基于以方测证的阿尔茨海默病脾肾亏虚、痰瘀互结证动物模型的构建及评价OA

This study aims to establish a standardized mouse model of Alzheimer's disease(AD)with spleen-kidney deficiency and stagnant phlegm syndrome(AD-SKDSP)based on TCM theory,so as to provide a disease-syndrome combined model that aligns with the TCM diagnosis and treatment paradigm of"disease-syndrome-formula-efficacy"for modern research on AD prevention and treatment.Four-month-old male double-transgenic APP/PS1 mice were used as AD model animals.A standardized animal model of AD-SKDSP was constructed by high-sugar and high-fat diet feeding combined with ice-water bath and tail-clamping stimulation.The mice were randomly divided into an AD model group,an AD-SKDSP group,an AD Zhinao Capsule group,and a normal control group consisting of same-litter and age-matched male C57BL/6J mice.Corresponding drug treatments were administered at designated time points.During the eight-week modeling period,the following parameters were measured:physical sign scores,grip strength,body weight,24-hour food intake,24-hour fecal water content,female mouse fertility,Morris water maze performance,nose-tongue-collateral-foot color,hippocampus detected by hematoxylin-eosin(HE)staining,Aβ1-42 and brain-derived neurotrophic factor(BDNF)detected by immunohistochemistry,whole blood and plasma viscosity,2-hour D-xylose,testosterone(T),estradiol(E2),calcium(Ca),phosphorus(P),bone Gla protein(BGP),hippocampal synapsin(SYN)and postsynaptic density protein 95(PSD-95)mRNAs,and SYN,PSD-95,and BDNF proteins.The results showed that by the end of the 4th week,compared with the normal control group,the AD model group,AD-SKDSP group,and AD Zhinao Capsule group exhibited progressively increased physical sign scores and 24-hour fecal water content,and progressively decreased grip strength,body weight,and 24-hour food intake(P<0.05,P<0.01).Compared with the AD model group,the AD-SKDSP group and AD Zhinao Capsule group showed significantly increased physical sign scores and 24-hour fecal water content,along with significantly reduced grip strength,body weight,and 24-hour food intake(P<0.05,P<0.01).From the 5th week onward,compared with the AD-SKDSP group,the AD Zhinao Capsule group demonstrated significant reductions in physical sign scores and 24-hour fecal water content,as well as significant increases in grip strength,body weight,and 24-hour food intake with prolonged intragastric administration of Zhinao Capsule(P<0.05,P<0.01).By the end of the 8th week,compared with the normal control group,the AD model group and AD-SKDSP group exhibited significantly decreased female fertility,corrected R/G/B values of nose-tongue-collateral-foot,hippocampal BDNF expression,levels of 2-hour D-xylose,T,E2,Ca,P,and BGP,hippocampal SYN and PSD-95 mRNA expression,and SYN,PSD95,and BDNF protein expression.Meanwhile,platform latency,hippocampal Aβ1-42 expression,and whole blood and plasma viscosity(low,medium,and high shear rates)were significantly increased,while platform crossings and target quadrant swimming time were markedly reduced(P<0.05,P<0.01).Hippocampal CA1 neurons in these groups displayed partial loss of normal morphology,with pyknotic or swollen nuclei,deep blue staining,disorganized distribution,and a thickness of"3-5"layers.Compared with the AD model group,the AD-SKDSP group showed significant reductions in female fertility,corrected R/G/B values of nose-tongue-collateral-foot,hippocampal BDNF expression,levels of 2-hour D-xylose,T,E2,Ca,P,and BGP,hippocampal SYN and PSD-95 mRNA expression,and SYN,PSD95,and BDNF protein expression,significant increases in platform latency,hippocampal Aβ1-42 expression,and whole blood and plasma viscosity(low,medium,and high shear rates),and significant decreases in platform crossings and target quadrant swimming time(P<0.05,P<0.01).The hippocampal CA1 neurons exhibited irregular shapes,increased nuclear pyknosis,intensified deep blue staining,a thickness of"1-3"layers,and chaotic distribution.Compared with the AD-SKDSP group,the AD Zhinao Capsule group demonstrated significant increases in female fertility,corrected R/G/B values of nose-tongue-collateral-foot,hippocampal BDNF expression,levels of 2-hour D-xylose,T,E2,Ca,P,and BGP,hippocampal SYN and PSD-95 mRNA expression,and SYN,PSD95,and BDNF protein expression,significant decreases in platform latency,hippocampal Aβ1-42 expression,and whole blood and plasma viscosity(low,medium,and high shear rates),and significant increases in platform crossings and target quadrant swimming time(P<0.05,P<0.01).The hippocampal CA1 neuronal pathology was markedly alleviated.In summary,guided by the holistic concept and syndrome differentiation theory of TCM and on the basis of characteristics of"spleen deficiency","kidney deficiency",and"intermingled phlegm and blood stasis",this study successfully established a standardized AD-SKDSP animal model by combining a high-sugar and high-fat diet with ice-water bath and tail-clamping stimulation for eight weeks.This modeling method exhibits strong controllability,minimal physicochemical stimulation,reproducibility,and verifiability,providing a stable and standardized disease-syndrome combined animal model for future research on the"disease-syndrome-formula-efficacy"paradigm in AD-SKDSP.