Expression Levels Of Collagen Research Articles

Huoxue Jiedu Huayu recipe inhibits macrophage-secreted vascular endothelial growth factor-a on angiogenesis and alleviates renal fibrosis in the contralateral kidneys of unilateral ureteral obstruction rats.

Male Wistar rats were randomly divided into 4 groups: the sham group, UUO group (180 d of left ureter ligation), UUO plus eplerenone (EPL) group, and UUO plus HJHR group. After 180 d of oral drug administration, blood and contralateral kidneys were collected for analysis. Angiogenesis- and fibrosis-related indexes were detected. HJHR and EPL improved structural damage and renal interstitial fibrosis in the contralateral kidney and reduced the protein expression levels of α-smooth muscle actin (α-SMA), vimentin and collagen I. Moreover, these treatments could reduce the expression of vascular endothelial growth factor-A (VEGFA) by inhibiting the infiltration of macrophages. Furthermore, HJHR and EPL significantly reduced the expression of CD34 and CD105 by downregulating VEGFA production, which inhibited angiogenesis. Finally, the coexpressions of CD34, CD105 and α-SMA were decreased in the HJHR and EPL groups, indicating that endothelial-to-mesenchymal transition was inhibited. These findings confirm that HJHR alleviates contralateral renal fibrosis by inhibiting VEGFA-induced angiogenesis, encourage the use of HJHR against renal interstitial fibrosis and provide a theoretical basis for the clinical management of patients with CKD.

Overexpression of zinc-finger protein 418 inhibits pathological cardiac remodelling after acute myocardial infarction.

Zinc-finger protein 418 (ZNF418) has been confirmed to be expressed in myocardial tissue. However, the role and mechanism of ZNF418 in pathological myocardial remodelling after myocardial infarction (MI) have not been reported. This study was to elucidate the effect and mechanism of ZNF418 on ventricular remodelling after MI in mice. MI mice and H9c2 cardiomyocytes were used to conduct in vivo and in vitro experiments, respectively. ZNF418 expression was regulated by adeno-associated virus 9 and adenovirus vectors. Pathological analysis, echocardiography, and molecular analysis were performed. ZNF418 was down-regulated in the left ventricular tissues of post-MI mice. In contrast, ZNF418 overexpression decreased mortality and improved cardiac function in MI mice. The MI mice exhibited a significantly increased cross-sectional area of myocardial cells and elevated protein expression levels of myocardial hypertrophy markers ANP, BNP, and β-MHC (all P<0.05). Moreover, a significantly increased area of myocardial fibrosis and protein expression levels of myocardial fibrosis markers collagen I, collagen III, and CTGF were observed in MI mice (all P<0.05) in MI mice. All of the above negative effects in MI mice were ameliorated in ZNF418 overexpressed mice (all P<0.05). Mechanistically, ZNF418 overexpression inhibited the activation of the MAPK signalling pathway, as evidenced by the in vivo and in vitro experiments. Overexpression of ZNF418 could improve cardiac function and inhibit pathological cardiac remodelling by inhibiting the MAPK signalling pathway in post-MI mice.

The Preventive Effect of Endostar on Radiation-induced Pulmonary Fibrosis.

Radiation-induced pulmonary fibrosis (RIPF) is a long-term complication of thoracic radiotherapy without effective treatment available. This study aimed to establish a RIPF mouse model and explore the therapeutic effects and mechanisms of recombinant human endostatin (Endostar). C57BL/6 mice received a 16-Gy dose of X-rays to the whole thorax with or without the administration of Endostar for 24 weeks. Radiation-induced body weight loss was partially attenuated by Endostar (P<0.05). Endostar significantly reduced alveolar inflammation (P<0.05) and pulmonary fibrosis (P<0.001), as indicated by a decrease in the expression levels of collagen I and collagen IV in lung tissue (both P<0.001). Angiogenesis (as shown by CD31 immunohistochemistry) was also decreased (P<0.01). In irradiated mice, Endostar inhibited the transforming growth factor-β1 (TGF-β1)/drosophila mothers against the decapentaplegic 3 (Smad3)/extracellular regulated protein kinases (ERK) signaling pathway (all P<0.05). In vitro, Endostar treatment decreased the radiation-induced expression of TGF-β1, vascular endothelial growth factor (VEGF), p-Smad3, and p-ERK in alveolar epithelial cells and vascular endothelial cells (all P<0.05). Endostar could alleviate RIPF through decreased antiangiogenic activity and inhibition of the TGF-β1/Smad3/ERK pathway.

Silymarin and MSC-exosomes ameliorate thioacetamide-evoked renal fibrosis by inhibiting TGF-β/SMAD pathway in rats

BackgroundTGF-β1 and SMAD3 are particularly pathogenic in the progression of renal fibrosis.AimThis study aimed to evaluate the kidney protective potentials of silymarin (SM) and exosomes of mesenchymal stem cells against the nephrotoxin thioacetamide (TAA) in rats.Methods32 female rats were randomly assigned into four groups: the control group, the TAA group, the TAA + SM group, and the TAA + Exosomes group. The kidney homogenates from all groups were examined for expression levels of TGF-β receptors I and II using real-time PCR, expression levels of collagen type I and CTGF proteins using ELISA, and the expression levels of nuclear SMAD2/3/4, cytoplasmic SMAD2/3, and cytoplasmic SMAD4 proteins using the western blot technique.ResultsCompared to the control group, the injection of TAA resulted in a significant increase in serum levels of urea and creatinine, gene expression levels of TβRI and TβRII, protein expression levels of both collagen I and CTGF proteins, cytoplasmic SMAD2/3 complex, and nuclear SMAD2/3/4 (p-value < 0.0001), with significantly decreased levels of the co-SMAD partner, SMAD4 (p-value < 0.0001). Those effects were reversed considerably in both treatment groups, with the superiority of the exosomal treatment regarding the SMAD proteins and the expression levels of the TβRI gene, collagen I, and CTGF proteins returning to near-control values (p-value > 0.05).ConclusionUsing in vitro and in vivo experimental approaches, the research discovered a reno-protective role of silymarin and exosomes of BM-MSCs after thioacetamide-induced renal fibrosis in rats, with the advantage of exosomes.

Development of alginate-collagen interpenetrating network for osteoarthritic cartilage by in situ softening

This study presents an alginate-collagen interpenetrating network (IPN) matrix of incorporating collagen fibrils into an alginate hydrogel by physical mixing and controlled gelation. The resulting matrix closely mimics the physiological and pathological stiffness range of the chondrocyte pericellular matrix (PCM). Chondrocytes were cultured within three-dimensional (3D) alginate-collagen IPN matrices with varying stiffness, namely Firm, Medium, and Soft. Alginate lyase was introduced to study the effects of the changes in stiffness of the Firm on chondrocyte response by in situ softening. The developed alginate-collagen IPN matrix displayed good cell-biocompatibility. Compared with stiffer tissue culture plastic (TCP), chondrocytes grown within Firm displayed a stabilized differentiated phenotype characterized by higher expression levels of aggrecan, collagen II, and SOX-9. Moreover, the developed alginate-collagen IPN matrix exhibited a gradually increased percentage of propidium iodide (PI)-positive dead cells with decreasing stiffness. Softer matrices directed cells towards higher proliferation rates and spherical morphologies while stimulating chondrocyte cluster formation. Furthermore, reducing Firm stiffness by in situ softening decreased aggrecan expression, contributing to matrix degradation similar to that seen in osteoarthritis (OA). Hence, the 3D alginate-collagen IPN constructs hold significant potential for in vitro replicating PCM stiffness changes observed in OA cartilage.

Polymeric nanofiber leveraged co-delivery of anti-stromal PAK1 inhibitor and paclitaxel enhances therapeutic effects in stroma-rich 3D spheroid models

The role of tumor stroma in solid tumors has been widely recognized in cancer progression, metastasis and chemoresistance. Cancer-associated fibroblasts (CAFs) play a crucial role in matrix remodeling and promoting cancer cell stemness and resistance via reciprocal crosstalk. Residual tumor tissue after surgical removal as well as unresectable tumors face therapeutic challenges to achieve curable outcome. In this study, we propose to develop a dual delivery approach by combining p21-activated kinase 1 (PAK1) inhibitor (FRAX597) to inhibit tumor stroma and chemotherapeutic agent paclitaxel (PTX) to kill cancer cells using electrospun nanofibers. First, the role of the PAK1 pathway was established in CAF differentiation, migration and contraction using relevant in vitro models. Second, polycaprolactone polymer-based nanofibers were fabricated using a uniaxial electrospinning technique to incorporate FRAX597 and/or PTX, which showed a uniform texture and a prolonged release of both drugs for 16 days. To test nanofibers, stroma-rich 3D heterospheroid models were set up which showed high resistance to PTX nanofibers compared to stroma-free homospheroids. Interestingly, nanofibers containing PTX and FRAX597 showed strong anti-tumor effects on heterospheroids by reducing the growth and viability by > 90 % compared to either of single drug-loaded nanofibers. These effects were reflected by reduced intra-spheroidal expression levels of collagen 1 and α-smooth muscle actin (α-SMA). Overall, this study provides a new therapeutic strategy to inhibit the tumor stroma using PAK1 inhibitor and thereby enhance the efficacy of chemotherapy using nanofibers as a local delivery system for unresectable or residual tumor. Use of 3D models to evaluate nanofibers highlights these models as advanced in vitro tools to study the effect of controlled release local drug delivery systems before animal studies.

Antrodia cinnamomea extract alleviates bleomycin-induced pulmonary fibrosis in mice by inhibiting the mTOR pathway

BackgroundPulmonary fibrosis is a progressive diffuse parenchymal lung disorder with a high mortality rate. Studies have indicated that injured lung tissues release various pro-inflammatory factors, and produce a large amount of nitric oxide. There is also accumulation of collagen and oxidative stress-induced injury, collectively leading to pulmonary fibrosis. Antrodia cinnamomea is an endemic fungal growth in Taiwan, and its fermented extracts exert anti-inflammatory effects to alleviate liver damages. Hence, we hypothesized and tested the feasibility of using A. cinnamomea extracts for treatment of pulmonary fibrosis. MethodsThe TGF-β1-induced human lung fibroblast cells (MRC-5) in vitro cell assay were used to evaluate the effects of A. cinnamomea extracts on the collagen production in MRC-5. Eight-week-old ICR mice were intratracheally administered bleomycin and then fed with an A. cinnamomea extract on day 3 post-administration of bleomycin. At day 21 post-bleomycin administration, the pulmonary functional test, the expression level of inflammation- and fibrosis-related genes in the lung tissue, and the histopathological change were examined. ResultsThe A. cinnamomea extract significantly attenuated the expression level of collagen in the TGF-β1-induced MRC-5 cells. In the A. cinnamome-treated bleomycin-induced lung fibrotic mice, the bodyweight increased, pulmonary functions improved, the lung tissues expression level of inflammatory factor and the fibrotic indicator were decreased, and the histopathological results showed the reduction of thickening of the inter-alveolar septa. ConclusionsThe Antrodia cinnamomea extract significant protects mice against bleomycin-induced lung injuries through improvement of body weight gain and lung functions, and attenuation of expression of inflammatory and fibrotic indicators.

Ferroptosis Mediates Pulmonary Fibrosis: Implications for the Effect of Astragalus and Panax notoginseng Decoction.

To investigate the mechanism through which Astragalus and Panax notoginseng decoction (APD) facilitates the treatment of ferroptosis-mediated pulmonary fibrosis. First, the electromedical measurement systems were used to measure respiratory function in mice; the lungs were then collected for histological staining. Potential pharmacologic targets were predicted via network pharmacology. Finally, tests including immunohistochemistry, reverse transcription-quantitative polymerase chain reaction, and western blotting were used to evaluate the relative expression levels of collagen, transforming growth factor β, α-smooth muscle actin, hydroxyproline, and ferroptosis-related genes (GPX4, SLC7A11, ACSL4, and PTGS2) and candidates involved in the mediation of pathways associated with ferroptosis (Hif-1α and EGFR). APD prevented the occurrence of restrictive ventilation dysfunction induced by ferroptosis. Extracellular matrix and collagen fiber deposition were significantly reduced when the APD group compared with the model group; furthermore, ferroptosis was attenuated, expression of PTGS2 and ACSL4 increased, and expression of GPX4 and SLC7A11 decreased. In the APD group, the candidates related to the mediation of ferroptosis (Hif-1α and EGFR) decreased compared with the model group. Discussion and Conclusions. APD may ameliorate restrictive ventilatory dysfunction through the inhibition of ferroptosis. This was achieved through the attenuation of collagen deposition and inflammatory recruitment in pulmonary fibrosis. The underlying mechanisms might involve Hif-1α and EGFR.

Abstract A096: Blockade of LAIR1-collagen interaction enhances the therapeutic activity of BiTE-secreting T cells in ovarian cancer

Abstract Introduction: The tumor microenvironment (TME), abundant with collagen produced by cancer associated fibroblasts (CAFs), plays a critical role in evading the immune system and limiting the efficacy of cell therapies. High levels of collagen and CAF expression correlate with malignant potential, decreased T cell accumulation, and overall unfavorable clinical outcomes in ovarian cancer (OC). Finding ways to address this immunosuppressive TME is therefore critical to improving therapeutic options. We recently reported on the therapeutic potential of adoptively transferred T cells engineered to secrete a folate receptor alpha (FRα)-targeted bispecific T cell engager (FR-B T cells) to target a tumor antigen commonly overexpressed in OC. Transcriptional profiling following ACT revealed that effector-like FR-B T cells (FR-BE T cells) upregulated genes associated with extracellular matrix interaction, suggesting interaction with the tumor stroma may limit antitumor immunity by FR-B T cells. Our goal was to improve the effectiveness of FR-B T cells by inhibiting LAIR-1, a widely present immune checkpoint receptor that sends T cell inhibitory signals after binding to collagen-like domains, leading to T cell exhaustion. We hypothesized that interrupting the LAIR1-collagen pathway using a LAIR1 decoy (NC410, supplied by NextCure Inc.) could amplify the FR-BE T cell effector function, thereby improving the response. Methods: We utilized in vitro co-culture assays to understand the phenotype and mechanistic changes occurring at the level of the T cell/tumor interface when exposed to collagen, as well as effects of LAIR 1 pathway blockade with the addition of NC410. In vivo murine models were used to determine disease progression and overall survival (OS) following treatment with bispecific T cell engager-armed T cells ± LAIR1 blockade. Results: Phenotypic analysis of FR-B T cells revealed elevated LAIR-1 expression on FR-BE T cells. FRα+ OC lysis by FR-BE T cells was diminished in the presence of collagen, suggesting LAIR-1 signaling can suppress tumor attack. In vitro, the addition of NC410 enhanced the activity of FR-BE T cells when exposed to collagen, improving both FRα+ target cell lysis and partially restoring the production of IFN-γ by FR-BE T cells. In vivo, the combination of NC410 with FR-BE T cells led to improved tumor control compared to FR-BE T cell therapy alone. Conclusions: Our results highlight understanding the interactions between infiltrating immune cells and the immunosuppressive TME to better design combination therapies that improve duration and magnitude of immune attack on tumors. By blocking T cell/stromal interactions, FR-B T cell therapy can be further enhanced, creating a promising opportunity to improve the impact of adoptive cell therapy in OC. Citation Format: Sarah Werner, Nicole Gaulin, Jessie Chiello, Theja Giridharan, Anm Nazmul Khan, Sora Suzuki, Suzanne Hess, Sol Langermann, Emese Zsiros, Brahm H. Segal, A.J. Robert McGray. Blockade of LAIR1-collagen interaction enhances the therapeutic activity of BiTE-secreting T cells in ovarian cancer [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr A096.

Investigation of the Mechanism of Action of Qingzaojiufei Decoction on Idiopathic Pulmonary Fibrosis Based on Network Pharmacology and Experimental Validation

Objective: The Traditional Chinese Medicine Qingzao Jiufei Tang decoction (QZJFD) is effective for the treatment of idiopathic pulmonary fibrosis (IPF). In this study, we explored the anti-pulmonary fibrosis effect of QZJFD and the underlying mechanism. Methods: The effects of QZJFD at low, medium, and high doses were investigated in a rat model of lung fibrosis induced by tracheal injection of bleomycin; pirfenidone was included as a positive control. Serum levels of interleukin-6 (IL-6), tumor necrosis factors (TNF-α), and IL-1β in rats were detected by enzyme-linked immunosorbent assay (ELISA). Expression of α-smooth muscle actin (α-SMA), TGF-β1, p-Jun N-terminal kinase (p-JNK), JNK, p-P38 mitogen-activated protein kinase (MAPK), P38 MAPK, collagen I, and fibronectin-1 (FN1) in lung tissues was detected by immunofluorescence labeling and western blot analysis. Results: QZJFD contained 209 main components and 575 corresponding targets. In total, 3875 disease action targets were related to IPF, with 308 common targets shared by drugs and diseases. The key targets included albumin (ALB), recombinant protein, TNF, IL-6, and tumor protein p53. In total, 3061 items were identified in the gene ontology enrichment analysis ( P &lt; .05) and 197 signaling pathways in the Kyoto encyclopedia of genes and genomes ( P &lt; .05), including MAPK, calcium, advanced glycosylation end products - receptors, TNF, and IL-17. Molecular docking simulation showed that the 2 predominant compounds of QZJFD, naringenin, and kaempferol, bound with high affinity to ALB. Serum levels of IL-6, TNF-α, and IL-1β and the expression levels of α-SMA, TGF-β1, p-JNK, p-P38 MAPK, collagen I, and FN1 in lung tissues were significantly increased in the model rats ( P &lt; .001). After treatment with pirfenidone and QZJFD at the medium and high doses, serum levels of IL-6, TNF-α, and IL-1β and expression levels of α-SMA, p-JNK, p-P38 MAPK, collagen I, and FN1 in lung tissues of rats were significantly lower than those in the model group ( P &lt; .05). Conclusions: QZJFD may exert antifibrotic and anti-inflammatory effects that improve the status of IPF by regulating the MAPK signaling pathway.

Mechanism of Kuntai Capsules in treatment of polycystic ovary syndrome rat models based on AGE-RAGE signal pathway

This study aims to investigate the impact of Kuntai Capsules(KTC) on polycystic ovarian syndrome(PCOS) rat models and explore the underlying mechanism. Fifty female SD rats were randomly divided into five groups(10 rats in each group), including control group, model group, low-, medium-, and high-dose KTC group. Except for the control group, the other groups were injected with dehydroepiandrosterone(DHEA) combined with a high-fat diet(HFD) to induce the PCOS rat model for 28 days. 0.315, 0.63, and 1.26 g·kg~(-1)·d~(-1) KTC was dissolved in the same amount of normal saline and given to low-, medium-, and high-dose KTC groups by gavage. Both control group and model group were given the same amount of normal saline for 15 days. After administration, fasting blood glucose(FBG) was measured by a glucose meter. Fasting insulin(FINS), luteinizing hormone(LH), testosterone(T), and follicle-stimulating hormone(FSH) were detected by enzyme-linked immunosorbent assay(ELISA), and LH/FSH ratio and insulin resistance index(HOMA-IR) were calculated. The pathological morphology of ovarian tissue was observed by hematoxylin-eosin(HE) staining. The expression levels of collagen α type Ⅲ 1 chain(COL3A1), apoptotic factors Bax, and Bcl-2 were detected using Western blot and immunofluorescence. The mRNA expressions of COL3A1, Bax, and Bcl-2 in ovarian tissue were performed by real-time PCR(RT-PCR). The results show that compared with the control group, the body weight, serum levels of FBG, FINS, LH, T, LH/FSH, and HOMA-IR are higher in model group(P&lt;0.05 or P&lt;0.01), and the level of FSH is lower(P&lt;0.05). In model group, a large number of white blood cells are found in the vaginal exfoliated cells, mainly in the interictal phase. There are more cystic prominences on the surface of the ovary. The thickness of the granular cell layer is reduced, and oocytes are absent. COL3A1 and Bax protein expression levels are increased(P&lt;0.01), while Bcl-2 protein expression levels are decreased(P&lt;0.05) in the ovarian tissue COL3A1 and Bax mRNA expression levels are increased in ovarian tissue(P&lt;0.05). Compared with the model group, the body weight, FBG, FINS, LH, T, LH/FSH, and HOMA-IR in low-, medium-, and high-dose KTC groups are decreased(P&lt;0.05 or P&lt;0.01), while the levels of FSH in medium-, and high-dose KTC groups are increased(P&lt;0.05 or P&lt;0.01). Low-, medium-, and high-dose KTC groups gradually show a stable interictal phase. The surface of the ovary is smooth. Oocytes and mature follicles can be seen in ovarian tissue, and the thickness of the granular cell layer is increased. The expression level of COL3A1 protein decreases in low-and medium-dose KTC groups(P&lt;0.05 or P&lt;0.01), and that of Bax protein decreases in low-dose KTC group(P&lt;0.05 or P&lt;0.01), and the expression level of Bcl-2 protein increases in low-dose KTC group(P&lt;0.01). The expression levels of COL3A1 and Bax mRNA decreased in the low-dose KTC group(P&lt;0.05), while the expression levels of Bcl-2 mRNA increased(P&lt;0.05). In summary, KTC can inhibit ovarian granulosa cell apoptosis and reduce follicular atresia by regulating the AGE-RAGE signaling pathway. It can promote insulin secretion, reduce blood sugar and body weight, restore serum hormone levels, improve symptoms of PCOS, alleviate morphological damage of the ovary, and restore ovarian function, which is of great value in the treatment of PCOS.

Effect of electroacupuncture on ventricular structure and function in rats with myocardial ischemia-reperfusion injury.

To observe the effect of electroacupuncture (EA) on changes of ventricular structure and function in rats with myocardial ischemia-reperfusion injury (MIRI), so as to explore its potential mechanisms underlying improvement of ventricular remodeling after MIRI. Forty male SD rats were randomly divided into 4 groups：sham operation group, model group, EA group and medication (sacubactril valsartan, LCZ696) group, with 10 rats in each group. The MIRI model was established by ligation of the left anterior descending coronary artery and reperfusion. EA (2 Hz/100 Hz, 2 mA) was applied to bilateral "Neiguan" (PC6) for 20 min, once every other day for 21 d. Rats of the medication group received gavage of LCZ696 (60 mg·kg-1·d-1). After the intervention, echocardiography was used to detect the ejection fraction (EF) and fractional shortening (FS) of the left ventricle, and the contents of serum tumor necrosis factor-α(TNF-α), vascular cell adhesion molecule-1(VCAM-1) and intercellular cell adhesion molecule-1(ICAM-1) were assayed by enzyme-linked immunosorbent assay. The pathological changes of myocardial tissue were observed after HE staining. The Masson staining was used to evaluate the myocardial collagen deposition and myocardial fibrosis. The mRNA expression levels of collagen Ⅰ and Ⅲ and connective tissue growth factor (CTGF) in the myocardial tissue were detected by quantitative real-time PCR, and the expression levels of IL-1β and IL-18 were detected by Western blot. In contrast to the sham operation group, the EF and FS levels of the left ventricle were ob-viously decreased (P<0.001), while the contents of serum TNF-α, VCAM-1 and ICAM-1, the proportion of myocardial fibrosis area, the mRNA expression levels of myocardial collagen Ⅰ, collagen Ⅲ and CTGF, the expression levels of IL-1β and IL-18 were significantly increased (P<0.001, P<0.000 1, P<0.05, P<0.01) in the model group. Compared with the model group, the EF and FS levels were remarkably increased (P<0.01), whereas the contents of serum TNF-α, VCAM-1 and ICAM-1, the proportion of myocardial fibrosis area, the mRNA expression levels of myocardial collagen Ⅰ, collagen Ⅲ and CTGF, and the expression levels of IL-1β and IL-18 were significantly down-regulated (P<0.001, P<0.01, P<0.05) in both the medication and EA groups. No significant differences were found between the EA and medication groups in all the indexes mentioned above. EA can improve the left-ventricular fibrosis and function, delay or reverse ventricular remodeling in MIRI rats, which may be related to its functions in down-regulating myocardial inflammatory response and mRNA expression levels of myocardial collagen Ⅰ, collagen Ⅲ and CTGF.

Effects of an Ultra-Polished Scalpel on Incisional Wounds in a Diabetic Model.

Inflammation after surgical incisions is related to the degree of tissue damage. Healing with low inflammation is desirable, especially in patients with compromised healing potential. This experimental study was conducted to assess the degree of inflammatory reaction and scar formation from incisions made by an ultra-polished scalpel (UPS). Two paravertebral incisions were made with a conventional scalpel (CS) and a UPS in 18 individual rats with diabetes. The fibrotic tissue (scar) area and expression levels of collagen, transforming growth factor, and matrix metalloproteinases were quantified on postoperative days 3, 7, and 30. The scar widths and areas were significantly lower in the UPS group than in the CS group. The scar widths were 64.3 ± 14.7 µm and 86.8 ± 12.1 µm in the UPS and CS groups, respectively (P = 0.03). The scar areas were 11,398 ± 1595 µm2 in the UPS group and 17,433 ± 3487 µm2 in the CS group (P = 0.014). The UPS group had less inflammation on day 3, less transforming growth factor synthesis on days 3 and 7, lower levels of matrix metalloproteinases, and less collagen synthesis on day 7 than did the CS group. The UPS achieved less local inflammation by reducing the local tissue damage in diabetic rat models, enabling better healing, and resulting in less scar formation. The UPS warrants further clinical study as it may bring beneficial outcomes for patients with impaired healing capability and patients who seek to reduce scarring.

A novel and efficient murine model for investigating tendon-to-bone healing

BackgroundTendon-to-bone healing is a critical challenge in sports medicine, with its cellular and molecular mechanisms yet to be explored. An efficient murine model could significantly advance our understanding of this process. However, most existing murine animal models face limitations, including a propensity for bleeding, restricted operational space, and a steep learning curve. Thus, the need for a novel and efficient murine animal model to investigate the cellular and molecular mechanisms of tendon-to-bone healing is becoming increasingly evident.MethodsIn our study, forty-four 9-week-old male C57/BL6 mice underwent transection and reattachment of the Achilles tendon insertion to investigate tendon-to-bone healing. At 2 and 4 weeks postoperatively, mice were killed for histological, Micro-CT, biomechanical, and real-time polymerase chain reaction tests.ResultsHistological staining revealed that the original tissue structure was disrupted and replaced by a fibrovascular scar. Although glycosaminoglycan deposition was present in the cartilage area, the native structure had been destroyed. Biomechanical tests showed that the failure force constituted approximately 44.2% and 77.5% of that in intact tissues, and the ultimate tensile strength increased from 2 to 4 weeks postoperatively. Micro-CT imaging demonstrated a gradual healing process in the bone tunnel from 2 to 4 weeks postoperatively. The expression levels of ACAN, SOX9, Collagen I, and MMPs were detected, with all genes being overexpressed compared to the control group and maintaining high levels at 2 and 4 weeks postoperatively.ConclusionsOur results demonstrate that the healing process in our model is aligned with the natural healing process, suggesting the potential for creating a new, efficient, and reproducible mouse animal model to investigate the cellular and molecular mechanisms of tendon-to-bone healing.Graphical abstract

Therapeutic effect and mechanism of Yougui Wan in rats with intervertebral disk degeneration

ObjectiveTo explore the potential mechanism of Yougui Wan on deformed lumbar intervertebral disk structure in rats.MethodsThirty male Sprague–Dawley rats were randomly divided into 3 groups, with 10 rats in each group. The animals in the blank control group were healthy rats without specific treatment, and those in the model group and traditional Chinese medicine (TCM) group were used to establish the intervertebral disk degeneration (IDD) model by puncturing the annulus. Four weeks after modeling, rats in the TCM group were administered Yougui Wan by gavage for 2 consecutive weeks. Serum interleukin-6 (IL-10), macrophage migration inhibitory factor (MIF) and tumor necrosis factor alpha (TNF-α) levels were measured by ELISA, and the protein expression levels of collagen II and Notch1 in intervertebral disk tissues were examined by Western blotting. Apoptosis was detected by the TUNEL method.ResultsCompared with those in the blank group, IL-10, MIF and TNF-α levels in the model group and TCM group were increased (P < 0.05), the protein expression levels of collagen II were decreased, and the protein expression levels of Notch1 were increased. Compared with those in the model group, the levels of IL-10 in the TCM group were increased (P < 0.05), the levels of MIF and TNF-α were decreased (P < 0.05), the protein expression levels of collagen II were increased, and the protein expression levels of Notch1 were decreased.ConclusionYougui Wan can inhibit the inflammatory response in IDD rats, reduce the degradation of extracellular matrix, reduce apoptosis in nucleus pulposus cells, and alleviate intervertebral disk degeneration. The mechanism may be related to the regulation of the Notch signaling pathway.

An RORα agonist, ODH-08, inhibits fibrogenic activation of hepatic stellate cells via suppression of SMAD3

AimsHepatic fibrosis is a dynamic process characterized by the net accumulation of an extracellular matrix resulting from chronic liver injury such as nonalcoholic steatohepatitis. Activation of hepatic stellate cells (HSCs) plays a role in transdifferentiation of quiescent cells into fibrogenic myofibroblasts. We aimed to examine the function of retinoic acid receptor-related orphan receptor alpha (RORα) and its novel agonistic ligand, 1-(4-benzyloxybenzyl)-3-(2-dimethylaminoethyl)-thiourea (ODH-08) against activation of HSCs using hepatic fibrosis mouse models. Main methodsChemical synthesis, a reporter gene assay, surface plasmon resonance analysis, and a docking study were performed to evaluate ODH-08 as a ligand of RORα. In vivo experiments with mice fed a Western diet were performed to evaluate the effect of ODH-08. The human HSC line, Lx-2, and primary mouse HSCs were employed to identify the molecular mechanisms underlying the antifibrogenic effect of ODH-08. Key findingsA novel RORα-selective ligand, ODH-08, was developed based on modification of JC1-40, an analog of N-methylthiourea. Administration of ODH-08 to the Western diet-fed mice reduced hepatic collagen deposition and expression levels of fibrogenic markers such as α-smooth muscle actin and collagen type I alpha 1 chain. Activation of RORα—either by transient overexpression of RORα or treatment with ODH-08—suppressed the expression of fibrogenic proteins in HSCs. The activation of RORα suppressed the activity of SMAD2 and 3, which are the primary downstream proteins of transforming growth factor β. SignificanceRORα and its agonist ODH-08 have a potent antifibrotic effect, which could provide a novel antifibrotic strategy against hepatic fibrosis.

Phomopsterone B Alleviates Liver Fibrosis through mTOR-Mediated Autophagy and Apoptosis Pathway.

Liver fibrosis is the initial pathological process of many chronic liver diseases. Targeting hepatic stellate cell (HSC) activation is an available strategy for the therapy of liver fibrosis. We aimed to explore the anti-liver fibrosis activity and potential mechanism of phomopsterone B (PB) in human HSCs. The results showed that PB effectively attenuated the proliferation of TGF-β1-stimulated LX-2 cells in a concentration-dependent manner at doses of 1, 2, and 4 μM. Quantitative real-time PCR and Western blot assays displayed that PB significantly reduced the expression levels of α-SMA and collagen I/III. AO/EB and Hoechst33342 staining and flow cytometry assays exhibited that PB promoted the cells' apoptosis. Meanwhile, PB diminished the number of autophagic vesicles and vacuolated structures, and the LC3B fluorescent spots indicated that PB could effectively inhibit the accretion of autophagosomes in LX-2 cells. Moreover, rapamycin and MHY1485 were utilized to further investigate the effect of mTOR in autophagy and apoptosis. The results demonstrated that PB regulated autophagy and apoptosis via the mTOR-dependent pathway in LX-2 cells. In summary, this is the first evidence that PB effectively alleviates liver fibrosis in TGF-β1-stimulated LX-2 cells, and PB may be a promising candidate for the prevention of liver fibrosis.

Leech extract alleviates idiopathic pulmonary fibrosis by TGF-β1/Smad3 signaling pathway

Ethnopharmacological relevanceLeech, as a traditional Chinese medicine for the treatment of blood circulation and blood stasis, was also widely used to cure pulmonary fibrosis in China. In clinical practice, some traditional Chinese medicine preparation such as Shui Zhi Xuan Bi Hua Xian Tang and Shui Zhi Tong Luo Capsule composed of leech, could improve the clinical symptoms and pulmonary function in patients with idiopathic pulmonary fibrosis (IPF). However, the material basis of the leech in the treatment of IPF were not yet clear. Aim of the studyScreen out the components of leech that have the anti-pulmonary fibrosis effects, and further explore the therapeutic mechanism of the active components. Materials and methodsIn this study, the different molecular weight components of leech extract samples were prepared using the semi-permeable membranes with different pore sizes. The therapeutic effects of the leech extract groups with molecular weight greater than 10 KDa (>10 KDa group), between 3 KDa and 10 KDa (3–10 KDa group), and less than 3 KDa (<3 KDa group) on pulmonary fibrosis were firstly investigated by cell proliferation and cytotoxicity assay (MTT), cell wound healing assay, immunofluorescence staining (IF) and Western blot (WB) assay through the TGF-β1-induced fibroblast cell model. Then bleomycin-induced pulmonary fibrosis (BML-induced PF) mouse model was constructed to investigate the pharmacological activities of the active component group of leech extract in vivo. Pathological changes of the mouse lung were observed by hematoxylin-eosin staining (H&E) and Masson's trichrome staining (Masson). The hydroxyproline (HYP) content of lung tissues was quantified by HYP detection kit. The levels of extracellular matrix-related fibronectin (FN) and collagen type Ⅰ (Collagen Ⅰ), pyruvate kinase M2 (PKM2) monomer and Smad7 protein were determined via WB method. PKM2 and Smad7 protein were further characterized by IF assays. ResultsUsing TGF-β1-induced HFL1 cell line as a PF cell model, the in vitro results demonstrated that the >10 KDa group could significantly inhibited the cell proliferation and migration, downregulated the expression level of cytoskeletal protein vimentin and α-smooth muscle actin (α-SMA), and reduced the deposition of FN and Collagen Ⅰ. In the BML-induced PF mouse model, the >10 KDa group significantly reduced the content of HYP, downregulated the expression levels of FN and Collagen Ⅰ in lung tissues, and delayed the pathological changes of lung tissue structure. The results of WB and IF assays further indicated that the >10 KDa group could up-regulate the expression level of PKM2 monomer and Smad7 protein in the cellular level, thereby delaying the progression of pulmonary fibrosis. ConclusionsOur study revealed that the >10 KDa group was the main material basis of the leech extract that inhibited pulmonary fibrosis through TGF-β1/Smad3 signaling pathway.

Atrial fibrillation in human patients is associated with increased collagen type V and TGFbeta1

Background and aimAtrial fibrosis is an important factor in initiating and maintaining atrial fibrillation (AF). Collagen V belongs to fibrillar collagens. There are, however no data on collagen V in AF. The aim of this work was to study the quantity of collagen V and its relationship with the number of fibroblasts and TGF- b 1 expression in patients in sinus rhythm (SR) and in patients with atrial fibrillation (AF). MethodsWe used quantitative immuhistochemistry to study collagen V in right and left atrial biopsies obtained from 35 patients in SR, 35 patients with paroxysmal AF (pAF) and 27 patients with chronic, long-standing persistent AF (cAF). In addition, we have quantified the number of vimentin-positive fibroblasts and expression levels of TGF-β1. ResultsCompared to patients in SR, collagen V was increased 1.8- and 3.1-fold in patients with pAF and cAF, respectively. In comparison with SR patients, the number of vimentin-positive cells increased significantly 1.46- and 1.8-fold in pAF and cAF patients, respectively.Compared to SR patients, expression levels of TGF-ß1, expressed as fluorescence units per tissue area, was significantly increased by 77 % and 300 % in patients with pAF and cAF, respectively. Similar to intensity measurements, the number of TGFß1-positive cells per 1 mm2 atrial tissue increased significantly from 35.5 ± 5.5 cells in SR patients to 61.9 ± 12.4 cells in pAF and 131.5 ± 23.5 cells in cAF. In both types of measurements, there was a statistically significant difference between pAF and cAF groups. ConclusionsThis is the first study to show that AF is associated with increased expression levels of collagen V and TGF-ß1indicating its role in the pathogenesis of atrial fibrosis. In addition, increases in collagen V correlate with increased number of fibroblasts and TGF-β1 and are more pronounced in cAF patients than those in pAF patients.

Study on the molecular mechanism of dihydromyricetin in alleviating liver cirrhosis based on network pharmacology.

Dihydromyricetin (DHM) is a bioactive flavonoid extracted from Hovenia dulcis, which has various activities. In the present study, the molecular mechanism of dihydromyricetin (DHM) in relieving liver cirrhosis was investigated through network pharmacology and experimental verification. The cell model was induced by TGF-β1 activating the human hepatic stellate cell line (HSC; LX-2). The protein levels of α-SMA, collagen I, and collagen III and pathway-related proteins within LX-2 cells were detected using Western blot. EdU staining was conducted to detect cell proliferation. Immunofluorescence staining was performed to detect the expression levels of α-SMA and collagen I. Next, the drug targets of DHM were screened from the PubChem database. The differentially expressed genes in the liver cirrhosis dataset GSE14323 were identified. The expression of the identified drug targets in LX-2 cells was verified using qRT-PCR. The results showed that TGF-β1 treatment notably increased LX-2 cell viability, promoted cell proliferation, and elevated α-SMA, collagen I, and collagen III protein contents. DHM treatment could partially eliminate TGF-β1 effects, as evidenced by the inhibited cell viability and proliferation and reduced α-SMA, collagen I, and collagen III contents. After network pharmacology analysis, nine differentially expressed target genes (MMP2, PDGFRB, PARP1, BCL2L2, ABCB1, TYR, CYP2E1, SQSTM1, and IL6) in liver cirrhosis were identified. According to qRT-PCR verification, DHM could inhibit the expression of MMP2, PDGFRB, PARP1, CYP2E1, SQSTM1, and IL6, and enhance ABCB1 expression levels within LX-2 cells. Moreover, DHM inhibited mTOR and MAPK signaling pathways in TGF-β1-induced HSCs. In conclusion, DHM could inhibit HSC activation, which may be achieved via acting on MMP2, PDGFRB, PARP1, CYP2E1, SQSTM1, IL6, and ABCB1 genes and their downstream signaling pathways, including mTOR and MAPK signaling pathway.