Methylene blue attenuates renal ischemia-reperfusion injury by negative regulation of NLRP3 signaling pathway.

OBJECTIVE: To investigate the effect of methylene blue (MB) on renal ischemia-reperfusion (IR) injury in mice and its possible relevant mechanisms.

MATERIALS AND METHODS: A total of 30 male C57/BL6 mice aged 4 months old were randomly divided into the following three groups: Sham group (n=10), IR group (n=10), and MB group (n=10). Mice in MB group were treated with gavage continuously using methylene blue solution (dosage: 25 mg·kg-1·d-1) until they were 7 months old. Mice in the other two groups were administrated with the same amount of normal saline for gavage. After that, the abdomen of mice in Sham group was opened and closed, and bilateral renal pedicles of mice in IR group and MB group were occluded using a micro-artery clamp for 45 min for modeling. After modeling, the renal tissues and blood samples of mice were taken for detection. The levels of serum creatinine (Scr), blood urea nitrogen (BUN), and malondialdehyde (MDA), and activity of superoxide dismutase (SOD) in mice in each experimental group were detected and statistically analyzed, respectively. The degree of renal tubular necrosis in renal tissues of mice was observed under an optical microscope. Enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of inflammatory factors [interleukin-1β (IL-1β), IL-18, IL-10 and transforming growth factor-β1 (TGF-β1)] in renal tissues of mice in each experimental group, followed by relevant statistical analyses. The expressions of relevant proteins [NACHT, LRR, and PYD domains-containing protein 3 (NLRP3), nuclear factor-κB (NF-κB), caspase-1, pro-IL-1β, and IL-1β] in renal tissues of mice in each experimental group were detected via Western blotting, and the gray value of each band was detected for statistical analysis.

RESULTS: It was found in this experimental study that Scr and BUN levels in MB group were significantly lower than those in IR group, and the differences were statistically significant (p<0.05). Compared with that in IR group, the degree of renal tubular necrosis in MB group was significantly alleviated, and the difference was statistically significant (p<0.05). Compared with those in IR group, the levels of inflammatory factors (IL-1β and IL-18) in MB group were significantly decreased, but the levels of IL-10 and TGF-β1 in MB group were significantly increased, and the differences were statistically significant (p<0.05). Compared with those in IR group, the activity of SOD in MB group was increased significantly, but the level of MDA was decreased, and the differences were statistically significant (p<0.05). The protein expressions of NLRP3, NF-κB, caspase-1, and pro-IL-1β in MB group were decreased compared with those in IR group, but the expression of IL-1β in MB group was increased, and the differences were statistically significant (p<0.05).

CONCLUSIONS: We showed that methylene blue can alleviate the apoptosis and inflammatory response induced by renal IR injury in mice, and its relevant mechanism may be related to the fact that methylene blue can negatively regulate NLRP3 signaling pathway.