Xixin Decoction's novel mechanism for alleviating Alzheimer's disease cognitive dysfunction by modulating amyloid-β transport across the blood-brain barrier to reduce neuroinflammation

Abstract

Purpose: Xixin Decoction (XXD) is a classical formula that has been used to effectively treat dementia for over 300 years. Modern clinical studies have demonstrated its significant therapeutic effects in treating Alzheimer's disease (AD) without notable adverse reactions. Nevertheless, the specific mechanisms underlying its efficacy remain to be elucidated. This investigation sought to elucidate XXD's impact on various aspects of AD pathology, including blood-brain barrier (BBB) impairment, neuroinflammatory processes, and amyloid-β (Aβ) deposition, as well as the molecular pathways involved in these effects.

Methods: In vitro experiments were conducted using hCMEC/D3 and HBVP cell coculture to establish an in vitro blood-brain barrier (BBB) model. BBB damage was induced in this model by 24-h exposure to 1 μg/mL lipopolysaccharide (LPS). After 24, 48, and 72 h of treatment with 10% XXD-medicated serum, the effects of XXD were assessed through Western blotting, RT-PCR, and immunofluorescence techniques. In vivo, SAMP8 mice were administered various doses of XXD via gavage for 8 weeks, including high-dose XXD group (H-XXD) at 5.07 g kg^-1·d^-1, medium-dose XXD group (M-XXD) at 2.535 g kg^-1·d^-1, and low-dose XXD group (L-XXD) at 1.2675 g kg^-1·d^-1. Cognitive function was subsequently evaluated using the Morris water maze test. BBB integrity was evaluated using Evans blue staining, and protein expression levels were analyzed via ELISA, Western blotting, and immunofluorescence.

Results: In vitro experiments revealed that XXD-containing serum, when cultured for 24, 48, and 72 h, could upregulate the expression of P-gp mRNA and protein, downregulate CB1 protein expression, and upregulate CB2 and Mfsd2a protein expression. In vivo studies demonstrated that XXD improved spatial learning and memory abilities in SAMP8 mice, reduced the amount of Evans blue extravasation in brain tissues, modulated the BBB-associated P-gp/ECS axis, RAGE/LRP1 receptor system, as well as MRP2 and Mfsd2a proteins, and decreased the accumulation of Aβ in the brains of SAMP8 mice. Additionally, XXD upregulated the expression of TREM2, downregulated IBA1, TLR1, TLR2, and CMPK2 expression, and reduced the levels of pro-inflammatory factors NLRP3, NF-κB p65, COX-2, TNF-α, and IL-1β in the hippocampal tissues.

Conclusion: XXD may exert its effects by regulating the P-gp/ECS axis, the RAGE/LRP1 receptor system, and the expression of MRP2 and Mfsd2a proteins, thereby modulating the transport function of the BBB to expedite the clearance of Aβ, reduce cerebral Aβ accumulation, and consequently inhibit the activation of microglia induced by Aβ aggregation. This process may suppress the activation of the CMPK2/NLRP3 and TLRs/NF-κB pathways, diminish the production of inflammatory cytokines and chemokines, alleviate neuroinflammation associated with microglia in the brain of AD, and ultimately improve AD pathology.

Keywords: Alzheimer’s disease; Xixin Decoction; amyloid-beta; blood-brain barrier; neuroinflammation.

FIGURE 1

Schematic diagram of the mechanism by which BBB damage in the AD brain aggravates Aβ accumulation and promotes neuroinflammation.

FIGURE 2

Screening of optimal concentrations of LPS and XXD medicated serum. (A) Effects of different concentrations of LPS on the viability of hCMEC/D3 cells after 24, 48, and 72 h of incubation; (B) Effects of different concentrations of LPS on the viability of HBVP cells after 24, 48, and 72 h of incubation; (C) Effects of different concentrations of XXD medicated serum on the viability of LPS-induced hCMEC/D3 cells; (D) Effects of different concentrations of XXD medicated serum on the viability of LPS-induced HBVP cells. MTT assay; **p < 0.01; *p < 0.05.

FIGURE 3

XXD medicated serum promotes P-gp expression in the blood-brain barrier cell model. (A) Comparison of P-gp mRNA expression; (B) and (C) Comparison of relative P-gp protein expression; (D) and (F) Comparison of P-gp protein positive expression in hCMEC/D3 cells; (E) and (G) Comparison of P-gp protein positive expression in HBVP cells. Scale bar: 20 μm; images at ×200 magnification; **p < 0.01, *p < 0.05.

FIGURE 4

XXD medicated serum upregulates CB2 and Mfsd2a expression and downregulates CB1 expression in the blood-brain barrier cell model. (A) and (B) Comparison of relative CB1 protein expression; (C) and (D) Comparison of relative CB2 protein expression; (E) and (F) Comparison of relative Mfsd2a protein expression; **p < 0.01, *p < 0.05.

FIGURE 5

XXD improves learning and spatial memory impairments in SAMP8 mice and reduces Evans blue extravasation. (A) Escape latency (s) in the place navigation test (n = 8); (B) Time spent in the target quadrant (s) in the spatial probe test (n = 8); (C) Number of crossings over the platform location in the spatial probe test; (D) and (E) Comparison of Evans blue extravasation in the brains of different groups of mice (n = 4); images at ×400 magnification; **p < 0.01, *p < 0.05.

FIGURE 6

XXD Modulates the P-gp/eCBs Axis in the Hippocampal CA1 Region of SAMP8 Mice; (A) and (B) Comparison of relative P-gp protein expression levels; (C) and (D) Comparison of relative CB1 protein expression levels; (E) and (F) Comparison of relative CB2 protein expression levels; (G) and (H) Immunohistochemical staining for P-gp in the hippocampal CA1 region of mice; (I) and (J) Immunohistochemical staining for CB1 in the hippocampal CA1 region of mice; (K) and (L) Immunohistochemical staining for CB2 in the hippocampal CA1 region of mice; (n = 6); scale bar: 20μm; images captured at ×400 magnification; **p < 0.01, *p < 0.05.

FIGURE 7

XXD Regulates the RAGE/LRP1 Receptor System and MRP2, Mfsd2a Proteins, Accelerating Aβ_1-42 Clearance in the Brains of SAMP8 Mice; (A), (B), and (C) Comparison of relative RAGE and LRP1 protein expression levels; (D) and (E) Comparison of RAGE protein positive expression in the hippocampal CA1 region; (F) Comparison of hippocampal Aβ_1-42 content; (G), (H), and (I) Comparison of relative MRP2 and Mfsd2a protein expression levels; (n = 6); Scale bar: 20μm; images captured at ×400 magnification; **p < 0.01, *p < 0.05.

FIGURE 8

XXD Upregulates TREM2 Expression and Downregulates IBA1, TLR1, and TLR2 Expression, Inhibiting Microglial Activation; (A) and (E) Analysis of relative IBA1 protein expression levels and average fluorescence intensity of IBA1 in the hippocampal CA1 region of mice; (B) and (F) Analysis of relative TLR1 protein expression levels and average fluorescence intensity of TLR1 in the hippocampal CA1 region of mice; (C) and (G) Analysis of relative TLR2 protein expression levels and average fluorescence intensity of TLR2 in the hippocampal CA1 region of mice; (D) and (H) Analysis of relative TREM2 protein expression levels and average fluorescence intensity of TREM2 in the hippocampal CA1 region of mice; (n = 6); Scale bar: 20μm; images captured at ×400 magnification; **p < 0.01, *p < 0.05.

FIGURE 9

XXD reduces the levels of inflammation-related molecules and cytokines in the hippocampus: (A) Comparison of relative expression levels of CMPK2 protein in the hippocampus of different groups of mice; (B) Comparison of NLRP3 protein levels in the hippocampus of different groups of mice; (C) Comparison of IBA1 protein levels in the hippocampus of different groups of mice; (D) Comparison of COX-2 protein levels in the hippocampus of different groups of mice; (E) Comparison of NF-κB p65 protein levels in the hippocampus of different groups of mice; (F) Comparison of IL-1β protein levels in the hippocampus of different groups of mice; (G) Comparison of TNF-α protein levels in the hippocampus of different groups of mice. (n = 6); **p < 0.01, *p < 0.05.