Insufficiency of collagenases in establishment of primary chondrocyte culture from cartilage of elderly patients receiving total joint replacement.

Background Collagenases are frequently used in chondrocyte isolation from articular cartilage. However, the sufficiency of this enzyme in establishing primary human chondrocyte culture remains unknown. Methods Cartilage slices shaved from femoral head or tibial plateau of patients receiving total joint replacement surgery (16 hips, 8 knees) were subjected to 0.02% collagenase IA digestion for 16 h with (N = 19) or without (N = 5) the pre-treatment of 0.4% pronase E for 1.5 h. Chondrocyte yield and viability were compared between two groups. Chondrocyte phenotype was determined by the expression ratio of collagen type II to I. The morphology of cultured chondrocytes was monitored with a light microscope.Results Cartilage with pronase E pre-treatment yielded significantly higher chondrocytes than that without the pre-treatment (3,399 ± 1,637 cells/mg wet cartilage vs. 1,895 ± 688 cells/mg wet cartilage; P = 0.0067). Cell viability in the former group was also significantly higher than that in the latter (94% ± 2% vs. 86% ± 6%; P = 0.03). When cultured in monolayers, cells from cartilage with pronase E pre-treatment grew in a single plane showing rounded shape while cells from the other group grew in multi-planes and exhibited irregular shape. The mRNA expression ratio of collagen type II to I was 13.2 ± 7.5 in cells isolated from cartilage pre-treated with pronase E, indicating a typical chondrocyte phenotype. Conclusions Collagenase IA was not sufficient in establishing primary human chondrocyte culture. Cartilage must be treated with pronase E prior to collagenase IA application.

The cardioprotective and anxiolytic effects of Chaihujialonggumuli granule on rats with anxiety after acute myocardial infarction is partly mediated by suppression of CXCR4/NF-κB/GSDMD pathway.

AIMS: Over-expression of CXCR4 activates nuclear translocation of NF-κB, induces high expression of NLRP3, GSDMD, IL-1ß and IL-18, which promotes severe inflammatory response following myocardial infarction. Previous studies revealed inflammation induces anxiety after myocardial infarction. The Chaihujialonggumuli granule has anti-inflammatory properties and could tranquillize mind. But the mechanism of its efficacy remains unknown. This study was to investigate the possible mechanism of BFG on cardioprotective and anxiolytic. METHODS: The expression of CXCR4, NF-κB, NLRP3and GSDMD was measured with western-blot, QRT-PCR. The expression location of CXCR4, NLRP3, GSDMD were determined by immunohistochemistry. IL-1ß、IL-18 in the peripheral blood were measured by ELISA. HE staining, Masson staining and transmission electron microscopy were used to observe morphological changes of cardiomyocytes. Echocardiography was used to assess cardiac function after cardiac surgery. Elevated cross maze test and open field test were used to evaluate behaviours. Western blot was used to detect the protein expressions of 5-HT, DA, IL-1ß, IL-18 and neuron damage was investigated by Nissl staining in the hippocampus. RESULTS: The up-regulation of CXCR4, NF-κB, NLRP3 and GSDMD were found in the infarcted area after left coronary artery ligation. Pathological staining and analysis showed that more severe inflammatory cytokines infiltration, myocardial fibrosis, were found in myocardial tissue of the complex group rats. And when compared to the sham group, the levels of IL-1ß, IL-18 was increased of the complex group in both peripheral blood and brain. Behavioural test and echocardiography indicated that the rats in complex group exploration behaviours was significantly reduced, and with poor cardiac functional recovery. The AMD3100 had an inhibitory impact of CXCR4 on the activition of its downstream effectors, alleviating inflammatory reaction. Furthermore, the BFG decreased the expression level of CXCR4, NF-κB, GSDMD, NLRP3 in the infarcted area after myocardial infarction, when compared to the complex group. The assays in the brain indicated the BFG suppressed expression and activity of IL-1ß, IL-18, and improved 5-HT and DA synthesis. CONCLUSIONS: In sum, our study indicated that BFG may reduce inflammation, treat co-existing anxiety after myocardial infarction through inhibition of CXCR4/NF-κB/GSDMD signalling.