Suppressive effect of Aurantii Fructus Immaturus and Atractylodis Macrocephalae Rhizoma on glutamic acid-induced autophagy of interstitial cells of Cajal

Abstract

Objective: To investigate the effects of Aurantii Fructus Immaturus (Zhishi, ZS) and Atractylodis Macrocephalae Rhizoma (Baizhu, BZ)-containing serum on glutamate-induced autophagy in rat colonic interstitial cells of Cajal (ICCs) and to analyze the underlying mechanism.

Methods: Rat colonic ICCs cultured in vitro were identified by fluorescence and then stimulated with glutamic acid (5 mmol/L) for 24 h to establish a cell model of autophagy. The cells were then treated with different concentrations of ZSBZ-containing serum or rat serum. The viability of the ICCs was detected with cell counting kit-8 assays, and cell apoptosis rates were examined with flow cytometry. The ultrastructure and autophagosomes in the ICCs were observed using transmission electron microscopy. The effects of ZSBZ-containing serum on apoptosis-associated mediators were assessed by Western blotting and real-time quantitative polymerase chain reaction. In addition, microtubule-associated protein light chain 3 (LC3), p-phosphoinositide 3-kinase (p-PI3K), p-Akt and p-mammalian target of rapamycin (p-mTOR) expression was detected via Western blotting analysis.

Results: Compared to those in the model group, ICC viability and apoptosis rates were significantly increased by ZSBZ-containing serum (P < 0.05). In addition, the expression levels of Beclin-1, LC3, p-PI3K, p-Akt and p-mTOR were significantly lower (P < 0.05) and Bcl-2 expression was higher in the ZSBZ-containing serum treatment groups than in the model group (P < 0.05).

Conclusion: Our findings demonstrated that ZSBZ protects glutamic acid-stimulated ICCs, and this beneficial effect may be mediated by a reduction in autophagy via inhibition of the PI3K/Akt/mTOR pathway.

Keywords: Autophagy; Herbal medicine, Chinese; Phosphoinositide 3-kinase; Signal pathway.