Objective

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To construct and evaluate the animal model of Alzheimer's disease （AD） with deficiency-impairment-stasis-toxin syndrome using a multifactorial combined induction approach.

Methods

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Twenty-four 2-month-old SPF C57BL/6J mice were randomly divided into a blank control group and low- （0.02%）， medium- （0.04%）， and high （0.06%）-dose carrageenan groups （n=6 per group）. A carrageenan-induced blood stasis model was established to screen for optimal carrageenan intervention concentrations. Thirty 5-month-old APP/PS1 mice were randomly divided into toxin-impairment group，

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-deficiency group， kidney-deficiency group， deficiency-impairment syndrome group， and deficiency-impairment-stasis-toxin syndrome group， with 6 mice per group. Additionally， six 5-month-old C57BL/6J mice from the same litter served as the litter-matched control group. A multifactorial combined induction experiment was conducted using sleep deprivation， carrageenan， and hydrocortisone. Specifically， sleep deprivation induced

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deficiency for mice in

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-deficiency group， deficiency-impairment syndrome group， and deficiency-impairment-stasis-toxin syndrome group； hydrocortisone solution gavage induced kidney deficie

ncy for mice in kidney-deficiency group， deficiency-impairment syndrome group， and deficiency-impairment-stasis-toxin syndrome group； carrageenan solution intraperitoneal injection induced blood stasis for mice in deficiency-impairment-stasis-toxin syndrome group. Throughout the modeling period， general status and body weight were observed and recorded weekly. On the day after modeling concluded， grip index was detected by grip meter； learning and memory ability was detected by water maze； organ indexes were calculated by combining the weights of spleen， kidney， testis and body weight； hippocampal histopathology was detected by Nissl staining； aortic pathology was analyzed by HE staining； Aβ

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deposition in the hippocampus was detected by immunofluorescence； The expression levels of vascular adhesion molecule （VCAM-1） and intercellular adhesion molecule （ICAM-1）， tumor necrosis factor （TNF-α）， interleukin-6 （IL-6）， interleukin-1β （IL-1β）， cortisol， and adrenocorticotropic hormone （ACTH） were detected by enzyme-linked immunosorbent assay （ELISA）； the expression levels of amyloid precursor protein （APP）， progeroid protein-1 （PS1） and β-site amyloid precursor protein cleaving enzyme （BACE1） were detected by Western blot.

Results

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The 0.06% concentration of carrageenan was selected for subsequent experiments. Multifactorial combined induction experiment： compared with the same-litter control group， mice in deficiency-impairment-stasis-toxin syndrome group exhibited reduced body weight and grip index after modeling， shorter platform stay time， fewer platform crossings， decreased spleen index， kidney index， and testis index， elevated hippocampal tissue Aβ

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fluorescence expression， and increased serum VCAM-1， TNF-α， IL-6， IL-1β， and cortisol levels in serum increased， while hippocampal tissue expression of APP， BACE1， and PS1 proteins elevated （

P

＜0.05 or

P

＜0.01）. Neuronal damage was observed in the hippocampal region， characterize

d by nuclear condensation and dissolution or disappearance of Nissl bodies.

Conclusion

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The mouse models of AD with deficiency-impairment-stasis-toxin syndrome were established through combined induction by sleep deprivation， carrageenan， and hydrocortisone， which exhibits the syndrome characteristics of

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deficiency， kidney deficiency， and blood stasis.