Apoptosis In Fibroblasts Research Articles

Supramolecular Assembly‐Enabled Transdermal Therapy of Hypertrophic Scarring Through Concurrent Ferroptosis‐Apoptosis

AbstractHypertrophic scar (HS) remains a major challenge for clinicians due to unsatisfactory therapeutic performance. Although inducing apoptosis of HS fibroblasts (HSFs) has proved to be an effective nonsurgical treatment strategy, potential chemotherapy resistance to apoptosis of HSFs makes the development of new and efficient strategies highly demanding. Herein, a ferroptosis‐apoptosis combined therapeutic strategy for the treatment of HS is developed through supramolecular self‐assembly between cucurbit[7]uril (CB[7]) and two bioactive agents, dihydroartemisinin (DHA) and gold nanoclusters (AuNCs). The resulting self‐assembled supramolecular nanoparticles, named CAD NPs, showed high guest loading efficiency and prominent pH‐responsive degradability in the acidic lysosomes of HSFs. Importantly, both DHA and AuNCs act synergistically to generate excessive reactive oxygen species and lipid peroxidation, leading to mitochondrial damage, and ultimately inducing concurrent ferroptosis and apoptosis on HSFs. Upon further loading into hydrogel microneedles to facilitate their transdermal delivery, these CAD NPs showed superior antiscar therapeutic effects in shortening the treatment span to 3 weeks and improving the HS appearance, as demonstrated at a rabbit ear model of HS. The present supramolecular assembly‐based ferroptosis‐apoptosis strategy provides an innovative guideline for efficiently treating HS as well as other diseases.

Diagnostic Value of lncRNA FGD5-AS1 Sponge miR-223-3p in Infantile Pneumonia and Its Prognostic Effect on Rehabilitation

Abstract Objective This study aimed to uncover the value of long noncoding RNA FGD5-AS1 (lncRNA FGD5-AS1) in the diagnosis of infantile pneumonia and explore its pathological mechanism in lung fibroblasts. This research may provide a potential biomarker for diagnosing patients and predicting their rehabilitation outcomes. Methods This study included 92 children with infantile pneumonia as the research object, and an equal number of healthy children were introduced as controls. The FGD5-AS1 content was detected using real-time quantitative polymerase chain reaction (RT-qPCR) assay. The diagnostic value of FGD5-AS1 was evaluated by receiver operating characteristic (ROC) and logistic analysis. The molecular mechanism of FGD5-AS1 and miR-223-3p was studied by luciferase activity assays. The impact of abnormal FGD5-AS1 expression on the proliferation and apoptosis of lung fibroblasts was analyzed using the cell counting kit-8 (CCK-8) and flow cytometry. Results FGD5-AS1 expression was decreased in the serum of infantile pneumonia patients, which may be a diagnostic marker for children with pneumonia. Furthermore, FGD5-AS1 has the ability to predict patient outcomes. FGD5-AS1 levels in lung fibroblasts (WI-38) decreased when induced by lipopolysaccharide (LPS). This decline resulted in reduced cell proliferation ability, increased apoptosis rate, and elevated inflammatory factor content. However, upregulated FGD5-AS1 counteracted the effects of LPS on WI-38 cells activity and inflammatory factors. Conclusion FGD5-AS1 may act as a potential marker in infantile pneumonia, and regulate the biological activity and inflammation level of lung fibroblasts by targeting miR-223-3p.

The therapeutic potential of PX-478 in a murine model of pelvic organ prolapse

Background Pelvic organ prolapse (POP), characterised by the downward displacement of pelvic organs, is a prevalent disorder that affects adult women. This study explored the therapeutic potential of PX-478, a selective hypoxia-inducible factor-1α (HIF-1α) inhibitor, in a murine POP model. Methods A murine POP model was established through ovariectomy, mimicking oestrogen deprivation. Fifteen C57BL/6J mice were randomly assigned to control, POP, and PX-478 groups. PX-478, targeting HIF-1α, was administered intravaginally. The analysis of fibroblasts, macrophage and inflammation was performed through Masson staining, immunofluorescence, and ELISA. Collagen distribution was assessed using Sirius Red staining. Expression levels of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMP-1) were determined through immunohistochemistry and western blot. Fibroblast proliferation and apoptosis were evaluated by CCK-8 assay and flow cytometry. Results PX-478 treatment significantly reduced vaginal length, indicating a therapeutic effect on POP severity. Masson staining revealed reduced fibrotic changes and collagen disruption in PX-478-treated mice. Immunofluorescence showed increased fibroblast markers (Vimentin, α-SMA) and collagen fibres by PX-478. Sirius Red staining indicated PX-478 mitigated damage to Type I and Type III collagen fibres. PX-478 significantly reduced MMP-2 and MMP-9 expression while increased TIMP-1. In macrophages, PX-478 decreased M1 and M2 markers (CD80, CD206) and IL-18 secretion. Fibroblasts exhibited increased proliferation, reduced apoptosis, and altered MMP/TIMP expression under PX-478 influence. Conclusion PX-478 demonstrates a therapeutic potential in the mice POP model. It reduces vaginal length, attenuates fibrosis, and modulates collagen synthesis. Its immunomodulation is evident through reduced M1 and M2 macrophages and suppressed IL-18 secretion.

Inhibition of Small Extracellular Vesicles by GW4869 Does not Disrupt the Paracrine Regulation of Adipose-Derived Mesenchymal Stem Cells Over Keloid Fibroblasts.

Keloid, scar caused by atypical wound repair, represents a significant difficulty for specialists in plastic surgery and dermatology. Adipose-derived mesenchymal stem cells (ADSCs) can regulate fibrotic phenotypes of keloid fibroblasts (KFs) in a paracrine fashion, but whether small extracellular vesicles (SEVs) are the key functional carrier in ADSC paracrine regulation of KFs remains unknown. This study aims to explore whether the regulatory effects of conditioned medium (CM) obtained from ADSCs on KFs can be impaired by decreased SEV content in the ADSC-CM. Clinical specimens were utilized to extract keloid fibroblasts (KFs), normal fibroblasts (NFs), and adipose-derived stem cells (ADSCs). Fibroblasts were cultured with CM obtained from ADSCs untreated or treated with the sphingomyelinase inhibitor GW4869. The features of SEVs derived from ADSC-CM were characterized, and fibroblast proliferation, migration, apoptosis, and expression of ECM proteins were analyzed. The sphingomyelinase inhibitor GW4869 successfully reduced the SEV content in ADSC-CM, and both control ADSC-CM and ADSC-CM with reduced SEV content significantly inhibited KF proliferation, migration, and α-SMA synthesis but not KF apoptosis, whereas only NF proliferation was inhibited by ADSC-CM. The reduced SEV content only affected the inhibition of KF proliferation induced by ADSC-CM. ADSC-CM inhibits various fibrotic phenotypes of KFs, but decreasing the SEV content in ADSC-CM did not significantly alter the antifibrotic effects of ADSC-CM. Thus, SEVs may not be the key mediator of ADSCs paracrine regulation of KFs. This journal requires that authors assign a level of evidence to each submission to which Evidence-Based Medicine rankings are applicable. This excludes Review Articles, Book Reviews, and manuscripts that concern Basic Science, Animal Studies, Cadaver Studies, and Experimental Studies. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors . www.springer.com/00266 .

Administration of a combination of COX-2/TGF-β1 siRNAs induces hypertrophic scar fibroblast apoptosis through a TP53 mediated caspase pathway

Hypertrophic scar (HTS) formation is a pathological fibrotic skin disease, with no satisfactory treatments available currently. Inducing apoptosis of HTS-derived fibroblasts (HSFs) are becoming promising approaches. In this research, we aim to improve the technology with co-delivery COX-2 and TGF-β1 siRNAs and further investigate the underlying mechanism. Firstly, the HSFs were transfected with 1 µg/ml COX-2 and/or TGF-β1 siRNAs, and proved that the apoptosis of HSFs was greater induced by COX-2/TGF-β1 siRNAs than either COX-2 or TGF-β1 siRNA alone by flow cytometry. To investigate the impact of co-silencing TGF-β1 and COX-2 mRNA expression in vivo, we established HTSs model in rat tails. Our results confirmed that co-silencing of TGF-β1 and COX-2 mRNA expression could significantly alleviate the HTS formation in vivo. Furthermore, we explored the potential molecular mechanism and revealed that the protein levels of TP53, Bcl-2 and Caspase-3 were downregulated while Bax and Cleaved Caspase-3 were upregulated in the COX-2/TGF-β1 siRNA groups compared with HKP group. Taken together, our results demonstrated that simultaneous silencing of COX-2 and TGF-β1 expression by siRNAs induced HSF apoptosis through a TP53 mediated caspase pathway. Therefore, COX-2/TGF-β1 siRNAs might serve as a novel and effective therapeutic alternative for HTSs treatments.

Manuka Essential Oil Triggers Apoptosis and Activation of c-Jun N-Terminal Kinase in Fibroblasts and Fibrosarcoma Cells.

Manuka essential oil has long been used in traditional medicine, though the effects of the oil on cancer cells have limited studies. The goal of this project was to treat cancer cell lines with manuka essential oil at different concentrations and to ascertain the effects on the cell proliferation of normal fibroblast (CUA-4) and on fibrosarcoma (HT-1080) cells. Cell lines were grown on 24-well plates, and subconfluent cultures were treated with varying concentrations of manuka oil for 24 h. The effect of the oil on proliferation and viability was measured through direct cell counting using trypan blue dye exclusion and through the use of an MTT assay. As the concentration of oil increased, proliferation of all cell lines tested decreased with increasing dosage, concurrently with a decrease in MTT activity. To determine if the decrease in cell numbers observed from manuka oil treatment is the result of apoptosis, PARP cleavage assays were performed, confirming that the treatment caused apoptosis in both normal fibroblasts and fibrosarcoma cells. The stress-activated MAPK protein, JNK, was activated by manuka essential oil treatment, occurring synergistically with a decrease in MKP-1 expression.

5-Demethylnobiletin Ameliorates Isoproterenol-Induced Cardiac Fibrosis and Apoptosis by Repressing the Sirt1/FOXO3a/NF-κB and Wnt/β-Catenin Pathways.

Apoptosis and fibrosis are two main factors leading to heart failure. 5-Demethylnobiletin (5-OH-Nob) is a natural polymethoxyflavone derived from the peel of citrus fruits that has many biological effects, such as antioxidative stress and anti-inflammatory effects. Here, we aimed to probe the function and mechanism of 5-OH-Nob in myocardial damage. Primary rat cardiac fibroblasts were exposed to isoproterenol (ISO, 10 µM) to establish an in vitro model of cardiac damage, and ISO (30 mg/kg/d) was used to induce myocardial fibrosis in mice. 5-OH-Nob was used for treatment in vivo and ex vivo. Functional assays revealed that 5-OH-Nob alleviated the apoptosis and fibrosis of cardiac fibroblasts treated with ISO and increased cell viability (p < 0.05). In vivo, 5-OH-Nob treatment ameliorated cardiac injury in ISO-treated mice (p < 0.05). Mechanistically, 5-OH-Nob treatment enhanced Sirt1 expression and suppressed ISO-mediated activation of the FOXO3a/nuclear transcription factor-κB (NF-κB) and Wnt/β-catenin pathways. Furthermore, Sirt1 inhibition attenuated the protective effect of 5-OH-Nob on ISO-induced cardiac apoptosis and fibrosis. Overall, 5-demethylnobiletin mediates the Sirt1/FOXO3a/NF-κB and Wnt/β-catenin pathways to mitigate ISO-induced myocardial fibrosis and apoptosis.

STIL overexpression shortens lifespan and reduces tumor formation in mice.

Centrosomes are the major microtubule organizing centers of animal cells. Supernumerary centrosomes are a common feature of human tumors and associated with karyotype abnormalities and aggressive disease, but whether they are cause or consequence of cancer remains controversial. Here, we analyzed the consequences of centrosome amplification by generating transgenic mice in which centrosome numbers can be increased by overexpression of the structural centrosome protein STIL. We show that STIL overexpression induces centrosome amplification and aneuploidy, leading to senescence, apoptosis, and impaired proliferation in mouse embryonic fibroblasts, and microcephaly with increased perinatal lethality and shortened lifespan in mice. Importantly, both overall tumor formation in mice with constitutive, global STIL overexpression and chemical skin carcinogenesis in animals with inducible, skin-specific STIL overexpression were reduced, an effect that was not rescued by concomitant interference with p53 function. These results suggest that supernumerary centrosomes impair proliferation in vitro as well as in vivo, resulting in reduced lifespan and delayed spontaneous as well as carcinogen-induced tumor formation.

Molybdenum temporary epicardial pacing wires: function and biodegradation

Abstract Cardiac pacing with temporary epicardial pacing wires (TEPW) is used to treat rhythm disturbances after cardiac surgery. Wires typically begin to fail around postoperative day four and are extracted. Occasionally, TEPW cannot be mechanically removed, have to stay in the thorax and may rarely cause serious complications like migration and infection. We aim to develop novel, bioresorbable TEPW which will dissolve over time, even if postoperative removal is unsuccessful. We manufactured prototypical braided molybdenum (Mo) leads (16 Mo wires of 40 µM diameter each, length 18 cm for ex vivo, 7.5 cm for in vivo experiments) coated with the biodegradable polymers poly(lactide-co-glycolic acid) (PLGA, inner coating) and polycaprolactone (PCL, outer coating) for shaping and electrical insulation. Mo electrodes showed similar pacing and sensing properties to conventional steel electrodes (Osypka) in Langendorff-perfused rat hearts, even with somewhat lower stimulation thresholds. In artificial body fluid at 37°C, the polymer-coated Mo electrodes dissolved at a rate of 1.6 ± 0.3 µg/cm2·d (n = 5) compared to uncoated electrodes with 30.3 ± 0.8 µg/cm2·d (n = 4, p &amp;lt; 0.001). Assessing apoptosis and necrosis in human cardiomyocytes and cardiac fibroblasts, we detected no toxicity at Mo concentrations up to 0.52 mM. To test the in vivo properties of Mo TEPW, we sutured them epicardially onto the anterior wall of the heart of female Wistar rats, led them out of the thorax through an intercostal space and placed them in a subcutaneous pocket. We tested electrophysiological properties directly after implantation and after different time periods. Mo TEPW showed similar pacing and sensing properties directly upon implantation and after 2 weeks, with only impedance and slew rate of the sensed R-wave decreasing time-dependently. After one month, all but one pair of Mo electrodes were mechanically broken at their exit from the thorax, the site of assumedly highest mechanical stress. Progressive Mo degradation led to multiple fragmentation of the Mo TEPW after 6 months. The conventional steel TEPW we used as control had similar electrical properties directly after implantation, after 2 weeks and one month without signs of broken electrodes. After 6 months, all but one pair of steel electrodes were mechanically broken at their thorax exit site. Comparing urinary Mo concentration of Mo TEPW treated rats to controls, we saw similar values of 1.9 ± 1.9 µM (n = 6) vs. 0.6 ± 0.2 µM (n = 5, n.s.) after one week. In contrast, we saw a significant increase of 12.1 ± 9.7 µM (n = 5) vs. 1.0 ± 0.9 µM (n = 5, p &amp;lt; 0.001) after 6 months, reflecting the degradation progress. We demonstrate that Mo TEPW are a feasible option for epicardial pacing in vivo for up to 2 weeks and observed the progress of Mo degradation up to 6 months. These findings represent an important step in the development of bioresorbable TEPW as a novel and even safer approach to temporary epicardial pacing.Graphical AbstractIn Vivo Degradation Progress

Silencing of ERRα gene represses cell proliferation and induces apoptosis in human skin fibroblasts.

Estrogen‑related receptor (ERR) is an orphan nuclear receptor structurally akin to the estrogen receptor. ERR is expressed in tissues with active energy metabolism and regulates intracellular metabolic functions. Additionally, ERRs are known to be strongly expressed in the epidermis of skin tissue, but their functions are unknown. The present study investigated the function of ERRα in human skin fibroblasts. ERRα expressed in human dermal fibroblast TIG113 was knocked down using small interfering (si)RNA and gene expression was comprehensively analyzed using microarrays 48 h later. Pathway analysis was performed using Wikipathways on genes exhibiting expression changes of ≥1.5‑fold. Expression of cell cycle‑related and apoptosis‑related genes was compared using reverse transcription‑quantitative PCR. After treating TIG113 cells with siERRα for 72 h, cell proliferation was assessed using the Cell Counting Kit‑8 or a scratch wound healing assay and apoptotic cells were measured using the Poly Caspase Assay Kit. Cell cycle analysis was performed using flow cytometry. The expression of the ERRα gene was suppressed by siRNA. The expression of genes associated with cell cycle‑related pathways were decreased while that of those associated with apoptosis‑related pathways increased. Furthermore, the expression of cell cycle‑related genes such as cell division cycle 25C, cyclin E and cyclin B1 was decreased and the expression of apoptosis‑related genes such as caspase3 and Fas cell surface death receptor was increased. Cell proliferation was suppressed and the number of apoptotic cells increased ~2‑fold in ERRα‑knockdown TIG113 cells. Cell cycle analysis revealed that the number of cells in the Sub‑G1 phase increased and that in the S and G2/M phases decreased. The present study suggested that ERRα is an essential for the survival of human skin fibroblasts.

A New Strategy to Inhibit Scar Formation by Accelerating Normal Healing Using Silicate Bioactive Materials.

Inspired by the scar-free wound healing in infants, an anti-scar strategy is proposed by accelerating wound healing using silicate bioactive materials. Bioglass/alginate composite hydrogels are applied, which significantly inhibit scar formation in rabbit ear scar models. The underlining mechanisms include stimulation of Integrin Subunit Alpha 2 expression in dermal fibroblasts to accelerate wound healing, and induction of apoptosis of hypertrophic scar fibroblasts by directly stimulating the N-Acylsphingosine Amidohydrolase 2 expression in hypertrophic scar fibroblasts, and indirectly upregulating the secretion of Cathepsin K in dermal fibroblasts. Considering specific functions of the bioactive silicate materials, two scar treatment regimes are tested. For severe scars, a regenerative intervention is applied by surgical removal of the scar followed by the treatment with bioactive hydrogels to reduce the formation of scars by activating dermal fibroblasts. For mild scars, the bioactive dressing is applied on the formed scar and reduces scar by inducing scar fibroblasts apoptosis.

Compression-induced apoptosis of fibroblasts and myofibroblasts in an in vitro model of pulmonary fibrosis by alginate/gelatin scaffold

Pulmonary fibrosis leads to increased mortality but is poorly understood. Fibrotic progression is associated with abnormal wound repair and an increase in myofibroblast cell populations. Here we investigate how the myofibroblast population is impacted by unique compression-induced apoptosis derived from mechanical strain characteristic of asthma. Using a mechanical device, both static and dynamic mechanical strains were applied to alginate/gelatin/CaCl2 scaffolds containing fibroblasts and myofibroblasts. As cell groups were stimulated with 30 % static strain for 12 h, fibroblast and myofibroblast cell groups showed increased cell apoptosis by 5.55 % and 19.56 %, respectively, compared to control groups. Additionally, myofibroblasts exhibited higher susceptibility to apoptosis induction than did fibroblasts. Comparing dynamic and static loading modes, dynamic loading resulted in a higher apoptosis rate of fibroblast and myofibroblast cells, indicating its potential to induce apoptosis effectively. These findings suggest that mechanical stimulation can be considered a promising approach to induce apoptosis in myofibroblasts, thus offering the potential for future approaches to treating pulmonary fibrosis. Moreover, mechanical loads can be designed for other diseases, selectively reducing or increasing apoptosis in either hard or soft cell groups, based on specific application needs.

Design, synthesis, and evaluation of a novel TRAIL-activated HDAC6 inhibitor for the treatment of pulmonary fibrosis

Pulmonary fibrosis (PF) is a common, severe, chronic, and progressive pulmonary interstitial disease characterized by rapid disease progression and high mortality. Despite the Food and Drug Administration (FDA)’s approval of two antifibrotic drugs, nintedanib and pirfenidone, effectively halting the progression of pulmonary fibrosis remains challenging. Histone deacetylase (HDAC) inhibitors have indeed emerged as an important class of antitumour drugs. However, their application in the treatment of fibrotic diseases is still relatively limited. Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) has the potential to inhibit fibrotic processes by inducing fibroblast apoptosis. In this study, we designed and synthesized a series of histone deacetylase 6 (HDAC6) inhibitors that activate TRAIL, among which compound 7e exhibited potent inhibitory activity against HDAC6, with an IC50 of 42.90 ± 4.96 nM and superior antiproliferative effects on fibroblasts. Therefore, we further investigated its anti-pulmonary fibrosis effect in mouse models of both idiopathic pulmonary fibrosis (IPF) and silicosis. Our results suggest that compound 7e is a promising candidate for the treatment of pulmonary fibrosis.

Mitochondrial functional impairment in ARL3-mutation related rod-cone dystrophy.

Mitochondria are vital for retinal cell function and survival, and there is growing evidence linking mitochondrial dysfunction to retinal degenerations. Although ARL3 mutations have been linked to multiple forms of retinal degeneration, the relationship between ARL3 and mitochondria remains unexplored. Herein, we investigated the effects of ARL3 T31A , ARL3 C118F , and ARL3 T31A/C118F mutations on mitochondrial function in fibroblasts obtained from patients with ARL3-related rod-cone dystrophy. Our findings revealed that these mutations led to a decrease in mitochondrial respiration, an increase in the accumulation mitochondrial reactive oxygen species (ROS), and induction of apoptosis in fibroblasts. Additionally, we conducted a comparative analysis of the effects of ARL3T31A, ARL3C118F, and ARL3T31A/C118F proteins on mitochondria in ARPE-19 cells. Results showed that ARL3T31A and ARL3T31A/C118F not only affected mitochondrial function but also induced apoptosis in ARPE-19 cells. Conversely, ARL3C118F primarily influenced cell apoptosis with minimal effects on mitochondrial function in ARPE-19 cells. Transcriptome analysis further suggested the involvement of respiratory electron transport, response to ROS, and apoptotic signaling pathways in ARL3T31A/C118F cells. Our study demonstrated that ARL3-related mutations play a significant role in the diversity of mitochondrial function, providing novel insights into the functional analysis of ARL3-related mutations.

Anti-Heat Shock Protein 70 Autoantibodies from Patients with Idiopathic Pulmonary Fibrosis Epigenetically Enhance Lung Fibroblast Apoptosis Resistance and Bcl-2 Expression.

IgG autoantibodies to heat shock protein 70 (HSP70) are found in many immune-mediated clinical syndromes, and their presence among patients with idiopathic pulmonary fibrosis (IPF) portends especially poor outcomes. However, pathological effects of IPF anti-HSP70 have not been studied extensively. IPF lung fibroblasts are apoptosis resistant, and this dysregulation contributes to the accumulation of fibroblasts that characterizes the disease. During stress, HSP70 protein is exported extracellularly, where it binds to cognate cell surface receptors that mediate a variety of functional effects, including apoptosis inhibition. We hypothesized anti-HSP70 could engage HSP70-receptor complexes on fibroblasts that alter their apoptosis susceptibility. We found HSP70 is ubiquitously expressed on primary human lung fibroblasts. Treatment with anti-HSP70 isolated from patients with IPF with acute exacerbations increased Bcl-2 expression in human lung fibroblasts and reduced their susceptibility to staurosporine-induced apoptosis. Chromatin immunoprecipitation assays showed Bcl-2 gene promoter regions are enriched with the active histone mark H4 lysine 16 acetylation, and this was increased in the autoantibody-treated fibroblasts. When H4 lysine 16 acetylation was decreased by knocking down its acetyltransferase, MOF (males absent on the first), the anti-HSP70 treatments failed to upregulate Bcl-2. This study describes a heretofore unknown, to our knowledge, pathogenic consequence of autoimmunity in which autoantibodies affect the epigenetic regulation of fibroblast apoptosis. In addition to IPF, this autoimmune process could also have relevance in other immunological syndromes characterized by anti-HSP70 autoimmunity. These findings lend credence to the importance of autoimmunity in IPF and illustrate pathways that could be targeted in innovative therapies for this morbid, medically refractory lung disease.

Effects of low dose skin tissue derived peptides on the function and collagen expression of keloid fibroblasts

This study aims to investigate the effects of the skin tissue derived peptides on proliferation, apoptosis, migration and collagen expressions in keloid fibroblasts. From January 2015 to January 2017, patients with hypertrophic scar who underwent surgical excision in department of plastic surgery of Nanjing maternal and child health hospital were included in this retrospective study. Four peptides were selected from the differential peptides between human hypertrophic scar and normal skin tissue. They were named as peptide deregulated in hypertrophic scar 2-5 (PDHPS2-5). Bioinformatics and functional analysis were performed. A low dose of 10 μmol/L of four peptides were respectively added to the culture medium of human primary keloid fibroblasts for 24 h. Cell counting kit-8 (CCK-8) were used to detect the changes in cell viability. Cell apoptosis was detected by flow cytometry. Cell migration ability was checked by Transwell chamber. The protein expressions of collagen COL1A2 (Collagen type I alpha 2) and the myofibroblast marker gene ACTA2 (Actin alpha 2) were analyzed by Western blot. The results showed that bioinformatics prediction analysis revealed that peptide PDHPS4 has the longest half-life and the highest thermal stability. Compared with the control group, low dose of four peptides had no significant effect on the survival rate and apoptosis of keloid fibroblasts tested by CCK-8 assay and flowcytometry. Transwell analysis showed that one peptides (PDHPS5) can significantly inhibit the cell migration ability (The optical density value in Control is 0.81±0.11, in PDHPS5 is 0.27±0.03, t=8.61, P=0.001). Western blot analysis showed that four peptides (PDHPS2, PDHPS3, PDHPS4, PDHPS5) can significantly inhibit the protein expressions of COL1A2 (The relative protein band intensity in Control is 1.02±0.02, in PDHPS2 is 0.21±0.04, in PDHPS3 is 0.26±0.03, in PDHPS4 is 0.53±0.04, in PDHPS5 is 0.73±0.04, t=31.38, 38.54, 18.88, 11.07 respectively, all P value are less than 0.01). Three peptides (PDHPS2, PDHPS3, PDHPS5) can significantly inhibit the protein expressions of ACTA2 (The relative protein band intensity in Control is 1.02±0.02, in PDHPS2 is 0.64±0.05, in PDHPS3 is 0.77±0.06, in PDHPS5 is 0.47±0.07, t=12.08, 6.38, 14.06 respectively, all P value are less than 0.01). In conclusion, the differentially expressed peptides in human hypertrophic scar tissue can affect the function of keloid fibroblasts and collagen expressions to varying degrees. Among them, two peptides (PDHPS2,PDHPS3) significantly inhibit the protein expressions of COL1A2 and ACTA2. The peptide PDHPS5 has high stability, significantly suppresses cell migration, and reduces the protein expressions of COL1A2 and ACTA2, which may provide a new strategy for scar prevention and treatment.

Formononetin alleviates thermal injury-induced skin fibroblast apoptosis and promotes cell proliferation and migration

The aim of this study was to explore the effect and mechanism of formononetin (FMNT) in thermal-injured fibroblast proliferation, apoptosis, and oxidative stress. After thermal injury, human skin fibroblast (HSF) cells showed inhibited proliferation, migration, extracellular matrix (ECM) synthesis; and increased apoptosis, reactive oxygen species (ROS) production, and inflammation. Specifically, after thermal injury, cell viability, migration distance, and protein levels of collagen I, collagen III, α-SMA, MMP1, and MMP3 were reduced; cell apoptosis rate and TUNEL-positive cell numbers were increased; the levels of Bax and cleaved caspase-3 were elevated, while Bcl-2 level was reduced. Moreover, the thermally injured HSF cells showed increased levels of ROS, MDA, LDH, TNF-α, and IL-1β, and decreased GSH, SOD, GSH-Px, and CAT. FMNT levels can partially eliminate the effects of thermal injury on HSF cells, as shown by promoting thermally injured HSF cell proliferation and migration, and inhibiting cell apoptosis, ROS production, and inflammation. FMNT exerted no significant effect on normal HSF cells. Additionally, the levels of the P13K/AKT/mTOR signaling-related proteins (p-P13K, p-AKT, and p-mTOR) were reduced in thermally injured HSF cells, whereas FMNT could promote p-P13K, p-AKT, and p-mTOR levels. FMNT can partially alleviate the thermal injury-induced inhibition of fibroblast proliferation and migration; FMNT also inhibited the apoptosis, ROS level, and inflammation in thermal-injured cells. The effects of FMNT may be mediated by regulating the P13K/AKT/mTOR pathway.

The GBA1 K198E Variant Is Associated with Suppression of Glucocerebrosidase Activity, Autophagy Impairment, Oxidative Stress, Mitochondrial Damage, and Apoptosis in Skin Fibroblasts.

Parkinson's disease (PD) is a multifactorial, chronic, and progressive neurodegenerative disorder inducing movement alterations as a result of the loss of dopaminergic (DAergic) neurons of the pars compacta in the substantia nigra and protein aggregates of alpha synuclein (α-Syn). Although its etiopathology agent has not yet been clearly established, environmental and genetic factors have been suggested as the major contributors to the disease. Mutations in the glucosidase beta acid 1 (GBA1) gene, which encodes the lysosomal glucosylceramidase (GCase) enzyme, are one of the major genetic risks for PD. We found that the GBA1 K198E fibroblasts but not WT fibroblasts showed reduced catalytic activity of heterozygous mutant GCase by -70% but its expression levels increased by 3.68-fold; increased the acidification of autophagy vacuoles (e.g., autophagosomes, lysosomes, and autolysosomes) by +1600%; augmented the expression of autophagosome protein Beclin-1 (+133%) and LC3-II (+750%), and lysosomal-autophagosome fusion protein LAMP-2 (+107%); increased the accumulation of lysosomes (+400%); decreased the mitochondrial membrane potential (∆Ψm) by -19% but the expression of Parkin protein remained unperturbed; increased the oxidized DJ-1Cys106-SOH by +900%, as evidence of oxidative stress; increased phosphorylated LRRK2 at Ser935 (+1050%) along with phosphorylated α-synuclein (α-Syn) at pathological residue Ser129 (+1200%); increased the executer apoptotic protein caspase 3 (cleaved caspase 3) by +733%. Although exposure of WT fibroblasts to environmental neutoxin rotenone (ROT, 1 μM) exacerbated the autophagy-lysosomal system, oxidative stress, and apoptosis markers, ROT moderately increased those markers in GBA1 K198E fibroblasts. We concluded that the K198E mutation endogenously primes skin fibroblasts toward autophagy dysfunction, OS, and apoptosis. Our findings suggest that the GBA1 K198E fibroblasts are biochemically and molecularly equivalent to the response of WT GBA1 fibroblasts exposed to ROT.

Role of hsa_circ_0007482 in pterygium development: insights into proliferation, apoptosis, and clinical correlations.

To investigate the impact of hsa_circ_0007482 on the proliferation and apoptosis of human pterygium fibroblasts (HPFs) and its correlation with the severity grades of pterygium. Pterygium and normal conjunctival tissues were collected from the superior area of the same patient's eye (n=33). The correlation between pterygium severity and hsa_circ_0007482 expression using quantitative reverse-transcription polymerase chain reaction (RT-qPCR) were analyzed. Three distinct siRNA sequences targeting hsa_circ_0007482, along with a negative control sequence, were transfected into HPFs. Cell proliferation was assessed using the cell counting kit-8. Expression levels of Ki67, proliferating cell nuclear antigen (PCNA), Cyclin D1, Bax, B-cell lymphoma-2 (Bcl-2), and Caspase-3 were measured via RT-qPCR. Immunofluorescence staining was employed to detect Ki67 and vimentin expressions. Apoptosis was evaluated using flow cytometry. Hsa_circ_0007482 expression was significantly higher in pterygium tissues compared to normal conjunctival tissues (P<0.001). Positive correlations were observed between hsa_circ_0007482 expression and pterygium severity, thickness, and vascular density. Knockdown of hsa_circ_0007482 inhibited cell proliferation, reducing the mRNA expression of Ki67, PCNA, and Cyclin D1 in HPFs. Hsa_circ_0007482 knockdown induced apoptosis, increasing mRNA expression levels of Bax and Caspase-3, while decreasing Bcl-2 expression in HPFs. Additionally, hsa_circ_0007482 knockdown attenuated vimentin expression in HPFs. The downregulation of hsa_circ_0007482 effectively hampers cell proliferation and triggers apoptosis in HPFs. There are discernible positive correlations detected between the expression of hsa_circ_0007482 and the severity of pterygium.

Heat Shock Related Protein Expression in Abdominal Testes of Asian Elephant (Elephas maximus).

The abdominal testes of Asian elephants show normal spermatogenesis. Heat shock in cryptorchid testes elevates heat shock factor (HSF) expression, leading to germ cell apoptosis, while increased heat shock proteins (HSPs) levels provide protection. To investigate how heat shock affects elephant spermatogenic cells, focusing on heat shock-related molecules and the cell death mechanism, immunohistochemistry and TUNEL staining were employed to assess the immunoexpression of several heat shock-related molecules and the status of apoptosis in elephant fibroblasts (EF) induced by heat shock stimulus. Additionally, the immunoexpression of heat shock-related molecules and cell proliferation status in the elephant spermatogenic cells. Our finding indicated that heat shock-induced HSF1 immunoexpression in EF leads to apoptosis mediated by T-cell death-associated gene 51 (TDAG51) while also upregulating HSP70 to protect damaged cells. In elephant spermatogenic cells, immunostaining revealed a predominance of proliferating cell nuclear antigen (PCNA)-positive cells with minimal TDAG51- and TUNEL-positive cells, suggesting active proliferation and apoptosis suppression during normal spermatogenesis in the abdominal testis. Interestingly, spermatogonia co-immunoexpressed HSF1 and HSP90, potentially reducing apoptosis through protective mechanisms different from those observed in other mammals. Spermatogenic cells did not show immunolocalisation of HSP70, and hence, it may not contribute to protecting the spermatogonia from heat shock because the transcriptional activity of HSF1 is suppressed by HSP90A binding. This study provides insight into the specific heat shock response and defence mechanisms in elephant spermatogenic cells and may contribute to our understanding of species-specific adaptation to environmental stresses of the testis.