[Directed differentiation of human induced pluripotent stem cells into midbrain].

OBJECTIVE: To establish an efficient protocol for directed differentiation of human induced pluripotent stem cells (hiPSCs) into functional midbrain dopaminergic progenitor cells (DAPs) in vitro. METHODS: hiPSCs were induced to differentiate into DAPs in two developmental stages. In the first stage (the first 13 days), hiPSCs were induced into intermediate cells morphologically similar to primitive neuroepithelial cells (NECs) in neural induction medium containing a combination of small molecule compounds. In the second stage, the intermediate cells were further induced in neural differentiation medium until day 28 to obtain DAPs. After CM-DiI staining, the induced DAPs were stereotactically transplanted into the right medial forebrain bundle (MFB) of rat models of Parkinson's disease (PD). Eight weeks after transplantation, the motor behaviors of PD rats was evaluated. Immunofluorescence assay of brain sections of the rats was performed at 2 weeks after transplantation to observe the survival, migration and differentiation of the transplanted cells in the host brain microenvironment. RESULTS: hiPSCs passaged stably on Matrigel showed a normal diploid karyotype, expressed the pluripotency markers OCT4, SOX2, and Nanog, and were positive for alkaline phosphatase. The primitive neuroepithelial cells obtained on day 13 formed dense cell colonies in the form of neural rosettes and expressed the neuroepithelial markers (SOX2, Nestin, and PAX6, 91.3%-92.8%). The DAPs on day 28 highly expressed the specific markers (TH, FOXA2, LMX1A and NURR1, 93.3-96.7%). In rat models of PD, the hiPSCs-DAPs survived and differentiated into TH+, FOXA2+ and Tuj1+ neurons at 2 weeks after transplantation. Eight weeks after transplantation, the motor function of PD rats was significantly improved as shown by water maze test (P < 0.0001) and apomorphine-induced rotation test (P < 0.0001) compared with rats receiving vehicle injection. CONCLUSION: HiPSCs can be effectively induced to differentiate into DAPs capable of differentiating into functional neurons both in vivo and in vitro. In rat models of PD, the transplanted hiPSCs-DAPs can survive for more than 8 weeks in the MFB and differentiate into multiple functional neurocytes to ameliorate neurological deficits of the rats, suggesting the potential value of hiPSCs-DAPs transplantation for treatment of neurological diseases.

O2 variant chip to simulate site-specific skeletogenesis from hypoxic bone marrow.

The stemness of bone marrow mesenchymal stem cells (BMSCs) is maintained by hypoxia. The oxygen level increases from vessel-free cartilage to hypoxic bone marrow and, furthermore, to vascularized bone, which might direct the chondrogenesis to osteogenesis and regenerate the skeletal system. Hence, oxygen was diffused from relatively low to high levels throughout a three-dimensional chip. When we cultured BMSCs in the chip and implanted them into the rabbit defect models of low-oxygen cartilage and high-oxygen calvaria bone, (i) the low oxygen level (base) promoted stemness and chondrogenesis of BMSCs with robust antioxidative potential; (ii) the middle level (two times ≥ low) pushed BMSCs to quiescence; and (iii) the high level (four times ≥ low) promoted osteogenesis by disturbing the redox balance and stemness. Last, endochondral or intramembranous osteogenesis upon transition from low to high oxygen in vivo suggests a developmental mechanism-driven solution to promote chondrogenesis to osteogenesis in the skeletal system by regulating the oxygen environment.

Synthesis and anti-prion aggregation activity of acylthiosemicarbazide analogues.

Prions are infectious protein particles known to cause prion diseases. The biochemical entity of the pathogen is the misfolded prion protein (PrPSc) that forms insoluble amyloids to impair brain function. PrPSc interacts with the non-pathogenic, cellular prion protein (PrPC) and facilitates conversion into a nascent misfolded isoform. Several small molecules have been reported to inhibit the aggregation of PrPSc but no pharmacological intervention was well established thus far. We, here, report that acylthiosemicarbazides inhibit the prion aggregation. Compounds 7x and 7y showed almost perfect inhibition (EC50 = 5 µM) in prion aggregation formation assay. The activity was further confirmed by atomic force microscopy, semi-denaturing detergent agarose gel electrophoresis and real-time quaking induced conversion assay (EC50 = 0.9 and 2.8 µM, respectively). These compounds also disaggregated pre-existing aggregates in vitro and one of them decreased the level of PrPSc in cultured cells with permanent prion infection, suggesting their potential as a treatment platform. In conclusion, hydroxy-2-naphthoylthiosemicarbazides can be an excellent scaffold for the discovery of anti-prion therapeutics.

In Vitro Characteristics of Canine Primary Tracheal Epithelial Cells Maintained at an Air-Liquid Interface Compared to In Vivo Morphology.

Culturing respiratory epithelial cells at an air-liquid interface (ALI) represents an established method for studies on infection or toxicology by the generation of an in vivo-like respiratory tract epithelial cellular layer. Although primary respiratory cells from a variety of animals have been cultured, an in-depth characterization of canine tracheal ALI cultures is lacking despite the fact that canines are a highly relevant animal species susceptible to various respiratory agents, including zoonotic pathogens such as severe acute respiratory coronavirus 2 (SARS-CoV-2). In this study, canine primary tracheal epithelial cells were cultured under ALI conditions for four weeks, and their development was characterized during the entire culture period. Light and electron microscopy were performed to evaluate cell morphology in correlation with the immunohistological expression profile. The formation of tight junctions was confirmed using transepithelial electrical resistance (TEER) measurements and immunofluorescence staining for the junctional protein ZO-1. After 21 days of culture at the ALI, a columnar epithelium containing basal, ciliated and goblet cells was seen, resembling native canine tracheal samples. However, cilia formation, goblet cell distribution and epithelial thickness differed significantly from the native tissue. Despite this limitation, tracheal ALI cultures could be used to investigate the pathomorphological interactions of canine respiratory diseases and zoonotic agents.

Fermented Lettuce Extract Containing Nitric Oxide Metabolites Attenuates Inflammatory Parameters in Model Mice and in Human Fibroblast-Like Synoviocytes.

Lettuce (Lactuca sativa L.) contains various bioactive compounds that can reduce the severity of inflammatory diseases. This study aimed to identify therapeutic effects and underlying mechanisms of fermented lettuce extract (FLE) containing stable nitric oxide (NO) on collagen-induced arthritis (CIA) in mice and fibroblast-like synoviocytes (MH7A line) from patients with rheumatoid arthritis (RA). DBA/1 mice were immunized with bovine type II collagen and orally administered FLE for 14 days. On day 36, mouse sera and ankle joints were collected for serological and histological analysis, respectively. Consuming FLE inhibited RA development, suppressing pro-inflammatory cytokine productions, synovial inflammation, and cartilage degradation. The therapeutic effects of FLE in CIA mice were similar to those of methotrexate (MTX), which is typically used to treat RA. In vitro, FLE suppressed the transforming growth factor-ß (TGF-ß)/Smad signaling pathway in MH7A cells. We also demonstrated that FLE inhibited TGF-ß-induced cell migration, suppressed MMP-2/9 expression, inhibited MH7A cell proliferation, and increased the expression of autophagy markers LC3B and p62 in a dose-dependent manner. Our data suggest that FLE could induce autophagosome formations in the early of stages of autophagy while inhibiting their degradation in the later stages. In conclusion, FLE is a potential therapeutic agent for RA.

Heterogeneity of Phenotypic and Functional Changes to Porcine Monocyte-Derived Macrophages Triggered by Diverse Polarizing Factors In Vitro.

Swine are attracting increasing attention as a biomedical model, due to many immunological similarities with humans. However, porcine macrophage polarization has not been extensively analyzed. Therefore, we investigated porcine monocyte-derived macrophages (moMΦ) triggered by either IFN-γ + LPS (classical activation) or by diverse "M2-related" polarizing factors: IL-4, IL-10, TGF-ß, and dexamethasone. IFN-γ and LPS polarized moMΦ toward a proinflammatory phenotype, although a significant IL-1Ra response was observed. Exposure to IL-4, IL-10, TGF-ß, and dexamethasone gave rise to four distinct phenotypes, all antithetic to IFN-γ and LPS. Some peculiarities were observed: IL-4 and IL-10 both enhanced expression of IL-18, and none of the "M2-related" stimuli induced IL-10 expression. Exposures to TGF-ß and dexamethasone were characterized by enhanced levels of TGF-ß2, whereas stimulation with dexamethasone, but not TGF-ß2, triggered CD163 upregulation and induction of CCL23. Macrophages stimulated with IL-10, TGF-ß, or dexamethasone presented decreased abilities to release proinflammatory cytokines in response to TLR2 or TLR3 ligands: IL-10 showed a powerful inhibitory activity for CXCL8 and TNF release, whereas TGF-ß provided a strong inhibitory signal for IL-6 production. While our results emphasized porcine macrophage plasticity broadly comparable to human and murine macrophages, they also highlighted some peculiarities in this species.

Contractility-induced self-organization of smooth muscle cells: from multilayer cell sheets to dynamic three-dimensional clusters.

Smooth muscle cells (SMCs) are mural cells that play a vital contractile function in many tissues. Abnormalities in SMC organization are associated with many diseases including atherosclerosis, asthma, and uterine fibroids. Various studies have reported that SMCs cultured on flat surfaces can spontaneously form three-dimensional clusters whose organization resembles that encountered in some of these pathological settings. Remarkably, how these structures form remains unknown. Here we combine in vitro experiments and physical modeling to show that three-dimensional clusters initiate when cellular contractile forces induce a hole in a flat SMC sheet, a process that can be modeled as the brittle fracture of a viscoelastic material. The subsequent evolution of the nascent cluster can be modeled as an active dewetting process with cluster shape evolution driven by a balance between cluster surface tension, arising from both cell contractility and adhesion, and cluster viscous dissipation. The description of the physical mechanisms governing the spontaneous emergence of these intriguing three-dimensional clusters may offer insight into SMC-related disorders.

VX-765 ameliorates CKD VSMC calcification by regulating STAT3 activation.

BACKGROUND: Recent clinical evidences show that caspase-1 inhibitor-VX-765 attenuates atherosclerosis in ApoE deficient mice. However, there is rarely information about the effect of VX-765 on hyperphosphatemia-induced vascular smooth muscle cells (VSMCs) calcification or vascular calcification in chronic kidney disease (CKD) rats. Here we investigate the effect of VX-765 on vascular calcification in uremia circumstances. METHODS: Hyperphosphatemia-induced VSMC calcification were evaluated by Alizarin Red S. Aortas from CKD rats which were gavaged with VX-765 were examined for calcification signal using micro-CT. Levels of NLRP3, caspase-1, and GSDMD were measured by quantitative real-time PCR, western blotting, immunofluorescence assay, and immunohistochemistry. RESULTS: We demonstrated for the first time that the levels of NLRP3, caspase-1, GSDMD, IL-1ß, and IL-18 were up-regulated in hyperphosphatemia-induced calcifying VSMCs. Blockade of caspase-1 activation by VX-765 inhibited pyroptosis-related molecules and VSMC calcification in a concentration-dependent manner in vitro. Further analysis of aortas from calcified CKD rats showed an up-regulation of caspase-1 and GSDMD expression compared with those non-calcified vascular tissue from control rats or with those decreased-calcified vascular tissue from CKD rats treated with 50 mg/kg/d, which indicated that pyroptotic indicators were tightly correlated with CKD arterial calcification. In vitro studies further demonstrated that VX-765 ameliorated hyperphosphatemia-induced VSMCs calcification through inhibiting the STAT3 activation. CONCLUSIONS: Our findings indicated that VX-765 could inhibit hyperphosphatemia-induced calcifying VSMCs and ameliorate vascular calcification in CKD rats. VX-765 might be a potential treatment strategy for CKD vascular calcification.

In Vitro Generation of Murine Bone Marrow-Derived Dendritic Cells.

Dendritic cells (DCs) are mononuclear phagocytes of hematopoietic origin residing in lymphoid and nonlymphoid tissues. DCs are often referred as the sentinels of the immune system as they can sense pathogens and danger signals. Upon activation, DCs migrate to the draining lymph nodes and present antigens to naïve T cells to trigger adaptive immunity. Hematopoietic progenitors for DCs reside in the adult bone marrow (BM). Therefore, BM cell culture systems have been developed to generate large amounts of primary DCs in vitro conveniently enabling to analyze their developmental and functional features. Here, we review various protocols enabling to generate DCs in vitro from murine BM cells and discuss the cellular heterogeneity of each culture system.

[Research of epigallocatechin gallate in delaying chondrocyte senescence].

Objective: To investigate the effect of epigallocatechin gallate (EGCG) on chondrocyte senescence and its mechanism. Methods: The chondrocytes were isolated from the articular cartilage of 4-week-old Sprague Dawley rats, and cultured with type Ⅱcollagenase and passaged. The cells were identified by toluidine blue staining, alcian blue staining, and immunocytochemical staining for type Ⅱ collagen. The second passage (P2) cells were divided into blank control group, 10 ng/mL IL-1ß group, and 6.25, 12.5, 25.0, 50.0, 100.0, and 200.0 µmol/L EGCG+10 ng/mL IL-1ß group. The chondrocyte activity was measured with cell counting kit 8 after 24 hours of corresponding culture, and the optimal drug concentration of EGCG was selected for the subsequent experiment. The P2 chondrocytes were further divided into blank control group (group A), 10 ng/mL IL-1ß group (group B), EGCG+10 ng/mL IL-1ß group (group C), and EGCG+10 ng/mL IL-1ß+5 mmol/L 3-methyladenine (3-MA) group (group D). After cultured, the degree of cell senescence was detected by ß-galactosidase staining, the autophagy by monodansylcadaverine method, and the expression levels of chondrocyte-related genes [type Ⅱ collagen, matrix metalloproteinase 3 (MMP-3), MMP-13] by real-time fluorescent quantitative PCR, the expression levels of chondrocyte-related proteins (Beclin-1, LC3, MMP-3, MMP-13, type Ⅱ collagen, P16, mTOR, AKT) by Western blot. Results: The cultured cells were identified as chondrocytes. Compared with the blank control group, the cell activity of 10 ng/mL IL-1ß group significantly decreased ( P<0.05). Compared with the 10 ng/mL IL-1ß group, the cell activity of EGCG+10 ng/mL IL-1ß groups increased, and the 50.0, 100.0, and 200.0 µmol/L EGCG significantly promoted the activity of chondrocytes ( P<0.05). The 100.0 µmol/L EGCG was selected for subsequent experiments. Compared with group A, the cells in group B showed senescence changes. Compared with group B, the senescence rate of chondrocytes in group C decreased, autophagy increased, the relative expression of type Ⅱ collagen mRNA increased, and relative expressions of MMP-3 and MMP-13 mRNAs decreased; the relative expressions of Beclin-1, LC3, and type Ⅱ collagen proteins increased, but the relative expressions of P16, MMP-3, MMP-13, mTOR, and AKT proteins decreased; the above differences were significant ( P<0.05). Compared with group C, when 3-MA was added in group D, the senescence rate of chondrocytes increased, autophagy decreased, and the relative expressions of the target proteins and mRNAs showed an opposite trend ( P<0.05). Conclusion: EGCG regulates the autophagy of chondrocytes through the PI3K/AKT/mTOR signaling pathway and exerts anti-senescence effects.

Circ_0000479 promotes proliferation, invasion, migration and inflammation and inhibits apoptosis of rheumatoid arthritis fibroblast-like synoviocytes via miR-766/FKBP5 axis.

Circular RNAs have been demonstrated to play a critical role in the progression of autoimmune diseases. This study aimed to investigate the function of circ_0000479 in rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLSs). Circ_0000479 was found to be upregulated in RA-FLSs. Flow cytometry analysis, cell counting Kit-8, transwell, wound-healing and enzyme-linked immunosorbent assays were conducted to evaluate RA-FLS apoptosis, proliferation, invasion, migration and inflammation. The results confirmed that circ_0000479 knockdown suppressed pathogenic properties of RA-FLSs. Through bioinformatics analysis and screening, we obtained 18 miRNAs that can bind to circ_0000479, of which miR-766 was most significantly up-regulated after circ_0000479 knockdown. MiR-766 was confirmed to be down-regulated in RA-FLSs and the combination between circ_0000479 and miR-766 was verified by dual-luciferase reporter assays. Moreover, the inhibitory effect of circ_0000479 knockdown in RA-FLS progression was attenuated by miR-766 inhibitor. By intersecting the target genes of miR-766 with the up-regulated genes in RA, we obtained 8 genes, of which FKBP5 was most significantly down-regulated after miR-766 overexpression. The results of dual-luciferase reporter assays also verified that FKBP5 was the target gene of miR-766. In addition, FKBP5 overexpression abated the inhibition of RA-FLS progression caused by circ_0000479 silencing. In summary, circ_0000479 binds to miR-766 to promote RA progression via FKBP5.

Residual periodontal ligament in the extraction socket promotes the dentin regeneration potential of DPSCs in the rabbit jaw.

BACKGROUND: Because of the low regeneration efficiency and unclear underlying molecular mechanism, tooth regeneration applications are limited. In this study, we explored the influence of residual periodontal ligament on the dentin regeneration potential of dental pulp stem cells (DPSCs) in the jaw. METHODS: To establish a tooth regeneration model, the incisors of New Zealand white rabbits were extracted while preserving residual periodontal ligament, followed by the implantation of DPSCs. After 3 months, micro-computed tomography (micro-CT), stereomicroscopy and scanning electron microscopy (SEM) were used to observe the volume, morphology and microstructure of regenerated tissue. Histological staining and immunostaining analyses were used to observe the morphological characteristics and expression of the dentin-specific proteins DMP1 and DSPP. To explore the mechanism, DPSCs and periodontal ligament stem cells (PDLSCs) were cocultured in vitro, and RNA was collected from the DPSCs for RNA-seq and bioinformatic analysis. RESULTS: The results of micro-CT and stereomicroscopy showed that the number of sites with regeneration and the volume of regenerated tissue in the DPSCs/PDL group (6/8, 1.07 ± 0.93 cm3) were larger than those in the DPSCs group (3/8, 0.23 ± 0.41 cm3). The results of SEM showed that the regenerated dentin-like tissue in the DPSCs and DPSCs/PDL groups contained dentin tubules. Haematoxylin and eosin staining and immunohistochemical staining indicated that compared with the DPSCs group, the DPSCs/PDL group showed more regular regenerated tissue and higher expression levels of the dentin-specific proteins DMP1 and DSPP (DMP1: P = 0.02, DSPP: P = 0.01). RNA-seq showed that the coculture of DPSCs with PDLSCs resulted in the DPSCs differentially expressing 427 mRNAs (285 upregulated and 142 downregulated), 41 lncRNAs (26 upregulated and 15 downregulated), 411 circRNAs (224 upregulated and 187 downregulated), and 19 miRNAs (13 upregulated and 5 downregulated). Bioinformatic analysis revealed related Gene Ontology function and signalling pathways, including extracellular matrix (ECM), tumour necrosis factor (TNF) signalling and chemokine signalling pathways. CONCLUSIONS: Residual periodontal ligament in the extraction socket promotes the dentin regeneration potential of DPSCs in the jaw. RNA-seq and bioinformatic analysis revealed that ECM, TNF signalling and chemokine signalling pathways may represent the key factors and signalling pathways.

ATP2B1-AS1 exacerbates sepsis-induced cell apoptosis and inflammation by regulating miR-23a-3p/TLR4 axis

Background: Sepsis is a life-threatening disease with dominant mortality. Its early diagnosis and treatment can improve prognosis and reduce mortality. Long noncoding RNAs (lncRNAs) ATPase plasma membrane Ca2+ transporting 1 antisense RNA 1 (ATP2B1-AS1) is dysregulated and is involved in the progression of various diseases. Nevertheless, the role of ATP2B1-AS1 in sepsis remains unclear. Methods: A human monocytic cell line, THP-1 cells, was stimulated to induce a model of sepsis in vitro. The levels of ATP2B1-AS1, miR-23a-3p, and TLR4 were assessed by real-time quantitative polymerase chain reaction. The role of ATP2B1-AS1 in cell apoptosis and inflammation was explored by flow cytometry, Western blot analysis and enzyme-linked immunosorbent serologic assay. The binding sites between ATP2B1-AS1 and miR-23a-3p, and between miR-23a-3p and TLR4 were predicted by BiBiServ and the Encyclopedia of RNA Interactomes (ENCORI) online sites, respectively, and confirmed by the luciferase assay. Results: The level of ATP2B1-AS1 was increased in lipopolysaccharide (LPS)-treated THP-1 cells. LPS increased apoptosis ratio, relative protein expressions of pro-apoptotic factors, and relative messenger RNA (mRNA) level and concentrations of pro-inflammatory cytokines, but decreased the relative expression of anti-apoptosis protein and relative mRNA level and concentrations of anti-inflammatory factor. All these alterations were reversed with transfection of shATP2B1-AS1 into THP-1 cells. Moreover, ATP2B1-AS1 directly bound miR-23a-3p and negatively modulated the level of miR-23a-3p. Meanwhile, TLR4 was directly targeted by miR-23a-3p, and negatively and positively modulated by miR-23a-3p and ATP2B1-AS1, respectively. Conclusion: ATP2B1-AS1 aggravated apoptosis and inflammation by modulating miR-23a-3p/TLR4 axis in LPS-treated THP-1 cells (AU)

Effect of transduced mesenchymal stem cells with IL-10 gene on control of allergic asthma

Asthma is an important pulmonary disease associated with T helper lymphocyte (Th)2 dominant immune response, which can initiate allergic and inflammatory reactions. Interleukin (IL)-10 is the main immune suppressor cytokine, and mesenchymal stem cells (MSCs) have an immune-modulatory potential that can be transduced with the expression of the IL-10 gene to control pathophysiology of allergic asthma. Bone marrow’s MSCs were isolated and transduced with the expression vector that contains the expressible IL-10 gene. Then, allergic asthma mouse model was produced and treated with manipulated MSCs. Methacholine challenge test; measurement of IL-4, IL-5, IL-8, IL-13, IL-25, and IL-33; and total and ovalbumin (OVA)-specific immunoglobulin (Ig)E levels were done. Hyperplasia of the goblet cell, secretion of mucus, and peribronchiolar and perivascular eosinophilic inflammation were evaluated in lung pathological sections. IL-25, IL-33, and total IgE levels; AHR; eosinophilic inflammation; hyperplasia of the goblet cell; and secretion of mucus could be controlled in M, MV, and MV-10 groups, and the control in the MV-10 group was strong compared to M and MV groups. MSCs have immune-modulatory capacity that can control allergic asthma pathophysiology, and this effect can be strengthened and reinforced by the expression of IL-10 gene (AU)

Tectorigenin inhibits inflammation in keratinocytes by inhibition of NLRP3 inflammasome regulated by the TLR4/NF-kB pathway

Background: Psoriasis is a prevalent inflammatory skin disease characterized by excessive proliferation and abnormal differentiation of keratinocytes, and infiltration of inflammatory cells into the epidermis. However, the underlying mechanisms remain unclear. Tectorigenin is an active ingredient in traditional medicines and has anti-inflammatory activity. This research explored the effects of tectorigenin on the anti-inflammatory property, autophagy, and the underlying mechanisms in M5 ([IL-22, IL-17A, oncostatin M, IL-1α, and TNF-α])–stimulated HaCaT cells. Methods: The in vitro model of mixed M5 cytokines–stimulated HaCaT keratinocytes was established to investigate the phenotypic features in psoriasis. Cell viability was assessed by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay, cell proliferative rate by EdU (5-ethynyl-2'-deoxyuridine) assay, and autophagy was detected by immunofluorescence staining. After M5 exposure, the proliferative rate, protein expression of autophagy, and signaling activities of NLR family pyrin domain containing 3 (NLRP3) inflammasome and toll-like receptor 4 (TLR4)/nuclear factor-κB (NF-κB) were measured. The latter were quantitated using quantitative PCR and western blot, respectively. The inflammatory response was detected by enzyme-linked immunosorbent assay (ELISA). Results: Tectorigenin exerted a protective effect in ameliorating the hyperproliferation and inflammation of HaCaT keratinocytes induced by M5 cytokines. Furthermore, tectorigenin on keratinocytes seemed to inactivate NLRP3 inflammasome and inhibit cell proliferation and inflammation response via suppression of TLR4/NF-κB pathway. Conclusion: This study proves that tectorigenin may be a potential therapeutic candidate for psoriasis treatment in future (AU)

CHAC1 exacerbates LPS-induced ferroptosis and apoptosis in HK-2 cells by promoting oxidative stress

Background: Sepsis-induced acute kidney injury (AKI) is a singularly grievous and life-threatening syndrome. Its pathogenesis is closely related to inflammatory response, apoptosis, oxidative stress, and ferroptosis. Cation transport regulator-like protein 1 (CHAC1), as a proapoptic factor, may be involved in apoptosis, oxidative stress, and ferroptosis. This study aimed to explore the role of CHAC1 in the lipopolysaccharide (LPS)-induced the human renal proximal tubular epithelial (HK-2) cells. Methods: HK-2 cells were challenged with LPS to construct a model of sepsis-induced AKI in vitro. The role of CHAC1 in the LPS-induced HK-2 cells was explored using Western blot assay, cell counting kit-8 (CCK-8), flow cytometry, and colorimetric assays. Additionally, N-acetyl cysteine (NAC) was incubated with HK-2 cells to define deeply the relation between oxidative stress and apoptosis or ferroptosis. Results: The expression of CHAC1 was enhanced in the kidney tissues of mice with sepsis--induced multiple organ dysfunction syndrome (MODS), through the Gene Expression Omnibus database (GSE60088 microarray dataset), and in the LPS-induced HK-2 cells. The cell viability was significantly reduced by LPS treatment, which was at least partly restored by the transfection of siCHAC1#1 and siCHAC1#2 but not siNC. In addition, down-regulation of CHAC1 counteracted the LPS-induced reactive oxygen species level and malonaldehyde concentrations while restored the LPS-induced glutathione concentrations. Meanwhile, interference of CHAC1 neutralized LPS-induced apoptosis rate, and the relative level of cleaved poly(ADP-ribose) polymerase (PARP)/PARP, and cleaved caspase-3/caspase-3(AU)

MiR-328-3p promotes TGF-B1-induced proliferation, migration, and inflammation of airway smooth muscle cells by regulating the PTEN/Akt pathway

Background: Recent studies have shown that the up-regulation of microRNA miR-328-3p expression increases seasonal allergy and asthma symptoms in children, but the specific mechanism remains unclear. Therefore, the aim of this study was to explore the role and mechanism of -miR-328-3p in transforming growth factor (TGF)-β1-induced airway smooth muscle cells (ASMCs). Methods: The effect of TGF-β1 on the expression of miR-328-3p in ASMCs was examined by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). Cells proliferation, migration, and inflammatory factors in TGF-β1-induced ASMCs were measured by cell counting kit-8 (CCK-8), transwell, and enzyme-linked immunosorbent assay (ELISA), respectively. Besides, TargetScan was used to predict phosphatase and tensin homolog (PTEN), the downstream target of miR-328-3p; double-luciferase reporter assay, western blot, and qRT-PCR were used to verify the targeting relationship between miR-328-3p and PTEN; western blot was also used to examine the effects of PTEN and miR-328-3p knockdown on the expression levels of PTEN, Akt, and p-Akt proteins. Results: The expression of miR-328-3p was up-regulated in TGF-β1-induced ASMCs. Knockdown of miR-328-3p significantly inhibited proliferation, migration, and inflammation of ASMCs induced by TGF-β1 and decreased levels of tumor necrosis factor (TNF)-α and interleukin (IL)-1β. The dual--luciferase reporter assay results confirmed that PTEN was a target gene of miR-328-3p. Moreover, inhibition of PTEN expression reversed the inhibitory effect of low miR-328-3p expression on -TGF-β1-induced ASMC’s proliferation, migration, and inflammation (AU)

Triamcinolone acetonide induces the autophagy of Ag85B-treated WI-38 cells via SIRT1/FOXO3 pathway

Background: Tracheobronchial stenosis due to tuberculosis (TSTB) seriously threatens the health of tuberculosis patients. The inflammation and autophagy of fibroblasts affect the development of TSTB. Triamcinolone acetonide (TA) can regulate the autophagy of fibroblasts. Nevertheless, the impact of TA on TSTB and underlying mechanism has remained unclear. Objective: To study the impact of TA on TSTB and underlying mechanism. Material and Methods: In order to simulate the TSTB-like model in vitro, WI-38 cells were exposed to Ag85B protein. In addition, the cell counting kit (CCK)-8 assay was applied to assess the function of TA in Ag85B-treated WI-38 cells. Quantitative real-time polymerase chain reaction was applied to detect the mRNA level of sirtuin 1 (SIRT1) and forkhead box O3 (FOXO3a), and autophagy-related proteins were evaluated by Western blot analysis. Vascular endothelial growth factor (VEGF) level was investigated by immunohistochemical staining. Enzyme-linked immunosorbent serologic assay was applied to detect the secretion of inflammatory cytokines. Furthermore, hematoxylin and eosin staining was applied to observe tissue injuries. Results: Ag85B affected WI-38 cell viability in a limited manner, while TA notably suppressed Ag85B-treated WI-38 cell viability. TA induced the apoptosis of Ag85B-treated WI-38 cells in a dose-dependent manner. In addition, Ag85B-treated WI-38 cells demonstrated the upregulation of interleukin (IL)-6, tumor necrosis factor-α (TNF-α), interferon gamma (IFN-γ), and fibrotic proteins (transforming growth factor-beta [TGF-β] and vascular endothelial growth factor [VEGF]), which can be significantly destroyed by the TA. Meanwhile, TA reversed Ag85-induced inhibition of cell autophagy by mediation of p62, LC3, and Beclin1 (AU)

MiR-760 targets HBEGF to control cartilage extracellular matrix degradation in osteoarthritis.

The present study was developed to explore whether microRNA (miR)-760 targets heparin-binding EGF-like growth factor (HBEGF) to control cartilage extracellular matrix degradation in osteoarthritis. Both miR-760 and HBEGF expression levels were analysed in human degenerative cartilage tissues and in interleukin (IL)-1ß/tumour necrosis factor (TNF)-α-treated chondrocytes in vitro. A series of knockdown and overexpression assays were then used to gauge the functional importance of miR-760 and HBEGF in OA, with qPCR and western immunoblotting analyses. Bioinformatics assays were used to identify putative miR-760 target genes, with these predictions then being validated through RNA pulldown and luciferase reporter assays. A murine anterior cruciate ligament transection model of OA was then established to prove the in vivo relevance of these findings. These experiments revealed that human degenerative cartilage tissues exhibited significant increases in miR-760 expression with a concomitant drop in HBEGF levels. IL-1ß/TNF-α-treated chondrocytes also exhibited significant increases in miR-760 expression with a concomitant drop in HBEGF expression. When chondrocytes were transfected with either miR-760 inhibitor or HBEGF overexpression constructs, this was sufficient to interfere with degradation of the extracellular matrix (ECM). Moreover, miR-760 was confirmed to control chondrocyte matrix homeostasis by targeting HBEGF, and the overexpression of HBEGF partially reversed the effects of miR-760 mimic treatment on the degradation of the cartilage ECM. When OA model mice were administered an intra-articular knee injection of an adenoviral vector encoding a miR-760 mimic construct, cartilage ECM degradation was aggravated. Conversely, the overexpression of HBEGF in OA model mice partially reversed the effects of miR-760 overexpression, restoring appropriate ECM homeostasis. In summary, these data indicated that the miR-760/HBEGF axis plays a central role in orchestrating the pathogenesis of OA, making it a candidate target for therapeutic efforts in OA.

Silencing of long non-coding RNA SOX2-overlapping transcript relieves myocardial ischemia/reperfusion injury through up-regulating miRNA-146a-5p.

Long non-coding RNAs (lncRNAs) are involved in the development of myocardial ischemia/reperfusion injury (MIRI). In this study, we aimed to explore the regulatory effect and mechanism of lncRNA SOX2-overlapping transcript (SOX2-OT) in MIRI. The viability of oxygen and glucose deprivation/reperfusion (OGD/R)-treated H9c2 cells was detected by MTT assay. The levels of interleukin (IL)-1ß, IL-6, tumor necrosis factor (TNF)-α, malondialdehyde (MDA), and superoxide dismutase (SOD) were measured by ELISA. The target relationship between SOX2-OT and miR-146a-5p was predicted by LncBase, and subsequently confirmed by Dual luciferase reporter assay. The effects of SOX2-OT silencing on myocardial apoptosis and function were further validated in MIRI rats. The expression of SOX2-OT was increased in OGD/R-treated H9c2 cells and myocardial tissues of MIRI rats. Silencing of SOX2-OT increased the viability and inhibited the inflammation and oxidative stress of OGD/R-treated H9c2 cells. SOX2-OT negatively regulated its target miR-146a-5p. Silencing of miR-146a-5p reversed the effects of sh-SOX2-OT on OGD/R-treated H9c2 cells. In addition, silencing of SOX2-OT also alleviated myocardial apoptosis and improved myocardial function in MIRI rats. Silencing of SOX2-OT relieved the apoptosis, inflammation, and oxidative stress of myocardial cells via up-regulating miR-146a-5p, contributing to the remission of MIRI.