Bone marrow mesenchymal stem cells attenuate pyroptosis lipopolysaccharide-induced renal injury rats / 中华危重病急救医学

ABSTRACT

Objective:To investigate the effect and mechanism of bone marrow mesenchymal stem cell (BMSC) on pyroptosis of rats with kidney injury.Methods:Bone marrow of 4-5 week-old female Sprague-Dawley (SD) rats was isolated in vitro and BMSC was obtained. The third generations of BMSC were used to further experiments. Fifteen 6 week-old SD rats were cluster-randomized divided into control group, kidney injury group and BMSC group (5 rats in each group). Rats in kidney injury group were injected with lipopolysaccharide (LPS) 1 mg/kg via tail vein; the control group was given the same amount of normal saline. BMSC group was injected with 0.5 mL BMSC (including 2×10 6 BMSC) via tail vein after modeling; the kidney injury group received the same amount of normal saline. On day 3 after these injections, serum creatinine (SCr) was detected by picric acid method, and blood urea nitrogen (BUN) was detected by diacetyl monoxime. The levels of cystatin C (Cys C), interleukins (IL-1β and IL-18) in blood were detected by enzyme-linked immunosorbent assay (ELISA). The rats were then sacrificed and their kidneys were removed for subsequent detection. The mRNA expression levels of NOD-like receptor protein 3 (NLRP3) and cysteinyl aspartate-specific protease-1 (caspase-1) of kidney were detected by quantificational real-time quantitative polymerase chain reaction (qRT-PCR). The protein expression levels of NLRP3 and caspase-1 of kidney were detected by Western blotting. Results:In vitro, after bone marrow cell suspension was cultured for 24 hours, a large number of round adherent cells and suspended cells appeared in each culture flask. After 4-5 days of culture, a large number of long spindle cells adhered to the wall, and there were still obvious impurity cells. After trypsin digestion and passage to the third generation, the long spindle adherent cells grew mainly in the culture flask and were basically purified as BMSC. In vivo, compared with the control group, the levels of SCr, BUN, Cys C, IL-1β and IL-18 in kidney injury group were increased [SCr (μmol/L) 85.22±2.29 vs. 21.80±0.59, BUN (mmol/L) 11.50±0.64 vs. 5.86±0.83, Cys C (mg/L) 0.13±0.01 vs. 0.11±0.02, IL-1β (ng/L) 31.49±1.42 vs. 4.74±0.49, IL-18 (ng/L) 29.01±1.95 vs. 1.52±0.03, all P < 0.05]. The mRNA and protein expression levels of NLRP3, caspase-1 were significantly increased [NLRP3 mRNA (2 -&Delta;&Delta;Ct) 3.635±0.296 vs. 1.000±0.002, caspase-1 mRNA (2 -&Delta;&Delta;Ct) 4.020±0.228 vs. 1.001±0.003; NLRP3 protein (NLRP3/β-actin) 1.560±0.868 vs. 0.902±0.036, caspase-1 protein (caspase-1/β-actin) 1.392±0.097 vs. 0.895±0.046, all P < 0.05]. Compared with kidney injury group, the levels of SCr, BUN, IL-1β and IL-18 in BMSC group were significantly decreased [SCr (μmol/L) 51.64±3.84 vs. 85.22±2.29, BUN (mmol/L) 9.90±0.46 vs. 11.50±0.64, IL-1β (ng/L) 24.20±1.45 vs. 31.49±1.42, IL-18 (ng/L) 12.97±1.25 vs. 29.01±1.95, all P < 0.05]. The mRNA and protein expression levels of NLRP3, caspase-1 were significantly decreased [NLRP3 mRNA (2 -&Delta;&Delta;Ct) 1.488±0.136 vs. 3.635±0.296, caspase-1 mRNA (2 -&Delta;&Delta;Ct) 1.643±0.143 vs. 4.020±0.228; NLRP3 protein (NLRP3/β-actin) 1.227±0.053 vs. 1.560±0.868, caspase-1 protein (caspase-1/β-actin) 1.159±0.107 vs. 1.392±0.097, all P < 0.05]. Conclusion:In vivo, BMSC may attenuate pyroptosis in LPS-induced kidney injury rats.