Shikonin inhibited the proliferation and migration of the renal tubular cells after the renal ischemia-reperfusion injury

doi:10.3877/cma.j.issn.2095-3216.2022.01.003

Abstract

Abstract:

Objective

To investigate the effect of pyruvate kinase M2 (PKM2) inhibitor shikonin on renal tubular repair after renal ischemia-reperfusion injury (IRI).

Methods

Thirty 8-week-old male C57BL/6J mice were randomly divided into 6 groups: sham solvent group (sham-operation plus solvent), sham shikonin group (sham-operation plus shikonin); bIRI solvent 1 d group (bilateral IRI plus solvent 1 d) and bIRI shikonin 1 d group (bilateral IRI plus shikonin 1 d); bIRI solvent 3 d group (bilateral IRI plus solvent 3 d), bIRI shikonin 3 d group (bilateral IRI plus shikonin 3 d). To establish the renal IRI model, bilateral renal pedicles were clipped for 28 min, and 3 mg/kg shikonin or solvent was intraperitoneally injected 4 h after the operation. The mice were sacrificed 1 or 3 days after the operation for samples collection. The serum creatinine and blood urea nitrogen levels of mice in each group were measured. The renal histopathological changes were observed by PAS staining. The expressions of PKM2, proliferating cell nuclear antigen (PCNA), and Lotus tetragonolobus lectin (LTL) were detected by immunofluorescence. Western blotting was used to detect renal PCNA expression. For the in vitro experiments, the human proximal tubular epithelial HK2 cells were divided into 8 groups: control group (without hypoxia treatment), model group (with hypoxia/reoxygenation treatment), shikonin doses groups (hypoxia/reoxygenation plus shikonin of 0.5 μM, 1.0 μM, 1.5 μM, 2.0 μM, 3.0 μM, and 7.0 μM). The cell survival rate and cell growth inhibition rate were detected by the cell counting kit 8 (CCK-8). The scratch wound healing assay was applied to detect cell migration ability.

Results

The renal tubular injury area scores in the bIRI shikonin 1 d group and the bIRI shikonin 3 d group, were higher than those in the bIRI solvent 1 d group and bIRI solvent 3 d group, respectively (P=0.0337), while the renal tubular injury area scores in the sham solvent group and the sham shikonin group did not differ from each other significantly. Both immunofluorescence (P=0.0331) and western blotting (P=0.0228) results showed that the expression of PCNA in the kidneys of mice was significantly lower in the bIRI shikonin 3 d group than in the bIRI solvent 3 d group. During the in vitro experiments, compared with the model group, the shikonin doses groups (1.5 μM, 2.0 μM, 3.0 μM, and 7.0 μM) showed significantly lower survival rates of HK2 cells (P<0.001), as well as higher rates of cell growth inhibition (P<0.001). The scratch wound healing assay displayed that shikonin of 1.5 μM could obviously inhibit the migration of renal tubular HK2 cells after reoxygenation (P<0.001).

Conclusion

After renal IRI injury, shikonin may inhibit the renal tubular repair process through reducing the regeneration and migration of renal tubular epithelial cells, thereby aggravating the renal injury.

Key words: Acute kidney injury, Tubular epithelial cell, Shikonin, Proliferation, Migration

Yulan Chen, Jianwen Chen, Fei Zhu, Tiantian Wang, Yan Zhang, Jiaona Liu, Mengjie Huang, Lingling Wu, Xiangmei Chen. Shikonin inhibited the proliferation and migration of the renal tubular cells after the renal ischemia-reperfusion injury[J]. Chinese Journal of Kidney Disease Investigation(Electronic Edition), 2022, 11(01): 15-21.

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References 26

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