| Objective:Cancer-related fatigue(CRF)occurs frequently in various stages of cancer treatment and cancer metastasis.Among them,up to 90% of cancer patients are troubled by it,and the current treatment of cancer-related fatigue is scarce.Therefore,the fatigue of cancer patients often lasts for several years and cannot be relieved,which seriously reduces the quality of life of patients.In fact,Chinese medicine has been treating cancer-related fatigue for a long time,and it is quite effective in improving the symptoms of fatigue.It has gradually attracted the attention and recognition of many researchers at home and abroad.Shengqi Fuzheng Injection(SFI),as an adjuvant drug for tumor treatment,has carried out clinical treatment research on tumors and complications as early as the early 20 th century,especially for improving the fatigue symptoms of cancer patients.However,there are few studies on the molecular mechanism of the therapeutic effect of Shenqi Fuzheng injection on cancer-related fatigue,which makes it difficult to promote the clinical promotion and social recognition of Shenqi Fuzheng injection.This article intends to sort out and analyze the clinical research literature of Shenqi Fuzheng Injection in the treatment of cancer-related fatigue,verify the clinical efficacy of Shenqi Fuzheng Injection,and provide evidence-based medical evidence for its clinical application.At the same time,through animal experiments,cell experiments,data mining,and molecular docking studies,the regulatory effects of Shenqi Fuzheng Injection on the maintenance of skeletal muscle mitochondria quantity and functional homeostasis were explored,in order to preliminarily clarify the molecular mechanism of the therapeutic effect of Shenqi Fuzheng injection.Methods:1.Through meta-analysis,verify the clinical efficacy of Shenqi Fuzheng Injection in treating cancer-related fatigueBy searching PubMed,Cochrane Library,Weipu and other databases(the search time is from the time the database was established to January 31,2021),to collect randomized controlled trials(RCT)on the treatment of cancer-related fatigue with Shenqi Fuzheng injection,and Cochrane Handbook 5.1.0 provides for back-to-back evaluation,using Rev Man5.3 software statistical analysis to evaluate fatigue score,clinical effectiveness,quality of life,and adverse events.2.Through animal experiment research,compare the degree of fatigue between different tumor-bearing mice,and explore the therapeutic effect of Shenqi Fuzheng injection on mice with cancer-induced fatigue and its influence on gastrocnemius muscle and mitochondriaUse mouse-derived colon cancer cell CT26 to construct subcutaneous transplanted tumor mouse models and abdominal metastasis mouse models,observe and record the body weight and tumor weight of different model mice,and compare the fatigue behavior of exhaustive swimming,tail suspension experiment and open field experiment.Detect mouse hemoglobin,hematocrit,serum liver and kidney function and blood lipid level,compare organ index,Elisa detects SOD,MDA,T-AOC content and glycogen level of gastrocnemius muscle,HE staining observes tumor tissue,gastrocnemius muscle and thigh The head muscle is pathological,and the cross-sectional area of the gastrocnemius and quadriceps muscle is calculated,and the ultrastructural changes of the gastrocnemius muscle tissue are observed under an electron microscope.A mouse model of abdominal metastasis was used as a positive control model for cancer-related fatigue,and Shenqi Fuzheng injection was used for intervention.Observe and record the body weight,tumor weight and survival time of mice,compare the fatigue behavior indicators of exhaustive swimming,open field experiment,and tail suspension experiment,compare the weight and index of the gastrocnemius muscle of mice,Elisa detects the SOD,MDA,and total ATPase of the gastrocnemius muscle Content,RT-PCR detection of gastrocnemius mitochondrial mt DNA expression level,HE staining to observe tumor tissue and gastrocnemius pathology,IHC detection of tumor tissue proliferation marker protein Ki-67(proliferation marker protein Ki-67)expression,and calculation of gastrocnemius cross-sectional area,The ultrastructure changes of gastrocnemius muscle tissue were observed under electron microscope.3.Through cell experiment research,it is clear that Shenqi Fuzheng injection maintains the regulation mechanism of healthy mitochondria quantity and functional homeostasis of myoblastsA co-culture system of mouse-derived colon cancer cell CT26 and murine-derived myoblasts C2C12 was constructed to simulate the microenvironment of tumor metabolism in vivo.The effects of Shenqi Fuzheng injection were determined by CCK8 cell proliferation detection and flow cytometry apoptosis detection.Drug time and concentration,flow cytometry to detect myoblast ROS and mitochondrial membrane potential,electron microscope to observe myoblast ultrastructure,RT-PCR to detect myoblast mitochondrial mt DNA,Elisa to detect myoblast SOD,MDA,total ATPase The content and ADP/ATP ratio,Seahorse Energy Metabolism Meter detects the oxidative respiration rate of myoblast mitochondria.4.Based on metabolomics and network pharmacology research,explore the potential medicinal compounds and molecular regulation mechanisms of Shenqi Fuzheng Injection to maintain the number and functional homeostasis of healthy mitochondria of myoblastsExtract the metabolites of all cells,use Thermo Ultimate 3000 for chromatographic analysis,Thermo Q Exactive for mass spectrometry analysis,convert the obtained raw data format through Proteowizard software,and perform data standardization processing,import the software package SIMCA-P(v13.0)And R language ropls package for multivariate statistical analysis,screening and identification of differential metabolites,importing into the Met PA database,using hypergeometric test algorithm and Relative-betweeness Centrality to analyze the metabolic pathways.Chinese medicine system pharmacology analysis platform database,traditional Chinese medicine component database and literature review collection of chemical components of Shenqi Fuzheng injection,oral bioavailability(OB),Caco2 cell model and drug similarity(DL)screening of key compounds with high activity,Uniprot database and Genecard database searches for related targets,intersects with cancer-related fatigue disease targets,constructs a "component-target" network diagram and PPI protein interaction diagram,compares the toxicology genetics database and the DAVID database for enrichment analysis,and uses R3.5.3 The software draws charts.5.Based on molecular targeted docking and Western bolt research,screening and verifying the regulatory proteins and signal pathways that Shenqi Fuzheng injection maintains the number and functional homeostasis of myoblast mitochondriaThe protein structure screened from the PDB protein database search network pharmacology combined with metabolomics,Pubchem database is used to search and find the chemical structure information of the compound of Shenqi Fuzheng injection with good ADME properties,and use Chem Bio Draw Ultra software to carry out the structure of the pharmacological compound Modification,using Py MOL to edit the three-dimensional structure of the protein,using Autodock 4.2 software to pre-treat the medicinal compounds and proteins,and using the Lamarck genetic algorithm to complete the molecular docking of the medicinal compound small molecule structure and the protein structure,and using Western bolt to verify the molecular docking The selected regulatory protein of Shenqi Fuzheng injection.Results:1.Meta-analysis results of Shenqi Fuzheng injection in the treatment of cancer-related fatigueIn this study,a total of 139 literatures on the treatment of cancer-related fatigue with Shenqi Fuzheng injection were retrieved.According to the literature screening process developed by Cochrane,7 literatures were finally included.The data of the included literature was extracted and analyzed,and the outcome indicators were summarized.The combined analysis effect of liquid to improve the treatment of cancer-induced fatigue is RR of 1.30(95%CI: 1.14~1.50),and the combined analysis effect of improving PFS(Piper Fatigue Score)score is SMD of-1.00(95%CI:-1.43~-0.57),especially for physical fatigue and emotional fatigue,the combined analysis effect of improving the quality of life is-12.05(95%CI:-12.76~-11.34).2.In animal experiments,the fatigue of abdominal metastasis model mice is even worse,and Shenqi Fuzheng injection can significantly reduce the fatigue of model mice,and maintain gastrocnemius muscle mass by protecting the function of gastrocnemius mitochondriaIn the experiment comparing the fatigue degree of different tumor-bearing mouse models,compared with the normal control group,the active exercise time(autonomous activity)and passive exercise time(exhausted swimming)of subcutaneous transplanted tumor model mice and abdominal cavity metastasis model mice were both Significantly decreased,but there was no significant difference in the activity time and absolute resting time of the tail suspension experiment.The gastrocnemius and quadriceps visceral indexes were decreased,the ultrastructure of muscle cells were destroyed to varying degrees,and the content of muscle glycogen was both Compared with subcutaneously transplanted tumor model mice,the time of active exercise(autonomous activity)and passive exercise time decreased more significantly,HGB and HCT decreased even more in abdominal metastasis model mice,and AST in liver function increased significantly,TP,ALB and GLOB were significantly reduced,BUN in renal function was significantly increased,TC in blood lipid level was significantly decreased,gastrocnemius area was reduced even more,gastrocnemius MDA content was significantly increased.In the experiment of Shenqi Fuzheng injection intervention in mice with cancer-induced fatigue,the active exercise time(autonomous activity)and passive exercise time(exhausted swimming)of mice in the Shenqi Fuzheng injection intervention group were more obvious than those in the cancer-induced fatigue model group Longer,but there is no significant difference in the activity time of the tail suspension experiment;Shenqi Fuzheng injection can significantly increase the gastrocnemius weight of mice with abdominal metastasis,increase the cross-sectional area of the gastrocnemius muscle,reduce the number of damaged mitochondria in the gastrocnemius muscle,and increase the expression level and total mitochondrial mt DNA The content of ATPase reduces the MDA content of gastrocnemius muscle and significantly increases the SOD content.3.The regulation mechanism of Shenqi Fuzheng Injection to maintain healthy mitochondrial quantity and functional homeostasis in myoblasts was clarified by cell experimentTo construct a co-culture system of rat colon cancer cell CT26 and rat myoblast C2C12,Simulates the tumor metabolic microenvironment in vivo,Determination of time and concentration of Shenqi Fuzheng injection by CCK8 cell proliferation and apoptosis detection by flow cytometry,Flow cytometry to detect changes in myoblast ROS and mitochondrial membrane potential,The ultrastructural changes of myoblasts were observed by transmission electron microscopy,Elisa detection of total ATP enzyme content and ADP/ATP ratio in myoblasts,RT-PCR detection of mitochondrial mt DNA,in myoblasts Seahorse energy metabolizer to detect the oxidative respiration rate of myoblast mitochondria.4.Metabolomics and network pharmacology explore the potential pharmacodynamic compounds and molecular mechanisms of Shenqi Fuzheng Injection to maintain the number and functional homeostasis of myoblasts in healthy mitochondriaPrincipal component analysis and orthogonal-partial least square discriminant analysis both showed that the sample distribution of the model group and the intervention group of Shenqi Fuzheng injection was more discrete,indicating that the two groups had greater metabolic differences and comparability.According to the screening requirements of P ?0.05+VIP ? 1 and P ? 0.05(Multiple Groups),a total of 40 different metabolites were screened among cells in each group,including 2-hydroxybutyric acid,L-methionine,and D-Fructose and phosphorylcholine,among which,the relative content of 18 different compounds in the Shenqi Fuzheng injection intervention group and the model group were significantly reduced,including 2-hydroxybutyric acid,L-serine,and L-asparagine,2-phenylacetamide,acetylcholine,imidazole-5-ylpyruvate,L-histidine,etc.;the relative content of 4 different compounds increased significantly,including ?-dimorphic acid,phenylacetylglycine,vanillinic acid And L-sorbose;there are 6 different compounds that can be screened for related genes,including L-serine,tyrosine and citrulline,etc.;enriched through metaboanalyst pathway,phenylalanine,tyrosine and tryptophan The biosynthesis and phenylalanine metabolism of Shenqi Fuzheng injection may be the key metabolic pathways related to the protection of skeletal muscle mitochondrial function;the KEGG enrichment analysis of the differential compound-related genes revealed 19 related pathways,including metabolic pathways,Tyrosine metabolism and regulation of lipolysis in adipocytes,etc.The comparison of pathway gene enrichment shows that the metabolic pathway may be a key signal pathway related to the function of Shenqi Fuzheng injection in protecting skeletal muscle mitochondria.A total of 191 components of Astragalus and 428 components of Codonopsis were obtained through the TCMSP database search.A total of 619 compounds were obtained,with OB ?30%,DL ? 0.18,and Caco-2 values as positive.The screening required a total of 15 components of Astragalus and 21 components of Codonopsis A total of 36 potential active compounds with good ADME properties,and a total of 936 human potential targets were found.The intersection analysis of targets matched with cancer-related fatigue diseases yielded 393 relevant targets,and the potential target genes of the drug were identified.Through the Bioinformatics&Evolutionary Genomics platform to match disease genes,244 key targets were obtained.Cytoscape 3.2.1 was used to construct a network diagram of "Astragalus and Codonopsis-cancer-induced fatigue targets".The GO function enrichment analysis was carried out through DAVID database.The related biological processes of Shenqi Fuzheng injection in the treatment of cancer-related fatigue include signal transduction,negative regulation of apoptosis process and positive regulation of transcription.The CTD database was used to carry out KEGG pathway enrichment analysis and found that Shenqi Fuzheng injection treats carcinogenicity Possible signaling pathways for fatigue include metabolic pathways,cancer small RNA,PI3K-Akt signaling pathways,etc.;comparison of pathway gene enrichment shows that metabolic pathways may be the key signaling pathways that interact with the medicinal substances of Shenqi Fuzheng injection5.Molecular targeted docking screened out the possible regulatory proteins AMPK,SIRT1 and Akt for Shenqi Fuzheng injection.Western bolt verified the drug's influence on regulatory proteinsWe collected related proteins enriched in metabolic pathways and pathway proteins related to apoptosis enriched in KEGG,and molecularly docked with the pharmacodynamic compounds of Shenqi Fuzheng injection with good ADME properties,and found that there are15 proteins with The 36 medicinal compounds have good binding free energy scores.Among them,5 proteins with the largest number of medicinal compounds are docked,namely AMPK,SIRT1,Akt1,NOS2 and RXRA.A total of 33 medicinal compounds are docked,and the remaining proteins are They can only be docked with 32 compounds,and the compound with more protein than other proteins is Chrysanthemaxanthin.According to the scores of binding free energy of the above 5 proteins and Chrysanthemaxanthin,the 3 proteins with the best binding free energy are AMPK(-7.5kacl/mol),SIRT1(-7.1kacl/mol),Akt1(-6.1kacl/mol),And the molecular geometry of Chrysanthemaxanthin and the hydrophobic cavities inside AMPK,SIRT1 and Akt1 proteins have good shape complementarity.The hydrophobic residues that constitute AMPK binding pockets include TYR-181,ASN-226,LEU-172,ASN-175,etc.,the hydrophobic residues constituting the SIRT1 binding pocket include LEU-478,ILE-243,ILE-485,LYS-236,etc.,and the hydrophobic residues constituting the Akt1 pocket include TRP-345,SER-474,ARG-103,LEU-473,etc.Western bolt results show that Shenqi Fuzheng injection can activate AMPK signaling pathway by increasing the expression level of AMPK phosphorylation sites;increase the expression level of p-Akt phosphorylation sites of Akt and decrease the expression level of p-PI3 K phosphorylation sites of PI3 K.Inhibition of the PI3K/Akt signaling pathway increased the expression of Bcl2 and decreased the expression of Bax,and activated the different phosphorylation sites of Foxo3 a,increasing the expression of the mitochondrial protein Mn SOD.Conclusion:Shenqi Fuzheng injection has a clear curative effect in the treatment of cancer-related fatigue,and its mechanism of effect is related to improving the increase of skeletal muscle and mitochondrial dysfunction.Shenqi Fuzheng injection can reduce the oxidative stress of muscle cells by activating AMPK pathway and inhibiting PI3K/Akt pathway.Injury,and activate the skeletal muscle autophagy/lysosomal pathway,maintain a high level of healthy mitochondria in muscle cells and the steady state of mitochondrial function,achieve the curative effect of increasing skeletal muscle mass and alleviating cancer-related fatigue. |
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