OL-FS13 Alleviates Cerebral Ischemia-reperfusion Injury by Inhibiting miR-21-3p Expression

Abstract

Background: OL-FS13, a neuroprotective peptide derived from Odorrana livida, can alleviate cerebral ischemia-reperfusion (CI/R) injury, although the specific underlying mechanism remains to be further explored.

Objective: The effect of miR-21-3p on the neural-protective effects of OL-FS13 was examined.

Methods: In this study, the multiple genome sequencing analysis, double luciferase experiment, RT-qPCR, and Western blotting were used to explore the mechanism of OL-FS13.

Results: Showed that over-expression of miR-21-3p against the protective effects of OL-FS13 on oxygen- glucose deprivation/re-oxygenation (OGD/R)-damaged pheochromocytoma (PC12) cells and in CI/R-injured rats. miR-21-3p was then found to target calcium/calmodulin-dependent protein kinase 2 (CAMKK2), and its overexpression inhibited the expression of CAMKK2 and phosphorylation of its downstream adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK), thereby inhibiting the therapeutic effects of OL-FS13 on OGD/R and CI/R. Inhibition of CAMKK2 also antagonized up-regulated of nuclear factor erythroid 2-related factor 2 (Nrf-2) by OL-FS13, thereby abolishing the antioxidant activity of the peptide.

Conclusion: Our results showed that OL-FS13 alleviated OGD/R and CI/R by inhibiting miR-21-3p to activate the CAMKK2/AMPK/Nrf-2 axis.

Keywords: CAMKK2/AMPK pathway; Cerebral ischemia-reperfusion; microRNA; neuroprotective; oxidative stress; peptide.

Fig. (1)

RNA profile and targeting relationship in rat cerebral cortex after I/R. (A and B) Heat map of differentially expressed miRNA (A) and mRNA (B) associated with neuroprotection of OL-FS13.The miRNA and mRNA which most strongly associated with neuroprotection were circled by red rectangular boxes; (C and D). Dual luciferase reporter assay showed that miR-21-3p targeted CAMKK2. **P<0.01, ^nsP ≥ 0.05.

Fig. (2)

Expression of miR-21-3p, CAMKK2, and AMPK in vivo and in vitro after OL-FS13 intervention. (A-D). RT-qPCR results for the expression level of miR-21-3p (A and C) and CAMKK2 mRNA (B and D) in the cerebral cortex of I/R rats after OL-FS13 intervention and in PC12 cells after OGD/R; (E-H). Expression level of CAMKK2, pAMPK/AMPK in vitro and in vivo. ****P < 0.0001, ***P < 0.001, **P < 0.01, *P<0.05.

Fig. (3)

Overexpression of miR-21-3p inhibited the protective effect of OL-FS13 on PC12 cells. (A) Viability of PC12 cells. The treatment of OL-FS13 alleviated OGD/R-induced cell injury and was inhibited by overexpression of miR-21-3p; (B and C). The expression level of CAMKK2 and pAMPK/AMPK in vitro and in vivo. ****P < 0.0001, ***P < 0.001, **P < 0.01.

Fig. (4)

The effect of miR-21-3p overexpression inhibited the improvement of OL-FS13 on cerebral infarct volume and neurological function in I/R rats. (A and B) TTC staining; (C) Score of neurological function injury in rats. ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05, ^nsP ≥ 0.05.

Fig. (5)

Overexpression of miR-21-3p interfered with the neuroprotection of OL-FS13 at the tissue level. (A and B) Nissl staining and H&E staining (red rectangle boxes indicated the enlarged observation areas; red arrows indicated normal cells; black arrows indicated abnormal individuals; black bar: 100 μm; yellow bar: 50 μm); (C and D) Bars showed the proportion of surviving neurons in hippocampal (C) and cortical (D) areas based on the results of Nissl staining. ****P < 0.0001,^nsP ≥ 0.05.

Fig. (6)

Overexpression of miR-21-3p inhibited the activation of CAMKK2/AMPK and Nrf-2 by OL-FS13. Control, I/R, I/R + OL-FS13, and I/R + OL-FS13 + agomiR were used for grouping. The expression levels of CAMKK2 (A), pAMPK/AMPK (B), Nrf-2 (C and D), Bax (E), Bcl-2 (F), Cleaved caspase 3/caspase 3 (G) (n=3) were evaluated. ***P < 0.001, **P < 0.01, *P < 0.05, ^nsP ≥ 0.05.

Fig. (7)

OL-FS13 activated Nrf-2 by increasing CAMKK2 expression. (A) The inhibitor of CAMKK2 (STO-609) and Nrf-2 (ML385) antagonized the cytoprotective effect of OL-FS13. (B-E) Control, OGD/R, OGD/R + OL-FS13, OGD/R + OL-FS13 + STO-609, and OGD/R + OL-FS13 + ML385 were used for grouping. The expression levels of CAMKK2 (B), pAMPK/AMPK (C), and Nrf-2 (D and E) (n=3) were evaluated. ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05, ^nsP ≥ 0.05.

Fig. (8)

OL-FS13 attenuated CI/R injury by activating the CAMKK2/AMPK/Nrf-2 axis. Control, OGD/R, OGD/R + OL-FS13, OGD/R + OL-FS13 + STO-609, and OGD/R + OL-FS13 + ML385 were used for grouping. The expression levels of CAMKK2 (A), pAMPK/AMPK (B), Nrf-2 (C and D), Bax (E), Bcl-2 (F), Cleaved caspase 3/ caspase 3 (G) (n=3) were evaluated. ***P < 0.001, **P < 0.01, *P < 0.05, ^nsP ≥ 0.05.