The Effectiveness And Mechanism Of Tonifying Kidney And Spleen Method On Preventing And Treatment Of Myelosuppression Induced By Adjuvant Chemotherapy Of Colorectal Cancer

Myelosuppression induced by chemotherapy is a problem that cannot be ignored in cancer therapies. Chinese  Medicine   (CM) has been used to prevent and treat myelosuppression caused by adjuvant chemotherapy in patients with colorectal cancer  (CRC)for years. Clinical trials have been designed to evaluate the effectiveness of CM  on chemotherapy-induced myelosuppression  (CIM)in  CRC  patients. However,  high-quality evidence is required for decision-making in clinical practice and the underlying mechanism of CM remains unclear. Thus, a systematic review and meta-analysis were conducted to provide high-quality evidence on the effectiveness of CM on bone marrow suppression caused by adjuvant chemotherapy in    CRC   patients. Secondly,  the gut microbiota of CRC patients diagnosed with stage II (high-risk)/III, going to receiving adjuvant chemotherapy, and qualified as spleen and kidney deficiency was investigated.  Finally, we tried to explore the mechanism of Qitu Erzhi Decoction (QED), which has been used for preventing CIM in patients with CRC by Professor Yufei Yang, based on network pharmacology and experimental.

NEW HERB FOR TREATING KIDNEY DEFICIENCY

Part1 Effectiveness of Oral Chinese Medicine for Myelosuppression Induced by Adjuvant Chemotherapy in Patients with CRC: A Systematic Review and Meta-analysis

Objective: To evaluate the efficacy of oral CM on the prevention and treatment of myelosuppression induced by adjuvant chemotherapy in patients with CRC.

Methods: 8 electronic databases were searched from their inception to January 20, 2021. The screening and data extraction were independently carried out by two reviewers. Two investigators assessed the methodological quality of qualified researchers using the Risk of Bias (ROB)tool.RevMan 5.3 was applied for the meta-analysis.

Results: A total of 37 documents were included in the study, all of which were randomized controlled trials (RCTs), involving a total of 2705 subjects. Meta-analysis results show that oral CM significantly reduced the incidence of leukopenia(RR=0.67, 95%CI[0.61,0.75], P<0.00001), hemoglobin         reduction         (RR=0.75,95%CI[0.63, 0.89], P<0.0009), thrombocytopenia                  (RR=0.71,95%CI[0.60,0.82], P<0.0001), neutropenia(RR=0.60,95%CI[0.46,0.79], P=0.0003), grade       3/4        leukopenia(RR= 

0.36,95%CI[0.21,0.64],P=0.04),grade    3/4    hemoglobin    reduction     (RR=0.36,95% CI[0.13,0.95],P=0.04),and      grade       3/4     neutropenia(RR=0.38,95%CI[0.16,0.89], P=0.03)induced by  adjuvant  chemotherapy  in  patients  with  CRC.However, oral  CM can't reduce the incidence of erythropenia, grade 3/4 thrombocytopenia, or grade 3/4 erythropenia. Furthermore, oral  CM  seems not helpful in increasing white blood cell count(WBC), red blood cell count  (RBC), platelet  (PLT), or hemoglobin  (HGB).

Conclusions:  Oral CM can be effective in reducing the incidence of leukopenia, anemia, thrombocytopenia, neutropenia, grade   3/4   leukopenia, grade    3/4   hemoglobin reduction, and grade  3/4  neutropenia induced by adjuvant chemotherapy in  CRC patients. It can be considered to recommend oral CM for the prevention and treatment of myelosuppression caused by adjuvant chemotherapy in patients with colorectal cancer.

Part  2 Study on Characteristics of Gut Microbiota of Patients with Stage II (high risk)/II Colon Cancer and Going to Receive Adjuvant Chemotherapy and Diagnosed  of Yang Deficiency of Spleen and Kidney Syndrome Based on Chinese Medicine

Objective:  To investigate the characteristics of gut microbiota in patients with colon cancer, who are going to receive adjuvant chemotherapy and were diagnosed ofYang deficiency of spleen and kidney syndrome(D group)or not (N group).

Methods: Subjects from Xiyuan Hospital of China Academy of Chinese Medical Sciences, Jiangsu Province Hospital of Chinese Medicine, and so on were recruited, and were divided into two groups according to CM syndrome differentiation. Feces specimens of qualified patients were collected and analyzed by using 16s rDNA high-throughput sequencing technology.

Results:  32  patients were qualified, with  11  and  21  in the D  group and  N  group, respectively. There was no statistical difference between the two groups in terms of gender, age, or primary site of cancer. There was no statistical difference between the two groups in terms of community richness, diversity, or evenness. Firmicutes to Bacteroidetes ratio (FBR)in the D group was lower than that of the N group, though both Firmicutes and Bacteroidetes are the preponderant populations of the two groups. Enterobacteriaceae increased in the D group while sutterellaceae increased in the N group.  Furthermore, both   Clostridia   and   Flavobacteriales   increased   in the D   group   with

Statistical difference

Conclusions:  There was no difference between Yang deficiency of spleen and kidney syndrome and other TCM Syndrome in patients with colon cancer and going to undertaking adjuvant chemotherapy as for the community richness, diversity, or evenness.FBR may differ between the two TCM syndromes. Sutterellaceae increased in the N group while Enterobacteriaceae, clostridia, and Flavobacteriales increased in the D group.

Part   3  Study   on  the  Molecular  Mechanism   of  QED   in  the   Treatment  of Chemotherapy-induced Myelosuppression Based on Network Pharmacology

Objective: To explore the molecular mechanism of QED in the treatment of CIM.

Methods:  (1)Main ingredients of  QED  were searched in the Traditional  Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP)and The Encyclopedia of  Traditional   Chinese  Medicine  (ETCM). (2)Upload the potential effective ingredients to Swiss Target Prediction to obtain the targets. (3)Disease-related targets were obtained by querying the TTD  database, Gene   Card database, Drugbank Online, and OMIM database. (4)Wayne analysis was used to obtain the intersection of CIM  disease-related targets and the potential targets of  QED. A  protein-protein interaction network(PPI)was carried out in string database. (5)Gene ontology  (GO) analysis and enrichment pathway analysis of Kyoto gene and genome encyclopedia (KEGG)of key target proteins were conducted through the Metascape data platform. Both the results of GO and KEGG analysis were presented.

Results: 15,6,10,10,11 and 49 ingredients were identified as the potential active ingredients of Hedysarum Multijugum Maxim (Huang qi), Angelicae Sinensis Radix (Dang GUI), Ecliptae  Herba(Mo han lian), Fructus  Ligustri  Lucidi  (Nv zhen zi), Cuscutae Semen (Tu si zi)and Fructus Psoraleae (Bu gu zhi), respectively.A total of

575 targets were defined as the main targets of QED.3205 CIM disease-related targets were obtained using the 4 databases.347 targets of QED could have therapeutic effects on CIM. The PPI network map analysis found that many targets may be involved in this process, such as PIK3CA ,PIK3R1 ,MAPK1 ,SRC ,MAPK3 ,AKT1 ,STAT3.GO analysis and KEGG enrichment pathway analysis showed that QED may prevent and/or treat CIM via cellular senescence, PI3K-Akt signaling pathway, and NF-kB signaling 

pathway.

Conclusions: QED may influence the  PI3K-Akt  signaling pathway, cellular senescence, and/or NF-kB signaling pathway to prevent and/or treat CIM.

Part    4     Establishment     and     Comparison     of    Chemotherapy-induced Myelosuppression Model in BALB/c Mice by 5-Fu/5-Fu plus L-OHP

Objective:  To establish a CIM model in BALB/mice using 5-Fu plus L-OHP, and compare the new model with a pre-existing model which used 5-Fu alone.

Methods:  100 male BALB/c mice were randomly divided into 5 groups:5-Fu (high-dose)(M-H)group(n=20),5-Fu(low-dose)(M-L)group(n=20),5-Fu+L-OHP

(high-dose)(D-H)group     (n=20),5-Fu+L-OHP     (low-dose)(D-L)group(n=20)and control group(n=20)and myelosuppression was induced using 5-Fu 180 mg/kg,5-Fu 150 mg/kg,5-Fu 150 mg/kg plus L-OHP 7.2 mg/kg,5-Fu 112.5 mg/kg plus L-OHP 5.4 mg/kg and 0.9%sodium chloride solution by intraperitoneal injection, respectively. General condition, weight, WBC, RBC, PLT, and HGB in the peripheral blood were measured. Bone marrow nucleated cells (BMNCs)were tested on d6 and d13 after the mice were sacrificed.

Results: (1)Mortality: There are 4 and 6 mice who died in the M-H group and D-H group from d7 to d13, respectively. (2)Weight: compared to the control group,  the weight of mice in model groups declined firstly, especially in both the D-H group and the D-L group, and then the weight increased. (3)Compared with the control group, both WBC and PLT in the four model groups declined on d6(P<0.01).WBC of mice in the M-H group recovered to a level close to the control group(P>0.05), while the WBC of mice in the ML group, D-H group, and D-L group surpassed both the baseline and control group on d13, though the difference was not statistically significant(P>0.05).PLT of mice in the four model groups increased significantly and was higher than both the baseline and control groups (P<0.01). The experiment witnessed a sustained downward trend of RBC and HGB in the four model groups during the whole process. (4)Compared to the control group, BMNCs of mice in the four model groups decreased significantly(P< 0.01)on d6. There was an escalating trend of BMNCs on d13 compared to d6, though lower than the BMNCs in the control group(P<0.01).BMNCs of mice in the D-L group were more than those in the M-L group and D-H group(P<0.01).10vwW.

Conclusions:  (1)All of the four regimens may induce BALB/c mice to develop myelosuppression. (2)The mortality in the M-H group and D-H group were over 30% and 50%, respectively, which indicated excessive dosage in the two groups. (3)There were no differences between the M-L group and the D-L group in terms of myelosuppression,  weight, and mortality.

Part5  An Experimental Study of Effect and Mechanism of QED in the Treatment of Chemotherapy-induced Myelosuppression

Objective: To evaluate the effectiveness of QED on myelosuppression induced by 5-Fu, and to investigate the mechanism.

Methods: 120 male BALB/c mice were randomly divided into 6 groups, with 20  mice in each group: control group(C group), model group(M group), G-CSF group(G  group), high dosage of QED(QED-H)group, middle dosage of QED  (QED-M)group and low dosage of QED (QED-M)group. Different dosages of QED were given to the  QED-H  group, QED-M  group, and  QED-L  group from  d-6  to  d13  by intragastric administration. On d0, myelosuppression was induced by injection of 5-Fu in all the 6  groups, except for mice in the C group. Mice in the G group were administrated to G-  CSF by intraperitoneal injection from d1 to d13.On d-7,d0, and every other day after modeling, mice were weighed and sampling blood was collected using the tail-cuff method to test the WBC, RBC, HGB, and  PLT  of mice.10  mice in each group were euthanatized on  d6  and  d13, respectively.BMNCs,  the cell  cycle of  BMNCs,  and TNF-a/IL-  6/ROS/INF-y/SCF in the plasma were tested. In addition, the bone marrow tissues of 3 mice in the C group, M group, and QED-M group were collected to study the expression of p38, E2F, and c-myc using Western blot and qPCR methods.

Results: (1):①On d6, compared to C group, WBC of M group were declined (p <0.01), WBC of both QED-H and QED-M group were higher than M group(P<0.01 or  P<0.05); on  d13, WBC  of  M  group  was  lower  than  C  group(P<0.01), WBC  of QED-M group was increased compared with the M group(P<0.05).②Both RBC and HGB of mice in the M group decreased compared with the C group both on d6 and d13 (P<0.05  or  P<0.01); there  was  no  difference  between  the  M  group  and  the  QED groups  in  terms  of RBC  or  HGB.③PLT  declined  in  the  M  group(P<0.01)on  d6

while it increased ond13 compared with the C group. There was no statistically significant 11

Preface

The latest cancer statistics from the International Agency for Research on Cancer (IARC) show that in 2020, there were 1.9 million new cases of colorectal cancer and 930,000 deaths from colorectal cancer worldwide; there were 560,000 new cases of colorectal cancer in China and about 290,000 deaths from colorectal cancer. The incidence and mortality of colorectal cancer are still on the rise worldwide and in my country.

40%-50% of colorectal cancer patients are diagnosed in the early and middle stages, at which time, radical surgery is the key to treatment. For patients with postoperative pathological stage II (high-risk)/stage III, or patients after radical surgery for stage IV, adjuvant chemotherapy can further improve the patient's disease-free survival (DFS) and/or overall survival (OS). However, bone marrow suppression caused by adjuvant chemotherapy often leads to a reduction in chemotherapy drugs, delay in chemotherapy, or even termination. Hematopoietic growth factors (HGFs), which are widely used in clinical practice, can be used to prevent and treat bone marrow suppression caused by chemotherapy, but some patients have poor responses to HGFs. At the same time, the use of HGFs may bring new clinical problems.

In my country, the prevention and treatment of bone marrow suppression caused by chemotherapy by traditional Chinese medicine is accepted by many clinical physicians and patients.

However, most of the existing related clinical studies are small sample studies. The efficacy of traditional Chinese medicine in preventing and treating bone marrow suppression caused by chemotherapy needs to be objectively evaluated, and its mechanism of action needs to be further studied.

Professor Yang Yufei, the tutor, summarized his many years of clinical experience and, based on the guiding principle of "benefiting those who are damaged" in the Yellow Emperor's Classic of Internal Medicine, formulated the Qitu Erzhi Recipe to prevent and treat bone marrow suppression caused by adjuvant chemotherapy for colorectal cancer by strengthening the spleen and kidney. Clinical application has significant efficacy. However, the mechanism of action of the Qitu Erzhi Recipe is still unclear.

This study (1) used systematic review and meta-analysis to evaluate the efficacy of traditional Chinese medicine in preventing and treating bone marrow suppression caused by adjuvant chemotherapy for colorectal cancer, in order to provide evidence-based medicine for clinical practice; (2) preliminarily explored the distribution characteristics of intestinal flora in patients with spleen and kidney yang deficiency after radical surgery for stage II (high-risk)/stage III colon cancer; (3) explored the efficacy mechanism of Qitu Erzhi prescription in preventing and treating bone marrow suppression caused by chemotherapy based on network pharmacology; (4) explored the administration method and optimal dose of 5-Fu combined with oxaliplatin-induced bone marrow suppression animal model; (5) explored and verified the efficacy mechanism of Qitu Erzhi prescription in preventing and treating 5-Fu-induced bone marrow suppression through animal experiments.

This study was conducted based on the key project of the National Key R&D Program of the Ministry of Science and Technology of the People's Republic of China "Modernization Research of Traditional Chinese Medicine" "Study on the synergistic effect and mechanism of sequential prescription of invigorating spleen and tonifying kidney on adjuvant chemotherapy of colon cancer (2017YFC1700604)" and the National Natural Science Foundation of China "Study on the mechanism of sequential therapy of invigorating spleen and tonifying kidney in improving chemotherapy-induced digestive tract and bone marrow toxicity based on stem cell regeneration regulated by Wnt/β-catenin (8197142427)".