Fibroblast-like Cell Research Articles

Mitochondrial Metabolism in X-Irradiated Cells Undergoing Irreversible Cell-Cycle Arrest.

Irreversible cell-cycle-arrested cells not undergoing cell divisions have been thought to be metabolically less active because of the unnecessary consumption of energy for cell division. On the other hand, they might be actively involved in the tissue microenvironment through an inflammatory response. In this study, we examined the mitochondria-dependent metabolism in human cells irreversibly arrested in response to ionizing radiation to confirm this possibility. Human primary WI-38 fibroblast cells and the BJ-5ta fibroblast-like cell line were exposed to 20 Gy X-rays and cultured for up to 9 days after irradiation. The mitochondrial morphology and membrane potential were evaluated in the cells using the mitochondrial-specific fluorescent reagents MitoTracker Green (MTG) and 5,5',6,6'-tetraethyl-benzimidazolylcarbocyanine iodide (JC-1), respectively. The ratio of the mean MTG-stained total mitochondrial area per unit cell area decreased for up to 9 days after X-irradiation. The fraction of the high mitochondrial membrane potential area visualized by JC-1 staining reached its minimum 2 days after irradiation and then increased (particularly, WI-38 cells increased 1.8-fold the value of the control). Their chronological changes indicate that the mitochondrial volume in the irreversible cell-cycle-arrested cells showed significant increase concurrently with cellular volume expansion, indicating that the mitochondria-dependent energy metabolism was still active. These results indicate that the energy metabolism in X-ray-induced senescent-like cells is active compared to nonirradiated normal cells, even though they do not undergo cell divisions.

Hydrogen sulfide alleviates ischemia induced liver injury by repressing the SPHK1/S1P pathway.

Ischemia/reperfusion (I/R) induced liver injury is a severe pathological process which frequently occurs during clinical hepatic operations. The current study investigated the protective function and underlying mechanisms of hydrogen sulfide (H2S) in I/R induced liver injury. The effects of H2S were examined using the fibroblast-like rat liver cell line BRL-3A (the name of normal hepatocytes in rats) cultured under hypoxic conditions and an I/R rat model. The viability of BRL-3A cells was assessed using the methylthiazolyldiphenyl-tetrazolium (MTT) assay and Hoechst analysis. The expression of C/EBP homologous protein (CHOP), sphingosine kinase 1 (SPHK1), and sphingosine 1-phosphate (S1P) were determined in hypoxic BRL-3A cells with or without H2S treatment. CHOP was overexpressed in hypoxic BRL-3A cells to further evaluate whether H2S protected the liver against I/R injury by decreasing endoplasmic reticulum (ER) stress. Finally, the inflammation levels in the serum and the histopathological changes of liver were examined in the I/R rat model to evaluate the therapeutic function of H2S on I/R induced liver injury in vivo. H2S alleviated hypoxic damage in BRL-3A cells. In addition, hypoxia increased the expression of CHOP, SPHK1, and S1P in BRL-3A cells, and this was abolished by H2S pretreatment. Notably, overexpression of CHOP significantly inhibited the effect of H2S on the viability of BRL-3A cells during hypoxia. Overall, H2S effectively protected against I/R induced liver injury, decreased the inflammatory responses, and attenuated apoptosis of hepatocyte via inhibiting the ER stress response. These findings demonstrated that pre-treatment of H2S protected against I/R induced liver injury by repressing the SPHK1/S1P pathway via inhibition of ER stress, suggesting an effective therapeutic method for the treatment of I/R induced liver injury.

Distinguishing between ante factum and post factum properties of animal cell lines and demonstrating their use in grouping ray-finned fish cell lines into invitromes.

In this review, animal cell lines are considered to have two classes of attributes: "before-the-fact" (ante factum) and "after-the-fact" (post factum) properties. Fish cell lines from Actinopterygii (ray-finned fishes) are used to illustrate this distinction and to demonstrate how these properties can be used in various ways to categorize cell lines into groups or invitromes. Before-the-fact properties are set at initiation and are properties of the sample and species from which the cell line arose and of the scientist(s) who developed the cell line. On the basis of the Actinopterygii sample, invitromes exist for embryos, larvae, juveniles, adults, and spawning fish, and for most solid organs but rarely for biological fluids. For species, invitromes exist for only a small fraction of the Actinopterygii total. As to their development, scientists from around the world have contributed to invitromes. By contrast, after-the-fact properties are limitless and become apparent during development, characterization, use, and storage of the cell line. For ray-finned invitromes, cell lines appear to acquire immortality during development, are characterized poorly for differentiation potential, have numerous uses, and are stored formally only sporadically. As an example of applying these principles to a specific organ, the skeletal muscle invitrome is used. For ante factum properties, the cell lines are mainly from trunk muscle of economically important fish from 11 orders, 15 families, 19 genera, and 21 species of ray-finned fishes. For post factum properties, fibroblast-like and myogenic cell lines have been described but epithelial-like FHM is most widely used and curated. Considering cell lines by their before- and after-the-fact properties should facilitate integration of new cell lines into the literature and help incorporate the discipline of cell biology into other research areas, particularly the natural history of fishes.

Regulatory Fibroblast-Like Synoviocytes Cell Membrane Coated Nanoparticles:A Novel Targeted Therapy for Rheumatoid Arthritis.

Fibroblast-like synoviocytes (FLS) are the main cell component in the inflamed joints of patients with rheumatoid arthritis (RA). FLS intimately interact with infiltrating T cells. Fibroblasts have potent inhibitory effects on T cells, leading to the resolution of inflammation and immune tolerance. However, this "regulatory" phenotype is defect in RA, and FLS in RA instead act as "proinflammatory" phenotype mediating inflammation perpetuation. Signals that orchestrate fibroblast heterogeneity remain unclear. Here, it is demonstrated that different cytokines can induce distinct phenotypes of FLS. Interferon-gamma (IFN-γ) is pivotal in inducing the regulatory phenotype of FLS (which is termed FLSreg ) characterized by high expressions of several inhibitory molecules. Rapamycin enhances the effect of IFN-γ on FLS. Based on the characteristics of FLSreg , a novel biomimetic therapeutic strategy for RA is designed by coating cell membrane derived from FLSreg induced by IFN-γ and rapamycin on nanoparticles, which is called FIRN. FIRN show good efficacy, stability, and inflammatory joint targeting ability in an RA mouse model. The findings clarify how fibroblast phenotypes are modulated in the inflammatory microenvironment and provide insights into novel therapeutic designs for autoimmune diseases based on regulatory fibroblasts.

In vitro "benefit-risk" evaluation and network regulation mechanism of Colquhounia Root Tablets and its key material basis

Colquhounia Root Tablets, prepared from Tripterygium, is effective for rheumatoid arthritis, diabetic nephropathy, and membranous nephropathy. However, the adverse reactions, such as liver injury, nausea, and vomiting, limit its application. This study aims to evaluate the advantages and risk of Colquhounia Root Tablets and its key active components in the treatment of rheumatoid arthritis, diabetic nephropathy, and membranous nephropathy and explore the potential mechanism in treating different diseases based on in vitro efficacy and toxicity assessment and biomolecular network analysis. First, the components of Colquhounia Root Tablets absorbed in blood were detected via ultra-performance liquid chromatography-quadrupole-time of flight mass spectrometry, and the influence of Colquhounia Root Tablets and its key components triptolide and celastrol on viability of human hepatocyte L02, human rheumatoid fibroblast-like synovial cell MH7 A, human renal tubular epithelial cell HK-2, and mouse podocyte MPC-5 was detected by cell counting kit 8(CCK8) assay. Then the expression of inflammatory cytokines of MH7 A and HK-2 cells was detected by enzyme-linked immunosorbent assay(ELISA). Moreover, the expression of nephrin and podocin in MPC-5 cells was measured by Western blot, and the expression of cytoskeletal protein by immunofluorence assay. Candidate targets of components from Colquhounia Root Tablets absorbed in blood were retrieved from TCMIP v2.0, and targets of the three diseases from GEO. The "disease-related genes-drug targets" network was constructed based on STRING, followed by pathway enrichment. Finally, molecular docking was performed by AutoDock Vina to explore the binding affinity of triptolide and celastrol with putative targets in the key signaling pathway. RESULTS:: showed that Colquhounia Root Tablets, triptolide, and celastrol can obviously reduce the levels of inflammatory cytokines in supernatant of MH7 A and HK-2 cells and enhance the expression of nephrin and podocin in MPC-5 cells. In addition, triptolide had the strongest toxicity to L02 cells, while Huobahuagen Tablets had the least toxicity to hepatocytes. Network analysis revealed that Colquhounia Root Tablets may intervene the three diseases through PI3 K/HIF1α/NOS signaling pathway. Both triptolide and celastrol had high binding affinities to corresponding targets in this signaling pathway. In conclusion, Colquhounia Root Tablets exerts similar effects on rheumatoid arthritis, diabetic nephropathy, and membranous nephropathy to triptolide and celastrol, but the toxicity was lower. PI3 K/HIF1α/NOS signaling pathway may be the common pathway of Colquhounia Root Tablets in the treatment of the three diseases.

Extracellular matrix: Brick and mortar in the skeletal muscle stem cell niche.

The extracellular matrix (ECM) is an interconnected macromolecular scaffold occupying the space between cells. Amongst other functions, the ECM provides structural support to tissues and serves as a microenvironmental niche that conveys regulatory signals to cells. Cell-matrix adhesions, which link the ECM to the cytoskeleton, are dynamic multi-protein complexes containing surface receptors and intracellular effectors that control various downstream pathways. In skeletal muscle, the most abundant tissue of the body, each individual muscle fiber and its associated muscle stem cells (MuSCs) are surrounded by a layer of ECM referred to as the basal lamina. The core scaffold of the basal lamina consists of self-assembling polymeric laminins and a network of collagens that tether proteoglycans, which provide lateral crosslinking, establish collateral associations with cell surface receptors, and serve as a sink and reservoir for growth factors. Skeletal muscle also contains the fibrillar collagenous interstitial ECM that plays an important role in determining tissue elasticity, connects the basal laminae to each other, and contains matrix secreting mesenchymal fibroblast-like cell types and blood vessels. During skeletal muscle regeneration fibroblast-like cell populations expand and contribute to the transitional fibronectin-rich regenerative matrix that instructs angiogenesis and MuSC function. Here, we provide a comprehensive overview of the role of the skeletal muscle ECM in health and disease and outline its role in orchestrating tissue regeneration and MuSC function.

Establishment and characterization of a new fibroblast-like cell line from the skin of a vertebrate model, zebrafish (Danio rerio).

The available fully sequenced genome and genetic similarities compared to humans make zebrafish a prominent in vitro vertebrate model for drug discovery & screening, toxicology, and radiation biology. Zebrafish also possess well developed immune systems which is ideal for studying infectious diseases. Fish skin confers immunity by serving as a physical barrier against the invading pathogens in the aquatic habitat. Therefore in vitro models from the skin tissue of zebrafish help to study the physiology, functional genes in vitro, wound healing, and pathogenicity of microbes. Hence the study aimed to develop and characterize a skin cell line from the wild-type zebrafish Danio rerio. A novel cell line designated as DRS (D. rerio skin) was established and characterized from the skin tissue of wild-type zebrafish, D. rerio, by the explant technique. The cells thrived well in the Leibovitz's -15 medium supplemented with 15% FBS and routinely passaged at regular intervals. The DRS cells mainly feature fibroblast-like morphology. The culture conditions of the cells were determined by incubating the cells at varying concentrations of FBS and temperature; the optimum was 15% FBS and 28°C, respectively. Cells were cryopreserved and revived with 70-75% viability at different passage levels. Two extracellular products from bacterial species Aeromonas hydrophila and Edwardsiella tarda were tested and found toxic to the DRS cells. Mitochondrial genes, namely COI and 16S rRNA PCR amplification and partial sequencing authenticated the species of origin of cells. The modal diploid (2n) chromosome number of the cells was 50. The cell line DRS was found to be free from mycoplasma. The cells were transfected with pMaxGFP plasmid and tested positive for green fluorescence at 24-48h post-transfection. The findings from this study thus confirm the usefulness of the developed cell line in bacterial susceptibility and transgene expression studies.

Protein arginine methyltransferase 1 upregulates matrix metalloproteinase-2/9 expression via Zeste Homolog 2 to promote human rheumatoid arthritis fibroblast-like synovial cell survival and metastasis.

To explore the role of protein arginine methyltransferase 1 (PRMT1) in the development of rheumatoid arthritis (RA). Fibroblast-like synoviocytes (FLS) were isolated from synovial tissues, cultured and transfected with plasmid vector or short hairpin RNA (shRNA). The morphology and surface markers of FLS were investigated by light microscopy and flow cytometry. The expression levels of PRMT1, Zeste Homolog 2 (EZH2), matrix metalloproteinase-2 (MMP-2) and MMP-9 were detected by real-time polymerase chain reaction and Western blotting. Cell viability was tested by MTT assay, cell proliferation was assessed by EdU assay, cell invasion was evaluated by Transwell invasion test, cell migration was detected by wound healing assay, and cell apoptosis was detected by flow cytometry. The expression of PRMT1 was elevated in RA synovial tissues compared with control tissues. FLS from control tissues showed a lower level of PRMT1 than FLS from RA tissues, and FLS from RA tissues had a stronger ability of cell survival and metastasis than those from control tissues. When silencing PRMT1 expression, FLS from RA tissues showed a decreased ability of cell survival and metastasis. Additionally, FLS from RA tissues expressed a higher level of MMP-2 and MMP-9. When silencing PRMT1 expression, the expression of MMP-2 and MMP-9 of FLS was suppressed. Furthermore, the effect of PRMT1 on FLS was mediated by EZH2. We found that PRMT1 had an overall effect on FLS via EZH2, which contributed to the development of RA. Hence, PRMT1 and EZH2 provide potential targets for RA treatment.

Multilayer Electrospun-Aligned Fibroin/Gelatin Implant for Annulus Fibrosus Repair: An In Vitro and In Vivo Evaluation.

Annulus fibrosus (AF) damage is proven to prompt intervertebral disc (IVD) degeneration, and unrepaired AF lesions after surgical discectomy may boost herniation of the nucleus pulposus (NP) which may lead to further compression of neural structures. Moreover, vascular and neural ingrowth may occur within the defect which is known as a possible reason for discogenic pain. Due to a limited healing capacity, an effective strategy to repair and close the AF defect is necessary. In this study, using electrospinning technology, two nature polymers, silk fibroin and gelatin, were linked to imitate the unique lamellae structure of native AF. Our findings revealed that a multilayer electrospun-aligned fibroin/gelatin scaffold with mechanical and morphological properties mimicking those of native AF lamellae have been developed. The average diameter of the nanofiber is 162.9 ± 38.8 nm. The young’s modulus is around 6.70 MPa with an ultimate tensile strength of around 1.81 MP along preferred orientation. The in vitro test confirmed its biocompatibility and ability to maintain cell viability and colonization. Using a porcine model, we demonstrated that the multilayer-aligned scaffold offered a crucial microenvironment to induce collagen fibrous tissue production within native AF defect. In the implant-repaired AF, H&E staining showed homogeneous fibroblast-like cell infiltration at the repaired defect with very little vascular ingrowth, which was confirmed by magnetic resonance imaging findings. Picrosirius red staining and immunohistochemical staining against type I collagen revealed positively stained fibrous tissue in an aligned pattern within the implant-integrated site. Relative to the intact control group, the disc height index of the serial X-ray decreased significantly in both the injury control and implant group at 4 weeks and 8 weeks (p < 0.05) which indicated this scaffold may not reverse the degenerative process. However, the results of the discography showed that the effectiveness of annulus repair of the implant group is much superior to that of the untreated group. The scaffold, composed with nature fibroin/gelatin polymers, could potentially enhance AF healing that could prevent IVD recurrent herniation, as well as neural and neovascular ingrowth after discectomy surgeries.

Heterogeneity of the NIH3T3 Fibroblast Cell Line.

The embryonic mouse fibroblast cell line NIH3T3 is widely used in life science research, including the study of cell cycle control and primary cilia. Fibroblasts are the most important cell type in connective tissue, as they produce components of the extracellular matrix and determine tissue architecture. However, they are very heterogeneous and consist of subtypes specific to their organ of residence, among others. The NIH3T3 cell line was derived from whole mouse embryos that developed to pre-birth and is therefore most likely composed of different fibroblast subtypes. Furthermore, prolonged proliferation may have influenced their cellular composition. A heterogeneous cell population is unsuitable for any sophisticated research project. We found that the proportion of ciliated cells in the total NIH3T3 cell population was highly variable and asked whether this was a consequence of cellular heterogeneity and what molecular signatures were associated with it. We have established sub-cell lines by clonal expansion of single cells and characterized them morphologically and molecularly. Eventually, a myofibroblast-like and a fibroblast-like cell line were generated that differ in ciliation and proliferation. These homogeneous cell lines are valuable for a more detailed study of their molecular signatures, not least to uncover further the molecular pathways that contribute to the formation of the primary cilium.

Natural medicines of targeted rheumatoid arthritis and its action mechanism.

Rheumatoid arthritis (RA) is an autoimmune disease involving joints, with clinical manifestations of joint inflammation, bone damage and cartilage destruction, joint dysfunction and deformity, and extra-articular organ damage. As an important source of new drug molecules, natural medicines have many advantages, such as a wide range of biological effects and small toxic and side effects. They have become a hot spot for the vast number of researchers to study various diseases and develop therapeutic drugs. In recent years, the research of natural medicines in the treatment of RA has made remarkable achievements. These natural medicines mainly include flavonoids, polyphenols, alkaloids, glycosides and terpenes. Among them, resveratrol, icariin, epigallocatechin-3-gallate, ginsenoside, sinomenine, paeoniflorin, triptolide and paeoniflorin are star natural medicines for the treatment of RA. Its mechanism of treating RA mainly involves these aspects: anti-inflammation, anti-oxidation, immune regulation, pro-apoptosis, inhibition of angiogenesis, inhibition of osteoclastogenesis, inhibition of fibroblast-like synovial cell proliferation, migration and invasion. This review summarizes natural medicines with potential therapeutic effects on RA and briefly discusses their mechanisms of action against RA.

Therapeutic mechanism of emodin for treatment of rheumatoid arthritis: a network pharmacology-based analysis

To investigate the therapeutic mechanism of emodin in the treatment of rheumatoid arthritis (RA) using a network pharmacology-based method and validate this mechanism in a fibroblast-like synovial cell line. The PubChem, Targetnet, SwissTargetPrediction, Genecards, OMIM, and DisGeNET databases were searched to obtain emodin targets and RA-related genes. A protein-protein interaction (PPI) network was constructed, and GO and KEGG pathway enrichment analyses were carried out to analyze the intersection genes. AutoDock4.2.6 software was used to simulate molecular docking between emodin and its candidate targets. In a cultured fibroblast-like synovial cell line (MH7A), the effects of different concentrations of emodin on proliferation of tumor necrosis factor-α (TNF-α)-induced cells were investigated using CCK-8 assay, cell scratch experiment and flow cytometry; the changes in the expressions of nuclear factor-κB (NF-κB) pathway proteins were detected using Western blotting, and the mRNA expressions of the hub genes were examined with RT-qPCR. We identified 32 intersection genes of emodin and RA, and the key targets including CAPS3, ESR1, and MAPK14 involved mainly the NF-κB signaling pathway. Cell scratch experiment and flow cytometry demonstrated a strong inhibitory effect of emodin on MH7A cell proliferation. Treatment with TNF-α significantly increased the cellular expressions of the NF-κB pathway proteins, which were obviously lowered by treatment with 80 μmol/L emodin. The results of RT-qPCR showed that TNF-α treatment obviously up-regulated the expressions of the hub genes COX2 and P38MAPK, and emodin treatment significantly down-regulated the expressions of MAPK and PTGS2 and up-regulated the expression of CASP3. The therapeutic effect of emodin on RA is mediated mainly through regulation of cell proliferation, apoptosis, and the NF-κB pathway.

Epigenetic lockdown of CDKN1A (p21) and CDKN2A (p16) characterises the neoplastic spindle cell component of giant cell tumours of bone.

Giant cell tumour of bone (GCTB) comprises the eponymous osteoclastic multinucleated giant cells eliciting bone lysis, an H3F3A-mutated neoplastic mononucleated fibroblast-like cell population, and H3F3A wild-type mononucleated stromal cells. In this study, we characterised four new cell lines from GCTB. Furthermore, we compared the genome-wide DNA methylation profile of 13 such tumours and three further cell lines with giant cell-rich lesions comprising three H3F3B-mutated chondroblastomas, three USP6-rearranged aneurysmal bone cysts, three non-ossifying fibromas, two hyperparathyroidism-associated brown tumours as well as mesenchymal stem cells, osteoblasts, and osteoclasts. In an unsupervised analysis, we delineated GCTB and chondroblastomas from the other analysed tumour entities. Using comparative methylation analysis, we demonstrated that the methylation pattern of the cell lines approximately equals that of H3F3A-mutated stromal cells in tissue. These patterns more resemble that of osteoblasts than that of mesenchymal stem cells, which argues for the osteoblast as the cell of origin of giant cell tumours of bone. Using enrichment analysis, we detected distinct hypermethylated clusters containing histone and collagen genes as well as target genes of the tumour suppressor p53. We found that the promotor regions of CDKN1A, CDKN2A, and IGFBP3 are methylated more strongly in GCTB than in the other giant cell-containing lesions, mesenchymal stem cells, osteoblasts, and osteoclasts (p < 0.001). This hypermethylation correlates with the lower gene expression at the mRNA level for these three genes in the cell lines, the lack of p16 and p21 in these cell lines, and the lower expression of p16 and p21 in GCTB. Overall, our analysis reveals characteristic DNA methylation patterns of giant cell tumours of bone and chondroblastomas and shows that cell lines of giant cell tumours of bone are a valid model for further analysis of H3F3A-mutated tumour cells. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Sphk1 and Sphk2 Differentially Regulate Erythropoietin Synthesis in Mouse Renal Interstitial Fibroblast-like Cells.

Erythropoietin (Epo) is a crucial hormone regulating red blood cell number and consequently the hematocrit. Epo is mainly produced in the kidney by interstitial fibroblast-like cells. Previously, we have shown that in cultures of the immortalized mouse renal fibroblast-like cell line FAIK F3-5, sphingosine 1-phosphate (S1P), by activating S1P1 and S1P3 receptors, can stabilize hypoxia-inducible factor (HIF)-2α and upregulate Epo mRNA and protein synthesis. In this study, we have addressed the role of intracellular iS1P derived from sphingosine kinases (Sphk) 1 and 2 on Epo synthesis in F3-5 cells and in mouse primary cultures of renal fibroblasts. We show that stable knockdown of Sphk2 in F3-5 cells increases HIF-2α protein and Epo mRNA and protein levels, while Sphk1 knockdown leads to a reduction of hypoxia-stimulated HIF-2α and Epo protein. A similar effect was obtained using primary cultures of renal fibroblasts isolated from wildtype mice, Sphk1−/−, or Sphk2−/− mice. Furthermore, selective Sphk2 inhibitors mimicked the effect of genetic Sphk2 depletion and also upregulated HIF-2α and Epo protein levels. The combined blockade of Sphk1 and Sphk2, using Sphk2−/− renal fibroblasts treated with the Sphk1 inhibitor PF543, resulted in reduced HIF-2α and Epo compared to the untreated Sphk2−/− cells. Exogenous sphingosine (Sph) enhanced HIF-2α and Epo, and this was abolished by the combined treatment with the selective S1P1 and S1P3 antagonists NIBR-0213 and TY52156, suggesting that Sph was taken up by cells and converted to iS1P and exported to then act in an autocrine manner through S1P1 and S1P3. The upregulation of HIF-2α and Epo synthesis by Sphk2 knockdown was confirmed in the human hepatoma cell line Hep3B, which is well-established to upregulate Epo production under hypoxia. In summary, these data show that sphingolipids have diverse effects on Epo synthesis. While accumulation of intracellular Sph reduces Epo synthesis, iS1P will be exported to act through S1P1+3 to enhance Epo synthesis. Furthermore, these data suggest that selective inhibition of Sphk2 is an attractive new option to enhance Epo synthesis and thereby to reduce anemia development in chronic kidney disease.

About 4-day rhythm of proliferative activity of fibroblast-like cell cultures isn\u2019t endogenous and don\u2019t depend from the variations of Earth\u2019s magnetic field

A study of the 4-day rhythm of the proliferative activity of the embryonic fibroblast-like cells in the logarithmic growth phase was carried out. It was shown that in cell cultures obtained on different days from embryos of different ages, the phase of the 4-day rhythm coincides. In vitro the maxima of the proliferative activity were consistent with the minima of the motor activity of mice. Freezing the culture for 2 or 6 days does not cause a shift in the phase of the 4-day rhythm of cell proliferative activity compare with the unfreezing culture. That indicates the existence of an external synchronizer, which determines the 4-day infradian rhythm of the proliferative activity of embryonic cells. Then we daily thawed samples of single L929 culture of mice fibroblast-like cells for 22 and 17 days and researched the dynamics of its proliferative activity. We also showed 4-day rhythm of the simultaneous increase in the number of cells for all thawed samples. Taking into account that deep freezing of a culture leads to the cessation of all life processes, the fact we obtained indicates an exogenous mechanism of the formation of about a 4-day rhythm of the proliferative activity of cell culture. Variations of the Earth's magnetic field could be one of the external synchronizers of the infradian rhythm. We studied the increase in number of L929 cell in conditions of a magnetic permalloy screen and showed that the magnetic shielding no affect the parameters of the infradian rhythm of L929 cell proliferative activity. So further searches of the external synchronizers are need.

An estrogen-sensitive fibroblast population drives abdominal muscle fibrosis in an inguinal hernia mouse model.

Greater than 25% of all men develop an inguinal hernia in their lifetime, and more than 20 million inguinal hernia repair surgeries are performed worldwide each year. The mechanisms causing abdominal muscle weakness, the formation of inguinal hernias, or their recurrence are largely unknown. We previously reported that excessively produced estrogen in the lower abdominal muscles (LAMs) triggers extensive LAM fibrosis, leading to hernia formation in a transgenic male mouse model expressing the human aromatase gene (Aromhum). To understand the cellular basis of estrogen-driven muscle fibrosis, we performed single-cell RNA sequencing on LAM tissue from Aromhum and wild-type littermates. We found a fibroblast-like cell group composed of 6 clusters, 2 of which were validated for their enrichment in Aromhum LAM tissue. One of the potentially novel hernia-associated fibroblast clusters in Aromhum was enriched for the estrogen receptor-α gene (Esr1hi). Esr1hi fibroblasts maximally expressed estrogen target genes and seemed to serve as the progenitors of another cluster expressing ECM-altering enzymes (Mmp3hi) and to upregulate expression of proinflammatory, profibrotic genes. The discovery of these 2 potentially novel and unique hernia-associated fibroblasts may lead to the development of novel treatments that can nonsurgically prevent or reverse inguinal hernias.

Isoprenaline induced Takotsubo syndrome: Histopathological analyses of female rat hearts.

BackgroundTakotsubo syndrome (TTS) is a stress-induced disorder affecting mostly postmenopausal women. The aim of the study was to evaluate isoprenaline (ISO) dependent female rat model and histopathological characteristics in TTS.MethodsForty-nine Sprague Dawley female rats, 12 weeks old, were injected intraperitoneally with a single dose of ISO at doses 50 (n = 8), 75 (n = 6), 100 (n = 3), 150 (n = 27) and 200 (n = 5) mg/kg body weight (bw). The control group (n = 6) was injected with physiological saline. The echocardiographic examination to assess wall motion abnormalities took place 24, 48, 72 h, and 7 days post-ISO. Histopathological analysis was performed on the basis of hematoxylin-eosin staining.ResultsThe total mortality rate was 3/49 (6.12%). The optimum dose of ISO to induce TTS was 150 mg/kg bw and 21/27 (77.77%) rats showed apical ballooning. Histopathological analysis revealed focal necrosis/apoptosis of cardiomyocytes with inflammatory and fibroblast-like cell infiltration. Foci were the most numerous in the central muscle layer with apical-basal gradient 24, 48, 72 h post-ISO (p < 0.05). Significant differences were noted 48 h post-ISO in the central layer in apical vs basal segments (p = 0.0032), in the endocardial layer in apical vs basal segments (0.00024) and in mid-cavital vs. basal segments (p = 0.0483). The number of foci in endocardium of apical region differ 48 h post-ISO in rats with a dose of 150 vs. 200 mg/kg bw (p = 0.0084).ConclusionsThe ISO female rat model of TTS is associated with higher optimum dose and lower mortality in comparison with the male TTS model. TTS presents as a singles cardiomyocyte disorder, foci concerned mainly central muscle layer with apical-basal gradient.

The protective effect of gentisic acid on rheumatoid arthritis via the RAF/ERK signaling pathway

BackgroundRAF and ERK pathways are known to be activated in human rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS), which play an important role in the pathogenesis and destruction of RA. Gentisic acid (GA) was a natural product derived from plants, which has been reported can attenuate pressure overload‐induced cardiac hypertrophy and fibrosis in mice through inhibition of the ERK1/2 pathway. Whether GA can inhibit the occurrence and development of RA through RAF/ERK signaling pathway has not been reported. The purpose of this study is to determine whether GA may have a certain therapeutic effect on RA-FLS.MethodBovine type II collagen was used to establish a rat model of rheumatism. Enzyme-linked immunosorbent assay was used to detect inflammatory factors, anti-inflammatory mediators, and rheumatoid factor. Hematoxylin and eosin and TUNEL staining were used to detect the effect of GA on histochemical with rheumatoid arthritis. RAF, ERK, and p-ERK expressions in synovial tissue were measured by western blot and immunohistochemical. Besides, human rheumatoid arthritis fibroblast-like synoviocytes cell line MH7A was used to investigate the biological behavior influenced by GA. Apoptosis assay was performed to detect apoptosis of GA on MH7A cells. Transwell invasion assay was performed to detect the ability of cell migration.ResultThe result showed that GA could reduce joint swelling and inflammation. At the same time, it can also promote the apoptosis of synovial cells and down-regulate the RAF/ERK pathway.ConclusionGA may ameliorate inflammatory factors’ abnormality, synovial hyperplasia, and apoptosis of synovium via inhibiting the RAF/ERK signaling pathway.

A Preliminary Inquiry Into the Potential Mechanism of Huang-Lian-Jie-Du Decoction in Treating Rheumatoid Arthritis via Network Pharmacology and Molecular Docking.

Huang-Lian-Jie-Du decoction (HLJDD) has been widely applied to treat inflammation-associated diseases for thousands of years in China. However, the concrete molecular mechanism of HLJDD in the treatment of rheumatoid arthritis (RA) remains unclear. In this work, network pharmacology and molecular docking were applied to preliminarily analyze the potential active ingredients, drug targets, and related pathways of HLJDD on treating RA. A total of 102 active compounds with corresponding 189 targets were identified from HLJDD, and 41 common targets were further identified by intersecting with RA-related targets. Functional enrichment analysis was performed to screen the biological pathways associated with RA. Ten hub targets were further identified through constructing the protein–protein interaction (PPI) network of common targets, which were mainly enriched in the interleukin-17 (IL-17) signaling pathway, tumor necrosis factor (TNF) signaling pathway, and Toll-like receptor signaling pathway. Furthermore, a complex botanical drugs-ingredients-hub-targets-disease network was successfully constructed. The molecular docking results exhibited that these vital ingredients of HLJDD had a stable binding to the hub targets. Among these ingredients, quercetin (MOL000098) was the most common molecule with stable binding to all the targets, and PTGS2 was considered the most important target with multiple regulations by the most active ingredients. In vitro, we successfully validated the inhibitory role of quercetin in the cellular proliferation of human RA fibroblast-like synoviocyte cell line (MH7A cells). These findings indicated that the potential mechanisms of HLJDD for RA treatment might be attributed to inhibiting the immune-inflammatory response, reducing the release of chemokines, and alleviating the destruction of extracellular matrix (ECM) in the synovial compartment.

Effects of Gene Delivery Approaches on Differentiation Potential and Gene Function of Mesenchymal Stem Cells.

Introduction of a gene to mesenchymal stem cells (MSCs) is a well-known strategy to purposely manipulate the cell fate and further enhance therapeutic performance in cell-based therapy. Viral and chemical approaches for gene delivery interfere with differentiation potential. Although microinjection as a physical delivery method is commonly used for transfection, its influence on MSC cell fate is not fully understood. The current study aimed to evaluate the effects of four nonviral gene delivery methods on stem cell multi-potency. The four delivery methods are robotic microinjection, polyethylenimine (PEI), cationic liposome (cLipo), and calcium phosphate nanoparticles (CaP). Among the four methods, microinjection has exhibited the highest transfection efficiency of ∼60%, while the three others showed lower efficiency of 10-25%. Robotic microinjection preserved fibroblast-like cell morphology, stress fibre intactness, and mature focal adhesion complex, while PEI caused severe cytotoxicity. No marked differentiation bias was observed after microinjection and cLipo treatment. By contrast, CaP-treated MSCs exhibited excessive osteogenesis, while PEI-treated MSCs showed excessive adipogenesis. Robotic microinjection system was used to inject the CRISPR/Cas9-encoding plasmid to knock out PPARγ gene in MSCs, and the robotic microinjection did not interfere with PPARγ function in differentiation commitment. Meanwhile, the bias in osteo-adipogenic differentiation exhibited in CaP and PEI-treated MSCs after PPARγ knockout via chemical carriers. Our results indicate that gene delivery vehicles variously disturb MSCs differentiation and interfere with exogenous gene function. Our findings further suggest that robotic microinjection offers a promise of generating genetically modified MSCs without disrupting stem cell multi-potency and therapeutic gene function.