关键词: 骨质疏松症, 骨疏康, 含药血清, 破骨细胞, Nrf2, c-Fos/NFATc1通路, RAW 264.7细胞

Abstract: BACKGROUND: It has been shown that Gushukang affects bone metabolism by regulating nucleotide and amino acid metabolism and immune mechanisms. Current research on the mechanism of Gushukang in the treatment of osteoporosis primarily focuses on osteoblast regulation and requires further improvement from the perspective of osteoclasts.
OBJECTIVE: To investigate the mechanism by which Gushukang interferes with osteoclasts in the treatment of osteoporosis using RAW264.7 cells as the research model. 
METHODS: Twenty-four 8-week-old female Sprague-Dawley rats were randomly divided into four groups (n=6 per group): the three experimental groups were given 1, 2 and 4 g/kg osteoporosis solution by gavage (2 times per day), and the control group was given an equal amount of distilled water by gavage (2 times per day). After 7 days of intragastric administration, aortic blood samples were extracted to collect serum samples using centrifugation, and serum samples from the same groups were combined to obtain the low-, medium-, and high-concentration Gushukang-containing and normal sera for the subsequent experiments. (1) RAW264.7 cells were cultured in six groups: normal serum was added to the control group; low, medium, and high concentration groups were added with low, medium, and high concentrations of Gushukang-containing serum, respectively; ML385, a nuclear factor erythroid 2-related factor 2 (Nrf2) inhibitor was given in the Nrf2 inhibitor group; and t-BHQ, a Nrf2 activator, was added in the Nrf2 activator group. Cell viability was detected using the cell counting kit-8 assay. (2) The 3rd generation RAW 264.7 cells were cultured and divided into five groups: the blank control group was added with normal serum, the osteoclast group was added with receptor activator of nuclear factor κB ligand (RANKL), and the low-, medium-, and high-concentration groups were added with low-, medium-, and high-concentration Gushukang-containing serum based on the addition of RANKL. Tartrate-resistant acid phosphate staining was performed after 5 days of culture. (3) RAW264.7 cells were cultured and divided into five groups: blank control group was cultured with normal serum, osteoclast group cultured with normal serum and RANKL, high concentration+osteoclast group cultured with RANKL+high concentration Gushukang-containing serum, osteoclast+Nrf2 agonist group cultured with RANKL+t-BHQ, and high concentration+osteoclast+Nrf2 inhibitor group cultured with RANKL+high concentration Gushukang-containing serum+ML385. Western blot assay and determination of reactive oxygen content were performed after 5 days of culture.
RESULTS AND CONCLUSION: The cell counting kit-8 results indicated that Gushukang-containing serum, NRF2 inhibitor or agonist had no significant effect on RAW264.7 cell viability. Tartrate-resistant acid phosphate staining results demonstrated that Gushukang-containing serum exhibited a concentration-dependent inhibitory effect on osteoclast differentiation. Western blot analysis and determination of reactive oxygen species revealed that compared with the blank control group, Nrf2 protein expression was decreased in the osteoclast group (P < 0.05), while c-Fos and NFATc1 protein expression and reactive oxygen species content were elevated (P < 0.05); compared with the osteoclast group, Nrf2 protein expression was elevated and reactive oxygen species content was decreased in the high-concentration+osteoclast group, osteoclast+Nrf2 agonist group, and high-concentration+osteoclast+Nrf2 inhibitor group (P < 0.05), while c-Fos and NFATc1 protein expression was decreased in the high concentration+osteoclast group and osteoclast+Nrf2 agonist group (P < 0.05); compared with the high concentration+osteoclast group, Nrf2 protein expression was decreased (P < 0.05) and reactive oxygen species content was elevated (P < 0.05) in the high concentration+osteoclast+Nrf2 inhibitor group. To conclude, Gushukang reduces reactive oxygen species production by activating Nrf2, thereby inhibiting downstream of the c-Fos/NFATc1 pathway and suppressing osteoclast differentiation.

Key words:  osteoporosis, Gushukang, drug-containing serum, osteoclast, Nrf2, c-Fos/NFATc1 pathway, RAW 264.7 cells