Collagen Expression Research Articles

Effect of moderate-intensity aerobic exercise on bladder TGF-β1 and type I collagen expressions in diabetic rat model

Transforming growth factor-beta 1 (TGF-β1) and type I collagen play crucial roles in the pathogenesis of diabetic bladder disease (DBD). Moderate-intensity aerobic exercise increases antioxidant activity to help manage DBD. The aim of this study was to evaluate the effect of moderate-intensity aerobic exercise on the expression of TGF-β1 and type I collagen in the detrusor and lamina propria of the bladder in a type 2 diabetes mellitus (T2DM) rat model. A true experimental design with a post-test-only control group design was conducted with white rats (Rattus norvegicus), divided into three groups: a T2DM model group sacrificed after T2DM induction and diagnosed with T2D from a fasting blood glucose (FBC) test (Group C), a T2DM model group that did not receive exercise (Group NE), and a T2DM model group that received moderate-intensity aerobic exercise (Group E). Moderate-intensity aerobic exercise was conducted over six weeks, with a frequency of five days per week for 60 minutes per session. The findings revealed a significant reduction in TGF-β1 expression in the lamina propria in Group E compared to Group C (p=0.004) Additionally, both Group E (p=0.002) and Group NE (p=0.028) showed a significant reduction in type I collagen expression in the lamina propria compared to Group C. These findings provide a basis for further investigation regarding the mechanism of non-pharmacologic DBD management by employing moderate-intensity exercise.

Human umbilical cord mesenchymal stem cells restore chemotherapy-induced premature ovarian failure by inhibiting ferroptosis in vitro ovarian culture system

BackgroundMesenchymal stem cells (MSCs) have shown potential in repairing chemotherapy-induced premature ovarian failure (POF). However, challenges such as stem cell loss and immune phagocytosis post-transplantation hinder their application. Due to easy and safe handling, in vitro ovarian culture is widely available for drug screening, pathophysiological research, and in vitro fertilization. MSCs could exhibit therapeutic capacity for ovarian injury, and avoid stem cell loss and immune phagocytosis in vitro tissue culture system. Therefore, this study utilizes an in vitro ovarian culture system to investigate the reparative potential of human umbilical cord mesenchymal stem cells (hUCMSCs) and their mechanism.MethodsIn this study, a chemotherapy-induced POF model was established by introducing cisplatin in vitro ovarian culture system. The reparative effects of hUCMSCs on damaged ovarian tissue were validated through Transwell chambers. Tissue histology examination, immunohistochemical staining, Western blotting, and RT-PCR were employed to evaluate the expression effects of hUCMSCs on ferroptosis and fibrosis-related genes during the process of repairing cisplatin-induced POF.ResultsCisplatin was found to activate ovarian follicles in vitro POF model. Transcriptomic sequencing analysis revealed that cisplatin could activate genes associated with ferroptosis. hUCMSCs alleviated cisplatin-induced POF by suppressing the expression of ferroptosis. Moreover, inhibiting ferroptosis by hUCMSCs also ameliorated ovarian hormone levels and reduced the expression of fibrosis-related factors α-SMA and COL-I in the ovaries.ConclusionsThis study confirms that cisplatin-induced ovarian damage via ferroptosis in vitro POF model, and hUCMSCs repair ovarian injury by inhibiting the ferroptosis pathway and suppressing fibrosis. This research contributes to evaluating the effectiveness of hUCMSCs in treating chemotherapy-induced POF by inhibiting ferroptosis in an in vitro ovarian culture system and provides a potential therapeutic strategy for chemotherapy-induced POF.

Inhibition of GDNF-Driven Macrophage-to-Myofibroblast Transition Protects Against Colitis-Associated Intestinal Fibrosis.

Glial cell line-derived neurotrophic factor (GDNF) has been demonstrated to promote the development of liver fibrosis, but its role in intestinal fibrosis is unknown. Macrophage-to-myofibroblast transition (MMT) is an important pathway contributing to fibrosis diseases. However, whether MMT cells, characterized by co-expressing both macrophage (CD68 or F4/80) and myofibroblast (α-SMA) markers, occurs in intestinal fibrosis remain to be addressed. Here, we showed that GDNF expression and the infiltration of MMT cells in intestinal tissues from patients with fibrostenotic Crohn's disease (CD) and a mouse model of chronic dextran sodium salt-induced intestinal fibrosis were significantly increased. GDNF induced bone marrow-derived macrophages (BMDMs) differentiation into MMT cells in vitro. Mechanistically, the Src pathway was activated by GDNF stimulation and contributed to GDNF-induced MMT in BMDMs. Moreover, pharmacological inhibition of GDNF by using antibody markedly decreased the infiltration of MMT cells following the decrease of collagen deposition and α-SMA and Col1 expression in the mouse model of colitis-associated intestinal fibrosis. In conclusion, GDNF is able to induce MMT and contributes to intestinal fibrosis in the context of chronic intestinal inflammation. Pharmacological inhibition of GDNF-driven MMT might provide a novel approach for the treatment of fibrosis complication in CD.

Short-Term Culture of Human Hyalocytes Retains Their Initial Phenotype and Displays Their Contraction Abilities

Background: Hyalocytes are the main vitreal cell types with critical functions in health and vitreoretinal diseases. Our aim was to develop cultures of human hyalocytes and verify the retention of their initial cellular features after 3 and 6 days of culturing (3 d and 6 d) by analyzing and comparing a few morphological and functional parameters. Methods: Vitreous samples (n = 22) were collected and vitreous cells and bead-enriched hyalocytes were developed and compared (3 d vs. 6 d cultures). Vitreous and conditioned media were tested for collagen, vascular endothelial growth factor (VEGF), transforming growth factor β1 (TGFβ1), nerve growth factor (NGF), matrix metalloproteinases (MMPs)/tissue inhibitors of metalloproteinases (TIMPs) and alpha-smooth muscle actin (αSMA) expression (ELISA, array/IP/WB, RT-PCR). Cells were observed at light and fluorescent microscopy (phenotypical properties) and tested for their 3D collagen gel contraction abilities. Results: An increased expression of collagens, vimentin, fibronectin, and the MMP9/TIMP1 ratio were observed in vitreous tissues. In 3 d cultures, collagens and MMP9 were upregulated while the related tissue-enzymes were deregulated. Vitreous samples also showed high levels of TGFβ1, VEGF, and NGF, and this protein signature was retained at 3 d while decreased at 6 d. The original phenotype (low αSMA) was retained at 3 d from seeding while an increased αSMA expression was observed at 6 d; NGF/trkANGFR was expressed in cultured hyalocytes and partially drives the collagen retraction. Conclusions: The vitreous print comparison between untouched and cultured hyalocytes allowed us, on one side, to select 3 d cultures and, on the other, to highlight the neuroprotective/contractile NGF in vitro hyalocytes effects. The possibility of scoring reactive hyalocytes would represent an interesting aspect of screening the vitreoretinal interface severity.

Cartilage extracellular matrix collagen type II and aggrecan expressions in rabbit cartilage following mesenchymal stem cell implantation

Collagen II and aggrecan are the major components of the cartilage extracellular matrix (ECM) that determine the structure and function of the hyaline articular cartilage. Therefore, their examination should be included in the evaluation of the repair tissue, which is formatted into osteochondral defects (ODs). In this study, the effect of allogeneic-derived stem cells (DSCs), including bone marrow-DSCs (BMDSCs), adipose-DSCs (ADSCs), and synovial membrane-DSCs (SMDSCs), implantation on collagen II and aggrecan production was evaluated. Forty-eight New Zealand rabbits underwent the creation of 3×3mm sized ODs in the femoral patellar groove on both knees and were then randomly assigned to three groups. In all groups, the ODs in the right limbs were filled with fibrin glue (FG), whereas the ODs in the left limbs were filled with ADSCs+FG in group A, with BMDSCs+FG in group B and with SMDSCs+FG in group C. After 12 weeks the repair tissues were immunohistologically assessed for collagen II and aggrecan. Accordingly, FG and ADCSs (p=0.003), BMDSCs (p=0.017), and SMDSCs (p=0.003) implantation resulted in significantly stronger expression of collagen II in the reparative tissue compared with the FG groups. Also, the newly formed tissue in the BMDSCs+FG (p=0.015) and SMDSCs+FG (p=0.003) groups expressed significantly higher aggrecan compared with the FG groups, whereas BMDSCs+FG (p=0.030) and SMDSCs+FG (p=0.002) transplantation promoted significantly higher aggrecan composition compared with ADSCs+FG. These findings indicate the profound impact of BMDSCs and SMDSCs on the ECM quality of the repair tissue. Additionally, the inefficiency of FG to induce a well-developed ECM in the repair area was highlighted.

Study on the mechanism of OSM participating in myocardial fibrosis by inhibiting TGFβ-induced EndMT of cardiac microvascular endothelial cells through SPARC/SMAD signaling.

Cardiac fibrosis constitutes a crucial element in the progression of diverse chronic cardiac conditions. Notably, a significant correlation has been observed between the endothelial-to-mesenchymal transition (EndMT) and the emergence of cardiac fibrosis. To investigate mechanisms, we employed immunofluorescence for α-SMA and CD31 analysis, Western blotting for CD34, vimentin, and SPARC overexpression. CCK8, wound healing, and transwell assay-assessed cell viability, invasion, and migration. SPARC overexpression plasmid was constructed and validated by Western blotting. Fibrosis levels were quantified via Masson staining, and collagen 1 and 3 expressions were measured using ELISA assays. Notably, in TGF-β-induced H5V cells, the downregulation of CD31 and CD34 expression, along with the upregulation of α-SMA and vimentin, suggests the induction of EndMT in cardiac fibrosis. Interestingly, OSM treatment mitigated EndMT progression, cell invasion, migration, and the expression of p-SMAD2, p-SMAD3, and SPARC in TGF-β-treated H5V cells. Further analysis revealed that OSM alleviated TGFβ-induced EndMT, invasion, and migration of cardiac microvascular endothelial cells by suppressing SPARC/SMAD signaling. Moreover, OSM therapy notably mitigated myocardial tissue fibrosis, along with a reduction in the expression of collagen 1, collagen 3, α-SMA, and CD34, while augmenting CD31 and vimentin expression in ISO-induced myocardial tissue. Additionally, OSM exhibited the ability to suppress myocardial tissue fibrosis and the expression of EndMT markers as well as SPARC/SMAD signals in ISO-induced myocardial tissue. Our comprehensive analysis unveiled that OSM contributes significantly to myocardial fibrosis modulation by inhibiting TGFβ-mediated EndMT in myocardial microvascular endothelial cells via SPARC/SMAD signaling.

Quercetin Alleviates Scleral Remodeling Through Inhibiting the PERK-EIF2α Axis in Experiment Myopia.

This study aims to investigate the effect of quercetin (QUE) on scleral remodeling by inhibiting the PERK-EIF2α signaling pathway and to evaluate its potential role in slowing myopia. Lens-induced myopia (LIM) guinea pigs were obtained and treated with QUE. After 4 and 6 weeks of treatments, ocular biological measurements were conducted. Hematoxylin and eosin (H&E) staining was used to observe the changes in scleral morphology and thickness, and Masson staining was used to examine scleral collagen fiber arrangement. Quantitative PCR (qPCR) and Western bolt were utilized to detect the mRNA and protein expression of PERK, EIF2α, MMP-2, TIMP-2, and collagen I in the scleral tissues. Calcium ion flow in each group was measured using noninvasive micro-test technology, and reactive oxygen species levels were detected by flow cytometry. Compared with the LIM group, the ocular measurements showed that the refractive errors and axial length of the eyes were significantly reduced in the LIM+QUE group (P < 0.01). H&E and Masson staining showed that sclera in the LIM+QUE group was thickened, collagen was dense, and the fiber gap was reduced. In the LIM+QUE group, the expression levels of PERK, EIF2α, and MMP-2 were decreased, whereas the expression levels of TIMP-2 and collagen I were increased. Calcium release and reactive oxygen species (ROS) in the LIM+QUE group were decreased. Quercetin ameliorates scleral remodeling in myopic guinea pigs by inhibiting the PERK-EIF2α signaling pathway, thereby alleviating the progression of myopia. These findings provide new experimental evidence for the potential application of quercetin in myopia prevention and treatment.

Incorporating Hydrogel (with Low Polymeric Content) into 3D-Printed PLGA Scaffolds for Local and Sustained Release of BMP2 in Repairing Large Segmental Bone Defects.

Treating large bone defects remains a considerable challenge for clinicians: bone repair requires scaffolds with mechanical properties and bioactivities. Herein, based on crosslinking o-phthalaldehyde (OPA) with amine groups, 4-arm polyethylene glycol (4armPEG)-OPA/Gelatin hydrogel loaded with bone morphogenetic protein 2 (BMP2) is prepared and a three dimensional (3D)-printed poly (lactic-co-glycolic acid) (PLGA) porous scaffold is filled with the hydrogel solution. The composite scaffold, with a compression modulus of 0.68 ±0.097GPa similar to the cancellous bone, has a porosity of 56.67 ± 4.72% and a pore size of about 380µm, promoting bone growth. The hydrogel forms a porous network at low concentrations, aiding protein release and cell migration. The hydrogel degrades in approximately three weeks, and the scaffold takes five months, matching bone repair timelines. BMP2 release experiment shows a sustained BMP2 release with a 72.4 ± 0.53% release ratio. The ALP activity test and alizarin red staining shows effective osteogenic promotion, while RT-PCR confirms BMP2@Gel enhanced COL-1 and OPN expression. Animal experiments further validate the composite scaffold's bone repair efficacy. This study demonstrates the effectiveness of the hydrogel in releasing BMP2 and the mechanical support of the 3D-printed PLGA porous scaffold, providing a new treatment for bone defects.

Odd-Numbered Agaro-Oligosaccharides Produced by α-Neoagaro-Oligosaccharide Hydrolase Exert Antioxidant Activity in Human Dermal Fibroblasts

Agarases produce agar oligosaccharides with various structures exhibiting diverse physiological activities. α-Neoagaro-oligosaccharide hydrolase (α-NAOSH) specifically cleaves even-numbered neoagaro-oligosaccharides, producing 3,6-anhydro-l-galactose (l-AHG) and odd-numbered agaro-oligosaccharides (OAOSs). In this study, α-NAOSH from the agar-degrading marine bacterium Gilvimarinus agarilyticus JEA5 (Gaa117) was purified and characterized using an E. coli expression system to produce OAOSs and determine their bioactivity. Recombinant Gaa117 (rGaa117) showed maximum activity at pH 6.0 and 35 °C. rGaa117 retained &gt;80% of its initial activity after 120 min at 30 °C. The activity was enhanced in the presence of Mn2+. Km, Vmax, and Kcat/Km values of the enzyme were 22.64 mM, 246.3 U/mg, and 15 s−1/mM, respectively. rGaa117 hydrolyzed neoagarobiose, neoagarotetraose, and neoagarohexaose, producing OAOSs that commonly contained l-AHG. Neoagarobiose and neoagarotetraose mixtures, designated NAO24, and mixtures of l-AHG and agarotriose, designated AO13, were obtained using recombinant rGaa16B (β-agarase) and rGaa117, respectively, and their antioxidant activities were compared. AO13 showed higher hydrogen peroxide-scavenging activity than NAO24 in human dermal fibroblasts in vitro because of structural differences: AOSs have d-galactose at the non-reducing end, whereas NAOSs have l-AHG. In conclusion, OAOSs exhibited high ROS-scavenging activity in H2O2-induced human dermal fibroblasts. They may be applicable in cosmetics and pharmaceuticals for prevention of skin aging.

Unilateral “Inactive” Condylar Hyperplasia: New Histological Data

Background: Unilateral condylar hyperplasia (UCH) is characterized by slow progression and enlargement of the condyle, accompanied by elongation of the mandibular body, resulting in facial asymmetry, occlusal disharmony, and joint dysfunction. This condition can be defined as “active” or “inactive”: the active form is characterized by continuous growth and dynamic histologic changes, whereas the inactive form indicates that the growth process has stabilized. Since there are few microscopic studies on the inactive form, this study aims to investigate the histological features and expression of key proteins and bone markers in patients diagnosed with inactive UCH. Methods: A total of 15 biopsies from patients aged 28 to 36 years were examined by light microscopy and immunofluorescence for collagen I and II, metalloproteinases 2 (MMP-2) and 9 (MMP-9), receptor activator of nuclear factor- kappa B (RANK), and osteocalcin. Results: Our findings indicate that during inactive UCH, the ongoing process is not entirely stopped, with moderate expression of collagen, metalloproteinases, RANK, and osteocalcin, although no cartilage islands are detectable. Conclusions: The present study shows that even if these features are moderate when compared to active UCH and without cartilage islands, inactive UCH could be characterized by borderline features that could represent an important trigger-point to possible reactivation, or they could represent a long slow progression that is not “self-limited”.

Application of Alginate-Collagen Hydrogel and Its Related Mechanisms in the Repair of Rat Cartilage Injuries

This research investigated the application of alginate-collagen hydrogel (ACH) in rat cartilage injury (CI) repair and its related mechanisms through in vitro and in vivo experiments. Alginate solution and modified collagen solution were mixed to prepare the ACH. The obtained samples were characterized and their sustained release properties were determined. L929 mouse fibroblasts were assigned into a blank control group (BC group, untreated), a positive control group (PC group, treated with 6.4% phenol), a negative control group (NC group, treated with tissue culture polystyrene), and an ACH group (treated with ACH). Cell viability was assessed using the MTT assay. Meanwhile, the cytotoxicity of the ACH and its impact on cell proliferation and cell attachment capabilities were analyzed. Thirty 4-week-old specific pathogen-free (SPF) Sprague Dawley (SD) rats were randomly grouped into a chondrocyte group (5 rats, chondrocytes collected and cultured), a cPAGE group (5 rats, chondrocytes treated with ACH), a model group (10 rats, CI model), and a mPAGE group (10 rats, CI model treated with CAH). The influence of CAH on chondrocyte proliferation and the type II collagen (COLII) expression were assessed using the cell counting kit 8 (CCK-8) assay and immunofluorescence staining. Furthermore, the impacts of CAH on chondrocyte tissue and COL-II expression were analyzed using Safranin O-Fast Green (SOFG) staining, the International Cartilage Repair Society (ICRS) score, and COL-II tissue staining. Immunoblotting was utilized to detect the TNF-α, NF-κB, matrix metalloproteinase 13 (MMP-13), and COL-II proteins in cartilage tissue. The results showed that release rate (RR) of the constructed ACH reached over 85% within 72 hours and achieved degradation equilibrium within 5 days with a weight loss (WL) rate of over 60%. L929 fibroblasts culture results showed that the ACH posed no significant cytotoxicity, and cell attachment results were not obviously different from those on tissue culture plates (P &gt;0.05). MTT assay results demonstrated that the ACH exerted no adverse effects on L929 cell proliferation and growth, indicating a good biocompatibility. CCK-8 and COL-II results indicated that chondrocyte proliferation in cPAGE group was much higher in contrast to that in chondrocyte group (P &lt;0.05). Besides, the animal experiments unveiled that in comparison to the CI group, the mPAGE group had more chondrocytes, upregulated COL-II, and higher ICRS scores (P &lt;0.05). MMP-13, NF-κB, and TNF-α were downregulated, and COL-II was upshifted in the mPAGE group, with remarkable differences (P &lt;0.05). In conclusion, ACH exhibited excellent biocompatibility and low cytotoxicity, promoted cell proliferation and growth, facilitated chondrocyte proliferation in rat CI repair, offering a valuable research foundation for CI treatment.

Hemin attenuates bleomycin-induced lung fibrosis in mice by regulating the TGF-β1/MAPK and AMPK/SIRT1/PGC-1α/HO-1/NF-κB pathways.

The objective of this study was to investigate the protective effect and potential mechanism of action of hemin on bleomycin-induced pulmonary fibrosis in mice. Male C57BL/6 mice were randomly divided into control, bleomycin and bleomycin + hemin groups. Mice in the bleomycin and bleomycin + hemin groups were injected intratracheally with bleomycin to establish the pulmonary fibrosis model. The bleomycin + hemin group mice were injected intraperitoneally with hemin starting 7 days before modeling until the end of Day 21 after modeling. Pathological changes in lung tissue were assessed by HE and Masson staining. Malondialdehyde (MDA), superoxide dismutase (SOD) and catalase (CAT) levels were determined in lung tissue. Immunohistochemistry was performed to assess the expression of α-SMA and collagen I. The serum levels of IL-6 and TNF-α were measured via ELISA. Western blotting was used to determine the expression of TGF-β1, SIRT1, PGC-1α and HO-1 and the phosphorylation levels of p38, ERK1/2, JNK, AMPK and NF-κB p65 in lung tissue. Hemin significantly reduced lung indices, increased terminal body weight. It also significantly increased SOD and CAT activities; decreased MDA, IL-6 and TNF-α levels; reduced the levels of α-SMA and collagen I-positive cells; upregulated SIRT1, PGC-1α and HO-1 expression; promoted AMPK phosphorylation; and downregulated TGF-β1 expression and p38, ERK1/2, JNK and NF-κB p65 phosphorylation. Hemin might attenuate oxidative damage and inflammatory responses and reduces extracellular matrix deposition by regulating the expression and phosphorylation of proteins associated with the TGF-β1/MAPK and AMPK/SIRT1/PGC-1α/HO-1/NF-κB pathways, thereby alleviating bleomycin-induced pulmonary fibrosis.

Exploring the Osteogenic Effects of Simiao Wan through Activation of the PI3K/AKT Pathway in Osteoblasts

Ethnopharmacological relevanceOsteoporosis (OP) is a degenerative bone disease commonly associated with reduced bone density and increased fracture risk. Aim of the studyThis study aimed to validate the therapeutic effects of Simiao wan (SMW) on OP and explore the underlying mechanism, particularly focusing on the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway. Materials and methodsThe chemical components of SMW were identified using UPLC-Q-TOF-MS/MS. The obtained compounds were then input into the TCMSP, TargetNet, and SwissTargetPrediction databases to predict potential targets. OP-related targets were collected from the GeneCards and DisGeNET databases, and intersecting targets were identified through a Venn diagram. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed on the intersecting targets using the Database for Annotation, Visualization and Integrated Discovery (DAVID). SMW extract was subsequently used to treat osteoblasts in vitro, and its toxicity on osteoblasts was assessed using Cell Counting Kit-8 (CCK-8) and lactate dehydrogenase (LDH) release assays. Osteoblast differentiation and activity were further evaluated using alizarin red staining, alkaline phosphatase staining, and western blot analyses to validate the activation of network pharmacological signaling pathways. ResultsA total of 121 potential targets were identified for SMW in the treatment of OP, with AKT1 as the primary target. The PI3K/AKT pathway emerged as a key signaling pathway potentially involved in SMW’s therapeutic effects o OP. Toxicity assessments showed no significant toxicity of SMW on osteoblasts. Additionally, SMW promoted osteoblast proliferation, alkaline phosphatase activity, calcium nodule deposition, and the expression of osteogenic markers (osteocalcin (OCN), runt-related transcription factor 2 (RunX2), and collagen I), and activated the PI3K/AKT signaling pathway. The PI3K/AKT pathway inhibitor LY294002 partially reversed the SMW-induced mineral deposition and expression of OCN, RunX2, and collagen I. ConclusionSMW demonstrated effective multi-target and multi-pathway therapeutic potential in the treatment of OP, with a significant impact on the PI3K/AKT signaling pathway.

Single intra-articular injection of human synovial membrane MSC from grade IV knee osteoarthritis patient improve cartilage repair in OA rat model

AimThis study aims to assess the effectiveness of therapy of human synovial membrane-derived MSCs (SM-MSC) from OA grade IV patients in treating knee OA.MethodsSM-MSC were isolated from patients undergoing total knee replacement surgery, cultured to the fourth passage, and characterized using flow cytometry. Differentiation potential was assessed through lineage-specific staining. Osteoarthritis was induced in 24 Wistar rats via monosodium iodoacetate (MIA). The rats were divided into three groups: negative control, OA control, and OA treated with SM-MSC. Radiological, histopathological, and molecular analyses were conducted to evaluate cartilage repair and gene expression.ResultsFlow cytometry confirmed the MSC phenotype of SM-MSC, and successful differentiation was observed. Radiological and histopathological analyses showed significant improvement in the SM-MSC treated group, with reduced cartilage damage and higher Safranin O staining compared to the OA control group. Gene expression analysis indicated increased type-2 collagen (COL-2) expression in the SM-MSC treated group, although MMP-13 levels remained unchanged across all groups.ConclusionHuman SM-MSCs from OA grade IV patients significantly improved cartilage repair in an OA rat model, demonstrating their potential as a therapeutic option for OA. To enhance long-term efficacy and anti-inflammatory effects, further studies are needed to optimize treatment protocols, including injection frequency and dosage.

Polydatin protects against articular cartilage degeneration by regulating autophagy mediated by the AMPK/mTOR signaling pathway.

Knee osteoarthritis (KOA) is one of the leading causes of disability. Polydatin has a potential effect on KOA treatment but the therapeutic mechanism is not clear. This study aims to investigate the therapeutic action of polydatin in KOA and its mechanism in activating autophagy via the AMP-activated protein kinase (AMPK)/mTOR signaling pathway. After a KOA rat model was established by anterior cruciate ligament transection surgery, model rats were treated with polydatin 40 mg/kg for 30 days. Subsequently, cartilage tissues were collected, and hematoxylin-eosin (HE), Safranin-O, and TUNEL staining, and western blotting were performed to evaluate the pathological damage and autophagy-related protein expression. Then, human chondrocyte C28/I2 cells were stimulated with lipopolysaccharide (LPS), and the effects of polydatin on C28/I2 cell viability, apoptosis, and autophagy-related protein expression were detected by MTT, Flow Cytometry, and western blot. In addition, an AMPK inhibitor (Dorsomorphin 2HCl) was used to probe the cell proliferation and apoptosis of polydatin-administered C28/I2 cells. Polydatin ameliorated the pathological damage in rat cartilage tissues and inhibited cell apoptosis in KOA rats. Meanwhile, in C28/I2 cells, polydatin promoted viability and reduced apoptosis. In addition, the protein expression of collagen II, LC3II/LC3I, Beclin-1, and p-AMPK/AMPK were upregulated, and p62 and p-mTOR/mTOR were downregulated by polydatin treatment. Interestingly, relative results showed that the protective effect of polydatin in LPS-stimulated-C28/I2 cells was blocked by the AMPK/mTOR inhibitor, dorsomorphin 2HCl. Our research showed that polydatin reduced apoptosis and activated autophagy both in vivo and in vitro by the AMPK/mTOR signaling pathway to protect against KOA, which provided the basis for further investigation into the potential therapeutic impact of polydatin on KOA.

Olive mill wastewater: From by-product to smart antioxidant material

Olive mill wastewater (OMWW) is a byproduct of olive oil extraction that represents a critical environmental concern due to its potential adverse effects on ecosystems. Given these premises, spray-dried microparticles were designed and developed using maltodextrins as carriers to encapsulate OMWW bioactive compounds. The microparticles were manufactured using an easily scalable and sustainable spray-drying process. The resulting microparticles were smooth, spherical, and exhibited a mean particle size of about 18 μm. The systems demonstrated notable antioxidant properties with a DPPH radical scavenging activity higher than 60 %, due to the polyphenolic compounds of OMWW (about 24 gallic acid equivalents per g of sample). In addition, the microparticles supported fibroblast and macrophage viability at concentrations up to 1 mg/mL. They also determined a 4-fold inflammation reduction in macrophages, improved collagen expression in fibroblasts, and modulated oxidative stress on aged fibroblasts. In conclusion, these microparticles could be considered as promising medical devices in wound healing, while offering a sustainable solution for valorizing OMWW.

Optimizing prolyl hydroxylation for functional recombinant collagen in Escherichia coli

Collagen, a key extracellular matrix component, is renowned for its biocompatibility, biodegradability, and bioactivity, finding wide applications in food, medicine, cosmetics, and industry. Recombinant collagen expression in Escherichia coli offers advantages such as shorter production cycles and lower costs compared to extraction from animal tissues, though it is known to lack essential post-translational modifications, such as proline hydroxylation, which are crucial for its stability and biological function. Studies have shown that certain prolyl hydroxylases, including BaP4H, DsP4H, and L593, exhibit relatively high modification efficiency in the E. coli expression system. However, structures and functions of recombinant human type III collagen after modification by three prolyl hydroxylases remain uncertain. In this study, we investigated the percentage of proline hydroxylation, hydroxylation sites, circular dichroism spectra, and biological functions of recombinant human type III collagen modified by various prolyl hydroxylases. The results indicated that the L593 exhibited the highest percentage of proline hydroxylation, and the percentage of proline hydroxylation was closely associated with the formation of the collagen triple helix, while the hydroxylation ratio of prolines is not positively correlated with the stability of the collagen triple helix structure. The biological function results showed that the cell adhesion of recombinant collagen 3-3(BaP4H) and 3-3(L593) was significantly enhanced, which was closely related to the triple helix structure of recombinant human type III collagen. Our study provides valuable insights into the industrial production and biological applications of collagen, enhancing its functional research and scalability.

Ononin delays the development of osteoarthritis by down-regulating MAPK and NF-κB pathways in rat models.

Osteoarthritis (OA) is featured as cartilage loss, joint pain and loss of labor, which the inflammatory reaction may play critical roles. Ononin is an isoflavone isolating from medicinal plants and has anti-inflammatory effects. Our study investigated the anti-inflammation response of ononin on OA. Anterior cruciate ligament transection (ACLT)-induced OA operation was used to establish research model, then treated with ononin for 8 weeks. The condition of joint injury was assessed using pathological staining. The concentration of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6) in serum were measured by Elisa kit. The expression of collagen II and matrix metalloproteinase 13 (MMP-13) proteins to assess cartilage metabolism level by immunohistochemistry and Western blot. We detected the expression of proteins involved in the MAPK and NF-κB signaling pathways. Finally, we used molecular docking to assess the affinity of ononin for the target proteins ERK1/2, JNK1/2, p38 and p65. Our results confirmed that ononin ameliorated cartilage impairment through histopathological analysis by improving the morphological structures and cartilage tidal lines and decreasing Osteoarthritis Research Society International (OARSI) scores in OA rats. Moreover, ononin inhibited the secretion of above factors in OA rats. Furthermore, ononin has been shown to improve cartilage content levels in OA rats. In addition, ononin inhibited the reactivity of MAPK and NF-κB pathways in OA rats. And molecular docking indicated the ligand molecules could stably bind to the proteins of above receptors. Our results demonstrated that ononin may ameliorate cartilage damage and inflammatory response in OA rats by downgrading MAPK and NF-κB pathways, thus identifying ononin as a potential novel drug to treat OA.

MiR-126 accelerates renal injury induced by UUO via inhibition PI3K/ IRS-1/ FAK signaling induced M2 polarization and endocytosis in macrophages

To investigate the role and molecular mechanism of miR-126 in unilateral ureteral occlusion (UUO). We used bioinformatics to analyse miRNAs specifically expressed in UUO. The mouse model of UUO was established using RAW264.7 cells cultured in vitro and in vivo. The mice were divided into control group, miR-126-NC (negative control) group and miR-126-KD (knockdown) group. Then the relative expression of miR-126 was detected by quantitative reverse transcription-polymerase chain reaction (qRT-PCR), the renal fibrosis was detected by Masson staining, and the protein expression of CD68, collagen I and collagen III in the kidney was detected by immunofluorescence assay. Immunohistochemistry detects α-SMA expression. Moreover, Western blotting was performed to measure the expressions of p-PI3K, CD163, CD206, CD86, iNOS, IL-1β, p-FAK, p-Rac-1, p-IRS-1 and MMP9. The relative fluorescence intensity of F-actin and p-FAK was detected by immunofluorescence assay, and the phagocytosis ability of macrophages was determined by phagocytosis assay with fluorescent microspheres. Bioinformatics analysis reveals miR-126-specific overexpression in UUO. Successful transfection of miR-126-NC and miR-126-KD was confirmed by RT-PCR. The selective reduction of miR-126 was validated by Masson, immunohistochemistry and immunofluorescence staining to decrease the area of UUO-induced renal fibrosis and to lower the expression of CD68, α-SMA, collagen I, and collagen III. The reduction of iNOS expression may also be achieved with selective knockdown of miR-126, as verified by cell tests. enhances the phagocytic ability of macrophages and the expression of p-PI3K, CD206, p-FAK, F-actin, p-Rac-1, p-IRS-1 and MMP9. MiR-126 can inhibit the PI3K signaling pathway, promote M1 macrophage polarization, and suppress the activation of FAK and Rac-1, thus accelerating the progression of UUO.

A novel 3D cardiac microtissue model for investigation of cardiovascular complications in rheumatoid arthritis

BackgroundRheumatoid arthritis (RA) is a chronic inflammatory disease that affects not only the joints but also has significant cardiovascular (CV) manifestations. The mechanistic interplay between RA and cardiovascular complications is not yet well understood due to the lack of relevant in vitro models. In this study, we established RA cardiac microtisses (cMTs) from iPSC-derived cardiomyocytes (CMs), endothelial cells (ECs) and cardiac fibroblasts (CFs) to investigate whether this fully human 3D multicellular system could serve as a platform to elucidate the connection between RA and CV disorders.MethodsPBMC and FLS from healthy and RA donors were reprogrammed to hiPSCs with Sendai vectors. hiPSCs pluripotency was assessed by IF, FACS, spontaneous embryoid bodies formation and teratoma assay. hiPSCs were differentiated to cardiac derivatives such as CMs, ECs and CFs, followed by cell markers characterizations (IF, FACS, qRT-PCR) and functional assessments. 3D cMTs were generated by aggregation of 70% CMs, 15% ECs and 15% CFs. After 21 days in culture, structural and metabolic properties of 3D cMTs were examined by IF, qRT-PCR and Seahorse bioanalyzer.ResultshiPSCs demonstrated typical colony-like morphology, normal karyotype, presence of pluripotency markers, and ability to differentiate into cells originating from all three germ layers. hiPSC-CMs showed spontaneous beating and expression of cardiac markers (cTnT, MYL7, NKX2.5, MYH7). hiPSC-ECs formed sprouting spheres and tubes and expressed CD31 and CD144. hiPSC-CFs presented spindle-shaped morphology and expression of vimentin, collagen 1 and DDR2. Self-aggregation of CMs/ECs/CFs allowed development of contracting 3D cMTs, demonstrating spherical organization of the cells, which partially resembled the cardiac muscle, both in structure and function. IF analysis confirmed the expression of cTnT, CD31, CD144 and DDR2 in generated 3D cMTs. RA cMTs exhibited significantly greater formation of capillary-like structures, mimicking enhanced vascularization—key RA feature—compared to control cMTs. Seahorse examination of cMTs revealed changes in mitochondrial and glycolytic rates in the presence of metabolic substrates and inhibitors.ConclusionsThe cMTs model may represent an advanced human stem cell-based platform for modeling CV complications in RA. The highly developed capillary-like structures observed within RA cMTs highlight a critical feature of inflammation-induced CV dysfunction in chronic inflammatory diseases.