阿尔茨海默病(AD)是一种常见的神经退行性疾病，其部分特征是聚集性过度磷酸化的Tau蛋白形成神经原纤维缠结，促进AD的发病机制。丹参中丹参酮IIA(Tan IIA)可靶向降解AD Tau。在N2A细胞、Tau过表达细胞和3×Tg-AD中，Tan IIA能降低Tau的表达，减弱小鼠原代神经元细胞的Tau磷酸化。此外，Tan IIA增加了多泛素化Tau的积累，并诱导蛋白酶的降解。此外，Tan IIA还可以与Tau蛋白结合，抑制肝素诱导的Tau纤维的形成。总之，Tan IIA可增加多泛素化Tau的积累，诱导蛋白酶体降解Tau蛋白和Tan IIA与Tau蛋白结合体，抑制Tau纤维的形成。Tan IIA可能会进一步作为AD治疗的潜在候选者。

 Figure 1. 丹参酮IIA的抑制表达效果Inhibitory effect of Tan IIA on the expression and phosphorylation of Tau protein in N2a and HEK293/Tau cells. N2a (A) or HEK293/Tau (C) cells were treated for 24 h with various concentrations of Tan IIA (0, 0.5, 1.0, 2.5, 5.0, and 10 μM), and the cell viability was determined by the CCK-8 assay. No significant difference was observed between the groups. The expression levels of Tau, pS396-Tau, and pS404-Tau proteins in N2a (B) or HEK293/Tau (D) cells were detected by western blot analysis after Tan IIA treatment. β-Actin was used as a loading control. The data were the results of three independent experiments, **p < 0.01, ***p < 0.001. (E) Immunofluorescence staining of HEK293/Tau cells was performed using the Tau antibody (in green). The nuclei were stained with DAPI (in blue). Scale bar, 10 μm.

Figure 2. Tan IIA介导的抑制Tau和Tau磷酸化水平作用Rescue effect of MG132 in Tau and Tau phosphorylation levels from Tan IIA-mediated inhibition. HEK293/Tau cells were treated with Tan IIA in the absence or presence of 50 nM Baf-A1 for 24 h (A) or 10 μM MG132 (B). Cell lysates were immunoblotted with the indicated antibodies. β-Actin was used as a loading control. All data obtained were from three independent experiments, ***p <0.001.

Figure 4.Tan IIA对3×Tg-AD小鼠原代神经元细胞Tau和磷酸化Tau水平的抑制作用 Inhibitory effect of Tan IIA on Tau and phosphorylated Tau levels in primary neuron cells of 3×Tg-AD mice. (A) After treatment with Tan IIA (5.0 μM, 24 h), the Tau, pS396-Tau, and pS404-Tau protein expression levels in primary neuron cells of the 3×Tg-AD mice were detected by western blot analysis. β-Actin was used as a loading control. The data were summarized from three independent experiments, ***p < 0.001. (B) Immunofluorescence staining was performed via double-staining of Map 2 (in red) and Tau (in green) in Tan IIA-treated primary neuron cells of 3×Tg-AD mice. The nuclei were stained with DAPI (in blue). Scale bar, 50 μm.

Figure 5. Tan IIA对Tau纤维的聚集抑制作用和TanIIA在Tau中的结合位点Aggregation inhibition of Tau fibrils by Tan IIA and the putative binding site of Tan IIA in Tau. (A) Relative ThT fluorescence intensity of 3R Tau aggregation with or without Tan IIA incubation, ***p < 0.001. (B) TEM images of 3R Tau aggregation with or without Tan IIA incubation. Scale bar, 500 nm. (C) rmsd values of all the atoms of the Tau−Tan IIA complex with respect to its initial structure as a function of time. (D) Predicted binding mode of Tan IIA in the Tau binding pocket obtained from MD simulation.

总之，在本研究中，观察到Tan IIA可以降低Tau的表达，在N2A细胞中降低Tau磷酸化，Tau过表达细胞和3×Tg-AD小鼠原代神经元细胞。基于分析，假设Tan IIA可能是增加多泛素化Tau积累，诱导Tau蛋白的蛋白酶体降解。此外，Tan IIA抑制体内肝素诱导Tau纤维前体的形成和Tan IIA结合到Tau。因此，预防或治疗使用TanIIA可进一步探讨AD和头病理相关的神经退行性疾病。

原文详见：

https://pubs.acs.org/doi/10.1021/acs.jafc.9b07022