Results Of Cell Counting Kit-8 Research Articles

Application of Alginate-Collagen Hydrogel and Its Related Mechanisms in the Repair of Rat Cartilage Injuries

This research investigated the application of alginate-collagen hydrogel (ACH) in rat cartilage injury (CI) repair and its related mechanisms through in vitro and in vivo experiments. Alginate solution and modified collagen solution were mixed to prepare the ACH. The obtained samples were characterized and their sustained release properties were determined. L929 mouse fibroblasts were assigned into a blank control group (BC group, untreated), a positive control group (PC group, treated with 6.4% phenol), a negative control group (NC group, treated with tissue culture polystyrene), and an ACH group (treated with ACH). Cell viability was assessed using the MTT assay. Meanwhile, the cytotoxicity of the ACH and its impact on cell proliferation and cell attachment capabilities were analyzed. Thirty 4-week-old specific pathogen-free (SPF) Sprague Dawley (SD) rats were randomly grouped into a chondrocyte group (5 rats, chondrocytes collected and cultured), a cPAGE group (5 rats, chondrocytes treated with ACH), a model group (10 rats, CI model), and a mPAGE group (10 rats, CI model treated with CAH). The influence of CAH on chondrocyte proliferation and the type II collagen (COLII) expression were assessed using the cell counting kit 8 (CCK-8) assay and immunofluorescence staining. Furthermore, the impacts of CAH on chondrocyte tissue and COL-II expression were analyzed using Safranin O-Fast Green (SOFG) staining, the International Cartilage Repair Society (ICRS) score, and COL-II tissue staining. Immunoblotting was utilized to detect the TNF-α, NF-κB, matrix metalloproteinase 13 (MMP-13), and COL-II proteins in cartilage tissue. The results showed that release rate (RR) of the constructed ACH reached over 85% within 72 hours and achieved degradation equilibrium within 5 days with a weight loss (WL) rate of over 60%. L929 fibroblasts culture results showed that the ACH posed no significant cytotoxicity, and cell attachment results were not obviously different from those on tissue culture plates (P >0.05). MTT assay results demonstrated that the ACH exerted no adverse effects on L929 cell proliferation and growth, indicating a good biocompatibility. CCK-8 and COL-II results indicated that chondrocyte proliferation in cPAGE group was much higher in contrast to that in chondrocyte group (P <0.05). Besides, the animal experiments unveiled that in comparison to the CI group, the mPAGE group had more chondrocytes, upregulated COL-II, and higher ICRS scores (P <0.05). MMP-13, NF-κB, and TNF-α were downregulated, and COL-II was upshifted in the mPAGE group, with remarkable differences (P <0.05). In conclusion, ACH exhibited excellent biocompatibility and low cytotoxicity, promoted cell proliferation and growth, facilitated chondrocyte proliferation in rat CI repair, offering a valuable research foundation for CI treatment.

Effects of BRCA1 overexpression via the NRF2 / HO1 / NQO1 pathway on oral cancer cells proliferation, migration, and apoptosis

ObjectiveHerein, we explored the influences of breast cancer susceptibility gene 1 (BRCA1) overexpression on oral cancer cells proliferation, migration, and apoptosis via evaluation of its interactions with nuclear factor erythroid 2-like 2 (NRF2). DesignCAL-27 and DOK cells were transfected with a BRCA1 overexpressing lentivirus. Next, we utilized Western blot and quantitative real-time polymerase chain reaction (qRT-PCR) analyses to evaluate BRCA1, NRF2, and their target gene expressions. Using cell counting kit-8 (CCK-8) assessment, we assessed cell proliferation and a scratch test detected CAL-27 cell migration. Additionally, flow cytometry was employed used to examine cell apoptosis, while an enzyme-linked immunosorbent assa (ELISA) was employed for evaluation of 8-hydroxy-2′- deoxyguanosine (8-OHdG) expression. An immunohistochemical analysis was employed to determine the NRF2 target genes and Ki-67 expressions. ResultsBRCA1 overexpression increased the NRF2 and its target gene transcript and protein expressions. CCK-8 and scratch test results showed that BRCA1 overexpression decreased cell proliferation and weakened CAL-27 cell migratory ability. Flow cytometry results showed that BRCA1 overexpression promoted cell apoptosis in a time-dependent manner, while enzyme-linked immunosorbent assay results showed that BRCA1 overexpression decreased 8-OHdG expression levels in CAL-27 and DOK cells. Immunohistochemical analysis results showed higher expression of NRF2 target genes and Ki-67 in oral squamous cell carcinoma cells. ConclusionsExperiments involving oral cancer cells confirmed that BRCA1 overexpression could up-regulate the NRF2 signalling pathway, reduce oxidative damage, and inhibit cell proliferation and other biological behaviours. The BRCA1 and NRF2 pathways might be associated with oral cancer occurrence and development.

The role of β2-AR/PI3K/AKT pathway in the proliferation, migration and invasion of THLE-2 cells induced by nicotine

Nicotine, the primary constituent of tobacco, is one of the important factors that induce the occurrence of hepatocellular carcinoma (HCC). The β2-adrenergic receptor (β2-AR) is implicated in the growth and advancement of tumors. However, the role of β2-AR and its mediated cascades in nicotine-induced HCC remains unclear. This present study aims to observe the effects of nicotine on the proliferation, migration, and invasion of immortalized human liver epithelial (THLE-2) cells, as well as to explore the underlying mechanisms of action. The results of cell counting kit-8 (CCK-8) assay showed that 0.3125 μM nicotine had the ability to promote the proliferation of THLE-2 cells with a significant time-dependent manner. Therefore, THLE-2 cells were mainly selected for chronic treatment with 0.3125 μM nicotine in the later stage to cause transformation. After 30 passages of THLE-2 cells with 0.3125 μM nicotine treatment, chronic exposure to nicotine significantly enhanced the proliferation, metastasis, and invasion of cells. Besides, it also upregulated the intracellular levels of β2-AR, phosphoinositide 3-kinase (PI3K), AKT, matrix metalloproteinase-2 (MMP-2) and Cyclin D1, as well as downregulated the expression of p53. More importantly, the β2-AR/PI3K/AKT pathway was found to mediate the expression of MMP-2, Cyclin D1, and p53 in THLE-2 cells, playing a crucial role in their proliferation, migration, and invasion after continuous exposure to nicotine. Simply put, it demonstrated the role of β2-AR/PI3K/AKT pathway in the transformation of THLE-2 cells induced by nicotine. This study could provide valuable insights into the relationship between nicotine and HCC. Additionally, it lays the groundwork for investigating potential anticancer treatments for liver cancer linked to tobacco consumption.

Dexmedetomidine hydrochloride plus sufentanil citrate inhibits glucose metabolism and epithelial‑mesenchymal transition in human esophageal squamous carcinoma KYSE30 cells by modulating the JAK/STAT3/HIF‑1α axis.

Dexmedetomidine hydrochloride (DEX-HCl) and sufentanil citrate (SFC) are commonly used anesthetic drugs for esophageal cancer (EC) surgery. The present study was performed to investigate the effect of DEX-HCl and SFC treatment on glucose metabolism and epithelial-mesenchymal transition in EC. Cell counting kit-8 (CCK8), clonogenic, wound healing and Transwell migration assays were performed to assess the effects of the DEX-HCl and SFC on KYSE30 cell proliferation, invasion and migration. Changes in lactate and glucose levels in KYSE30 cells were also detected. Western blot analysis was used to determine the protein expression levels of the JAK/STAT signaling pathway and glucose metabolism-related proteins. The results of CCK8, clonogenic and wound healing assays demonstrated that DEX-HCl and SFC inhibited KYSE30 cell proliferation, invasion and migration. Similarly, the combined DEX-HCl and SFC treatment significantly reduced lactate production, ATP production and glucose levels in KYSE30 cells. Western blotting indicated that DEX-HCl and SFC could reduce JAK/STAT and metastasis-related protein expression. Demonstrating a reduction in Hexokinase 2, matrix metallopeptidase 2 and 9, N-cadherin and lactate dehydrogenase A protein expression levels. The effects of DEX-HCl and SFC combined treatment were counteracted by the addition of JAK/STAT pathway activator RO8191, which suggested that DEX-HCl and SFC could serve a role in mediating the JAK/STAT signaling pathway in KYSE30 cells.

Effects of hypoxia on the growth of gastric cancer and the chemotherapeutic efficacy of 5-fluorouracil.

Hypoxia is an important cause of chemotherapy resistance in gastric cancer. However, little is known about the growth of gastric cancer under purely hypoxia conditions. This study aims to study the effect of hypoxia on the growth patterns of gastric cancer cells and explore the response of gastric cancer cells to the chemotherapeutic drug 5-fluorouracil (5-FU) in a hypoxic environment. Gastric cancer cells MKN45 were cultured under 1% oxygen hypoxia and conventional air conditions. An intervention group with the addition of the chemotherapeutic drug 5-FU was also established. The proliferation and apoptosis of gastric cancer cells under different oxygen conditions and intervention groups were detected using the cell counting kit-8 (CCK-8) method, JC-1 mitochondrial membrane potential assay, and Annexin-V/PI double staining method. Cell cycle changes were detected by flow cytometry, and mitochondrial changes were detected using electron microscopy. In the absence of 5-FU intervention, compared with the normoxia group, the hypoxia group showed higher rates of early and late apoptosis and higher cell death rates as indicated by the JC-1 mitochondrial membrane potential assay, Annexin-V/PI double staining, and CCK-8 results. Flow cytometry results showed that the cell cycle was arrested in the G0/G1 phase without progression. Electron microscopy revealed more severe mitochondrial destruction. However, with 5-FU intervention, the hypoxia group showed lower apoptosis rates, more cell cycle progression, and less mitochondrial destruction compared with the normoxia group. Hypoxic environments promote apoptosis and even death in gastric cancer cells, but hypoxia counteracts the efficacy of the chemotherapeutic drug 5-FU, which may contribute to 5-FU chemotherapy resistance.

Circ_0003764 Regulates the Osteogenic Differentiation of Periodontal Ligament Stem Cells

Specific circular RNAs (circRNAs) have been proven to play crucial roles in osteogenesis in vitro and in vivo. This study aims to identify a certain circRNA involved in the osteogenic differentiation of periodontal ligament stem cells (PDLSCs) and explore its regulatory role. The expression of 5 candidate circRNAs (circ_0026344, circ_ACAP2, circ_0003764, circ_0008259, and circ_0060731) was detected by real-time quantitative reverse-transcription polymerase chain reaction (qRT-PCR) after PDLSCs were cultured in the osteogenic induction medium or medium supplemented with tumour necrosis factor-α (TNF-α, 10 ng/mL) for 3 and 7 days. The circRNA significantly decreased in both 3 and 7 days of osteogenic induction in PDLSCs and markedly increased in TNF-α-induced PDLSCs for 3 and 7 days screened. Identified circRNA was knocked down or overexpressed, and the effect on the osteogenic differentiation of PDLSCs was investigated by qRT-PCR, western blot, alkaline phosphatase (ALP) staining, and alizarin red S (ARS) staining. Cell counting kit-8 (CCK-8) assay and 5-ethynyl-2'-deoxyuridine (EdU) assay were applied to detect the effect of the circRNA on the proliferation of PDLSCs. qRT-PCR results showed that the expression of circ_0003764 was significantly decreased when PDLSCs were cultured in the osteogenic induction medium for 3 or 7 days, whereas it was dramatically increased in TNF-α-induced PDLSCs. Knockdown of circ_0003764 promoted the expression of the osteogenesis-related genes (RUNX2, ALP, OCN) and proteins (RUNX2, OCN), enhanced the ALP activity, and elevated the mineralization by PDLSCs, as shown by ARS staining. However, with the overexpression of circ_0003764, the osteogenic differentiation capacity of PDLSCs was significantly reduced. The CCK-8 and EdU results indicated that circ_0003764 could inhibit the proliferation of PDLSCs. Circ_0003764 is involved in the osteogenesis process and inhibits the osteogenic differentiation and proliferation of PDLSCs. This study indicates that circ_0003764 can serve as a diagnostic and therapeutic target in bone regeneration-related diseases treated by PDLSCs-based tissue engineering.

Picroside II suppresses chondrocyte pyroptosis through MAPK/NF-κB/NLRP3 signaling pathway alleviates osteoarthritis.

Picroside II (P-II) is the main bioactive constituent of Picrorhiza Kurroa, a traditional Chinese herb of interest for its proven anti-inflammatory properties. Its beneficial effects have been noted across several physiological systems, including the nervous, circulatory, and digestive, capable of treating a wide range of diseases. Nevertheless, the potential of Picroside II to treat osteoarthritis (OA) and the mechanisms behind its efficacy remain largely unexplored. This study aims to evaluate the efficacy of Picroside II in the treatment of osteoarthritis and its potential molecular mechanisms. In vitro, we induced cellular inflammation in chondrocytes with lipopolysaccharide (LPS) and subsequently treated with Picroside II to assess protective effect on chondrocyte. We employed the Cell Counting Kit-8 (CCK-8) assay to assess the impact of Picroside II on cell viability and select the optimal Picroside II concentration for subsequent experiments. We explored the effect of Picroside II on chondrocyte pyroptosis and its underlying molecular mechanisms by qRT-PCR, Western blot (WB) and immunofluorescence. In vivo, we established the destabilization of the medial meniscus surgery to create an OA mouse model. The therapeutic effects of Picroside II were then assessed through Micro-CT scanning, Hematoxylin-eosin (H&E) staining, Safranin O-Fast Green (S&F) staining, immunohistochemistry and immunofluorescence. In in vitro studies, toluidine blue and CCK-8 results showed that a certain concentration of Picroside II had a restorative effect on the viability of chondrocytes inhibited by LPS. Picroside II notably suppressed the expression levels of caspase-1, IL-18, and IL-1β, which consequently led to the reduction of pyroptosis. Moreover, Picroside II was shown to decrease NLRP3 inflammasome activation, via the MAPK/NF-κB signaling pathway. In vivo studies have shown that Picroside II can effectively reduce subchondral bone destruction and osteophyte formation in the knee joint of mice after DMM surgery. Our research suggests that Picroside II can inhibit chondrocyte pyroptosis and ameliorate osteoarthritis progression by modulating the MAPK/NF-κB signaling pathway.

Genipin relieves diabetic retinopathy by down-regulation of advanced glycation end products via the mitochondrial metabolism related signaling pathway.

Glycation is an important step in aging and oxidative stress, which can lead to endothelial dysfunction and cause severe damage to the eyes or kidneys of diabetics. Inhibition of the formation of advanced glycation end products (AGEs) and their cell toxicity can be a useful therapeutic strategy in the prevention of diabetic retinopathy (DR). Gardenia jasminoides Ellis (GJE) fruit is a selective inhibitor of AGEs. Genipin is an active compound of GJE fruit, which can be employed to treat diabetes. To confirm the effect of genipin, a vital component of GJE fruit, in preventing human retinal microvascular endothelial cells (hRMECs) from AGEs damage in DR, to investigate the effect of genipin in the down-regulation of AGEs expression, and to explore the role of the CHGA/UCP2/glucose transporter 1 (GLUT1) signal pathway in this process. In vitro, cell viability was tested to determine the effects of different doses of glucose and genipin in hRMECs. Cell Counting Kit-8 (CCK-8), colony formation assay, flow cytometry, immunofluorescence, wound healing assay, transwell assay, and tube-forming assay were used to detect the effect of genipin on hRMECs cultured in high glucose conditions. In vivo, streptozotocin (STZ) induced mice were used, and genipin was administered by intraocular injection (IOI). To explore the effect and mechanism of genipin in diabetic-induced retinal dysfunction, reactive oxygen species (ROS), mitochondrial membrane potential (MMP), and 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxy-d-glucose (2-NBDG) assays were performed to explore energy metabolism and oxidative stress damage in high glucose-induced hRMECs and STZ mouse retinas. Immunofluorescence and Western blot were used to investigate the expression of inflammatory cytokines [vascular endothelial growth factor (VEGF), SCG3, tumor necrosis factor-alpha (TNF-α), interleukin (IL)-1β, IL-18, and nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing 3 (NLRP3)]. The protein expression of the receptor of AGEs (RAGE) and the mitochondria-related signal molecules CHGA, GLUT1, and UCP2 in high glucose-induced hRMECs and STZ mouse retinas were measured and compared with the genipin-treated group. The results of CCK-8 and colony formation assay showed that genipin promoted cell viability in high glucose (30 mmol/L D-Glucose)-induced hRMECs, especially at a 0.4 μmol/L dose for 7 d. Flow cytometry results showed that high glucose can increase apoptosis rate by 30%, and genipin alleviated cell apoptosis in AGEs-induced hRMECs. A high glucose environment promoted ATP, ROS, MMP, and 2-NBDG levels, while genipin inhibited these phenotypic abnormalities in AGEs-induced hRMECs. Furthermore, genipin remarkably reduced the levels of the pro-inflammatory cytokines TNF-α, IL-1β, IL-18, and NLRP3 and impeded the expression of VEGF and SCG3 in AGEs-damaged hRMECs. These results showed that genipin can reverse high glucose induced damage with regard to cell proliferation and apoptosis in vitro, while reducing energy metabolism, oxidative stress, and inflammatory injury caused by high glucose. In addition, ROS levels and glucose uptake levels were higher in the retina from the untreated eye than in the genipin-treated eye of STZ mice. The expression of inflammatory cytokines and pathway protein in the untreated eye compared with the genipin-treated eye was significantly increased, as measured by Western blot. These results showed that IOI of genipin reduced the expression of CHGA, UCP2, and GLUT1, maintained the retinal structure, and decreased ROS, glucose uptake, and inflammation levels in vivo. In addition, we found that SCG3 expression might have a higher sensitivity in DR than VEGF as a diagnostic marker at the protein level. Our study suggested that genipin ameliorates AGEs-induced hRMECs proliferation, apoptosis, energy metabolism, oxidative stress, and inflammatory injury, partially via the CHGA/UCP2/GLUT1 pathway. Control of advanced glycation by IOI of genipin may represent a strategy to prevent severe retinopathy and vision loss.

Mouse Embryonic Fibroblasts-Derived Extracellular Matrix Facilitates Expansion of Inner Ear-Derived Cells.

Previous reports showed that mouse embryonic fibroblasts (MEFs) could support pluripotent stem cell selfrenewal and maintain their pluripotency. The goal of this study was to reveal whether the decellularized extracellular matrix derived from MEFs (MEF-ECM) is beneficial to promote the proliferation of inner ear-derived cells. In this experimental study, we prepared a cell-free MEF-ECM through decellularization. Scanning electron microscope (SEM) and immunofluorescent staining were conducted for phenotype characterization. Organs of Corti were dissected from postnatal day 2 and the inner ear-derived cells were obtained. The identification of inner ear-derived cells was conducted by using reverse transcription-polymerase chain reaction (RT-PCR). Cell counting kit-8 (CCK-8) was used to evaluate the proliferation capability of inner ear-derived cells cultured on the MEFECM and tissue culture plate (TCP). The MEF-ECM was clearly observed after decellularization via SEM, and the immunofluorescence staining results revealed that MEF-ECM was composed of three proteins, including collagen I, fibronectin and laminin. Most importantly, the results of CCK-8 showed that compared with TCP, MEF-ECM could effectively facilitate the proliferation of inner ear-derived cells. The discovery of the potential of MEF-ECM in promoting inner ear-derived cell proliferation indicates that the decellularized matrix microenvironment may play a vital role in keeping proliferation ability of these cells. Our findings indicate that the use of MEF-ECM may serve as a novel approach for expanding inner ear-derived cells and potentially facilitating the clinical application of inner ear-derived cells for hearing loss in the future.

Expression and effect of heterogeneous nuclear ribonucleoprotein A2/B1 in tongue squamous cell carcinoma.

Tongue squamous cell carcinoma (TSCC) is a common cancer in the oral and maxillofacial region, which seriously endangers people's life and health.Heterogeneous nuclear ribonucleoprotein A2/B1(hnRNP A2/B1) is an RNA-binding protein that regulates the expression of a variety of genes and participates in the occurrence and development of a variety of cancers. This study aims to investigate the role of hnRNP A2/B1 in TSCC progression. The differential expression of hnRNP A2/B1 in oral squamous cell carcinoma (OSCC) and normal oral mucosa cells and tissues was analyzed based on the gene expression profiles of GSE146483 and GSE85195 in the Gene Expression Omnibus (GEO) database. The correlation between hnRNP A2/B1 expression and disease-free survival of TSCC patients was analyzed based on TSCC related chip of GSE4676. TSCC cancer and paracancerous tissue samples of 30 patients were collected in Hunan Cancer Hospital from July to December 2021. Real-time RT-PCR and Western blotting were used to verify the mRNA and protein expression of hnRNP A2/B1 in TSCC patients'samples, respectively. Human TSCC Tca-8113 cells were transfected with hnRNP A2/B1 empty vector (a sh-NC group), knockdown plasmid (a sh-hnRNP A2/B1 group), empty vector overexpression plasmid (an OE-NC group) and overexpression plasmid (an OE-hnRNP A2/B1 group), respectively. The knockdown or overexpression efficiency of hnRNP A2/B1 was detected by Western blotting. The proliferation activity of Tca-8113 cells was detected by cell counting kit-8 (CCK-8), and the apoptosis rate of Tca-8113 cells was detected by flow cytometry. Based on the analysis of OSCC-related chips of GSE146483 and GSE85195 in the GEO database, it was found that hnRNP A2/B1 was differentially expressed in the OSCC and normal oral mucosa cells and tissues (all P<0.01). Meanwhile, the analysis of TSCC related chip GSE4676 confirmed that the expression of hnRNP A2/B1 was negatively correlated with the disease-free survival of TSCC patients (P=0.006). The results of real-time RT-PCR and Western blotting showed that the relative expression levels of hnRNP A2/B1 mRNA and protein in TSCC tissues were significantly up-regulated compared with those in adjacent tissues (all P<0.01). The results of Western blotting showed that the expression level of hnRNP A2/B1 in Tca-8113 cells was significantly inhibited or promoted after knockdown or overexpression of hnRNP A2/B1 (all P<0.01). The results of CCK-8 and flow cytometry showed that inhibition of hnRNP A2/B1 expression in Tca-8113 cells reduced cell proliferation activity (P<0.05) and increased cell apoptic rate (P<0.01). Overexpression of hnRNP A2/B1 in Tca-8113 cells significantly increased cell proliferation (P<0.05) and decreased cell apoptosis (P<0.01). HnRNP A2/B1 is a key factor regulating the proliferation and apoptosis of TSCC cells. Inhibition of hnRNP A2/B1 expression can reduce the proliferation activity of TSCC cells and promote the apoptosis of TSCC cells.

Role of ADAM33 short isoform as a tumor suppressor in the pathogenesis of thyroid cancer via oncogenic function disruption of full-length ADAM33

Thyroid cancer is the most prevalent endocrine malignancy globally; however, its underlying pathogenesis remains unclarified. Reportedly, alternative splicing is involved in processes such as embryonic stem and precursor cell differentiation, cell lineage reprogramming, and epithelial-mesenchymal transitions. ADAM33-n, an alternative splicing isoform of ADAM33, encodes a small protein containing 138 amino acids of the N-terminal of full-length ADAM33, which constructs a chaperone-like domain that was previously reported to bind and block the proteolysis activity of ADAM33. In this study, we reported for the first time that ADAM33-n was downregulated in thyroid cancer. The results of cell counting kit-8 and colony formation assays showed that ectopic ADAM33-n in papillary thyroid cancer cell lines restricted cell proliferation and colony formation. Moreover, we demonstrated that ectopic ADAM33-n reversed the oncogenic function of full-length ADAM33 in cell growth and colony formation in the MDA-T32 and BCPAP cells. These findings indicate the tumor suppressor ability of ADAM33-n. Altogether, our study findings present a potential explanatory model of how the downregulation of the oncogenic gene ADAM33 promotes the pathogenesis of thyroid cancer.

UBE2C-mediated Autophagy Inhibition via Ubiquitination of SIRT1 Contributes to Endometrial Cancer Progression.

Our study highlights the tumorigenic role and regulatory mechanism of UBE2C in endometrial cancer; UBE2C inhibits endometrial cancer cell apoptosis through autophagy-related mechanisms and our findings provide new insights into the treatment of endometrial cancer.

Baicalin attenuates dexamethasone-induced apoptosis of bone marrow mesenchymal stem cells by activating the hedgehog signaling pathway.

Perturbations in bone marrow mesenchymal stem cell (BMSC) differentiation play an important role in steroid-induced osteonecrosis of the femoral head (SONFH). At present, studies on SONFH concentrate upon the balance within BMSC osteogenic and adipogenic differentiation. However, BMSC apoptosis as well as proliferation are important prerequisites in their differentiation. The hedgehog (HH) signaling pathway regulates bone cell apoptosis. Baicalin (BA), a well-known compound in traditional Chinese medicine, can affect the proliferation and apoptosis of numerous cell types via HH signaling. However, the potential role and mechanisms of BA on BMSCs are unclear. Thus, we aimed to explore the role of BA in dexamethasone (Dex)-induced BMSC apoptosis in this study. Primary BMSCs were treated with 10 -6 mol/L Dex alone or with 5.0 μmol/L, 10.0 μmol/L, or 50.0 μmol/L BA for 24 hours followed by co-treatment with 5.0 μmol/L, 10.0 μmol/L, or 50.0 μmol/L BA and 10 -6 mol/L Dex. Cell viability was assayed through the Cell Counting Kit-8 (CCK-8). Cell apoptosis was evaluated using Annexin V-fluorescein isothiocyanate/propidium iodide (PI) staining followed by flow cytometry. The imaging and counting, respectively, of Hochest 33342/PI-stained cells were used to assess the morphological characteristics and proportion of apoptotic cells. To quantify the apoptosis-related proteins (e.g., apoptosis regulator BAX [Bax], B-cell lymphoma 2 [Bcl-2], caspase-3, and cleaved caspase-3) and HH signaling pathway proteins, western blotting was used. A HH-signaling pathway inhibitor was used to demonstrate that BA exerts its anti-apoptotic effects via the HH signaling pathway. The results of CCK-8, Hoechst 33342/PI-staining, and flow cytometry showed that BA did not significantly promote cell proliferation (CCK-8: 0 μmol/L, 100%; 2.5 μmol/L, 98.58%; 5.0 μmol/L, 95.18%; 10.0 μmol/L, 98.11%; 50.0 μmol/L, 99.38%, F = 2.33, P > 0.05), but it did attenuate the effect of Dex on apoptosis (Hoechst 33342/PI-staining: Dex+ 50.0 μmol/L BA, 12.27% vs. Dex, 39.27%, t = 20.62; flow cytometry: Dex + 50.0 μmol/L BA, 12.68% vs. Dex, 37.43%, t = 11.56; Both P < 0.05). The results of western blotting analysis showed that BA reversed Dex-induced apoptosis by activating the HH signaling pathway, which down-regulated the expression of Bax, cleaved-caspase 3, and suppressor of fused (SUFU) while up-regulating Bcl-2, sonic hedgehog (SHH), and zinc finger protein GLI-1 (GLI-1) expression (Bax/Bcl-2: Dex+ 50.0 μmol/L BA, 1.09 vs. Dex, 2.76, t = 35.12; cleaved caspase-3/caspase-3: Dex + 50.0 μmol/L BA, 0.38 vs . Dex, 0.73, t = 10.62; SHH: Dex + 50.0 μmol/L BA, 0.50 vs . Dex, 0.12, t = 34.01; SUFU: Dex+ 50.0 μmol/L BA, 0.75 vs . Dex, 1.19, t = 10.78; GLI-1: Dex+ 50.0 μmol/L BA, 0.40 vs . Dex, 0.11, t = 30.68. All P < 0.05). BA antagonizes Dex-induced apoptosis of human BMSCs by activating the HH signaling pathway. It is a potential candidate for preventing SONFH.

Influence of antimicrobial peptide biofunctionalized TiO2 nanotubes on the biological behavior of human keratinocytes and its antibacterial effect

Objective: To fabricate TiO2 nanotube material functionalized by antimicrobial peptide LL-37, and to explore its effects on biological behaviors such as adhesion and migration of human keratinocytes (HaCaT) and its antibacterial properties. Methods: The TiO2 nanotube array (NT) was constructed on the surface of polished titanium (PT) by anodization, and the antimicrobial peptide LL-37 was loaded on the surface of TiO2 nanotube (LL-37/NT) by physical adsorption. Three samples were selected by simple random sampling in each group. Surface morphology, roughness, hydrophilicity and release characteristics of LL-37 of the samples were analyzed with a field emission scanning electron microscope, an atomic force microscope, a contact angle measuring device and a microplate absorbance reader. HaCaT cells were respectively cultured on the surface of three groups of titanium samples. Each group had 3 replicates. The morphology of cell was observed by field emission scanning electron microscope. The number of cell adhesion was observed by cellular immunofluorescence staining. Cell counting kit-8 (CCK-8) assay was used to detect cell proliferation. Wound scratch assay was used to observe the migration of HaCaT. The above experiments were used to evaluate the effect of each group on the biological behavior of HaCaT cells. To evaluate their antibacterial effects, Porphyromonas gingivalis (Pg) was respectively inoculated on the surface of three groups of titanium samples. Each group had 3 replicates. The morphology of bacteria was observed by field emission scanning electron microscope. Bacterial viability was determined by live/dead bacterial staining. Results: A uniform array of nanotubes could be seen on the surface of titanium samples in LL-37/NT group, and the top of the tube was covered with granular LL-37. Compared with PT group [the roughness was (2.30±0.18) nm, the contact angle was 71.8°±1.7°], the roughness [(20.40±3.10) and (19.10±4.11) nm] and hydrophilicity (the contact angles were 22.4°±3.1° and 25.3°±2.2°, respectively) of titanium samples increased in NT and LL-37/NT group (P<0.001). The results of in vitro release test showed that the release of antimicrobial peptide LL-37 was characterized by early sudden release (1-4 h) and long-term (1-7 d) slow release. With the immunofluorescence, more cell attachment was found on NT and LL-37/NT than that on PT at the first 0.5 and 2.0 h of culture (P<0.05). The results of CCK-8 showed that there was no significant difference in the proliferation of cells among groups at 1, 3 and 5 days after culture. Wound scratch assay showed that compared with PT and NT group, the cell moved fastest on the surface of titanium samples in LL-37/NT group at 24 h of culture [(96.4±4.9)%] (F=35.55, P<0.001). A monolayer cells could be formed and filled with the scratch in 24 h at LL-37/NT group. The results of bacterial test in vitro showed that compared with the PT group, the bacterial morphology in the NT and LL-37/NT groups was significantly wrinkled, and obvious bacterial rupture could be seen on the surface of titanium samples in LL-37/NT group. The results of bacteria staining showed that the green fluorescence intensity of titanium samples in LL-37/NT group was the lowest in all groups (F=66.54,P<0.001). Conclusions: LL-37/NT is beneficial to the adhesion and migration of HaCaT cells and has excellent antibacterial properties, this provides a new strategy for the optimal design of implant neck materials.

Inhibition of NUCB2 suppresses the proliferation, migration, and invasion of rheumatoid arthritis synovial fibroblasts from patients with rheumatoid arthritis in vitro

Rheumatoid arthritis (RA) is an autoimmune polyarthritis in which synovial fibroblasts (SF) play a major role in cartilage and bone destruction through tumorlike proliferation, migration, and invasion. Nesfatin-1, an 82-amino-acid-long peptide discovered by Oh-I in 2006, is derived from the precursor protein nucleobindin-2 (NUCB2). NUCB2/nesfatin-1 promotes cell proliferation, migration, and invasion in various tumors. We have previously shown that increased nesfatin-1 levels in the synovium may be associated with disease severity in patients with RA. However, the effect of NUCB2 on the tumorlike transformation of RASF has not yet been reported. The expression of NUCB2 mRNA in the synovium of RA and non-RA patients was further confirmed using three individual datasets from the NCBI GEO database. Gene set enrichment analysis (GSEA) was employed to explore the association between NUCB2 mRNA and RA-related gene signatures or signaling pathways in the GSE77298 dataset. Cell proliferation, migration, and invasion abilities were determined using Cell Counting Kit-8 (CCK-8), 5-ethynyl-2'-deoxyuridine (EdU), wound healing, and transwell assays, respectively. The results showed that the levels of NUCB2 mRNA in the synovium were significantly elevated in patients with RA. Moreover, GSEA showed that high expression of NUCB2 mRNA was related to gene signatures, including those involved in the cell cycle, DNA replication, extracellular matrix–receptor interaction, and focal adhesion. Furthermore, the results of CCK-8 and EdU assays indicated that inhibition of NUCB2 markedly repressed RASF proliferation. Additionally, the results of wound healing and transwell assays demonstrated that inhibition of NUCB2 significantly suppressed the migratory and invasive abilities of RASFs. Our findings are the first to demonstrate that the inhibition of NUCB2 suppresses the proliferation, migration, and invasion of RASFs in vitro.

M2 macrophage-derived exosomal miR-145-5p protects against the hypoxia/reoxygenation-induced pyroptosis of cardiomyocytes by inhibiting TLR4 expression.

Exosomes carrying micro ribonucleic acids (miRNAs) protect against myocardial ischemic injury. In the study, we sought to investigate the protective effect mechanism of M2 macrophage-derived exosome miR-145-5p in hypoxia-reoxygenation (H/R)-induced cardiomyocytes. M2 macrophages were isolated and induced from blood donated by healthy donors. M2 macrophages were transfected with or without miR-145-5p. Exosomes derived from M2 macrophages were isolated and identified by flow cytometry, nanoparticle tracking analysis, and transmission electron microscopy (TEM). AC16 cells were used to establish an H/R model, and cell activity was detected using a Cell Counting Kit 8 (CCK-8). Western blot was used to detect the expression of gasdermin D (GSDMD), nucleotide-binding domain-like receptor protein 3 (NLRP3), and caspase-1 in the H/R-induced AC16 cells to evaluate pyroptosis. Immunofluorescence staining was used to detect the positive rates of PKH26 and caspase-1. Combined with database prediction, dual luciferase reporter assays were used to validate toll-like receptor 4 (TLR4) as a downstream target molecule of miR-145-5p. A real-time quantitative polymerase chain reaction (RT-qPCR) analysis and western blot were used to detect the expression of TLR4 in the AC16 cells. Flow cytometry, western blot, nanoparticle tracking and TEM results confirmed the successful isolation of M2 macrophage-derived exosomes. CCK-8 results showed M2 macrophage-derived exosomes decreased the viability of the H/R-induced cells. Western blot results showed the expressions of GSDMD, caspase-1, and NLRP3 were significantly downregulated in the H/R group. Moreover, CCK-8 results showed the M2 macrophage-derived exosome miR-145-5p significantly ameliorated H/R-induced AC16 cellular activity. Western blot results confirmed the expressions of GSDMD, NLRP3, and caspase-1 were significantly downregulated in the macrophage-derived exosome miR-145-5p group compared to the M2 macrophage-derived exosome NC (normal control) group. Immunofluorescence staining results displayed the same trend in terms of the caspase-1 positivity rate. Further, we demonstrated overexpression of TLR4 partially reversed the protective effect of M2 macrophage-derived exosome miR-145-5p in the H/R-induced AC16 cells. Additionally, overexpression of TLR4 reversed the protein expression associated with pyroptosis in M2 macrophage-derived exosome miR-145-5p in the H/R-induced AC16 cells. Our study indicated M2 macrophage-derived exosomes carrying miR-145-5p inhibited H/R-induced cardiomyocyte pyroptosis by downregulating the expression of TLR4.

Sodium cantharidate promotes autophagy in breast cancer cells by inhibiting the PI3K-Akt-mTOR signaling pathway.

Sodium cantharidate (SCA) is a derivative of cantharidin obtained by its reaction with alkali. Studies have shown that it inhibits the occurrence and progression of several cancers. However, therapeutic effects of SCA on breast cancer are less well studied. This study aimed to clarify the effect of SCA on breast cancer cells and its mechanism, and to provide a scientific basis for the clinical use of SCA for the treatment of breast cancer. The results of cell counting kit-8, colony formation assay, and 5-ethynyl-2'-deoxyuridine staining showed that SCA inhibited breast cancer cell proliferation. Wound-healing and transwell assays demonstrated that SCA inhibited the migration and invasion of breast cancer cells. Transmission electron microscopy revealed that SCA induced autophagy in breast cancer cells. RNA sequencing technology showed that SCA significantly regulated the phosphoinositide 3-kinase-Akt-mammalian target of rapamycin (PI3K-Akt-mTOR) pathway, which was further verified using western blotting. The inducing effect of SCA on breast cancer autophagy was reversed by the mTOR activator MHY1485. In addition, subcutaneous xenograft experiments confirmed that SCA significantly inhibited tumor growth in vivo. Hematoxylin-eosin, TdT-mediated dUTP nick-end labeling, and immunohistochemical staining indicated that SCA induced tumor cell autophagy and apoptosis in nude mice without causing organ damage. In summary, we found that SCA promoted breast cancer cell apoptosis by inhibiting the PI3K-Akt-mTOR pathway and inducing autophagy.

MiR-181a-5p can inhibit the proliferation and promote the differentiation of chicken primary myoblasts

ABSTRACT 1. Myoblast proliferation and differentiation is one of the most important biological processes in the development of skeletal muscle. MicroRNAs (miRNAs) play a crucial role in this process. 2. In this study, the expression level of miR-181a-5p was detected, which found that miR-181a-5p was expressed differently in different tissues, different embryonic ages, and different differentiation stages of primary myoblasts in Gushi chickens. 3. The effect of miR-181a-5p was further investigated on chicken primary myoblasts (CPMs). The results of cell counting kit-8 (CCK-8), 5-ethynyl-2’-deoxyuridine (EdU) and cell cycle showed that miR-181a-5p could inhibit the proliferation of CPM. The miR-181a-5p promoted the expression of MYOD, MYOG, and MYHC. MYHC protein immunofluorescence experiments showed that miR-181a-5p increased the area of myotubes. 4. In total, 63 potential target genes of miR-181a-5p in mRNA transcriptome data analysis were identified. Functional enrichment analysis was performed on these target genes, and ASNS, SMYD1, and FOS were found to play regulatory roles in biological processes such as muscle development. It was speculated that miR-181a-5p played a role in myoblast development through these genes. 5. In conclusion, miR-181a-5p can inhibit the proliferation of chicken myoblasts and promote the differentiation of chicken myoblasts. This study laid the foundation for further research on the regulatory mechanism of miR-181a-5p in the development of skeletal muscle and the formation of excellent meat quality traits in Gushi chicken.

LncRNA BIRF Promotes Brain Ischemic Tolerance Induced By Cerebral Ischemic Preconditioning Through Upregulating GLT-1 via Sponging miR-330-5p

Long noncoding RNAs (lncRNAs) play an important regulatory role in various diseases. However, the role of lncRNAs in brain ischemic tolerance (BIT) induced by cerebral ischemic preconditioning (CIPC) is still unknown. The lncRNA profile of rat cortical astrocytes pretreated with ischemic preconditioning was analyzed by high-throughput sequencing. The results of Cell-Counting Kit-8 (CCK-8) assay showed that a novel lncRNA, NONRATT009133.2, which we referred to as brain ischemia–related factor (BIRF), was highly correlated with BIT. Through bioinformatics analysis, we predicted that BIRF, miR-330-5p, and GLT-1 (also named Slc1a2) might constitute a ceRNA regulatory network in the induction of BIT. We found that BIRF was upregulated by CIPC, which promoted GLT-1 expression and BIT induction. BIRF could directly bind to miR-330-5p. Furthermore, miR-330-5p directly targeted GLT-1, and miR-330-5p inhibited both GLT-1 expression and BIT induction in vitro and in vivo. Moreover, BIRF acts as a molecular sponge to competitively bind to miR-330-5p with GLT-1 mRNA, while the miR-330-5p inhibitor reversed all the effects of BIRF siRNA on GLT-1 expression and neuronal vitality. Taken together, our results demonstrate the important roles of the BIRF/miR-330-5p/GLT-1 axis in the induction of BIT by CIPC. BIRF may be a potentially effective therapeutic strategy against stroke injury.

An Integrated Approach Based on Network Analysis Combined With Experimental Verification Reveals PI3K/Akt/Nrf2 Signaling Is an Important Way for the Anti-Myocardial Ischemia Activity of Yi-Qi-Tong-Luo Capsule.

Background: Yiqi-Tongluo Capsule (YTC) is a Chinese traditional patent medicine that has been used in the treatment of myocardial ischemia (MI). However, its molecular mechanisms against MI have not been clear. Methods: Network analysis and experimental verification were used to explore the potential molecular mechanisms of YTC for MI treatment. Firstly, the main components in the capsules and the potential targets of these components were predicted by online databases. The MI related genes were collected from Genecards and Online Mendelian Inheritance in Man (OMIM) databases. The drug targets and disease targets were intersected, and then the protein-protein interaction (PPI) and Drug-Molecular-Target-Disease Network (DMTD) were constructed, and GO enrichment analysis and KEGG pathway enrichment analysis were performed. Based on the H2O2-stimulated H9c2 cells, flow cytometry, western blot (WB) and immunofluorescence experiments were performed to verify the network analysis prediction. Results: A total of 100 active components and 165 targets of YTC were predicted, in which there were 109 targets intersected with the targets of MI. GO and KEGG analysis showed that these potential targets were related to a variety of biological processes and molecular mechanisms, including oxidative stress and PI3K/AKT pathway. Astragaloside IV (AS IV) and paeoniflorin (PAE) might be the main active components in YTC. The results of cell counting kit-8 (CCK-8) showed that YTC alleviated the damage of H2O2 to H9c2 cells. The results of flow cytometry, DAPI staining and JC-1 probe showed that YTC alleviated H2O2 induced apoptosis in H9c2 cells. In addition, YTC reduced the level of intracellular superoxide anion, increased the activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px), and reduced the content of malondialdehyde (MDA) in H2O2-induced H9c2 cells. The results of immunofluorescence and WB showed that the phosphorylation of PI3K and Akt were increased, the expression of Bcl-2 was up-regulated and the expression of cleaved caspase-3 and Bax were down-regulated. Besides, the nuclear translocation of Nrf2 were increased. Conclusion: In conclusion, the results of this study showed that YTC might alleviate MI by suppressing apoptosis induced by oxidative stress via the PI3K/Akt/Nrf2 signal pathway.