Yishen Huazhuo decoction regulates microglial polarization to reduce Alzheimer's disease-related neuroinflammation through TREM2

Abstract

Background: Aging is the primary risk factor for the onset of Alzheimer's disease (AD). Inflamma-aging is a major feature in the process of aging, and the chronic neuroinflammation caused by inflamma-aging is closely related to AD. As the main participant of neuroinflammation, the polarization of microglia (MG) could influence the development of neuroinflammation.

Objective: This study aims to observe the impact of YHD on microglia (MG) polarization and neuroinflammation to delay the onset and progression of AD.

Methods: In vivo experiment, four-month senescence accelerated mouse prone 8 (SAMP8) were used as the model group, the SAMR1 mice of the same age were used as the control group. In YHD group, 6.24 g/kg YHD was intragastrically administrated continuously for 12 weeks, and Ibuprofen 0.026 g/kg in positive control group. Morris Water Maze test was used to evaluate the learning and memory ability, Nissl's staining and immunofluorescence double staining for neuron damage and MG M1/M2 polarization, Enzyme-Linked Immunosorbent Assay (ELISA) for neuroinflammation biomarkers in hippocampus, Western blot for key protein expression of TREM2/NF-κB signaling pathway. In vitro experiments, 10 μM/l Aβ_1-42 induced BV-2 cell model was used to re-verify the effect of YHD regulating MG polarization to reduce neuroinflammation. Also, TREM2 small interfering RNA (siRNA) was used to clarify the key target of YHD.

Results: YHD could improve the learning and memory ability of SAMP8 mice evaluated by the Morris Water Maze test. Like Ibuprofen, YHD could regulate the M1/M2 polarization of MG and the levels of neuroinflammatory markers TNF-α and IL-10 in hippocampus, and relieve neuroinflammation and neuron loss. In addition, YHD could also regulate the expression of PU.1, TREM2, p-NF-κB P65 in the TREM2/NF-κB signaling pathway. Further in vitro experiments, we found that YHD had a significant regulatory effect on Aβ_1-42-induced BV-2 cell polarization, and it could significantly increase PU.1, TREM2, decrease p-NF-κB P65, p-IKKβ, TNF-α, IL-6, IL-1β. At the same time, using siRNA to inhibit TREM2, it proved that TREM2 was a key target for YHD to promote Aβ_1-42-induced BV-2 cell M2 polarization to reduce neuroinflammation.

Conclusions: YHD could regulate the TREM2/NF-κB signaling pathway through TREM2, thereby to adjust MG polarization and reduce AD-related neuroinflammation.

Keywords: Alzheimer's disease; Microglial polarization; Neuroinflammation; TREM2; Yishen Huazhuo decoction (YHD).

Fig. 1

Typical chromatograms of mixed standard compound (A), YHD lyophilized powder (B) and Shichangpu (Acorus gramineus) water solution (C). 1.Echinacoside, 2.Verbascoside, 3.2,3,5,4-Tetrahydroxystilbene-2-O-β-D-glucoside, 4.Ferulicacid. Ligustroflavone, 6.Icariin.

Fig. 2

YHD improves the learning and memory ability of SAMP8 mice in MWM. (A) Swimming speed on the first day. (B) Swimming path in the navigation experiment on the fifth day. (C, D) Escape latency changes of each group. (E) Swimming path in the spatial probe test. (F) The numbers of platform crossing. The number of crossing platforms are expressed as Median (IQR), swimming speed and escape latency are expressed as Mean ± SD (n = 15), *P < 0.05 vs R1 group; #P < 0.05 vs P8 group.

Fig. 3

Influence of YHD on M1/M2 polarization, neuroinflammation and neuron loss in hippocampus of SAMP8 mice. (A) The Nissl's staining results of the neurons in hippocampal CA1 area (scale bar = 50 μm), the red arrow indicating the lack of neurons. (B) Quantifications of neuron count in hippocampal CA1 area. (C, D) The ELISA test results of TNF-α and IL-10. (E) The immunofluorescence double staining results of MG in hippocampal CA1 area (scale bar = 100 μm). Blue fluorescence indicating nucleus, green fluorescence for iNOS (white arrow), and red fluorescence for Arg-1 (yellow arrow). (F, G) Quantifications of the average optical density value of iNOS and Arg-1. The data are represented by Mean ± SD (n = 3), *P < 0.05 vs R1 group; #P < 0.05 vs P8 group.

Fig. 4

Influence of YHD on PU.1, TREM2, and p–NF–κB p65 proteins in hippocampus of SAMP8 mice. The data are represented by Mean ± SD (n = 3), *P < 0.05 vs R1 group; #P < 0.05 vs P8 group.

Fig. 5

YHD reducing the toxic effect of Aβ_1-42 on BV-2 cells. (A) CCK-8 method to detect the vitality of BV-2 cells after YHD treatment. (B) CCK-8 detection of the vitality of BV-2 cells after treated by Aβ_1-42. (C) Using 10 μM/l Aβ_1-42 to induce BV-2 cells to establish cell models, and CCK-8 method to detect the effects of YHD on the vitality of cell models. (D) The effects of 50 μg/ml and 100 μg/ml YHD on the LDH release of BV-2 cells after treated by10 μM/l Aβ_1-42. The data are represented by Mean ± SD (n = 3), *P < 0.05 vs control group; #P < 0.05 vs Aβ group; △ P < 0.05 vs Aβ+YHD50 group.

Fig. 6

YHD inhibiting the activation of BV-2 cells induced by Aβ_1-42 and promoting its polarization from M1 to M2. (A) Inverted optical microscope of BV-2 cells (scale bar = 100 μm). (B–D) The ELISA test results of TNF-α, IL-1β, and IL-6 (E) BV-2 cells immunofluorescence double-stained results (scale bar = 100 μm). Blue fluorescence indicating the nucleus, green fluorescent for iNOS, and red fluorescence for Arg-1. (F, G) Quantifications of the relative fluorescence density of iNOS and Arg-1. (H) The mRNA expression of PU.1 and TREM2 in BV-2 cells by qRT-PCR. (I, J) Quantification of protein expression levels of PU.1, TREM2, p-IKKβ, p–NF–κB P65 in BV-2 cells by Western blot. The data are represented by Mean ± SD (n = 3), *P < 0.05 vs control group; #P < 0.05 vs Aβ group; △ P < 0.05 vs Aβ+YHD50 group.

Fig. 7

TREM2 siRNA transfection partially reversed the effect of YHD improving BV-2 cells polarization and neuroinflammation. (A–C) Western blot and qRT-PCR for TREM2 by Control siRNA and different TREM2 siRNA transfection. (D–F) The ELISA test results of TNF-α, IL-1β, and IL-6. (G) The immunofluorescence double staining results of Aβ_1-42-induced BV-2 cells after jointly treated by 100 μg/ml YHD and TREM2 siRNA (scale bar = 100 μm). Blue fluorescence indicating the nucleus, green fluorescent for iNOS, and red fluorescence for Arg-1. (H, I) Quantifications of the relative fluorescence density of iNOS and Arg-1. (J) qRT-PCR for TREM2 mRNA of Aβ_1-42-induced BV-2 cells after jointly treated by 100 μg/ml YHD and TREM2 siRNA. (K, L) Quantification of protein expression levels of TREM2, p-IKKβ, p–NF–κB P65. The data are represented by Mean ± SD (n = 3), *P < 0.05 vs Aβ+Con siRNA group; #P < 0.05 vs Aβ+Con siRNA + YHD100 group; △ P < 0.05 vs Aβ+TREM2 siRNA group.

Fig. 8

Schematic diagram depicting that YHD can regulate the TREM2/NF-κB signaling pathway through TREM2, thereby to adjust MG polarization and reduce AD-related neuroinflammation, and ultimately playing a role in neuroprotection and delaying learning and memory ability decline.