Soy isoflavones alleviates calcium overload in rats with cerebral ischemia-reperfusion by inhibiting the Wnt/Ca2+ signaling pathway

doi:10.12122/j.issn.1673-4254.2024.06.05

Abstract

Abstract:

Objective To explore the mechanism by which soybean isoflavone (SI) reduces calcium overload induced by cerebral ischemia-reperfusion (I/R). Methods Forty-eight SD rats were randomized into 4 groups to receive sham operation, cerebral middle artery occlusion for 2 h followed by 24 h of reperfusion (I/R model group), or injection of adeno-associated virus carrying Frizzled-2 siRNA or empty viral vector into the lateral cerebral ventricle after modeling. Western blotting was used to examine Frizzled-2 knockdown efficiency and changes in protein expressions in the Wnt/Ca²⁺ signaling pathway. Calcium levels and pathological changes in the ischemic penumbra (IP) were measured using calcium chromogenic assay and HE staining, respectively. Another 72 SD randomly allocated for sham operation, I/R modeling, or soy isoflavones pretreatment before modeling were examined for regional cerebral blood flow using a Doppler flowmeter, and the cerebral infarct volume was assessed using TTC staining. Pathologies in the IP area were evaluated using HE and Nissl staining, and ROS level, Ca²⁺ level, cell apoptosis, and intracellular calcium concentration were analyzed using immunofluorescence assay or flow cytometry; the protein expressions of Wnt5a, Frizzled-2, and P-CaMK II in the IP were detected with Western blotting and immunohistochemistry. Results In rats with cerebral I/R, Frizzled-2 knockdown significantly lowered calcium concentration (P<0.001) and the expression levels of Wnt5a, Frizzled-2, and P-CaMK II in the IP area. In soy isoflavones-pretreated rats, calcium concentration, ROS and MDA levels, cell apoptosis rate, cerebral infarct volume, and expression levels of Wnt/Ca²⁺ signaling pathway-related proteins were all significantly lower while SOD level was higher than those in rats in I/R model group. Conclusion Soy isoflavones can mitigate calcium overload in rats with cerebral I/R by inhibiting the Wnt/Ca²⁺ signaling pathway.

Key words: soy isoflavones, cerebral ischemia-reperfusion, calcium overload, Wnt/Ca²⁺ signaling pathway

Li LI, Mengzhe WANG, Saisai LIU, Xiaonan ZHANG, Jie CHEN, Weiting TAO, Shai LI, Zhiwen QING, Quanfang TAO, Yi LIU, Li Huang, Shidi ZHAO. Soy isoflavones alleviates calcium overload in rats with cerebral ischemia-reperfusion by inhibiting the Wnt/Ca²⁺ signaling pathway[J]. Journal of Southern Medical University, 2024, 44(6): 1048-1058.

Figures/Tables 6

Fig.1 Effect of inhibition of Wnt/Ca2+ signaling pathway on cerebral ischemia-reperfusion (I/R) injury in rats. A, B: Expression of Frizzled-2 in the brain tissue of sham-operated rats detected by Western blotting (n=3). C-F: Western blotting for detecting expressions of Frizzled-2, Wnt5a and P-CaMK II in the brain tissues of rats in each group (n=3). G: Ca2+ level in the brain tissues of the rats (n=6). H: HE staining of the brain tissues of the rats. *P<0.05, **P<0.01, ***P<0.001 vs Sham group; #P<0.05, ###P<0.001 vs I/R group.

Fig.2 Dynamic changes of regional cerebral blood flow (rCBF). A: Changes of rCBF in I/R group. B: Changes of rCBF in soy isoflavones (SI)-pretreated group.

Fig.3 Brain infarct volume and neurological deficit scores of the rats with sham operation, cerebral I/R injury and soy isoflavones (SI) pretreatment. A, B: Brain infarct volume (n=5). C: Neurological deficit scores (n=6). ***P<0.001 vs Sham group; ##P<0.01, ###P<0.001 vs I/R group.

Fig.4 HE staining (A) and Nissl staining (B) of the rats with sham operation, cerebral I/R injury and soy isoflavones (SI) pretreatment before modeling (scale bar=20 μm).

Fig.6 Changes of oxidative stress level in the brain tissue of the rats in the 3 groups. A, B: Immunofluorescence assay for detecting ROS level in the brain tissues (n=3). C: SOD activity in the brain tissue (n=6); D: Content of MDA in the brain tissue (n=6). ***P<0.001 vs Sham group; ###P<0.001 vs I/R group.

Fig.8 Western blotting (A) and immunohistochemistry (B, original magnification:×400) for detecting protein expression of Wnt5a, Frizzled-2 and P-CaMK II in the brain tissues of the rats in the 3 groups. **P<0.01, ***P<0.001 vs Sham group; #P<0.05 vs I/R group.

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Soy isoflavones alleviates calcium overload in rats with cerebral ischemia-reperfusion by inhibiting the Wnt/Ca²⁺ signaling pathway

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