The Treatment Effects And Target Molecular Network Exploration Of Chaihu-shugan San On APP/PS1 AD Mice

Background:Alzheimer’s disease(AD),known as senile dementia,is a chronic degenerative disease of the central nervous system characterized by memory loss and cognitive dysfunction,which is progressively aggravated.The main pathologic features of AD are senile plaques,neurofibrillary tangles,amyloid angiopathy,granular vacuolar degeneration and loss of neurons.Previous studies found that among the patients over 65 years of age,the incidence of female patients is almost three times that of male.Studies have shown that this was associated with decreased estrogen levels in women after menopause.At present,there is no effective treatment for AD.During past decades,single-target drugs showed limited efficacyn or intolerable side effects.Thus,developing multicomponent and multi-target drugs,such as Traditional Chinese medicine,is a potential and promising method to treat AD.Chinese medicine compound Chaihu-Shugan San(CSS)from "Jing Yue Quan Shu",containing Bupleurum,Cyperus rotundus,Chuanxiong,Citrus aurantium,Peony,Dried tangerine peel andLicorice seven herbs,is widely used to treat gastrointestinal diseases,gynecological diseases and mental neuropathy.In this study,the effects of Chaihu-ShuganSan on the cognitive and learning and memory ability of APP/PS1 Alzheimer’s disease transgenic mice were studied by modern techniques and methods based on the perspectives ofpharmacology,pharmacodynamics and intestinal microbiology,and detected ingredient of Chaihu-ShuganSan.And provide the theoretical basis for drug quality control,preparation and clinical application of Chaihu-Shugan San.Methods:4-month-old male APP/PS1 transgenic mice were randomly divided into model group(V),Chaihu-Shugan San high dose group(H,1.4g/kg),low dose group(L,0.7g/kg)and western medicine Aricept(A,2mg/kg)and the negative control group(C).Themice were administrated once daily for 8 weeks.Effects of Chaihu-Shugan San on cognition of mice were detectednew object recognition and Morris water maze.Immunohistochemistry was used to detect the deposition of senile plaques and estrogen receptor expression in APP/PS1 mice.The levels of estrogen in hippocampus and cortex of mice were detected by ELISA.The 16sRNA gene V3-V4 region of the experimental mice was subjected to high-throughput sequencing to detect the changes of intestinal microbial flora.The MRI was used to detect the biochemical indexes such as NAA,Cr,Cho and Tau in the brain.The liver and kidney function was evaluatedby serum biochemical indexs.Then,UFLC-MS/MS combined method was used to detect the effective chemical composition and quality of Chaih-Shugan San prepared by steam extraction and ultrasonic extraction.The effective chemical composition of Chaihu-Shugan San was established byaccurate means of quality testing,which provides a new method for quality control of Chaihu-Shugan San.Finally,according to the network pharmacologyand database analysis,we established the disease-target network and found AD-related targets and pathways.Results:Behavioral experimental results showed that Chaihu-Shugan San significantly improved thecognitive ability of APP/PS1,andreduced the deposition of senile plaques in the brain.MRI showed no difference in brain biochemical markers.Liver and kidney function indicators showed that the dosage of Chaihu-Shugan San had no side effects on APP/PS1 mice.The results of the screening of intestinal flora showed that,compared with the wild group,Clostridium,Roseburia,Polospore,Oscillospira,Prevotella,Butyricicoccus significantly decreasedin model group.Pullicaecorumwas significantly higher in the high and low dose groups,the wild group and the Aricept group compared with the model group at the species level.The results showed that theChaihu-Shugan San-treated mice had higher flora abundance and diversity than the wild group,and the changes of the flora of the model mice could be reversed after the drug intervention.It was concluded that Chaihu-Shugan San could change the intestinal flora of APP/PS1 mice.The results of UFLC-MS/MS mass spectrometry showed that the technology had high efficiency.The results showed that the content of paeoniflorin and methyl gallate prepared by steam extraction was 1.5 times higher than that of ultrasonic extraction.In the samples prepared by steam extraction,the main contents of Bupleurum were the saikosaponin B1 and B2,while the ultrasonic extraction of samples of Bupleurum D is higher.Chaihu-Shugan San 7 Chinese medicine has been reported to consist of 670 kinds of ingredients,butin line with oral use and drug-like screening conditions,only 40 speciesin our detection of Chaihu-Shugan San Recipe 17 ingredients.Andthere are 8 ingredientsto meet the network pharmacology screening conditions.The results showed that the active target of quercetin was the most relevant and there were384 in the active ingredient of Chaihu-Shugan San.Among the 40 components of Chaihu-Shugan San,there were 17 components targeting ER(α/β).Construction of disease-target network results showed that there were 59 AD-related targets,and the best target was CASP3,followed by MAPK1,MAPK3,CASP7,CASP8 and FAS.Conclusion:Chaihu-ShuganSanmay improve the cognitive function of APP/PS1 transgenic mice through regulating the expression of estrogen and estrogen receptor(ERα/β)in the brain.Meanwhile,it can reverse thealtered intestinal microflora of APP/PS1 mice.Additionally,network pharmacology analysis indicated that Chaihu-ShuganSan may play a positive role in AD-related disease by targeting other proteins,such as CASP3 and FAS.All the results may provide theoretical basis for further investigation and clinical application of Chaihu-Shugan San.