Cell Scratch Research Articles

Circular RNA circ-ABCB10 Promotes Proliferation and Inhibits Apoptosis of Laryngeal Carcinoma by Inhibiting KLF6.

Objective To explore the effect of circular RNA circ-ABCB10 on the proliferation and apoptosis of laryngeal carcinoma via inhibiting KLF6. Methods RT-qPCR assay was adopted to detect the expression of circ-ABCB10 and KFL6 in laryngeal carcinoma tissues and cell lines. Cell counting kit-8 (CCK-8) and clone formation assay were employed to detect laryngeal cancer cell viability and proliferation when circ-ABCB10 was silenced or upregulated. In this study, the apoptosis rate was detected by flow cytometry and the protein expression was detected by Western blotting. Wound healing and cross-hole invasion were used to study the migration and invasion of laryngeal cancer cells when circ-ABCB10 was silenced or upregulated. Results The results of RT-qPCR detection indicated that the expression of circ-ABCB10 in all three laryngeal carcinoma cells was downregulated by 3.2 times compared with that of HaCat cells. There is low expression of circ-ABCB10 in most laryngeal carcinoma tissues, the diagnostic cutoff value of circ-ABCB10 is 0.0008, the area under the curve is 0.718, the sensitivity is 0.981, and the specificity is 0.556. The expression level of KLF6 in laryngeal carcinoma is on the rise, which is significantly higher compared to healthy tissues (P < 0.05); 48 hours after transfection, RT-qPCR analysis confirmed the transfection efficiency, and upregulation of circ-ABCB10 could significantly promote cell proliferation. Compared with the control group, silencing circ-MTCL1 could inhibit cell proliferation, overexpression of circ-ABCB10 promoted cell migration, and downregulation of circ-ABCB10 significantly inhibited cell movement (P < 0.001). Upregulation of circ-ABCB10 significantly enhanced the invasiveness and motility of laryngeal cancer cells, while downregulation of circ-ABCB10 was the opposite. Compared with the KLF6 NC group, KLF6 level increased significantly in the KLF6 group, while cell viability, colony formation, scratch healing rate, invasive cell number, and Bcl-2 expression level decreased significantly in the KLF6 group, while apoptosis rate and Bax expression level increased significantly (P < 0.05). KLF6 level in the si-circ-ABCB10+anti-KLF6 group was significantly lower than that in the si-circ-ABCB10+anti-KLF6-NC group (P < 0.05). Meanwhile, the cell activity, colony formation number, cell scratch healing rate, number of invaded cells, and Bcl-2 all indicated an upward trend, while the cell apoptosis rate and Bax expression indicated a downward trend (P < 0.05). Conclusion The expression of circ-ABCB10 in laryngeal carcinoma was significantly higher compared to that in paracancerous tissues. Silencing circ-ABCB10 could significantly inhibit the growth and proliferation of laryngeal adenocarcinoma cells, while overexpression of circ-ABCB10 could significantly promote the growth of laryngeal adenocarcinoma cells, probably by inhibiting KLF6 to enhance the proliferation of laryngeal carcinoma and inhibit apoptosis.

Bioinspired hyaluronic acid based nanofibers immobilized with 3, 4- difluorobenzylidene curcumin for treating bacterial infections

Curcumin (Cur) is a natural polyphenol with multifaceted pharmacological functions, exploited extensively for biomedical applications. Traditionally curcumin is being used as an antimicrobial agent. However, to improvise the pharmacological properties, it is being modified synthetically. One of such modified Cur is 3, 4- difluorobenzylidene curcumin (CDF) which is aimed for enhancing the anti-cancer properties. Though there are reports on the studies of anti-cancer properties involving CDF, the anti-bacterial property is yet to be demonstrated. Accordingly, in our studies, we prepared bioinspired hyaluronic acid blends immobilized with CDF and fabricated non-woven nanofiber mats. These nanofiber mats were characterized and demonstrated in vitro cell culture studies, which involved cell viability, hemolysis, anti-bacterial and cell scratch assay to understand their efficacy in treating bacteria. The molecular docking studies of CDF and Cur were performed on the dihydrofolate reductase (DHFR) enzyme receptor, which is an essential protein of S.auerus (Staphylococcus aureus). The results of MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) assay, and hemolysis of the respective nanofiber mats with Cur and CDF showed non-toxicity and were compatible with blood cells. Further, the cell proliferation and adherence recorded >60% fibroblast cells for the nanofiber mats. The anti-bacterial property of Cur and CDF was similar. The in vitro release studies for the respective Cur and CDF loaded nanofiber mats recorded a release of 25 and 37%, respectively. From these studies, we concluded that the CDF sustained its anti-bacterial property in addition to the improved anti-cancer property; hence CDF being synergetic, it will have a better scope in cancer therapy.

18β-glycyrrhetinic acid regulates mitochondrial ribosomal protein L35-associated apoptosis signaling pathways to inhibit proliferation of gastric carcinoma cells.

BACKGROUNDGastric carcinoma (GC) is a common gastrointestinal malignancy worldwide. Based on the cancer-related mortality, the current prevention and treatment strategies for GC still show poor clinical results. Therefore, it is important to find effective drug treatment targets. AIMTo explore the mechanism by which 18β-glycyrrhetinic acid (18β-GRA) regulates mitochondrial ribosomal protein L35 (MRPL35) related signal proteins to inhibit the proliferation of GC cells. METHODSCell counting kit-8 assay was used to detect the effects of 18β-GRA on the survival rate of human normal gastric mucosal cell line GES-1 and the proliferation of GC cell lines MGC80-3 and BGC-823. The apoptosis and cell cycle were assessed by flow cytometry. Cell invasion and migration were evaluated by Transwell assay, and cell scratch test was used to detect cell migration. Furthermore, a tumor model was established by hypodermic injection of 2.5 × 106 BGC-823 cells at the selected positions of BALB/c nude mice to determine the effect of 18β-GRA on GC cell proliferation, and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was used to detect MRPL35 expression in the engrafted tumors in mice. We used the term tandem mass tag (TMT) labeling combined with liquid chromatography–tandem mass spectrometry to screen for differentially expressed proteins (DEPs) extracted from GC cells and control cells after 18β-GRA intervention. A detailed bioinformatics analysis of these DEPs was performed, including Gene Ontology annotation and enrichment analysis, Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis, and so on. Moreover, STRING database (https://string-db.org/) was used to predict protein-protein interaction (PPI) relationships and Western blot was used to detect the expression of proteins of interest in GC cells. RESULTSThe results indicated that 18β-GRA could inhibit the proliferation of GC cells in a dose- and time-dependent manner. It could induce GC cell apoptosis and arrest the cell cycle at G0/G1 phase. The proportion of cells arrested at S phase decreased with the increase of 18-GRA dose, and the migration and invasiveness of GC cells were inhibited. The results of animal experiments showed that 18β-GRA could inhibit tumor formation in BALB/c nude mice, and qRT-PCR results showed that MRPL35 expression level was significantly reduced in the engrafted tumors in mice. Using TMT technology, 609 DEPs, among which 335 were up-regulated and 274 were down-regulated, were identified in 18β-GRA intervention compared with control. We found that the intervention of 18β-GRA in GC cells involved many important biological processes and signaling pathways, such as cellular processes, biological regulation, and TP53 signaling pathway. Notably, after the drug intervention, MRPL35 expression was significantly down-regulated (P = 0.000247), TP53 expression was up-regulated (P = 0.02676), and BCL2L1 was down-regulated (P = 0.01699). Combined with the Retrieval of Interacting Genes/Proteins database, we analyzed the relationship between MRPL35, TP53, and BCL2L1 signaling proteins, and we found that COPS5, BAX, and BAD proteins can form a PPI network with MRPL35, TP53, and BCL2L1. Western blot analysis confirmed the intervention effect of 18β-GRA on GC cells, MRPL35, TP53, and BCL2L1 showed dose-dependent up/down-regulation, and the expression of COPS5, BAX, and BAD also increased/decreased with the change of 18β-GRA concentration.CONCLUSION18β-GRA can inhibit the proliferation of GC cells by regulating MRPL35, COPS5, TP53, BCL2L1, BAX, and BAD.

A Novel Hypoxia-inducible Factor 1α Inhibitor KC7F2 Attenuates Oxygen-induced Retinal Neovascularization

PurposeKC7F2 is a novel molecule compound that can inhibit the translation of hypoxia-inducible factor 1α (HIF1α). It has been reported to exhibit potential antiangiogenic effect. We hypothesized that KC7F2 could inhibit oxygen-induced retinal neovascularization (RNV). The purpose of this study was to investigate this assumption.MethodsOxygen-induced retinopathy (OIR) models in C57BL/6J mice and Sprague-Dawley rats were used for in vivo study. After intraperitoneal injections of KC7F2, RNV was detected by immunofluorescence and hematoxylin and eosin staining. Retinal inflammation was explored by immunofluorescence. EdU incorporation assay, cell counting kit-8 assay, scratch test, transwell assay, and Matrigel assay were used to evaluate the effect of KC7F2 on the proliferation, migration and tube formation of human umbilical vein endothelial cells (HUVEC) induced by vascular endothelial growth factor (VEGF) in vitro. Protein expression was examined by Western blot.ResultsKC7F2 treatment (10 mg/kg/d) in OIR mice significantly attenuated pathological neovascularization and decreased the number of preretinal neovascular cell nuclei, without changing the avascular area, which showed the same trends in OIR rats. Consistently, after the KC7F2 intervention (10 µM), cell proliferation was inhibited in VEGF-induced HUVEC, which was in agreement with the trend observed in the retinas of OIR mice. Meanwhile, KC7F2 suppressed VEGF-induced HUVEC migration and tube formation, and decreased the density of leukocytes and microglia colocalizing neovascular areas in the retinas. Moreover, the HIF1α–VEGF pathway activated in retinas of OIR mice and hypoxia-induced HUVEC, was suppressed by KC7F2 treatment.ConclusionsThe current study revealed that KC7F2 was able to inhibit RNV effectively via HIF1α–VEGF pathway, suggesting that it might be an effective drug for RNV treatment.

FTO Prevents Thyroid Cancer Progression by SLC7A11 m6A Methylation in a Ferroptosis-Dependent Manner.

N6 methyladenosine (m6A) modification serves as a novel epigenetic regulatory mechanism that is heavily implicated in the heredity of tumors. Meanwhile, fat mass and obesity-associated protein (FTO) has the potential to affect the regulation of m6A modification in the mRNA of key oncogenes as well as tumor suppressor genes that facilitate tumor progression. In our study, FTO was downregulated in papillary thyroid carcinoma (PTC) tissues. The role of FTO in PTC was assessed by Cell Counting Kit-8 analysis, cell scratch, migration, invasion experiment, flow cytometry apoptosis analysis, and nude mouse experiment. In addition to RNA-Seq and meRIP-Seq, luciferase reporting and mutation analysis have also identified SLC7A11 as the potential FTO regulatory gene. Moreover, X-ray electron microscopy, glutathione (GSH)/oxidized GSH, GPX, malondialdehyde determination, and western blot helped confirmed that FTO inhibited the development of PTC by downregulating the expression of SLC7A11 through ferroptosis. Finally, a rescue experiment was employed to clarify the relationship between FTO and its specific target gene SLC7A11. FTO is able to inhibit the occurrence of PTC by downregulating SLC7A11 in m6A independently, and it functions as a tumor suppressor gene in PTC. These findings could contribute to our understanding of the tumor malignancy regulated by m6A and might lead to new insights for potential biomarkers and therapeutic targets for the treatment of thyroid papillary carcinoma.

Effect of SOX2 gene on the biological characteristics of salivary adenoid cystic carcinoma cells.

This study aimed to investigate the effect of transfecting SOX2-shRNA vector lentivirus to SACC cell lines on the biological behavior of salivary adenoid cystic carcinoma (SACC)-LM and SACC-83. Three types of SOX2-shRNA lentiviral vectors (817, 818, and 819) were constructed and transfected successfully. The shRNA with the best inhibitory effect was screened out and transfected into SACC cells. Real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) and Western blot were used to detect the expressions of Survivin, E-cadherin, and N-cadherin of SACC-LM and SACC-83. CCK-8 and flow cytometry were used to detect SACC-LM and SACC-83 proliferation and apoptosis. Cell scratch test and Transwell method were used to detect the migration and invasion capabilities of SACC-LM and SACC-83. SOX2-shRNA-819 had the best interference effect among the three lentiviruses. After transfecting SOX2-shRNA-819 into SACC-LM and SACC-83, the expressions of SOX2, Survivin, and N-cadherin were significantly reduced, and that of E-cadherin was significantly increased (P<0.05). The cell proliferation ability decreased, and the number of apoptotic cells increased (P<0.05). The cell migration and invasion ability decreased (P<0.05). shRNA interference technology reduced SOX2 expression while downregulating Survivin expression. These two expressions may be related. Low SOX2 expression inhibits the proliferation, migration, and invasion of SACC cells and promotes the apoptosis of SACC cells. SOX2 may be involved in the epithelial⁃mesenchymal transition process. This study provided a relevant theoretical basis for targeting SOX2 gene therapy in SACC.

Sphingomyelin Phodiesterase Acid-Like 3A Promotes Hepatocellular Carcinoma Growth Through the Enhancer of Rudimentary Homolog.

BackgroundHepatocellular carcinoma (HCC) is one of the most common malignant tumors worldwide, with unclear pathogenesis. Sphingomyelin phodiesterase acid-like 3A (SMPDL3A) affects cell differentiation and participates in immune regulation. However, its molecular biological function in HCC has not yet been elucidated.MethodsData from 180 HCC patients were analyzed the relationship between the expression of SMPDL3A in liver cancer tissues and the prognosis of liver cancer patients. Crispr-Cas9 dual vector lentivirus was used to knock out SMPDL3A in HCC cell lines. The effects of SMPDL3A on cell viability were determined by CCK8 assay, clone formation experiment, cell cycle assay, cell scratch, TUNEL experiment and flow cytometry. Xenograft tumor assays in BALB/c nude mice confirmed that SMPDL3A promoted tumor growth and in vivo. Preliminary exploration of SMPDL3A interacting protein by mass spectrometry analysis and co-immunoprecipitation.ResultsThis study showed that the expression of SMPDL3A in HCC tissue differed from that in tumor-adjacent tissues. Moreover, the overall survival rate and tumor-free survival rate of patients with high-SMPDL3A expression were significantly lower than those with low-SMPDL3A expression. SMPDL3A expression was closely related to the level of protein induced by PIVKA-II, liver cirrhosis, tumor diameter, microvascular invasion, and Barcelona clinic liver cancer staging. Thus, SMPDL3A is an independent risk factor that affects the tumor-free survival rate and overall survival rate of HCC patients. In vitro study using Crispr-Cas9 genome editing technology revealed the knockout effect of SMPDL3A on cell proliferation, apoptosis, and migration. Cell counting kit-8 assay and clone formation experiment showed that sgSMPDL3A inhibited tumor cell proliferation and migration. Flow cytometry and TUNEL assay showed that sgSMPDL3A promoted apoptosis in tumors. Moreover, sgSMPDL3A inhibited tumor growth during subcutaneous tumor formation in nude mice. Immunohistochemistry of Ki67 and PNCA also indicated that sgSMPDL3A inhibited subcutaneous tumor proliferation in tumor-bearing nude mice. Further experiments showed that SMPDL3A interacts with the enhancer of rudimentary homolog (ERH).ConclusionsHigh-SMPDL3A expression was related to poor prognosis of patients with HCC. Knockout of SMPDL3A inhibited the proliferation and migration and accelerated the migration of HCC cells. SMPDL3A interacted with ERH to affect the tumorigenesis and progression of HCC.

LINC00941 promoted in vitro progression and glycolysis of laryngocarcinoma by upregulating PKM via activating the PI3K/AKT/mTOR signaling pathway.

BackgroundLINC00941 has been proved to be related to various tumors, but its relationship with laryngocarcinoma remains vague.MethodsLINC00941 expression in laryngocarcinoma tumor and laryngocarcinoma cells was determined by real time‐quantitative polymerase chain reaction (RT‐qPCR). Besides, the five‐year survival of laryngocarcinoma patients with different LINC00941 expression was analyzed with Kaplan–Meier survival analysis, and the clinical characteristics of laryngocarcinoma patients were also recorded. After transfection, cell viability, cell proliferation, apoptosis, cell cycle, migration, and invasion were detected by cell counting kit‐8 (CCK‐8), colony formation, flow cytometry, cell scratch, and Transwell assays, respectively. Glycolysis was assessed by the colorimetric method. Expressions of proliferation‐associated proteins, migration‐associated proteins, glycolysis‐associated proteins, and phosphatidylinositol 3‐kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) signal pathway‐associated proteins were detected by Western blot.ResultsIn laryngocarcinoma tumor tissues and cells, LINC00941 was highly expressed. High expression of LINC00941 decreased the 5‐year survival of laryngocarcinoma patients, and it was positively related to lymph node metastasis and clinical stages. LINC00941 overexpression decreased apoptosis but promoted cell viability, proliferation, cell‐cycle progression, migration, and invasion, and glucose consumption and lactate production in laryngocarcinoma cells. Moreover, LINC00941 overexpression elevated expressions of Ki‐67, PCNA, MMP2, N‐Cadherin, HK2, PFKFB4, and PKM, activated the PI3K/AKT/mTOR signal pathway but reduced E‐Cadherin expression, while LINC00941 silencing had the opposite effects. PKM overexpression reversed the effects of LINC00941 silencing on cellular and glycolytic phenotypes.ConclusionLINC00941 promoted in vitro progression and glycolysis of laryngocarcinoma cells by upregulating PKM via activating the PI3K/AKT/mTOR signaling pathway.

MicroRNA-144-3p Represses the Growth and EMT of Thyroid Cancer via the E2F2/TNIK Axis in Cells and Male BALB/c Nude Mice.

microRNA (miR/miRNA)-144-3p has been implicated in thyroid cancer (TC) progression with poorly identified mechanisms. Furthermore, E2F2 has been documented to assume a role in the development of various cancers. This research sought to ascertain the role of miR-144-3p in growth and epithelial-mesenchymal transition (EMT) in TC in cells and male BALB/c nude mice. In the obtained TC cells, miR-144-3p expression was detected by quantitative reverse transcription polymerase chain reaction, and E2F2 and TNIK expression by Western blot analysis. After gain- and loss-of-function assays, cell viability, clone formation, migration, and invasion were assessed by cell counting kit-8, clone formation, scratch, and Transwell assays. The expression of EMT-related proteins (Snail, Vimentin, N-cadherin, and E-cadherin) was tested by Western blot analysis. The targeting relationship between miR-144-3p and E2F2 was evaluated by dual-luciferase reporter and radioimmunoprecipitation assays, and the binding relationship between E2F2 and TNIK by dual-luciferase reporter and chromatin immunoprecipitation assays. TC cell growth in vivo was determined by subcutaneous tumorigenesis assays in nude mice. miR-144-3p was downregulated, whereas E2F2 and TNIK were upregulated in TC cells. Mechanistically, miR-144-3p inversely targeted E2F2, which increased TNIK expression by binding to TNIK promoter in TC cells. Overexpression of miR-144-3p reduced proliferation, migration, invasion, and EMT of FRO and KTC3 cells, which was nullified by overexpressing E2F2 or TNIK expression. Upregulation of miR-144-3p diminished FRO cell growth and EMT in nude mice, which was abrogated by overexpressing TNIK. miR-144-3p inhibits cell growth and EMT in TC through E2F2/TNIK axis inactivation in cells and male BALB/c nude mice.

Disulfiram Exerts Antifibrotic and Anti-Inflammatory Therapeutic Effects on Perimysial Orbital Fibroblasts in Graves' Orbitopathy.

Fibrosis of extraocular muscles (EOMs) is a marker of end-stage in Graves’ orbitopathy (GO). To determine the antifibrotic and anti-inflammatory therapeutic effects and the underlying molecular mechanisms of disulfiram (DSF) on perimysial orbital fibroblasts (pOFs) in a GO model in vitro, primary cultures of pOFs from eight patients with GO and six subjects without GO (NG) were established. CCK-8 and EdU assays, IF, qPCR, WB, three-dimensional collagen gel contraction assays, cell scratch experiments, and ELISAs were performed. After TGF-β1 stimulation of pOFs, the proliferation rate of the GO group but not the NG group increased significantly. DSF dose-dependently inhibited the proliferation, contraction, and migration of pOFs in the GO group. Additionally, DSF dose-dependently inhibited fibrosis and extracellular matrix production markers (FN1, COL1A1, α-SMA, CTGF) at the mRNA and protein levels. Furthermore, DSF mediates antifibrotic effects on GO pOFs partially through the ERK-Snail signaling pathway. In addition, DSF attenuated HA production and suppressed inflammatory chemokine molecule expression induced by TGF-β1 in GO pOFs. In this in vitro study, we demonstrate the inhibitory effect of DSF on pOFs fibrosis in GO, HA production, and inflammation. DSF may be a potential drug candidate for preventing and treating tissue fibrosis in GO.

Inhibitory Effects and Mechanism of the Natural Compound Diaporthein B Extracted from Marine-Derived Fungi on Colon Cancer Cells.

This study aimed to investigate the inhibitory effects and mechanism of diaporthein B (DTB), a natural compound extracted from the fungus Penicillium sclerotiorum GZU-XW03-2, on human colon cancer cells. The inhibitory effect of DTB at different concentrations on the proliferation of colon cancer cells HCT116 and LOVO was detected at 24 and 48 h. The effect of cell migration and clone formation ability were detected by cell scratch and plate cloning experiments. Morphological changes were observed by Hoechst 33342 and Annexin-V/PI staining, and flow cytometry was used to detect the proportion of apoptotic cells. DTB significantly inhibited colon cancer cell proliferation, migration, and apoptosis in a dose-dependent manner without significant effects on normal colonic epithelial cells NCM460. The IC50 inhibition effect can be achieved after treatment with 3 μmol/L DTB for 24 h. Compared with the blank group, the migration and clonal-forming ability of colon cancer cells in the DTB group was significantly decreased (p < 0.01), while the apoptotic cells were significantly increased (p < 0.01) in a concentration-dependent manner. DTB can inhibit the proliferation and migration of human colon cancer cells HCT116 and LOVO and promote the apoptosis of human colon cancer cells.

Integrin α10 regulates adhesion, migration, and osteogenic differentiation of alveolar bone marrow mesenchymal stem cells in type 2 diabetic patients who underwent dental implant surgery

ABSTRACT Type 2 diabetes mellitus (T2DM) is a clinically important risk factor for dental implant treatment failure. An imbalance in the microenvironment of the jawbone of diabetic patients can impair the functions of bone marrow mesenchymal stem cells (BMSCs), thereby interfering with implant osseointegration during the healing phase. This study aims to investigate potential molecular factors associated with abnormal BMSC biological functions in diabetic patients with dental implant failure and identify intervention targets to improve implant osseointegration. The results of cell adhesion, cell scratch migration, alkaline phosphatase (ALP) activity, alizarin red staining, and real-time PCR assays showed that the adhesion, migration, and osteogenic differentiation abilities were significantly lower in alveolar BMSCs isolated from diabetic patients with implant failure (DM-F group) than in those isolated from diabetic patients with implant success (DM-S group) and normal patients (Nor group). Also, bioinformatics analysis and verification of whole-cell proteomics results revealed that integrin subunit alpha10 (ITGA10) expression in BMSCs was significantly lower in the DM-F group than in the DM-S group and much lower than that in the Nor group. In addition, lentiviral mediated short hairpin RNA (shRNA) or complementary DNA (cDNA) was used to knockdown or overexpress ITGA10 in BMSCs from diabetic patients, and the results revealed that ITGA10 knockdown significantly reduced the adhesion and migration abilities of BMSCs and inhibited their osteogenic differentiation potential by disturbing the FAK/PI3K/AKT/GSK3β/β-catenin pathway. ITGA10 overexpression produced the opposite results. In summary, this study revealed that low ITGA10 expression in BMSCs from the DM-F group is a major cause of cell dysfunction, including reduction in the adhesion, migration, and osteogenic differentiation abilities of BMSCs, and provided insight into the underlying mechanism. Additionally, ITGA10 was identified as a potential target protein for improving the biological functions of BMSCs and dental implant osseointegration in T2DM patients.

P-18 Impact of fluoroquinolones and aminoglycosides on P. aeruginosa virulence factor production and cytotoxicity.

Expression levels of pcrV (T3SS needle component) from ExoU-expressing PA103 and ExoS-expressing PA76026 after 16h incubation in each antimicrobial was detected using western blotting. qRT PCR detected mRNA levels of ExoU, ExoS, pcrV and ExsA (T3SS activating factor) after PA103 and PA76026 were exposed to tobramycin and moxifloxacin. LIVE/DEAD and LDH assays after 24h evaluated how the antimicrobials influenced acute cytotoxicity in a HCE-T cell scratch and infection model. Tobramycin significantly reduced pcrV in both strains by 50.5-74.0% compared to the fluoroquinolones (p=0.001 and 0.003), even at low concentrations. Fluoroquinolones significantly increased pcrV by 57.0-81.8% (p=0.004 and 0.003). mRNA levels of ExoU, ExoS, pcrV and ExsA were reduced by tobramycin but moxifloxacin increased pcrV, ExsA and ExoS. Tobramycin, despite more bacterial expansion compared to the same relative concentrations of fluoroquinolones, reduced ExoU/ExoS cytotoxicity and allowed complete wound healing. Tobramycin downregulates T3SS expression and reduces ExoS/ExoU mediated cytotoxicity which protects infected HCE-T cells even at low concentrations. Fluoroquinolones however upregulated T3SS and do not negate the cytotoxic effects.

Construction of A Model Used for Mechanisms of the Antitumoral Effects of Traditional Chinese Medicine Compounds Using A High-content Imaging System

Objective: To construct a high-content imaging system analysis model to study the effects of the traditional Chinese medicine compounds puerarin on proliferation and migration of cervical cancer (HeLa) cells. Methods: The digital phase-contrast mode unique to the high-content imaging system was used to study the proliferation and migration effects of puerarin on HeLa cells. Results: Puerarin inhibited HeLa cell proliferation and migration. The high-content analysis model constructed in this study enabled efficiently and accurately determining cell proliferation and migration parameters in batches and allowed detecting multiple phenotypic parameters in one experiment. These parameters provide a theoretical basis for subsequent research. Conclusion: Puerarin inhibited HeLa cell proliferation and migration. The high-content analysis model constructed in this study uses a digital phase-contrast mode rather than traditional fluorescence staining and cell scratch tests for label-free analysis, thus providing a more rapid, efficient and accurate analysis method for tumor research.

The therapeutic potential of chondroitin sulfate in Aspergillus fumigatus keratitis

PurposeTo explore the therapeutic effect of chondroitin sulfate (CS) on Aspergillus fumigatus (A. fumigatus) keratitis. MethodsThe nontoxic concentration of CS was determined using the Draize eye test and cell counting kit-8. Cell scratch test and cell proliferation test were evaluated the impact of CS on the proliferation and migration of human corneal epithelial cells (HCECs). Adherence assay and plate counting were used to detect fungal load in vivo and in vitro, respectively. Clinical score and hematoxylin-eosin (HE) staining were applied to assess the therapeutic effects of CS in an A. fumigatus keratitis mice model. The neutrophil infiltration and activity were detected by flow cytometry (FCM), immunofluorescence staining, and myeloperoxidase (MPO) assay. Toll-like receptor 4 (TLR-4) expression in RAW 264.7 cells and mouse cornea was detected by immunofluorescence staining. Real-time PCR (RT-PCR), western blot, and enzyme-linked immunosorbent assay (ELISA) were applied to examine the expression of inflammatory mediators. ResultsCS at 400 μg/mL (non-cytotoxic) significantly promoted proliferation and migration of HCECs. In an A. fumigatus keratitis mice model, CS treatment alleviated fungal keratitis (FK) severity by decreasing corneal fungal load and inhibiting neutrophil infiltration. In RAW 264.7 cells, the mRNA and protein levels of TLR-4, phosphorylated nuclear factor (NF)-κB (p-NF-κB), interleukin-1β (IL-1β), interleukin-6 (IL-6), tumor necrosis factor-a (TNF-α), cyclooxygenase 2 (COX-2), and macrophage inflammatory protein-2 (MIP-2) were remarkably lower in the siTLR-4 treated group, while higher in rTLR-4 treated group than in the corresponding control group. CS treatment suppressed rTLR-4 induced the mRNA and protein expression of TLR-4, p-NF-κB, IL-1β, IL-6, COX-2, TNF-α, and MIP-2 in RAW cells. ConclusionCS may ameliorate the prognosis of A. fumigatus keratitis by promoting corneal epithelial proliferation, inhibiting the recruitment and activity of neutrophils, and inhibiting the inflammatory response by down-regulation of the TLR-4/NF-κB signaling.

Ruyi Jinhuang Powder accelerated diabetic ulcer wound healing by regulating Wnt/β-catenin signaling pathway of fibroblasts In Vivo and In Vitro

Ethnopharmacological relevanceDiabetic ulcer is a common complication of diabetes. Therapies of diabetic ulcer are still challenging due to the complicated aetiology. Ruyi Jinhuang Powder (RJP) is gradually adopted to treat diabetic ulcer and has a significant therapeutic effect.Aim of the study: To investigate the therapeutic potential for diabetic ulcer in vivo and in vitro, we explored whether and how RJP influences wound healing in mice and fibroblasts at the tissular, cellular and molecular levels. Materials and methodsThe chemical composition of RJP was identified by HPLC. Streptozotocin (STZ) induced diabetic mice were used to confirm the curative effect of RJP in vivo. Besides, the impact of RJP in stimulating fibroblasts proliferation, migration and reducing inflammation was studied through CCK-8 assay, cell scratch assay, PCR, WB, etc. ResultsA total of 17 compounds were identified in RJP by HPLC. Our data indicated that RJP promoted fibroblasts proliferation and migration via activating Wnt/β-catenin signaling pathway. Consistently, RNA-seq analysis of mice skin samples also showed that the shared differentially expressed genes (DEGs) between RJP group and control group were most enriched in wnt signaling pathway. These DEGs were closely related with wound repair. In addition, the anti-inflammation effect of RJP was also confirmed through downregulation of IL-1α, IL-1β, IL-6 and IL-10 expression levels. These biological effects were reduced when the Wnt/β-catenin signaling was blocked. The in vivo study also demonstrated the effect of RJP in improving epidermal wound closure, which was consistent with the in vitro results. ConclusionsTopical application of RJP was effective in treating diabetic ulcer. This research is helpful to provide new insights and evidence into the role of RJP in accelerating unhealing wound and reducing wound inflammation.

CXCR7 Agonist TC14012 Improves Angiogenic Function of Endothelial Progenitor Cells via Activating Akt/eNOS Pathway and Promotes Ischemic Angiogenesis in Diabetic Limb Ischemia.

Endothelial progenitor cells (EPCs) play a critical role in repairing damaged vessels and triggering ischemic angiogenesis, but their number is reduced and function is impaired under diabetic conditions. Improving EPC function has been considered a promising strategy to ameliorate diabetic vascular complications. In the present study, we aim to investigate whether and how CXCR7 agonist TC14012 promotes the angiogenic function of diabetic EPCs. High glucose (HG) treatment was used to mimic the hyperglycemia in diabetes. Tube formation, cell scratch recovery and transwell assay, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, and cleaved-caspase3 expression were used to evaluate the angiogenic capability, cell migration, and apoptosis of EPCs, respectively. Hind limb ischemia (HLI) model was used to appraise the ability of TC14012 in promoting diabetic ischemic angiogenesis in vivo. HG treatment impaired EPC tube formation and migration, and induced EPC apoptosis and oxidative damage, while TC14012 rescued tube formation and migration, and prevented HG-induced apoptosis and oxidative damage of EPCs. Furthermore, these beneficial effects of TC14012 on EPCs were attenuated by specific siRNAs against CXCR7, validating that CXCR7 is a functional target of TC14012 in EPCs. Mechanistic studies demonstrated that HG treatment reduced CXCR7 expression in EPCs, and impaired Akt and endothelial nitric oxide synthase (eNOS) phosphorylation and nitric oxide (NO) production; similarly, these signal impairments in HG-exposed EPCs could be rescued by TC14012. However, the protective effects of TC14012 on tube formation and migration, Akt and eNOS phosphorylation, and NO production in HG-treated EPCs were almost completely abolished by siRNAs against CXCR7 or Akt specific inhibitor wortmannin. More importantly, in vivo study showed that TC14012 administration enhanced blood perfusion recovery and angiogenesis in the ischemic hind limb and increased the EPC number in peripheral circulation of db/db mice, demonstrating the capability of TC14012 in promoting EPC mobilization and ischemia angiogenic function. TC14012 can prevent EPCs from HG-induced dysfunction and apoptosis, improve eNOS activity and NO production via CXCR7/Akt signal pathway, and promote EPC mobilization and diabetic ischemia angiogenesis.

Influence of Femtosecond Laser Modification on Biomechanical and Biofunctional Behavior of Porous Titanium Substrates.

Bone resorption and inadequate osseointegration are considered the main problems of titanium implants. In this investigation, the texture and surface roughness of porous titanium samples obtained by the space holder technique were modified with a femtosecond Yb-doped fiber laser. Different percentages of porosity (30, 40, 50, and 60 vol.%) and particle range size (100–200 and 355–500 μm) were compared with fully-dense samples obtained by conventional powder metallurgy. After femtosecond laser treatment the formation of a rough surface with micro-columns and micro-holes occurred for all the studied substrates. The surface was covered by ripples over the micro-metric structures. This work evaluates both the influence of the macro-pores inherent to the spacer particles, as well as the micro-columns and the texture generated with the laser, on the wettability of the surface, the cell behavior (adhesion and proliferation of osteoblasts), micro-hardness (instrumented micro-indentation test, P–h curves) and scratch resistance. The titanium sample with 30 vol.% and a pore range size of 100–200 μm was the best candidate for the replacement of small damaged cortical bone tissues, based on its better biomechanical (stiffness and yield strength) and biofunctional balance (bone in-growth and in vitro osseointegration).

Xihuang pills induce apoptosis in hepatocellular carcinoma by suppressing phosphoinositide 3-kinase/protein kinase-B/mechanistic target of rapamycin pathway.

BACKGROUNDThe phosphoinositide 3-kinase/protein kinase-B/mechanistic target of rapamycin (PI3K/Akt/mTOR) signalling pathway is crucial for cell survival, differentiation, apoptosis and metabolism. Xihuang pills (XHP) are a traditional Chinese preparation with antitumour properties. They inhibit the growth of breast cancer, glioma, and other tumours by regulating the PI3K/Akt/mTOR signalling pathway. However, the effects and mechanisms of action of XHP in hepatocellular carcinoma (HCC) remain unclear. Regulation of the PI3K/Akt/mTOR signalling pathway effectively inhibits the progression of HCC. However, no study has focused on the XHP-associated PI3K/Akt/mTOR signalling pathway. Therefore, we hypothesized that XHP might play a role in inhibiting HCC through the PI3K/Akt/mTOR signalling pathway.AIMTo confirm the effect of XHP on HCC and the possible mechanisms involved.METHODSThe chemical constituents and active components of XHP were analysed using ultra-performance liquid chromatography-quadrupole time of flight mass spectrometry (UPLC-Q-TOF-MS). Cell-based experiments and in vivo xenograft tumour experiments were utilized to evaluate the effect of XHP on HCC tumorigenesis. First, SMMC-7721 cells were incubated with different concentrations of XHP (0, 0.3125, 0.625, 1.25, and 2.5 mg/mL) for 12 h, 24 h and 48 h. Cell viability was assessed using the CCK-8 assay, followed by an assessment of cell migration using a wound healing assay. Second, the effect of XHP on the apoptosis of SMMC-7721 cells was evaluated. SMMC-7721 cells were stained with fluorescein isothiocyanate and annexin V/propidium iodide. The number of apoptotic cells and cell cycle distribution were measured using flow cytometry. The cleaved protein and mRNA expression levels of caspase-3 and caspase-9 were detected using Western blotting and quantitative reverse-transcription polymerase chain reaction (RT-qPCR), respectively. Third, Western blotting and RT–qPCR were performed to confirm the effects of XHP on the protein and mRNA expression of components of the PI3K/Akt/mTOR signalling pathway. Finally, the effects of XHP on the tumorigenesis of subcutaneous hepatocellular tumours in nude mice were assessed.RESULTSThe following 12 compounds were identified in XHP using high-resolution mass spectrometry: Valine, 4-gingerol, myrrhone, ricinoleic acid, glycocholic acid, curzerenone, 11-keto-β-boswellic acid, oleic acid, germacrone, 3-acetyl-9,11-dehydro-β-boswellic acid, 5β-androstane-3,17-dione, and 3-acetyl-11-keto-β-boswellic acid. The cell viability assay results showed that treatment with 0.625 mg/mL XHP extract decreased HCC cell viability after 12 h, and the effects were dose- and time-dependent. The results of the cell scratch assay showed that the migration of HCC cells was significantly inhibited in a time-dependent manner by the administration of XHP extract (0.625 mg/mL). Moreover, XHP significantly inhibited cell migration and resulted in cell cycle arrest and apoptosis. Furthermore, XHP downregulated the PI3K/Akt/mTOR signalling pathway, which activated apoptosis executioner proteins (e.g., caspase-9 and caspase-3). The inhibitory effects of XHP on HCC cell growth were determined in vivo by analysing the tumour xenograft volumes and weights.CONCLUSIONXHP inhibited HCC cell growth and migration by stimulating apoptosis via the downregulation of the PI3K/Akt/mTOR signalling pathway, followed by the activation of caspase-9 and caspase-3. Our findings clarified that the antitumour effects of XHP on HCC cells are mediated by the PI3K/Akt/mTOR signalling pathway, revealing that XHP may be a potential complementary therapy for HCC.

Corrigendum

CNS Neuroscience & TherapeuticsEarly View CORRIGENDUMOpen Access This article corrects the following: RGS16 promotes glioma progression and serves as a prognostic factor Ruoyu Huang, Guanzhang Li, Zheng Zhao, Fan Zeng, Kenan Zhang, Yanwei Liu, Kuanyu Wang, Huimin Hu, Volume 26Issue 8CNS Neuroscience & Therapeutics pages: 791-803 First Published online: April 22, 2020 First published: 12 April 2022 https://doi.org/10.1111/cns.13844AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat In Huang et al1 the authors noticed that the same figure was accidently presented in Figure 1C and 1D. In addition, the authors also found the Figure 7 caption to be incorrect. FIGURE 1Open in figure viewerPowerPoint Expression pattern of RGS16 in different grades of gliomas. (A,B) In CGGA microarray and TCGA sequencing database, the mRNA expression level of RGS16 increased with tumor grade. (C,D) In CGGA microarray and TCGA sequencing database, the mRNA expression level of RGS16 was higher in IDH1 wildtype gliomas than gliomas with mutated IDH1 in each grade, though some groups have no statistically significant. K–S test of normality was used to assess the distribution of RGS16 expression in CGGA microarray and TCGA sequencing database (p = 0.479 and p = 0.085, respectively). * p < 0.05, **p < 0.01, **** p < 0.0001, ns: no statistically significant The correct Figure 1 is given below: The correct Figure 7 legend is given below: Figure 7. RGS16 played an oncogene role in glioma cell lines. (A) The expression of RGS16 protein in the human astrocytes (HA) cell line and glioma cell lines. (B) The real-time quantitative PCR (qPCR) assay of RGS16 mRNA expression in LN229, U87 and U251 cell lines after infection with RGS16 siRNA or negative control. (C) The western blot analysis of RGS16, TCF8, N-cadherin and β-catenin protein expression in LN229, U87 and U251 cell lines after applying RGS16 siRNA or negative control. (D, E) Transwell assay of LN229 and U251 cell lines treated with RGS16 siRNA or negative control. (F, G) Cell scratch assay of LN229 and U87 cell lines treated with RGS16 siRNA or negative control. (H, I) Clonogenic assay of LN229 and U251 cell lines treated with RGS16 siRNA or negative control. It earlier read as, RGS16 played an oncogene role in glioma cell lines. (A) The expression of RGS16 protein in the human astrocytes (HA) cell line and glioma cell lines. (B) The real-time quantitative PCR (qPCR) assay of RGS16 mRNA expression in LN229, U87, and U251 cell lines after infection with RGS16 siRNA or negative control. (C) The Western blot analysis of RGS16, TCF8, N-cadherin, and β-catenin protein expression in LN229, U87, and U251 cell lines after applying RGS16 siRNA or negative control. (D, E) Transwell assay of LN229 and U251 cell lines treated with RGS16 siRNA or negative control. (D) Cell scratch assay of LN229 and U87 cell lines treated with RGS16 siRNA or negative control. (E) Clonogenic assay of LN229 and U251 cell lines treated with RGS16 siRNA or negative control. The authors apologize for these errors. REFERENCE 1Huang R, Li G, Zhao Z, et al. RGS16 promotes glioma progression and serves as a prognostic factor. CNS Neurosci Ther. 2020; 26: 791- 803. Wiley Online LibraryCASPubMedWeb of Science®Google Scholar Early ViewOnline Version of Record before inclusion in an issue FiguresReferencesRelatedInformation