miR-22 alleviates sepsis-induced acute kidney injury via targeting the HMGB1/TLR4/NF-κB signaling pathway

Abstract

Background: Acute kidney injury (AKI) is a severe complication of sepsis, and is strongly correlated with MicroRNAs (miRNAs). However, the mechanism of miR-22 on sepsis-induced AKI is not clearly understood. The study aimed to explore the role and mechanism of miR-22 on AKI.

Methods: The AKI models were established by cecal ligation and puncture (CLP) surgery in SD rats and lipopolysaccharide (LPS) induction in HBZY-1 cells. In AKI rats, the content of serum creatinine (SCr) and blood urea nitrogen (BUN) were detected. Kidney tissues were pathologically examined by H&E and PAS staining. The LPS-induced HBZY-1 cells were transfected with mimics miR-22, si-HMGB1, or oe-HMGB1. miR-22 and HMGB1 expression was detected in vivo and in vitro. In transfected cells, HMGB1/TLR4/NF-κB pathway-related protein expressions were measured by Western blot. The relationship between miR-22 and HMGB1 was assessed by a dual-luciferase gene report. Inflammatory cytokine levels in serum and cells were assessed by ELISA.

Results: In AKI rats, kidney injury was observed, accompanied by the down-regulated miR-122 expression and up-regulated HMBG1 expression. The dual-luciferase report found miR-22-3p could targetly regulate HMBG1. Furthermore, both in vitro and in vivo experiments revealed that the releases of inflammatory cytokine were increased after AKI modeling, but the situation was reversed by mimics miR-22 or si-HMGB1 in vitro. In HBZY-1 cells, mimics miR-22 could suppress LPS-induced overexpression of HMGB1/TLR4/NF-κB signaling pathway-related proteins. However, the oe-HMGB1 addition reversed the effect of mimics miR-22.

Conclusion: miR-22 can inhibit the inflammatory response, target the HMGB1, and inhibit the HMGB1/TLR4/NF-kB pathway, to attenuate the sepsis-induced AKI, which indicates that miR-22 may serve as a potential treatment target in sepsis-induced AKI.

Keywords: Acute kidney injury; HMGB1; Sepsis; Signaling pathway; microRNA.

Fig. 1

The contents of serum creatinine (SCr) and blood urea nitrogen (BUN) were detected in cecal ligation and puncture (CLP)-induced acute kidney injury (AKI) rats. The contents of SCr (A) and BUN (B) were compared between the sham group and the sepsis group. ^#p < 0.05, ^##p < 0.01 vs. Sham. n = 6

Fig. 2

The kidney histopathological structure was damaged in CLP-induced AKI rats. Hematoxylin and eosin (H&E, A) and periodic acid-schiff (PAS, B) were used to perform histopathological analysis of kidneys in sham and sepsis groups. Original magnification 100 × or 400 ×

Fig. 3

The expression of miR-22 was down-regulated and the expression of HMGB1 was up-regulated in CLP-induced rats. Q-PCR (quantitative PCR) was conducted to detect the expression of miR-22 and HMGB1 mRNA in rats (A) and western blot was performed to detect the expression of HMGB1 protein in rats (B). ^#p < 0.05, ^##p < 0.01 vs. Sham. n = 6

Fig. 4

HMGB1 was a direct target of miR-22. Double luciferase gene reporter analysis showed that there was a binding site between miR-22 and the 3’-UTR of HMBG1 mRNA (A). The luciferase activity was assessed to approve the relationship between HMGB1 and miR-22 (B). ^&&p < 0.01 vs. NC

Fig. 5

miR-22 attenuated LPS-induced inflammatory response in the AKI model. ELISA was applied to detected the content of inflammatory factors in model rats (A) and HBZY-1 cells (B), ^#p < 0.05, ^##p < 0.01 vs. Sham or control; *p < 0.05, **p < 0.01 vs. lipopolysaccharide (LPS)

Fig. 6

miR-22 inhibited HMGB1 protein expression in AKI vitro model. Q-PCR was applied to measure the expression of miR-22 and HMGB1 mRNA in HBZY-1 cells (A) and western blot was used to detect the expression of HMGB1 protein in HBZY-1 cells (B), ^#p < 0.05, ^##p < 0.01 vs. Control; *p < 0.05, **p < 0.01 vs. LPS

Fig. 7

miR-22 decreased the expression of TLR4, TLR2, MyD88, and p-NF-κB p65 protein in the AKI vitro model. Western blot was conducted to measure the protein expression of TLR4, TLR2, MyD88, TM and p-NF-κB p65 in HBZY-1 cells. ^#p < 0.05, ^##p < 0.01 vs. Control. *p < 0.05, **p < 0.01 vs. LPS

Fig. 8

si-HMGB1 decreased the expression of TLR4, TLR2, MyD88, and p-NF-κB p65 protein in the AKI vitro model. Western blot was used to measure the protein expression of TLR4, TLR2, MyD88, and p-NF-κB p65 in HBZY-1 cells. ^#p < 0.05, ^##p < 0.01 vs. Control. *p < 0.05, **p < 0.01 vs. LPS

Fig. 9

miR-22 alleviated AKI by targeting the HMGB1/TLR4/NF-κB signaling pathway. Western blot was carried out to measure the protein expression of TLR4, TLR2, MyD88, and p-NF-κB p65 in HBZY-1 cells. *p < 0.05, **p < 0.01 vs. LPS, ^★p < 0.05, ^★★p < 0.01 vs. LPS + mimics miR-22