| Objectives:Liu Jun An Wei Formula(LJAW),as an empirical formula developed by Professor Yufei Yang,is widely used in clinical practice for gastrointestinal reactions caused by tumor chemotherapy.This study relied on the " 13th Five-Year Plan" National key research and development Program of the Ministry of Science and Technology-Key Special Project for Modernization of Traditional Chinese Medicine,to evaluate the clinical efficacy of LJAW in treating gastrointestinal reactions during adjuvant chemotherapy after colon cancer surgery.Relying on the National Natural Science Foundation of China,we will further apply ultra-high liquid chromatography mass spectrometry technology,network pharmacology methods,small intestine organoid experiments,and animal experiments to explore the efficacy and mechanism of LJAW in improving chemotherapy induced intestinal injury.Methods:Clinical study:Relying on the national key research and development plan-the key special project of the modernization of traditional Chinese medicine:"Study on the synergistic effect and mechanism of LJAW sequential Qitu Erzhi formula on adjuvant chemotherapy for colon cancer"(Registered with ClinicalTrials.gov,No.NCT03716518),a prospective,multi-center,stratified group randomized 1:1 placebo parallel control,double-blind clinical study was conducted.From August 2018 to July 2021,a total of 400 patients with high-risk stage Ⅱ andⅢ colon cancer who were to receive chemotherapy under CapeOX protocol were enrolled in the outpatient departments and wards of 13 Grade-A TCM and Western medicine hospitals nationwide.The treatment group was given LJAW granules on days 0-6 of chemotherapy,and the control group was given Liu Jun An Wei placebo granules on days 0-6 of chemotherapy.Basic data such as gender,age,site of disease,pathological type and TNM stage were collected before chemotherapy.Liver and kidney function were collected from 3 days to 0 days before chemotherapy,and gastrointestinal adverse reactions such as diarrhea,constipation,nausea and vomiting were recorded at 0,3 and 6 days after chemotherapy.Chi-square test,Fisher’s exact probability test,t test.Logistic regression and other statistical methods were used to evaluate the clinical effect of gastrointestinal reactions during adjuvant chemotherapy after colon cancer treatment with LJAW and analyze the influencing factors of gastrointestinal reactions.Component analysis of LJAW:The main chemical components of LJAW intestinal absorption liquid were analyzed by ultra high liquid chromatography and mass spectrometry.Network pharmacology:Based on the chemical components analyzed and identified by ultra high liquid chromatography and mass spectrometry,the component-target network and PPI network were constructed by network pharmacology,and further GO and KEGG analysis were performed to predict the core pharmacodynamic components,key targets and related pathways of gastrointestinal reactions induced by LJAW chemotherapy for colon cancer.Small intestine organoid experiments:Small intestine of C57BL/6 mice was taken,small intestine stem cells were isolated,small intestine organoids were cultured in small intestine organoids medium and Matril in an incubator.Small intestinal organoids were divided into normal control group(NC),model group(5-FU),LJAW treatment group(LJAW),glutamine treatment group(Gln),namely,positive control group.Organoid activity was detected by MTT.The intestinal barrier function of organoids was detected by fluorescein isothiocyanate permeability test.mRNA and protein expressions of Occludin and Claudin-1 related to barrier function and PUMA related to apoptosis were detected by RT-qPCR and Western blot.The protein expression of PI3K/AKT and MAPK signaling pathways was detected by Western blot.Animal experiments:Male C57BL/6J mice were randomly divided into normal control group(NC),model group(5-FU),LJAW high dose group(LJAW-H),LJAW medium dose group(LJAW-M)and LJAW low dose group(LJAW-L).The mRNA and protein expression of PUMA were detected by RT-qPCR and Western blot.The protein expression of PI3K/AKT and MAPK signaling pathways was detected by Western blot.Results:Clinical research:(1)A total of 400 patients were included,including 15 patients who had not completed one treatment.including chemotherapy or experimental drug treatment,so 385 patients in the full analysis set,193 patients in the experimental group and 192 patients in the control group.There was no statistical significance in the baseline data(gender,age,stage,etc.)between the two groups(P>0.05).(2)Compared with the two groups,LJAW could reduce the number and incidence of grade 1 and above diarrhea from the first cycle to the eighth cycle,and LJAW could reduce the number and incidence of grade 2 and above diarrhea on the third day of the first cycle,with statistical significance(P<0.05).(3)Compared with the two groups,LJAW reduced the number of patients with grade 1 and above constipation on the 3rd and 6th days from the first cycle to the eighth cycle,and the differences was statistically significant(P<0.05);LJAW reduced the number of patients with grade 1 and above constipation and the number of patients with grade 1 constipation on day 0+day 3+day 6 from cycle 1 to Cycle 8,and the difference was statistically significant(P<0.05).(4)Compared with the occurrence of vomiting in the two groups,LJAW could reduce the number of patients with grade 2 or above vomiting on the 3rd day from the 1st cycle to the 8th cycle,and the difference was statistically significant(P<0.05).LJAW decreased the number of patients with grade 2 or above vomiting from cycle 1 to Cycle 8 on day 0+ day 3+ day 6,with statistically significant difference(P<0.05).(5)There was no significant difference in the incidence of nausea between the two groups at each observation time point in the whole chemotherapy cycle(P>0.05).(6)Compared with the number of gastrointestinal reactions in the two groups,LJAW decreased the number of gastrointestinal reactions of grade 1 and above in the third day of the first cycle,with statistical significance(P<0.05).LJAW decreased the incidence of grade 1 and above gastrointestinal reactions on day 0+day 3+ day 6 from cycle 1 to Cycle 8,with statistical significance(P<0.05).(7)Subgroup analysis showed that the incidence of diarrhea in patients younger than 65 years old,experimental group and control group was statistically significant(P<0.05).In patients with right colon cancer,the incidence of diarrhea in experimental group and control group was statistically significant(P<0.05).(8)The influencing factors of diarrhea occurrence:Binary logistic regression univariate and multivariate analysis showed that D0 diarrhea status,group and age were statistically significant(P<0.05).Patients with diarrhea in D0 had a higher risk of diarrhea during chemotherapy than those without diarrhea in D0(OR=104.771),and patients in control group had a higher risk of diarrhea than patients in test group(OR=1.665).Patients younger than 65 years had a higher risk of diarrhea than those older than 65 years(OR=1.622).(9)Factors influencing the occurrence of constipation:The results showed that gender and stage were statistically significant(P<0.05).Female patients had a higher risk of constipation than male patients(OR=1.802),and stage Ⅲ patients had a higher risk of constipation than stage Ⅱpatients(OR=1.504).(10)Factors influencing the occurrence of vomiting:The results showed that gender was statistically significant(P<0.05),and female patients had a higher risk of vomiting than male patients(OR=1.455).(11)Factors influencing the occurrence of nausea:The results showed that age and degree of differentiation were statistically significant(P<0.05).Patients under 65 years old had a higher risk of developing nausea than those above 65 years old(OR=2.078),and undifferentiated patients had a lower risk of developing nausea than well differentiated patients(OR=0.047).(12)Safety analysis:The incidence of adverse events such as liver and kidney dysfunction in the experimental group and control group was 7.3%and 8.9%.respectively,with no statistically significant difference between the two groups(P>0.05).Component analysis of LJAW:97 compounds were identified in LJAW intestinal absorption liquid,including 7 compounds in Pseudostellariae Radix,6 compounds in Atractylodis Macrocephalae Rhizoma,5 compounds in Poria.24 compounds in Pinelliae Rhizoma Praeparatum Cum Zingibere Et Alumine,17 compounds in Citri Reticulatae Pericarpium and 50 compounds in Glycyrrhizae Radix Et Rhizoma.Network pharmacology:Component-target analysis showed that Galangin,Licochalcone B,Ergosterol-5,8-oxidant and other 18 compounds could be the core components.PPI network analysis showed that STAT3,STAT1,PIK3CA,PIK3R1,MAPKI,MAPK14 and AKT1 were the key targets.The enrichment results of KEGG pathway suggest that LJAW may affect cell apoptosis by regulating PI3K/AKT,MAPK and other signaling pathways.Small intestine organoid experiments:The culture system of intestinal organoids,5-FU and LJAW intestinal absorption liquid was established successfully.The organoids in NC group grew well and showed good villus-recess structure.Compared with NC group,5-FU group had serious organoid morphological damage,fewer buds emerged and more deaths(P<0.05).Compared with 5-FU group,the morphology of organoids in LJAW group and Gln group was improved,germination increased and death decreased(P<0.05).Permeability test showed that the mean fluorescence intensity of 5-FU group was significantly increased compared with NC group(P<0.05).Compared with 5-FU group,the mean fluorescence intensity in LJAW group was significantly decreased(P<0.05).Western blot results showed that compared with NC group,the expression level of Occludin and Claudin-1 in 5-FU group was significantly downregulated(P<0.05),and the expression level of PUMA was significantly up-regulated(P<0.05).Compared with 5-FU group,the expression level of Occludin and Claudin-1 in LJAW group was significantly up-regulated(P<0.05),and the expression level of PUMA was significantly down-regulated(P<0.05).Similar results were obtained for mRNA expression levels of Occludin,Claudin-1 and PUMA.Compared with NC group,the ratios of p-PI3K/PI3K,p-AKT1/AKT1,p-MAPK1/MAPK1 and p-MAPK14/MAPK14 in 5-FU group were significantly increased(P<0.05).Compared with 5-FU group,the ratios of p-PI3K/PI3K,pAKT1/AKT1,p-MAPK1/MAPK1 and p-MAPK14/MAPK14 in LJAW group were significantly decreased(P<0.05).Animal experiments:Compared with NC group,the ratios of p-PI3K/PI3K,p-AKT1/AKT1,p-MAPK1/MAPK1 and p-MAPK14/MAPK14 in 5-FU group were significantly increased(P<0.05).Compared with 5-FU group,the ratios of p-PI3K/PI3K,p-AKT1/AKT1,pMAPK1/MAPK1 and p-MAPK14/MAPK14 in LJAW-H and LJAW-M groups were significantly decreased(P<0.05).Compared with NC group,the relative expression of PUMA protein in 5-FU group was significantly increased(P<0.05).Compared with 5-FU group,the relative expression level of PUMA in LJAW-H and LJAW-M groups was significantly decreased(P<0.05),and similar results were obtained in terms of PUMA mRNA expression level.Conclusions:Clinical research:1(1)LJAW can improve the occurrence of diarrhea throughout adjuvant chemotherapy after colon cancer surgery,and reduce the occurrence of grade 2 and above diarrhea on day 3 of the first cycle.(2)It can reduce the occurrence of constipation during adjuvant chemotherapy of colon cancer.(3)Reduce the incidence of grade 2 and above vomiting throughout colon cancer adjuvant chemotherapy.(4)Improve the occurrence of gastrointestinal reactions throughout colon cancer adjuvant chemotherapy.(5)Subgroup analysis suggests that LJAW may have better clinical efficacy for chemotherapy-induced diarrhea in patients younger than 65 years of age and in patients with right colon cancer.2(1)D0 diarrhea status,group and age were the independent factors affecting the occurrence of diarrhea.(2)Gender and stage are independent influencing factors for the occurrence of constipation.(3)Gender is an independent influencing factor on the occurrence of vomiting.(4)Age and degree of differentiation are independent influencing factors for the occurrence of nausea.Experimental research:3 LJAW can alleviate the morphological damage of small intestinal organoids and intestinal barrier function induced by 5-FU,and promote the recovery of barrier function by up-regulating the expression of Occludin and Claudin-1.LJAW can reduce intestinal cell apoptosis by regulating PI3K/AKT and MAPK signaling pathways,thus ameliorating intestinal injury caused by chemotherapy.Integration of clinical and experimental researches:4 LJAW can improve the clinical symptoms of gastrointestinal reactions during adjuvant chemotherapy after colon cancer surgery,providing a higher level of evidence-based medicine evidence for clinical application.The composition analysis,network pharmacology study,small intestine organoid study and mouse experiment of LJAW provide the pharmacodynamic material basis for the efficacy of LJAW,and explain the mechanism of its therapeutic effect.The evidence system of clinical study,in vitro study and in vivo study has been formed,which provides a relatively complete evidence chain for the further development and utilization of LJAW. |
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