Experimental verification about treatment of Bu-Shen-Yi-Jing-Fang in Alzheimer's disease by the analysis of the feasible signaling pathway of network pharmacology.

CONTEXT: Bu-shen-yi-jing-fang (BSYJF) has been reported to reduce amyloid-ß (Aß)1-42 deposition in the brain of APP/PS1 mice and ameliorate cognitive function. However, its neuroprotective mechanism remains unclear. OBJECTIVE: This study aims to investigate whether BSYJF exerts a protective effect on Aß1-42-induced oxidative stress injury and explore its possible mechanism. MATERIALS AND METHODS: The platform databases TCMSP, Swiss, TTD, DrugBank, and GeneCards were used to mine the targets of Alzheimer's disease (AD) and BSYJF. The platform databases STRING and Metascape were used to build the interaction network of the target protein, and Cytoscape software was used to analyze this network and screen out the key pathways. Aß1-42-treated SKNMC cells were established to verify the mechanism of BSYJF and the key proteins. The downstream proteins and antioxidants as well as apoptosis and ferroptosis of the PI3K/AKT/Nrf2 signaling pathway were validated using an in vitro SKNMC cell model experiment. The expression levels of related proteins were detected using Western blotting. Flow cytometry and immunofluorescence staining were used to analyze apoptosis and ferroptosis. RESULTS: Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis considered the key signal pathways, mainly involving the PI3K/AKT signaling pathway. Experimental validation demonstrated that BSYJF treatment markedly increased the activity of the PI3K/AKT pathway, which could exert anti-AD effects. CONCLUSIONS: Our data provided compelling evidence that the protective effects of BSYJF might be associated with their regulation of the PI3K/AKT/Nrf2 signaling pathway. These studies offered a potential therapy for natural herbal medicine treatment of AD.

Analysis of risk factors for fatty liver disease in children with Wilson's disease.

BACKGROUND AND AIMS: Many children with Wilson's disease are complicated with dyslipidemia. The aim of this study was to investigate the risk factors for the development of fatty liver disease (FLD) in children with Wilson's disease. METHODS: We evaluated sex, age, weight, the disease course, treatment course, clinical classification, alanine transaminase (ALT), aspartate transaminase, γ-glutamyl transpeptidase, total biliary acid, triglyceride, total cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, homocysteine, uric acid, fibrinogen (FBG), creatinine, procollagen III N-terminal propeptide, laminin, hyaluronic acid, type IV collagen, and performed receiver operating characteristic curve analysis to investigate the forecast value of individual biochemical predictors and combined predictive indicators to evaluate FLD in Wilson's disease. RESULTS: The multivariate logistic regression analysis revealed that ALT [odds ratio (OR), 1.011; 95% confidence interval (CI), 1.004-1.02; P  = 0.006], uric acid (OR, 1.01; 95% CI, 1.002-1.018; P  = 0.017), FBG (OR, 3.668; 95% CI, 1.145-13.71; P  = 0.037), creatinine (OR, 0.872; 95% CI, 0.81-0.925; P  < 0.001), and laminin (OR, 1.01; 95% CI, 1.002-1.018; P  = 0.017) acted as independent risk factors in Wilson's disease complicated with FLD. The receiver operating characteristic curves for combined predictive indicators demonstrated an area under the curve values of 0.872, which was found to be a significant predictors for FLD in Wilson's disease. CONCLUSIONS: We screened out the most important risk factors, namely ALT, uric acid, creatinine, FBG, and laminin for Wilson's disease complicated with FLD. The joint prediction achieved is crucial for identifying children with Wilson's disease complicated with FLD.

Impact of antifibrotic therapy on disease progression, all-cause mortality, and risk of acute exacerbation in non-IPF fibrosing interstitial lung diseases: evidence from a meta-analysis of randomized controlled trials and prospective controlled studies.

BACKGROUND: Nintedanib and pirfenidone are preferred pharmacological therapies for patients with idiopathic pulmonary fibrosis (IPF). However, evidence favoring antifibrotic therapy in patients with non-IPF fibrosing interstitial lung diseases (ILD) is limited. OBJECTIVE: To investigate the effects of antifibrotic therapy on disease progression, all-cause mortality, and acute exacerbation (AE) risk in patients with non-IPF fibrosing ILDs. DESIGN: Meta-analysis. DATA SOURCES AND METHODS: Electronic databases were searched for articles published before 28 February 2023. Studies that evaluated the efficacy of antifibrotic agents in patients with fibrosing ILDs were selected. The primary outcome was the disease progression risk, and the secondary outcomes included all-cause mortality and AE risk. The GRADE criteria were used for the certainty of evidence assessment. RESULTS: Nine studies with 1990 participants were included. Antifibrotic therapy reduced the rate of patients with disease progression (five trials with 1741 subjects; relative risk (RR), 0.56; 95% CI, 0.42-0.75; p < 0.0001; I2 = 0; high-certainty evidence). Antifibrotic therapy did not significantly decrease all-cause mortality (nine trials with 1990 subjects; RR, 0.76; 95% CI, 0.55-1.03; p = 0.08; I2 = 0; low-certainty evidence). However, in patients with progressive fibrosing ILDs (PF-ILD), antifibrotic therapy decreased all-cause mortality (four trials with 1100 subjects; RR, 0.69; 95% CI, 0.48-0.98; p = 0.04; I2 = 0; low-certainty evidence). CONCLUSION: Our study supports the use of antifibrotic agents in patients with PF-ILDs, which could slow disease progression and decrease all-cause mortality. TRIAL REGISTRATION: This study protocol was registered with PROSPERO (registration number: CRD42023411272).

Tetrahydroalstonine possesses protective potentials on palmitic acid stimulated SK-N-MC cells by suppression of Aß1-42 and tau through regulation of PI3K/Akt signaling pathway.

Alzheimer's disease (AD) is the most common neurodegenerative disease. The morbidity of Alzheimer's disease is currently on the rise worldwide, but no effective treatment is available. Cornus officinalis is an herb and edible plant used in traditional Chinese medicine, whose extract has neuroprotective properties. In this investigation, we endeavored to refine a systems pharmacology strategy combining bioinformatics analysis, drug prediction, network pharmacology, and molecular docking to screen tetrahydroalstonine (THA) from Cornus officinalis as a therapeutic component for AD. Subsequent in vitro experiments were validated using MTT assay, Annexin V-PI flow cytometry, Western blotting, and immunofluorescence analysis. In Palmitate acid-induced SK-N-MC cells, THA restored the impaired PI3K/AKT signaling pathway, regulated insulin resistance, and attenuated BACE1 and GSK3ß activity. In addition, THA significantly reduced cell apoptosis rate, down-regulated relative levels of p-JNK/JNK, Bax/Bcl-2, cytochrome C, active caspase-3 and caspase-3, and attenuated Palmitate acid-induced Aß1-42 and Tau generation. THA may regulate the phenotype of AD and reduce cell apoptosis by modulating the PI3K/AKT signaling pathway. This systematic analysis provides new ramifications concerning the therapeutic utility of tetrahydroalstonine for AD.

Network pharmacology analysis and experimental validation of Anemarrhenae Rhizoma in treating Alzheimer's disease. / çŸ¥æ¯æ²»ç–—é˜¿å°”èŒ¨æµ·é»˜ç— ç½‘ç»œè¯ç†å­¦ç ”ç©¶åŠå®žéªŒéªŒè¯.

OBJECTIVES: To explore the mechanism of Anemarrhenae Rhizoma in treatment of Alzheimer's Disease (AD). METHODS: The active ingredients and targets of Anemarrhenae Rhizoma for treatment of AD were screened with network pharmacology methods, the protein-protein interaction (PPI) network was constructed and the core targets were analyzed. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways enriching analysis was performed. The peripheral blood lymphocytes were extracted and lymphoblastoid cell lines (LCL) were constructed and an in vitro cell model of LCL-SKNMC was established. MTT and CCK-8 methods were used to quantify SKNMC/LCL cells, 2 ´, 7 ´-dichlorodihydrofluorescein diacetate (DCFH-DA) probe was used to detect reactive oxygen species (ROS), and immunofluorescence staining was used to detect the generation of Aß1-42 in a co-cultured model. Western blotting was used to detect protein expression in the co-culture model. The lifespan of N2 nematodes was observed under oxidative stress, normal state, and heat stress; ROS generated by N2 nematodes was detected by DCFH-DA probes. The paralysis time of CL4176 N2 nematodes was evaluated by paralysis assay, and Aß deposition in the pharynx was detected by Thioflavin S staining. RESULTS: Through network pharmacology, 15 potential active ingredients and 103 drug-disease targets were identified. PPI analysis showed that the Anemarrhenae Rhizoma might play anti-AD roles through albumin, Akt1, tumor necrosis factor, epidermal growth factor receptor (EGFR), vascular endothelial growth factor A (VEGFA), mammalian target of rapamycin (mTOR), amyloid precursor protein (APP) and other related targets. KEGG analysis showed that the pharmacological effects of Anemarrhenae Rhizoma might involve the biological processes of Alzheimer's disease, endocrine resistance, insulin resistance; and neuroactive ligand-receptor interaction, phosphatidylinositol 3-kinase (PI3K)-Akt signaling pathway, calcium signaling pathway, AGE-RAGE signaling pathway in diabetes complications, neurotrophic factor signaling pathway and others. The in vitro cell experiments showed that Anemarrhenae Rhizoma was able to reduce the production of ROS and Aß1-42 (both P<0.01), inhibit the expression of ß-secretase 1 (BACE1), APP and Aß1-42 proteins (all P<0.05), up-regulate the expression of p-PI3K/PI3K, p-AKT/AKT, p-GSK3ß/GSK3ß in SKNMC cells (all P<0.05). The in vivo studies further confirmed that Anemarrhenae Rhizoma prolonged the lifespan of C. elegans under stress and normal conditions, reduced the accumulation of ROS and the toxicity of Aß deposition. CONCLUSIONS: Anemarrhenae Rhizoma may reduce the production of Aß in AD and inhibit its induced oxidative stress, which may be achieved by regulating the PI3K/Akt/GSK-3ß pathway.

Harpagide alleviate neuronal apoptosis and blood-brain barrier leakage by inhibiting TLR4/MyD88/NF-κB signaling pathway in Angiotensin II-induced microglial activation in vitro.

Angiotensin II, the effector peptide of the renin-angiotensin system, is not only a pivotal peptide implicated in the regulation of blood pressure but also a key mediator of the inflammatory processes that play an important role in the pathology of hypertension-related cSVD. Harpagide is the major bioactive constituent of Scrophulariae Radix widely used in traditional Chinese medicine for numerous diseases including hypertension. The present study aimed to investigate the effect of harpagide on Ang II-induced neuroinflammation and the potential mechanism. Pretreated with harpagide or resatorvid (the TLR4 pathway inhibitor), BV2 cells were treated with Ang II or LPS (the TLR4 activator). NO, pro-inflammatory cytokines, the proteins on TLR4/MyD88/NF-κB signaling pathway and the expression of CD86, CD206, TREM2 in BV2 cells were detected respectively. Subsequently, the effects of harpagide on neurotoxicity and BBB destruction triggered by Ang II-induced neuroinflammation were investigated in the co-cultures of BV2 microglia/HT22 hippocampal neurons, BV2 microglia/bEnd.3 endotheliocyte and BV2 microglia/BBB monolayer model. We found that Ang II converted microglia into M1 state and resulted in neuroinflammation through activating TLR4/MyD88/NF-κB signaling pathway. It also triggered the imbalance of TLR4/TREM2 in microglia. Ang II-mediated inflammation microglia further led to neuronal apoptosis and BBB damage. Harpagide showed the effect of alleviating Ang II-mediated neuroinflammation as well as the resulting neurotoxicity and BBB destruction through inhibiting the TLR4/MyD88/NF-κB pathway. The anti-inflammatory and neuroprotective effect of harpagide suggested that it might be a potential therapeutic strategy in hypertensive cSVD.

Effects of on behavior and blood-brain barrier in Alzheimer's disease mice.

To investigate the effects of on behavior and blood brain barrier (BBB) in Alzheimer's disease mice. Thirty-eight 4-month-old APP/PS1 double transgenic mice were randomly divided into three groups: model group, low-dose group and high-dose group. Saline, and 12 g·kg·d were given to each group by continuous gavage once a day for respectively. The changes in activities of daily live and fear conditioning memory behavior of mice were examined by nesting behavior test and fear conditioning test, respectively. The ß-amyloid protein (Aß) depositions in cortex and hippocampal CA1 area of mice were detected by thioflavin T staining. The CD34 and activities fibrinogen (Fib) immunofluorescence double staining were used to determine the vascular endothelial integrity and BBB exudation. Compared with model mice, activities of daily live were significantly improved in low-dose and high-dose groups (both <0.01), the fear memory ability was significantly increased in high-dose group (<0.01). The amount of Aß deposition in cortex and hippocampal CA1 decreased significantly in high-dose group, the area ratio decreased significantly; the area ratio of Aß deposition in hippocampal CA1 region in low-dose group also decreased (all <0.05). The proportions of CD34 positive area of cortex in low and high dose groups increased, the percentage of fibrinogen positive area decreased (all <0.05). The proportion of CD34 positive area in hippocampal CA1 region in high-dose group was significantly increased, the percentage of fibrinogen positive area decreased significantly (both <0.05). especially high-dose can improve the activities of daily live and fear conditioning memory function of APP/PS1 mice, reduce the deposition of Aß in brain. The mechanism may be related to the reduction of BBB permeability and the protection of the integrity of BBB.

Cycloastragenol inhibits Aß1-42-induced blood-brain barrier disruption and enhances soluble Aß efflux in vitro.

This study aimed to evaluate the effect Cycloastragenol (CAG), a triterpenoid saponin isolated from the Radix astragali, on Aß-induced BBB damage. An immortalized endothelial cell line (bEnd.3) was employed to mimic a BBB. The Western blot, TUNEL staining, Flow cytometric analysis and Enzyme-linked immunosorbent assay were performed. The present results showed that CAG (10, 50, 75 µM) can alleviate oligomer Aß1-42 induced bEnd.3 cell apoptosis and increase tight junction scaffold proteins expression. The result also indicated that CAG increased soluble Aß efflux across BBB via upregulation of the P-gp and downregulation of RAGE expression.[Formula: see text].

Gandouling Tablets Inhibit Excessive Mitophagy in Toxic Milk (TX) Model Mouse of Wilson Disease via Pink1/Parkin Pathway.

OBJECTIVE: Gandouling (GDL) tablet is a Chinese patent medicine approved by the National Medical Product Administration, which is used to treat Wilson disease (WD) in China. In this study, we aimed to investigate the effects of GDL on mitophagy in the hippocampus in the toxic milk (TX) mouse model of WD. METHODS: Mice were randomly divided into the following four groups: control, Wilson (model group), D-penicillamine (DPA), and GDL groups. The animal behaviors were evaluated by the water maze experiment, traction test, and pole test. Transmission electron microscopy was used for the detection of mitochondrion structure. An enzyme-linked immunosorbent assay (ELISA) was performed for the analysis of the changes in liver function. Colocalization of mitophagy-related proteins was detected by fluorescence microscopy. Western blotting (WB) and reverse transcription-polymerase chain reaction (RT-PCR) were conducted for the detection of protein expression and mRNA levels, respectively. RESULTS: Significant reduction in neurological impairments was observed in the WD model group. All of these results were significantly reversed by GDL intervention. Compared with the levels in the Wilson group, the levels of alanine aminotransferase (ALT), aspartate transaminase (AST), total bilirubin (TBIL), and albumin (ALB) changed obviously. Colocalization between mitophagy-related proteins pink1, parkin, and mitochondria was changed significantly. The mitophagy-related mRNA (pink1, parkin, and LC3II) and protein expression levels (pink1, parkin, and the rate of LC3II/LC3I) were decreased significantly, while p62 was remarkably increased after GDL intervention. CONCLUSION: Our findings indicated that the neuroprotective mechanism of GDL may occur via the inhibition of excessive mitophagy through the regulation of the pink1/parkin pathway in the TX mouse brain of WD.

Hyperoside protects the blood-brain barrier from neurotoxicity of amyloid beta 1-42.

Mounting evidence indicates that amyloid ß protein (Aß) exerts neurotoxicity by disrupting the blood-brain barrier (BBB) in Alzheimer's disease. Hyperoside has neuroprotective effects both in vitro and in vivo against Aß. Our previous study found that hyperoside suppressed Aß1-42-induced leakage of the BBB, however, the mechanism remains unclear. In this study, bEnd.3 cells were pretreated with 50, 200, or 500 µM hyperoside for 2 hours, and then exposed to Aß1-42 for 24 hours. Cell viability was determined using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide assay. Flow cytometry and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay were used to analyze cell apoptosis. Western blot assay was carried out to analyze expression levels of Bax, Bcl-2, cytochrome c, caspase-3, caspse-8, caspase-9, caspase-12, occludin, claudin-5, zonula occludens-1, matrix metalloproteinase-2 (MMP-2), and MMP-9. Exposure to Aß1-42 alone remarkably induced bEnd.3 cell apoptosis; increased ratios of cleaved caspase-9/caspase-9, Bax/Bcl-2, cleaved caspase-8/caspase-8, and cleaved caspase-12/caspase-12; increased expression of cytochrome c and activity of caspase-3; diminished levels of zonula occludens-1, claudin-5, and occludin; and increased levels of MMP-2 and MMP-9. However, hyperoside pretreatment reversed these changes in a dose-dependent manner. Our findings confirm that hyperoside alleviates fibrillar Aß1-42-induced BBB disruption, thus offering a feasible therapeutic application in Alzheimer's disease.

Morroniside prevents H2O2 or Aß1-42-induced apoptosis via attenuating JNK and p38 MAPK phosphorylation.

Amyloid-ß peptide (Aß) plays a causal role in the development and progression of Alzheimer's disease (AD). Oxidative stress and activation of mitogen-activated protein kinase (MAPK) are involved in Aß-induced neurotoxicity. Morroniside, one active monomer of dry ripe sarcocarp of Cornus officinalis, has shown antioxidant properties in several cell lines. The present study investigated the protective actions of morroniside against the cytotoxicity produced by exposure to H2O2 or Aß1-42 in rat pheochromocytoma (PC12) cells. Exposure of PC12 cells to 150 µM H2O2 or 20 µM Aß1-42 down-regulated anti-apoptotic protein expression (Bcl-2), up-regulated pro-apoptotic protein expression (Bax, cytochrome C, and cleaved caspase-3), increased JNK and p38 MAPK phosphorylation and finally caused significant cell death. This effect was reversed by pretreatment with morroniside in a dose-dependent manner. Among the selective inhibitors of MAPKs, the JNK inhibitor (SP600125) and p38 MAPK inhibitor (SB203580) showed steady preventive effect against H2O2 or Aß1-42-induced apoptosis. The results suggest that different from the selective inhibitors of MAPKs, morroniside can inhibit H2O2 or Aß1-42-induced apoptotic pathway activation through suppressing its upstream signaling components of JNK and p38 MAPK phosphorylation simultaneously.

Catalpol provides a protective effect on fibrillary Aß1-42 -induced barrier disruption in an in vitro model of the blood-brain barrier.

Excessive amyloid beta (Aß) deposition in brain is mainly responsible for cell damage and blood-brain barrier (BBB) disruption in Alzheimer's disease (AD). Catalpol, an iridoid glucoside extracted from the root of Rehmannia glutinosa Libosch, has neuroprotective effect against AD. It is unclear whether catalpol has a protective effect on Aß-induced BBB leakage. We employed an immortalized endothelial cell line (bEnd.3) and astrocytes co-culture to mimic a BBB model in vitro and investigated the effect of catalpol on BBB. We found that treatment with catalpol decreased BBB hyperpermeability induced by fibrillar Aß1-42 . Data from western blotting showed that catalpol prevented fibrillar Aß1-42 -induced bEnd.3 cell apoptosis through mitochondria-dependent and death receptor pathways; decreased the levels of matrix metalloproteinases (MMPs), MMP-2, MMP-9, and the receptor for advanced glycation end products; and increased the levels of tight junction proteins (ZO-1, occludin, and claudin-5), low-density lipoprotein receptor-related protein 1, and P-glycoprotein in fibrillar Aß1-42 -treated bEnd.3 cells. Moreover, catalpol also enhanced soluble Aß efflux across the fibrillar Aß1-42 -treated bEnd.3 cells BBB monolayer model. Altogether, our results suggest that catalpol alleviate fibrillar Aß1-42 -induced BBB disruption, enhance soluble Aß clearance, and offer a feasible therapeutic application in AD treatment.

Sortilin-related VPS10 domain containing receptor 1 and Alzheimer's disease-associated allelic variations preferentially exist in female or type 2 diabetes mellitus patients in southern Han Chinese.

BACKGROUND: Both in vitro and in vivo, overexpression of the sortilin-related VPS10 domain containing receptor 1 (SORCS1) protein lowers amyloid-ß generation. Recent studies have shown that SORCS1 variations in intron 1 are associated with sporadic Alzheimer's disease (SAD), but the results remain inconsistent. METHODS: In order to clarify the role of the SORCS1 gene in southern Han Chinese, we genotyped eight single nucleotide polymorphisms (SNP) of SORCS1 in 128 SAD patients and 92 healthy controls. RESULTS: By dividing patients and controls according to apolipoprotein status, sex and whether they had type 2 diabetes mellitus, we found that rs7907690 C allele frequencies were significantly higher in the Alzheimer's disease (AD) patients with type 2 diabetes mellitus than in the controls (P=0.041). Also, the rs600879 GG genotype and G allele worked as protective factors of SAD in women (GG genotype, P=0.007; G allele, P=0.009). In multilocus analysis, the frequency of an eight-single nucleotide polymorphism rs601883/rs7907690/rs600879/rs17277986/rs2900717/rs10884399/rs11193170/rs4918280 CCGGACGG haplotype was significantly higher in AD patients (6.3%), especially in female AD patients (9.5%), than in the controls (0.5%) (P=0.003; P=0.0002). However, the CTGGACGG haplotype was significantly lower in AD patients (9.3%) than in controls (20.3%) (P=0.001). The association remained significant even after Bonferroni correction for the number of haplotypes. CONCLUSION: This study provides evidence that variations in the SORCS1 gene influence susceptibility to SAD in southern Han Chinese. The genetic link between AD and SORCS1 gene variations are influenced by ethnic background, sex and whether an individual has type 2 diabetes mellitus.

Is there an association of regulatory region polymorphism in the alpha-1-antichymotrypsin gene with sporadic Alzheimer's disease in the northern Han-Chinese population?

Both invitro and invivo alpha-1-antichymotrypsin (ACT) directly inhibits amyloid beta peptide (Abeta) degradation and promotes Abeta deposition. However, whether the genetic variants in the regulatory region (including the promoter and the two enhancers) of the ACT gene affect susceptibility to Alzheimer's disease (AD) remains controversial. Here, we screened ACT promoter and enhancers in 244 patients with sporadic Alzheimer's disease (SAD) and 205 control patients, both of north Han-Chinese origin. Four single nucleotide polymorphisms (SNP) were found: (i) 11510T/C (rs10145747, named as ACT1); 11496G/A (rs4375593, ACT2); (iii) 11491T/C (rs4508366, ACT3); and (iv) 51G/T (rs1884082, ACT4). Neither individual SNP nor haplotypes were associated with AD onset. We concluded that the effect of the variations in the ACT regulatory region must be very limited, if occurring at all.