p21 Improving Osteoarthritis by Regulating the Pyroptosis of Cartilage Cells

Abstract

Background: The regulation of p21 in the pyroptosis of cartilage cells still needs to be further clarified. We aimed to explore the regulation of p21 on the pyroptosis of cartilage cells and to reveal the improvement of osteoarthritis.

Methods: Chondrocytes were collected and isolated from patients with osteoarthritis (average age 58.64 ± 4.32) in Xuzhou Third People's Hospital, China in 2019, and healthy volunteers (average age 58.23 ± 3.91) were enrolled as the control group. mRNA expression levels of p21 and pyroptosis-related proteins (NLRP3, ASC, total caspase1 and cleaved-Caspase1) were detected by Western blot and real-time PCR. Cell activity, total number of cells and number of dead cells were calculated with CCK-8, MTT. And the regulation of p21 on the pyroptosis of cartilage cells was verified with overexpression and knockdown of p21 in cartilage cells.

Results: In cartilage cells of patients with osteoarthritis, the transcription and translation levels of pyrolysis-related genes (NLRP3, cleaved-caspase 1, and ASC) significantly increased (P<0.01). p21 expression was up-regulated and positively correlated with the changing trend of pyrolysis-related proteins (P<0.01). Overexpressing p21 genes in normal cartilage cells significantly increased the expression of pyrolysis-related proteins (P<0.01).

Conclusion: The pyroptosis of cartilage cells is causally related to the process of osteoarthritis, and can be regulated by transcription factor p21, which is a potential therapeutic target for osteoarthritis.

Keywords: Cartilage cells; Osteoarthritis; Pyroptosis; p21.

Fig. 1:

Changing expression of pyroptosis-related proteins of cartilage cells in patients with osteoarthritis Note: A–B: mRNA levels of pyroptosis-related genes, Nlrp3 and Asc, of cartilage cells in each group was detected by real-time method, n=5, **P<0.01

Fig. 2:

Trends in expression of transcription factor p21 positively correlated with the pyroptosis of cartilage cells Note: A: Expression levels of p21 in normal cartilage cells and osteoarticular cartilage cells were detected with real-time PCR method, n=5, ** P<0.01; B–E: Correlation between p21 and the pyroptosis-related factors of cartilage cells was analyzed by regression curve, with the correlation degree presented by r², n=10

Fig. 3:

p21 overexpression significantly increased pyroptosis levels of cartilage cells Note: A, B: Transfection efficiency of p21 overexpression vectors were verified with Western blot, real-time PCR method, n=5, ***P<0.001; C–E: Levels of NLRP3, caspase 1, and ASC in cartilage cells of each group were detected by Western blot method, n=5, ***P<0.001; G, H: Levels of Nlrp3 and Asc in cartilage cells of each group were detected by Nlrp3, of the pyroptosis of cartilage cellsrelated genes in cartilage cells of each group were detected with real-time method, n=5, ***P<0.001; I–K: Cell activity, cell number and number of dead cells were detected with CCK-8 and trypan blue staining, n=5, ***P<0.001

Fig. 4:

Knockdown of transcription factor p21 inhibited the pyroptosis levels of osteoarthritis cartilage cells Note: A,B: The transfection efficiency of p21 knockdown was checked with Western blot, namely, real-time PCR method, n=5, ***P<0.001; C–E: Levels of NLRP3, caspase 1, and ASC in cartilage cells of each group were detected by Western blot method, n=5, ***P<0.001; G,H: mRNA levels of the pyroptosis of Nlrp3 and Asc in cartilage cells of each group were detected with real-time PCR method, n=5, ***P<0.001; I–K: Cell activity, cell number and number of dead cells were detected with CCK-8 and trypan blue staining, n=5, ***P<0.001