Excessive Collagen Deposition Research Articles

Spatiotemporal modulation of immune microenvironment via composite hydrogel brakes for diabetic wound healing

Uncontrolled programmed polarization of macrophages plays an important role in chronic diabetic wound healing. Therefore, correcting programmed macrophage polarization while inducing new tissue remodelling through materials and preventing scar formation is desirable. Herein, a composite hydrogel brake that enables spatiotemporal regulation of the immune microenvironment was constructed to promote skin tissue regeneration. The system was based on thiolated hyaluronic acid to reshape the programmed polarization of macrophages, methacrylate gelatin as a mechanical support, and poly (L-lactic acid) nanoparticles loaded with TGF-β receptor antagonists to inhibit scar formation. Notably, the composite hydrogel acts as a double brake: in the early stage, self-induced M2-type polarization is achieved with the help of thiols; the TNF and NLRP signalling pathways are downregulated; and the immune microenvironment is regulated. During tissue remodeling, the long-term biological effect of TGF-β secretion by M2-type macrophages is interrupted by the slow release of antagonists, which inhibits excessive collagen deposition and prevents scar formation. The dual effect of restraint on reprogramming macrophage polarization and inhibiting scar formation was further confirmed in a diabetic rat skin defect model. Therefore, based on the immunological mechanism, a composite hydrogel brake was specifically designed to regulate the immune microenvironment, induce new tissue remodelling, and provide a promising clinical strategy for treating chronic diabetic wounds.

Ultrastructural and immunohistochemical evaluation of hyperplastic soft tissues surrounding dental implants in fibular jaws

In reconstructive surgery, complications post-fibula free flap (FFF) reconstruction, notably peri-implant hyperplasia, are significant yet understudied. This study analyzed peri-implant hyperplastic tissue surrounding FFF, alongside peri-implantitis and foreign body granulation (FBG) tissues from patients treated at the Department of Oral and Maxillofacial Surgery, Seoul National University Dental Hospital. Using light microscopy, pseudoepitheliomatous hyperplasia, anucleate and pyknotic prickle cells, and excessive collagen deposition were observed in FFF hyperplastic tissue. Ultrastructural analyses revealed abnormal structures, including hemidesmosome dilation, bacterial invasion, and endoplasmic reticulum (ER) swelling. In immunohistochemical analysis, unfolded protein-response markers ATF6, PERK, XBP1, inflammatory marker NFκB, necroptosis marker MLKL, apoptosis marker GADD153, autophagy marker LC3, epithelial–mesenchymal transition, and angiogenesis markers were expressed variably in hyperplastic tissue surrounding FFF implants, peri-implantitis, and FBG tissues. NFκB expression was higher in peri-implantitis and FBG tissues compared to hyperplastic tissue surrounding FFF implants. PERK expression exceeded XBP1 significantly in FFF hyperplastic tissue, while expression levels of PERK, XBP1, and ATF6 were not significantly different in peri-implantitis and FBG tissues. These findings provide valuable insights into the interconnected roles of ER stress, necroptosis, apoptosis, and angiogenesis in the pathogenesis of oral pathologies, offering a foundation for innovative strategies in dental implant rehabilitation management and prevention.

Gelatin/heparin coated bio-inspired polyurethane composite fibers to construct small-caliber artificial blood vessel grafts

Long-term patency and ability for revascularization remain challenges for small-caliber blood vessel grafts to treat cardiovascular diseases clinically. Here, a gelatin/heparin coated bio-inspired polyurethane composite fibers-based artificial blood vessel with continuous release of NO and biopeptides to regulate vascular tissue repair and maintain long-term patency is fabricated. A biodegradable polyurethane elastomer that can catalyze S-nitrosothiols in the blood to release NO is synthesized (NPU). Then, the NPU core-shell structured nanofiber grafts with requisite mechanical properties and biopeptide release for inflammation manipulation are fabricated by electrospinning and lyophilization. Finally, the surface of tubular NPU nanofiber grafts is coated with heparin/gelatin and crosslinked with glutaraldehyde to obtain small-caliber artificial blood vessels (ABVs) with the ability of vascular revascularization. We demonstrate that artificial blood vessel grafts promote the growth of endothelial cells but inhibit the growth of smooth muscle cells by the continuous release of NO; vascular grafts can regulate inflammatory balance for vascular tissue remodel without excessive collagen deposition through the release of biological peptides. Vascular grafts prevent thrombus and vascular stenosis to obtain long-term patency. Hence, our work paves a new way to develop small-caliber artificial blood vessel grafts that can maintain long-term patency in vivo and remodel vascular tissue successfully.

Recruitment or activation of mast cells in the liver aggravates the accumulation of fibrosis in carbon tetrachloride-induced liver injury

Liver diseases caused by viral infections, alcoholism, drugs, or chemical poisons are a significant health problem: Liver diseases are a leading contributor to mortality, with approximately 2 million deaths per year worldwide. Liver fibrosis, as a common liver disease characterized by excessive collagen deposition, is associated with high morbidity and mortality, and there is no effective treatment. Numerous studies have shown that the accumulation of mast cells (MCs) in the liver is closely associated with liver injury caused by a variety of factors. This study investigated the relationship between MCs and carbon tetrachloride (CCl4)-induced liver fibrosis in rats and the effects of the MC stabilizers sodium cromoglycate (SGC) and ketotifen (KET) on CCl4-induced liver fibrosis. The results showed that MCs were recruited or activated during CCl4-induced liver fibrosis. Coadministration of SCG or KET alleviated the liver fibrosis by decreasing SCF/c-kit expression, inhibiting the TGF-β1/Smad2/3 pathway, depressing the HIF-1a/VEGF pathway, activating Nrf2/HO-1 pathway, and increasing the hepatic levels of GSH, GSH-Px, and GR, thereby reducing hepatic oxidative stress. Collectively, recruitment or activation of MCs is linked to liver fibrosis and the stabilization of MCs may provide a new approach to the prevention of liver fibrosis.

Abstract LB320: Runx2 as a key transcription factor regulating contractility and extracellular matrix remodeling in abnormally activated fibroblasts: Implications for tumorigenesis and keloid pathogenesis

Abstract Cancer associated fibroblasts (CAFs) express a variety of cytokines and factors that activate and contribute to pathways that favor tumorigenesis. CAFs disrupt normal cellular functions, such as cell cycle regulation and cell death including apoptosis, or signal certain types of cells to abnormally activate pro-tumor activities. In addition, when it is abnormally activated, it is characterized by increased collagen deposition and more contractility. Likewise, This pattern of abnormal activation is often observed in keloid disease. Keloid constitutes an abnormal fibroproliferative wound healing response with excessive collagen deposition causing abnormal wound healing. To better understand the CAF subtypes with abnormal activation and cancer-promoting properties and the subtypes of keloid fibroblasts with similar activation properties, it is essential to find the master transcription factors (TFs) that determine their identity. To identify functionally important TFs in abnormal CAFs and keloid fibroblasts, we analyzed H3K27ac ChIP-seq of CAFs and keloid fibroblasts and observed that Runx2 significantly enriched the two types of abnormal fibroblasts.To investigate the biological function of Runx2 , we established CAFs from colon cancer patients and performed ShRNA-mediated knockdown of the RUNX2 gene. We confirmed that knockdown of RUNX2 in CAFs suppressed the migration or invasion of cancer cells. Additionally, using a gel contraction assay, we confirmed that fibroblast contractility was significantly reduced by RUNX2 knockdown. Similarly, in keloid fibroblasts, it was confirmed that the migration and gel contraception abilities of fibroblasts were significantly reduced following knockdown of RUNX2 [A1] gene. Last but not least, expression of ECM elements, such as collagens and fibronectin, were significantly as a result of the knockdown of the RUNX2 gene in both abnormally activated fibroblasts. To conclude, we believe that Runx2 acts as a key TF that maintains the contractility and ECM remodeling ability of abnormally activated fibroblasts. Our findings indicate that Runx2 is a attractive therapeutic target not only for pro-tumorigenesis in the cancer microenvironment, but also for keloid fibroblasts. Citation Format: Keun-Woo Lee, Insuk Sohn, Seok-Hyung Kim, So-Young Yeo. Runx2 as a key transcription factor regulating contractility and extracellular matrix remodeling in abnormally activated fibroblasts: Implications for tumorigenesis and keloid pathogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB320.

Effects of lactobacillus pentosus postbiotics on fibrotic response in arecoline-induced oral fibrogenesis

Background/purposeOral submucous fibrosis (OSF), characterized by excessive collagen deposition by myofibroblasts, is often linked to Areca nuts consumption. Probiotics consumption has shown protective effects against fibrotic diseases, and recently, their metabolic byproducts, known as postbiotics, have demonstrated superior advantages over probiotics. However, studies on the therapeutic impact of postbiotics on OSF have been scarce. Therefore, this study aims to examine the effect of PostBio GK4, a postbiotic derived from Lactobacillus pentosus GK4, on OSF and explore its underlying mechanisms. Materials and methodsThe cytotoxicity of GK4 in normal buccal mucosal fibroblasts (BMFs) and fibrotic BMFs (fBMFs) were assessed. Following this, we evaluated the effects of GK4 on collagen contraction, migratory, and wound healing capacities in arecoline-induced fibrotic BMFs. Next, Western blotting and ELISA were employed to assess GK4's impact on fibrosis-related proteins such as COL1A1, and α-SMA, as well as on TGF-β and Smad2/3 signaling pathway. ResultsArecoline was shown to stimulate cell migratory, contractile and wound healing abilities as well as the expression of α-SMA and COL1A1 in BMFs. Treatment with GK4 reduced all arecoline-induced phenomena in BMFs. Moreover, GK4 diminished the increased expression of TGF-β and Smad2/3. ConclusionOur findings proposed that GK4 may exert a suppressive effect on arecoline-induced myofibroblast activities via the inhibition of TGF-β and Smad2/3 signaling pathway. Therefore, GK4 holds promise as an adjunct therapeutic approach for intervening in OSF. Further in-vivo and clinical studies are warranted to validate these observations.

MST1/2 regulates fibro/adipogenic progenitor fate decisions in skeletal muscle regeneration

Defective skeletal muscle regeneration is often accompanied by fibrosis. Fibroblast/adipose progenitors (FAPs) are important in these processes, however, the regulation of FAP fate decisions is unclear. Here, using inducible conditional knockout mice, we show that blocking mammalian Ste20-like kinases 1/2 (MST1/2) of FAPs prevented apoptosis and reduced interleukin-6 secretion invivo and invitro, which impaired myoblast proliferation and differentiation, as well as impaired muscle regeneration. Deletion of Mst1/2 increased co-localization of Yes-associated protein (YAP) with Smad2/3 in nuclei and promoted differentiation of FAPs toward myofibroblasts, resulting in excessive collagen deposition and skeletal muscle fibrosis. Meanwhile, inhibition of MST1/2 increased YAP/Transcriptional co-activator with PDZ-binding motif activation, which promoted activation of the WNT/β-catenin pathway and impaired the differentiation of FAPs toward adipocytes. These results reveal a new mechanism for MST1/2 action in disrupted skeletal muscle regeneration and fibrosis via regulation of FAP apoptosis and differentiation. MST1/2 is a potential therapeutic target for the treatment of some myopathies.

Prefeeding of Clarias gariepinus with Spirulina platensis counteracts petroleum hydrocarbons-induced hepato- and nephrotoxicity

Petroleum aromatic hydrocarbons are considered one of the most dangerous aquatic pollutants due to their widespread across water bodies, persistence, and extension to the food chain. To our knowledge, there hasn’t been any research investigating the hepatorenoprotective effects of Spirulina platensis (SP) against toxicity induced by these environmental toxicants in fish. Thus, we decided to explore its potential safeguarding against benzene and toluene exposure in adult Clarias gariepinus. To achieve this objective, fish were divided into five groups (60 per group; 20 per replicate). The first group served as a control. The second and third groups were intoxicated with benzene and toluene at doses of 0.762 and 26.614 ng/L, respectively for 15 days. The fourth and fifth groups (SP + benzene and SP + toluene, respectively) were challenged with benzene and toluene as previously mentioned following dietary inclusion of SP at a dose of 5 g/kg diet for 30 days. The marked increase in liver metabolizing enzymes, glucose, total protein, albumin, globulin, albumin/globulin ratio, and creatinine confirmed the hepato- and nephrotoxic impacts of benzene and toluene. These outcomes were coupled with cytopathological affections and excessive collagen deposition. The incorporation of SP in ration formulation, on the contrary, restored the previously mentioned toxicological profile due to its antioxidant and cytoprotective attributes. Regardless of SP intervention, the renal tissues still displayed histo-architectural lesions, because of insufficient dose and timeframe. Additional research will be required to identify the ideal SP remediation regimen.

RNA-seq combined network pharmacology reveals that Fu-Gan-Wan (FGW) inhibits liver fibrosis via NF-κB/CCL2/CCR2 and lipid peroxidation via Nrf2/HMOX1 signaling pathway

Ethnopharmacological relevanceLiver fibrosis is a serious complication of liver disease characterized by excessive collagen deposition, without effective therapeutic agents in the clinic. Fu-Gan-Wan (FGW) is an empirical formula used for the clinical treatment of hepatitis and cirrhosis. It has been shown to reverse experimental liver fibrosis. However, its corresponding mechanisms remain unclear. Aim of the reviewThis study aimed to elucidate the key pathways and target genes of FGW in attenuating liver fibrosis. Materials and methodsThe therapeutic effects of different doses of FGW on liver fibrosis were investigated using a 2 mL/kg 15% CCl4-induced mouse model. Then, RNA-seq combined with network pharmacology was used to analyze the key biological processes and signaling pathways underlying the anti-liver fibrosis exertion of FGW. These findings were validated in a TGF-β1-induced model of activation and proliferation of mouse hepatic stellate cell line JS-1. Finally, the key signaling pathways and molecular targets were validated using animal tissues, and the effect of FGW on tissue lipid peroxidation was additionally observed. ResultsWe found that 19.5 g/kg FGW significantly down-regulated CCl4-induced elevation of hepatic ALT and AST, decreased collagen deposition, and inhibited the expression of pro-fibrotic factors α-SMA, COL1α1, CTGF, TIMP-1, as well as pro-inflammatory factor TGF-β1. Additionally, FGW at doses of 62.5, 125, and 250 μg/mL dose-dependently blocked JS-1 proliferation, migration, and activation. Furthermore, RNA-seq identified the NF-κB signaling pathway as a key target molecular pathway for FGW against liver fibrosis, and network pharmacology combined with RNA-seq focused on 11 key genes. Significant changes were identified in CCL2 and HMOX1 by tissue RT-PCR, Western blot, and immunohistochemistry. We further demonstrated that FGW significantly attenuated CCl4-induced increases in p-p65, CCL2, CCR2, and HMOX1, while significantly elevating Nrf2. Finally, FGW significantly suppressed the accumulation of lipid peroxidation products MDA and 4-HNE and reconfigured the oxidation-reduction balance, including promoting the increase of antioxidants GPx, GSH, and SOD, and the decrease of peroxidation products ROS and GSSG. ConclusionsThis study demonstrated that FGW exhibits potential in mitigating CCl4-induced hepatic fibrosis, lipid peroxidation, and iron metabolism disorders in mice. This effect may be mediated through the NF-κB/CCL2/CCR2 and Nrf2/HMOX1 pathways.

Protective Effect of CD137 Deficiency Against Postinfarction Cardiac Fibrosis and Adverse Cardiac Remodeling by ERK1/2 Signaling Pathways.

Myocardial fibrosis, a common complication of myocardial infarction (MI), is characterized by excessive collagen deposition and can result in impaired cardiac function. The specific role of CD137 in the development of post-MI myocardial fibrosis remains unclear. Thus, this study aimed to elucidate the effects of CD137 signaling using CD137 knockout mice and in vitro experiments. CD137 expression levels progressively increased in the heart after MI, particularly in myofibroblast, which play a key role in fibrosis. Remarkably, CD137 knockout mice exhibited improved cardiac function and reduced fibrosis compared with wild-type mice at day 28 post-MI. The use of Masson's trichrome and picrosirius red staining demonstrated a reduction in the infarct area and collagen volume fraction in CD137 knockout mice. Furthermore, the expression of alpha-smooth muscle actin and collagen I, key markers of fibrosis, was decreased in heart tissues lacking CD137. In vitro experiments supported these findings because CD137 depletion attenuated cardiac fibroblast differentiation, and migration, and collagen I synthesis. In addition, the administration of CD137L recombinant protein further promoted alpha-smooth muscle actin expression and collagen I synthesis, suggesting a profibrotic effect. Notably, the application of an inhibitor targeting the extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway attenuated the profibrotic effects of CD137L. To conclude, this study provides evidence that CD137 plays a significant role in promoting myocardial fibrosis after MI. Inhibition of CD137 signaling pathways may hold therapeutic potential for mitigating pathological cardiac remodeling and improving post-MI cardiac function.

WSB1, a Hypoxia-Inducible E3 Ligase, Promotes Myofibroblast Accumulation and Attenuates Alveolar Epithelial Regeneration in Mouse Lung Fibrosis

Idiopathic pulmonary fibrosis is a progressive interstitial lung disease for which there is no curative therapy available. Repetitive alveolar epithelial injury repair, myofibroblast accumulation, and excessive collagen deposition are key pathologic features of idiopathic pulmonary fibrosis, eventually leading to cellular hypoxia and respiratory failure. The precise mechanism driving this complex maladaptive process remains inadequately understood. WD repeat and suppressor of cytokine signaling box containing 1 (WSB1) is an E3 ubiquitin ligase, the expression of which is associated strongly with hypoxia, and forms a positive feedback loop with hypoxia-inducible factor 1α (HIF-1α) under anoxic condition. This study explored the expression, cellular distribution, and function of WSB1 in bleomycin (BLM)-induced mouse lung injury and fibrosis. WSB1 expression was highly induced by BLM injury and correlated with the progression of lung fibrosis. Significantly, conditional deletion of Wsb1 in adult mice ameliorated BLM-induced pulmonary fibrosis. Phenotypically, Wsb1-deficient mice showed reduced lipofibroblast to myofibroblast transition, but enhanced alveolar type 2 proliferation and differentiation into alveolar type 1 after BLM injury. Proteomic analysis of mouse lung tissues identified caveolin 2 as a potential downstream target of WSB1, contributing to BLM-induced epithelial injury repair and fibrosis. These findings unravel a vital role for WSB1 induction in lung injury repair, thus highlighting it as a potential therapeutic target for pulmonary fibrosis.

Myofibroblast and pro-fibrotic cytokines in fibrosis of IgG4-related disease (IgG4-RD) patients from South Asia: preliminary data.

Fibrosis is a typical pathological characteristic in IgG4-RD patients and often irreversible. There exists a lack of suitable markers for detection of earlier onset of fibrosis in various organs in IgG4-RD patients. Hence, this study aims at analysing ambispectively the myofibroblasts and the pro-fibrotic cytokines, IFN gamma and IL-33 involved in IgG4-RD associated fibrosis in South Asian patients. Archived biopsy samples of definite/probable/possible cases of IgG4-RD, classified according to diagnostic criteria, taken from patients who attended the OPD and IPD of our tertiary care centre during January 2015-January 2020 were chosen for this study.The paraffin sections were examined qualitatively for fibrosis and the excessive collagen deposition by Hematoxylin & Eosin and Masson's Trichrome staining. Also, the presence of alpha-Smooth muscle actin (α-SMA) expressing myofibroblasts and the involvement of pro-fibrotic cytokines (IFN-gamma, IL-33) were assessed by Immunohistochemistry and scored semi-quantitatively (+mild, ++moderate, +++ severe). Serum IL-33 levels were analysed by indirect Elisa (R & D Systems). Myofibroblasts were present in 10/12 biopsy samples, in moderate levels in 4 (33%) and very high levels (+++) in 3 (25%) of the patients. IFN-gamma was expressed at low levels in 6 (50%) and absent in 6 (50%). All patients showed IL-33 expression with very high levels in tissue (6, 50%), as well as in serum samples. The findings of this study reinforce the role of myofibroblasts and profibrotic cytokines like IL-33 in fibrosis of Ig4-RD patients, pointing to their potential as earlier predictive markers of onset and extent of fibrosis.

Schwann cell derived pleiotrophin stimulates fibroblast for proliferation and excessive collagen deposition in plexiform neurofibroma.

Neurofibromatosis type 1 associated plexiform neurofibroma (pNF) is characterized by abundant fibroblasts and dense collagen, yet the intricate interactions between tumor-origin cells (Schwann cells) and neurofibroma-associated fibroblasts (NFAFs) remain elusive. Employing single-cell RNA sequencing on human pNF samples, we generated a comprehensive transcriptomics dataset and conducted cell-cell communication analysis to unravel the molecular dynamics between Schwann cells and NFAFs. Our focus centered on the pleiotrophin (PTN)/nucleolin (NCL) axis as a pivotal ligand-receptor pair orchestrating this interaction. Validation of PTN involvement was affirmed through coculture models and recombinant protein experiments. Functional and mechanistic investigations, employing assays such as CCK8, EdU, Western Blot, ELISA, Hydroxyproline Assay, and Human phospho-kinase array, provided critical insights. We employed siRNA or inhibitors to intercept the PTN/NCL/proline-rich Akt substrate of 40 kDa (PRAS40) axis, validating the associated molecular mechanism. Our analysis highlighted a subset of Schwann cells closely linked to collagen deposition, underscoring their significance in pNF development. The PTN/NCL axis emerged as a key mediator of the Schwann cell-NFAF interaction. Furthermore, our study demonstrated that elevated PTN levels enhanced NFAF proliferation and collagen synthesis, either independently or synergistically with TGF-β1 in vitro. Activation of the downstream molecule PRAS40 was noted in NFAFs upon PTN treatment. Crucially, by targeting NCL and PRAS40, we successfully reversed collagen synthesis within NFAFs. In conclusion, our findings unveil the pivotal role of the PTN/NCL/PRAS40 axis in driving pNF development by promoting NFAFs proliferation and function. Targeting this pathway emerges as a potential therapeutic strategy for pNF. This study contributes novel insights into the molecular mechanisms governing pNF pathogenesis.

The Role of MicroRNAs in Mesenchymal Stem Cell-Based Modulation of Pulmonary Fibrosis.

Pulmonary fibrosis is a complex and multifactorial condition that involves a cascade of events, including lung injury, damage of alveolar epithelial cells (AECs), generation of immune cell-driven inflammation, and activation of fibroblasts and their differentiation into myofibroblasts, resulting in the excessive production and deposition of collagen and progressive scarring and fibrosis of the lung tissue. As lung fibrosis advances, the scarring and stiffening of lung tissue can significantly hinder the exchange of oxygen and carbon dioxide, potentially leading to respiratory failure that can be life-threatening. Anti-inflammatory and immunosuppressive drugs are used to slow down the progression of the disease, manage symptoms, and enhance the patient's quality of life. However, prolonged immunosuppression could increase the susceptibility to severe bacterial, viral, or fungal pneumonia in lung-transplant recipients. Therefore, there is an urgent need for new therapeutic agents that can effectively reduce lung inflammation and fibrosis without compromising the protective immune response in patients with severe lung fibrosis. Results obtained in recently published studies demonstrated that mesenchymal stem/stromal cell-derived microRNAs (MSC-miRNAs) could attenuate detrimental immune response in injured lungs and prevent progression of lung fibrosis. Through the post-transcriptional regulation of target mRNA, MSC-miRNAs modulate protein synthesis and affect viability, proliferation, and cytokine production in AECs, fibroblasts, and lung-infiltrated immune cells. In order to delineate molecular mechanisms responsible for beneficial effects of MSC-miRNAs in the treatment of lung fibrosis, in this review article, we summarized current knowledge related to anti-fibrotic and anti-inflammatory pathways elicited in immune cells, AECs, and myofibroblasts by MSC-miRNAs.

Inhibitory effect of a novel Curcumin derivative DMC-HA on keloid fibroblasts.

Keloids pose a significant dermatological challenge, marked by abnormal fibroblast proliferation and excessive collagen deposition in response to skin injury or trauma. In the present study, we introduce DMC-HA, a derivative of Curcumin, as a promising candidate for keloid treatment. DMC-HA is poised to provide superior therapeutic benefits compared to Curcumin due to its structural modifications. Examining the comparative effects of DMC-HA and Curcumin on keloid fibroblasts can offer insights into their potential as therapeutic agents and the underlying mechanisms in keloid pathogenesis. In our study, CCK-8 experiments revealed that, at equivalent concentrations, DMC-HA demonstrated greater efficacy in inhibiting the proliferation of keloid fibroblasts compared to Curcumin. Flow cytometry analysis indicated that DMC-HA induced fibroblast apoptosis more significantly than Curcumin at the same concentration. Further data demonstrated that DMC-HA notably increased the production of reactive oxygen species (ROS), upregulated the expression levels of Bax, cleaved PARP, and cleaved Caspase-3. Interestingly, the impact of DMC-HA was reversed upon the application of the antioxidant NAC. Additionally, DMC-HA could suppress IL-6-induced increased expression of p-STAT3. Collectively, our findings suggest that DMC-HA is more effective than Curcumin in inhibiting the proliferation of keloid fibroblasts. The underlying mechanism of its action appears to be associated with the augmentation of ROS induction and the concurrent inhibition of STAT3 activation.

Effect and mechanism of action of botulinum toxin type A on hypertrophic scar <i>in vitro</i>

Purpose: To examine the effect of botulinum toxin type A (BTXA) on the formation of hypertrophic scar and to unravel its mechanism of action.
 Methods: HSF cells were isolated from hypertrophic scars and cultured. Immunohistochemistry (IHC) assays were performed to determine TGF-β1, FN, and Col1 expressions in hypertrophic scar and normal tissues while the expressions and phosphorylation of p38, ERK, JNK, as well as the expressions of α-SMA, Col1 and FN1 in HSF cells were evaluated by immunoblot techniques. CCK-8 and Transwell assays were used to assess the effect of BTXA on the viability and motility of HSF cells.
 Results: BTXA suppressed MAPK pathway in hypertrophic scar fibroblasts (p < 0.01). It also suppressed excessive collagen deposits in hypertrophic scar through MAPK pathway (p < 0.01), and restrained HSF growth and motility via MAPK pathway (p < 0.01).
 Conclusion: BTXA suppresses hypertrophic scarring via MAPK pathway and thus, can potentially be developed as a drug for the treatment of hypertrophic scarring.

Protective effect of Moringa oleifera leaf ethanolic extract against uranyl acetate-induced testicular dysfunction in rats

Uranyl acetate (UA) is used in civilian and military applications, predisposing it to wide dispersion in ecosystems. Using high-performance liquid chromatography, gas chromatography–mass spectrometry, and 2,2-Diphenyl-1-picrylhydrazyl scavenging radical analysis, we confirmed that Moringa oleifera leaf ethanolic extract (MLEE) is rich in biologically active phytochemicals. Thus, this study aims to investigate the possible defensive effect of MLEE against UA-induced testicular dysfunction. To achieve this, rats were divided randomly and evenly into three groups for 14 days. The control group received no treatment, while the UA group received a single intraperitoneal injection of UA at a dose of 5 mg/kg BW dissolved in saline on the 12th day of the experiment, followed by no treatment the following day. The MLEE + UA group received daily oral administration of MLEE (300 mg/kg BW) dissolved in distilled water before exposure to UA intoxication. The disruption observed in the pituitary–gonadal axis of UA-intoxicated rats was characterized by a significant decrease in luteinizing hormone, follicle-stimulating hormone, testosterone, and estradiol 17beta levels. Additionally, there was a notable increase in malondialdehyde and a decrease in catalase, superoxide dismutase, reduced glutathione, and nitric oxide, accompanied by an up-regulation in the immuno-expression of nuclear factor-kappa B, indicating a disturbance in the redox balance. The TUNEL assay confirmed a substantial rise in apoptotic cell numbers in the UA group. Testicular histopathological changes, excessive collagen deposition, and reduced glycogen content were evident following UA exposure. However, supplementation with MLEE effectively countered these mentioned abnormalities. MLEE is proposed to combat the toxicological molecular targets in the UA-affected testis by restoring the balance between oxidants and antioxidants while obstructing the apoptotic cascade. MLEE contains an abundance of redox-stabilizing and cytoprotective phytochemicals that have the potential to counteract the mechanistic pathways associated with UA exposure. These findings encourage further research into other plausible protective aspects of Moringa oleifera against the UA challenge.

Amniotic membrane modulates MMP9 and MMP12 gene and protein expression in experimental model of the hepatic fibrosis.

Hepatic fibrosis is characterized by excessive deposition of collagen in the hepatic parenchyma, which disturbs the normal architecture and function. We have shown that human amniotic membrane (AM) can be used as a patch on the whole liver surface, resulting in an extremely significant reduction in collagen deposition. The aim of this study was to investigate the effects of AM on the matrix metalloproteinase 9 (MMP9) and matrix metalloproteinase 12 (MMP12) genes and proteins expression by real time quantitative PCR and immunohistochemistry, respectively, as well as image analysis on biliary fibrosis induced in rats by the bile duct ligation (BDL).Two weeks after the BDL, an AM fragment was applied onto the liver, and four weeks later, the liver samples were collected. MMP9 and MMP12 genes were significantly over expressed in group treated with AM. The immunoexpression of MMP9 and MMP12 was observed in all groups. However, the quantitative image analysis demonstrated an increase of the area occupied only by MMP12 in the livers of AM-treated rats with respect to BDL rats. These findings suggest that the AM exerts its beneficial effects on biliary fibrosis by increasing the MMP12, which in turn reduces the excessive collagen deposition on liver tissue.

Inhibition of the ITGB1 gene attenuates crystalline silica-induced pulmonary fibrosis via epithelial-mesenchymal transformation.

Silicosis is a systemic disease caused by long-term exposure to high concentrations of free silica dust particles in the workplace. It is characterized by a persistent inflammatory response, fibroblast proliferation, and excessive collagen deposition, leading to pulmonary interstitial fibrosis. Epithelial interstitial transformation (EMT) can cause epithelial cells to lose their tight junctions, cell polarity, and epithelial properties, thereby enhancing the properties of interstitial cells, which can lead to the progression of fibrosis and the formation of scar tissue. Integrin 1 (ITGB1) is considered an important factor for promoting EMT and tumor invasion in a variety of tumors and also plays an important role in the progression of fibrotic diseases. Therefore, ITGB1 can be used as a potential target for the treatment of silicosis. In this study, we found that silica exposure induced epithelial-mesenchymal transformation in rats and that the expression of integrin ITGB1 was elevated along with the EMT. We used CRISPR/Cas9 technology to construct integrin ITGB1 knockdown cell lines for in vitro experiments. We compared the expression of the EMT key proteins E-cadherin and vimentin in the ITGB1 knockdown cells and wild-type cells simultaneously stimulated by silica and detected the aggregation point distribution of E-cadherin and vimentin in the cells using laser confocal microscopy. Our results showed that ITGB1 knockout inhibited the ITGB1/ILK/Snail signaling pathway and attenuated the EMT occurrence compared to control cells. These results suggested that ITGB1 is associated with silica-induced EMT and may be a potential target for the treatment of silicosis.

The Role of Tenascin-C in Hypertrophic Scar Formation: Insights from Cell and Animal Experiments.

Hypertrophic scars (HS) are dermal diseases characterized by excessive fibroblast proliferation and collagen deposition following burns or trauma. While Tenascin-C (TNC)'s role in promoting visceral fibrosis has been established, its impact on skin tissue fibrosis remains unclear. This study aims to investigate the effects of TNC on HS. RNA sequence and IHC techniques were used to examine the upregulation of TNC gene in human hypertrophic scar tissue compared to normal tissues. Knockdown of TNC in Human skin fibroblasts (HFF-1) cells was achieved, and expression of Col1 and Col3 was evaluated using qPCR. Sirius red collagen staining assessed impact on total collagen content and ECM deposition. Effects on cell proliferation and migration were investigated through cck-8 and cell scratch experiments. Lentivirus infection was used to knock out TNC, and resulting samples were injected into ear wound of rabbits. Effects of TNC knockout on ear scar formation were measured using digital morphology, ultrasound, SEI, H&E, and Masson trichrome methods. Cell experiments: downregulation of TNC decreased Col1 and Col3 expression, leading to reduced collagen production and extracellular matrix deposition. It did not affect HFF-1 cell proliferation and migration. Animal experiments: TNC knockdown promoted wound healing and reduced collagen deposition in rabbit ears. This study suggests that knocking down TNC inhibits collagen formation and extracellular matrix deposition, thereby inhibiting hypertrophic scar formation. Therefore, TNC can be considered a potential biomarker for HS formation and may offer promising treatment strategies for clinical management of hypertrophic scars.