| Myelosuppression induced by chemotherapy is a problem that cannot be ignored in cancer therapies.Chinese Medicine(CM)have 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 was conducted to provide high-quality evidence on the effectiveness of CM on bone marrow suppression caused by adjuvant chemotherapy in CRC patients.Secondly,gut microbiota of CRC patients diagnosed with stage ?(high-risk)/?I,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.Part 1 Effectiveness of Oral Chinese Medicine for Myelosuppression Induced by Adjuvant Chemotherapy in Patients with CRC:A Systematic Review and Meta-analysisObjective: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 was independently carried out by two reviewers.Two investigators assessed the methodological quality of qualified researches 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 ?(high risk)/? Colon Cancer and Going to Receive Adjuvant Chemotherapy and Diagnosed of Yang Deficiency of Spleen and Kidney Syndrome Based on Chinese MedicineObjective:To investigate the characteristics of gut microbiota in patients with colon cancer,who are going to receiving adjuvant chemotherapy and were diagnosed of Yang 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,who 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 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 group in terms of community richness,diversity or evenness.Firmicutes to Bacteroidetes ratio(FBR)in D group was lower than that of N group,though both Firmicutes and Bacteroidetes are the preponderant populations of the two groups.Enterobacteriaceae increased in D group while sutterellaceae increased in N group.Furthermore,both Clostridia and Flavobacteriales increased in 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 N group while Enterobacteriaceae,clostridia and Flavobacteriales increased in D group.Part 3 Study on the Molecular Mechanism of QED in the Treatment of Chemotherapy-induced Myelosuppression Based on Network PharmacologyObjective:To explore the molecular mechanism of QED in the treatment of CIM.Methods:(1)Main ingredients of QED were searched in 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 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 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 was obtained using the 4 databases.347 targets of QED could have therapeutic effect 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-?B signaling pathway.Conclusions:QED may influence the PI3K-Akt signaling pathway,cellular senescence and/or NF-?B 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-OHPObjective:To establish CIM model in BALB/c 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,and 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 died in the M-H group and D-H group from d7 to d13,respectively.(2)Weight:compared to the control group,weight of mice in model groups declined firstly,especially in both D-H group and 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 M-H group recovered to the level close to the control group(P>0.05),while WBC of mice in M-L 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 were higher than both the baseline and control group(P<0.01).The experiment witnessed 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 was more than that in the M-L group and D-H group(P<0.01).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 M-L group and D-L group in terms of myelosuppression,weight and mortality.Part 5 An Experimental Study of Effect and Mechanism of QED in the Treatment of Chemotherapy-induced MyelosuppressionObjective: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 dl 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 WBC,RBC,HGB and PLT of mice.10 mice in each group were euthanatized on d6 and d13,respectively.BMNCs,cell cycle of BMNCs,TNF-?/IL-6/ROS/INF-?/SCF in the plasma were tested.In addition,the bone marrow tissues of 3 mice in 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):?On 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).?Both 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.?PLT declined in the M group(P<0.01)on d6 while it increased on d13 compared with C group.There was no statistically significant difference in platelets between the M group and the QED groups both on d6 and d1 3(P>0.05).(2)On d6,compared to C group,BMNCs of mice in M group decreased(P<0.01),while BMNCs of QED groups were more than that of M group(P<0.01 or p<0.05);on d13,BMNCs of mice in M group was lower than C group(P<0.01),while BMNCs of QED groups increased in comparison to M group(P<0.01).(3)On d6,both spleen index and thymus index of the model group significantly decreased(P<0.01);the spleen index and thymus index of QED-H group increased compared with M group(P<0.01).On d13,the spleen index of M group was higher than that of C group(P<0.01),while thymus index of M group was lower than C group(P<0.01).There was statistical difference between QED groups and M group in terms of neither spleen index nor thymus index(P>0.05).(4)On d6,percentage of BMSCs at phase G0/G1 increased(P<0.01)while percentages of BMSCs at phase S,G2/M were decreased(P<0.05)and proliferation index(PI)declined(P<0.01)compare with C group;percentage of BMSCs at phase G0/G1 decreased(P<0.01 or P<0.05),while percentages cells at phase S,G2/M increased(P<0.05 or P<0.01)in QED groups.(5)On d6,the level of TNF-?/IL-6/ROS/SCF(P<0.01)increased while the level of INF-y in plasma decreased(P<0.05)in QED group;opposite changes happened in QED groups.(6)Western blot:Compared to C group,the expression of both p38 and E2F2 in bone marrow of M group increased,but there was no significant difference(P>0.05);the expression of both p38 and E2F2 in bone marrow of QED-M group were lower than M group,though the difference was insignificant(P>0.05).(7)qPCR:Compared to C group,the expression of E2F in bone marrow of M group increased(P<0.05),while that of QED-H group decreased in comparison to M group,though there was no significant difference(P>0.05).The expression of c-myc increased in M group(P<0.05),while that of QED-H group decreased(P<0.01).Conclusions:(1)QED alleviated the reduction of BMNCs induced by 5-Fu;both QED-H and QED-M can effectively prevent and treat chemo-induced leukopenia,which may indicate a dose-effect relationship.(2)QED-H can improve the immune function of mice suffering from myelosuppression.(3)QED can adjust the ratio of percentages BMNCs in GO/G phase,S phase and G2/M phase,and increase PI of BMNCs.(4)QED may enhance the hematopoiesis of bone marrow via regulating TNF-?,IL-6,SCF,ROS,INF-? in the plasma.(5)QED may exert anti-apoptosis effect of HSCs in bone marrow through downregulation of p38,E2F2 and/or c-myc. |
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