Cordyceps sinensis alleviates β-glycerophosphate-induced vascular smooth muscle cell calcification through promoting autophagy

Abstract

Objective To investigate the influence mechanism of Cordyceps sinensis (CS) on β-glycerophosphate-induced vascular smooth muscle cell (VSMC) calcification. Methods The effect of CS on VSMC cell viability was detected by CCK-8. The cellular models of rat VSMC calcification were established by treating with β-glycerophosphate (β-GP, 10 mmol/L); then CS (10 mg/L), autophagy inhibitor 3-methyladenine (3-MA, 5 mmol/L), and AMPK inhibitor compound C (CC, 10 μmol/L) were added to the cell cultures. There were a total of 5 experiment groups: VSMC cultured in normal medium (Control), VSMC treated with β-GP, VSMC treated with β-GP and CS, VSMC treated with 3-MA, β-GP and CS, and VSMC treated with CC, β-GP and CS. The calcium nodules and calcium content were examined with alizarin red S staining and the O-cresolphthaleincomplexone method, respectively. The autophagosomes within the VSMC were observed using transmission electron microscope (TEM). Immunofluorescence showed the accumulation of microtubule-associated protein 1 light chain 3 (LC3) puncta. In addition, levels of osteogenic related proteins, autophagy related proteins, and AMPK/mTOR pathway related proteins were evaluated by Western blotting. Results CS increased the number of autophagosomes and the accumulation of LC3 puncta within VSMC. It also up-regulated the protein levels of LC3Ⅱ/LC3Ⅰ, beclin1, α-SMA, and p-AMPK; whereas, the protein levels of Runx2 and p-mTOR, as well as calcium nodules and calcium content were reduced (all P<0.01). When the cells were pretreated with 3-MA before treating with β-GP and CS, the autophagosomes, accumulation of LC3 puncta, and protein levels of LC3Ⅱ/LC3Ⅰ, beclin1, and α-SMA were decreased (all P<0.01); however, the protein level of Runx2, and the calcium nodules and calcium content were increased (all P<0.01). Nevertheless, when the cells were pretreated with CC before giving β-GP and CS, the autophagosomes, the accumulation of LC3 puncta, and the expression levels of p-AMPK, LC3Ⅱ/LC3Ⅰ, beclin1, and α-SMA were significantly down-regulated (all P<0.01); whereas, the expression levels of Runx2 and p-mTOR, as well as calcium nodules and calcium content were increased (all P<0.01). Conclusions CS can effectively alleviate β-GP-induced VSMC calcification, which may be due to the activation of autophagy by AMPK/mTOR signaling pathway. Key words: Cordyceps sinensis; Autophagy; Muscle, smooth, vascular; Phosphorus; Renal insufficiency, chronic; Calcification