Search

Objective To explore the regulative effect of autophagy-related protein ATG5 on the expression of type Ⅱ collagen in human chondrocytes through affecting autophagy function. Methods Transcriptome data of 13 human cartilage tissue samples were downloaded from GEO database to analyze the correlation between ATG5 and COL2A1 expression.Western blotting and immunofluorescence assay were used to detect the expression of type Ⅱ collagen (Collagen Ⅱ, COL2) in human chondrocytes C28/I2 after rapamycin (Rapa) and bafilomycin A1(BafA1) treatment.Human COL2A1 promoter plasmid was constructed, and dual-luciferase reporter assay was applied to detect the effect of overexpression of ATG5 on COL2A1 promoter activity.mRFP-GFP-LC3 autophagy dual fluorescent virus was used to dynamically monitor the changes in autophagic flux of C28/I2 cells with overexpression and knockdown of ATG5.Western blotting was used to detect the effects of Rapa and BafA1 on COL2, LC3Ⅱ/LC3Ⅰ expression levels after overexpression or knockdown of ATG5. Results The results of correlation analysis showed a positive correlation between the mRNA expression of ATG5 and COL2A1 in human chondrocytes samples.Rapa and BafA1 significantly promoted and inhibited the expression of COL2 in C28/I2 cells (P < 0.05), respectively.The human COL2A1 promoter plasmid was successfully constructed, and the transcriptional activity of the COL2A1 promoter was up-regulated by the overexpression of ATG5(P < 0.01).Treatment with autophagy dual-fluorescence virus resulted in LC3-GFP fluorescence quenching after overexpression of ATG5(P < 0.01), suggesting autophagy activation; and a greater fluorescence intensity of LC3-GFP was observed after knockdown of ATG5(P < 0.05), demonstrating autophagy inhibition.Rapa obviously promoted the expression of COL2 and LC3Ⅱ/LCⅠ, which were up-regulated by ATG5(P < 0.05), but BafA1 showed the opposite effect of Rapa (P < 0.05). Conclusion Overexpression of ATG5 up-regulates the expression of type Ⅱ collagen, and the regulation of ATG5 on the expression of type Ⅱ collagen depends on autophagy in chondrocytes.

Objective To investigate the effect of the regulator of ribosome synthesis 1 (RRS1) gene on the proliferation and migration of breast cancer BT549 cells and its mechanism of action. Methods Western blot was used to measure the protein expression of RRS1 in human breast cancer cell lines (MDA-MB-231, MDA-MB-468, BT549, and MCF-7 cells) and normal human breast epithelial MCF-10A cells, and immunofluorescence assay was used to observe the localization of RRS1 in BT549 cells. The lentivirus infection method was used to establish an RRS1-knockdown BT549 cell line, and quantitative real-time PCR and Western blot were used to measure the relative mRNA and protein expression levels of RRS1 in the Con group transfected with negative control lentivirus and the sh-RRS1 group transfected with shRNA-RRS1 lentivirus. CCK8 assay and colony-forming assay were used to observe the effect of RRS1 on the proliferation ability of BT549 cells, and wound healing assay and Transwell assay were used to observe the effect of RRS1 on the migration ability of BT549 cells. Western blot was used to measure the relative expression levels of AKT-mTOR signaling pathway proteins and their downstream proteins hypoxia-inducible factor 1α (HIF-1α) and vascular endothelial growth factor (VEGF) in the two groups. Results The relative expression level of RRS1 in breast cancer cell lines MDA-MB-231, MDA-MB-468, BT549, and MCF-7 was significantly higher than that in normal human breast epithelial MCF-10A cells (F=48.92,P

Objective To investigate the effect of FXX-W-12-4, a novel heat shock protein 90 (Hsp90)/histone deacetylase (HDAC) dual-target inhibitor, on the proliferation, invasion, and migration of breast cancer MDA-MB-231 cells. Methods CCK-8 assay was used to observe the effect of FXX-W-12-4 on the viability of MDA-MB-231 cells, and its half-maximal inhibitory concentration (IC50) was calculated as the reference value for concentration in follow-up experiment. On the basis of the IC50 value, FXX-W-12-4 with a final concentration of 0, 100, 200, and 300 nmol/L was added to MDA-MB-231 cells and was established as groups A, B, C, and D, respectively. Cell scratch assay and Transwell assay were used to observe the effect of FXX-W-12-4 on the invasion and migration of MDA-MB-231 cells in each group. Western blot was used to measure the relative expression levels of proteins such as ac-H3 and Hsp70 in MDA-MB-231 cells. Results CCK-8 assay showed that the viability of MDA-MB-231 cells gradually decreased with the increase in FXX-W-12-4 concentration (F=2 663.00,P

Objective To investigate the mechanism of kaempferol in the treatment of rheumatoid arthritis (RA) based on network pharmacology and in vitro experiments. Methods Kaempferol targets were queried by TCMSP and SwissTarget databases, and RA related functional targets were searched in the GeneCards and DisGeNET databases. After the cross between kaempferol and RA targets through Venny2.1.0, STRING database was used for protein-protein interaction (PPI) analysis, and molecular docking between kaempferol and the targets was conducted. Mouse mononuclear leukemia RAW264.7 cells were treated by lipopolysaccharide (LPS) and kaempferol, and divided into control group, LPS group, LPS+Kaempferol group. MTT assay was used to determine the cytotoxicity of RAW264.7 cells after kaempferol treatment. LPS-induced RAW264.7 cells were used to construct a cellular inflammatory model. The change in aging and aging-related inflammatory factors were detected in each treatment group. The content of β-galactosidase was detected by SA-β-Gal, and the expression of IL-6 was detected by cellular immunofluorescence assay. Results A total of 138 targets of kaempferol and 76 potential targets of kaempferol against RA were obtained. PPI analysis showed that TNF, EGFR, PTGS2 and AKT1 may be the core targets of kaempferol against RA. The results of molecular docking indicated that kaempferol could form stable complexes with the core targets of TNF, EGFR, PTGS2 and AKT1. The results of MTT assay displayed that the treatment of 0.125 to 16 μmol/L kaempferol for 24 h promoted the proliferation of RAW264.7 cells (P < 0.05), the doses of 0.125 to 8 μmol/L for 48 h exerted similar effect on the proliferation (P < 0.05), but the dose of 0.125 to 16 μmol/L for 72 h had no such significant effect. The results of qPCR and Western blotting showed that kaempferol decreased the expression of cell senescence markers p16 and p21 and promoted the expression of cell cycle markers CCND1 and CCNE1 (P < 0.05). Cellular immunofluorescence assay indicated that kaempferol reduced the expression of IL-6 (P < 0.05). SA-β-Gal staining displayed that LPS treatment promoted β-galactosidase level in RAW264.7 cells (P < 0.05), and this effect was reversed by kaempferol treatment (P < 0.05). Conclusion Kaempferol may regulate the expression of IL-1β, IL-6 and TNF-α and p16/p21 senescence related genes, prevent premature aging of immune cells, and thus play a therapeutic role for RA. [

The aim of this study was to investigate the rheological properties, particle size distribution, color change, and stability of lily juice under different ultrasonic treatment conditions (152 W, 304 W, 456 W, 608 W, and 760 W). The results showed that the lily juice exhibited non-Newtonian shear thinning behavior, and the viscosity decreased with the increase in ultrasonic power. Under ultrasonic treatment conditions, there was no significant change in the pH value and zeta potential value of the samples. The content of cloudy value and total soluble solids (TSS) increased gradually. However, both the sedimentation components and centrifugal sedimentation rate showed a downward trend and an asymptotic behavior. In addition, high-power ultrasound changed the color index (L* value decreased, a* value increased), tissue structure, and particle distribution of the sample, and small particles increased significantly. To sum up, ultrasonic treatment has great potential in improving the physical properties and suspension stability of lily juice.

Objective To investigate the effect of the long non-coding RNA (lncRNA) small nucleolar RNA host gene 1 (SNHG1) on the migration, invasion, and epithelial-mesenchymal transition (EMT) of breast cancer cells and its mechanism. Methods Breast cancer tissue samples and adjacent normal tissue samples were collected from 20 patients with breast cancer, and normal human breast epithelial cell MCF-10A and breast cancer cell lines MCF-7, BT-549, MDA-MB-231, and MDA-MB-468 were cultured. RT-qPCR was used to measure the expression levels of SNHG1 and miR-641 in tissue samples and cells. BT-549 cells were divided into groups A to D and were transfected with si-NC (group A), si-SNHG1 (group B), si-SNHG1+anti-miR-641-NC (group C), and si-SNHG1+anti-miR-641 (group D), respectively. RT-qPCR was used to measure the expression levels of SNHG1 and miR-641 in cells; Transwell assay was used to observe cell invasion and migration abilities; Western blot was used to measure the expression levels of the EMT-related proteins E-cadherin, N-cadherin, and Vimentin. BT-549 cells were divided into groups E to H and were co-transfected with SNHG1-WT and miR-641 mimics (group E), SNHG1-WT and miR-NC (group F), SNHG1-MUT and miR-641 mimics (group G), and SNHG1-MUT and miR-NC (group H), respectively, and dual-luciferase reporter assay was used to determine the interaction between SNHG1 and miR-641. Results SNHG1 was highly expressed in breast cancer tissue (t=4.81,P0.05). Conclusion cSNHG1 is highly expressed in breast cancer tissue and cells, and downregulation of its expression can inhibit the migration, invasion, and EMT of breast cancer cells by targeting miR-641.