The Theory And Experimental Study Of Treatment Of Cancer-Related Fatigue In Colorectal Cancer From Liver And Spleen

Objective:To explore the etiology and pathogenesis of cancer-related fatigue and the theory of differentiation and treatment in TCM,to put forward the viewpoint of treating cancer-related fatigue from liver and spleen,to prepare the animal model of cancerrelated fatigue caused by chemotherapy,to verify the therapeutic effect of invigorating the spleen,soothing the liver and detoxifying therapy on fatigue by intestinal flora and liver metabolomic analysis,To understand the possible mechanism of the treatment of cancer-related fatigue of colorectal cancer by invigorating the spleen and soothing the liver.To give a new way for clinical treatment of cancer-related fatigue of colorectal cancer.Methods:Theoretical research: The progress of Western medicine diagnosis and treatment of cancer-related fatigue was summarized by consulting ancient books and analyzing literature,which was carried out from four aspects: etiology,pathogenesis,diagnosis and evaluation,and treatment.Then,the name,etiology,pathogenesis,syndrome type and treatment of the disease were summarized.On this basis,the theory of treating cancer-related fatigue of colorectal cancer from the perspective of liver and spleen was analyzed.Experimental study: Tumor bearing and chemotherapy(5-FU)method was used to construct cancer-related fatigue mice model,and then the established model was used for pharmacodynamics and corresponding mechanical research.The experiment was randomly divided into 5 groups: blank control group,tumor bearing group,model group(tumor bearing + 5-FU),spleen invigorating and detoxifying group,also called Jian Pi Jie Du group(tumor bearing + 5-FU + Jian Pi Jie Du formula),spleen invigorating and liver invigorating and detoxifying group,also called Jian Pi Shu Gan Jie Du group(tumor bearing + 5-FU + Jian Pi Shu Gan Jie Du formula),(n=10).The first three groups(blank control group,tumor bearing group and model group)were used to evaluate whether the model is successfully constructed.By comparing the difference among the three groups including model group,Jian Pi Jie Du group and Jian Pi Shu Gan Jie Du group,the efficacy and mechanism of Jian Pi Jie Du treatment and Jian Pi Shu Gan Jie Du treatment in the model mice can be evaluated.The other four groups except the blank control group were inoculated with CT26 cancer cells to form xenograft colorectal tumor,and the intervention was started after tumor formation.The dosage regimen was as following:The mice in the blank control group and the tumor bearing group were not received chemotherapy,and the mice in the other three groups were received chemotherapy,meanwhile,the mice in the Jian Pi Jie Du group and Jian Pi Shu Gan Jie Du group were also intragastrically administrated with TCM formula.Furthermore,the blank control group and the tumor bearing group were intraperitoneal injected with normal saline(0.25 m L,1 time / 2 days for two weeks).The model group,Jianpi Jiedu group and Jianpi Shugan Jiedu group were intraperitoneal injected with fluorouracil(20 mg/kg,1 time / 2 days for two weeks).In addition,the model group was intragastrically administrated with 0.5m L normal saline once daily for 14 days;the Jianpi Jiedu group was intragastrically administrated with 0.5m L Jianpi Jiedu formula once daily for 14 days;the Jianpi Shugan Jiedu group was intragastrically administrated with 0.5m L Jianpi Shugan Jiedu formula once daily for 14 days.The model was evaluated by behavioral tests such as exhaustive swimming test,tail suspension test,and biochemical indicators such as liver glycogen and myoglycogen.The transplanted tumor body,left lower limb muscle,liver,feces and other samples were extracted after anesthesia with isoflurane and well preserved for the follow-up analysis.The left lower limb muscle and liver tissue were used to detect glycogen content,and liver tissue was also used for liver metabonomic analysis,then feces were used for intestinal flora analysis.Results:Theoretical research results: Cancer-related fatigue of colorectal cancer is a typical physical and mental disease.It is not only physical fatigue,but also mental fatigue,and can affect emotional and cognitive functions.The incidence rate of this disease is high,which affects the treatment and prognosis of patients.The pathogenesis and mechanism of Western medicine is not clear,involving inflammatory factors,metabolic disorders,mitochondrial dysfunction,muscle atrophy,immune balance damage,neuroendocrine system disorders,gene polymorphism,etc.The disease can not be alleviated by sleeping,relaxing and rest.At present,there are no effective drugs and intervention methods.There are two kinds of clinical interventions: one is drug-induced intervention,including central stimulant(methylphenidate),and the use of cortical sterol in the end-stage patients;the other is non drug-induced intervention,including physical activity,massage therapy,psychosocial intervention,nutrition counseling and sleep cognitive therapy,and bright white light therapy,which are mostly symptomatic treatment,with poor overall effect.The treatment of cancer-related fatigue in traditional Chinese medicine mainly focuses on the whole body and mind of patients,referring to the etiology,pathogenesis and treatment of "virtual fatigue".Based on the experience and discussion of the doctors,the syndrome differentiation of this disease is mainly based on deficiency,the syndrome differentiation of viscera as the program,and the syndrome differentiation of essence,Qi,blood and body fluid as the meridians and latitudes.Deficiency is the leading type of syndrome.When we clearly distinguish the deficiency of yin and Yang of Qi and blood in the five zang organs,we should also take into account the syndrome of deficiency with excess.Here,the excess mainly refers to four kinds of cancer poisons that have been generated by the four kinds of cancer poisons: Qi stagnation,blood stasis,phlegm turbidity and heat toxin.Specifically,the cancerrelated fatigue caused by colorectal cancer is not easy to be treated early because of its hidden incidence;the diet is not regular,spleen deficiency is often present;the emotion is abnormal,liver depression is inevitable;the cancer toxin is persistent,the deficiency and the actual are mixed,and its clinical manifestations are also consistent with the syndrome of liver depression and spleen deficiency.Therefore,the viewpoint of treating cancer-related fatigue from the perspective of liver and spleen is put forward,and the pathogenesis of the disease is spleen deficiency,liver depression and toxin retention It can cure Chong,invigorate the spleen and Qi,dredge the liver and nourish the liver,and clear away the remaining poison.Experimental results:1.Model evaluationThe three groups including blank group,tumor bearing group and model group were compared to evaluate the success of the model construction.There was no significant difference about the weight,fur,drinking and eating,mental state,activity and other general conditions of mice in each group(P = 0.667)before the start of chemotherapy.Moreover,there was no significant difference in tail suspension time(P = 0.471)and exhausted swimming time(P = 0.424)among the three groups before the start of chemotherapy.At the end of the experiment,the general situation of the blank mice was good,while the mice in the tumor bearing group and the model group showed sparse hair,inactivity,sluggish action,reduced food intake,loose stool,etc,which was more serious in the model group.The mice body weight in each group increased compared with the weight before the experiment,however,the mice body weight in blank group,tumor bearing group and model group decreased respectively(P = 0.022).Moreover,the mice body weight between model group and blank group also showed statistical difference(P = 0.006).The mean tumor weight in tumor bearing group and model group was 1.885 g and 0.66 g,respectively(P < 0.001).The exhausted swimming time of mice in tumor bearing group and model group was shorter than before,and the tail suspension time was longer than before.The difference between model group and blank group was statistically significant(P < 0.001).The content of glycogen of liver and muscle in model group,tumor bearing group and blank group increased respectively,and the content of glycogen in model group was the lowest(P < 0.001).2.Therapeutic effects of two TCM formulaThe model group,Jian Pi Jie Du group and Jian Pi Shu Gan Jie Du group were compared to evaluate the therapeutic effects of two TCM formula.There was no significant difference in weight,skin color and appearance,water and food intake,mental state,activity and other general conditions before the start of experiment(P = 0.738).There was no significant difference about exhaustive swimming time(P = 0.879)and tail suspension immobility time(P = 0.484)before the start of experiment.After the end of the experiment,the mice body weight in each group increased compared with the weight before the experiment.The weight of mice in the model group,Jian Pi Jie Du group and Jian Pi Shu Gan Jie Du group increased gradually,however there was no significant difference among the groups(P = 0.553).In general,the mice in each group showed sparse hair,inactivity,sluggish action,reduced food intake,loose stool and so on.The exhaustive swimming time of the three groups was shorter than the time before the experiment,the most shortest in the model group(P = 0.011).The tail suspension time of three groups was longer than that before the experiment.Among them,the model group was the longest,Jian Pi Jie Du group was the second,and Jian Pi Shu Gan Jie Du group was the shortest.The difference between each of the two groups was statistically significant(P < 0.0.05).The content of liver glycogen and muscle glycogen in the model group,Jian Pi Jie Du group and Jian Pi Shu Gan Jie Du group increased gradually,and the difference was statistically significant between the model group and Jian Pi Jie Du group(P = 0.005,P = 0.013),the model group and Jian Pi Shu Gan Jie Du group(P < 0.001,P < 0.001).The difference between Jian Pi Jie Du group and Jian Pi Shu Gan Jie Du group was also statistically significant(P = 0.016 for glycogen in liver,P = 0.014 for glycogen in muscle).3.Intestinal flora analysisThe amplified region of the original sequence is 338 f R,the length of the inserted fragment is 400 bp,the length of the sequence is pe300,the number of the original sequence is 2356505* 2,and the total base number is 1418616010 bp.The amplified region of the optimized sequence was 338 f R,the number of samples was 40,the number of effective sequences was 1692356,the number of effective bases was 70295722 bp,the sequence length was between 401 and 440 bp,with the average length 415.37 bp.The trend of dilution curve tends was flat.Pan / core analysis showed that the decreasing amplitude of the number of core OTUs gradually decreased with the increase of the number of sequenced samples.The dominant species are the same at the level of phylum,class,order,family and species,however,the abundance of dominant species is different.The dominant phylum with the highest abundance was Bacteroides,sclerenchyma and Proteus.Bacteroides accounted for 20%,21%,18%,19%,22% and sclerenchyma for 19%,19%,22%,22% and 28% in blank group,tumor bearing group,model group,Jian Pi Jie Du group and Jian Pi Shu Gan Jie Du group.The dominant classes at the class level are clostridia,bacteroidea and bacteroidea.Clostridia were 19%,21%,17%,19%,23% and Bacteroides were 19%,19%,22%,22%,28%,respectively.At the level of order,the dominant orders are clostridia,Bacteroides and lactobacilli.At the family level,the dominant families are trichospirillaceae,group S24-7 bacteroideae,scientific research department,verrucaceae and lactobacteraceae.At the genus level,the dominant genera are Bacteroides S24-7 and trichospirillaceae NK4A136.The OUT numbers of blank group,tumor bearing group,model group,Jian Pi Jie Du group and Jian Pi Shu Gan Jie Du group were 624,660,664,654 and 671 respectively.Among them,the total number of OUT in each group was 547,accounting for more than 87.67%.According to the analysis of community Heatmap,the composition and abundance of the samples in each group were different at the genus level.After sequencing and 97% similarity regression,the coverage index of each group was greater than 0.99,and the sequencing coverage depth of samples was more than 99%.Compared with the model group,Jian Pi Jie Du formula can increase ace index,Chao index and Shannon index,reduce Simpson index,while Jian Pi Shu Gan Jie Du formula showed better effects.The hierarchical cluster analysis of samples was shown by thermogram,and the results showed that the samples in this study were well grouped.PCA analysis and PCo A analysis showed that the two groups(Jian Pi Jie Du group and Jian Pi Shu Gan Jie Du group)were close to each other,with similar species composition,which was significantly different from the other groups.PLS-DA analysis showed that the yellow group(Jian Pi Jie Du group)and purple group(Jian Pi Shu Gan Jie Du group)were separated on the Comp1 axis,while the yellow group(Jian Pi Jie Du group)and purple group(Jian Pi Shu Gan Jie Du group)were separated on the Comp2 axis from the red group(blank group)and green group(model group),The factors represented by Comp1 axis are whether the traditional Chinese medicine used containing liver soothing medicine,and Comp2 axis is whether the traditional Chinese medicine is used.It can be concluded that the use of spleen strengthening and detoxification TCM and spleen strengthening,Liver Soothing and detoxification TCM can affect the composition of intestinal flora,and there are differences between Jian Pi Jie Du group and Jian Pi Shu Gan Jie Du group.The results of Lefse multi-level species difference discriminant analysis showed that there were differences in intestinal microflora among different groups,and there were also differences between Jian Pi Jie Du group and Jian Pi Shu Gan Jie Du group.Among which,there were three significantly different strains in Jian Pi Jie Du group including acidophilus,anaerobe,Eubacterium which was different from Jian Pi Shu Gan Jie Du group.Also,there were three significantly different strains in Jian Pi Shu Gan Jie Du group including Saccharification bacillus,desulphurization vibrio,moon bacteria acidophilus,which was different from Jian Pi Jie Du group(P=0.039).At the class level,Deferribacteres was significantly different between the two TCM formula groups(P = 0.0167).At the order level,Deferribacteres was significantly different between the two TCM formula groups(P = 0.0167).At the level of genus and family,the abundance of Bacteroides s24-7 and Spirillum was higher than that of other groups(P < 0.05).There were differences in the abundance of the strains near the front at the Family level in the Jian Pi Jie Du group and Jian Pi Shu Gan Jie Du group,among which the difference of bacteroides was the most obvious.The main biological functions related to the sequenced flora in this study included carbohydrate transport and metabolism,amino acid transport and metabolism,nucleotide transport and metabolism,inorganic ion transport and metabolism,participation in the biogenesis,replication,reorganization and repairing of cell membrane,transcription process,translation,ribosome structure and biogenesis,lipid transport and metabolism,biosynthesis of secondary metabolites,transport and catabolism,energy production and transformation,etc.4.Metabonomic analysis of liverThe total ion chromatogram of GC / MS was different among the five groups.Firstly,unsupervised PCA analysis was carried out,however,the metabolism spectrum of blank group and tumor bearing group could not be well distinguished.Next,supervised PLS-DA analysis was carried out,and the liver metabolites of blank group and tumor bearing group showed a good distinction.And that the model showed a high interpretation rate and prediction rate(the cumulative interpretation rate of the two main components R2Xcum=0.72,Q2Ycum=0.66),which indicated that the metabolism of mice has changed after tumor loading.The PLS-DA model was established between the tumor bearing group and the blank group by using the partial least square method of interactive verification.104 different variables were identified,29 metabolites were identified as butyric acid,valine,phosphate,L-isoleucine,proline,glycine,2-buteneic acid,threonine,alanine,malic acid,aspartic acid,butyrate,acetic acid,glutamyl Amines,L-phenylalanine,phosphoric acid,aminolevulinic acid,glucose,d-Gluconic acid,talose,hexadecanoic acid,D-galactose,aniline,aspirin,maltose,1H indole,heptanose,inositol,docosahexaenoic acid.Changes of different metabolites in each treatment group can be completely separated.Meanwhile,Jian Pi Jie Du group and Jian Pi Shu Gan Jie Du group are close to the blank group,which shows that Jian Pi Jie Du group and Jian Pi Shu Gan Jie Du group corresponding to the two treatment methods can reverse the metabolic disorder caused by tumor loading and chemotherapy.By analyzing the changes of metabolites among different groups,the results showed that there were six metabolites(butyric acid,acetic acid,phosphoric acid,aminolevulinic acid,dimethylamine,docosahexaenoic acid)in difference in the model group;There were 9 metabolites(phosphate,L-isoleucine,Lproline,glycine,2-buteneic acid,glutamine,d-Gluconic acid,benzoylamine and docosahexaenoic acid)in difference in Jian Pi Jie Du group;There were 21 metabolites(butyric acid,valine,phosphate,L-isoleucine,L-proline,glycine,2-buteneic acid,lthreonine-alanine,malic acid,aspartic acid,acetic acid,glutamine,L-phenylalanine,D-glucosamine,hexadecane acid,Dgalactose,benzoylamine,aspirin,1H indole,heptanose,inositol docosahexaene acid)in difference in Jian Pi Shu Gan Jie Du group;It can be drawn that two kinds of TCM formula with different treatment methods could make the metabolic disorder return to normal,and the effect of Jian Pi Shu Gan Jie Du group is more obvious.Metabolic pathway analysis showed that the metabolic pathway of tumor bearing mice changed,involving seven important metabolic pathways: phenylalanine,tyrosine and tryptophan biosynthesis,starch and sucrose metabolism,phenylalanine metabolism,alanine,aspartic acid and glutamic acid metabolism,glycine,serine and threonine metabolism,phosphoinositol metabolism,glyoxylate and dicarboxylic acid metabolism.There are three metabolic pathways involved in the changes of tumor bearing mice after chemotherapy: pyruvate metabolism,glycolysis / gluconeogenesis,porphyrin and chlorophyll metabolism.The changes of metabolic pathway after the treatment of Jian Pi Jie Du prescription also involve three pathways: glycine,serine and threonine metabolism,alanine,aspartic acid and glutamic acid metabolism,glyoxylate and dicarboxylic acid metabolism.After the treatment of Jian Pi Shu Gan Jie Du formula,the metabolic pathway changes involing six pathways: phenylalanine,tyrosine and tryptophan biosynthesis,phenylalanine metabolism,alanine,aspartic acid and glutamic acid metabolism,glycine,serine and threonine metabolism,inositol phosphate metabolism,glyoxylate and dicarboxylic acid metabolism.Compared with Jian Pi Jie Du group,Jian Pi Shu Gan Jie Du group has three more metabolic pathways,indicating that adding soothing medicine group can adjust metabolic disorders through more channels.Conclusions:1.Cancer related fatigue of colorectal cancer can be treated from the perspective of liver and spleen.The pathogenesis of the disease is spleen deficiency,liver depression and toxin retention.The treatment method is invigorating the spleen and replenishing qi,soothing the liver and nourishing the liver,and clearing away the remaining toxin;2.The cancer-related fatigue model of mice can be successfully induced by chemotherapy intervention in the xenograft model;however,the fatigue degree of the mice model made by tumor bearing plus chemotherapy is more obvious3.Routine therapy of invigorating the spleen and detoxifying can improve the fatigue degree and glycogen level in cancerrelated fatigue mice model,and the therapeutic effects can be improved after adding TCM which can soothe the liver,which indicated that the principle of invigorating the spleen,soothing the liver and detoxifying will be better.4.The two TCM treatment of Jianpi Jiedu and Jianpi Shugan Jiedu can affect the composition and abundance of intestinal flora and the metabolism of the model mice with cancer-related fatigue.However,the effects of the two groups are different.Jianpi Shugan Jiedu treatment are more able to reverse the metabolic disorder of the model mice,which may be one of the mechanisms of its therapeutic effect.