Protective effect and mechanism of Ribociclib on sepsis induced-acute kidney injury / 中华危重病急救医学

ABSTRACT

Objective:To investigate the role of Ribociclib in sepsis induced-acute kidney injury (AKI) and its possible mechanisms.Methods:① Twenty adult male C57BL/6 mice were divided into sham operation group (Sham group; only open the abdomen without ligating or perforating the cecum, administered with sodium lactate buffer 12 hours before the sham operation), Ribociclib control group (administered with 150 mg/kg Ribociclib), cecal ligation and puncture (CLP) group (sepsis model induced by CLP; lactate buffer was given by intragastric administration 12 hours before CLP), and Ribociclib pretreatment group (administered with 150 mg/kg Ribociclib 12 hours before CLP) according to random number table, with 5 mice in each group. Kidneys were harvested 12 hours after the operation. Pathological changes in kidney were observed by hematoxylin-eosin (HE) staining. Tumor necrosis factor-α(TNF-α) and interleukin-6 (IL-6) levels in mice kidney homogenate were measured by enzyme linked immunosorbent assay (ELISA). Western Blot was used to detect the expression of cell cycle-related protein phosphorylate retinoblastoma protein (p-Rb), apoptosis-related protein Bcl-2 and Bax. ② Mouse renal tubular epithelial (TCMK-1) cell line was used for in vitro experiment. The cells were divided into control group, Ribociclib group (treated with 5 μmol/L Ribociclib for 24 hours), lipopolysaccharide (LPS) group (treated with 200 mg/L LPS for 6 hours), Ribociclib+LPS group (replaced with the medium containing 5 μmol/L Ribociclib and 200 mg/L LPS for 6 hours after exposing with 5 μmol/L Ribociclib for 18 hours). Inflammatory cytokines in cell culture medium were detected by ELISA. The expression of p-Rb, Bcl-2 and Bax, autophagy-related proteins microtubule associated protein 1 light chain LC3b (LC3bⅡ, LC3bⅠ) and p62, phosphate protein kinase B (p-AKT), phosphorylated mammalian target of rapamycin (p-mTOR) were measured by Western Blot. Results:① Animal experiments showed that, compared with the Sham group, the kidney tissue of mice were significantly damaged, the levels of TNF-α and IL-6 were increased, the expressions of p-Rb and Bcl-2/Bax ratio were decreased in kidney tissue in CLP group; but there was no significant difference in indexes between Ribociclib control group and Sham group. Compared with the CLP group, kidney injury in mice pretreated with Ribociclib was significantly ameliorated, the pathological score was significantly decreased (1.48±0.16 vs. 2.68±0.16, P < 0.01), the levels of TNF-α and IL-6 in kidney homogenate were significantly decreased [TNF-α(ng/g) 340.55±34.96 vs. 745.08±58.86, IL-6 (mg/g) 17.33±1.01 vs. 114.20±20.49, both P < 0.01], the expression of p-Rb was furtherly decreased (p-Rb/β-tubulin 0.14±0.01 vs. 0.73±0.06, P < 0.01), Bcl-2/Bax ratio was increased (0.89±0.06 vs. 0.62±0.10, P < 0.01). ② In vitro experiments showed that, compared with the control group, the releases of TNF-α and IL-6 were increased, the expression of p-Rb was decreased, the ratios of Bcl-2/Bax and LC3bⅡ/Ⅰ were decreased, the expressions of p62, p-AKT and p-mTOR were increased in LPS group; the expression of p-Rb was decreased after Ribociclib treatment in TCMK-1 cells. Compared with the LPS group, TNF-α and IL-6 were decreased [TNF-α (ng/L) 2.73±0.23 vs. 4.96±0.10, IL-6 (ng/L) 36.05±5.83 vs. 53.78±24.08, both P < 0.01], the expression of p-Rb was furtherly decreased (p-Rb/β-tubulin 0.25±0.05 vs. 0.65±0.05, P < 0.01), the ratios of Bcl-2/Bax and LC3bⅡ/Ⅰ were increased (Bcl-2/Bax 1.01±0.07 vs. 0.73±0.05, LC3bⅡ/Ⅰ 2.08±0.31 vs. 1.04±0.01, both P < 0.05), the expressions of p62, p-AKT and p-mTOR were decreased (p62/β-tubulin 0.59±0.01 vs. 1.09±0.08, p-AKT/β-tubulin 0.61±0.03 vs. 1.20±0.06, p-mTOR/β-tubulin 0.50±0.05 vs. 1.15±0.08, all P < 0.01) in the Ribociclib+LPS group. Conclusion:Ribociclib pretreatment ameliorated sepsis-induced AKI and AKT/mTOR pathway may be involved in the protective role of Ribociclib on kidney.