Objective To observe the effect of pilose antler polypeptides(PAP)on the apoptosis of rabbit marrow mesenchymal stem cells (MSCs) differentiated into chondrogenic phenotype by interleukin 1β (IL-1β) so as to optimize the seeding cells in cartilage tissue engineering. Methods The MSCs were separated from the nucleated cells fraction of autologus bone marrow by density gradient centrifuge and cultured in vitro. The MSCs were induced into chondrogenic phenotype by transforming growth factor β1(TGF-β1) and basic fibroblast growth factor(bFGF). According to different medias, the MSCs were randomly divided into four groups: group A as black control group, group B(100 ng IL-1β)，group C(10 μg/ml PAP+100 ng IL-1β) and group D(100 ng/ml TGF-β1 +100 ng IL-1β). The samples were harvested and observed by morphology, flow cytometry analysis, RT-PCR and ELISA at 24, 48 and 72 hours. Results The intranuclear chromatin agglutinated into lump and located under nulear membranes which changed into irregular shapeat 24 hours. The intranuclear chromatin agglutinated intensifily at 48 hours. Then the nucear fragments agglutinated into apoptosic corpuscles at 72 hours in group B. The structure change of cells in groups C and D was later than that in group B, and the number of cells changed shape was fewer than that in group B. The structure change of cells in group A was not significant. The apoptosic rate of cells, the mRNA expression of Caspase-3 and the enzymatic activity of Caspase-3 gradually increased in group B, and there were significant differences compared with groups A,C and D(Plt;0.01). Conclusion Caspase-3 is involved in aoptosis of the MSCs differentiated into chondrogenic phenotype cultured in vitro. PAP could prevent from or reverse apoptosis of these MSCs by decreasing the expression of Caspase-3 and inhibiting the activity of Caspase-3.
OBJECTIVE To investigate the possibility of repairing the cartilage cartilage defect with homogeneous chondrocytes combined with Pluronic. METHODS: Homogeneous cartilage chondrocytes of adult New Zealand rabbits were harvested and cultured in vitro, which were marked by 3H-TdR and mixed with Pluronic. The medial or lateral condyle defects were made (phi 4 mm, extending down to the calcified zone) in 20 rabbits. In the experimental group, the right defects were repaired by homogeneous chondrocytes combined with Pluronic; in the control group, the left defects were repaired by Pluronic only or were left un-repaired. The animals were sacrificed in the 4th, 8th and 16th weeks after operation respectively. The repair results were observed and the cell source of repair tissue was distinguished. RESULTS: In the experimental group, the cartilage defects were repaired by the cartilage-like tissue after 8 weeks of operation; the defects were completely filled with mature cartilage tissue, which integrated smoothly with articular cartilage 16 weeks later. In the control group, only a small amount fibrous tissues were seen on the surface of defects. Autoradiographic assessment showed that the repair cells came from the implants, but not from self-chondrocytes. CONCLUSION: It is a good way to repair articular cartilage defects with homograft of tissue engineering cartilage. It is a convenient method to mark with 3H-TdR to discriminate the resource of the repair cells.
Objective To investigate the inhibitory effects and related mechanisms of NOD like receptor protein 3 (NLRP-3) inflammasome inhibitor MCC950 on oxidative stress, inflammation, and pyroptosis in human esophageal epithelial cells (HEECs). MethodsHEECs cells were passaged and divided into blank control group, acid stimulation group (stimulated 3 times a day with pH 4 acidic medium for 15 minutes each time, cultured for 48 hours), bile salt stimulation group (stimulated 3 times a day with 400 μmol/L bile salt mixture for 15 minutes each time, cultured for 48 hours), lipopolysaccharide (LPS) group (stimulated with 10 μL of 100 ng/mL LPS for 48 hours), MCC950 group (stimulated HEECs cells with 10 μL of 7.5 ng/mL MCC950 for 4 hours, then stimulated with acid, bile hydrochloric acid, and LPS for 48 hours), and N-acetyl-L-cysteine (NAC) group (stimulated HEECs cells with 1 mmol/L NAC for 4 hours, then stimulated with acid, bile hydrochloric acid, and LPS and incubated for 48 hours). Three culture dishes were used in each group to detect the mRNA and protein expression levels of oxidative protein/antioxidant protein [Nox-4 (NADPH oxidase 4), nuclearfactor erythroidderived 2-like 2 (Nrf-2), heme oxygenase-1 (HO-1)], NLRP-3 signaling pathway [NLRP3/caspase-1/intereukin-1β (IL-1β)/intereukin-18 (IL-18)], and cell apoptosis pathway [caspase-4/caspase-5/GSDMD] using real-time reverse transcription polymerase chain reaction (RT-PCR) and Western blotting experiments. Cell apoptosis were observed through Hoechst 33342 staining. ResultsMCC950 intervention (average optical density: 0.023) and NAC intervention (average optical density: 0.031) effectively inhibited HEECs apoptosis induced by acid (average optical density: 0.042), bile salt (average optical density: 0.047), and LPS (average optical density: 0.054). The results of RT-PCR and Western blotting experiments showed that MCC950 intervention and NAC intervention significantly inhibited the high expression of Nox-4 mRNA (MCC950:1.68±0.18, NAC: 1.62±0.17) and protein in HEECs cells induced by acid (1.00±0.05), bile salt (3.07±0.25), and LPS (3.52±0.37); And significantly upregulated the mRNA and protein expression levels of antioxidant proteins Nrf-2 (MCC950: 0.72±0.12, NAC: 0.57±0.12) and HO-1 (MCC950: 0.74±0.12, NAC: 0.57±0.12). MCC950 intervention and antioxidant NAC intervention effectively inhibited the mRNA and protein expression levels of acid stimulated, bile salt stimulated, and LPS induced HEECs cell NLRP-3 (MCC950 intervention: 1.58±0.06, NAC intervention: 1.47±0.09), ASC (MCC950 intervention: 1.56±0.09, NAC intervention: 1.93±0.17), caspase-1 (MCC950 intervention: 1.64± 0.13, NAC intervention: 1.96±0.20), IL-1β (MCC950 intervention: 1.66±0.18, NAC intervention: 1.82±0.20), IL-18 (MCC950 intervention: 1.58±0.13, NAC intervention: 1.84±0.16) and other indicators. MCC950 intervention and antioxidant NAC intervention effectively inhibited the mRNA and protein expression levels of apoptosis pathway markers such as caspase-4 (MCC950 intervention:1.51±0.03, NAC intervention: 1.61±0.12), caspase-5(MCC950 intervention: 1.38±0.13, NAC intervention: 1.64±0.21), and GSDMD (MCC950 intervention: 1.41±0.04, NAC intervention: 1.54±0.10) induced by acid stimulation, bile salt stimulation, and LPS in HEECs cells. ConclusionAcid, bile salts, and LPS can all induce the overexpression of oxidative stress markers in HEECs, reduce the expression of antioxidant proteins, and activate the NLRP3 inflammasome signaling pathway and cell pyroptosis pathway, promoting cellular inflammatory damage, but MCC950 has a protective effect.