Part III: Study On The Mechanism Of Qitu Erzhi Prescription in Preventing And Treating Chemotherapy-induced Bone Marrow Suppression Based On Network Pharmacology

1 Research background

Based on many years of clinical experience, Professor Yang Yufei, the supervisor, summarized the common adverse reactions of adjuvant chemotherapy for colorectal cancer and their timing of occurrence. Combining the principle of "reinforcing the damaged and replenishing the deficient" in the Yellow Emperor's Classic of Internal Medicine, he proposed a "two-stage three-step" approach to bone marrow suppression caused by adjuvant chemotherapy for colorectal cancer [I], which accurately responds to gastrointestinal reactions and bone marrow suppression caused by adjuvant chemotherapy for colorectal cancer. Qitu Erzhi Recipe is a prescription designed for the second stage of bone marrow suppression. It has the function of strengthening the spleen and tonifying the kidney. The recipe is derived from Erzhi Pills combined with Danggui Buxue Decoction. The ingredients include Astragalus, Angelica, Ligustrum lucidum, Ecliptae, Psoralea corylifolia, Cuscuta australis, etc.

NEW HERBS FOR KIDNEY YIN AND YANG DEFICIENCY

Erzhi Pills originated from the Ming Dynasty's "Fushou Jing Recipe". Because Ligustrum lucidum is "harvested on the winter solstice" and Ecliptae is "harvested on the summer solstice", it is named Erzhi Pills. The winter solstice is the time when yang rises, and the summer solstice is the time when yin is born. Collecting herbs at these two times can obtain the yin and yang of the four seasons, which is unique in nourishing the kidneys, the "innate foundation". The "Shennong Bencao Jing" says that the fruit of Ligustrum lucidum can nourish the middle, calm the five internal organs, nourish the spirit, and eliminate all diseases. The "Compendium of Materia Medica" calls it a "superior non-toxic and wonderful medicine". The herb of Eclipta prostrata was first recorded in the "Tang Materia Medica". Li Shizhen believed that the herb of Eclipta prostrata can "blacken the beard and hair, and benefit the kidney yin". Wang Ang's "Yi Fang Ji Jie" in the Qing Dynasty recorded that it can "tonify the waist and knees, strengthen the tendons and bones, strengthen the yin kidney, and blacken the beard and hair". The Chinese angelica blood-replenishing soup was first recorded in "Neiwai Shang Bianhuo Lun". It is a classic Chinese medicine prescription for nourishing qi and generating blood, and can also be used for fever caused by blood deficiency. The "Shennong Bencao Jing" lists Astragalus as a top-grade product, and the original text states: It tastes sweet and slightly warm. It is mainly used to...tonify deficiency and treat all kinds of diseases in children. Its color is yellow, its taste is sweet and it enters the spleen. It is an important medicine for strengthening the spleen, replenishing qi, and tonifying deficiency. Zhang Zhongjing discussed in "Golden Chamber Synopsis · Blood Bi Deficiency Disease Pulse Symptom and Treatment Chapter 6": Deficiency and labor, urgency, and various deficiencies, Huangqi Jianzhong Decoction is the main treatment. The effect of Huangqi in tonifying deficiency is obvious. Zhang Xichun's "Medical Zhong Can Xi Lu" believes that it "has the best effect of replenishing qi, so it is regarded as the best tonic." The blood-tonifying effect of angelica can be found in "Rihuazi Bencao": "It treats...all blood, replenishes all fatigue...nourishes new blood." Zhang Xichun's "Medical Zhong Can Xi Lu" records that it "is the main medicine for generating blood and activating blood circulation, and it can also promote the flow of qi so that qi and blood can each go to their place, so it is named angelica." The two medicines are used together to form Danggui Buxue Decoction. The prescription is named after Angelica sinensis, but in fact, the amount of Astragalus membranaceus in the original prescription is five times that of Angelica sinensis. It is a true reflection of the idea in Jingyue Quanshu that "tangible blood cannot be produced quickly, and intangible Qi should be consolidated urgently", and has a good effect on blood loss and Qi loss with blood. Although bone marrow suppression caused by chemotherapy is not like acute blood loss, it is still a large loss of hematopoietic cells in a short period, which is as dangerous as acute blood loss. Therefore, Angelica sinensis blood-replenishing soup is used to replenish Qi and produce blood. Psoralea corylifolia and Cuscuta chinensis are both fruits of plants and are used to nourish the kidney. Cuscuta chinensis was first recorded in Shennong's Herbal Classic: it tastes spicy and neutral and is mainly used to heal injuries, replenish deficiencies, replenish Qi and strength, and make the body healthy. Suwen · Zangqi Fashi Lun: "The kidney is bitter and dry, so eat spicy food to moisten it." If the kidney yin and kidney essence are insufficient, spicy products should be used to promote the four Qi, and the original Yang and fire will slowly grow. It contains the meaning of "seeking Yin in Yang", and is a top-grade product for nourishing the kidney and replenishing essence. Psoralea corylifolia, also known as Po Gu Zhi, tastes bitter and pungent, is warm, and enters the kidney and spleen meridians. The Kaibao Materia Medica discusses its function: "It is used to treat five kinds of labor and seven kinds of injuries, wind deficiency and cold, bone marrow damage, kidney cold and semen flow, and women's blood and qi abortion." It is an important medicine for nourishing the kidney and marrow, warming the spleen and replenishing deficiency. The famous Chinese medicine practitioner Li Ke combined Psoralea corylifolia, dodder seed, celery, and wolfberry into the "four kidney flavors", calling them "moist but not dry, nourishing but not greasy." Psoralea corylifolia and dodder seed can enter the spleen and kidney meridians, nourish the spleen and kidney and nourish blood without nourishing and greasy to the stomach. They are pungent in taste and warm in nature, without the risk of blood movement. They are very beneficial for the situation of spleen and kidney damage during chemotherapy.

The Qitu Erzhi formula is exquisitely composed, the whole formula is small and light, and it nourishes the kidney without the risk of nourishing and greasy to the stomach. It has achieved good results in clinical application. However, its mechanism of action in preventing and treating bone marrow suppression caused by adjuvant chemotherapy is still unclear. This study used network pharmacology to explore the mechanism of Qituerzhi prescription in preventing and treating chemotherapy-induced bone marrow suppression.

2 Data and methods

2.1 Screening of active ingredients

With the help of Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform [2] (TCMSP), we searched for Hedysarum Multijugum Maxim, Angelicae Sinensis Radix, Fructus Ligustri Lucidi, Ecliptae Herba, Cuscutae Semen, and Fructus Psoraleae to collect the main chemical components of the Qitu Erzhi prescription. We supplemented and improved the information based on literature reports. Potential active ingredients were screened according to oral bioavailability (OB) ≥ 30%I³] and drug-likeness (DL) ≥ 0.18 [4]. For Chinese medicines not included in the TCMSP database, we searched with the help of the ETCM (The Encyclopedia of

Traditional Chinese Medicine) database5, and combined with relevant literature to search for drug chemical components. For the retrieved chemical components, based on their chemical structures, potential active ingredients were screened on the Swiss ADME online platform based on GI absorption and DL (including five aspects such as Lipinski, Ghose, Vibe, Egan, and Muegge).

2.2 Target screening and component-target network construction

With the help of the Swiss Target Prediction online platform, target prediction was performed for the potential active ingredients of each Chinese medicine, and the species type was selected as "Homo sapiens". If there are many potential targets, the median of Probability was used for screening. The target of the potential active ingredient of each medicine was obtained, and the target dataset of the potential active ingredient of the Qitu Erzhi prescription was constructed.

2.3 Disease target collection

Using "chemotherapy-induced bone marrow suppression", "chemotherapy-induced marrow depression", "chemotherapy-induced Myelosuppression", "chemotherapy-induced thrombocytopenia", "chemotherapy-induced anemia", "chemotherapy-induced erythropenia", "chemotherapy-induced neutropenia", "chemotherapy-induced leukopenia" and other search terms, we searched for disease-related targets of chemotherapy-induced bone marrow suppression in four databases, including OMIM (Online Mendelian Inheritance in Man, OMIM, https://www.omim.org/), GeneCards (https://www.genecards.org/), TTD database.

[6](Therapeutic Target Database, http://db.idrblab.net/td/), and Drugbank (https://go.drugbank.com/)[7,8]. In the GeneCards database, the score was set as Targets with values greater than the median are potential targets[9]. For the retrieved disease targets, duplicate values were deleted and their union was taken as the disease target.

2.4 Drug-disease Target screening and PPI network construction

Venny 2.1.0 (https://bioinfogp.cnb.csic.es/tools/venny/) was used to intersect the potential drug targets and disease targets and draw a Venn diagram. The targets obtained from the intersection were uploaded to the String11.0 (https://string-db.org/) database[10 for protein-protein interaction (PPI) network construction.

2.5 GO function and KEGG pathway enrichment analysis

Gene Ontology (GO) covers three aspects: molecular function (MF), biological process (BP), and cellular component (CC), which can be used to interpret biological functions; Kyoto Encyclopedia of Genes and Genomes (Kyoto Encyclopedia of Genes and Genomes) KEGG (Kenoy et al., 2011) can be used to study the high-level functions of biological systems. In this study, GO and KEGG analysis was performed on candidate genes using the Metascape database [11] (http://metascape.org/). Twenty GO and KEGG genes with significant differences were screened.

3 Research results

3.1 Screening of active ingredients in traditional Chinese medicine

(1) Astragalus

87 components of Astragalus were retrieved, and 20 potential active ingredients were obtained after screening. The specific components are shown in Table 3-1.

(2) Angelica sinensis

A preliminary search of 125 components of Angelica sinensis revealed that only two potential active components were obtained after screening: beta-sitosterol and Stigmasterol. Considering that Angelica sinensis is a "blood-replenishing medicine", it contains a variety of components such as volatile oils, organic acids, polysaccharides, and flavonoids [12-14]. Among them, volatile oils and ferulic acid are important indicators for identifying the quality of Angelica sinensis [15]. At the same time, combined with the research of other scholars [16], the remaining 10 components such as ferulic acid were included for target prediction. (Table 3-2)