Cistanoside A mediates p38/MAPK pathway to inhibit osteoclast activity Ⅱ
May 16, 2024
2 Results Results
2.1 Cistancheside A inhibits RANKL-induced osteoclast differentiation.
The results of TRAP staining showed that compared with the control group, the number of osteoclasts was significantly higher when the concentration of Cistancheside A was 5, 10, 20, 40, 80, and 160 μmol/L. decreased, and Cistancheside A had the strongest inhibitory effect on osteoclastogenesis at a concentration of 160 μmol/L (Figure 1A, B). The results of the CCK-8 cytotoxicity experiment showed that Cistancheside A had no toxic effect on bone marrow macrophages at concentrations below 160 μmol/L ( Figure 1C ).
2.2 Cistancheol
A inhibits the formation of actin rings on the surface of osteoclasts and the bone resorption function of osteoclasts. See Figure 2. Phalloidin and DAPI fluorescent staining results showed that compared with the positive control group, the area of osteoclast actin rings in the low- and high-dose groups of Cistancheside A was significantly reduced, and the actin ring of osteoclasts in the high-dose group of Cistancheside A was significantly reduced. The ring area was smaller than that of the Cistancheside A low-dose group (Figure 2A, C). The results of toluidine blue staining of bone grinding discs showed that compared with the positive control group, the area of bone lacunae on bone grinding discs in the low- and high-dose groups of Cistancheol A was significantly reduced, and the area of bone lacunae in the high-dose Cistancheol A group was significantly reduced. The area was smaller than that of the Cistancheside A low-dose group (Figure 2B, D).
Figure 1 ｜ Cistanoside A (Cis A) inhibits osteoclast differentiation induced by receptor activator of nuclear factor kappa-B ligand (RANKL)
Figure 2 ｜ Cistanoside A (CisA) inhibits F-actin ring formation in osteoclasts and bone resorption function
2.3 Cistancheside A down-regulates the expression of key osteoclast genes.
Using the average expression level of the GAPDH gene as the control standard, the results showed that compared with the positive control group, the low-dose group of Cistancheside A could significantly downregulate the osteoclast regulatory differentiation-related genes TRAP, Expression levels of Nfatc -1, DC-STAMP and bone resorption-related genes c-Fos and ctsk (Figure 3).
2.4 Cistancheside A inhibits the expression of downstream proteins of the JNK/MAPK pathway. Western blot results show that compared with the positive control group, Cistancheside A at concentrations of 40, 80 and 160 μmol/L can significantly inhibit the expression of Nfatc1 and c-Fos proteins. amount, and this effect was dose-dependent (Figure 4).
2.5 Cistanche A inhibits the expression of p-JNK protein. Western blot results show that in the time gradient of 0, 5, 10, 20, 30, and 60 min, the expression of p-JNK protein shows a trend of first high and then low, and Compared with the positive control group, Cistancheside A at a concentration of 160 μmol/L can significantly inhibit the expression of p-JNK protein at 10, 20, 30 and 60 minutes of intervention (P < 0.01, P < 0.000 1), see figure 5.
Figure 5 ｜ Cistanoside A (Cis A) inhibits the expression of p-JNK protein
