Upregulation of miR-216a-5p by Lentinan Targeted Inhibition of JAK2/STAT3 Signaling Pathway to Reduce Lung Adenocarcinoma Cell Stemness, Promote Apoptosis, and Slow Down the Lung Adenocarcinoma Mechanisms

To investigate the effect of Lentinan (LNT) on lung adenocarcinoma (LUAD) cell stemness and its mechanism. In this study, we founded that LNT significantly reduce the cell proliferation, activity, migration, invasion, and stemness of LUAD cells, and promote their apoptosis compared with the control group in vitro. Moreover, LNT significantly inhibited the volume and weight of tumors of nude mice in vivo. At the same time, LNT can significantly up-regulate miR-216a-5p levels and reduce the protein expression of phospho-JAK2 (Y1007/1008) and phospho-STAT3 (Tyr705), thereby inhibiting the JAK2/STAT3 signaling pathway. Interfering with miR-216a-5p expression and activating the JAK2/STAT3 signaling pathway can significantly reverse LNT inhibitory effects on LUAD. Collectively, LNT can inhibit the JAK2/STAT3 signaling pathway by up-regulating miR-216a-5p, reducing stemness, and promoting LUAD cells apoptosis, then slow down LUAD occurrence and development, providing concepts and experimental foundation treating patients with LUAD.

Immunomodulation Effect of Kaempferia galanga Ethanolic Extract On In Vitro Lymphocyte Cell Proliferation

<p><em>Kaempferia galanga</em> L. is a traditional medicine with antitumor properties, as indicated by its immunomodulatory activities. This study aimed to determine the effect of <em>K. galanga</em> on lymphocyte cell proliferation activity as an indicator of immunomodulatory properties. This study was conducted at the Indonesian Research Center for Veterinary Science (February to April 2018). The immunomodulatory activity of the extract was evaluated with an <em>in vitro</em> splenocyte proliferation assay. The assay was based on cellular enzymatic synthesis to transform the XTT from formazan tetrazolium as an indicator. The <em>K. galanga</em> extract was obtained by 96% ethanol extraction. The test was conducted in an aseptic condition, consisted of five treatment groups with three replications each. Three groups of splenocyte cell culture, each with extract concentration of 2.5 µg.ml^-1, 25 µg.ml^-1, and 250 µg.ml^-1, as well as a positive (Concanavalin A/Con A) and negative (cell only) control. The cell suspension (10x10⁴cells/ml) was distributed on 96-well plates and cultured following the treatment groups. The same five plates were made for five days of observation and retrieved daily by observing an Elisa reader at 450 nm. The extract of <em>K. galanga </em>at 2.5 µg.ml^-1, 25 µg.ml^-1, and 250 µg.ml^-1 significantly (P &lt;0.05) promoted splenocyte proliferation compared to control. Therefore, it was expected that <em>K. galanga </em>has a high potential to be used as immunomodulators. Hence, further investigations should be done to clarify the mechanisms of the immunomodulatory effect of <em>K. galanga</em> as an antitumor <em>in vivo</em>.</p>

KMT2D deficiency disturbs the proliferation and cell cycle activity of dental epithelial cell line (LS8) partially via Wnt signaling

KMT2D, as one of the key histone methyltransferases responsible for histone 3 lysine 4 methylation (H3K4me), has been proved to be the main pathogenic gene of Kabuki syndrome disease. Kabuki patients with KMT2D mutation frequently present various dental abnormalities, including abnormal tooth number and crown morphology. However, the exact function of KMT2D in tooth development remains unclear. In this report, we systematically elucidate the expression pattern of KMT2D in early tooth development and outline the molecular mechanism of KMT2D in dental epithelial cell line. KMT2D and H3K4me mainly expressed in enamel organ and Kmt2d knockdown led to the reduction of cell proliferation activity and cell cycling activity in dental epithelial cell line (LS8). RNA-seq and KEGG enrichment analysis screened out several important pathways that affected by Kmt2d knockdown including Wnt signaling. Consistently, Top/Fop assay confirmed the reduction of Wnt signaling activity in Kmt2d knockdown cells. Nuclear translocation of β-catenin was significantly reduced by Kmt2d knockdown, while lithium chloride (LiCl) partially reversed this phenomenon. Moreover, LiCl partially reversed the decrease of cell proliferation activity and G1 arrest, and the downregulation of Wnt-related genes in Kmt2d knockdown cells. In summary, this study uncovered a pivotal role of histone methyltransferase KMT2D in dental epithelium proliferation and cell cycle homeostasis partially through regulating Wnt/β-catenin signaling. The findings are important for understanding the role of KMT2D and histone methylation in tooth development.

Effect of key enzyme inhibition of glycolysis on synovial fibroblasts in Rheumatoid Arthritis

Objective: To observe effects of glycolysis on human rheumatoid arthritis Fibroblast-like synoviocytes (HFLS-RA) by inhibiting glycolysis. Methods: Hexokinase inhibitor (3-bromopyruvate, 3-BrPa), 6-phosphofructokinase 1 inhibitor citric acid and pyruvate kinase inhibitor shikonin were applied to HFLS-RA respectively. Cell count 8 Kit detects cell proliferation activity, the activity of hexokinase, 6-phosphofructokinase 1, and pyruvate kinase, as well as the cellular glucose, lactate and ATP content were detected by kits, and the ELISA kit detects the expression of cellular inflammatory factors TNF-α and TGF-β. Results: 10 μg/mL 3-BrPa, 160 μg/mL citric acid and 5 μg/mL shikonin significantly inhibited cell proliferation activity (P<0.001); and significantly inhibited HFLS-RA hexokinase and fructose 6-phosphate Kinase 1 and pyruvate kinase activity; Glucose, lactate and ATP content decreased; TNF-α expression decreased, while TGF-β expression increased. Conclusion: This study explored the changes in glucose metabolism and the expression of inflammatory factors in HFLS-RA by inhibiting the key enzymes of glycolysis, further confirming the important role of glycolysis in HFLS-RA, and laying a theoretical basis for a deep understanding of the pathogenesis of RA.

Compatibility of Composite Biomaterials in Sports Injury Repair

With the rapid development of sports, sports injuries have become an inevitable problem in sports. Repairing sports injuries has always been the focus of attention in the field of sports and medicine. The wide application of biomedical materials leads to the innovation and development of medical technology. It is of great significance to use them to repair sports injuries, but the current research results are not ideal. In this paper, the biocompatibility of composite biomaterials used to repair sports injuries was studied. First, the rat bone marrow mesenchymal stem cells were cultured to prepare a cell suspension. Then, the tricalcium phosphate-gelatin composite scaffold material and the collagen material were scraped into powder to make the material extract, which are, respectively, the experimental group and the control group. An apoptosis detection kit and a flow cytometer were used to detect apoptosis rate and survival rate of all samples, and a CKK-8 kit was used to detect cytotoxicity and calculate cell proliferation activity. The absorbance value was read with a microplate reader, and the coagulation performance and hemolysis rate of different groups were calculated. The experimental results showed that the cell survival rate of the experimental group was 91.41%, the early apoptosis rate was 2.81%, the stem cell proliferation activity was 0.83, and the anticoagulant performance and hemolysis rate were 64.8% and 0.28%, respectively. This indicates that although the anticoagulant performance of the tricalcium phosphate-gelatin composite scaffold is weak, its cell proliferation activity is strong, which is more suitable for cell adhesion and proliferation, and the cell apoptosis and hemolysis rate are lower. Therefore, it has better biocompatibility than a single collagen material.

miR-34c inhibits PDGF-BB-induced HAVSMCs phenotypic transformation and proliferation via PDGFR-β/SIRT1 pathway

The purpose of this study was to explore the effect of miR-34c on PDGF-BB-induced HAVSMCs phenotypic transformation and proliferation via PDGFR-β/SIRT1 pathway, so as to find a new method for early diagnosis and treatment of cardiovascular disease. HA-VSMCs were treated with platelet-derived growth factor-BB (PDGF-BB) at 0 h, 12 h, 24 h, 48 h or 36 h to explore the optimal time for phenotypic transformation of VSMCs. And then, PDGF-BB-induced HA-VSMCs were transfected with miR-34c mimics/mimics NC and pcDNA3.1-PDGFR-β/pcDNA3.1-NC to observe cell biological behaviour. CCK8 was used to detect cell proliferation activity. Transwell chamber assay was used to detect cell invasion. Early apoptosis was analyzed by flow cytometry. The expression of α-SMA and Smemb was detected by immunofluorescence staining. The expressions of PDGFR-β, IRF9, Acetyl-NF-κB/p65, Acetyl-p53 and CyclinD1 were analyzed by Western blot analysis. The expression of miR-34a, miR-34b and miR-34c was detected by RT-PCR, and the targeting relationship between miR-34c and PDGFR-β was detected by luciferase reporting assay. The results indicated the proliferation and migration of PDGF-BB-induced HA-VSMCs significantly increased, and apoptosis significantly decreased. Besides, α-SMA decreased significantly, while Smemb increased significantly. Furthermore, expressions of PDGFR-β, IRF9, Acetyl-NF-κB/p65, Acetyl-p53 and CyclinD1 increased significantly, and SIRT1 decreased significantly. Experimental results showed that, miR-34c mimics significantly inhibited cell proliferation and migration, and promoted cell apoptosis, and miR-34c inhibitor had the opposite effects. MiR-34c mimics significantly increased α-SMA expression and decreased Smemb expression, while the opposite effects were reflected after transfection with miR-34c inhibitor. Moreover, miR-34c mimics significantly decreased the expressions of PDGFR-β, IRF9, Acetyl-NF-κB/p65, Acetyl-p53 and CyclinD1, and significantly increased the expression of SIRT1, while miR-34c inhibitor had the opposite effects. Luciferase assay confirmed that PDGFR-β was a potential target of miR-34c. Subsequently, PDGF-BB-induced HA-VSMCs were co-transfected with miR-34c mimics and pcDNA3.1-PDGFR-β. The results indicated that PDGFR-β reversed the biological function of miR-34c mimic. The results revealed the potential application value of miR-34c as a marker molecule of phenotypic transformation, providing a potential target for improving phenotypic transformation.

Aqueous Polyphenol Semi-Purified Fractions from Galls of Quercus infectoria Increase ALP Activity and Mineralisation of hFOB 1.19 Cells

The galls of Quercus infectoria (QI) have been reported to possess numerous medicinal values and give a positive effect on bone metabolism. This study investigated the effects of semi-purified fractions of QI gall extract on the proliferation, alkaline phosphatase (ALP) activity, and mineralisation of human foetal osteoblast cell line (hFOB1.19). The semi-purified fractions (fraction A and B) were prepared by column chromatography methods. MTT assay was used to measure cell proliferation activity to obtain half maximal concentration (EC50) of the cells treated with fraction A (phenolic components and contained amide), fraction B (phenolic components with the presence of alkene), and pamidronate (drug control). The most potent or lowest EC50 was further used to measure ALP activity in the treated and untreated cells at day 1, 3, 7, 10, and 14 by ELISA. Cell mineralisation was determined by von Kossa staining for phosphate depositions and Alizarin Red S staining for calcium depositions. The EC50 values for fraction A and B were 8.86 and 9.92 μg/mL, respectively, which showed a greater effect compared to the pamidronate (15.27 μg/mL). The ALPactivity of both fractions in the treated cells were also greater compared to the two control groups (cells treated with pamidronate and untreated cells), starting from day 3 onwards. The calcium depositions appeared as red spots, while phosphate depositions appeared as black spots. Interestingly, the calcium depositions of cells treated with both fractions were higher than those of the two control groups. In conclusion, semi-purified fractions of QI gall extract enhanced proliferation, improved mineralisation, and increased ALP activity of hFOB 1.19 cells.

The antimalaria drug artemisinin displays strong cytotoxic effect on leukaemia lymphocytes in combination with vitamin C and pro-vitamin K3

This study investigated the anticancer effect of the anti-parasitic drug artemisinin in combination with two redox modulators: vitamin C and pro-vitamin K3 (C/K3) The experiments were conducted on leukaemia cells Jurkat. Cells were treated with either artemisinin or C/K3 alone and with all three compounds. Cell proliferation and viability were analysed using trypan blue stating and automated cell counting. The results showed that artemisinin (>10 mM) suppressed cell proliferation activity, but did not induce cell death up to 500 mM. The drug demonstrated a clear cytostatic effect at concentrations 250- 500 mM – Jurkat cells did not proliferate, but were alive. The combination C/K3 (200:2, 300:3 mM/mM) applied alone did not affect cell proliferation and viability. Vitamins C/K3 in concentration ratio 500:5 (μM/mM) decreased cell proliferation activity by ~10%. The triple combination artemisinin/C/K3 manifested synergistic anti-proliferative effects at all concentration ratios analysed. This synergistic effect increased with increasing C/K3 concentration. Based on literature data, it was assumed that the anti-proliferative effect of the triple combination was mediated by changes in the redox-homeostasis of cancer cells. The C/K3 redox system likely acted on cancer mitochondria and increased superoxide production and activation of pro-apoptotic signals, specific for cancer cells. On the other hand, artemisinin could generate hydroxyl radicals as a result of activation of Fenton reactions, depleting intracellular reducing equivalents. Both redox mechanisms lead to activation of signal pathways for induction of cancer cell death.