Targeted inhibition of frizzled- 2 significantly attenuates Wnt5a/Ca<sup>2+</sup> mediated intracellular calcium accumulation signaling after traumatic brain injury

doi:10.3877/cma.j.issn.2095-9141.2015.01.008

Objective

To investigate the role of Wnt5a/Frizzled-2 signal in the process of nerve cell calcium overload after traumatic brain injury (TBI).

Methods

In vivo experience: adult Sprague-Dawley rats (n=96) were randomly divided into Sham group A (n=32), Pure injury group B(n=32) and RNAi inhibition group C (n=32). Moderate TBI modal was made in group B and C by Fenny method.RNAi was stereotactic hippocampal injected 48 hours before modal were made in group C to inhibit the expression of Frizzled-2.Rats were killed 24 h after injury,the levels of Frizzled-2 and Wnt5a in the injured side of hippocampal tissue were tested by western blotting method, the levels of calcium concentration were tested by immunofluorescent staining and laser confocal microscope. The data among groups was compared by single factor analysis of variance.

Results

In group A, the expression of Wnt5a/Frizzled-2 signal in the hippocampal tissue cells were stable. Compared with group A, the expression of Wnt5a and Frizzled-2 in the hippocampal tissue cells after TBI were increased by 2 times and 5 times (P＜0.01) respectively in group B, and the level of calcium ion increased significantly (P＜0.01). Compared with group B, with the inhibition of Frizzled-2, the expression of Wnt5a and Frizzled-2 in the hippocampal cells were decreased by 1 times and 3.55 times(P＜0.01) respectively in group C, meanwhile the level of calcium ion were significantly decreased 1.5 times, close to the group A (P＜0.01).

Conclusion

Under the physiological and pathological conditions, Wnt5a/Frizzled-2 signal plays an important role in the change of calcium concertration in the regulation of nerve cells. Our research suggests that the important factor related to the signal of Wnt5a, Frizzled-2, P-CamKII and RNAi which specific designed for Frizzled-2 RNAi could become potential therapeutic targets for further study in TBI.

关键词: Traumatic brain injury, Frizzled-2, Wnt5a, P-CamKII, Ca²

目的

探讨Wnt5a/Frizzled-2信号在创伤性颅脑损伤(TBI)后神经细胞钙超载过程中的作用。

方法

体内实验：成年Sprague-Dawley大鼠(96只)，随机分为正常组A(32只)，单纯损伤组B(32只)和RNAi抑制组C(32只)。B组和C组Fenny 式法制作中型颅脑损伤模型。其中C组模型建立前48 h采用立体定向海马区注射RNAi以抑制Frizzled-2的表达。伤后24 h处死大鼠，取伤侧海马组织，用western blotting 方法检测其中Frizzled-2 和Wnt5a 含量，用免疫荧光染色和激光共聚焦显微镜检测其中钙离子浓度。单因素方差分析比较各组间的差别。

结果

A组大鼠海马组织细胞中稳定表达着Wnt5a/Frizzled-2 信号。TBI 后，与A 组相比，B 组海马组织中的Wnt5a 和Frizzled-2的表达分别升高了2倍和5倍(P＜0.01)，海马细胞中钙离子的含量显著增高(P＜0.01)。与B组相比，随着Frizzled-2的抑制，C组海马细胞中的Wnt5a和Frizzled-2的表达分别降低1倍、3.5倍(P＜0.01)，同时细胞中的钙离子含量显著降低1.5 倍，接近A 组(P＜0.01)。

结论

在生理和病理条件下，Wnt5a/Frizzled-2信号在调节神经细胞中的钙离子浓度变化中起到重要的作用，同时我们的研究提示该信号的相关重要组成因子Wnt5a，Fzd2 和P-CamKII以及针对frizzled-2 设计的特异性RNAi可以作为日后TBI研究和治疗的相关靶点。

Key words: 脑损伤, Frizzled-2, Wnt5a, P-CamKII, Ca²⁺

Fig.1 Wnt5a and Fzd2 gene were highly expressed after TBI.

Fig.2 Expressions of Wnt5a,Fzd2 and p-CaMKII proteins in injured hippocampus with or without treatment of Stealth RNAi and Invivofectamine.

Fig.3 Expressions of Wnt5a and Fzd2 affected the accumulation of intracellular Ca²⁺in injured hippocampi with or without combined treatment of Stealth RNAi and Invivofectamine

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