Potential of Dragon’s Blood as a Space Radiation Protectant Especially on Brain-Liver Bystander Effect

During space manned missions, radiation is a serious health risk. Radiation can not only directly cause damage to target organs but also trigger side effects to affect nontarget organs. Dragon’s Blood (DB) is a traditional Chinese Dai medicine that has been proven to exhibit radioprotective effects in our lab previously. It can alleviate brain damage, hematopoietic dysfunction, and gastrointestinal damage caused by radiation in rats, but its mechanism of action is not clear yet. In order to study the effect of brain irradiation on the damage to the liver and the protective effects of Dragon’s Blood, herein, liquid chromatography coupled with a mass spectrometer was used to analyze the total differential protein expression in the rat liver after 30Gy Coγ-ray whole-brain irradiation with/without administration of Dragon’s Blood for 10 days before irradiation. A total of 4557 proteins were identified in the rat liver. A total of 299 coexpressed differential proteins were screened in the RAD/CON group indicating that brain radiation significantly affected the liver’s metabolic system (such as drug and arachidonic acid metabolism), chemical carcinogenesis, and peroxisome process. A total of 85 differential proteins were screened in the DB/ RAD group. Results indicated that Dragon’s Blood significantly regulated the expression of 26 proteins to normal levels (Msrb2, Txnrd2, Samm50, Pir, Pex11a, etc.) mainly through regulating the metabolism and redox homeostasis process. The results of molecular docking and network pharmacology found that the main effective radiation protection components in Dragon’s Blood are natural chalcones, flavan, and phenolic derivatives.


Introduction
Space flight affects the physiological and biochemical processes of astronauts and vertebrate models, including bone and muscle atrophy, immune dysfunction, neurobehavioral changes, and visual impairment [1]. Radiation is the main source of biological stress in the low-Earth orbit (LEO), which is the region encompassed by the Earth's magnetosphere where all manned missions currently occur. In the upcoming missions to the moon and Mars, deep space radiation is a serious health risk. In particular on long manned missions, radiation dose accumulation can be a very dangerous factor.
Low dose of ionizing radiation can cause dysfunction in the central nervous system, while larger doses of ionizing radiation cause pathological changes in neuronal cells. Mature neuronal cells and glial cells are relatively insensitive to radiation, but radiation damage may cause microcirculation disorders, ischemia, hypoxia, and metabolic disorders [2]. This will in turn cause neuron and neuroglia cells to undergo degeneration, necrosis, and apoptosis. After the nervous system is damaged by large doses of radiation, the early brain tissue may show edema, inflammation, and chromatin dissolution, and then, apoptosis and vacuolar degeneration will happen, and nerve fibers will swell and demyelinate [3].
As the largest internal organ in the human body, the liver has multiple functions, including metabolism of ingested nutrients, elimination of waste, glycogen storage, and synthesis of plasma proteins. The bile secreted by the liver plays a huge role in digestion. Radiotherapy can cause classic and nonclassical liver diseases. Classic liver disease usually occurs between 2 weeks and 3 months after radiotherapy and involves hepatomegaly, elevated alkaline phosphatase, central lobular vein occlusion, retrograde congestion, and severe secondary hepatocyte necrosis, even leading to liver failure. Nonclassical liver disease usually occurs between 1 week and 3 months after radiotherapy and involves abnormally elevated liver transaminase [4]. The brain and liver can interact through inflammation, metabolism, immunity, and hormones. Stroke patients may develop liver damage related to changes in the glutamatemetabolizing enzymes in the liver that are involved in metabolizing glutamate released after cerebral ischemia [5]. At the same time, chronic hepatitis and acute liver failure can also lead to brain disease, associated with systemic inflammation and hyperammonemia [6].
Among the commonly used drugs for the treatment of cerebral and liver diseases, drugs that reduce ammonia content such as lactulose [7] and rifaximin [8] are still the first choice for the treatment of hepatic encephalopathy [9]. Anti-inflammatory drugs such as ibuprofen restore motor and learning abilities in animals with mild hepatic encephalopathy by reducing oxidative and/or nitrative stress [10]. N-Acetylcysteine normalizes glutathione levels in the brain while preventing liver damage from azomethane exposure [11].
In recent years, traditional Chinese medicine has been widely used due to its multicomponent, multitarget, low toxicity, and high safety characteristics. For example, Ganoderma lucidum polysaccharide-1 has been reported to play an important role in regulating inflammation of the liverbrain axis. Ganoderma lucidum polysaccharide-1 (GLP-1) can significantly improve cognitive impairment in Dgalactose (D-gal) rats, and its mechanism is related to decreased levels of TNF-α, IL-6, phospho-p38MARK, phospho-p53 and phospho-JNK1+JNK2+JNK3, increased IL-10 and TGF-β1 levels, and regulation of liver-brain axis metabolic disorders [12].
Dragon's Blood (DB) is a bright red resin extracted from Dracaena cochinchinensis (Lour.) (SC Chen, China). It has been used in China for more than 1500 years and is known as the "Holy Medicine for Promoting Blood" in the Compendium of Materia Medica [13]. Pharmacological studies have shown that Dragon's Blood has anti-inflammatory [14,15], antibacterial [16], antithrombotic [17], and analgesic effects [18] and immune-enhancing function [19]. Modern medical research has confirmed that Dragon's Blood can act on the microcirculation, relax and soften blood vessels, and can reverse hardening and narrowing of small arteries [20]. At the same time, it can reduce blood viscosity and reduce platelet aggregation. Dragon's Blood is also clinically effective in treating diseases such as gynecological uterine blood, upper gastrointestinal bleeding, and nasal cavity bleeding [21].
Herein, a quantitative label-free proteomic analysis method was used to evaluate the acute changes of total liver proteins (<7 days) after 30 Gy Co 60 γ-ray whole-brain irradiation in rats with/without the administration of Dragon's Blood 7 days before radiation. In order to characterize the functional consequences of proteins in the liver, GO functional enrichment and KEGG pathway analysis were per-formed on differentially expressed proteins. The results indicated that the administration of Dragon's Blood before brain radiation provided protective effects on drug metabolism, redox homeostasis, chemical carcinogenesis, and peroxisome process in brain-irradiated rats. The target proteins were then docked with the natural molecules in Dragon's Blood, and the active ingredients and their mechanism of protection were discovered. Based on the analysis of the target-drug interaction network, we systematically observe the intervention and influence of drugs on liver proteins, revealing the mystery of multimolecular synergistical drugs acting on the human body. This work provides important guidance on the by-strand effect of brain radiation and its protective method through the application of DB. The proteomic results also provide information to help the exploration of new targets for radiation protection drugs. (BIT-EC-SCXK2016-0006-M-2021065). All efforts were made to minimize animal suffering and to use as few animals as necessary.

Animal
Model of Radiation. The present study complied with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH publication no. 85-23, revised in 1996). The 7-week-old male Wistar wild-type rats were purchased from Sibeifu Biotechnology Co., Ltd. (NO. SCXK Jing 2019-0010). Upon arrival, rats were maintained in a sterile housing condition at 25°C, with 12-hour light-dark schedule with free access to food and water. After one week of adaptive feeding, 24 healthy male Wistar rats were randomly divided into three groups and had circulated light for 12 hours and had free access to food and water. Control group (CON) rats were given 10 mL/kg distilled water (containing 0.5% sodium carboxymethyl cellulose) daily to the day of sacrifice. The whole-brain irradiated group (RAD) was given 10 mL/kg of distilled water (containing 0.5% sodium carboxymethyl cellulose) daily to the day of execution on the third day after irradiation. Dragon's Blood administration group (DB) was given 10 mL/kg of 200 mg/mL Dragon's Blood aqueous suspension daily to the day of execution on the third day after irradiation. After intragastric administration for ten days, the rats were irradiated with Co 60 γ-ray whole brain in the cobalt source room of Peking University (RSL2089, REVISS, Peking University, Beijing). The rats were anesthetized by intraperitoneal injection of chloral hydrate at a dose of 2 Space: Science & Technology 350 mg/kg, and the position was fixed before radiation. A lead brick (5 cm) was used to shield the lower body from the neck of the rats, exposing the head of the rat to radiation. Under radiation exposure, the rat hangs vertically, the head is aligned with the radiation beam window, and γ-rays are irradiated at a time with a source distance of 200 cm, a dose rate of 2 Gy/min, and a total dose of 30 Gy. The animals in the CON group were also anesthetized but did not receive any irradiation. All efforts are made to minimize animal suffering and use as few animals as possible. After the irradiation, the experimental animals were transported back to the animal room and continued normal feeding.

Histomorphology Studies.
The mice were sacrificed on the third day after irradiation, and the rat liver was excised and partially immersed in 4% paraformaldehyde (Solarbio, Beijing, China) for histomorphological study. The remaining tissue was collected and stored at -80°C for further study. The liver tissue was fixed for 4-6 hours, dehydrated, and embedded in paraffin. A 4 μm liver tissue section was cut on glass slides and airdried. After the paraffin sections were deparaffinized, tissue was stained with hematoxylin for 3 minutes and then stained with eosin for 3 minutes. The slices were then dehydrated with alcohol, made transparent with xylene, and sealed. The cell morphology of the liver was observed under an optical microscope, and the atrophy morphology, hepatocyte necrosis, inflammatory cell infiltration, and other pathological conditions of liver slices in each group were counted.

Protein
Extraction and Digestion. Solution of 8 M urea and 10 mM DTT were made with 40 mM Tris-HCl (pH = 7:4) buffer. Protease inhibitor (50×, diluted 50 times for use) and phosphatase inhibitor mixture (50×) were added to the system to form a protein lysate. A total of 5 tissue samples were chosen randomly from each group for proteomic analysis (one sample was damaged in the CON group due to operational problems). 100 mg of tissues in each sample were accurately weighted and washed with precooled PBS. Protein lysates at 10 : 1 (v : w) were homogenized to obtain the tissue lysate with the help of ultrasonic disruption. The samples were then centrifuged at 12000 r/min for 15 min, and then, the supernatant was taken. The Coomassie brilliant blue method was used to determine the protein concentration of each sample. The protein samples were reacted in a water bath in 10 mM dithiothreitol (DTT) at 37°C for 60 minutes. The samples were then placed in a 50 mM IAA solution in the dark at room temperature for 30 min to make it fully alkylated. The samples were incubated with trypsin (Promega, Madison, WI, USA) at the ratio of 80 : 1 (protein : trypsin) at 37°C overnight and enzymatically digested for 16 hours. The mixed samples were purified by C18 column desalination. The samples after desalting and purification were quickly concentrated in a vacuum to obtain dry peptide powder. International standards were adopted with false discovery rate ð FDRÞ < 1%, dipeptide matching, screening, and identifying proteins. Fold changes ðFCÞ > 1:5 or <1/1.5 and p < 0:05 were used as the threshold to screen the differential proteins between the groups. The two groups of differential proteins were clustered and analyzed by the bioinformatics analysis tool DAVID (http://david.abcc.ncifcrf.gov/summary.jsp). The detailed procedure was described as follows. First, the differentially expressed protein data was imported into the DAVID database. Gene Ontology (GO) enrichment analysis was selected to obtain statistics on the three GO classifications. The three classifications were biological process (BP), cellular component (CC), and molecular function (MF). KEGG analysis was chosen for pathway enrichment analysis. The gplot package in R language was used to cluster the expression patterns of differential proteins. We use the protein database UniProt to understand the properties and functions of differential proteins. The distance calculation algorithm was used: Spearman between samples and Pearson between genes. The clustering method used was hcluster (complete algorithm). All the proteins identified in each group were imported into Origin software for principal component analysis. From the principal component analysis (PCA) diagram, the distance between different samples could be visually seen, and the similarity or difference of the samples could be distinguished based on this. Analyze proteinprotein interaction by using the STRING official website (https://string-db.org/).

Molecular
Docking and Network Pharmacology. The molecular structure of phytochemicals in Dragon's Blood was drawn by using ChemDraw software. AutoDock was used to perform small-molecule 3D structure optimization. The target protein crystal structures were obtained through the UniProt database. AutoDock tools were used to optimize the crystal structure. The optimization process included removal of ligands, removal of water molecules, and hydrogenation. The programmed batch molecular docking software AutoDock Vina and the script Msys2 were used to run the code. The Dragon's Blood radiation protection target protein library (75 proteins) and the Dragon's Blood phytochemical library (135 compounds) were used for the programmed batch molecular docking. The result was displayed by the binding energy score between the two molecules, and the successful docking condition was set to the binding energy≤−7 in order to obtain the interaction relationships between the above protein library and the com-pound library. Finally, the successfully docked proteins and compounds were summarized into a table for the subsequent construction of the "component-target" network of Dragon's Blood for radiation protection.
2.9. Statistical Methods. The experimental results were expressed as ±s. The data of each group were statistically processed by SPSS17.0 (IBM SPSS Company) software. The independent sample t-test was used to determine whether the difference between each indicator group was statistically significant.

Results
3.1. Pathological Analysis of the Rat Liver Tissue. To examine the effect of whole-brain Co 60 γ irradiation on liver damage, the histological features of the rat liver were determined by H&E staining after 3 days of whole-brain irradiation. As shown in Figure 1(a), the liver lobules of the CON group were clearly structured and the liver cords were neatly arranged. The liver cells were rich in the cytoplasm, and the morphology and structure were normal. The liver sinusoids were not significantly expanded or squeezed, and little  Space: Science & Technology bit of inflammatory cell infiltration foci (black arrows) were seen in the portal area. In the RAD group ( Figure 1(b)), hepatocyte necrosis (yellow arrow) was seen locally, the normal structure disappeared, and the nucleus fragmented and disappeared, accompanied by inflammatory cell infiltration (black arrow). In contrast, there was no observed hepatocyte necrosis in the DB group (Figure 1(c)). The same as shown in the CON group, little bit of inflammatory cell infiltration was seen in the portal area (black arrow). Pathological analysis showed that whole-brain Co 60 γ irradiation did cause rat liver damage. The administration of Dragon's Blood before irradiation helps in alleviating the liver damage.

Effects of Whole-Brain
Irradiation on the Levels of TNFα, IL-6, and IL-1β in the Rat Liver. From the results of Figure 2, it can be seen that after γ-ray irradiation, the level of TNF-α in the liver tissue decreases, and the level increases after the administration of Dragon's Blood, returning to normal levels. The concentration of IL-1β was significantly reduced, and it was slightly increased after Dragon's Blood was given but with no significant difference. The concentration of IL-6 in the liver was significantly increased after irradiation, and there was no significant change after the administration of Dragon's Blood.

Protein Expression and Identification.
Protein composition is of particular interest to study the pathology pathways and the protection effect of drugs. Liver tissue samples were collected on the 3rd day postirradiation, and label-free proteomics based on UPLC-MS technology was used to study the differential proteins in rats' liver to capture the early changes in protein profiles. In total, 4557 proteins were identified in rat liver tissues. The cut-off values of upregulation and downregulation fold change were set when the protein ratios in different groups were above 1.5 or below 1.5, and the p value was lower than 0.05 to be considered significant. In the RAD/CON group, 180 proteins were upregulated and 119 proteins were downregulated. In the DB/RAD group, 55 proteins were upregulated and 30 proteins were downregulated. The volcano plot analyzed the degree of differentially expressed protein between the different treatment groups ( Figure 3). Each group of differentially coexpressed proteins was located into the GO database and classified according to biological process, cellular component, and molecular function. The results are shown in Figure 4. The GO enrichment analysis showed that there were a large number of significant changes in biological processes between the proteomics of the three groups (RAD/CON group and DB/RAD group).  In the RAD/CON group, 23 proteins involved in redox biological processes were enriched, including glutathione peroxidase 3 (Gpx3), methionine-R-sulfoxide reductase B2 (Msrb2), glycine decarboxylase (Gldc), cytochrome P450 4B1 (Cyp4b1), thioredoxin reductase 2 (Txnrd2), and some other proteins. The remaining differential proteins were involved in biological processes such as the cytochrome P450 oxidase pathway, drug metabolism, and glutathione metabolism. These proteins were mainly located on the cytoplasm (119 proteins), mitochondrion (62 proteins), extracellular exosome (80 proteins), and membrane (57 proteins). They provided molecular functions such as poly (A) RNA binding, aromatase activity, iron ion binding, and heme binding. In the DB/RAD group, there were 12 proteins involved in the redox process, including 5 proteins that overlapped with the RAD/CON group, namely, Cyp3a9, Cyp2b2, Pirin (Pir), Cyp3a1, and thioredoxin reductase 2 (Txnrd2). Differential proteins were also involved in biological processes such as drug metabolism (4 proteins), response to drugs (9 proteins), and oxidative demethylation (3 proteins). These proteins were mainly located on intracellular membrane-bounded organelle (13 proteins), extracellular exosome (19 proteins), endoplasmic reticulum membrane (10 proteins), and mitochondrion (16 proteins). The molecular functions included oxidoreductase activity, aromatase activity, heme binding, monooxygenase activity, and poly (A) RNA binding.
The KEGG pathways were also analyzed for the differential proteins in each group as shown in Figure 5. The p value of the enrichment result of the KEGG pathway was indicated by color, and the number of genes was indicated by the size of the bubble. Comparing RAD and CON groups, 13 KEGG terms were significantly enriched (p < 0:05, Figure 5(a)), mainly including metabolism (45 proteins), ribosomes (13 proteins), chemical carcinogenesis (11 proteins), retinol metabolism (10 proteins), peroxisomes (10 proteins), and other pathways. Comparing DB and RAD groups, 8 KEGG terms were significantly enriched (p < 0:05, Figure 5(b)), including retinol metabolism (8 proteins), metabolism (21 proteins), steroid hormone biosynthesis (6 proteins), chemical carcinogenesis (6 proteins), linoleic acid metabolism (4 proteins), insulin resistance (4 proteins), tryptophan metabolism (3 proteins), and fatty acid degradation pathways.
3.3.1. Drug Metabolism. The metabolic transformation of most drugs in the body is mainly carried out in the liver, which can be divided into the first-phase metabolic reaction and the secondphase metabolic reaction. A large number of coexpressed differential proteins participating in the drug phase I and phase II metabolism were enriched in the RAD/CON group.
The proteins involved in the phase I metabolic process are summarized in Table 2(a), and the overall differential proteins showed a downward trend except Cyp2c7. The proteins whose expression decreased after irradiation included Cyp2c6v1, Cyp2a1, Cyp2b2, Cyp3a23/3a1, Cyp3a9, Cyp2c11, and Cyp2j3, and the protein whose expression increased after irradiation was Cyp2c7. Cyp2a1 is highly active in the 7-αhydroxylation of testosterone, progesterone, and androstenedione. Cyp2c11 is a cytochrome P450 monooxygenase involved in the metabolism of steroid hormones and fatty acids. It catalyzes the metabolism of testosterone to 2alphaand 16alpha-hydroxytestosterone and catalyzes the epoxidation of polyunsaturated fatty acid (PUFA) double bonds. Cyp2c7 metabolizes arachidonic acid predominantly via a NADPH-dependent olefin epoxidation mainly to 14,15-, 11,12-, and 8,9-epoxyeicosatrienoic acids. It also acts as an omega-1-hydroxylase by metabolizing arachidonic acid to 19-hydroxyeicosatetraenoic acid (19-OH-AA).
The proteins involved in the phase II metabolic process are shown in Table 2(b), and most of the differential proteins were increased. Proteins with increased expression levels after irradiation (italic marker font) included glutathione    Rdh16 can oxidize all-trans-retinol, 9-cis-retinol, 11-cis-retinol, and 13-cis-retinol to the corresponding aldehydes. The expression of Aox1 in the RAD group was upregulated by~1.79 times. Aox1 catalyzes the conversion of all-trans-retinal to all-transretinoic acid. Compared with the CON group, the expression of Cyp3a9 in the RAD group was downregulated by~0.36 times. On the other hand, the expression of the DB group was upregulated~2.25 times compared to that of the RAD group. Compared with the CON group, the expression of Cyp3a1 in the RAD group was downregulated by~0.34 times. However, the expression of the DB group was upregulated~2.97 times compared to that of the RAD group. Compared with the CON group, the expression of Cyp2b2 in the RAD group was downregulated by~0.25 times, while the expression of the DB group was upregulated~6.84 times compared with that of the RAD group. Cyp2a1, Cyp2b2, Cyp2c7, Cyp2c11, Cyp3a23/ 3a1, and Cyp3a9 can further oxidize retinoic acid to generate 4-oxyretinoic acid.
Cyp2c6v1, Cyp2c7, Cyp2c11, Gsta3, and Gstm4 participate in the process of aromatic hydrocarbon metabolism to generate DNA adducts (aromatic hydrocarbons). Cyp2c6v1 was increased by 1.87 times in the RAD/CON group and 1.20 times in the DB/RAD group. Cyp2c7 was upregulated by 2.92 times in the RAD/CON group and downregulated by 0.92 times in the DB/RAD group. Gsta3 was upregulated by 3.44 times in the RAD/CON group and downregulated by 0.84 times in the DB/RAD group. Gstm4 was increased by 2.15 times in the RAD/CON group but only 1.08 times in the DB/RAD group.
Cyp3a23/3a1 and Cyp3a9 are metabolized with azo dyes to form DNA adducts (Azo dyes). Cyp3a23/3a1 was downregulated by 0.34 times in the RAD/CON group and upregulated by 2.97 times in the DB/RAD group. Cyp3a9 was downregulated by 0.36 times in the RAD/CON group and upregulated by 2.25 times in the DB/RAD group.

Protective Effect of Dragon's Blood.
In order to analyze the protective effect of Dragon's Blood on protein more intuitively, a principal component analysis (PCA) chart was generated ( Figure 6(a)). The results showed that the distribution of RAD histones was significantly different from that of the CON group, while DB histones tended to migrate to the CON group, indicating the protective role of DB administration. Ten of the 180 upregulated proteins in the RAD/CON group overlapped with the downregulated proteins in the DB/RAD group, and 16 of the 119 downregulated proteins in the RAD/CON group overlapped with the downregulated proteins in the DB/RAD group (Figure 6(b)). As shown in Table 3, these 26 proteins were enriched as the proteins that Dragon's Blood exerted significant radioprotective effect.
Among the 26 proteins that Dragon's Blood significantly regulates, methionine-R-sulfoxide reductase B2 and mitochondrial (Msrb2) methionine-sulfoxide reductase specifically reduce methionine (R)-sulfoxide to methionine. Although in many cases, methionine oxidation is the result of random oxidation after oxidative stress, methionine oxidation is also a posttranslational modification that occurs at specific residues. Under oxidative stress, its scavenging effect reduces the accumulation of reactive oxygen species in cells, which may play a role in maintaining the integrity of mitochondria, thereby contributing to cell survival and protein maintenance. Polymerase (RNA) II (DNA directed) polypeptide H (Polr2h) and DNAdependent RNA polymerase catalyze the transcription of DNA into RNA using the four ribonucleoside triphosphates as substrates. Txnrd2 is involved in the control of reactive oxygen levels and the regulation of mitochondrial redox homeostasis. Its key role is to maintain mitochondrial thioredoxin in a reduced state. Sorting and assembly machinery component 50 homolog (Samm50) plays a vital role in maintaining the structure of the mitochondrial cristae and the correct assembly of the mitochondrial respiratory chain complex. The TOMM40 needs to be assembled into the TOM complex. Pir is a transcriptional coregulator of NF-kappaB, which can promote the binding of NF-kappaB protein to the targeted kappaB gene in a redoxdependent manner. It may be necessary for effective end myeloid maturation of hematopoietic cells. The above-mentioned proteins are involved in the redox process indicating that Dragon's Blood could regulate the redox balance. Peroxisomal bifunctional enzyme (Ehhadh) is a peroxisome trifunctional enzyme with 2-enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase, and delta 3,delta 2enoyl-CoA isomerase activity. It can catalyze two of the four reactions in the long-chain fatty acid peroxisome βoxidation pathway. The amount of medium-chain dibasic fatty acids can also be adjusted. These fatty acids are the basic regulators of all fatty acid oxidation pathways. They also participate in the degradation of long-chain dicarboxylic acids through peroxisome β-oxidation.
Death domain-containing membrane protein (Nradd) modulates NTRK1 signaling. It activates several intracellular signaling pathways, leads to the activation of JUN, promotes translocation of SORT1 to the cell membrane, and thereby hinders lysosomal degradation of SOTR1 and promotes its interaction with NGFR (by similarity). Both isoform 1 and isoform 2 promote apoptosis.
Peroxisomal membrane protein 11A (Pex11a) may be involved in peroxisomal proliferation and regulate peroxisome division. It may mediate binding of coatomer proteins to the peroxisomal membrane and promote membrane protrusion and elongation on the peroxisomal surface.
In addition, Nags and Acy1 are involved in the metabolism of 2-oxocarboxylic acid and linoleic acid. Cyp2b2, Cyp3a1, and Cyp3a9 participate in the regulation of steroid hormone biosynthesis, retinol metabolism, chemical carcinogenesis, and arginine biosynthesis.

Molecular Docking.
Compared with the CON group, the DB group obtained 85 differential proteins when FC > 1:5 or FC < 0:77. A total of 73 differential proteins with 3D structure were obtained through UniProt protein database search and download, and these 73 differential proteins with 3D structure were formed into the target protein library of Dragon's Blood for radiation protection.
In order to explore the interaction between the phytochemicals in Dragon's Blood and target proteins, the computer-aided drug design platform to molecularly dock each target protein in the Dragon's Blood compound library with the target protein library was used. According to the libdockscore value of the binding score of the target protein and the compound, 20% of the scored compounds of each target protein were extracted as lead compounds. The lead compounds of each target protein were summarized (up to 10, rounded to the nearest integer). Cytoscape software was used to construct the compound-target network (C-T network) model of Dragon's Blood. The interaction network model of various lead compounds and target proteins is shown as follows.
The chalcone derivatives in the lead compounds and the protein targets are constructed as the radiation protection chalcone component-target network (chalcone C-T network) model of Dragon's Blood, as shown in Figure 7.  Space: Science & Technology upregulated proteins) nodes and 15 compound nodes were included. The remaining nodes and edges belonged to protein-protein interactions. The compound node was represented by a blue background, the upregulated protein node was represented by a yellow background, and the downregulated protein node was represented by a green background. Compound 10 (socotrin-4 ′ -ol), compound 9 (2 ′ -methoxy socotrin-5 ′ -ol), and compound 1 (licochalcone A) were linked to 54, 51, and 39 target proteins, respectively. They were not only in an important position in the chalcone C-T network but also in the overall Dragon's Blood C-T network model. Through molecular docking and network pharmacology analysis, it was suggested that chalcone derivatives were the active antiradiation ingredients in Dragon's Blood. The dihydrochalcone derivatives in the lead compounds and the protein targets constructed the dihydrochalcone component-target network (dihydrochalcone CT network) model as shown in Figure 8. Among them, 63 nodes and 275 edges belonged to compound-protein interactions. Among the 63 nodes, 47 target protein (15 downregulated proteins, 32 upregulated proteins) nodes and 16 compound nodes were included. The remaining nodes and edges belonged to protein-protein interactions. The compound node was represented by a blue background, the upregulated protein node was represented by a yellow background, and the downregulated protein node was represented by a green background. Compound 15 (2 ′ ,4 ′ -dihydroxy-4-methoxydihydrochalcone) and compound 6 (loureirin B) were linked to 26 and 24 target proteins, respectively. They were not only in an important position in the dihydrochalcone C-T network but also in an important position in the overall Dragon's Blood C-T network model. Through molecular docking and network pharmacology analysis, it is suggested that dihydrochalcone compounds are the antiradiation active ingredients in Dragon's Blood.
The flavan derivatives in the lead compounds and their protein targets constructed the radiation protection flavan component-target point network (flavan C-T network) model of Dragon's Blood, as shown in Figure 9. Among them, there were 64 nodes and 292 edges that belonged to the compoundprotein interaction. Among the 64 nodes, 56 target protein (19 downregulated proteins, 37 upregulated proteins) nodes and 8 compound nodes were included. The remaining nodes and edges belonged to protein-protein interactions. The compound node was represented by a blue background, the upregulated protein node was represented by a yellow background, and the downregulated protein node was represented by a green background. Compound 2 (4 ′ ,7-dihydroxy-3 ′ -meoxylflavan), compound 3 (4 ′ -hydroxy-7-methoxy-8-methylflavan), and compound 8 (7,4 ′ -dihydroxy-8-methylflavan) were linked to 47, 37, and 37 target proteins, respectively, and they were not only in an important position in the flavan C-T network but also in the overall Dragon's Blood C-T network model. Through molecular docking and network pharmacological analysis, it is suggested that flavan components are the antiradiation active ingredients in dragon's blood. The phenol and ester derivatives in the lead compounds and the protein targets bound to them were used to construct the phenol and ester component-target network (phenol and ester C-T network) model as shown in Figure 10. Among them, there were 60 nodes and 106 edges that belong to the compound-protein interaction, and the 64 nodes included 51 target protein (18 downregulated proteins, 33 upregulated proteins) nodes and 9 compound nodes. The remaining nodes and edges belonged to protein-protein interactions. The compound node was represented by a blue background, the upregulated protein node was represented by a yellow background, and the downregulated protein node was represented by a green background. Compound 2 (7,10-dihydroxy-11-methoxydracaena) and compound 1 (10-hydroxy-11-methoxydracaena) were connected to 45 and 42 target proteins, respectively. They were not only in the important position of phenol and ester C-T network but also in the important position of the whole Dragon's Blood C-T network model. Through molecular docking and network pharmacology analysis, it is suggested that phenol and ester compounds are the antiradiation active ingredients in Dragon's Blood.

Discussion
Cells and tissues that have not been irradiated may undergo genetic mutations, genetic instability, DNA damage, cell proliferation and apoptosis, inflammation, and cancer for-mation due to the side effects of radiation. At present, the research on side effects of radiation is mainly done in vitro (cocultivation and medium transfer) and in vivo research. Radiation destroys the structure of biological macromolecules through direct or indirect action. Through the complex regulatory network within the cell, it affects the normal functions of tissues and organs and ultimately renders the body in an abnormal state.
Clinical studies have found that tumor radiotherapy will cause damage to the corresponding normal tissues, such as radiation pneumonitis and radiation pulmonary fibrosis after radiotherapy for lung cancer [22,23]. Research by Siva et al. found that DNA damage was found in unirradiated hair follicles during radiotherapy and chemotherapy for patients with non-small-cell lung cancer, indicating that local radiotherapy would cause DNA damage in unirradiated distant normal tissues [24]. In addition, radiotherapy to noncerebral parts will cause side effects in the brain tissues of the remote unexposed parts, leading to a decrease in basal metabolism and persistent neuroinflammation in the brain [25]. The above-mentioned studies indicate that the pararadiation effect in vivo caused by radiotherapy on unirradiated normal tissues is mainly manifested as a damage effect. Radiation damage to the central nervous system will not only cause damage to the thymus, spleen, and peripheral blood. It also affects peripheral organs including the myocardium, lungs, liver, trachea, kidneys, and stomach. Our previous research found that a rat's  Space: Science & Technology liver had been damaged after whole-brain radiation, and the damage was manifested as a significant increase in the number of apoptotic cells and blurred central vein boundaries [26]. Dragon's Blood is a traditional Chinese medicine that can reduce the risk of radiation damage due to its rich biologically active substances. Previous studies in our lab have found that Dragon's Blood has nerve repair and neuroprotection effects and at the same time can enhance the body's immunity and exert antioxidant effects. The results of previous studies in our lab have shown that Dragon's Blood can protect against radiation-induced brain damage by reducing oxidative stress [27] and inflammation [28], inhibiting mitochondrial dysfunction [29], and promoting nerve cell regeneration [30]. Dragon's Blood can significantly increase the levels of superoxide dismutase (SOD) and glutathione (GSH) in the brain and reduce the levels of malondialdehyde (MDA) and hydrogen peroxide (H 2 O 2 ) in the rat brain in the brain-irradiated rat model. It also significantly reduces the levels of inflammatory factors such as TNF-α, interferon-γ (INF-γ), and IL-6 in the brain tissue after irradiation. In addition, Dragon's Blood can improve the inflammatory reaction of the gastrointestinal tract such as congestion and edema after brain irradiation, increase the number of immune cells, and maintain the stability of the body's endocrine system. Dragon's Blood can also improve the symptoms of liver punctate necrosis, focal necrosis, and inflammatory cell infiltration in rats with whole-body radiation.
This study further explored the effect of Dragon's Blood on the liver of brain-irradiated rats and its potential molecular mechanism through proteomic analysis and network pharmacology. Previous research indicated that in the acute phase (within 7 days) after the irradiation, the weight of the rats dropped rapidly and the irradiated parts were depilated. The rat sugar water preference experiment showed that the rats exhibited depression-like symptoms after the Co 60 γ-ray brain irradiation, suggesting that the whole-brain irradiation induces function damage on the central nervous system. The pathological section results showed that the neurons in the sensitive areas of the brain (the hippocampus and the prefrontal cortex) had partial edema and vacuolization. The Dragon's Blood administration group showed protection against radiation brain injury in terms of general growth of rats, sugar water preference experiment, brain pathological slice analysis, oxidative stress level, and inflammatory factor level (unpublished results) in the brain and peripheral blood system. Herein, after 3 days of brain irradiation, the liver pathological section results showed that DB significantly improved the local hepatocyte necrosis, nuclear fragmentation, and disappearance caused by radiation. From the level of inflammatory factors, since we observed changes in the liver during the acute phase (3 days) and the liver has a certain ability to repair, the increase in the content of inflammatory factors in the liver during the acute phase after irradiation is not very obvious. Through proteomic analysis, 4557 proteins in the liver were identified. There were obvious differences in the protein expression between the three groups of rats. The principal Irradiation significantly affects the metabolic system of the liver. The first phase of drug metabolism includes oxidation, demethylation, and hydrolysis. It is mainly mediated by CYP450 enzymes. CYP450 is a family of enzymes composed of a series of oxidases containing heme coenzymes, which are the main metabolizing enzymes of exogenous substances such as drugs, poisons, and environmental carcinogens. After the drug undergoes the first phase of metabolism such as oxidation and demethylation, the nonpolar fat-soluble compound becomes a metabolite with higher polarity and water solubility but lower activity. Up to date, the effect of radiation on cytochrome P450 is still a topic of debate. It is generally believed that high-dose radiation increases the expression of CYP1A1 and CYP1B1 in rats, zebrafish, or humans. It also increases the expression of CYP1A2, CYP2B1, and CYP3A1 in rats and CYP2E1 in mice or rats and decreases CYP2C11 and CYP2D1 in rats' expression. Radiation also increases the expression of multidrug resistance protein and breast cancer resistance protein. During simultaneous chemotherapy and radiotherapy, the metabolism and clearance of certain drugs increase, while the half-life, average residence time, and area under the curve decrease. The expression changes of cytochrome P450 and drug transporter are consistent with the pharmacokinetic changes of certain drugs under radiation [31]. The contents of CYP2C29, CYP2E1, and CYP1A2 in the liver of mice exposed to space flight and microgravity for 30 days were significantly changed compared with the ground control group. Within 7 days after landing and during the corresponding recovery period, the content of CYP2C29 and CYP1A2 returned to normal levels, while the content of CYP2E1 continued to increase [32]. The induction of enzymes observed in mice under space conditions can lead to changes in the biotransformation and efficiency of pharmaceutical preparations, and they are metabolized by the corresponding CYP subtypes.
The second-phase reaction refers to the binding reaction between the drug or its first phase metabolite and the endogenous binding agent. After the combination, the toxicity or activity of the drug decreases, and the polarity increases. It is easy to be discharged. In this study, radiation mainly changed the expression of GST enzyme system (Gsta5), Gstm4, and Mgst3 in the two-phase reaction of the rat liver and also affected the expression of glucuronate UGT enzyme (Ugt1a7c) and aldehyde oxidase 1 (Aox1). GSTs are the key enzymes that catalyze the initial steps of the glutathione binding reaction. GSTs catalyze the binding reaction of nucleophilic glutathione and various electrophilic exogenous chemical substances to form more soluble and nontoxic derivatives and make them easy to excrete or further metabolize. According to the change trend of differential protein in the irradiated drug phase I (the differential proteins showed mostly a downward trend) and phase II metabolism (the differential protein showed mostly an upward trend), it is believed that the liver itself can slow down the toxicity by changing the relevant drug-metabolizing enzymes and metabolic active intermediates, promoting the body's detoxification and discharging of substances. At the same time, environmental pollution or radiation caused by clinical treatment can change the pharmacokinetic characteristics of drugs. Therefore, the pharmacokinetics of the drug should be rechecked after radiotherapy, and the optimal dose should be reassessed.  Figure 11: The process of retinol metabolism. Red asterisks represent differential proteins.
14 Space: Science & Technology Radiation also significantly affected liver retinol metabolism and peroxisome processes. The conversion of retinol into biologically active retinoic acid requires a two-step oxidation reaction. The first step involves retinol dehydrogenase (ADH) and alcohol dehydrogenase catalyzing retinol to retinal. The second step involves retinal dehydrogenase (ALDH) catalyzing retinal to produce retinoic acid ( Figure 11). Differential protein Aldh1a1 can catalyze alltrans retinal to all-trans-retinoic acid, which can be further oxidized by CYP enzymes (including Cyp2a1, Cyp2b2, Cyp2c7, Cyp2c11, Cyp3a23/3a1, and Cyp3a9) to produce 4-oxyretinoic acid. The storage of retinoic acid and its active metabolites, as well as heredity, can affect the development and progression of liver fibrosis and inflammation [33]. Trans-retinoic acid (ATRA) can cause a decrease in body fat and a decrease in circulating triglyceride levels. Previous research showed that mice that flew on the Space Transportation System-(STS-) 135 for 13 days suffered liver damage, manifested by accumulation of liver lipid droplets, high triglyceride levels, and loss of retinoids in hepatic stellate cell (HSC) lipid droplets [34]. These results had been related to the activation of peroxisome proliferator-activated receptor alpha (PPARα) [35]. PPARα is a transcriptional regulator of genes involved in peroxisome and mitochondrial β oxidation, fatty acid transport, and hepatic glucose production, with the latter being unique to rodents [36]. Proinflammatory and acute-phase response signaling pathways negatively regulate PPARα, which can be seen in rodent models of systemic inflammation, atherosclerosis, and nonalcoholic stea-tohepatitis (NASH) [23,37]. The dysregulation of the PPARα signaling pathway can lead to the production of markers that are considered to be the precursors of early nonalcoholic fatty liver-(NAFLD-) like symptoms [38,39].
The differential proteins in RAD/CON groups including Cyp2c6v1, Cyp2c7, Cyp2c11, Gsta3, Gstm4, Cyp3a23/3a1, and Cyp3a9 and other proteins are involved in the chemical carcinogenesis process. These proteins are involved in the formation of highly electrophilic chemical carcinogens. The highly electrophilic chemical carcinogens bind to electron-rich nucleophilic residues in cellular DNA, affect the normal coordination of base pairs, and cause changes in amino acids and proteins.
The mechanism of radiation-induced liver cancer has been studied on the molecular level in literature. The defect in the FHIT gene seems to be related to the increased frequency of liver cancer induced by radiation [40]. The FHIT gene is a tumor suppressor gene, which is said to be involved in the regulation of normal cell checkpoints and the progression of apoptosis [41]. According to reports, many cases of radiation-induced liver cancer are caused by acute radiation, but the occurrence of liver cancer may also be related to the radiation dose rate [42].
Radiation-induced epigenetic effects such as DNA methylation and microRNA regulation have received extensive attention. In the liver, miRNA21 appears to be involved in the formation of radiation-induced liver cancer [43]. Iron ion beam and high-line energy transfer (LET) particle radiation obviously induced the expression of miRNA21. In addition, it is reported that miRNA34 has also changed after   [44]. Other studies have examined changes in protein expression. In particular, proteins related to inflammation or apoptosis seem to contribute to the effects of radiation on the liver [45,46]. High doses of whole-body radiation usually induce inflammation, leading to fibrosis or cancer. Interestingly, the expression of inflammationrelated proteins in high-dose (4 Gy or 8 Gy) acute wholebody irradiation mice continued to change, while longterm irradiation with a total dose of 8 Gy at a low dose rate induced changes in the expression of many apoptosisrelated proteins [47]. It suggested that apoptosis induced inhibitory effect on the occurrence of liver cancer to some extent [48]. Changes in apoptosis-related proteins may indicate that the radiation-induced protective ability will continue after radiation damage [42,47].
As shown in Figures 12, 85 significantly changed proteins in the DB/RAD group affect each other, which are involved in processes such as retinol metabolism, steroid hormone biosynthesis, chemical carcinogenesis, and linoleic acid metabolism. Among them, 26 proteins have returned to normal levels by radiation. Dragon's blood regulates the CYP450 enzyme system (Cyp2b2, Cyp3a1, and Cyp3a9) to normal level. It also regulates mitochondrial homeostasis and maintains redox at normal levels (Msrb2, Txnrd2, and Samm50).
Due to the characteristics of multicomponent, multipathway, and multitarget synergistic effects of traditional Chinese medicine and its compound prescriptions, the component complexity makes the mechanisms of traditional Chinese medicines unclear. The lack of a scientific, reasonable, and effective efficacy and safety evaluation system hindered the development of traditional Chinese medicine. It is difficult to conduct a comprehensive and systematic study from the whole picture to the level of tissues, organs, cells, and molecules. This greatly limits the modernization and internationalization of Chinese medicine. Network pharmacology is developed on the basis of the rapid development of systems biology and computer technology. Through the docking and scoring of the main active ingredients in Dragon's Blood and the significantly different proteins, the main radiation protection components and target proteins in Dragon's Blood have been found. These results bring important reference value for the development of Dragon's Blood as a radiation protection drug. Dragon's Blood mainly contains chalcones, dihydrochalcones, flavans, phenols, and ester natural products. Through the docking results, it is found that 2 ′ -methoxy socotrin-5 ′ -ol, socotrin-4 ′ -ol, 4 ′ ,7-dihydroxy-3 ′ -meoxylflavan, 4 ′ -hydroxy-7-methoxy-8methylflavan, 10-hydroxy-11-methoxydracaena, and 7,10dihydroxy-11-methoxydracaena have the best binding effect with differential proteins.

Conclusion
Herein, the proteomic method combined with network pharmacology molecular docking technology is used to clarify the damage effect of radiation on the liver and the protective mechanism of Dragon's Blood. Proteomic analysis shows that 30 Gy γ-ray radiation of the whole brain causes liver metabolism disorders, including protein changes, which mainly involve drug metabolism, retinol metabolism, chemical carcinogenesis, and peroxisome pathways. The proteomics of the liver of rats exposed to brain radiation, the protective effect of Dragon's Blood, and its potential molecular mechanism were analyzed. A total of 26 key proteins (Msrb2, Txnrd2, Samm50, Pir, Ehhadh, Pex11a, etc.) were significantly regulated with the administration of Dragon's Blood for 10 days before radiation, mainly regulating metabolic processes and redox homeostasis. Network pharmacology shows that the main active ingredients in Dragon's Blood include chalcone (2 ′ -methoxy socotrin-5 ′ -ol and socotrin-4 ′ -ol), flavans (4 ′ ,7-dihydroxy-3 ′ -meoxylflavan, 4 ′ -hydroxy-7-methoxy-8methyl flavan), phenol and ester derivatives (10-hydroxy-11methoxy dracaena, 7,10-dihydroxy-11-methoxy dracaena), etc. Each compound corresponds to multiple protein targets, reflecting the multicomponent and multitarget characteristics of traditional Chinese medicine. In summary, Dragon's Blood can protect the liver of brain-irradiated rats by improving liver necrosis, regulating the level of inflammatory factors, and regulating the expression of liver proteins. This work provided insights into the pathways involved in the brain radiationinduced liver damage, as well as guidance that pharmacologists could potentially focused to develop drugs for radiotherapy protection.