Collagen Expression Research Articles

Sacubitril/valsartan preserves regional cardiac function following myocardial infarction in rats.

Sacubitril/valsartan (Sac/Val) is used for treatment of heart failure. The effect of Sac/Val on regional dysfunction following myocardial infarction (MI) remains uncertain. This study aimed at understanding the effects of Sac/Val on regional function after MI. MI or sham surgery was performed in Sprague-Dawley rats. Animals were randomized to treatment with Sac/Val, valsartan (Val) or vehicle (Veh). Magnetic resonance imaging was used to acquire left ventricular volumes and strain. Left ventricular tissue was obtained for wesern blotting, PCR and Masson's trichrome staining. Isolated cardiac fibroblasts were cultured with Veh, atrial natriuretic peptide (ANP), adrenomedullin (ADM) and sacubitrilat, and collagen expression assessed with droplet digital PCR. Sac/Val reduced ventricular end-diastolic volume by 18% compared with Veh, and preserved circumferential systolic strain in the zone proximal to infarction compared with sham after 42days of treatment (peak strain±SEM: sham: -0.19±0.01%; Sac/Val: -0.14±0.02%; Val: -0.10±0.02%; Veh: -0.10±0.02%). Masson's trichrome staining demonstrated lower fibrotic deposition in the intermediate zone with Sac/Val treatment than Veh (sham: 2.29±0.17%; Sac/Val: 2.31±0.27%; Val: 3.22±0.60%; Veh: 4.14±0.48%). The amounts of the pro-apoptotic caspase 3 cleavage fragments p19/17 were 89% higher in Val than sham, with Sac/Val showing no significant increase compared with sham. Collagen expression in human fibroblast culture was lower in cells co-treated with sacubitrilat and ANP, an effect not observed with sacubitrilat/ADM co-treatment. Sac/Val preserves in vivo myocardial function in the region most proximal to MI in rats and reduces left ventricular dilatation. These effects may be related to a reduction in both fibrosis and pro-apoptotic signalling.

Repair of Osteochondral Defect with Acellular Cartilage Matrix and Thermosensitive Hydrogel Scaffold.

In the present study, acellular cartilage matrix (ACM) was modified with poly-l-lysine/hyaluronic acid (PLL/HA) multilayers via detergent-enzyme chemical digestion and layer-by-layer self-assembly technology. This modified ACM was then loaded with Transforming Growth Factor Beta 3 (TGF-β3) and incorporated into a thermosensitive hydrogel (TH) to create a HA/PLL-ACM/TH composite scaffold with sustained-release function. This study aimed to evaluate the efficacy of this novel composite scaffold in promoting chondrogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) and facilitating osteochondral defect repair. In vitro, isolated, and cultured rat BMSCs were inoculated in equal amounts into TH, ACM/TH, and HA/PLL-ACM/TH groups, with or without TGF-β3 supplementation, for 21 days. Western blot (WB) analysis and immunofluorescence staining were employed to assess the expression levels of collagen II, aggrecan, and SOX-9. In vivo, osteochondral defect was created in the Sprague-Dawley rat trochlea using microdrilling. TH, ACM/TH, and HA/PLL-ACM/TH scaffolds, with or without TGF-β3, were implanted into the defect. After 6 weeks, the repairs were evaluated macroscopically, using Micro computed tomography (micro-CT), histological analysis, and immunohistochemistry. The results demonstrated that the HA/PLL-ACM/TH scaffold loaded with TGF-β3 significantly upregulated the expression of collagen II, aggrecan, and SOX-9 compared with the control and other experimental groups. Furthermore, at 6 weeks postsurgery, the HA/PLL-ACM/TH group loaded with TGF-β3 exhibited superior tissue formation on the joint surface, as confirmed by micro-CT and histological evidence, indicating improved osteochondral repair. These findings suggest that the HA/PLL-ACM/TH scaffold loaded with TGF-β3 holds promise as a therapeutic strategy for osteochondral defect and offers a novel approach for utilizing acellular cartilage microfilaments.

Loss of PTPN21 disrupted mitochondrial metabolic homeostasis and aggravated experimental pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is a high-mortality lung disease with unclear pathogenesis. Convincing evidence suggests that an imbalance in mitochondrial homeostasis resulting from repeated injury to alveolar epithelial type 2 cells (AEC2) underlies IPF. Non-receptor protein tyrosine phosphatase 21 (PTPN21) performs various functions in cancer; however, its role in IPF has not been studied. This study aimed to investigate the role of PTPN21 in lung fibrosis. The experimental results showed that loss of PTPN21 exacerbated lung fibrosis by increasing cell numbers in bronchoalveolar lavage fluid, lung hydroxyproline content, and extracellular matrix protein expression of fibronectin and α-smooth muscle actin (α-SMA) in bleomycin-challenged mouse lungs. In A549 cells (AEC2), knockdown of PTPN21 suppressed focal adhesion and migration, reduced mitochondrial fission and increased fusion, increased the level of mitochondrial superoxide, decreased mitochondrial membrane potential and ATP levels. Simultaneously, knockdown of PTPN21 impaired autophagy, and increased intracellular reactive oxygen species levels. Treatment of fibroblasts (MRC-5) and primary human lung fibroblasts (PHLF)) with the supernatant from PTPN21-knockdown A549 cells increased the expression of fibronectin, collagen 1 and α-SMA. Conversely, overexpression of PTPN21 in A549 cells produced opposite effects. However, treatment of MRC-5 and PHLF with the supernatant from PTPN21-overexpressing A549 cells only slightly reduced the expression of fibronectin, collagen 1 in MRC-5 cells, but did not change the expression of α-SMA. In summary, this study revealed that the loss of PTPN21 in epithelial cells disrupted mitochondrial metabolic homeostasis, leading to epithelial cell inactivation and increased the deposition of extracellular matrix proteins in fibroblasts, thereby exacerbating experimental pulmonary fibrosis.

Versican Proteolysis by ADAMTS: Understanding Versikine Expression in Canine Spontaneous Mammary Carcinomas

Background: The present study investigates VKINE, a bioactive proteolytic fragment of the proteoglycan VCAN, as a novel and significant element in the tumor extracellular matrix (ECM). Although VKINE has been recognized for its immunomodulatory potential in certain tumor types, its impact on ECM degradation and prognostic implications remains poorly understood. Objectives: This study aimed to evaluate VCAN proteolysis and its association with ADAMTS enzymes involved in extracellular matrix remodeling in spontaneous canine mammary gland cancer. Methods: The expression levels of VKINE, ADAMTS enzymes, and collagen fibers were comparatively analyzed in situ and in invasive areas of carcinoma in mixed tumor (CMT) and carcinosarcoma (CSS) with different prognoses. Results: VKINE was notably expressed in the stroma adjacent to the invasion areas in CMT, whereas ADAMTS-15 was identified as the enzyme associated with VCAN proteolysis. Inverse correlations were observed between type III collagen and VCAN expression in in situ areas. Conclusions: Our findings suggest that VKINE and ADAMTS-15 play crucial roles in the tumor microenvironment, influencing invasiveness and type III collagen deposition. This study contributes to a better understanding of the dynamics within the ECM, paving the way for potential new tools in diagnosing and treating human and canine mammary tumors.

Tendon-targeted knockout of collagen XI disrupts patellar and Achilles tendon structure and mechanical properties during murine postnatal development

ABSTRACT Background Collagen XI is a fibril-forming collagen typically associated with type II collagen tissues but is also expressed in type I collagen-rich tendons, especially during development. We previously showed that tendon-targeted (Scx-Cre) Col11a1 knockout mice have smaller tendons in adulthood with aberrant fibril structure and impaired mechanical properties. However, the manifestation of this phenotype is not clearly understood. Therefore, our objective is to define the spatiotemporal roles of collagen XI in tendon structure-function during postnatal development. Given the high expression of collagen XI during embryonic development, we hypothesized that collagen XI knockout leads to the deposition of weakened extracellular matrix during early postnatal timepoints, disrupting the establishment of tendon structure and function. Methods Patellar and Achilles tendons from postnatal (P) days 0, 10, 20, and 30 tendon-targeted scleraxis-Cre heterozygous and homozygous Col11a1 knockout mice were evaluated for morphology, nuclear organization, fibril morphology, mechanical properties, and gene expression. Results At P0, there were no differences in tendon length or fibril diameter of either tendon. By P10, striking structural and functional differences emerged, with collagen XI deficiency resulting in increased tendon length, a heterogeneous and larger diameter population of fibrils, and inferior mechanical properties in both patellar and Achilles tendons. Differences increased in magnitude through P30, supporting our hypothesis that impaired structure-function during postnatal development may drive tendon lengthening and reduced mechanical properties. Conclusions Though collagen XI is a quantitatively minor component of the tendon extracellular matrix, these results highlight the critical role of collagen XI in the acquisition of tendon structure-function.

Ginsenoside Rb1 affects mitochondrial Ca2+ transport and inhibits fat deposition and fibrosis by regulating the wnt signaling pathway to treat rotator cuff tears via docking with SFRP1

BackgroundRotator cuff tears (RCTs) are among the most common musculoskeletal disorders that affect quality of life. This study aimed to investigate the efficacy of ginsenoside Rb1 in RCTs and the mechanisms involved.MethodsFirst, a fibrotic model of FAPs was induced, and FAPs were cultured in media supplemented with different concentrations of ginsenoside Rb1. Next, a rat model of RCTs was constructed and treated with ginsenoside Rb1. Molecular docking was subsequently utilized to detect the binding of ginsenoside Rb1 and SFRP1. Finally, SFRP1 was knocked down and overexpressed in vivo and in vitro to investigate the mechanism of ginsenoside Rb1 and SFRP1 in RCTs.ResultsCompared with the Normal group, FAP viability was decreased, but Collagen II, FN and α-SMA levels were increased in the Control group. After treatment with different concentrations of ginsenoside Rb1, FAP viability increased, but Collagen II, FN and α-SMA levels decreased. Among them, 60 µM ginsenoside Rb1 had the best effect. In vivo experiments revealed that ginsenoside Rb1 improved RCTs in rats. Molecular docking revealed the binding of ginsenoside Rb1 to SFRP1. Additionally, SFRP1 levels were lower in the Control group than in the Normal group. After treatment with ginsenoside Rb1, SFRP1 levels increased. In vivo, overexpressing SFRP1 along with ginsenoside Rb1 treatment further alleviated tendon tissue fibroblast infiltration and fat accumulation and further reduced the expression of Collagen II, FN, and α-SMA. In vitro, overexpressing SFRP1 along with ginsenoside Rb1 treatment further decreased the expression of CaMKII, PLC, PKC, Wnt, and β-catenin, further decreased the Ca2+ fluorescence intensity and mitochondrial length, increased the red/green intensity, and decreased the MitoSOX fluorescence intensity. Additionally, overexpressing SFRP1 along with ginsenoside Rb1 treatment further increased cell proliferation, decreased apoptosis, reduced the protein expression of Collagen II, FN, and α-SMA in muscle tissue, and further reduced the levels of TNF-α, IL-1β, and IL-6 in the cell supernatant.ConclusionsGinsenoside Rb1 inhibited the activation of the Wnt signaling pathway by promoting SFRP1 expression, thereby inhibiting mitochondrial function and Ca2+ absorption to treat fat infiltration and muscle fibrosis caused by RCTs.

Bruton tyrosine kinase promotes wound healing after myocardial infarction by inhibiting the transcription of u-PA

BackgroundsBruton tyrosine kinase (BTK), which is highly expressed in immune cells, plays a critical role in regulating the function of macrophages. A growing body of evidence has demonstrated that the accumulation of macrophages in cardiac tissue after myocardial infarction (MI) significantly affects wound healing and ventricular remodeling during the early phase of repair after MI. However, the role of BTK in cardiac repair post-MI, especially in macrophage-mediated repair, remains unclear. MethodsMI was induced by permanent left anterior descending (LAD) artery ligation in wild-type (WT) mice and macrophage-specific BTK-knockout (BTKMAC-KO) mice. Expression of BTK and phosphorylated BTK were assessed by western blotting. Then, RNA sequencing and ChIP-qPCR assay were performed to explore potential BTK targets and transcriptional regulatory sites. ResultsBTK, which was mainly expressed in macrophages, was upregulated in mice after MI. Compared with WT mice, BTKMAC-KO mice had significantly greater mortality due to heart rupture, reduced wall thickness and severe impairment of left ventricular (LV) function after MI. In addition, increased matrix metalloproteinase-9 (MMP-9) expression and decreased α-SMA and collagen expression were observed in BTKMAC-KO mice after MI. Further experiments revealed that BTK deficiency in macrophages reduces the expression of VEGF and impairs angiogenesis after MI. By RNA sequencing, we found that Nf-kB family genes, as well as the urokinase-type plasminogen activator (uPA), were significantly upregulated in BTK-deficient macrophages. By ChIP-qPCR analysis, we confirmed that uPA was transcriptionally activated by the Nf-kB p65 subunit. Finally, the application of plasminogen activator inhibitor-1 (PAI-1), an uPA inhibitor, markedly protected against cardiac rupture, lowered the mortality rate, and improved cardiac function by increasing collagen deposition and promoting tissue healing in BTKMAC-KO mice after MI. ConclusionsThe present study identifies PAI-1 as a novel cardioprotective agent for cardiac repair post-MI that increases collagen deposition and promotes tissue healing. A therapeutic strategy targeting BTK may be a promising treatment for cardiac repair post-MI.

Oxygen-controllable injectable hydrogel alleviates intervertebral disc degeneration by balancing extracellular matrix metabolism

Nucleus pulposus (NP) cells, situated at the core of intervertebral discs, have acclimated to a hypoxic environment, orchestrating the equilibrium of extracellular matrix metabolism (ECM) under the regulatory influence of hypoxia inducible factor-1α (HIF-1α). Neovascularization and increased oxygen content pose a threat, triggering ECM degradation and intervertebral disc degeneration (IVDD). To address this, our study devised an oxygen-controllable strategy, introducing laccase into an injectable and ultrasound-responsive gelatin/agarose hydrogel. Laccase-mediated reactions were employed to deplete oxygen, establishing a hypoxic microenvironment that upregulated HIF-1α expression. The activation of hypoxia-inducible factors significantly enhanced the expression of aggrecan and collagen II, concurrently suppressing Matrix metalloproteinases (MMP13) and A Disintegrin and Metalloproteinase with Thrombospondin motifs (ADAMTS5) levels, thereby restoring the equilibrium of ECM metabolism. Simultaneously, the hydrogel facilitated the recruitment of stem cells into the NP through the controlled release of ATI2341, activating C-X-C chemokine receptor type 4 (CXCR4). Moreover, ultrasound amplification enhanced ATI2341 release, promoting the migration of NP stem cells. The hydrogel's efficacy in mitigating metabolic imbalances and inhibiting IVDD progression was substantiated in a rat puncture IVDD model through hydrogel injection into the discs. In conclusion, this hypoxia-inducible hydrogel, responsive to thermal stimuli from ultrasound, presents a promising avenue for IVDD treatment.

A multifunctional bacterial cellulose-based dressing modified by quaternized chitosan and grafted protocatechuic acid for diabetic ulcer.

Herein, we developed a multifunctional bacterial cellulose-based dressing (PHBC) modified by quaternized chitosan (HACC) along with protocatechuic acid (PA), through in situ biosynthesis combined with covalent immobilization. The obtained PHBC dressing maintained the excellent physicochemical characteristics of BC, such as high porosity (above 76 %); high water absorption ratio, >80 % of water absorption rate (approximately 30 g/g) has completed in half an hour; favorable hydrophilicity with contact angle of about 50° and excellent flexibility. The introduction of PA-grafted HACC endows exhibited outstanding antibacterial properties against, anti-inflammatory performance and antioxidant capacity. Furthermore, PHBC II, with the reaction solubility of PA was 3 mg/mL, could promote NIH3T3 and HUVECs proliferation and spread. In vivo experiments further verified that PHBC II can effectively promote new granulation tissue hyperplasia and collagen deposition and expression around diabetic ulcers, reduce the inflammatory phenomenon around the wound, and promote the internal capillaries of the wound. The repair and regeneration of the network can promote better and faster wound healing. These results illustrate that the PHBC functional dressing has an important reference value for the clinical treatment of diabetic ulcers.

Adipose stem-cell-derived microvesicles ameliorate long-term bladder ischemia-induced bladder underactivity.

The mechanism for long-term hypoxia/ischemia induced bladder underactivity is uncertain. It requires an effectively therapeutic treatment. Therefore, we determined the pathophysiologic mechanisms of long-term bilateral partial iliac arterial occlusion (BPAO)-induced bladder underactivity and explored the therapeutic potential of adipose-derived stem cells (ADSCs) and ADSC-derived microvesicles (MVs) on BPAO-induced bladder dysfunction. The study included four groups: sham, BPAO, BPAO+ADSCs, and BPAO+ADSC-MVs. ADSCs or ADSC-MVs were isolated, characterized with specific CD markers and injected through the femoral artery to the rat bladders. Real-time laser speckle contrast imaging evaluated bladder microcirculation after BPAO. The transcystometrogram, pelvic nerve activity, bladder histology, immunohistochemistry, and lipid peroxidation assays were conducted after 4-week BPAO induction. The molecular mechanisms of bladder expression of purinergic P2X2/P2X3 and cholinergic M2/M3 receptors for regulating bladder contractility, nerve growth factor (NGF) for nerve injury repair, and collagen-1 for fibrosis were evaluated. Long-term BPAO significantly reduced bladder microcirculation, prolonged the intercontraction interval, decreased voiding volume, increased residual urine volume, lengthened phase 1 contraction, shortened phase 2 contraction, increased leukocytes and CD68 infiltration, increased malondialdehyde levels, and decreased levels of P2X3 and M3 receptors. ADSC-MVs were more efficient than ADSCs in improving BPAO induced parameters, recovering P2X3 and M3 receptors, increasing NGF expression, and decreasing collagen-1 expression in the bladder. ADSC-derived MVs were better than ADSCs to improve long-term BPAO-induced detrusor underactivity, bladder ischemia, and oxidative stress. ADSC-MVs through the therapeutic action of ameliorating inflammation, improving purinergic/cholinergic signaling and neuronal regeneration, and decreasing fibrosis improved BPAO-induced bladder underactivity.

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.

ScRNA-Seq Analysis of Tongue Tissues in Chronic Hyperplastic Candidiasis.

Chronic hyperplastic candidiasis (CHC) is a rare but severe subtype of oral candidiasis distinguished by its potential malignant transformation and suboptimal response to antifungal therapies. However, the cells and mechanisms that play key roles in this process remain unclear. Therefore, we performed the first single-cell RNA sequencing (scRNA-seq) analysis of CHC-affected tongue tissues to reveal the microenvironmental changes and immunological etiology of CHC. First, the features of CHC lesions manifesting as thickening and hardening nodular lesions, including their pathological and microbiological characteristics, were elucidated. Then, a comprehensive cellular atlas and distinct immune landscape in CHC compared with healthy tissues were characterized using scRNA-seq, highlighting significant modifications in the cell number and functionality of fibroblasts and T/NK cells. Importantly, the central role of fibroblasts in cell-cell interactions in CHC was hinted, and possible ligand-receptor pairs mainly associated with inflammation and carcinogenesis were identified. Moreover, it was revealed that significant functional activation of fibroblasts, related to the activation of the epithelial-mesenchymal transition pathways and increased expression of collagen I, matrix metalloproteinase 1 (MMP1), and MMP2, could be a hallmark of CHC, correlating with CHC's clinical characteristics of tongue hardening and intense inflammation. Notably, there is sequencing evidence of the recruitment of CD8+Tex cells and activation of PD-1 and TIGIT immune checkpoint pathways. Moreover, cDC_LAMP3 cells exhibited high CD274 expression, suggesting immune exhaustion and an increased susceptibility to carcinogenesis. This pioneering study provides valuable insights into CHC pathogenesis and immune responses, enhancing our understanding of potential therapeutic strategies.

Study on the mechanism of Fuzheng Huayu formula against biliary fibrosis in mice

Objective: To investigate the effect and mechanisms of Fuzheng huayu formula (FZHY) on biliary fibrosis in mice. Methods: Mdr2 gene knowout (Mdr2-/-) mice were randomly divided into model group, FZHY-treated group and obeticholic acid (OCA)-treated group. Wide type C57BL/6J (WT) mice of the same age were used as the control group. Mdr2-/- mice were treated with the corresponding drugs by gavage from the first day of the 9th week old. The WT and model groups were given 0.3% sodium carboxymethyl cellulose by gavage, and the mice were sacrificed at the end of 12th week old. The activities of serum alkaline phosphatase (ALP) and alanine aminotransferase (ALT) were detected by high-speed biochemical analyzer. H&E staining and sirius red staining were used to observe the pathological changes of liver tissues. The hepatic hydroxyproline content was determined. The hepatic expressions of Col-Ⅰ and α-SMA, ductular reaction markers Epcam, CK7, CK19, and the expression of Pcna, Mki67 and Ccnd1; as well as the expression of inflammatory cytokines Ccl2, Ccl5, Tnf-α, Il10 and Cxcl4 were detected by immunohistochemical staining, western blot and qRT-PCR, respectively. Furthermore, the expression of PPARα and the phosphorylation NF-κB were detected by western blot. Results: Compared with WT mice, the serum ALT and ALP activities of Mdr2-/- mice were significantly increased (P<0.001). The percentage of SR positive stained area and hepatic Hyp content were significantly increased (P<0.01). The hepatic expression of Col-Ⅰ and α-SMA; Epcam, CK7, CK19, Pcna, Mki67 and Ccnd1; Ccl2, Ccl5, Tnf-α, Il10 and Cxcl4 were significantly increased (P<0.01). However, both FZHY and OCA significantly reversed the elevation of these aforementioned indicators (P<0.05; P<0.01). Further study showed that the hepatic expression of PPARα in Mdr2-/- mice was significantly lower than that of WT mice, however the phosphorylation of NF-κB was significantly enhanced (P<0.01). The expression of PPARα in liver tissues of FZHY mice was significantly increased (P<0.05), and the NF-κB phosphorylation was significantly inhibited compared with Mdr2-/- mice (P<0.05). Conclusion: FZHY significantly ameliorated liver fibrosis, ductular reaction and inflammation in Mdr2-/- spontaneous biliary fibrosis mice, and its mechanism was related to the regulation of PPARα/NF-κB pathway.

A Simple and Cost-Efficient Method for the Production of Recombinant Horseradish Peroxidase in E. coli.

Horseradish peroxidase (HRP) is a chromogenic glycoenzyme widely used in research, diagnostics, and therapeutics. Due to its high demand, various eukaryotic and prokaryotic expression systems have been employed for the production of recombinant HRP. Eukaryotic systems yield properly folded, fully functional enzymes with the necessary post-translational modifications. However, these systems can be costly, time-consuming, and prone to hyperglycosylation. In contrast, prokaryotic systems are simple, inexpensive, and readily available, but achieving proper folding and subsequent modifications can be challenging. In this study, we employed a simple and cost-effective method to produce recombinant HRP in soluble form, using the E. coli expression system. The produced enzyme demonstrated substantial activity (89.75 ± 3.25 U/mg) and resistance to heat (T1/2 = 5 min at 50°C), pH variations (up to 8), and H2O2 concentrations (up to 10 mM). Additionally, we systematically compared our method with those of other researchers, highlighting methodological details and outcomes of HRP production in E. coli.

Duhuo Jisheng Decoction in reduction of inflammatory response via Transforming growth factor-β/Smad signaling pathway for repairing rabbit articular cartilage Injury: A Randomized Controlled Trial

ObjectiveThis study aims to investigate the mechanism underlying the effect of Duhuo Jisheng Decoction on the repair of rabbit articular cartilage injury through a reduction in the inflammatory response mediated by the Transforming growth factor (TGF)-β/Smad signaling pathway. MethodsA rabbit articular cartilage injury model was constructed using a ring bone extraction drill. Twenty-four Japanese white rabbits were randomly divided into six groups, namely Sham operation, model, low-dose Duhuo Jisheng Decoction, medium-dose Duhuo Jisheng Decoction, high-dose Duhuo Jisheng Decoction, and positive control groups. The treatment lasted 12 weeks. Gross observation, International Cartilage Repair Society score, Wakitani score, and Micro-computed tomography analysis were used to evaluate the structural repair of cartilage injury. Histology and immunohistochemistry were used to observe the proteoglycan, P-TβRII, P-Smad2, and type II collagen expression levels. Enzyme-linked immunosorbent assay was used to analyze the concentrations of Matrix Metalloproteinase-13 and Syndecan-4 in the joint fluid; and RT-PCR and Western Blot were used to observe the mRNA and protein expressions of ALK5, Sox-9, P-Smad3, and TGF-β1 at the injury repair site. ResultsThe repair effect of cartilage injury, as seen through gross observation and quantitative scoring, was better in all the Duhuo Jisheng Decoction treatment groups than in the model group. The medium dose group of Duhuo Jisheng Decoction had the best repair effect. We observed remarkable structural restoration of cartilage injury in the medium-dose Duhuo Jisheng Decoction group, with the subchondral bone presenting a distinct hierarchy, and parameters such as bone volume fraction and trabecular separation/spacing being significantly augmented. We found high expression levels of proteoglycans, P-TβRII, P-Smad2, and type II collagen. The concentrations of Matrix Metalloproteinase-13 and Syndecan-4 in the joint fluid were significantly lower following treatment. The low gene expression levels of ALK5, Sox-9, P-Smad3, and TGF-β1 in the injury site of the model group could be reversed in the medium-dose Duhuo Jisheng Decoction group. ConclusionDuhuo Jisheng Decoction can repair rabbit cartilage injury and reverse the levels of inflammatory factors in the joint fluid. The mechanism underlying its therapeutic effect is related to the activation of the TGF-β/Smad signaling pathway. This study provides a reliable basis for using Duhuo Jisheng Decoction to treat cartilage injury following knee osteoarthritis.

Effect of microtextured titanium sheets using laser enhances proliferation and collagen synthesis of mouse fibroblasts via the TGF-β/Smad pathway

PurposeThis study investigates the effects of hexagonal microtextured titanium sheets on fibroblast growth and collagen synthesis, crucial factors in anastomotic healing.Materials and methodsHexagonal titanium sheets were fabricated using a laser microtextured machine. Mouse fibroblastic embryonic stem cells (NIH/3T3) were cultured on these sheets. Cell proliferation was assessed using a CCK8 assay, and expression of TGF-β/Smad pathway-related genes and collagen types I and III was evaluated through qRT-PCR and western blot.ResultsHexagonal titanium sheets significantly enhanced fibroblast growth and collagen synthesis. The 50–30 group, with the smallest contact angle (48 ± 2.3°), exhibited the highest cell growth rates by CCK8 assay. Gene expression analysis revealed that TGF-β1, Smad2, Smad3, Smad4, and COL1A1 were significantly upregulated in the 50–30 group on day 7. Meanwhile, type I collagen expression was significantly increased in the 50–30 group on day 7 by western blot analysis.ConclusionOur findings demonstrate that laser-fabricated hexagonal microtextured titanium sheets enhance hydrophilicity and promote fibroblast growth, activating the TGF-β/Smad pathway to facilitate collagen synthesis. These results have important implications for tissue repair and regeneration.

Atomic force microscopy reveals the influence of substrate collagen concentration and TGF-β on lung fibroblast mechanics

Understanding how extracellular matrix (ECM) stiffness and biochemical factors such as TGF-β affect cell behaviour is critical for elucidating mechanisms underlying several pathologic conditions such as tissue fibrosis and cancer metastasis. This study investigates the effects of varying collagen substrate concentration and consequently varying stiffness conditions along with TGF-β treatment on the morphology, nanomechanical properties, and gene expression of normal human lung fibroblasts (NHLF). Our results reveal that increased substrate stiffness leads to more elongated cell morphology, decreased cellular stiffness, and significant alterations in gene expression related to cytoskeletal organization and myofibroblast activation genes. TGF-β treatment further induces myofibroblast differentiation, as evidenced by increased α-SMA and collagen expression, while also reducing cellular stiffness and promoting a more elongated, invasive phenotype. These findings highlight the critical role of both mechanical and biochemical cues in modulating fibroblast behaviour, with significant implications in fibrosis development and cancer progression.

Collagen-1 and elastin expression in cervical tissue: A comparison across cervical elongation, pelvic organ prolapse, and combined conditions

HIGHLIGHTS Pathogenesis of cervical elongation is still limited, including the histological and molecular differences between a cervical elongation and a normal cervix. The expression of collagen-1 level in the cervical elongation group was stronger compared to the cervical elongation with POP and control group. ABSTRACT Objective: This study aimed to assess differences in the expression of Collagen-1 and Elastin in cervical tissues among patients with Cervical Elongation (CE), Pelvic Organ Prolapse (POP), a combination of CE with POP, and those without either condition. Materials and Methods: An analytical study with a cross-sectional design was conducted, using immunohistochemistry (IHC) to analyze cervical tissue samples preserved in paraffin blocks. Patient groups included those diagnosed with CE, POP, CE combined with POP, and a control group without CE or POP. All participants underwent surgery between January 2021 and April 2023. IHC was used to measure the expression levels of Collagen-1 and Elastin in each tissue sample. Observations were made under 400x magnification, focusing on five randomly selected visual-field areas in each sample to determine the area fraction. Two experienced pathologists conducted the analyses in a blinded manner to ensure objective evaluation. Results: Statistical analysis using the Kruskal-Wallis test revealed significant differences in the expression of Collagen-1 across the four groups (CE, POP, CE with POP, and control). Patients with CE showed a higher expression of Collagen-1 than those with CE and POP combined, as well as the control group. However, no significant differences in Elastin expression were observed among the groups. Conclusion: Collagen-1 expression differs significantly across patients with CE, POP, and CE combined with POP, suggesting a distinct role in cervical tissue remodeling in these conditions. Conversely, Elastin expression was consistent across all groups, indicating that it may not play a differentiating role in these pathologies. These findings highlight Collagen-1’s potential involvement in the structural changes associated with CE and POP.

Emodin Inhibits AIM2 Inflammasome Activation via Modulating K27-Linked Polyubiquitination to Attenuate Renal Fibrosis.

Chronic kidney diseases (CKD) is a serious threat to people's health with renal fibrosis as the major pathological feature. The absent in melanoma 2 (AIM2) has recently been proposed to play a critical role in CKD. Emodin is a major bioactive compound from rhubarb, which is widely used for clinical treatment of renal disease. The aim of this study is to elucidate the effect of emodin on unilateral ureteral obstruction (UUO) model mice and its association with the AIM2 inflammasome. In this study, we established the UUO-induced mice renal interstitial fibrosis invivo and bone marrow-derived macrophages (BMDMs) model invitro. The BUN, SCr, TNF-α, IL-1β in serum were examined. The degree of renal damage and fibrosis were determined by histological assessment. Immunofluorescence, western blot, and Co-IP were used to determine the mechanisms of emodin against CKD. Emodin could improve UUO-induced abnormal renal function and histopathological abnormalities. It could also ameliorate renal fibrosis, evidenced by inhibiting the expression of α-SMA, TGF-β1, FN, and collagen I. Mechanistically, emodin significantly suppressed AIM2 inflammasome as well as its components including ASC, cleaved caspase-1, and IL-1β both invivo and invitro. Further studies demonstrated that emodin inhibited K27-linked polyubiquitination of AIM2 by targeting on K64 sites of the lysine residues. In summary, emodin could hinder the activation of AIM2 inflammasome in UUO model mice through K27-linked polyubiquitination to reduce renal fibrosis. Emodin is a possible therapeutic option for CKD treatment.

Tumor-Colonizing E. coli Expressing Both Collagenase and Hyaluronidase Enhances Therapeutic Efficacy of Gemcitabine in Pancreatic Cancer Models.

Desmoplasia is a hallmark feature of pancreatic ductal adenocarcinoma (PDAC) that contributes significantly to treatment resistance. Approaches to enhance drug delivery into fibrotic PDAC tumors continue to be an important unmet need. In this study, we have engineered a tumor-colonizing E. coli-based agent that expresses both collagenase and hyaluronidase as a strategy to reduce desmoplasia and enhance the intratumoral perfusion of anticancer agents. Overall, we observed that the tandem expression of both these enzymes by tumor-colonizing E. coli resulted in the reduced presence of intratumoral collagen and hyaluronan, which likely contributed to the enhanced chemotherapeutic efficacy observed when used in combination. These results highlight the importance of combination treatments involving the depletion of desmoplastic components in PDAC before or during treatment.