Collagen Expression Research Articles

E'jiao and Cubilose Formula Induced Antioxidant Activity and Improved Collagen Expression of Human Skin Fibroblasts During Oxidation Damage

ABSTRACTBackgroundAs traditional Chinese medicinal materials, E'jiao and cubilose are rich in various bioactive substances with good antioxidant, anti‐inflammatory, and immune‐regulating effects.AimTo obtain the optimal ratio of synergistic effect between E'jiao and cubilose.MethodsThe antioxidant capacity of E'jiao and cubilose digestive fluid was evaluated in vitro, as well as the intracellular oxidation balance between HSF cells and 3D whole‐skin model induced by H2O2.ResultsE'jiao, cubilose, and their different ratios of composites had better scavenging ability against free radicals such as DPPH (2,2‐Diphenyl‐1‐picrylhydrazyl). Using HSF cells induced by H2O2 as an oxidative damage model, it was found that the combination of E'jiao and cubilose in a ratio of 2:3 significantly enhanced the activities of SOD, GSH‐PX, and CAT enzymes compared to the separate treatments of E'jiao and cubilose, as well as other combination ratios. It effectively reduced the accumulation of reactive oxygen species caused by H2O2 in HSF (Human Skin Fibroblast) cells and protected the integrity of the cells. Further analysis using flow cytometry and a 3D full‐thickness skin model revealed that the combination ratio of 2:3 increased the proportion of H2O2‐treated cells in the S%+G2% phase from 19.1% + 7.4%–22.1% + 28.8%, helping oxidatively damaged cells partially recover their proliferative capacity. It also promoted the expression of collagen I and collagen IV in the 3D full‐thickness skin model, with improvement rates reaching 168.00% and 123.68%, respectively.ConclusionsThese findings indicate that the combination of E'jiao and cubilose in a ratio of 2:3 exhibits good synergistic effects, enhancing the ability of cells to resist oxidative damage, promoting cellular renewal and metabolism, and improving skin antiwrinkle capacity.

Potential of epicatechin as antioxidant and antiaging in UV-induced BJ cells by regulating COL1A1, FGF-2, GPX-1, and MMP-1 gene, protein levels, and apoptosis

Background Oxidative stress caused by exposure to ultraviolet (UV) light on the skin can damage deoxyribonucleic acid (DNA) and cause keratinocytes to undergo apoptosis. Endogenous antioxidants which play a role in trapping free radicals are also unable to overcome excess reactive oxygen species (ROS) in the body due to UV exposure, so exogenous antioxidants are needed. Polyphenolic compounds extracted from natural ingredients such as flavonoids, quercetin, and epicatechin have quite strong antioxidant activity. This is influenced by the chemical structure of these compounds which are rich in hydroxyl groups and aromatic groups. This structure allows the compound to become an electron donor so that it can neutralize free radicals. In vitro research was used to see the potential effectiveness of epicatechin as an antiaging and antioxidant. The study aims to confirm the potential of epicatechin as an antiaging by in vitro assay. Methods The viability test of epicatechin on human skin fibroblast (BJ) cells was carried out using the water-soluble tetrazolium (WST) assay. BJ cells were UV-induced as a cell model of premature aging. Epicatechin 6.25, 12.5, and 25 µg/mL were administered to UV-induced BJ cells. The gene expression of Collagen I Alpha 1 (COL1A1), matrix metalloproteinase-1 (MMP-1), fibroblast growth factor-2 (FGF-2), and glutathione peroxidase-1 (GPX-1) were analyzed by quantitative real-time polymerase chain reaction (qRT-PCR). Elastin (ELN), hyaluronidase (HAase), cyclooxigenase-2 (COX-2), 8-hydroxydeoxyguanosine (8-OhdG), and melatonin (MT) protein levels were analyzed by enzyme-linked immunosorbent assay (ELISA). The apoptosis of BJ cells was analyzed using flow cytometry. Results Treatment with epicatechin increased relative gene expression including COL1A1 (5.94), FGF-2 (8.34), and GPX-1 (8.09), and also decreased MMP-1 (2.90) relative gene expression compared to the UV-induced BJ cells. Epicatechin also increased levels of ELN (107.7 ng/mg protein) and MT (830 ng/mg protein) levels compared to the UV-induced BJ cells. Epicatechin treatment decreased levels of HAase (505.96 ng/mg protein), COX-2 (33.69 ng/mg protein), and 8-OHdG (97.87 ng/mg protein) compared to the UV-induced BJ cells. Epicatechin also succeeded in maintaining the percentage of live cells and reducing apoptosis, necrotic of UV-induced skin fibroblast cells. Conclusions Epicatechin has the potential to be an antiaging agent by in vitro assay.

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.

Thrombopoietin mimetic reduces mouse lung inflammation and fibrosis after radiation by attenuating activated endothelial phenotypes.

Radiation-induced lung injury (RILI) initiates radiation pneumonitis and progresses to fibrosis as the main side effect experienced by patients with lung cancer treated with radiotherapy. There is no effective drug for RILI. Sustained vascular activation is a major contributor to the establishment of chronic disease. Here, using a whole thoracic irradiation (WTI) mouse model, we investigated the mechanisms and effectiveness of thrombopoietin mimetic (TPOm) for preventing RILI. We demonstrated that administering TPOm 24 hours before irradiation decreased histologic lung injury score, apoptosis, vascular permeability, expression of proinflammatory cytokines, and neutrophil infiltration in the lungs of mice 2 weeks after WTI. We described the expression of c-MPL, a TPO receptor, in mouse primary pulmonary microvascular endothelial cells, showing that TPOm reduced endothelial cell-neutrophil adhesion by inhibiting ICAM-1 expression. Seven months after WTI, TPOm-treated lung exhibited less collagen deposition and expression of MMP-9, TIMP-1, IL-6, TGF-β, and p21. Moreover, TPOm improved lung vascular structure, lung density, and respiration rate, leading to a prolonged survival time after WTI. Single-cell RNA sequencing analysis of lungs 2 weeks after WTI revealed that TPOm shifted populations of capillary endothelial cells toward a less activated and more homeostatic phenotype. Taken together, TPOm is protective for RILI by inhibiting endothelial cell activation.

RIP1 inhibition reduces chondrocyte apoptosis through downregulating nuclear factor-kappa B signaling in a mouse osteoarthritis model.

Excessive chondrocyte death is a critical player in the process of osteoarthritis (OA). The present study was aimed to study the role of receptor-interacting serine/threonine kinase (RIP) 1-mediated signaling for programmed cell death in OA. In the present study, RIP1 protein expression was evaluated in mouse OA cartilage and cultured primary murine chondrocytes exposed to tumor necrosis factor-alpha (TNF-α). Protein expression involved in necroptosis and apoptosis and chondrocyte-derived extracellular matrix were examined. Inhibition of RIP1 was conducted using the RNAi technique and pharmacological inhibition. Western blot, immunohistochemistry, and immunofluorescence examination were applied. The protein presence of RIP1, but not RIP3, was increased in the mouse OA tissue and cultured chondrocytes exposed to TNF-α. Knockdown of RIP1 increased protein expression of collagen II and sex-determining region Y-box transcription factor 9, and reduced protein expression of matrix metallopeptidases 13 and a disintegrin and metalloproteinase with thrombospondin motifs 5. Inhibition of RIP1 reduced the phosphorylated NF-κB signals, decreased cell apoptosis, and restored extracellular matrix expression in cultured chondrocytes. Both RNAi and pharmacological inhibition of RIP1 decelerated the progress of OA in mice. RIP1 regulates chondrocyte apoptosis through NF-κB signaling. Inhibition of RIP1 provides a novel therapeutic approach for OA therapy.

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.

Extracellular vesicles from cartilage progenitors stimulate type II collagen expression and wound healing in meniscal cells.

Knee meniscus tearing is a common orthopaedic injury that can heal poorly if left untreated, increasing the risk of post-traumatic Osteoarthritis. Intraarticular injection of human cartilage-derived progenitor cells (CPCs) has been shown to promote meniscus healing after injury. However, the mechanism by which CPCs stimulated this effect was unclear. The purpose of this study was to determine the paracrine effects that CPC-derived extracellular vesicles (EVs) have on native meniscal cells during healing. EVs from human CPCs and marrow-derived stromal cells were isolated via ultracentrifugation. EVs produced by each cell type were quantified, and their sizes were determined via NanoSight. EV protein expression was characterized via western blot. Meniscal fibrochondrocyte cellular metabolic activity (as an indicator of cell viability and proliferation) following treatment with EVs, was quantified using MTT and ATP assays. A 2D wound healing assay was used to determine the effects of treating inner meniscal fibrochondrocytes with EVs in a dose-dependent manner. Gene expression analysis for chondrogenesis genes was performed via RT-qPCR on inner meniscal fibrochondrocytes following treatment with EVs. Our results showed that CPCs produced a wide size range of EVs expressing CD9, CD81, and HSP70. Treatment of inner meniscal fibrochondrocytes with CPC-EVs improved 2D wound healing, in comparison to EVs isolated from marrow-derived stromal cell controls. CPC-EV treatment increased Type II Collagen mRNA expression in inner meniscal fibrochondrocytes. These findings demonstrate that CPC-EVs stimulate chondrogenic matrix production and wound healing in meniscal cells at the optimal dose of 1.0 × 107 particles/mL, significantly outperforming the effects of marrow stromal cell-derived EVs.

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.