| ObjectiveCancer-related fatigue(CRF)is a kind of physical,emotional,or cognitive fatigue associated with cancer and relevant treatment,which would bring long-lasting negative effects to patients;meanwhile,CRF patients could hard to relieve through rest,whose quality of life is greatly interfered.Jian Pi Sheng Sui Gao(JPSSG)consists of 17 kinds of traditional Chinese medicines(including tortoise shell,rhizoma polygonate,glossy privet fruit,eclipta,codonopsis pilosula,etc),which is mild and effective to treat CRF patients.However,the present studies that reprot the efficacy of JPSSG in treating CRF patients are all clinical studies,which lack of systematic analysis.Additionally,the underlying molecular mechanism of JPSSG in treating CRF needs further exploration.Hence,this study contains meta-analysis,clinical study and the animal/cell experiments based on network pharmacology,aiming to explore the clinical efficacy and potential molecular mechanism of JPSSG in treating CRF patients,and to provide reliable evidence for subsequent clinical application of JPSSG.Methods1.Meta-analysisRelevant databases(including PubMed,Embase,Cochrane Library database,Web of Science database,China Biomedical Literature Database(CBM),China Journal Full-text Database(CNKI),China Science and Technology Journal Database and Wanfang database)were selected for searching,from their inception to February,2022.The randomized controlled trials(RCTs)published in China and abroad were collected.The methodological quality of the included literatures was evaluated using the Cochrane Collaboration risk of bias assessment tool.The extracted data were meta-analyzed using Stata(version 14.0,StataCorp,USA).2.Clinical studyA total of 50 colorectal cancer patients who were going to receive chemotherapy were enrolled in this single-center,double-blind,randomized,prospective controlled clinical study.They were randomly divided into the intervention group(n=25)and the control group(n=25)at a 1:1 ratio.Patients in the intervention group received JPSSG plus chemotherapy.The specific protocol was as follows:Patients orally received JPSSG for 14 consecutive days at 3 weeks before the first chemotherapy(20g each time,twice per day),and the JPSSG treatment should end for one week before the first chemotherapy.Subsequently,on the first day of the first chemotherapy,patients should orally receive JPSSG treatment for 14 days again(20g each time,twice per day).As to the control group,patients were treated with placebo plus chemotherapy,the specific protocol was similar to the intervention group.At the third week(W3)and the fifth week(W5)since patients received JPSSG/placebo treatment,their body mass index(BMI),Karnofsky Performance Status(KPS),Chinese medicine symptom complex score,Piper Fatigue Scale(PFS),and Quality of Life Questionnaire-Core30-Fatigue(QLQ-C30-FA)were evaluated”.3.Animal and cell experiment based on network pharmacologyThe main components of JPSSG were retrieved from the traditional Chinese medicine System Pharmacology Analysis platform database,traditional Chinese medicine composition database,the bioinformatics analysis tool of traditional Chinese medicine molecular mechanism,and the Taiwan traditional Chinese medicine Database platform.The retrieved components were used for target prediction analyzed through the aforementioned databases.Then,CRF-related targets were searched under the guidance of Genecards disease Target Screening database and malacards Disease database.Cytoscape 3.2.1 software was used to analyze the common targets of JPSSG and CRF,and the proteinprotein interaction(PPI)network diagrams of active components and disease targets were performed.Finally,the candidate genes were analyzed by Kyoto Encyclopedia of Genes and Genomes(KEGG)analysis and Gene Ontology(GO)analysis;after that,the enrichment results were visualized.Eighteen mice were randomly divided into three groups:blank control group(control)(n=6),CRF-model group(model)(n=6)and JPSSG-intervention group(JPSSG)(n=6).Mouse colon cancer cell(CT26)was injected intraperitoneally to establish the intraperitoneal metastasis mouse model.The JPSSG group mice were given JPSSG by gavage(3.0g/kg body weight);the control group and model group mice were given the same amout of normal saline by gavage.ALL mice in the three groups were given drug once a day for 15 consecutive days.Behavioral tests were performed on all mice on the 15th day,including open field test,exhaustive swimming test and tail suspension test.At the end of the test cycle,peripheral blood samples of all mice were collected to determine hemoglobin(Hb)and hematocrit(HCT);serum samples of all mice were collected to determine albumin(ALB),globulin(GLOB),ALB/GLOB,aspartate aminotransferase(AST),alanine aminotransferase(ALT),total protein(TP),creatinine(CR),blood urea nitrogen(BUN),triglyceride(TG),and total cholesterol(TC);gastrocnemius muscle tissues were collected to determine adenosine triphosphate(ATP),superoxide dismutase(SOD),malondialdehyde(MDA),hepatic glycogen,hepatic lactate,and blood glucose(GLU)levels.Additionally,according to the network pharmacological analysis results,gastrocnemius muscle samples of mice in each group were collected to detect the expression of adenosine 5’-monophosphate-activated protein kinase(AMPK),hypoxia inducible factor-1(HIF-1)and phosphatidylinositol-4,5-bisphosphate 3-kinase-protein kinase B(PI3K-AKT)pathway related proteins via western blot assay.The co-culture system of murine colon cancer cell CT26 and murine myoblast C2C12 was constructed.After induction of myotube formation,the cells were randomly divided into 5 groups,including C2C12 cell group(control),CT26 cell medium intervention group(CM),CT26 cell medium plus JPSSG intervention group(CM+JPSSG),hydrogen peroxide intervention group(H2O2),and hydrogen peroxide plus JPSSG intervention group(H2O2+JPSSG).Cell viability,apoptosis,oxidative stress index,reactive oxygen species(ROS)level,mitochondrial membrane potential and protein expression of AMPK and HIF1 pathway were detected in C2C12 cells.Results1.Meta-analysisA total of 926 studies were found through database retrieval,and 13 articles were finally included in the study after removing the repeatedly published articles and inclusion/exclusion criteria-unmet articles.Among the included 13 articles,the total sample size was 1128(578 cases in the experimental group and 550 cases in the control group).The articles could be divided into non-JPSSG(9 articles)and JPSSG(4 articles)according to the intervention regimen.There were 3 articles for lung cancer and 3 for colorectal cancer among the articles that define the tumor type.The results showed that the PFS score[WMD=-0.97,95%CI(-1.17,-0.76),Z=-9.438,P<0.001],PFS improvement rate[RR=2.24,95%CI(1.75,1.87),Z=2.341,P=0.019],KPS score[WMD=3.88,95%CI(0.50,7.27),Z=2.247,P=0.025],health improvement rate[RR=1.64,95%CI(1.41,1.90),Z=2.784,P=0.005]were improved in the JPSSG group compared with the control group;meanwhile,the QLQ-C30 FA score displayed an improving trend(without statistical significance)in the JPSSG group compared with the control group[WMD=-6.92,95%CI(-15.15,1.30),Z=-1.651,P=0.099].The subgroup analysis of different cancer types showed that the improvement of KPS score was more obvious in non-lung cancer patients than in lung cancer patients(P=0.022);the improvement effect of PFS improvement rate in colorectal cancer patients is more significant than that in non-colorectal cancer patients(P=0.000).The cream can also effectively reduce the QLQ-C30 score of patients with colorectal cancer(P<0.001).Interestingly,the subgroup analysis exhibited that PFS score[WMD=-1.44,95%CI(-1.98,-0.89),Z=-5.193,P<0.001]were improved in JPSSG group compared to the control group,and KPS score[WMD=3.29,95%CI(-1.57,8.15),Z=1.325,P=0.185]showed an increasing trend(lacked statistical significance)in JPSSG group compared to the control group.Besides,Begg and Egger test were used to analyze the outcomes with bias risk,and the results showed that there was no risk of publication bias in each outcome indicator(P>0.05).2.Clinical studyThe KPS score(P<0.001)was elevated,while the traditional Chinese medicine syndrome score(P<0.001),total PFS score(P<0.001),PFS of behavior or severity dimension(P<0.001),PFS of emotional dimension(P<0.001),PFS of sensation dimension(P<0.001),PFS of cognitive dimension(P<0.001),and QLQ-C30-FA score(P<0.001)were all decreased in JPSSG group after treatment than those before treatment.The traditional Chinese medicine syndrome score(P<0.001)was reduced in the control group after treatment than that before treatment;while the KPS score,total PFS score and PFS score of specific dimension,and QLQ-C30-FA score were of no difference before and after treatment in control group(all P>0.050).Additionally,total PFS score(P=0.017)and QLQC30-FA score(P=0.043)at W3 were declined in JPSSG group compared with control group;KPS score(P=0.008)were elevated,while traditional Chinese medicine syndrome score(P=0.010),PFS score(P<0.001),and QLQ-C30-FA score(P=0.009)were all decreased in JPSSG group compared to control group.3.Animal and cell experiment based on network pharmacologyA total of 87 potential active compound were obtained after the network pharmacology analysis(mainly including Citromitin,Nobiletin,Frutinone A,Daturilin,Diosgenin,Demethylwedelolactone,Syringaresinol-diglucoside,etc),132 JPSSG-CRF interaction targets,10 potential pathways(mainly including PI3K-AKT,HIF-1,AMPK signaling pathways,etc),and 19 biological processes(mainly including hypoxia response,lipopolysaccharide response,negative cell proliferation,etc).The animal experiment showed that:compared with the model group mice,the journey(P<0.050),mobile time(P<0.001),mobile number(P<0.010),and swimming time(P<0.050)of JPSSG group mice were all increased,while the absolute rest time(P<0.050)were reduced.The body weight of JPSSG group mice was similar to model group mice,while the body weight at day 7,9,11,13,and 15 of JPSSG group mice showed a decreasing trend(lacked statistical significance)compared with model group mice.Furthermore,the gastrocnemius weight were elevated in JPSSG group mice compared with model group mice,while the weight of the main visceral organs(including heart,liver,spleen,lung,and kidney)were of no difference between JPSSG group and model group(all P>0.050).The hematological index determination displayed that:the TG(P<0.001),ALB(P<0.050),hepatic glycogen(P<0.050),and GLU(P<0.050)were elevated,ALT(P<0.050)and AST(P<0.050)were declined in JPSSG group mice compared to model group mice,while Hb,HCT,TC,BUN,CR,TP、GLOB,ALB/GLOB,and hepatic lactate were of no difference between JPSSG group and model group(all P>0.050).With regard to the oxidative stress,ATP(P<0.050)and SOD(P<0.050)were both up-regulated in JPSSG group mice than those in model group mice,whereas MDA only showed a decreasing trend but without statistical significance in JPSSG group compared with model group(P>0.050).Moreover,p-AMPK(P<0.050),Sirtl(P<0.050),and HIF-1α(P<0.010)were all increased in JPSSG group compared with model group,while p-PI3K(P>0.050)and p-AKT(P>0.050)only exhibited an increasing trend(without statistical significance)in JPSSG group than those in model group.Cell experiment displayed that,cell viability was not different in C2C12 cells with JPSSG of different concentrations(0 mg/mL,2.5 mg/mL,5 mg/mL,10 mg/mL,20 mg/mL)(all P>0.050).The cell viability(P<0.050),Bcl2(P<0.050),ATP(P<0.050),SOD(P<0.050),and mitochondrial membrane potential in skeletal muscle cells(P<0.010)were increased,while cell apoptosis(P<0.050),cleavcd-caspase3(P<0.050),MDA(P<0.050),and ROS(P<0.050)were declined in CM+JPSSG group(vs.CM group).Cell viability(P<0.050),Bcl2(P<0.050),ATP(P<0.050),SOD(P<0.050),and mitochondrial membrane potential in skeletal muscle cells(P<0.050)were up-regulated,whereas cell apoptosis(P<0.010),cleaved-caspase3(P<0.010),MDA(P<0.050),and ROS(P<0.050)were down-regulated in H2O2+JPSSG group(vs.H2O2 group).In addition,p-AMPK(P<0.050)and HIF-1α(P<0.010)were increased,and the Sirtl only displayed an increasing trend(without statistical significance)(P>0.050)in CM+JPSSG group(vs.CM group);p-AMPK(P<0.050),Sirtl(P<0.050),and HIF-1α(P<0.010)were all up-regulated in H2O2+JPSSG group(vs.H2O2 group).ConclusionJPSSG has pleasing clinical efficacy in treating CRF in colorectal cancer patients,whose potential mechanism contains oxidative stress response and mitochondrial damage amelioration,as well as the prevention of skeletal muscle cell apoptosis.In addition,AMPK-Sirtl pathway and HIF-α pathway may be the main signaling pathways of JPSSG. |
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