Investigating The Mechanism By Which Cistanche Deserticola Treats Alzheimer's Disease Using Network Pharmacology And Experimental Validation

2 Results

2.1 Screening of the main active ingredients and ingredient targets of Cistanche deserticola

Cistanche deserticola was searched through the TCMSP database and the Swiss ADME platform, and literature search resulted in 8 main active ingredients, namely suchilactone, yangambin, quercetin, arachidonate, marckine, acteoside, echinacoside, and beta-sitosterol, with a total of 307 ingredient targets.

2.2 Screening of disease targets and Venn diagram preparation, protein-protein interaction network (PPI) construction, and GO and KEGG

Enrichment analysis was performed through OMIM, GeneCards, and NCBI databases, and a total of 2 013 AD-related targets were obtained after screening. Import the Cistanche deserticola component targets and AD-related targets into the Venny diagram online website, and obtain a total of 207 drug-disease intersection targets, and draw a Venn diagram, as shown in Figure 1A. Import the 207 intersection targets into the STRING database to analyze the drug-disease interaction. Then import the data file into the Cytoscape software, and after the degree, closeness and betweenness numerical screening, 42 targets and 593 edges are obtained. According to the degree size sorting, TNF, Akt1, CASP3, PPARG, EGFR, MMP9, ESR1, HIF1A are selected as key target genes, and the protein-protein interaction network diagram is drawn (Figure 1B).

In order to explain the life process of the targets involved in the treatment of AD by Cistanche deserticola, the metascape database was used to perform GO enrichment analysis on the 42 targets obtained above, including three parts: cellular component (CC), molecular function (MF), and biological process (BP). The projects were screened with the standard of P≤0.05, and the top ten cellular components, molecular functions, and biological processes were drawn into bubble diagrams using the Microbiology Online Drawing Website (Figure 1C); the DAVID online tool was used to perform KEGG enrichment analysis on 42 common key targets of diseases and drugs, and the projects with P≤0.05 were screened. The results showed that a total of 172 signal pathways were enriched. The top 20 pathways were selected according to the P value, and the bubble diagram was drawn using the Microbiology Online Platform (Figure 1D). The enrichment results showed that the mechanism of Cistanche deserticola in treating AD may be closely related to the PI3K-Akt pathway, cancer pathway, adhesion focal and other pathways.

Fig 1 (A) Alzheimer's disease targets and cistanche component targets Venn chart; (B) Protein-protein interaction networks; (C) GO enrichment analysis bubble diagram; (D) KEGG Enrichment analysis bubble diagram

2.3 Preparation of drug-ingredient-disease-pathway-target map

By combining the KEGG enrichment results with the PPI network construction results, a drug-ingredient-disease-pathway-target network can be drawn (Figure 2). Blue triangle: drug Cistanche deserticola; orange triangle: disease AD; light green circle: main ingredient of the drug; yellow circle: main pathway related to the disease treated by Cistanche deserticola; dark green rectangle: drug-disease related target; edge: correlation between nodes

Fig 2 Total distance traveled by mice in each group of the place navigation test before going to the stage ( x  s, mm)

Tab 3 Escape latency of mice in each group of the place navigation test ( x  s, s)

*Note: Compared with SAMRI group, P < 0.05; Compared with Model group, &dagger;P < 0.05; L-GCS and H-GCS groups, &Dagger;P < 0.05.

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Fig 3 Percentage of time spent in the target quadrant and number of times the platform traversed by each group of mice in the probe trial testing

2.4.2 Y maze experiment

The results of the Y maze experiment showed that: compared with the SAMR1 group, the Model group's time to explore novel arms and the alternation rate were significantly reduced (P<0.05); compared with the Model group, the M-GCs group and H-GCs group's novel The arm exploration time was significantly increased (P<0.05); the spontaneous alternation rate of new arms in the GCs-administered group was significantly higher than that in the Model group (P<0.05), among which the spontaneous alternation rate of the M-GCs group was higher than that of the other administration groups ( P <0.05) (Fig. 4).

2.5 Effects of GCs on the number and morphology of mouse hippocampal neurons

Nissl staining results showed that the neuronal pyramidal cells in the SAMR1 group were round or oval, with complete shape and neat arrangement. The intracellular Nissl bodies were deeply stained and the number was larger. Compared with the SAMR1 group, the neuronal pyramidal cells in the Model group were The morphology of the somatic cells was broken, the arrangement was scattered, and Nissl bodies were lightly colored, and the number of complete pyramidal cells was significantly reduced (P<0.05); compared with the Model group, the morphology of the pyramidal cells in the GCs administration group was more complete and the arrangement was more complete. Relatively neat, the number of complete pyramidal cells increased significantly (P<0.05). Among them, most pyramidal cells in the M-GCs group were complete in shape and more neatly arranged. The number of complete pyramidal cells was significantly higher than that in other drug administration groups. increased (P <0.05), as shown in Figure 5.

2.4.2 Y maze experiment

The results of the Y maze experiment showed that: compared with the SAMR1 group, the Model group's time to explore novel arms and the alternation rate were significantly reduced (P<0.05); compared with the Model group, the M-GCs group and H-GCs group's novel The arm exploration time was significantly increased (P<0.05); the spontaneous alternation rate of new arms in the GCs-administered group was significantly higher than that in the Model group (P<0.05), among which the spontaneous alternation rate of the M-GCs group was higher than that of the other administration groups ( P <0.05) (Fig. 4).

2.5 Effects of GCs on the number and morphology of mouse hippocampal neurons

Nissl staining results showed that the neuronal pyramidal cells in the SAMR1 group were round or oval, with complete shape and neat arrangement. The intracellular Nissl bodies were deeply stained and the number was larger. Compared with the SAMR1 group, the neuronal pyramidal cells in the Model group were The morphology of the somatic cells was broken, the arrangement was scattered, and Nissl bodies were lightly colored, and the number of complete pyramidal cells was significantly reduced (P<0.05); compared with the Model group, the morphology of the pyramidal cells in the GCs administration group was more complete and the arrangement was more complete. Relatively neat, the number of complete pyramidal cells increased significantly (P<0.05). Among them, most pyramidal cells in the M-GCs group were complete in shape and more neatly arranged. The number of complete pyramidal cells was significantly higher than that in other drug administration groups. increased (P <0.05), as shown in Figure 5.

2.6 Effect of GCs on the expression of PI3K-AKT pathway related proteins and Tau protein in mouse hippocampus

Immunohistochemical staining results showed that compared with the SAMR1 group, the number of positive cells expressing PI3K and P-Akt in the hippocampal CA1 area in the Model group was significantly reduced (P<0.05), and the number of positive cells expressing P-Tau was significantly increased (P < 0.05); compared with the Model group, the number of positive cells in the hippocampal CA1 area of PI3K and P-Akt in the GCs group was significantly increased (P<0.05), and the number of positive cells expressing P-Tau was significantly reduced (P<0.05 ); among them, the number of positive cells expressing PI3K and P-Akt in the CA1 area of mouse hippocampus was significantly increased in the M-GCs group compared with other GCs administration groups (P<0.05), see Figure 6.

Fig 7 Western blot experimental strip chart

Tab 4 Strip absorbance value ( x  s )

*Note: Compared with SAMRI group, P < 0.05; Compared with Model group, &dagger;P < 0.05; L-GCS and H-GCS groups, &Dagger;P < 0.05.

3 Discussion

Alzheimer's disease is a common neurodegenerative disease. As a chronic disease with insidious onset and continuous progression, it is characterized by abnormal clinical manifestations such as memory, language, cognition, personality and behavioral functions[15,16]. At present, the pathogenesis of Alzheimer's disease is complex, and there are many theories such as A&beta; amyloid deposition, oxidative stress of neurons, neuroinflammation, abnormal phosphorylation of Tau protein and intestinal flora imbalance. Among them, the inflammatory apoptosis of neurons caused by Tau protein phosphorylation is an important mechanism of AD-induced cognitive impairment[17,18]. Tau protein is a microtubule-associated protein (MAPs) associated with neuronal axons. Its main function is to promote the formation of microtubules, maintain their stability and regulate axonal transport[19,20]. Excessive phosphorylation of Tau protein will lose its ability to bind to microtubules, causing microtubule depolymerization, axonal dysfunction and neurofibrillary tangles, leading to neuronal degeneration and apoptosis[21]. Studies have found that a variety of related signaling pathways are closely related to the phosphorylation of Tau protein, among which the intracellular phosphatidylinositol kinase/protein kinase B (PI3K/AKT) pathway plays a key role in the phosphorylation of Tau protein, and its abnormal expression can have a significant impact on the growth, development, and apoptosis of nerve cells [22].

In the above-mentioned network pharmacology study, GO and KEGG enrichment analysis showed that the I3K/Akt pathway plays a key role in the improvement of AD by Cistanche deserticola, and is closely related to cell apoptosis, senile plaque formation, and neuronal tangles; the common targets of GCs and AD were screened through the STRING database, among which Akt1 was an important key target and a key protein in the PI3K/Akt pathway. PI3K, as the upstream molecule of Akt, is a specific intracellular phosphatidylinositol kinase [23]. In mammals, the PI3K family has type I, II, and III isozymes, of which type I isozymes are the main regulatory targets in the PI3K-Akt pathway. When the IA type isozyme of PI3K binds to proteins such as tyrosine receptor kinases, PI3K can be phosphorylated and activated, phosphorylating the PIP2 target outside the cell membrane to become the PIP3 target, which attracts and phosphorylates Akt1, a subtype of Akt[24-26]. Members of the Akt family have the ability to regulate biological functions, including cell survival, proliferation, metabolism, and growth. On the one hand, phosphorylated Akt1 can phosphorylate the cell death agonist (BAD) associated with &beta;-cell CLL/lymphoma (Bcl-2) at serine residue 136, thereby inhibiting cell apoptosis[27]. On the other hand, it can regulate downstream proteins mTOR, GSK3&beta;, and FOXO, thereby reducing the production of P-Tau protein[28-30].
Since the increase of P-Tau protein can be regarded as a hallmark pathological feature of AD, it is believed to be closely related to cognitive decline and synaptic loss. Therefore, the activation of PI3K/Akt signaling pathway may play an important role in improving Alzheimer's disease.

Cistanche deserticola is the fleshy stem with dried scaly leaves of the plant Cistanche deserticola. It is warm in nature, sweet and salty in taste, and belongs to the kidney and large intestine meridians. It is a medicine for tonifying the kidney and strengthening yang, moistening the intestines and relieving constipation[31]. It has the effects of anti-apoptosis, anti-oxidation, regulating cell autophagy and neuroendocrine, enhancing physical strength, and improving learning and cognition. It has important value in the prevention and treatment of central nervous system diseases[32]. Many literatures record that Cistanche deserticola can protect and excite amino acids in the autophagy of neurons injured by cerebral ischemia-reperfusion, improve neuronal oxidative stress in dementia rats, protect microglial inflammatory response induced by lipopolysaccharide, and enhance the immune function of aging rats[33-36]. The above behavioral experiments showed that compared with the Model group, the mice in the GCs group had significant improvements in learning and cognition (P<0.05); Nissl staining experiments found that the number of pyramidal cells in the GCs group was significantly increased compared with the Model group, with complete morphology and neat arrangement (P<0.05); immunohistochemistry and Western blot experiments found that GCs could significantly increase the expression of PI3K and P-Akt proteins in the hippocampus of SAMP8 mice and significantly reduce the expression of P-Tau protein (P<0.05), which indicates that Cistanche deserticola may play a role in improving Alzheimer's disease by regulating the PI3K-Akt signaling pathway and affecting the production of P-Tau. This study used SAMP8 mice to construct an Alzheimer's disease model. Through network pharmacology, the main mechanism of action of Cistanche deserticola in treating Alzheimer's disease was explored, and a preliminary discussion of the mechanism of action and the optimal drug dose were explored. The subsequent exploration will continue to explore the mechanism of action and efficacy of Cistanche deserticola in improving the learning and cognitive function of AD model animals, in order to provide more modern scientific basis for the development of Cistanche deserticola products for the prevention and treatment of AD.