Therapeutic Effect of Ecdysterone Combine Paeonol Oral Cavity Direct Administered on Radiation-Induced Oral Mucositis in Rats

Radiation-induced oral mucositis represents an influential factor in cancer patients’ accepted radiation therapy, especially in head and neck cancer. This research investigates the treatment effect of Ecdysterone (a steroid derived from the dry root of Achyranthes bidentate) and Paeonol (a compound derived from Cortex Moutan) on radiation-induced oral mucositis and possible underlying mechanisms. Concisely, 20 Gy of X-rays (single-dose) irradiated the cranial localization in rats for the modeling of oral mucositis. The therapeutic effects of Ecdysterone-Paeonol oral cavity directly administered on radiation-induced oral mucositis were investigated by weight changes, direct observations, visual scoring methods, ulcer area/total area, and basic recovery days. Assessments of tumor necrosis factor α and interleukin-6 were performed to evaluate the inflammatory cytokines secretion in the damaged areas of tongues harvested post-treatment, and changes in signaling pathways were investigated by Western blotting. System Drug Target (SysDT) methods revealed the targets of Ecdysterone-Paeonol in order to support compound-target network construction. Four representative targets with different functions were chosen. The binding interactions between the compound and receptor were evaluated by molecular docking to investigate the binding affinity of the ligand to their protein targets. Ecdysterone-Paeonol, administered orally, effectively improved radiation-induced oral mucositis in rats, and the therapeutic effect was better than Ecdysterone administered orally on its own. In this study, calculational chemistry revealed that Ecdysterone-Paeonol affected 19 function targets associated with radiation-induced oral mucositis, including apoptosis, proliferation, inflammation, and wound healing. These findings position Ecdysterone-Paeonol as a potential treatment candidate for oral mucositis acting on multiple targets in the clinic.


Introduction
Radiation-induced oral mucositis (RIOM), a normal oral cavity tissue injury resulting from radiotherapy, represents an influential factor in cancer patients' accepted radiation therapy, especially in head and neck cancer patients [1][2][3]. Quality of life, economic cost, and radiotherapy continuity are affected by RIOM, which slows the cancer therapy and impoverishes tumor control [4]. Unfortunately, RIOM, throughout the whole course of radiotherapy, is a huge challenge for radiation oncologists, like climbing an endless mountain [5].

Introduction
Radiation-induced oral mucositis (RIOM), a normal oral cavity tissue injury resulting from radiotherapy, represents an influential factor in cancer patients' accepted radiation therapy, especially in head and neck cancer patients [1][2][3]. Quality of life, economic cost, and radiotherapy continuity are affected by RIOM, which slows the cancer therapy and impoverishes tumor control [4]. Unfortunately, RIOM, throughout the whole course of radiotherapy, is a huge challenge for radiation oncologists, like climbing an endless mountain [5].
Recent studies have proposed that radiation-induced oral mucositis involves five representative biologic progression phases [3,6]: Phases І, initiation stage characterized by large reactive oxygen species (ROS) production; Phases ІІ/Ⅲ, message generation/signaling and amplification stage characterized by large epithelial ablation caused by mass-apoptosis and halted cell proliferation; Phases ІѴ, ulceration stage characterized by a large area of mucositis and pro-inflammatory cytokines secretion; and Phases Ѵ, healing stage characterized by slow wound healing. As we can see, apoptosis, proliferation, inflammation, and wound healing present different functions in the pathological development course of RIOM.
A previous study by our research group revealed that Ecdysterone (a steroid derived from the dry root of Achyranthes bidentate) ( Figure 1A) accelerates the healing of radiation-induced oral mucositis in rats by increasing matrix cell proliferation [7]. However, after deeper research, we found that, even though Ecdysterone has an anti-inflammatory effect, the effect is only weak. Proinflammatory cytokines, such as TNF-α and IL-6, were lower than in the modeling rats, but there was no significantly difference. As the research went deeper, we found that the weak anti-inflammatory effect of Ecdysterone is a drawback in the course of treatment. Therefore, in order to increase the therapeutic effect, a suitable anti-inflammatory drug had to be used as a supplement.
Consequently, we hypothesized that a combination drug composed of an anti-inflammatory drug and Ecdysterone could increase the therapeutic effect. In the previous study, we used the oral direct administration method. Therefore, the combination anti-inflammatory drug had to meet the same conditions for better transdermal absorption. After preliminary screening, we found that Paeonol ( Figure 1B), a compound derived from Cortex Moutan, which has been used for its antiinflammatory, antioxidant, and antibacterial properties, could suit the supplement role [8][9][10][11]. In order to uncover the therapeutic effect and possible underlying mechanisms, we performed a systematic research approach as shown in Figure 2. In this study, we demonstrate that Ecdysterone-Paeonol combination drug administered orally effectively improves radiation-induced oral mucositis in rats, and the therapeutic effect is better than Ecdysterone administered orally on its own. A systems pharmacology approach uncovered the pharmacological characteristics of multiple targets, network pharmacology, and molecular docking between Ecdysterone-Paeonol and their receptors. These In order to uncover the therapeutic effect and possible underlying mechanisms, we performed a systematic research approach as shown in Figure 2. In this study, we demonstrate that Ecdysterone-Paeonol combination drug administered orally effectively improves radiation-induced oral mucositis in rats, and the therapeutic effect is better than Ecdysterone administered orally on its own. A systems pharmacology approach uncovered the pharmacological characteristics of multiple targets, network pharmacology, and molecular docking between Ecdysterone-Paeonol and their receptors. These findings position Ecdysterone-Paeonol as a potential treatment candidate for oral mucositis, acting on multiple targets in the clinic.

Ecdysterone-Paeonol Alleviates the Development of Radiation-Induced Oral Mucositis, and the Treatment Effect is Better than Ecdysterone Treatment Alone
After cranial irradiation, the average weight of the irradiated groups began to decline on the fifth day. The decreasing trend of each irradiated group was similar ( Figure 3A). However, actual weight loss in the 10% Ecdysterone and 10% Ecdysterone + 5% Paeonol groups was less than in the irradiated + vehicle group ( Figure 3C). This indicated that there was a decrease in the RIOM affected rats' eating desire in each irradiated group, but the situation was improved by treatment with different drugs.
Visual scoring observations every 2nd day throughout the course of treatment revealed that the development of RIOM was alleviated in each treatment group, and the effect in the 10% Ecdysterone + 5% Paeonol group was better than in the 10% Ecdysterone group after 12 days of treatment ( Figure 3B). Direct observations and ulcer area also evidenced this result ( Figure 3D).
According to the "visual scoring assessment of oral mucositis", the number of basic recovery days in the treatment group was less than in the irradiated + vehicle group. Better yet, the recovery time of the 10% Ecdysterone + 5% Paeonol group was shorter than the 10% Ecdysterone group ( Figure 3E).

Ecdysterone-Paeonol Alleviates Inflammatory Cytokines Secretion by Inhibiting the NF-κB Pathway
The therapeutic effect of 10% Ecdysterone + 5% Paeonol is better than Ecdysterone administered on its own. The results that expressions of TNF-α and IL-6, two representative inflammatory cytokines, were detected were explored more deeply using immunohistochemistry assay. The expression of the 10% Ecdysterone and the irradiated + vehicle group were higher than the unirradiated + vehicle group. Even though the expression of 10% Ecdysterone was lower than the irradiated + vehicle group, there was no significantly difference. However, 10% Ecdysterone + 5% Paeonol reduced the expression of inflammatory cytokines compared with the unirradiated + vehicle group ( Figure 4A). Meanwhile, the results of Western blotting assay indicated that the NF-κB signaling pathways were significantly down-regulated after 10% Ecdysterone + 5% Paeonol treatment ( Figure 4B).

Ecdysterone-Paeonol Alleviates Inflammatory Cytokines Secretion by Inhibiting the NF-κB Pathway
The therapeutic effect of 10% Ecdysterone + 5% Paeonol is better than Ecdysterone administered on its own. The results that expressions of TNF-α and IL-6, two representative inflammatory cytokines, were detected were explored more deeply using immunohistochemistry assay. The expression of the 10% Ecdysterone and the irradiated + vehicle group were higher than the unirradiated + vehicle group. Even though the expression of 10% Ecdysterone was lower than the irradiated + vehicle group, there was no significantly difference. However, 10% Ecdysterone + 5% Paeonol reduced the expression of inflammatory cytokines compared with the unirradiated + vehicle group ( Figure 4A). Meanwhile, the results of Western blotting assay indicated that the NF-κB signaling pathways were significantly down-regulated after 10% Ecdysterone + 5% Paeonol treatment ( Figure 4B).

Drug Targeting and Network Construction
Ecdysterone and Paeonol, two compounds isolated from traditional Chinese medicine herbs, might target multiple proteins. In order to probe the binding of their targets, SysDT methods were applied [12]. In the results shown in Table 1, 19 predicted targets associated with apoptosis, proliferation, inflammation, and wound healing, which play important roles in the development of RIOM, are screened out. For instance, Glycogen synthase kinase-3 beta (GSK3 β) is a negative regulator of apoptotic process protein [13]. Prothrombin (F2) is a positive regulator of cell growth and a negative regulator of cytokines production involved in inflammatory response protein [14,15]. Hematopoietic cell protein-tyrosine phosphatase 70Z-PEP (PTPN22) is a negative regulator of interleukin-6 secretion and the NIK/NF-kappaB signaling way [16]. Plectin (PLEC) is a wound healing regulator [17].

Drug Targeting and Network Construction
Ecdysterone and Paeonol, two compounds isolated from traditional Chinese medicine herbs, might target multiple proteins. In order to probe the binding of their targets, SysDT methods were applied [12]. In the results shown in Table 1, 19 predicted targets associated with apoptosis, proliferation, inflammation, and wound healing, which play important roles in the development of RIOM, are screened out. For instance, Glycogen synthase kinase-3 beta (GSK3 β) is a negative regulator of apoptotic process protein [13]. Prothrombin (F2) is a positive regulator of cell growth and a negative regulator of cytokines production involved in inflammatory response protein [14,15]. Hematopoietic cell protein-tyrosine phosphatase 70Z-PEP (PTPN22) is a negative regulator of interleukin-6 secretion and the NIK/NF-kappaB signaling way [16]. Plectin (PLEC) is a wound healing regulator [17]. To uncover the synergistic effects of Ecdysterone-Paeonol on multiple targets in the treatment of radiation-induced oral mucositis, as well as to assess their mechanisms, a compound−target (C-T) network, as shown in Figure 5, was constructed using detailed information of the targets provided in Table 1. All of these results indicated that Ecdysterone-Paeonol affected multiple targets and showed a synergistic effect [18][19][20]. To uncover the synergistic effects of Ecdysterone-Paeonol on multiple targets in the treatment of radiation-induced oral mucositis, as well as to assess their mechanisms, a compound−target (C-T) network, as shown in Figure 5, was constructed using detailed information of the targets provided in Table 1. All of these results indicated that Ecdysterone-Paeonol affected multiple targets and showed a synergistic effect [18][19][20].

Molecular Docking Simulations
To evaluate the reliability of Ecdysterone and Paeonol and their target interactions, we simulated four representative targets for each compound with the AutoDock approach. The binding cavity snapshots are depicted in Figures 6 and 7. The detailed view of the two-dimensional ligand interaction between Ecdysterone and Paeonol and four representative receptors as shown in Figures  8 and 9.
As we can see in Figures 10 and 8, eight receptors within the binding cavity are stable during the course of the molecular dynamics simulation, suggesting that the docking model was reliable. In addition, the results of the two-dimensional ligand interaction in Figures 6 and 7 demonstrate that Ecdysterone and Paeonol anchor into a hydrophobic pocket, which is formed by different residues.

Molecular Docking Simulations
To evaluate the reliability of Ecdysterone and Paeonol and their target interactions, we simulated four representative targets for each compound with the AutoDock approach. The binding cavity snapshots are depicted in Figures 6 and 7. The detailed view of the two-dimensional ligand interaction between Ecdysterone and Paeonol and four representative receptors as shown in Figures 8 and 9.
As we can see in Figures 8 and 10, eight receptors within the binding cavity are stable during the course of the molecular dynamics simulation, suggesting that the docking model was reliable. In addition, the results of the two-dimensional ligand interaction in Figures 6 and 7 demonstrate that Ecdysterone and Paeonol anchor into a hydrophobic pocket, which is formed by different residues. All of these demonstrate that the ligand fits well with the binding site of the receptors, and the complex may become more stable after a longer time of molecule docking simulations [18].

Binding Free Energy Analysis
Binding free energy reflect the binding affinity between the compounds and the target proteins. We chose four target proteins of Ecdysterone and Paeonol as being representative. As we can see from the details in Table 2, free energies (−4.14 to −9.43 kcal/mol) indicated that two compounds

Binding Free Energy Analysis
Binding free energy reflect the binding affinity between the compounds and the target proteins. We chose four target proteins of Ecdysterone and Paeonol as being representative. As we can see from the details in Table 2, free energies (−4.14 to −9.43 kcal/mol) indicated that two compounds

Binding Free Energy Analysis
Binding free energy reflect the binding affinity between the compounds and the target proteins. We chose four target proteins of Ecdysterone and Paeonol as being representative. As we can see from the details in Table 2, free energies (−4.14 to −9.43 kcal/mol) indicated that two compounds

Binding Free Energy Analysis
Binding free energy reflect the binding affinity between the compounds and the target proteins. We chose four target proteins of Ecdysterone and Paeonol as being representative. As we can see from the details in Table 2, free energies (−4.14 to −9.43 kcal/mol) indicated that two compounds showed high binding affinities to their targets and presented a high correlation with the bio-activities of Ecdysterone-Paeonol and the targets.

Discussion
Radiation-induced oral mucositis (RIOM), a normal oral cavity tissue injury resulting from radiotherapy and a huge challenge for radiation oncologists, represents an influential factor in cancer patients' accepted radiation therapy, especially in head and neck cancer patients [3,21]. According to incomplete statistics, RIOM treatment adds an economic cost increase of up to 17,000.00 USD for every head and neck cancer patient undergoing radiation therapy [22], which is a huge economic burden. Fortunately, many companies have an interest in developing interventions for RIOM. A range of compounds are in pre-clinical or clinical development, such as Mammalian target of rapamycin (mTOR) inhibitors and recombinant protein. Even so, the management of mucositis is still austere [23].
In previous research, we discovered that Ecdysterone accelerates the healing of radiation-induced oral mucositis in rats by increasing matrix cell proliferation. These findings position Ecdysterone as a potential treatment candidate for oral mucositis in the clinic. However, after deeper research we found that, although Ecdysterone has an anti-inflammatory effect, the effect is only weak. Pro-inflammatory cytokines such as TNF-α and IL-6 were lower than in the modeling rats, but there was no significantly difference. These pro-inflammatory factors increase the vascular permeability and recruit more inflammatory cells. With the accumulation of pro-inflammatory cytokines and their negative feedback regulation, inflammation will continuously develop [24,25].
Mechanism is the guiding principle for increasing the efficacy of any drug. A good therapeutic effect is the end result that drug workers desire. In previous research, we only found the advantages of Ecdysterone [8]. Subsequently, as the research went deeper, we found the weaknesses of Ecdysterone. Therefore, according to the discovered mechanism in previous research, and in order to improve the therapeutic effects on RIOM, we had to choose a suitable drug to play a supplemental role. Therefore, in the present study, we chose a suitable anti-inflammatory drug and demonstrated that Ecdysterone-Paeonol reduced the accumulation of pro-inflammatory cytokines by down-regulating the NF-κB signaling pathway. Paeonol made up for the deficiency in the treatment of RIOM compared with Ecdysterone oral cavity direct administrated on its own. In addition, Paeonol has a good transdermal absorption, which is suitable for oral direct administration. Moreover, Paeonol has been applied for its anti-inflammatory, antioxidant, and antibacterial properties for a long time in traditional Chinese medicine [26][27][28].
Systems pharmacology dissection indicated that the Ecdysterone-Paeonol targeted multiple-targets in the treatment of RIOM. All of these targets affected different functions such apoptosis, proliferation, inflammation, and wound healing, which present different functions in the course of the pathological development of RIOM. These two compounds works synergistically in improving the therapeutic effect. Meanwhile, the approach developed in this work offers a new understanding of the action mechanisms, which will promote the development and application of simple Chinese medicine monomer formulas [29,30].
However, in this research we only used Paeonol as the combination drug [31]. Even though the therapeutic effect was better than Ecdysterone administered on its own, the ratio test should be evaluated in the future, which might be another way to enhance the therapeutic effect. Moreover, there are a large number of anti-inflammatory drugs in traditional Chinese medicine, so whether Paeonol is the best one to use should also be researched in the future.

Animals
Male Sprague Dawley (SD) rats (180-220 g, 6-8 weeks) were provided by the Experimental Animal Center of Anhui Medical University (Hefei, China). Animal experiments were approved by the Institutional Animal Care and Use Committee of Hefei University of Technology (IACUC, Permit No.1809151121, date 09/01/2018).

Rats' Living and Feeding Conditions
Living conditions before and after irradiation: plastic cages filled with large sawdust on the bottom, 20 ± 2 • C, 60 ± 10% humidity, 12 h light-dark cycle with lights on at 7:00 a.m.
Feeding conditions before irradiation: Free access to tap water and a normal diet. Feeding conditions after irradiation: Free access to sterilized (not tap) water and sterilized food (sterilized with ultraviolet irradiation).

Radiation-Induced Oral Mucositis Rat Model
Rats were randomly divided into 4 groups including 1) unirradiated + vehicle, 2) irradiated + vehicle, 3) 10% Ecdysterone, and 4) 10% Ecdysterone + 5% Paeonol. There were 12 rats in each group. Before irradiation, the rats were anesthetized. Subsequently, they were kept in the 5 mm lead jig and exposed to a single dose of 20 Gy X-rays (Varian Medical System, 6 MV, dose rate: 250 cGy/min) ( Figure 9A), in accordance with previous research [32]. After irradiation, the rats were placed in separate boxes in order to prevent suffocation death caused by different anesthesia recovery times. Once all the rats were awake, the rats in different groups were gathered together and fed according to the conditions after irradiation mentioned above.

Treatment Administration
10% Ecdysterone and 5% Paeonol was dissolved in 0.5% CMC-na, and ultrasound was used to keep the compound in solution prior to administration. An in-house constructed syringe device was applied for the administration ( Figure 9B). Rats in each group were treated with vehicle or compounds three times daily, 2 mL each time (at 9:00 am, 2:00 pm, and 6:00 pm) [32]. After 12 days of treatment, some tongue samples in each group were harvested for direct observation, immunohistochemistry, and Western blotting analyses. During this course of treatment, rats were anesthetized and tongues were pulled for visual scoring observation every 2nd day, according to the "visual scoring assessment of oral mucositis" in previous research [32], and average weight was taken daily. Visual scoring assessment was done blind by the same three people in real time. Subsequently, the remaining rats were treated and evaluated for the total course of the treatment until the RIOM needed only basic rehabilitation.

Target Fishing and Network Construction
To obtain the molecular targets of Ecdysterone-Paeonol, SysDT methods were proposed, which efficiently integrated large scale information on targets [12]. Additionally, we characterized a compound−target (C−T) network to explore the biological effects of Ecdysterone-Paeonol on different function proteins in the treatment of RIOM [33].

Molecular Docking
Presently, to calculate the interactions between Ecdysterone-Paeonol and its targets, four important predicted and approved targets associated with apoptosis, proliferation, inflammation, and wound healing were selected for docking. By using the molecular docking program AutoDock (Scripps Research Institute, San Diego, USA), simulations were performed [34]. The unit uses an X-ray crystal structure of eight targets (F2, GSK3B, IDO1, PRSS1, ERN1, PARP1, Plectin, PTPN22), taken from the RCSB Protein Data Bank. Each protein was prepared using methods such as adding polar hydrogens, partial charges, and defining the rotatable bonds. The files were then input into AutoDock. Finally, the results were analyzed using the AutoDock Tools [35].

Statistical Analysis
All statistics are presented as mean ± standard deviation (std. dev.). A Student's t-test was used for statistical comparisons. Ulcer size measurements are given as mean ± standard error of the mean (SEM). A one-way ANOVA was done for the multiple groups.

Conclusions
In summary, the Ecdysterone-Paeonol combination drug alleviated the development of radiation-induced oral mucositis and reduced the total basic recovery days. As the guiding principle for increasing the efficacy of the drug, mechanism allowed investigators to demonstrate the weakness of Ecdysterone and made up for the deficiency through the combination administration method. Paeonol presents a suitable supplement drug. The Ecdysterone-Paeonol combination drug reduces pro-inflammatory cytokines production by inhibiting the NF-κB pathways. Systems pharmacology dissection results uncovered the detailed molecular mechanism and multiple targets of Ecdysterone-Paeonol. These findings position Ecdysterone-Paeonol as a potential treatment candidate. The development of twin medicines or multiple targets drug exploitation will be the future direction of our research group.