Cytotechnology最新文献

Acute kidney injury (AKI) is a common clinical condition and is associated with unacceptable morbidity and mortality. Obtusifolin is an anthraquinone extracted from the seeds of Cassia obtusifolia with anti-inflammatory properties. This study focused on the role and mechanism of obtusifolin in AKI. The mouse podocyte cell line MPC5 was exposed to lipopolysaccharide (LPS) to establish a cell model of AKI. The viability of MPC5 cells treated with obtusifolin and/or LPS was detected by 3-(4, 5-Dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide assay. Cell apoptosis was analyzed by flow cytometry. The levels of podocyte injury- and apoptosis-related proteins as well as the nuclear factor-kappaB (NF-κB) signaling pathway was examined using western blotting analysis. The renal protective effects of obtusifolin were determined using an LPS-induced mouse model of AKI. Serum creatinine and blood urea nitrogen levels were measured. Hematoxylin–eosin staining of kidney sections was performed to evaluate renal histology. We found that MPC5 cells treated with LPS showed suppressed cell viability (p < 0.01) and increased cell apoptosis (p < 0.001). LPS reduced the protein expression of Bcl-2, nephrin, and synaptopodin as well as increased the protein levels of Bax and Cleaved Caspase-3 in podocytes in a concentration-dependent manner (p < 0.01). In addition, 10 μg/ml LPS-repressed cell viability was rescued by obtusifolin in a concentration-dependent manner (p < 0.01). Moreover, LPS-induced increase in MPC5 cell apoptosis was reversed by obtusifolin treatment (p < 0.01). Obtusifolin administration ameliorated LPS-induced kidney injury and reduced blood urea nitrogen and serum creatinine levels in mice (p < 0.001). Additionally, obtusifolin inhibited LPS-induced activation of NF-κB signaling in vitro and in vivo (p < 0.01). Overall, obtusifolin was effective in protecting renal function against LPS-induced AKI via inactivation of NF-κB signaling, which suggested that obtusifolin may act as a valuable agent for AKI therapy.

In the fields of tissue engineering and regenerative medicine, extracellular vesicles (EVs) have become viable therapeutic tools. EVs produced from stem cells promote tissue healing by regulating the immune system, enhancing cell proliferation and aiding remodeling processes. Recently, EV has gained significant attention from researchers due to its ability to treat various diseases. Unlike stem cells, stem cell-derived EVs show lower immunogenicity, are less able to overcome biological barriers, and have a higher safety profile. This makes the use of EVs derived from cell-free stem cells a promising alternative to whole-cell therapy. This review focuses on the biogenesis, isolation, and characterization of EVs and highlights their therapeutic potential for bone fracture healing, wound healing, and neuronal tissue repair and treatment of kidney and intestinal diseases. Additionally, this review discusses the potential of EVs for the treatment of cancer, COVID-19, and HIV. In summary, the use of EVs derived from stem cells offers a new horizon for applications in tissue engineering and regenerative medicine.

Cardiovascular diseases remain as the most common cause of death worldwide. To reveal the underlying mechanisms in varying cardiovascular diseases, in vitro models with cells and supportive biomaterial can be designed to recapitulate the essential components of human heart. In this study, we analyzed whether 3D co-culture of cardiomyocytes (CM) with vascular network and with adipose tissue-derived mesenchymal stem/stromal cells (ASC) can support CM functionality. CM were cultured with either endothelial cells (EC) and ASC or with only ASC in hydrazide-modified gelatin and oxidized gellan gum hybrid hydrogel to form cardiovascular multiculture and myocardial co-culture, respectively. We studied functional characteristics of CM in two different cellular set-ups and analyzed vascular network formation, cellular morphology and orientation. The results showed that gellan gum-gelatin hydrogel supports formation of two different cellular networks and functional CM. We detected formation of a modest vascular network in cardiovascular multiculture and extensive ASC-derived alpha smooth muscle actin -positive cellular network in multi- and co-culture. iPSC-CM showed elongated morphology, partly aligned orientation with the formed networks and presented normal calcium transients, beating rates, and contraction and relaxation behavior in both setups. These 3D cardiac models provide promising platforms to study (patho) physiological mechanisms of cardiovascular diseases.

Pulmonary arterial smooth muscle cells (PASMCs) functions are associated with the pathogenesis of pulmonary hypertension (PH) which is a life-threatening complication of acute pulmonary embolism (APE). This study sought to explore the expression pattern of microRNA (miR)-221-3p in APE-PH patients and its role in PASMCs proliferation and migration. The clinical data and venous blood of APE-PH patients were collected. The expression levels of miR-221-3p and phosphatase and tensin homolog (PTEN) in serum were determined, followed by receiver operator characteristic curve analysis of miR-221-3p diagnostic efficacy. PASMCs were transfected with miR-221-3p mimics and PTEN-overexpressed vector, followed by assessment of cell viability, proliferation, and migration through cell counting kit-8, 5‐ethynyl‐2′‐deoxyuridine, Transwell, and wound healing assays. The binding between miR-221-3p and PTEN 3′UTR region was testified by the dual-luciferase assay. miR-221 was upregulated in the serum of APE-PH patients and presented with good diagnostic efficacy with 1.155 cutoff value, 66.25% sensitivity, and 67.50% specificity. miR-221 was negatively correlated with PTEN in APE-PH patients. miR-221 overexpression facilitated PASMCs proliferation and migration in vitro. miR-221-3p bound to PTEN 3′UTR region to decrease PTEN protein levels. PTEN overexpression abolished the promotive role of miR-221-3p in PASMCs. Overall, miR-221-3p targeted PTEN to facilitate PASMC proliferation and migration.

Pancreatic cancer is difficult to manage owing to the challenges involved in its treatment and nursing. This study aimed to clarify the roles and mechanisms of action of Poly (A)-binding protein cytoplasmic 1 (PABPC1) on pancreatic cancer. The expression of PABPC1 in pancreatic cancer tissues and cell lines was detected using RT-qPCR and western blotting. The effects of PABPC1 on proliferation, apoptosis, epithelial-mesenchymal transition (EMT), and the PI3K/AKT signaling pathway in pancreatic cancer cells were further investigated using MTT assays, flow cytometry, and western blotting. The expression of PABPC1 was significantly upregulated in pancreatic cancer tissues and cells, whereas PABPC1 downregulation inhibited pancreatic cancer cell proliferation, induced apoptosis, decreased the expression of EMT-associated proteins, and exerted a regulatory effect by inhibiting the PI3K/AKT signaling pathway. In addition, the findings indicated that PABPC1 over-expression significantly promoted pancreatic cancer cell proliferation, inhibited apoptosis, decreased the expression of E-cadherin, enhanced N-cadherin expression, and activating the PI3K/AKT signaling pathway. PABPC1 silencing significantly inhibited proliferation and EMT and induced apoptosis in pancreatic cancer cells. These findings provide novel insights into the role of PABPC1 in the development of pancreatic cancer.

Fulminant hepatitis (FH) is a life-threatening clinical liver syndrome characterized by substantial hepatocyte necrosis and severe liver damage. FH is typically associated with severe oxidative stress, inflammation, and mitochondrial dysfunction. Pyrroloquinoline quinone (PQQ), a naturally occurring redox cofactor, functions as an essential nutrient and antioxidant and reportedly inhibits oxidative stress and exerts potent anti-inflammatory effects. In the present study, we aimed to evaluate the therapeutic efficacy of PQQ in murine hepatitis virus strain 3 (MHV-3)-induced FH and examined the underlying mechanism. An MHV-3-induced FH mouse model was established for in vivo examination. Liver sinusoidal endothelial cells (LSECs) were used for in vitro experiments. Herein, we observed that PQQ supplementation significantly attenuated MHV-3-induced hepatic injury by suppressing inflammatory responses and reducing oxidative stress. Mechanistically, PQQ supplementation ameliorated MHV-3-induced hepatic damage by down-regulating the Keap1/Nrf2 signaling pathway in vivo and in vitro. Furthermore, Nrf2 small interfering RNA targeting LSECs abrogated the PQQ-mediated protective effects against MHV-3-related liver injury. Our results deepen our understanding of the hepatoprotective function of PQQ against MHV-3-induced liver injury and provide evidence that alleviating oxidative stress might afford a novel therapeutic strategy for treating FH.

We herein report two- (2D) and three-dimensional (3D) culture methods of cholangiocytes originating from extrahepatic bile ducts of biliary atresia (BA) patients. Cells were stabilized for in vitro analyses, and 3D culture by two different methods showed the structural and functional features of cholangiocytes in the gel scaffold. First, cells were obtained from gallbladder contents or resected tissues of patients at surgery and then cultured in our original conditioned medium with a cocktail of signaling inhibitors that maintains the immaturity and amplification of cells. Cells were immortalized by inducing SV40T and hTERT genes using lentivirus systems. Immunostaining with CK19 and Sox9 antibodies confirmed the cells as cholangiocytes. 3D organoids were formed in Matrigel in two different ways: by forming spheroids or via vertical growth from 2D cell sheets (2 + 1D culture). Organoids generated with both methods showed the uptake and excretion of rhodamine-123, and duct-like structures were also found. Our culture methods are simpler than previously reported methods and still show the structural and functional characteristics of cholangiocytes. Thus, this system is expected to be useful for the in vitro investigation of cholangiocyte damage or regeneration in BA patients.

Potential role and associated mechanisms of Annexin A8 (ANXA8), a member of the Annexins family, in cervical squamous cell carcinoma (CESC) are still unclear, despite being upregulated in various malignant tumors. Here, we observed a notably elevated expression of ANXA8 in CESC cells. The inhibition of ANXA8 amplified the susceptibility of CESC cells to Erastin and sorafenib-induced ferroptosis, whereas it exerted minimal influence on DPI7 and DPI10-induced ferroptosis. The results from the Fe²⁺ concentration assay showed no significant correlation between ANXA8 gene knockdown and intracellular Fe²⁺ concentration induced by ferroptosis inducers. Western blot analysis demonstrated that the knockdown of ANXA8 did not alter ACSL4 and LPCAT levels under ferroptosis-inducing conditions, but it did result in a reduction in intracellular GSH levels induced by the ferroptosis inducer. Subsequently, we identified TFAP2A as an upstream transcription factor of ANXA8, which plays a role in regulating cell ferroptosis. The knockdown of TFAP2A significantly elevated MDA levels and depressed GSH levels in the presence of a ferroptosis inducer, thereby inhibiting cell ferroptosis. However, this inhibitory effect could be reversed by ANXA8 overexpression. Therefore, our research suggests that the TFAP2A/ANXA8 axis exerts regulatory control over ferroptosis in CESC cells by mediating GSH synthesis in System Xc.

The aim of this study was to elucidate the anti-allergic effects of polymethoxyflavonoids in combination with milk proteins and the mechanism of inhibition. Three polymethoxyflavonoids and two milk proteins were exposed to the rat basophilic leukemia cell line RBL-2H3. β-hexosaminidase was used as an indicator of degranulation inhibition. The mechanism of inhibition was examined by measuring intracellular Ca²⁺ levels and western blot method. In the degranulation inhibition test with polymethoxyflavonoids and milk proteins alone, nobiletin was the strongest inhibitor in the polymethoxyflavonoid group and lactoferrin in the milk protein group. Next, co-stimulation with nobiletin and lactoferrin showed stronger synergistic degranulation inhibition than treatment with nobiletin or lactoferrin alone. Western blot analysis showed that co-stimulation with nobiletin and lactoferrin significantly downregulated the induction of phospholipase Cγ 1 phosphorylation. The degranulation response in RBL-2H3 cells was synergistically suppressed by co-stimulation of nobiletin and lactoferrin acting on both Ca²⁺-dependent and Ca²⁺-independent pathways.

Abstract

Psoriasis is a common chronic inflammatory skin disease. Abnormal proliferation of keratinocytes plays an important role in the pathogenesis of psoriasis. Long non-coding RNAs (lncRNAs) are involved in the regulation of a variety of cell biological processes. The purpose of this study was to investigate the potential role of lncRNA MIR181A2HG in the proliferation of human keratinocytes. qRT-PCR and Western blotting were performed to measure the expression levels of MIR181A2HG, SRSF1, KRT6, and KRT16 in tissue specimens and HaCaT keratinocytes. The effects of MIR181A2HG on HaCaT keratinocytes proliferation were evaluated using Cell Counting Kit-8 (CCK-8) assays, 5-Ethynyl-2’-deoxyuridine (EdU) incorporation, and cell-cycle assays. RNA pulldown-mass spectrometry (MS) was applied to identify the proteins interacting with MIR181A2HG. RNA pull-down-Western blotting and RNA immunoprecipitation coupled with real-time quantitative reverse transcription-PCR (RIP-qRT-PCR) assays were used to determine the interactions between MIR181A2HG and its RNA-binding proteins (RBPs). MIR181A2HG was down-regulated in psoriasis tissues. MIR181A2HG overexpression induced G0/G1 and G2/M phase cell cycle arrest and decreased the protein levels of KRT6, KRT16, Cyclin D1, CDK4, and Cyclin A2 in HaCaT keratinocytes. MIR181A2HG knockdown showed the opposite effect. By using RNA pulldown-MS, 356 proteins were identified to interact with MIR181A2HG potentially. Bioinformatics analysis showed that NOP56 and SRSF1 may be RNA binding proteins (RBPs) that may be interact with MIR181A2HG. Furthermore, by using RNA pull-down-Western blotting and RIP-qRT-PCR, SRSF1 was determined to interact with MIR181A2HG. Moreover, silencing of SRSF1 inhibited keratinocytes proliferation, which could be reversed with the knockdown of MIR181A2HG. Our findings indicated that MIR181A2HG can negatively regulate HaCaT keratinocytes proliferation by binding SRSF1, suggesting that MIR181A2HG and SRSF1 may serve as potential targets for the treatment of psoriasis.