328 (pLOXL2-328) were Chemically synthesized . For the LOXL2 reporter assay, HEK293T and LX-2 cells were seeded in 24-well plates. The LOXL2 promoter constructs and Renilla luciferase reporter were cotransfected with plasmid DNA of pcDNA3.1-vector (p3.1), pcDNA3.1-YAP (pYAP), and in some experiments, control shRNA (sh-Ctr) or YAP shRNA (sh-YAP). Lipofectamine 2000 reagent (Life Technologies) was used according to the manufacturer’s instructions. Both firefly and Renilla luciferase activity were measured using a dual-luciferase assay system  48 h after transfection. The LOXL2 promoter activity was normalized with the Renilla luciferase activity. Three biological repeats were used for each sample in the dual luciferase reporter.7 293T or LX-2 cells were lysed in sodium dodecyl sulfate lysis buffer. The cell lysate was sonicated to shear the DNA to 400- to 600-bp lengths. Chromatin samples were then precleared with a salmon sperm DNA/protein A agarose 50% slurry for 30 min at 4 °C and immunoprecipitated overnight with anti-IgG or anti-YAP . The purified DNA fragments were subjected to quantitative PCR using primers listed in Table Chromatin immunoprecipitation (ChIP) assay followed the manufacturer’s instructions . Briefly, formaldehyde was used to cross-link proteins with DNA, and 2 × 10http://targetscan.org/). The 3ʹ‑untranslated region (UTR) of the human YAP gene containing either wild-type (WT) or mutant-type (MT) binding sites of miR-27b-3p were synthesized and inserted in the pGL3 vector downstream of the firefly luciferase gene to form pGL3-YAP, named as YAP-WT and YAP-Mut. LX-2 cells (2.5 × 104/well) were seeded in 24-well plates and were co-transfected with miR-27b-3p mimics or negative control (NC) miRNA mimics and control reporter plasmids pGL3, YAP-WT or YAP-Mut using Lipofectamine 2000. The Dual-Luciferase Reporter assay system  was applied to examine the activities of Renilla and firefly luciferase based on the manufacturer’s protocols at 24 h post-transfection. Firefly luciferase activity was normalized to Renilla luciferase activity.YAP was predicted as a target of miR-27b-3p by TargetScan . 125 ng RNA was isolated from MSC-ex (104 particles). 500 ng RNA in 20 μL was used per reaction to generate cDNA by reverse transcription. The reaction mixture consisted of 2 μL QX200 ddPCR EvaGreen (Bio-Rad), 1 μL Laxa Fluor 647 dye (Thermo Fisher), 3.75 μL ddPCR™ supermix for probes (Bio-Rad), 1 μL primer, and 5 μL DNA template. Finally, RNase-free H2O was added to make a total reaction volume of 25 μL. The two-step method is used for ddPCR, and the cycling procedure consists of a pre-denaturation step at 95 °C for 5 min, followed by 45 cycles of denaturation at 95 °C for 10 s, annealing at 62 °C for 45 s, and 72 °C for 30 s. After the PCR cycle, the sapphire chip was transferred to a Naica Prism 3 fluorescence reader for imaging. Crystal Miner software (Stilla Technologies) was used for data analysis.Droplet digital PCR was performed using the naica5 cells were cultured in 24-well glass slide plates. 4% paraformaldehyde was used to fix the cells. 15 min of TRITON X-100 treatment was followed by incubation with blocking solution for 30 min (37 °C). Cy3-labeled miR-27b-3p fluorescent probe solution was incubated with cells in an in situ hybridizer for 14 h (37 °C). Tween 20 was used to wash the cells. For liver tissue, 7 mm frozen liver tissue sections were digested with proteinase K and incubated in a blocking buffer for 30 min (37 °C). Prepare the cy3-labeled miR-27b-3p fluorescent probe working solution at a volume ratio of 1:1. Incubate with liver tissue slices for 14 h (37 °C) in an in situ hybridization instrument. Wash sections with deionized formamide at 43 °C to denature unhybridized probes. Sections were washed three times with sodium citrate buffer (60 °C). FISH images were then captured by confocal microscopy.Cy3-labeled miR-27b-3p probe sequence was purchased from GenePharma, China. 3 × 10Human miR-27b-3p mimics, negative control mimics, and miR-27b-3p inhibitors, negative control inhibitors were purchased from GenePharma, China. According to the instruction of the manufacturer, miR-27b-3p mimics  and miR-27b-3p inhibitors  were transiently transfected into LX-2 cells using Lipofectamine 2000 in Opti-MEM™ medium  at 70–80% confluency in 6-well culture plates. At 4–6 h post-transfection, the culture medium was replaced with MEM with 10% FBS for another 48 h. The transfected cells were collected for further investigation.in-ex), or negative control inhibitors transfected MSC-ex (NCin-ex) were isolated, purified, and washed as our previously established method. After concentration and structure identification, miR-27bin-ex or NCin-ex were stored at − 70 °C for further use. Then LX-2 cells were treated with NCin-ex (200 μg/mL) or miR-27bin-ex (2000 μg/mL) for 48 h and collected for further investigation.Negative control inhibitors, miR-27b-3p inhibitors , were transiently transfected into MSC using Lipofectamine 2000 in Opti-MEM™ medium  at 70–80% confluency in 6-well culture plates. At 4–6 h post-transfection, the culture medium was replaced with an FBS-free medium for another 48 h. Then total RNA of inhibitors transfected MSC was collected for miR-27b-3p quantification. miR-27b-3p inhibitors transfected MSC-ex (miR-27boe-ex) or negative control mimics overexpressed MSC-ex (NCoe-ex) were washed with PBS 3 times to remove residual miRNA. After concentration and structure identification, miR-27boe-ex or NCoe-ex were stored at − 70 °C for further use. Then LX-2 cells were treated with NCoe-ex (50 μg/mL) or miR-27boe-ex (50 μg/mL) for 48 h and collected for further investigation.miR-27b-3p mimics were passively loaded into MSC-ex by the sonication method. MSC-ex was mixed with 50 nM miR-27b-3p mimics or negative control mimics and sonicated at 500 v, 2 kHz, 10% power, 6 cycles by 4 s pulse/2 s pause, cooled down on the ice for 2 min, and then sonicated again using Qsonica Sonicator Q700 . Then miR-27b-3p overexpressed MSC-ex (miR-27bANOVA) followed by Dunnett was used for studies involving more than two groups. A two-sided p < .05 was considered statistically significant.Statistical analyses were performed using GraphPad Prism version 8.3.0 . All the data is presented as mean ± SD. The Student’s t-test was used for comparisons between the two groups. One-way analysis of variance  injections for up to 6 weeks. We administered MSC-ex treatment concurrently with fibrosis induction for another 4 weeks. PBS or pan-Lox inhibitor BAPN was administered in parallel Fig. A. SurfacThe biodistribution of MSC-ex was investigated by labeling exosomes with DiR. In vivo, fluorescent imaging showed that DiR-labeled MSC-ex administered by tail vein targeted injured livers at 24 h post-injection Fig. E. Serum + endothelial cells, F4/80+ Kupffer cells, and albumin+ hepatocytes  are the primary source of collagen in established liver fibrosis, we sought to test whether HSC-activated myofibroblast functions as the cellular target of MSC-ex for anti-LOXL2 therapies. We also injected PKH26-labeled MSC-ex into liver fibrosis mice via tail vein injection. Immunofluorescence analysis showed that a small amount of PKH26-MSC-ex could localize in CD31To elucidate the key molecule and potential signaling pathways involved in LOXL2 regulation, we analyzed the LOXL2 promoter using the UCSC genome website and JASPAR database. YAP was screened out as the transcriptional factor binding to the LOXL2 promoter region. Indeed, we identified three potential YAP-binding sites in the promoter region of LOXL2 by motif analysis Fig. A. To valTGFβ is known to induce the activation of HSCs . We furt4 particles  compared with the exosomes isolated from negative control (NC) miRNA transfected MSC (NCin-ex). When NCin-ex or miR-27bin-ex were incubated with LX-2 cells, FISH showed that the knockdown of miR-27b-3p markedly reversed MSC-ex-induced up-regulation of miR-27b-3p    Fig. A. In miRex) Fig. B. NC-ex -ex Fig. C–F. West-ex Fig. G. These Liver fibrosis is a continuous wound-healing process of chronic injury. It results from excessive deposition and degreased degradation of the extracellular matrix (ECM) . LOXL2 iβ-aminopropionitrile (BAPN). In comparison with BAPN treatment, MSC-ex achieved better anti-LOXL2 results in vivo. Accordingly, MSC-ex (25 mg/kg) and BAPN treatment reduced collagen deposition, respectively. Our results provide, for the first time, evidence that MSC-ex could serve as a novel agent for anti-LOXL2 inhibition in liver fibrosis.Recently, inhibition of LOXL2 by small-molecule inhibitors and monoclonal antibodies has decreased fibrosis and increased survival in rodent models of liver fibrosis , 25. HowMSC can be obtained from bone marrow and other fetal or postnatal tissues, including adipose tissue, umbilical cord blood, and the Wharton’s jelly of the umbilical cord . Among tActivated HSC and myofibroblasts are primary cellular sources of LOXL2 in liver fibrogenesis . Our preFor the first time, we identified LOXL2 as a direct downstream target gene of YAP, which positively regulated LOXL2 transcription in HSC. YAP is a well-defined downstream effector in the Hippo pathway that promotes HSC activation when translocated to the nucleus during liver fibrosis . YAP actExosomes can specifically target recipient cells to exchange miRNAs, mRNAs, and proteins to trigger downstream signaling events . miRNAs While this study offers valuable insights, it is essential to acknowledge its limitations. One such challenge is the need for more knowledge regarding other active proteins or miRNAs in MSC-ex that may inhibit HSC activation and liver fibrosis. Furthermore, it remains to be seen which target molecules MSC-ex acts on besides LOXL2. Additionally, our study found that after in vivo injection, MSC-ex was localized in HSC and taken up by hepatocytes, endothelial cells, and macrophages. MSC-ex may carry other proteins or miRNAs, which may regulate these important target cells in the liver and inhibit liver fibrosis. Further research into these questions will significantly improve our understanding of the mechanisms behind the anti-fibrotic effects of MSC-ex.In summary, we reported that injected MSC-ex can locate in the HSC of fibrotic liver and substantially reduce LOXL2 expression, collagen matrix deposition, fibrosis progression, and direct HSC activation. MSC-ex-derived exosomal miR-27b-3p may target YAP, reducing YAP transcriptional-activated LOXL2 expression and inhibiting collagen crosslinking in vitro and in vivo Fig. .Fig. 9SAdditional file 1: Figure S1. Quantified fluorescence intensity of mice liver from PBS or DiR labeled MSC-ex group.Additional file 2: Figure S2. Safety evaluation of MSC-ex in health mice. A. Haematoxylin and eosin (HE) staining of cardiac, liver, spleen, lung, and kidney tissues in healthy mice intravenously injected with PBS or MSC-ex. Scale bars, 40 µm. B. The levels of liver function indicators , cardiac function indicators , and renal function indicators UREA in sera from PBS or MSC-ex treated mice.Additional file 3: Figure S3. Uptake of PKH26-MSC-ex in F4/80+ Kupffer cells, albumin+ hepatocytes, or CD31+ endothelial cells in fibrotic mouse liver. A. PKH26-labeled MSC-ex was taken up by F4/80+ Kupffer cells in fibrotic mouse liver 24 h post intravenous injection. Scale bars, 100 µm. B. PKH26-labeled MSC-ex was taken up by CD31+ endothelial cells in fibrotic mouse liver. Scale bars, 100 µm. C. PKH26-labeled MSC-ex was taken up by albumin+ hepatocytes in fibrotic mouse liver. Scale bar, 100 µm.Additional file 4: Figure S4. Number of miR27b-3p in MSC-ex detected by ddPCR. A. Fluorescence amplitude of miR-27b-3p in MSC-ex using ddPCR. 125 ng RNA from MSC-ex (104 particles) was used to generate cDNA by reverse transcription. ddPCR was performed using cDNA from 125 ng RNA in a total PCR system of 25 μL. The average concentration of miR-27b-3p is represented in copies/μL. B. ddPCR determined miR-27b-3p copies of MSC-ex (104 particles). Four parallel samples were tested.


Ex-PRESS glaucoma shunt stainless steel devices have been used worldwide for glaucoma treatment. The purpose of this study was to evaluate the safety of high-field magnetic resonance imaging (MRI) for Ex-PRESS–inserted eyes.Using rabbits, we performed Ex-PRESS shunt surgery in one eye in each rabbit and divided the rabbits into MRI and non-MRI groups. In the MRI group, 1 week after Ex-PRESS shunt surgery under low specific absorption rate (SAR) conditions and 1 week later under high SAR conditions, high-field 4.7-Tesla MRI was performed. Aqueous flare counts were measured before and after the Ex-PRESS shunt surgery and each MRI examination. The rabbits in the non-MRI group received only general anesthesia, and aqueous flare counts were measured as for those of the MRI group. Aqueous flare counts were expressed in photon counts per millisecond.P = 0.01). The day after MRI, the flare count improved spontaneously to the same level as that before MRI.No dislocation of the Ex-PRESS shunt device was observed after MRI. The flare count ratio (MRI/non-MRI) in Ex-PRESS–inserted eyes 2 hours after high SAR MRI increased significantly compared with that before MRI (0.8 ± 0.3 vs 2.7 ± 0.8; pre-high SAR MRI vs 2 hours after high SAR MRI, respectively; Our results indicate that high-field MRI can be performed relatively safely after Ex-PRESS shunt surgery.This study demonstrates the safety of high-field MRI for Ex-PRESS–inserted eyes using a rabbit model. The same observations and examinations were performed for both eyes in the rabbits in each group. The animals were housed and treated in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. All procedures were performed in accordance with the guidelines of the Committee on Animal Experimentation and the Committee of Research Facilities for Laboratory Animal Science, as well as the Natural Science Center for Basic Research and Development of Hiroshima University. All animals in this study were housed for at least 7 days before the experiment in an animal housing facility with a room temperature of 23°C and a 12-hour light/dark cycle (lights on at 8:00 am).Six pigmented male rabbits  were used for the experiments. Ex-PRESS shunt surgery was performed on only one eye of each rabbit. The rabbits were divided into two groups . The rabbits were sedated with an intramuscular injection of 40 mg/kg medetomidine and 1 mg/kg butorphanol. Anesthesia was maintained after intravenous catheterization with an intravenous injection of 50 mg/kg pentobarbital. The right eye was cleansed with topical 0.5% povidone-iodine, and a lid speculum was placed to hold the lids open. A 7-mm conjunctival incision was made along the superior corneal limbus with spring microscissors to make a fornix-based conjunctival flap. A 25G needle was inserted 1.2 mm from the corneal limbus into the anterior chamber, underneath the conjunctival flap. The Ex-PRESS shunt device was then placed in the anterior chamber through the ostium created by the needle. After fixation of the Ex-PRESS device, the conjunctival peritomy was closed using 10-0 nylon with wing sutures. Ofloxacin eye ointment  was applied at the end of the surgery, and no additional medications were administered postoperatively. The procedure required approximately 1 hour, including induction of anesthesia.All experiments were performed with a 4.7-T superconducting magnet system  with a transmit/receive orthogonal volume coil (154 mm inner diameter). The rabbits underwent rapid acquisition of refocused echo imaging  under the two conditions shown in Radiofrequency exposure produces an electric current in the body, creating a thermal effect owing to the absorption of electromagnetic wave energy. SAR is a measure of this thermal effect, usually indicated in watts per kilogram.Before the MRI examination, the rabbits were sedated with an intramuscular injection of 40 µg/kg medetomidine and 1 mg/kg butorphanol. Five minutes after sedation, the rabbits were masked and immobilized with isoflurane at 100 mL/min for 3 minutes in the surgical preparation room. The rabbits were then moved to the MRI room, and anesthesia was maintained with inhalational isoflurane at 60 mL/min. Inhalational anesthesia was maintained for 30 minutes for imaging under low SAR conditions and for 80 minutes under high SAR conditions. In the non-MRI group, anesthesia was administered at the same doses and concentrations as those in the MRI group and MRI was not performed.Aqueous flare was quantified by photometric measurements using a laser flare meter . Aqueous flare counts were expressed in photon counts per millisecond. The rabbit eye was placed in the center of the measurement section with no mydriasis and manually suppressed. Measurements were taken in an area measuring 0.3 × 0.5 mm at approximately the center of the anterior chamber. Measurements were taken by the same examiner; five measurements were taken, and the average value was used in the analysis.am, and the Ex-PRESS implant surgery was performed at noon. Aqueous flare was measured 2 hours after surgery and at 1:00 pm on postoperative days 1 and 2. One week after the surgery, after flare measurement at 1:00 pm, MRI was performed under low SAR conditions. Aqueous flare was measured 2 hours after a low-SAR MRI and at 1:00 pm on days 1 and 2 after a low-SAR MRI. One week after low-SAR MRI, after flare measurement at 1:00 pm, MRI was performed under high-SAR conditions. Aqueous flare was measured 2 hours after high-SAR MRI and at 1:00 pm on days 1 and 2 after high-SAR MRI. In the non-MRI group, flare measurements were taken at the same times as those for the MRI group, but without performing MRI.Preoperatively, aqueous flare was measured at 11:00 t-test. All statistical analyses were performed using JMP pro 16.0.0 , and a P value of less than 0.05 was considered statistically significant.The data were expressed as mean ± standard deviation. Non–Ex-PRESS-inserted eyes acted as controls. To analyze the effect of MRI scanning on aqueous flare counts, we compared the MRI group/non-MRI group ratios for the aqueous flare counts in each of the Ex-PRESS and non–Ex-PRESS-inserted eyes. The time course changes in aqueous flare were compared by Dunnett's test using pre-MRI values as a reference. The aqueous flare count ratios between the Ex-PRESS and non–Ex-PRESS-inserted eyes at each time point were compared by the Student The Ex-PRESS glaucoma filtration device was inserted into one eye of each of the three rabbits in the MRI and non-MRI groups. A photograph of the anterior segment of the eye after Ex-PRESS insertion is shown in P < 0.01). The flare counts in the Ex-PRESS eyes decreased spontaneously to 85.9 ± 78.9 pc/ms and 22.4 ± 12.2 pc/ms on days 1 and 2 after surgery, respectively. In the non–Ex-PRESS-inserted eyes, the flare counts ranged from 10.3 ± 3.2 pc/ms to 12.2 ± 2.4 pc/ms, with no significant change between the preoperative and postoperative day 2 values.Changes in flare counts before and after Ex-PRESS shunt surgery are shown in P = 0.01). Flare count ratios decreased the day after low-SAR MRI and were not significantly different compared with pre–low-MRI ratios. The mean flare count ratio 2 hours after the low-SAR MRI in the non–Ex-PRESS-inserted eyes also increased compared with that of pre–low-SAR MRI . There was no significant difference in the flare count ratios between the Ex-PRESS and non–Ex-PRESS-inserted eyes at all time points (Changes in flare counts and ratios before and after MRI are shown in e points A.P = 0.01). The flare count ratios decreased spontaneously the day after the high-SAR MRI. In contrast, there was no significant increase in flare count ratios after a high-SAR MRI in the non–Ex-PRESS-inserted eyes. Although the mean flare count ratio in the Ex-PRESS eyes 2 hours after a high-SAR MRI was approximately twice as high as that in the non–Ex-PRESS-inserted eyes, this difference was not statistically significant  (P = 0.50).The mean flare count ratio in the Ex-PRESS eyes 2 hours after MRI under high-SAR conditions increased significantly compared with that before high-SAR MRI  B. The meIn this study, we showed that, in Ex-PRESS–inserted eyes, the aqueous flare counts in the anterior chamber 2 hours after MRI increased with high-field MRI. Flare counts decreased spontaneously the day after MRI, and there were no findings indicating Ex-PRESS dislocation after high-field MRI.,,It has been shown that there was no deviation of the Ex-PRESS inserted in human cadaver eyes in a 3-T magnetic field.In this study, the anterior chamber flare counts were increased 2 hours after low-SAR MRI in the non–Ex-PRESS-inserted eyes, although less than those in the Ex-PRESS–inserted eyes. If MRI itself might be unsafe for the eye, the flare counts should still have been increased 1 and 2 days after MRI. However, there was no increase 1 and 2 days after MRI, only after 2 hours. Although the cause of this observation is unclear, the interaction between general anesthesia with isoflurane and MRI may be a potential cause. A study of pediatric patients who underwent MRI showed that serum IL-1β, a marker of systemic inflammation, was significantly elevated after 60 minutes of anesthesia with isoflurane.In this study, we investigated the safety of 4.7-T high-field MRI of Ex-PRESS–inserted eyes and noninserted eyes under two conditions: the low-SAR condition, which is SAR in accordance with the recommendations of international standards and safety guidelines, and the high-SAR condition, which is much higher than the guidelines recommendations. The results showed that intraocular inflammation was induced 2 hours after high-field MRI; however, this inflammation resolved spontaneously the next day without therapeutic intervention, such as with anti-inflammatory drugs. This temporary inflammation may have been influenced by inhalational anesthesia with isoflurane. However, inhalational anesthesia with isoflurane is not usually used in human MRI. This imaging is performed under awake conditions in adults who can remain at rest, and under oral or intravenous sedation in children and adults who cannot remain at rest. No deviation of the Ex-PRESS implants occurred during MRI imaging in either the low- or high-SAR conditions. This suggests that high-field MRI can be performed relatively safely in eyes implanted with Ex-PRESS shunt devices. MRI provides good visualization of soft tissues and is very useful for the early detection, diagnosis, and treatment of systemic diseases. Our study demonstrated the safety of high-field MRI in animal model after Ex-PRESS shunt surgery. This study suggests that high-field MRI imaging at 4.7 T could be relatively safe after Ex-PRESS shunt surgery.


HOPO was heated to 75 °C and mixed with mono- or diglycerides at those four concentrations. The thermomechanical properties of the melted oleogels were assessed using differential scanning calorimetry (DSC). The melted oleogels were cooled to final temperatures of 5 °C, 10 °C and 15 °C under identical cooling rates, after which a puncture test  was used to assess their textures. Finally, polarized light microscopy was used to assess the mechanical changes induced through emulsifier addition. The results showed that the use of mono- and diglycerides significantly modified the thermal and mechanical properties of the oleogels. The addition of saturated monoglycerides promoted a higher-temperature nucleation stage that did not previously occur in HOPO. The onset crystallization temperature increased with the addition of diglycerides, promoting crystallization at higher temperatures of the high-melting fraction of HOPO. The hardness of the oleogel generally increased with emulsifier addition and a reduction of the temperature. The effect of the temperature on the hardness was significantly greater in the diglyceride oleogel than in the monoglyceride oleogel. This study shows that the addition of mono- and diglycerides allows companies to customize their formulations to achieve desired results that may not previously have been possible, thereby facilitating novel uses for these oils within the industry.Alternatives to oils with high saturated fatty acid content are often liquid oils (high in unsaturated fatty acids) that have a modified structure created either through additives or processing. Emulsifiers are additives that can be used as structuring agents of liquid fats; this process results in products such as oleogels, which can broaden the applications of these oils. This study assessed and compared the effects of mono- and diglycerides at 3%, 5%, 7% and 10%  The American Heart Association recommends that vegetable oils and other fats comprise 20–35% of daily calorie consumption . In 2015E. guineensis and E. oleifera OxG; this palm has slower growth and is more resistant to bud rot disease, which increases its productive life and reduces the need for cut down and replanting, potentially decreasing deforestation rates [E. guineensis , resulting in a lower concentration of saturated fatty acids in HOPO (30%) without the need for fractionation. This oil is soft and semisolid at room temperature and contains high concentrations of bioactive compounds, such as vitamin A carotenes and Vitamin E α-tocopherol [w/w naturally occurring DAGs, which slow nucleation in regular palm oil, and a low concentration of fully saturated triglycerides (1.5%) [Common oils that are low in SFAs have been studied for potential oleogel applications, including health-contributing high-oleic varieties of soybean and canola ,11. Stanon rates ,14. HOPOcopherol . HOPO has (1.5%) . CommercAvailability, cost, health benefits and/or gelling properties are important considerations for structuring agents. The molecular structures of monoglycerides (MAGs) make them appealing for use as gelling agents . The hydThe processing conditions in oleogel manufacturing are as important to the stability of the oleogel as the ingredients. Critical processing parameters include shearing, crystallization temperature and conditioning. Additionally, supplemental processes, such as high-intensity ultrasound (HIU)-induced crystallization, have been shown to increase oil retention in oleogels . Da PievThe use of the herein proposed MAG and DAG oleogels has a widespread range found in the literature . ApplicaTo the knowledge of the authors, changes in the behavior of HOPO caused by the addition of emulsifiers have not been studied. The specific objectives of this study were to assess and compare the effects of added mono- and diglycerides on the mechanical and thermal properties of HOPO oleogels. This assessment was conducted through the use of DSC, texture analysis and microscopy tests.Melted MAG and DAG HOPO oleogels were assessed using DSC, and the resulting curves are shown in w/w. The standard first peak observed in HOPO  broadened in the presence of MAGs; even at the lowest concentrations of MAGs, 3% and 5%, a small peak was evidenced. Similar results have been obtained for olive oil oleogels containing 15% MAGs  and are The presence of an emulsifier that was rich in DAGs (63%) modified the first crystallization event of the HOPO, as seen in The previously described melting peak for HOPO “5A” was broadened with the increase in DAGs, suggesting that DAGs regulate polymorphic changes. The presence of DAGs delayed the offset melting point. At 31 °C, the HOPO was completely melted, increasing to 43.1 °C at 3% concentration and 49.3 °C at 10% concentration . These rw/w) and 1,3-OleicOleic (0.48% w/w) are the two most abundant structures [w/w). It has been previously reported that DAGS inhibit nucleation and retard crystal growth of triglycerides, although it seems to be concentration- and DAG-structure-dependent [The naturally occurring diglycerides in HOPO vary in their degrees of saturation. Unsaturated diglycerides 1,3-PalmiticOleic , see Conversely, w/w monoglyceride at a crystallization temperature of 5 °C; however, at these lower temperatures, MAG and DAG oleogels required similar energy levels for deformation, suggesting a more homogeneous structure in DAGs and a gradient of gel strength from the edges of the vials to the core with MAGs.The use of mono- and diglycerides changed the thermal and mechanical properties of HOPO , thereby broadening its potential industrial applications. The addition of commercially available saturated monoglycerides promoted an early nucleation stage that was not previously present in HOPO, and microscope images showed needle-like networks increasingly growing with higher concentrations of MAGs. The hardness of the oleogel generally increased with the addition of emulsifier. The hardest oleogel (4.6 kg) was obtained by using 10% w/w) diglyceride concentration at the lowest assessed temperature (5 °C).The commercially available saturated diglyceride DAGs also had major effects on the thermal and mechanical characteristics of HOPO by dominating the initial crystallization stage of the oleogel, as was also observed under microscopy as a promoter of small spherulite fat crystals. The onset crystallization temperature also increased with the addition of DAGs, promoting the early crystallization of the high-melting fraction of the HOPO. DAGs had less of an effect than MAGs on the hardness of the oleogel. The maximum hardness (3.8 kg) for DAG oleogels was also obtained at the highest , 0.05% maximum moisture, minimum iodine value (IV) of 67, maximum peroxide value (PV) of 1 and a maximum melting point of 27 °C. It had an SFC of 5–13% at 15 °C and 2–5% at 20 °C. This oil was provided by Thin Oil Products . The fatty acid composition of the HOPO used in this study is provided in The crude oil of the high-oleic palm oil (HOPO) was obtained using mechanical press extraction from the interspecific hybrid FA composition analysis was performed by FoodChain ID Testing Laboratories. HOPO is High-oleic palm oil. “Other FAs” include saturated, mono- and poly- unsaturated fatty acids which were not detected by the AOCS Ce-1b-89 method as reported by FoodChain ID Testing Laboratories.w/w dispersions of each emulsifier, for a total of 8 different samples: MAG_3%, MAG_5%, MAG_7%, MAG_10%, DAG_3%, DAG_5%, DAG_7% and DAG_10%. First, 1 L glass beakers of HOPO were immersed in a water bath and heated at 80 °C to melt all solid fatty acids and erase crystal memory. Aliquots of HOPO were portioned into 100 mL glass beakers. Each emulsifier was added in its corresponding percentage at 75 °C until the dispersion was clear; it was further stirred by hand with spatula every 2 min for a total of 10 min, and samples were allowed to heat up to 80 °C. The cooling of samples was critical for textural analysis, as described below. Aliquots of 15 g for each sample were transferred into 20 mL screw-capped glass vials (n = 4) and then transferred to sequential water baths at 45 °C, 40 °C, 35 °C and 30 °C to control the cooling transition temperature at ΔT = 25 °C. Upon reaching equilibrium, each sample was transferred to a second water bath at 15 °C, 10 °C and 5 °C, respectively, and conditioned for 24 h. This procedure was intended to create fat samples under identical cooling and shear conditions (0 s−1), carefully controlling the crystallization by temperature changes until equilibrium was reached. Before conducting thermomechanical tests, the confirmation of the formation of oleogels was achieved through the use of the inverted test tube method [HOPO was mixed with Alphadim 90 PBK or Trancendim 130 to form 3%, 5%, 7% and 10% A differential scanning calorimeter (DSC) Q2000 (TA Instruments) was used to assess the thermomechanical properties of both oils. Melted oleogel samples of between 6.0 and 9.0 mg were weighed into aluminum pans and hermetically sealed at 75 °C. An empty hermetically sealed aluminum pan was used as a baseline. All the samples followed the AOCS Cj 1–94, 2009 method described by Maruyama et al. —an 80 °CSamples were removed from their respective water baths at 15 °C, 10 °C and 5 °C .The microstructures of oleogels with MAGs and DAGs were captured at 10 °C to illustrate the differences in crystal shapes and abundance between both emulsifiers. These were imaged using PLM . A total of 10 μL of each melted oleogel sample was dropped on a clean glass slide, and a coverslip was carefully placed on the top to ensure uniform thickness. A controlled temperature platform was adapted to the microscope, with inner channels for coolant fluid flowing in and out from a recirculation water bath. The slides went through the same stepwise temperature cooling ramp as described in


PULP induced crystallinity and increased Vicat softening of the PLA/PULP and PLA/EPOXY/PULP composites. The EPOXY reaction of the PLA/PULP composites improved their tensile properties, toughness, thermal stability, and water resistance.A novel poly (lactic acid) (PLA) composite with excellent mechanical properties, toughness, thermal stability, and water resistance was developed using a reactive melt-blending technique. PLA was melt mixed with epoxy resin (EPOXY) and bamboo pulp (PULP) to improve its reaction and mechanical properties. FTIR analysis confirmed the successful reaction of the PLA/EPOXY/PULP composites; the epoxy groups of EPOXY reacted with the –COOH groups of PLA and the –OH groups of PULP. The PLA/EPOXY/PULP5 composite showed a high tensile strength (67 MPa) and high toughness of 762 folding cycles, whereas the highest tensile strength was 77 MPa in the PLA/EPOXY5/PULP20 sample. SEM images presented a gap between the PLA and PULP; gap size decreased with the addition of EPOXY. The T Acetobacter xylinum [Natural fibers are used to reinforce polymers, reduce polymer consumption, and increase the degradation ability of polymers. They are biodegradable, lightweight, inexpensive, non-toxic, recyclable, and possess good mechanical properties. Moreover, natural fibers are used as load bearings in polymers . The mai xylinum ) are useBamboo is a fast-growing and readily available plant in Southeast Asia that is commonly used as a natural fiber . Bamboo Poly (lactic acid) (PLA) is a popular biodegradable polymer used in various industries such as packaging, agricultural, and medical industries . PLA, hoEpoxy resin (EPOXY) is a possible crosslinking agent that induces reactions and interactions between the reactive functional groups of the fiber and PLA. EPOXY contains epoxy groups in its main structure, acting as crosslinking agents for various reactive functional groups such as hydroxyl, carboxylic, and amide groups . BisphenIn this study, bamboo pulp was mixed with PLA and EPOXY to induce chemical reactions and improve the composite properties. Bamboo pulp was utilized as a reinforcement for PLA, while EPOXY was employed as a crosslinking agent to establish crosslinks between PLA and the bamboo fibers. The mechanical properties, folding cycles, morphology, water resistance, heat resistance, and chemical reactions of the resulting composite were investigated.3, MW 100,000 g/mol, MFI 7 g/10 min at 210 °C. Epoxy resin (Diglycidyl ether of bisphenol A) grade 0302 was purchased from EASY Resin Co., Ltd. Nonthaburi, Thailand. Bamboo pulp  was purchased from Chiang Mai Province, Thailand. Sodium hydroxide (NaOH) analytical reagent grade was purchased from vs. chem house Ltd., Bangkok, Thailand.Polylactic acid 4032D (PLA) pellets were purchased from NatureWorks LLC  at a density of 1.24 g/cmBamboo pulp was prepared without bleaching process to reduce the strong chemical treatment process. The bamboo chips were dried in a hot air oven at 80 °C for 48 h, pretreated with NaOH (18 wt.%) aqueous solution at the pulp ratio of 1:20, and boiled at 85 °C for 3 h to eliminate the residual lignin, fatty acid, hemicellulose, and other impurities. The samples were then dissolved in distilled water and dried in an oven. Then, the dried samples (sizes lower than 250 microns) were sieved.The PLA was melt blended with epoxy resin using two-roll mills a,b with −1 with 4 cm−1 of resolution.The reaction mechanism was studied using ATR method by Fourier-transform infrared spectroscopy . The samples for the infrared spectra (FTIR) were prepared into thin films with a thickness of approximately 100 µm. The IR spectra were measured from 4000 to 500 cmThe tensile strength and elongation at break were determined using a mechanical testing machine . The shape of the dog-bone test was prepared according to JIS K 6251–7. The sample sheets were prepared with dimensions  of 5 mm, 30 mm, and 1 mm, respectively. The samples were kept at 25 °C with 50 ± 2% RH for 24 h. Samples were derived by averaging five experimental runs for each sample.−1.The thermal analysis was performed using a differential calorimeter . Approximately 5–10 mg of each sample was placed in a closed aluminum pan. Differential scanning calorimetry (DSC) analysis was performed from 0 to 200 °C at a heating and cooling rate of 10 °C minSoftening point measurements were performed using Vicat apparatus (c-090), Kijwisai, Bangkok, Thailand with a modified technique that allowed the standard ASTM D1525. The dimensions of the square test samples were 10 × 10 × 3 mm. All samples were derived by averaging five experimental runs for each sample.Contact angles were observed using a DSA30E Krüss GmbH instrument . Drop shape analysis was used to monitor the water contact angle. Water was dropped on the film surface and images were captured automatically every 20 s for 10 min.The morphology was examined using scanning electron microscopy  with a magnification of 2000× at 15 KV. The samples were broken in liquid nitrogen followed by coating with a thin layer of gold.3. A grip clearance of 10 mm was used . The dimensions of the rectangular test samples were 100 × 15 × 1 mmp < 0.05) were evaluated using Duncan’s test.To analyze the results, one-way ANOVA was performed using the Statistical Package for Social Sciences . Differences , C–H, and C–O–C, respectively [−1, aromatic C=C stretching vibrations at 1509 and 1608 cm−1, and aromatic C–H vibrations of the bisphenol A moiety in the hyperbranched epoxy resin at 3059 cm−1 [−1, a stretching vibration of C=O at 1748 cm−1, absorption peaks of C–O–C at 1086 cm−1 and 1178 cm−1, and –OH peaks at 3500–4000 cm−1 [−1 and the aromatic oxirane ring of EPOXY at 912 cm−1. EPOXY reacted with the carboxylic groups, and the reaction between the epoxy groups of EPOXY and the –COOH groups of PLA had been confirmed in a previous study [−1, C=O stretching of PLA at 1748 cm−1, a C–H peak of PLA at 1451 cm−1, aromatic C=C stretching of EPOXY at 1509 cm−1, and an aromatic oxirane ring at 912 cm−1. The intensity of the aromatic oxirane ring peak at 912 cm−1 in the PLA/EPOXY/PULP samples was lower than that in the PLA/EPOXY sample. This decrease indicated the reduction of aromatic epoxy groups, ring opening of epoxy groups, and reacting with OH groups of PULP. Reactions between the epoxy and –OH groups have also been reported [FTIR spectra of PLA, PLA/EPOXY, and PLA/EPOXY/PULP 1–20% are shown in ectively . EPOXY p000 cm−1 . The PLAThe tensile strengths and elongations at the break of the PLA, PLA/EPOXY, PLA/PULP, and PLA/EPOXY/PULP composites are shown in Folding endurance is the most useful index of the bending film test, crucial to evaluating the toughness of a sample. Hence, this method was used to clarify the relationship between the folding test results and the mechanical properties of the films. m, Tc, and Tg of the samples. g and Tm of PLA were 56 °C and 163 °C, respectively. PLA/PULP presented Tg and Tm that were almost identical to those of pure PLA. Adding EPOXY in PLA and PLA/PULP reduced the Tg of PLA from 56 °C to 51 °C  were investigated using DSC. DSC 2nd scan was performed to obtain the crystallinity, Tto 51 °C  owing toectively . The redThe Vicat softening temperature (VST) values of the PLA, PLA/EPOXY, and PLA/EPOXY/PULP composites are shown in Contact-angle measurements were used to evaluate the surface tension of the PLA composites. g, provide flexibility to the PLA sample, and induce crosslinking inside the PLA and PLA/PULP composites. This reaction enhances the crosslinking inside PLA and the interfacial adhesion between PLA and PULP, improving the tensile strength, toughness, thermal, and water resistance properties of the composites. Notably, the resulting PLA composites have excellent properties and can be used for packaging and agricultural applications.A PLA/EPOXY/PULP composite was successfully developed by melt-mixing PLA with PULP and EPOXY. FTIR confirmed the reaction between the epoxy groups of EPOXY and the –COOH of PLA, and the –OH groups of PULP. The tensile strength of PLA/EPOXY/PULP20 increased to 76.8 MPa compared to pure PLA (57.5 MPa). The addition of 20% bamboo pulp in PLA/EPOXY resulted in the highest tensile strength owing to the enhanced mechanical properties of bamboo pulp. However, it also led to a decrease in the elongation at break, indicating reduced flexibility, which can be attributed to the reaction facilitated by EPOXY. PLA/EPOXY/PULP5 showed the best improvement of high toughness properties with 764 folding cycles, 68 MPa tensile strength, and 6% elongation at break. EPOXY decreased the space and induced a connection between the PLA and PULP surfaces. Moreover, EPOXY enhanced the interfacial tension between PLA and the PLA/EPOXY/PULP composites via the EPOXY reaction, improving the water resistance of the composite. PULP acts as a nucleating agent to induce crystallinity in PLA, improving the thermal stability of the composite. Furthermore, EPOXY acts as a plasticizer for PLA to decrease T


APC that otherwise prevents prolonged stabilisation of β-catenin. Surprisingly, no inhibitor compounds for WNT/β-catenin signalling have reached clinical use in part owing to the lack of specific in vivo assays that discriminate between on-target activities and dose-limiting toxicities. Here, we present a simple in vivo assay with a binary outcome whereby the administration of candidate compounds to pregnant and phenotypically normal flox/floxApc mice can rescue in utero death of min/floxApc mutant conceptus without subsequent post-mortem assessment of WNT/β-catenin signalling. Indeed, the phenotypic plasticity of born min/floxApc conceptus enables future refinement of our assay to potentially enable dosage finding and cross-compound comparisons. Thus, we show for the first time the suitability of endogenous WNT/β-catenin signalling during embryonic development to provide an unambiguous and sensitive mammalian in vivo model to assess the efficacy and bioavailability of potential WNT/β-catenin antagonists. Specific signalling thresholds of the WNT/β-catenin pathway affect embryogenesis and tissue homeostasis in the adult, with mutations in this pathway frequently occurring in cancer. Excessive WNT/β-catenin activity inhibits murine anterior development associated with embryonic lethality and accounts for the driver event in 80% of human colorectal cancers. Uncontrolled WNT/β-catenin signalling arises primarily from impairment mutation in the tumour suppressor gene  The canonical WNT/β-catenin signalling pathway provides the core of an evolutionary conserved, fundamental pathway that controls growth and governs numerous developmental processes, while its aberrant regulation contributes to many diseases including cancer ,2,3. DesAPC gene, leading to uncontrolled stabilisation of β-catenin and the formation of constitutive complexes between β-catenin and members of the TCF family [The canonical WNT/β-catenin signalling pathway is activated by the interaction of WNT ligands with their cognate frizzled (FZD) receptors at the cell surface. Upon binding of WNT ligands to FZD, cytoplasmic AXIN proteins associate with phosphorylated lipoprotein receptor-related protein (LRP) to deactivate the adenomatous polyposis coli (APC)/AXIN destruction complex, leading to the stabilisation of β-catenin through phosphorylation and its subsequent translocation to the nucleus where it binds to T-cell factor (TCF) family members to upregulate target genes . Under hF family ,16.In the mouse embryo, the WNT/β-catenin signalling pathway is essential for the establishment of the anterior-posterior axis during gastrulation and for the development of the neural tube, somites, and limbs ,18. ThusApc mutant alleles, floxApc and minApc. In its naïve, unrecombined state, the floxApc allele harbours two loxP recombination sites flanking exon 14 of the Apc gene, thereby rendering this allele transcriptionally active albeit producing reduced expression of full-length 2843 amino acid APC protein [minApc allele encodes a nonsense mutation at codon 850, resulting in the expression of a non-functional, truncated APC protein that is unable to bind to β-catenin and therefore prevents effective degradation of β-catenin [min/flox;ctnnb1+/−Apc offspring [min/floxApc embryos could serve as a bimodal readout for a low-throughput screening platform to evaluate the specificity and biological availability of antagonists for canonical WNT signalling. Here, we provide proof-of-principle evidence for the validity of this hypothesis by showing that timed administration of WNT pathway inhibitors during pregnancy rescues embryonic lethality and anterior specification in min/floxApc embryos.In the present study, we harnessed genetic mouse models with various activation levels of canonical WNT signalling using two different hypomorphic  protein . Meanwhi-catenin . While bffspring . This obAll mouse experiments were carried out in compliance with every pertinent ethical regulation for animal research and were authorised by the local Animal Ethics Committee of Austin Health.min/+Apc and flox/floxApc mice have been previously described [escribed ,23 and ww/v) hydroxypropyl methylcellulose, 0.5% (v/v) benzyl alcohol, 0.4% (v/v) Tween 80) and given at 25 mg/kg by oral gavage.The tankyrase inhibitor G007-LK was administered to the diet by providing commercially prepared feed pellets  at an inclusion rate of 600 mg per kg chow. The daily food intake of individually housed mice is about 15–20 g per 100 g body weight , which e2O2 in PBS for 20 min at RT to block endogenous peroxidases followed by immersion in normal goat serum  for 1 h at RT to block nonspecific binding sites. Tissue sections were stained with various primary antibodies  in blocking buffer for 16 h at 4 °C. Excess antibody was removed by washing twice with 1× PBS/0.1% Tween and once with 1× PBS for a total of 30 min followed by incubation with secondary antibodies at RT for an additional 30 min. After three washes in 1× PBS, the sections were immersed in diluted (1:200 PBS) ABC solution  at RT for 30 min. Staining was developed by adding DAB to tissue sections, and the staining process was stopped by adding distilled water to the sections. Hematoxylin was used for counterstaining of tissue sections followed by incubation in ethanol gradients  and xylene for dehydration. Tissue sections were set in mounting media.The formalin-fixed and paraffin-embedded tissue samples were cleared in two successive xylene baths for 10 min each before being rehydrated in tiered ethanol baths . Tissue antigen retrieval was performed in sodium citrate buffer  or EDTA buffer at 95 °C for 18 min. Tissue sections were then incubated in 3% H® Reagent , and 2 µg of total RNA was used as input for cDNA preparation with the high-capacity cDNA reverse transcription kit  as per the manufacturer’s instructions. Quantitative (q)RT-qPCR analysis was conducted using the SensiMix SYBR kit  in technical triplicates on the ViiA 7 Real-Time PCR system .Total RNA was prepared from small intestines using Trizol2, 25 slices, and number of excitations = 2. Mice were imaged with a small animal camera .Magnetic Resonance Imaging (MRI) acquisition was performed using a T2-weighted sequence using a 2D Fast Spin Echo sequence (FSE). The parameters for 2D FSE are slice thickness = 1 mm, resolution= 0.21 × 0.21 mmt tests, and 2 × 2 contingency tables were calculated using Fisher’s exact test. Sample size calculation was carried out using the sample size calculator at ClinCalc.com . Data are expressed as mean ± standard error of the mean.Statistical analyses were carried out in Prism8 . Statistical significance was assessed by two-tailed unpaired min/floxApc embryos fail to develop anterior head structures from E10.5 onwards and subsequently die in utero at E15. Because this phenotype is prevented by monoallelic ablation β-catenin in corresponding min/floxApc embryos [min/floxApc embryos and overcome the associated embryonic lethality. Hence, we set up timed matings between female flox/floxApc with male min/+Apc mice to yield equal ratios of min/floxApc and flox/+Apc conceptions, with only the flox/+Apc embryos developing to term.WNT pathway inhibitors are a commodity in high demand owing to the large number of pathologies associated with aberrant, or excessive WNT/β-catenin signalling. In order to develop a sensitive assay with an unambiguous in vivo efficacy and toxicity readout for WNT pathway inhibitors, we made use of our previous observations that murine  embryos , we hypomin/+Apc mice without loss-of-heterozygosity of the wild-type Apc allele, and that monoallelic ablation of β-catenin rescued the anterior defect in min/floxApc embryos [flox/floxApc mice. When administered as G007-LK-containing chow over three consecutive days immediately prior to RNA expression analysis of the intestinal epithelium, this treatment reduced expression by 50–70% across Axin2, Sox9, Cd44, and Ccnd1  G007-LK ,10. This embryos ,27, we end Ccnd1 .minApcflox/ embryos.In light of the importance of the WNT/β-catenin pathway during gastrulation and many other subsequent stages of embryonic development ,30,31,32minApcflox/ embryos at the anterior border of the embryonic visceral endoderm [floxApcflox/ mice between E2–3 and E4–7 resulted in disrupted embryonic development as judged by the failure of pregnant mice to gain weight and develop an abdominal bulge despite the presence of a vaginal mating plug. We made a similar observation when G007-LK-containing chow was administered to “plugged” females during the corresponding E5–14 and E5–10 stages of embryonic development. Meanwhile, the administration of G007-LK-containing chow during the E5–6 stages yielded deformed and non-viable embryos at E15 that underwent subsequent spontaneous abortion  of the live pups and all the three stillborn pups returned minApcflox/ genotypes. Strikingly, all stillborn pups had cranial defects, while all live minApcflox/ pups had anophthalmia, including five with bilateral anophthalmia and three with a monolateral phenotype . This increase is statistically highly significant when compared to the background rate of anophthalmia in either wild-type or litters of treatment-naïve flox/floxApc mice  suggests that our model could be further refined to also provide insights into dose-activity relationships for putative inhibitors. Our data presented here provide striking evidence that our system faithfully records the activity of inhibitors that functionally act down-stream of Apc in the canonical WNT signalling cascade. However, we have previously found that min/floxApc cells remained responsive to WNT ligands, implying that the “rescue” of the developmental defects in min/floxApc embryos may potentially also result from interference with WNT signalling up-stream of Apc.Here, we show that in utero development of placenta . Moreovemin/floxApc mice here provide preliminary biological insights into the relationship between specific biological processes and the associated signalling thresholds. For instance, ectopic activation of WNT/β-catenin signalling in the lens placode prevents lens specification, and this is associated with deficiency of the Paired Box 6 (Pax6) gene [min/floxApc pups. We also observed expansion of the WNT target gene Cyp2e1 and the loss of periportal maker carbamoyl-phosphate synthase in 6-month-old min/floxApc mice  gene ,40,41. Ilox mice , suggestflox/floxApc and the highest in min/floxApc cells, with the activity in min/+Apc cells being intermediate [min/floxApc mice than in min/+Apc mice (6 mo vs. <3 mo; min/floxApc allele combination. By contrast, the signalling threshold enabling hepatocellular carcinomas is below that of flox/floxApc cells, since HCC occurs in the latter mice at 18 months of age [min/+Apc mice succumbed to intestinal tumours. Meanwhile, complete inactivation of Apc in the livers of CreTTR; flox/floxApc mice results in severe hyperammonemia and hyperglutaminemia due to changes of metabolic liver zonation changes resulting in the rapid onset of fatality [Comparing phenotypes across allelic series identifies threshold levels between physiological and pathological consequences, including the anterior–posterior axis formation discussed here. We have previously ranked the three genotypes discussed here with respect to responding to the WNT3a ligand, suggesting the lowest activity in rmediate . Interess of age  and notafatality . Thus, aCollectively, our findings here further support the existence of tissue and development-specific ‘just-right’ WNT signalling activation thresholds that demarcate between responses compatible with tissue homeostasis and those required for regenerative processes and/or underlying the development and progression of disease . A betteIn conclusion, we present a murine in vivo model based on a simple binomial readout to test the activity of putative WNT antagonists, thereby providing a mammalian development-based alternative to the popular Xenopus axis duplication assay that has been used for over 30 years to study the WNT pathway and assess activities of its signalling antagonists ,45,46.


Ferroptosis, a unique form of non-apoptotic cell death, is dependent on iron and lipoperoxidation, and has been shown to be associated with the pathogenesis of inflammatory bowel disease (IBD). Human umbilical cord mesenchymal stem cell-derived exosomes (hucMSC-Ex) are involved in cell survival, immune conditioning, and damage repair. However, the relationship between hucMSC-Ex, IBD, and ferroptosis is unknown. This paper explores the role of hucMSC-Ex in the repair of IBD through the regulation of the ferroptosis signaling pathway.In this study, we used small RNA sequencing to find that miR-129-5p was highly expressed in hucMSC-Ex, and by predicting its targeting to ACSL4, we verified the effect of miR-129-5p on mice IBD in vitro and human colonic epithelial cells (HCoEpiC) in vivo. We found that miR-129-5p reduces ferroptosis in intestinal epithelial cells by targeting ACSL4 to repair IBD, which provides new strategies for the prevention and treatment of IBD.In conclusion, our results demonstrate that hucMSC-Ex relieves IBD by targeting ACSL4 with miR-129-5p to inhibit lipid peroxidation (LPO) and ferroptosis, reducing intestinal inflammation and repairing damages.− mediates the transport of extracellular cystine into the cell, which gets reduced to cysteine to participate in GSH-mediated metabolism. GPX4 strongly inhibits ferroptosis by helping scavenge reactive oxygen species. The depletion of GSH correlates with decreased GPX4, and the imbalance of the antioxidant system leads to the formation of toxic phospholipid hydroperoxide, which promotes the occurrence of ferroptosis with the participation of irons. HucMSC-Ex has the ability to relieve GSH and GPX4 depletion and repair the intracellular antioxidant system. Ferric ions enter the cytosol through DMT1 and participate in lipid peroxidation. HucMSC-Ex can reduce the expression of DMT1 and alleviate this process. HucMSC-Ex-derived miR-129-5p targets ACSL4 and reduces the expression of ACSL4, an enzyme that mediates the conversion of PUFAs into phospholipids in intestinal epithelial cells, and is a positive regulator of lipid peroxidation. Abbreviations: GSH, glutathione; GPX4, glutathione peroxidase 4; GSSG, oxidized glutathione; DMT1, divalent metal transporter 1; ACSL4, acyl-CoA synthetase long-chain family member 4; PUFAs, polyunsaturated fatty acids; ALOXs, lipoxygenases; CoA, coenzyme A; PL, phospholipid; PLOOH, hydroperoxides, LOH, phospholipid alcohols; LPO, lipid peroxidation.Mechanism of hucMSC-Ex inhibiting ferroptosis in intestinal epithelial cells. System Xc Inflammatory bowel disease (IBD) is a chronic and persistent intestinal inflammatory disease with unknown etiology. It is usually believed to be related to intestinal microbiota imbalance, environmental, and genetic factors. IBD is mainly classified as ulcerative colitis (UC) and Crohn’s disease (CD) and has evolved with industrialization into a global disease, seriously affecting the living standards and well-being of patients . CurrentMesenchymal stem cells (MSCs), as ideal seed cells, have achieved significant efficacy in the treatment of IBD. Studies have shown that MSCs are mainly involved in tissue damage repair through the paracrine pathway, and extracellular vesicles (EVs) are the main effector components of MSCs to exert their paracrine effects. EVs are composed of exosomes and microvesicles (MVs), which are involved in physiological and pathological processes such as cell survival, immune regulation, angiogenesis, and tissue regeneration , 4. ExosMicroRNAs (miRNAs) are short-chain RNA molecules that regulate protein transcription in the translation stage and exert their biological effects by targeting different mRNAs. They induce gene silencing by complementarily targeting the 3 ‘UTR of the mRNA . This pr3+) enters the endosome of the cells and is reduced to divalent iron (Fe2+). Divalent metal transporter 1 (DMT1) mediates the release of iron ions from endosomes to the cytoplasm. Excessive iron accumulation produces reactive oxygen species (ROS) through the Fenton reaction and destroys cells. LPO is under steady-state metabolic control and peroxidation occurs only when the indispensable constraint conditions are met and the anti-peroxidation mechanism breaks down. The activity of selenoperoxidase glutathione peroxidase 4 (GPX4) is the cornerstone of the anti-peroxidation defense, which utilizes glutathione (GSH) synthesis to combat LPO [In addition to the two important forms of cell death, apoptosis and necrosis, non-apoptotic death, including necrosis, pyroptosis, ferroptosis, and other gene-regulated death processes, has attracted research attention . Ferroptmbat LPO . Depletimbat LPO . LPO prombat LPO , 21–23. mbat LPO . There ambat LPO .IBD is thought to be associated with diets rich in PUFAs because of the increased incidence of IBD in developed Western countries and the increasing incidence in developing countries, where people consume a PUFA-rich Western diet –28. Stud3 pieces and cultured in α-MEM (hyclone) medium supplemented with 15% fetal bovine serum  and 1% Penicillin-Streptomycin-Amphotericin B Solution (Procell), with the solution changed every other day. Around 7–10 days, hucMSCs were seen migrating from the tissue. HucMSC was cultured in α-MEM supplemented with 10% FBS and 1% Penicillin-Streptomycin-Amphotericin B Solution. After 24 h of culture, the supernatant was discarded and the medium was replaced with fresh 10% α-MEM. The FBS supplemented with the medium was processed by hyper-centrifugation to remove the exosomes. The supernatant was collected after 48 h of culture for subsequent experiments.Human umbilical cords were cut into 1mmAfter a series of centrifugation to remove dead cells and debris, the collected cell supernatant was placed in a 100 kDa ultrafiltration tube (Millipore) for further centrifugations. The supernatant obtained by centrifugation was further centrifuged at 100,000 g, then the supernatant was discarded, resuspended in PBS, filtered through a 0.22 μm filter (Millipore), and stored at -80 ° C for later use. NanoSight nanoparticle tracking analysis (NTA) was used to determine the particle number and particle size of exosomes. The morphology of exosomes was observed by transmission electron microscopy (TEM). Western blot was used for the identification of exosome surface markers CD9, CD81, Calnexin, and ALIX . Transfection of exosomes was performed using Exo-Fect™ Exosome Transfection Reagent (SBI) according to the manufacturer’s instructions.2. The transfection system was configured with mimics (Gamma gene) and Lipofectamine 2000 (Invitrogen). 1 × 106 HCoEpiC were plated in a 6-well plate. When the cell density reached 60-80%, the supernatant was changed into a serum-free DMEM medium, and the above transfection system was added. After 4 h of incubation in the dark, the medium was changed into a complete medium for another 24 h. Cells were harvested for subsequent experiments.Human colonic epithelial cells (HCoEpiC) were purchased from Nanjing Saihongrui Biotechnology Co., Ltd. HCoEpiC and human embryonic kidney cell line (HEK293T) were cultured in DMEM medium (hyclone) with 10% FBS and 1% Penicillin-Streptomycin-Amphotericin B Solution, and incubated at 37℃ with 5% COHCoEpiC were seeded into a 96-well plate at 1,500 cells/well with 100 µl of 10% FBS DMEM. Cells were divided into the NC group, LPS group, and hucMSC-Ex group. The NC group was replaced with fresh 10% FBS DMEM, while the LPS and hucMSC-Ex groups were replaced with 10% FBS DMEM containing 2 µg/ml LPS (Sigma). The hucMSC-Ex group was treated with an additional 400 µg/ml exosomes and all setups were incubated for 24 h. Cell viability was determined using a CCK-8 reagent (Vazyme).Male BABL/C mice  were purchased from the Animal Research Center of Jiangsu University . Relevant studies were carried out with the approval of the Ethics Committee and the Laboratory Animal Management and Use Committee of Jiangsu University. The mouse model of colitis was established by freely drinking 3% dextran sodium sulfate (DSS). Mice were randomly divided into three groups: NC group, DSS group, and hucMSC-Ex group. Mice in the NC group were given tap water, while mice in the DSS group and the hucMSC-Ex group were given tap water supplemented with 3% DSS. On the third, sixth, and ninth days after drinking 3% DSS, the NC and DSS groups were injected with sterile PBS, and the hucMSC-Ex group was injected with 1 mg/20 g weight of hucMSC-Ex. The mice were weighed at the same time every morning, and their living conditions and stool traits were observed. On day 10, mice were sacrificed and colorectal tissues were extracted for further experiments.Colonic tissues of each group were harvested and rinsed twice with pre-cooled PBS buffer, and the tissues were accurately weighed for later use. Colon tissue homogenate was prepared according to the instructions of the T-GSH detection kit . The concentration of T-GSH was obtained by measuring the absorbance at 405 nm with a microplate reader.Total proteins of cells and tissues were extracted with RIPA lysate (Solarbio) supplemented with protease inhibitors PMSF (Solarbio). The concentration of total proteins was measured using the BCA(Vazyme). The total protein was mixed with loading buffer (Life Technologies) at a ratio of 3:1 and boiled at 100℃ for 8 min. According to the concentration, the protein samples were added to the 10% SDS polyacrylamide gel  and the proteins were separated by electrophoresis according to their different molecular weights. The protein on the gel was transferred to polyvinylidene fluoride PVDF membrane (Millipore) and blocked in 5% skim milk for one and a half hours, followed by incubation with primary antibodies at 4℃ for one night. After washing with 1× TBS/T buffer, the membranes were incubated with the secondary antibody for 1 h at 25℃. The protein bands were visualized with a chemical gel imaging system (GE). The primary antibodies used were: anti-CD9 , anti-CD81 , anti-Alix , anti-Calnexin , anti-ACSL4 , anti-GPX4 , anti-DMT1 , anti-COX2  anti-PCNA , anti-β-actin , anti-Claudin-1 , and anti-Occludin . The secondary antibody used were: Goat anti-rabbit IgG, FITC conjugated , and Goat anti-rabbit IgG .Total mRNA from tissues and cells was extracted by Trizol (Vazyme). The mRNA was reverse transcribed using the reverse transcription kit (Vazyme). The real-time fluorescence quantification PCR (qRT-PCR) was performed to measure the transcript abundance of the genes with Synergy Brands (SYBR) Green detection(Vazyme). The relative expression level of the target gene was calculated using mRNA expression of β-actin as a control. The sequences of primers used are listed in Table The binding site between the 3’UTR of ACSL4 mRNA and miR-129-5p was predicted by Starbase. Plasmids containing wild-type and mutant 3’UTR of ACSL4 were obtained from Fenghbio. The mimics, mimics-NC, inhibitor, and inhibitor-NC of miR-129-5p and the plasmid carrying ACSL4 3’UTR were co-transfected into HEK293T cells. After 24 h, transfection efficiency and luciferase activity were measured by qRT-PCR and the dual luciferase assay kit (Vazyme).Colonic cell apoptosis was determined by TUNEL staining. After routine dewax, paraffin sections were incubated with 20 µg/ml proteinase K for 20–30 min before staining according to the instructions. The green fluorescence (FITC) at 520 ± 20 nm and blue fluorescence (DAPI) at 460 nm were observed by fluorescence microscope.The tissue in paraffin sections was dewaxed and then blocked with a 3% hydrogen peroxide solution for endogenous peroxidase at room temperature. The antigen was repaired with stimulation buffer by steaming in an electric oven for 30 min, and the non-specific antigen was blocked by incubation with 5% BSA solution. The paraffin sections were then incubated with an anti-ACSL4 antibody  at 4℃ overnight, and the secondary antibody was applied at 37℃ for 30 min. Subsequently, DAB (BOSTER) was used for color development and dehydrated in ethanol. Pictures were acquired using a pathology section scanner.Colonic tissues were fixed in 4% paraformaldehyde, dehydrated, and embed. The sections on slides were dewaxed and soaked in Perls Stain (Solarbio) for 25 min. After 1–2 min of washing with distilled water, the samples were immersed in a nuclear solid red dye solution for 5 min and washed. The samples were routinely dehydrated and resin sealed. Pictures were obtained with a pathological section scanner.The cells were plated at low density and fixed with 4% paraformaldehyde for 30 min at room temperature. The cells were incubated with Triton X-100 (0.1%) for 30 min to increase the membrane permeability to the antibody and blocked with 5% BSA. They were then incubated with the primary antibody at 4℃ overnight , followed by the fluorescent secondary antibody at 37℃ for 1 h. The slides were sealed with a prolonged live antifade reagent containing DAPI. Images were obtained by a confocal laser microscope (Nikon).The mice’s colonic tissues were fixed in 4% paraformaldehyde and embedded in paraffin. Theparaffin-embedded tissue was cut into 4-µm sections, dehydrated in ethanol, and stained with hematoxylin and eosin for light microscopy. Pictures were obtained with a pathological section scanner.All data were expressed as mean ± SEM. Statistical analysis was performed by using GraphPad Prism software for analysis of variance. Student’s t-test was performed for comparisons between any two groups. One-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison tests was performed for multiple groups. P ≤ 0.05 was considered statistically significant.11particles/mL and particle size between 30 and 300 nm . The results showed hucMSC-Ex concentration of 3.7 × 10To verify the repair effect of hucMSC-Ex on IBD mice, 3% DSS was used to induce IBD, and hucMSC-Ex was injected via the tail vein. The results showed that the weight of the mice in the NC group increased over time, while the weight of the DSS group did not. The hucMSC-Ex treated group increased in weight with time but began steadily decreasing on the sixth day Fig. a. Mice iNext, we analyzed the ferroptosis-related indicators of mice colon tissues to explore whether hucMSC-Ex can regulate ferroptosis. The results of qRT-PCR showed that the mRNA expression levels of divalent metal transporter 1 (DMT1), cyclooxygenase 2 (COX2), and ACSL4 significantly increased but GPX4 decreased in the DSS group, while hucMSC-Ex treatment downregulated DMT1, COX2, and ACSL4 and upregulated GPX4 Fig. a. Since After determining that ACSL4 is highly expressed in the intestinal epithelial cells of IBD mice, promoting LPO and iron transport/metabolism disorder, we selected human normal colon epithelial cell line HCoEpiC to verify the repair effect of hucMSC-Ex on IBD in vitro. An in vitro inflammatory model was established using LPS induction and treated with hucMSC-Ex. After LPS treatment, the expressions of pro-inflammatory factors TNF-α, IL-6, and IL-1β were increased, while the expression of anti-inflammatory factor IL-10 was decreased, and the trend was reversed after hucMSC-Ex treatment , miR-129-5p has the ability to target ACSL4 and we hypothesize that it can influence the expression of the ACSL4 protein (Fig. Studies have shown that hucMSC-Ex acts as an anti-inflammatory and repair agent by serving as a medium for information exchange between cells. Our research group conducted small RNA sequencing on hucMSC-Ex and HFL1-Ex, and found that miR-129-5p was highly expressed in hucMSC-Ex Fig. , we seleAfter identifying the relationship between miR-129-5p and ACSL4, we verified the relationship between miR-129-5p and ferroptosis in HCoEpiC. We transfected HCoEpiC with miR-129-5p mimics, inhibitors, or hucMSC-Ex, and compared their effects on LPS-induced ferroptosis. The results of immunofluorescence showed that the fluorescence intensity of ACSL4 protein in hucMSC-Ex and miR-129-5p mimics groups was inhibited, while the miR-129-5p mimics-NC, inhibitor, and inhibitor-NC groups were the same as that in LPS group, which indicated that hucMSC-Ex and miR-129-5p had the same inhibitory effect on the expression of ACSL4 protein Fig. a. Next, After the in vitro inhibitory effect of miR-129-5p on ferroptosis was verified, we wondered whether hucMSC-Ex played a role in alleviating IBD through miR-129-5p. We used hucMSC-Ex as a nanocarrier, transfected miR-129-5p into hucMSC-Ex using an exosome transfection kit, and injected hucMSC-Ex carrying miR-129-5p mimics and inhibitors into DSS induced IBD mice via tail vein. The results showed that the body weight of mice in the NC group increased steadily while that of the DSS group decreased significantly at the later stage. The body weight of the mimics group increased steadily up to day 6 and began to decrease at the last stage of the model. Weight gain in the mimics-NC and inhibitor groups was similar to that in the mimics group, while weight loss was significant in the inhibitor-NC group Fig. a. DAI scAfter demonstrating that miR-129-5p could alleviate DSS-induced IBD in mice using hucMSC-Ex as the vector, we further explored the effects of hucMSC-Ex transfected with miR-129-5p mimics and inhibitors on LPO and ferroptosis. We first analyzed the molecules related to ferroptosis in the colon tissues of each group of mice. The results were similar to in vitro experiments, as the miR-129-5p mimics group could significantly reduce the mRNA expression levels of DMT1, COX2, and ACSL4 and reverse the depletion of GPX4, while the other transfection groups increased the ferroptosis-related molecules compared to the mimics group Fig. a. In addIn this study, we explored the repair effect of hucMSC-Ex on IBD in vivo and in vitro and demonstrated that hucMSC-Ex inhibits LPO by reducing the markers of ferroptosis to repair IBD. In our previous studies, we reported that hucMSC-Ex, an emerging cell-free therapy, shows a strong IBD repair effect –38. RegaLPO is a key marker of ferroptosis, where PUFAs serve as the substrate and ACSL4 mediates the synthesis of phospholipids from CoA and PUFAs , 43. SevIron overload has long been reported in alcoholic liver disease, cardiomyopathy, and spinal cord injury –51. It hWe also examined how hucMSC-Ex functions as an inhibitor of ferroptosis in IBD. Exosomes are nanoscale vesicles containing a variety of bioactive substances, which have been shown to activate certain adenosine, mediate the activity of related pathways, and inhibit the expression of certain inflammatory factors , 54. ResIn conclusion, our results demonstrate that hucMSC-Ex relieves IBD by targeting ACSL4 with miR-129-5p to reduce LPO levels and ferroptosis, reducing intestinal inflammation and repairing the damage.