Research Strategies and Analysis of Traditional Chinese Medicine

Yuxuan Sun

University of California, Irvine, 32721 Arroyo Drive, U.S.A.

Keywords: Traditional Chinese Medicine, Phage Display, C-Fos Expression, Immunohistochemistry, Fluorescent In-Situ

Hybridization.

Abstract: Traditional Chinese medicines, beleaguered by researchers and scientists in the past for the ambiguity of

mechanism in the clinical field, can be proven by many recent strategies and technologies for their efficacy.

This article summarizes the role that Chinese medicine has recently played in the clinical field and also

outlines some of the modern research methods used to study Chinese medicine. Phage display, c-fos

expression, immunohistochemistry, and in-situ hybridization were summarized in the article. The advantages

and shortcomings of specific bacteriophages and the methodology of Immuno & FISH were discussed in the

article. A number of clinical examples were listed to provide the reference for the further analysis of TCMs.

1 INTRODUCTION

Traditional Chinese medicines (TCMs) are crucial

components in clinical aspects and occupy an

extremely important segment of the medical field.

For some diseases, TCM has received a great deal of

attention for its remarkable effectiveness. Chinese

medicine has a long history of wide use in China for

the prevention and treatment of various diseases by

targeting and modulating multiple disease-related

pathways with multiple effective components

(Zimmermann 2007). Unlike new psychoactive

substances (NPS) that would imperil mental

health

and contain addictive chemicals, TCM has fewer side

effects. Corydalis Yanhusuo (yhs) is widely used for

the treatment of pain and inflammation. In dopamine

D2 receptor knockout mice, its analgesic effects were

attenuated in acute and neuropathic pain, but not in

inflammatory pain assays. Thus, our results suggest

that YHS is effective in reducing acute,

inflammatory, and neuropathic pain without causing

tolerance (Wang 2016). Compared to the original

morphine, yhs which consisted of Corydalis and

Angelica dahurica that are clinical commonly used

classical prescriptions to alleviate pain (Wu 2015).

These effects could only be achieved with

psychotropic drugs such as morphine or opioids,

which have a huge addictive potential. Compared to

TCMs, chemical drugs act on a specific target of a

https://orcid.org/0000-0002-2699-6067

single drug, but a pair of herbal medicines consists of

many active ingredients. Different combinations of

drugs can have different effects on physical therapy

and herbal efficacy can change. For example, maoto

(mahuang-tang), containing both ephedra herb and

cinnamon bark, has a stronger diaphoretic effect and

warming property than ephedra herb alone (Hayashi

2009). On the molecular level, the basic theory of

TCM for the treatment or prevention of cancer is to

restore the patient to a healthy state by altering

multiple carcinogenic events. Wang et al. (Wang

2010) studied the oncogenic effects involving

multiple abnormal genes/pathways, the use of TCM

in cancer chemoprevention may be more

advantageous than drugs that target a single molecule

alone. In particular, resveratrol, curcumin, and

berberine have all been evaluated in a number of

clinical trials for the treatment of many types of

cancer (McCubrey 2017). Fei (Fei 2018) studied 345

patients with locally advanced colon glands

undergoing surgical resection divided equally into

three groups with placebo, twice-daily intraperitoneal

injections of 10 mg/kg of the herbal medicine catalase

(treatment group), and twice-daily intravenous

injections of 5 mg/kg of bevacizumab (control group)

for a total of 12 weeks. Patient overall survival (OS),

cancer-free survival (CFS) was significantly

increased in the catalase-treated group. The clinical

trial of the preliminary study concluded that treatment

Sun, Y.

Research Strategies and Analysis of Traditional Chinese Medicine.

DOI: 10.5220/0011157200003444

In Proceedings of the 2nd Conference on Artiﬁcial Intelligence and Healthcare (CAIH 2021), pages 23-28

ISBN: 978-989-758-594-4

with the herb catalpol showed beneficial clinical

outcomes, low cost, and no serious complications.

The study of herbal targets and their difficulties

are due to the fact that the target can be a protein,

DNA, or RNA that causes or contributes to disease.

its validation consists of demonstrating that

modulation of the target has a therapeutic effect.

Assay development follows target validation and is

an objective method for screening putative

compounds to determine interactions and/or

modifications with the target. Once the assay is

established, the next step is to find compounds that

can actively engage the target. From a library of

potential compounds, a select number of leads are

generated that demonstrate a relationship between

chemical structure and target-based activity in a

biochemical or cell-based assay (Improving and

Accelerating Therapeutic Development for Nervous

System Disorders: Workshop Summary 2014). The

difficulties of multifaceted research have led to the

achievement of this relatively difficult breakthrough

in mechanism as well as target analysis in Traditional

Chinese medicines. This article will focus on some

ways that can effectively contribute to the research of

TCMs and also exemplify the specific performance

of TCMs in clinical and therapeutic aspects.

2 RECENT TECHNOLOGY

2.1 C-fos Expression

The c-fos gene is expressed in the central nervous

system in neurons in response to various stimuli. The

effects of chronic morphine treatment and withdrawal

on c-fos mRNA in rat brains, especially in the

identified striatal neurons (Bullitt 1990).

Immunohistochemical methods may be used to

identify the protein product, c-fos protein. As a result,

after peripheral stimulation, c-fos expression may be

used as a proxy for neuronal activation in the neuraxis

(Bullitt 1990). Behavioral sensitization is believed to

be implicated in opioid-seeking activities in humans,

and is analogous to the intensification of drug craving

following prolonged exposure (Cruz 2015). Learned

connections between narcotics and the environment

are believed to be encoded within complex patterns

of sparsely spaced neurons called neuronal

ensembles, which play an important role in addiction

(Cruz 2015). Cellular imaging with the early gene c-

fos and its protein product, in particular. Fos has been

used to find sparsely scattered neurons that were

highly stimulated during programmed drug activities

like drug self-administration and context- and cue-

induced drug finding reinstatement. Using three

different experimental groups of saline, yhs, and

morphine for comparison and measured by the

combination of c-fos and immunohistochemistry, the

effect of herbal medicine can be obtained. To date, no

study has evaluated c-fos expression in vivo for YHS

alone or the combination of YHS and morphine. Fos

expression may help to understand more about the

mechanism underlying YHS mode action. In

addition, this study may help to understand new drugs

or systems to target with opioids to block the negative

side effects that come with opioids alone.

2.2 Immunohistochemistry And

Fluorescent In-situ Hybridization

(FISH)

Immunohistochemistry (IHC) binds fluorescent or

colorable chemicals to antibodies and uses the

immunological principle of specific binding reactions

between antigen and antibody to detect the presence

of target antigens in cells and tissues, which are then

examined using light microscopy. This method

allows observation not only of the amount of antigen

present but also of where the antigen is located in

brain sections. Fan (Fan 2014 )summarized five

milestones in the development and advancement of

the IHC field include (1) the discovery of monoclonal

antibodies that significantly improve diagnostic

specificity; (2) a thermally induced antigen repair

(AR) method that allows the most efficient IHC

detection of formalin-fixed and paraffin-embedded

surgical and cytological specimens; (3) a highly

sensitive secondary detection system that can detect

trace proteins on formalin-fixed and paraffin-

embedded tissues with virtually no background

staining; (4) an automated staining system that allows

hundreds of IHC slides to be run in the same lab on

the same day with reproducible and accurate results;

(5) a digital pathology with imaging analysis digital

pathology, allowing quality control using digital

slides (full slide scanning) and further reducing

turnaround time by making digital images (electronic

slides) available through the website. Fluorescence in

situ hybridization (FISH) is a molecular cytogenetics

technique that uses fluorescent probes to bind to

specific portions of nucleic acid sequences that have

a high degree of sequence complementarity.

CAIH 2021 - Conference on Artiﬁcial Intelligence and Healthcare

2.3 Combined Fluorescent In-Situ

Hybridization (FISH) And IHC

Staining of opioid receptors will be carried out using

fluorescence in-situ hybridization via RNA scope and

standard IHC as described previously (Grabinski

2015). Twenty-micron brain parts will be incubated

in a quenching solution containing H

at room

temperature for 45 minutes at the level of the

hypothalamus. After that, the sections will be put on

Fisherbrand Superfrost Plus Microscope Slides and

baked in the hybridization oven overnight at 60 °C.

Following the manufacturer's instructions,

RNAscope in situ hybridization will be performed

using the RNAscope Multiplex Fluorescent Kit

(Grabinski 2015). The brain sections will be blocked

for 1 hour at room temperature in a blocking buffer

containing 10% natural donkey serum and 0.3 percent

Triton X-100 (Phan 2020). The sections will incubate

with a MOR antibody overnight. The sections will be

cover-slipped using RNAscope DAPI, followed by

Invitrogen ProLong Gold Antifade-Mountant.

Images will then be captured using Leica Sp8 TCS

confocal microscope (Phan 2020). Once again,

ImageJ software will be used to analyze the number

of c-fos containing cells. By combining the two

approaches, the drug mechanism of yhs can be

studied in depth.

2.4 Phage Display

Phage display, established by smith in 1985, is a

unique genetic recombination technique to present

polypeptide on the surface of filamentous

bacteriophage. It is an effective screening method to

detect the displayed protein and peptide. Foreign

DNA fragments can be inserted into the gene (g3) of

phage fd-tet. The phage, fd-tet, can be used as a

cloning vector to produce cloned single-strained

DNA. This single-stranded form can be packed inside

the shell, indicating that phage particles (Smith

1985).

The basic principle of phage display technology

is a gene encoding an exogenous polypeptide or

protein is fused with a phage gene encoding a shell

protein and eventually presented on the surface of the

phage as a fusion protein.

Phages that have introduced various foreign genes

are called phage libraries. The molecule to be studied

is immobilized on the phage, and the phage library is

used to screen for phage-carrying ligands that bind

specifically to the molecule and isolate the phage in

the library for display. Such processing ensures the

biological function of the phage exogenous gene.

The advantages of phage display technology are

(1) high efficiency of panning and the ability to select

phages with high affinity. (2) The displayed

peptides and proteins are internally linked to the

phage, which facilitates the analysis of binding

peptide and protein sequences. (3) All peptides and

proteins encoded onto the exogenous genes are able

to maintain their original spatial structure and

biological activity. In recent years, this technology

has shown its unique advantages in finding tumor-

specific target molecules and targeted therapies.

Phage display derivatives play an important role in

the diagnosis and treatment of diseases and will be

used in a wide range of applications in different

medical technologies, including biosensing,

monitoring, molecular imaging, gene therapy,

vaccine development, and nanotechnology.

Bacteriophage, a class of viruses that use bacteria as

hosts, has a protein sshell and single-stranded or

double-stranded DNA wrapped inside the shell.

Based on the type of phages, phage displays can be

classified as filamentous, T4, T7, and lambda (λ)

phages. The structures of the phages are shown in

Fig.1.

Figure 1: The structure of M13 (A), T7 (B), λ (C), T4 (D).

2.4.1 Filamentous M13 Phage

Based on Fig 2, the M13 phage genome is 6407 bp

long and consists of structural proteins, replication

proteins, and morphogenetic proteins encoding a total

of 11 proteins. Among them, pⅧ is the main coat

protein of the phage display (Wezenbeek 1980). The

M13 phage enters the cell through the flagellum on

the surface of the host cell, therefore it only infects

“male” E. coli with flagella. The spacer regions II/IV

and VIII/III of the m13 phage genome can be used to

insert exogenous DNA, and the majority of other

regions are essential genes. The phage single-

stranded DNA is encapsulated in a tubular structure

consisting of approximately 2700-3000 molecules of

Research Strategies and Analysis of Traditional Chinese Medicine

the major capsid protein pVIII. 424 amino acid

residues of the pIII protein precursor can be inserted

into the exogenous gene, and the protein encoded by

the exogenous gene can be expressed as a fusion

protein on the phage surface without disturbing the

function of the phage (Shijie Huaren XiaoHua Zazhi

2003).

Figure 2: Single-stranded DNA of M13 bacteriophage.

2.4.2 M13KO7

The M13KO7 is a derivative of M13 bacteriophage

with gene II of M13mp1. The replication origin of

pl5A and the kanamycin resistance gene (K

, kanr) of

Tn 903 were inserted at the AvaI site (5825) of M13.

With the pl5A origin, the phage was able to replicate

independently of gIIp. This allows the phage to

overcome the effects of interference and maintain

sufficient genomic levels to express ssDNA in the

presence of the phage to produce the desired protein

(Vieira 1987). The pII protein introduced at M13KO7

is able to produce a stronger transcriptional effect

with the viral replication start point, ensuring the

preferential synthesis of the inserted gene.

2.4.3 T4 Phage

T4, the accessory proteins, HOC (highly antigenic

outer capsid protein) and SOC (small outer capsid

protein) connect to the capsid surface. SOC maintains

the stability of the head and HOC is an elongated

molecule protruding from the center of gp23*

hexamer (The detailed structure of T4 bacteriophage

is shown in Fig 3.). The T4 phage is characterized by

its ability to fuse two exogenous polypeptides or

proteins of completely different nature to the outer

shell proteins SOC and HOC on the T4 capsid and

display them directly on the surface of the T4 phage.

It contains a large system capacity and achieves site

display in vitro (Yap 2014). Although T4 phages can

successfully display the structure, they have some

limitations. Due to the restriction of SOC molecules

trimerization with C-terminal interactions, they are

not commonly used.

Figure 3: The structure of the T4 head in part.

2.4.4 T7 Phage

The double-stranded DNA of the T7 phage makes it

more stable and less prone to genetic mutations

during replication. The capsid of the T7 phage is

mainly composed of gp10A and gp10B, whose main

function is to protect the DNA inside. The multiple

properties of the T7 phage make it a better research

tool. The structure is shown (Fig. 4) can visually

show that the outer shell plays a crucial role in

protecting the DNA structure. Large protein

fragments can be incorporated into coat proteins at

low levels. Secondly, the coat shell consisting of

g10A and g10B can effectively survive under harsh

conditions such as high salinity, high pH value, and

even denatured environments. Its growth rate can

enable multiple rounds of selection in a single day.

However, its drawbacks are also more obvious,

because T7 phages are assembled in the cytoplasm

and released by lysing bacteria, which leads to the use

of T7 phages that are extremely destructive to the host

cells (Tan 2016).

Figure 4: The structure of T7 phage bacteria.

2.4.5 Lambda Phage (λ)

Lambda is a mild E. coli phage whose lytic nature and

CAIH 2021 - Conference on Artiﬁcial Intelligence and Healthcare

predominantly protein structure plays a significant

role during phage display. During lysis, the cyclic

DNA knows the synthesis of proteins required for

viral replication, phage particle assembly, and cell

lysis.

The advantage is that in the lysed state, the phage

integrates itself into the chromosome of the host cell

via lysogen and its lambda DNA resides in the host's

genome without causing significant harm to the host.

However, intracellular activity in the host cell is not

easily monitored.

2.5 Current Use for Locating Targets

Phage presentation technology can be well combined

with Chinese medicine into an effective target

screening tool for drugs. It can present exogenous

proteins and peptides on the phage surface and utilize

the specific affinity interaction of exogenous proteins

and peptides with the unknown to be screened. And

the phages that fail to bind will be discarded.

Afterward, the bound phages are eluted with reagents,

the eluted phages are collected and infected with

bacteria for amplification, and the resulting phages

are then repeated for the screening process. In this

way, after 3-5 rounds of "adsorption-elution-

amplification" cycle screening, phages that

specifically bind to the treated phage exogenous

peptide can be found. After gene sequence analysis,

the corresponding basic amino acid sequences, which

bind specifically to the target molecule, are obtained

based on the sequencing results. The proteins in the

organism containing the above amino acid sequences

are analyzed with the help of bioinformatics to obtain

the corresponding target proteins in the organism

(Feng 2021).

The advantage of this technique is that the phage

is easy to amplify and select, and the protein or

peptide obtained after screening can also be

determined by determining the DNA sequence of the

insert. Rodi (Rodi 1999) applied this method to

screen a random library of phage display peptides and

the binding of biotinylated derivatives of paclitaxel

(Taxol). The binding of paclitaxel to the anti-

apoptotic human protein Bcl-2 was confirmed by

ELISA assay. Zhang (Zhang 2017) studied that the

phage library of 15 peptide random sequences was

used to screen for specific cellular targets of

strychnine, interacting with cell-selective binding

peptides to investigate the mechanism of antitumor

activity of strychnine. Sun (Sun 2016) analyzed that

the specificity and molecular interactions between the

candidate binding protein Ubiquinol-cytochrome c

reductase binding protein (UQCRB) and oxymatrine

were investigated using the T7 phage technique. The

interaction between the two demonstrated that

UQCRB is a potential target for the treatment of

chronic hepatitis B (CHB).

3 CONCLUSION

Nowadays, TCM has triggered the research of many

scientists and there are many methods available to

determine the targets and active ingredients of TCM.

Each herbal medicine has its own composition and

validation methods. In the paper, a summarization of

the pros and cons of several common methods and

use cases would provide references for further

analysis. Considering the complexity and diversity of

the components and targets of TCM, it is difficult for

a single method to effectively analyze get an accurate

determination. The combination and practice of

multiple methods can better achieve the desired

results and can also improve the efficiency and

accuracy of the purpose.

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