Novel Compatibility of Huanglianjiedu Decoction in Behavioral and Psychological Symptoms of Dementia in Alzheimer's Disease1


 Background: Alzheimer's disease (AD) is characterized by progressive cognitive decline. Behavioral and psychological symptoms in dementia (BPSD) are another critical characterization of AD besides cognitive deficit. However, the pharmacological treatment of BPSD remains challenging. HuanglianJiedu decoction (HLJDD), which consists of four herbs, is applied to treat Alzheimer's disease in traditional Chinese medicine, especially AD with BPSD. While the frequently used compatibility of HLJDD, whose principal ingredient is Coptidis Rhizoma (Huang-lian, CR), may not be suitable for treating BPSD. Elucidating the mechanism underlying each herb is critical to HLJDD's pertinent compatibility.Methods: We utilized network pharmacology to analyze the herbs' targets and biological processes for treating BPSD in HLJDD and employed molecular docking to explore the binding activity between herbs' main active ingredients and neurotransmitter receptors. Results: Results showed that Scutellariae Radix (Huang-qin, SR) and Phellodendri Chinrnsis Cortex (Huang-bai, PCC) have better anti-BPSD effects than CR and Gardeniae Fructus (Zhi-zi, GF). SR has a better anti-neuroinflammation function, can better regulate blood vessels. PCC has a higher binding affinity with Dopamine D2 receptor (DRD2) and 5-hydroxytryptamine receptor 2A (HTR2A). CR and GF may be better for neuronal signaling.Conclusion: For treating BPSD, SR and PCC are the principal ingredients while CR and GF are the ancillary herbs.


Background
In traditional Chinese medicine (TCM), herbs combined into a formula to reinforce the overall effects or eliminate the adverse effects [1] , which is called the compatibility of TCM. Although formulas generally have conventional compatibility, it is essential to consider novel compatibility with different diseases. In our research, we proposed an innovative method for the compatibility of herbs based on molecular mechanisms. We investigate HLJDD's novel compatibility in BPSD.
BPSD appears in more than 90% of AD patients, including anxiety, agitation, aggression, irritation, depression, apathy, disinhibition, delusions, or hallucinations [2] . Molecules that involve in BPSD pathogenesis have been researched by mounting studies. Neuroin ammation, a response that involves neurons and microglia, has been reported to characterize many neurodegenerative diseases and neuropsychiatric conditions, resulting in the elevated production of pro-in ammatory cytokines, like IL-6, TNF-α, IL-8, IL-4, etc. Microglia activation is the rst sign of neuroin ammation. Activated microglia can release various oxidants such as reactive oxygen species and activate several genes and proteins, such as inducible nitric oxide synthase [3] .
Neurotransmitters and their receptors are considered to play a potential role in BPSD [4] . Genetic risk factors offer a powerful approach for the elucidation of mechanisms underlying BPSD. APOE epsilon4, the most recognized genetic risk factor for late-onset AD, increasing the risk of BPSD [5] . However, the e cacy of antipsychotics for the treatment of BPSD is scanty. Antipsychotic drugs that are often used to treat BPSD have extrapyramidal severe side effects (EPS).
Memantine, the NMDA receptor antagonist, a cure for treating moderate-to-severe AD, can reduce the antipsychotic-induced EPS [6] but had a controversial effect in treating BPSD. Acetylcholinesterase inhibitors, the current primary medication for AD, is controversial when treating BPSD [7] . Many researchers turn their attention to natural products as an alternative or complementary method to BPSD for their clinical e cacy with minimal side effects.
BPSD has different TCM syndrome differentiation. "Toxin damaging brain collaterals," an essential differentiation of BPSD, is put forward by academician Yongyan Wang in the study process of dementia. For this syndrome differentiation, the brain collaterals are injured by the toxins of re. For the treatment, purging re for removing toxins is commonly used. HLJDD is a classic prescription for heat-clearing away and detoxifying, which is composed of SR (Huangqin), CR (Huang-lian), PCC (Huang-bai), and Gardeniae Fructus (Zhi-zi). Studies show that HLJDD is frequently applied for Alzheimer's disease [8] , especially with BPSD [9] . While the TCM physicians seldom treat BPSD only with HLJDD. Making out the principal herbs is essential to BPSD, for the intrinsic compatibility of HLJDD maybe not suitable. To better understand HLJDD's compatibility in treating BPSD, we utilized network pharmacology and molecular docking to provide useful drugs for BPSD therapeutics by emphasizing their molecules' activities.

Material And Methods
Herbs' main active ingredients and their matched potential target proteins A natural plant contains various chemical compounds. Our study used the TCM systems pharmacology database and analysis platform (TCMSP, https://tcmspw.com/index.php) to detect herbs' main active ingredients. And ltered compounds by terms of oral bioavailability (OB) ≥ 30%, drug-likeness (DL) ≥ 0.18, drug half-life (HL) ≥ 4 h. Then predicted the active ingredients' potential molecular targets by the search tool for interactions of chemicals database (STITCH, http://stitch.embl.de/) with the species limited to "Homo sapiens." Potential targets prediction for behavioral and psychological symptoms of dementia in Alzheimer's disease We identi ed BPSD-associated protein targets by the GeneCards database (http://www.genecards.org/) [10] , with a higher rank score, higher correlation with BPSD. The searched keywords were "behavioral and psychological symptoms of dementia in Alzheimer's disease." We chose the top 50% of predicted targets as the potential targets for BPSD.

Network construction and analysis
We constructed the "compounds-targets-BPSD" networks by Cytoscape 3.7.2 software [11] . The .csv format les whose data combined compounds targets with BPSD targets were imported into Cytoscape. The node size was based on the target proteins score values provided by the GeneCards database. The overlapping targets between active compounds and BPSD were the herbs' putative targets related to BPSD and set with a red rectangle node. Memantine was applied to choose the potential neurotransmitter receptors in this process.

Annotation enrichment analysis of target proteins
We performed Gene Ontology (GO) functional enrichment analysis using the ClueGO plugin [12] , with the species limited to "Homo sapiens" and p-value < 0.05.

Molecular docking for active compounds with DRD2 and HTR2A
Molecular docking studies were conducted using AutoDock software [13] to evaluate the active compounds' binding a nity with neurotransmitter receptors.
The neurotransmitter receptors DRD2 and HTR2A were selected as the docking receptor. The memantine's mechanism on BPSD determined to choose them.
We downloaded the proteins' crystal structure from the RCSB Protein Data Bank (http://www.rcsb.org/). We chose 6CM4 and 6A93, whose ligand is Risperdal as the crystal structure. Then memantine, Risperdal, and the active compounds were docked with DRD2 and HTR2A. The binding pocket of DRD2 and HTR2A was set up regarding 6CM4 and 6A93.

Results
HLJDD's main active compounds and potential target proteins After searching the TCMSP database, We sifted out 80 compounds in HLJDD. SR, CR, PCC, and GF have 32, 10, 27, and 11 ingredients, respectively. Then import those compounds into the STITCH database to achieve the potential target proteins (Table 1).
"Compounds-targets-BPSD" network and GO biological process analysis In this network, the red rectangular nodes represent herbs' key protein targets of BPSD were the most in SR, followed by PCC, 24, and 15, respectively. For further research of the herbs' key protein targets, we applied GO biological process analysis. Fig. 1 b, c, d, and Tab.2 show the biological processes of the key target proteins of SR, include positive regulation of smooth muscle cell proliferation, lipopolysaccharide-mediated signaling pathway, regulation of neuroin ammatory response, etc. Fig. 2 b, c, d, and Tab.3 reveal the biological processes of the key target proteins of CR. Its biological processes are cellular response to cadmium ion, response to nicotine, and negative regulation of macroautophagy. Fig. 3 b, c, d, and Tab.4 illustrate PCC's key target proteins' biological processes. Include response to nicotine, glial cell apoptotic process, plasma lipoprotein particle, etc. Fig. 4 b, c, d, and Tab. 5 show the biological processes of the key target proteins of GF, which are mainly associated with regulation of amyloid-beta formation, regulation of membrane protein ectodomain proteolysis and regulation of nitric oxide biosynthetic process.
"Compounds-targets-BPSD" network of memantine Patients with moderate to severe AD exhibit relatively severe cognitive and psychological symptoms. N-Methyl-D-aspartic acid (NMDA) is one of the main treatments. Memantine is the most prevalent choice of NMDA [14] . In our research, we took memantine as a reference drug. We did the "compounds-targets-BPSD" network of memantine to search its potential mechanism on BPSD, especially for the neurotransmitter receptor. The network result showed that DRD2 and HTR2A were the neurotransmitter receptors that worked on BPSD. We chose DRD2 and HTR2A to do molecular docking for their crystal structure to be better docked and studied with Risperdal ligand in the PDB database.

Results of molecular docking
Serotonin and its receptors, particularly the HTR2A, are considered to play a potential role in cognitive behaviors and psychiatric conditions such as depression, schizophrenia, and AD [15] . A multitarget-directed ligand, acting on HTR2A and DRD2, exerting an anti-aggressive and antipsychotic activity, posing a promising strategy for the treatment of BPSD [16] . In our study, the active ingredients were selected as the docked compounds with DRD2 and HTR2A. The 3D structures of the active ingredients were downloaded in .mol2 format from the TCMSP database. They were later converted into .pdb format with the Open Babel GUI software [17] . We used the local search parameters and rigid lename of the macromolecule model when docking. We set the software's default values as our docking parameters. We set grid box size as follows: DRD2: x-dimension: 62, y-dimension: 84, z-dimension: 126, spacing: 0.375, X center:

Discussion
TCM plays an essential role in medical diagnosis and treatments. Based on the TCM theory. Chinese formulas contain a mixture of herbs, which combined the following compatibility principle "monarch, minister, assistant, and guide," meaning herbs play a primary, secondary, auxiliary, or harmonic roles, respectively [18] . The primary herbs are substances that provide the main therapeutic thrust. The second primary herbs enhance or assist the therapeutic actions of the rst.
The rest serve one of the following functions: treat accompanying symptoms, moderate the harshness or toxicity of the primary ones, guide the medicine to the proper organs, or exert a harmonizing effect [19] . In HLJDD, CR acts as the monarch role. SR plays the minister role. PCC and GF were the assistant and guide role. However, this compatibility is not suitable for treating BPSD. Our research studied drugs from the molecular perspective, which provides a novel method for the compatibility of formulas. Our results showed that SR and PCC are the principal herbs, CR and GF are the assistant herbs.
BPSD is a critical neuropsychiatric feature in Alzheimer's disease [4] . In Alzheimer's disease, abnormal accumulation of amyloid-β released from amyloid precursor protein and neuroin ammation are the partially pathologic hallmarks. Accumulation of amyloid-β also causes indirect injury to neurons by inducing neuroin ammation [20] . Microglia, the resident innate immune cells in the brain, is pivotal for the immune response observed in AD, acting as sentinel and protective cells, but may become inappropriately reactive in AD to drive neuropathology [21] . Lipopolysaccharide (LPS) is a gram-negative bacterial endotoxin released from the cell wall component that contributes to in ammation in the body. LPS is involved in the regulation of the expression of potent in ammatory factors [22] . Studies showed that with age, microglia exhibit enhanced sensitivity to in ammatory stimuli, similar to that observed in brains with ongoing neurodegeneration [23] . An increasing number of data has linked schizophrenia with neuroin ammatory conditions and microglia, which have been related to the pathogenesis of schizophrenia. Evidence suggests that neuroin ammatory changes observed in schizophrenia involve abnormal astrocyte functions [24] . In our research, Scutellaria baicalensis has anti-in ammatory roles in the biological process of treating BPSD, including regulating neuroin ammatory response, mediating lipopolysaccharide-mediated signaling pathway, cellular response to interleukin-6, and regulation of interferon -α production. GF can regulate amyloid-beta formation in treating BPSD.
Smooth muscle cell proliferation, especially vascular smooth muscle cells, is essential during cell growth or injury [25] . Blood vessels composed of vascular smooth muscle cells play a crucial role in normal brain function for its integrity structure and function and supplying adequate blood. Cerebral blood ow shortfalls and blood-brain barrier dysfunction are early ndings in neurodegenerative disorders. Cerebral blood ow reductions, impaired cerebrovascular reactivity, and impaired hemodynamic responses are increasingly recognized in AD's early stages [26] . Platelet, a critical factor for blood ow, is an anucleate cell in blood, whose principal function is to stop bleeding by forming aggregates for hemostatic reactions. Platelet aggregates are also involved in pathological thrombosis and play an essential role in in ammation [27] . Nitric oxide (NO) is a small free radical molecule with an endothelium-derived relaxing factor. Its adequate production levels in the vascular endothelium are critical for regulating blood ow and vasodilation. Besides, NO plays a vital neuronal signaling role [28] . Cadmium, a metal that resembles zinc and calcium, is also crucial for neuronal signaling. Cadmium exposure is linked to neurodegenerative diseases like Alzheimer's disease. It can alter neurotransmitters' release, cause oxidative stress, damage mitochondrion, and induce apoptosis [29] . In our study, SR can positively regulate smooth muscle cell proliferation, positively regulate the endothelial cell proliferation, and positively regulate the blood vessel endothelial cell migration. CR can mediate cellular response to cadmium ion. PCC can regulate platelet activation. GF can regulate the nitric oxide biosynthetic process.
Macroautophagy is an evolutionarily conserved dynamic pathway that functions primarily in a degradative manner. A wide range of diseases is associated with dysregulation of macroautophagy. Macroautophagy has a critical role in cellular homeostasis. Either insu cient or excessive macroautophagy can seriously compromise cell physiology, and thus, it needs to be regulated appropriately [30] . CR can negatively regulate macroautophagy. PCC can regulate the glial cell apoptotic process.
Research showed that Nicotine might be involved in the pathophysiology of psychosis. Smoking has a relationship with depression. In animal models, Nicotine shows anxiolytic properties. Depression people are more likely to smoke and more likely to develop severe depressive episodes upon smoking cessation. Nicotine has also been observed to produce similar cognitive improvements in AD patients [31] . However, the relationship between smoking and AD is still debatable [32] . In our herbs, CR, PCC has the function to respond to Nicotine.
It has been demonstrated that serotoninergic, dopaminergic, and cholinergic systems are mainly involved in the pathogenesis of BPSD, and the role of HTR2A and DRD2 as therapeutic targets appear to be evident [16] . Our research utilized molecular docking to nd potential active ingredients with good binding activities to DRD2 and HTR2A. The best-docked compound was beta-sitosterol. The free binding energy of beta-sitosterol with DRD2 and HTR2A was − 9.58 kcal/mol, -8.2 kcal/mol, respectively. Stigmasterol, chelerythrine, and campesterol also have good binding activities. Beta-sitosterol, Stigmasterol, chelerythrine, and campesterol are the active ingredients of PCC. It is the only herb containing these ve ingredients.
The TCM theory veri es our results. Triple energizers mean upper, middle, and lower energizer in TCM theory. They are the birth and channel to run for Qi, blood, thin, thick uids, and essence. Moreover, they also contact ve Zang-organs and six Fu-organs. SR affects the upper energizer, which is the brain and heart. PCC works on the lower energizer that is the kidney and liver. CR in uences the middle energizer that consists of the spleen and stomach. Kidney essence de ciency is a primary syndrome differentiation of AD in TCM theory. "Liver re" is the largest contributor to BPSD due to the imbalance between yin and yang of liver function [33,34] . PCC acts on the lower energizer can purge the liver re. "Su Wen" puts forward that "the mind is the monarch's o cial, and the gods come out of it." In the compendium of Materia Medica, Shizhen Li of the Ming Dynasty proposed that "the brain is the house of primordial God." SR works on the heart and brain, belongs to the upper energizer, is bene cial for the heart and brain. In conclusion, SR and PCC are HLJDD's primary herbs based on the TCM theory.

Conclusions
The therapeutic value of natural products in BPSD has increased in reputation due to their clinical impact and insigni cant side effects. Recently, different types of compounds were reviewed for their biological activities. In this review, we summarize the natural products of HLJDD for the molecules' targets and biological processes involved in the treatments of BPSD. Furthermore, put forward novel compatibility of HLJDD to BPSD. Our results showed that SR has more molecule targets and biological processes involved in BPSD, PCC contains more good-docked compounds: poriferast-5-en-3beta-ol (beta-sitosterol), Stigmasterol, chelerythrine, and campesterol, which have lower a nity energy with DRD2 and HTR2A. SR and PCC are the primary drugs in treating BPSD. SR plays an anti-in ammatory role; PCC can regulate the apoptotic process and respond to nicotine. All of them can regulate blood vessels. CR and GF play the   positive regulation of anion transport Group0 APOE|CFTR|TNFSF11 Table 5 The biological function of GF on BPSD Function Groups Group Genes regulation of amyloid-beta formation Group2 APOE|CASP3|TNF regulation of membrane protein ectodomain proteolysis Group1 APOE|IL10|TNF regulation of nitric oxide biosynthetic process Group0 ICAM1|IL10|TNF     biological processes analysis of GF. GF's key protein targets involved in 4 biological processes, they are the regulation of amyloid precursor protein catabolic process, regulation of membrane protein ectodomain proteolysis, regulation of nitric oxide biosynthetic process, and regulation of amyloid-beta formation.