Establishment and evaluation of pyroptosis model of rat chondrocytes induced by TNF-α
10.19405/j.cnki.issn1000-1492.2022.05.020
- Author:
Liang Xu
1
;
Yujiao Wu
1
;
Xiaoyang Yuan
1
;
Feng Zhang
1
;
Gang Cheng
1
;
Yunfang Zhang
1
;
Wei Wei
1
;
Shangxue Yan
1
Author Information
1. Institute of Clinical Pharmacology, Anhui Medical University, Key Laboratory of Anti⁃inflammatory and Immune Medicine , Ministry of Education , Anhui Engineering Research Center of Anti⁃inflammatory and Immune Drugs , Hefei 230032
- Publication Type:Journal Article
- Keywords:
TNF⁃α;
osteoarthritis;
chondrocyte;
pyroptosis
- From:
Acta Universitatis Medicinalis Anhui
2022;57(5):781-786
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To establish and evaluate the pyroptosis model of rat chondrocytes induced by tumor necrosis factor-α(TNF-α).
Methods:A two-step enzymatic digestion method was used to obtain rat articular chondrocytes, inverted phase contrast microscope was used to observe the morphological structure of chondrocytes. Toluidine blue staining and type Ⅱ collagen(Col Ⅱ) staining were used to identify chondrocytes. Different mass concentrations of TNF-α(5, 10, 20, 40 ng/ml) were used to establish the pyroptosis model with TNF-α(0 ng/ml) as the control group. Cell viability was detected by CCK-8 method and proteins of pyroptosis signal were detected by Western blot. The levels of IL-1β and IL-18 in cultured supernatants were examined by ELISA kits. The expression of gasdermin D(GSDMD) in chondrocytes was detected by immunofluorescence. Scanning electron microscope was used to observe the morphological changes of chondrocyte.
Results:Compared with the control group, the cell viability of rat chondrocytes gradually decreased with the increase of TNF-α concentration and the expression of nucleotide-binding and oligomerization domain-like receptor containing protein 3(NLRP3), caspase-1, GSDMD and Phospho-NF-κB p65(P-p65) proteins increased. Furthermore, TNF-α(20 ng/ml) could up-regulate the expression of matrix metalloproteinase-13(MMP-13), the fluorescence expression of GSDMD and the levels of IL-1β and IL-18 while the expression of Col Ⅱ was distinctly reduced. What′s more, the articular chondrocytes were swollen,and the microstructure was destroyed.
Conclusion:TNF-α(20 ng/ml) can cause the swelling and death of rat chondrocytes, degradation of cartilage matrix and activation of pyroptosis signaling pathway. The pyroptosis model of rat chondrocytes was successfully established.
- Full text:2025022015142045698TNF-α诱导大鼠软骨细胞焦亡模型的建立与评价_徐靓.pdf
