Fig. 6. ADM enhances cell autophagy to attenuate pyroptosis by inhibiting the AMPK/mTOR signalling pathway in LPS-exposed Leydig cells.

Western blot was used to detect the protein level of p-AMPK and AMPK (a) and p-mTOR and mTOR (b). Western blot was performed to evaluate the expression of LC3 and Beclin-1 in LPS-induced Leydig cells treated with ADM alone, rapamycin alone or ADM in combination with rapamycin (c). β-actin was used as internal control. Histogram displaying the densitometric analysis results of p-AMPK (d) and p-mTOR (e) and the ratio of LC3-II/LC3-I and Beclin-1 (f) normalised by the control group. TUNEL staining was used to evaluate the cell death (g) (scale bar: 40 µm). Statistical analysis of the percentage of TUNEL-positive cells normalised by the control group (h). i Flow cytometry was used to evaluate cell death. j Statistical analysis of the percentage of active caspase-1 and PI double-stained positive cells normalised by the control group. The normalised levels of gene expression are expressed as ratios of the copy number of mRNA and that of β-actin cDNA. Data were obtained from five independent experiments and expressed as mean ± SD. *P < 0.01 and ^#P < 0.05, compared with the corresponding control or treatment group