| Objective:Cyclophosphamide（CTX）,as a first-line tumor chemotherapy drug and powerful immunosuppressant,is widely used in the treatment of a variety of malignant tumors and autoimmune diseases.However,CTX can also cause a variety of serious adverse reactions,such as bone marrow suppression,cardiotoxicity,liver function damage,hemorrhagic cystitis and so on.In fact,CTX is often used to replicate animal models of Yang deficiency and blood deficiency syndrome in the field of basic scientific research of traditional Chinese medicine.At present,there is no specific drug for the toxic and side effects of CTX.It is of great clinical significance to prevent or alleviate the toxic and side effects of CTX chemotherapy for improving the therapeutic effect of tumor.Aikeqing granule（AKQ）,as a patent compound created by Institute of Tropical Medicine,Guangzhou University of traditional Chinese medicine,has been used in the treatment of AIDS for many years.It can enhance the immunity and improve the quality of life of AIDS patients.Our research group believes that the effect of AKQ on strengthening yang,Qi,kidney and spleen can be used to correct the side effects of deficiency of both qi and Yang and deficiency of both spleen and kidney caused by CTX.In this study,AKQ was selected as the research object.Through in vivo and in vitro experiments,combined with network pharmacology analysis,metabonomics and intestinal microflora analysis,the following research objectives were expected to be achieved1.To determine whether AKQ has attenuation effect on CTX induced toxicity in normal and tumor bearing mice and the main target organ of attenuation effect.2.To evaluate the protective effect of AKQ on H9c2 cardiomyocyte injury induced by acrolein（ACR）,a toxic metabolite of CTX.3.To explore the mechanism of AKQ in reducing CTX toxicity at the above animal and cell levels,and to provide experimental basis for the theory of treating different diseases with the same treatment.Methods:1.In vivo（animal）experiment 1:to observe the effect of AKQ on CTX induced toxicity in Kunming mice.（1）Establishment of CTX poisoning model in Kunming mice:eight week old healthy Kunming mice weighing 25±3g were injected intraperitoneally with CTX for four consecutive days:100 mg/kg/D on the first and second days,50 mg/kg/d on the third and fourth days.（2）Grouping treatment and general condition observation:24 healthy Kunming mice were randomLy divided into 3 groups,8 in each group:normal control group（intraperitoneal injection of normal saline 0.3 mL/time/d on day1 to day4+intragastric administration of pure water 0.3 mL/time/d from day1 to day7）,model group（CTX injection on day1 to day4+intragastric administration of pure water 0.3 mL/time/d from day1 to day7）The mice in Aikeqing group（CTX injection on day1 to day4+AKQ gavage 1.3 g/kg/d from dayl to day7）were observed and recorded daily for 8 days.AKQ 1.3 g/kg/d given to mice equivalents to 9 times of human clinical dose.（3）Determination of blood and biochemical indexes:after the experiment,the mice in each group were killed,and the whole blood and serum were taken.WBC,RBC,PLT,HGB,lym,Mon,AST,alt,CRE and CK were measured by blood cells and biochemical analyzer.（4）Histopathological observation of main organs:after the experiment,the mice in each group were killed,and the heart,liver,spleen,lung and kidney tissues of mice in each group were stained with HE-stained and observed.2.In vivo（animal）experiment 2:to observe the effect of AKQ on cardiac toxicity of CTX poisoned mice.（1）Establishment and grouping of cardiac toxicity model in CTX poisoned mice:Fifty healthy Kunming mice aged 8 weeks and weighing 25±3 g were randomLy divided into 5 groups with 10 mice in each group.CTX was injected intraperitoneally for 4 consecutive days（8-11 days）（the same scheme as in vivo experiment 1）.The animals were divided into normal group（intraperitoneal injection of N.S 0.3 mL/time/d on day 8-11+intragastric administration of pure water 0.3 mL/time/d from day 1-13）,model group（CTX injection on day 8-11+intragastric administration of pure water 0.3 mL/time/d from day 1-13）,AKQ low dose group（CTX intraperitoneal injection on day 8-11+AKQ gavage 0.65 g/kg/D from day 1-13）,AKQ medium dose group（CTX intraperitoneal injection on day 8-11+AKQ gavage 1.3 g/kg/D from day 1-13）,AKQ high dose group（CTX intraperitoneal injection on day 8-11+AKQ gavage 2.6 g/kg/D from day 1-13）.The experimental period was 14 days.After 14 days,the serum and heart were collected for relevant detection.The low,middle and high doses of Aikeqing given to mice were 4.5,9 and 18 times of human clinical dose.（2）Determination of heart index:the hearts of mice in each group were weighed and the heart index was calculated.（3）Determination of myocardial enzymes and oxidation indexes:serum CK,CK-MB,GSH,SOD and MDA were detected.（4）Histopathological observation of heart:the heart tissues of mice in each group were stained with HE-stained and observed.（5）The expression of Bax and Bcl-2 was detected by Western blot.3.In vitro（cell）experiment:to observe the effect of AKQ on H9c2 toxic injury induced by ACR and explore its mechanism（1）Observation on the effect of AKQ on H9c2 activity:the methanol extract of AKQ granules was dissolved with DMSO and diluted with cell culture medium to form different concentration gradients.H9c2 culture medium was added and incubated for 24h,48h and 72h.The cell activity was determined by CCK8 method.（2）Determination of IC50 of ACR on H9c2:different concentration gradient ACR solution was added to H9c2 culture medium for 24 hours,and cell viability was determined by CCK8 method.（3）Observation of the effect of AKQ on ACR induced H9c2 toxic injury:H9c2 cells were divided into five groups:control group（normal culture）,ACR group（ACR 20 μmol/1 intervention Culture）,ACR+AKQ low dose group（ACR+AKQ 20 μg/mL）,ACR+AKQ medium dose group（ACR+AKQ 40 μg/mL）,ACR+AKQ high dose group（ACR+AKQ 80 μg/mL）.After 24 hours of culture,the cell morphology was observed,and the expressions of Bax,caspase 3,caspase 9 and Bcl-2 were detected by CCK8,hochester staining,flow cytometry annexin V-FITC/PI double staining and WB.（4）Network pharmacology analysis of AKQ alleviating CTX cardiotoxicity:screening and mapping of CTX cardiotoxicity related disease targets and AKQ drug component therapeutic targets,constructing drug component target network and PPI network,and analyzing go and KEGG pathway enrichment of targets.（5）Experimental verification of network pharmacology analysis:RT qPCR and WB technology were used to verify the expression level of pathway targets enriched by network pharmacology analysis.（6）H9c2 cells were divided into four groups:control group（normal culture）,ACR group（ACR 20 μmol/L intervention Culture）,ACR+AKQ group（ACR+AKQ 80 μg/mL）,AKQ+LY294002 group（ACR+AKQ 80 μg/mL+LY294002 10 μmol/L）.After 24 hours of culture,RT qPCR and WB were used to detect the expression of related pathway proteins and apoptotic proteins.4.In vivo（animal）Experiment 3:the effect and mechanism of AKQ on the cardiotoxicity of CTX chemotherapy in tumor bearing mice（1）Establishment of tumor bearing mice model:S180 sarcoma cell suspension with density of 1 × 107 cells/mL was subcutaneously inoculated into the back neck of Kunming mice in the volume of 0.2 mL/mouse.（2）Animal grouping and treatment:twenty four S180 tumor bearing mice with tumor volume ranging from 40 mm3 to 100 mm3 were randomLy divided into three groups with 8 mice in each group:tumor model control group（0.3 mL/time/d pure water by gavage from dayl to day 13+8-11 days continuous intraperitoneal injection of N.S 0.3 mL/d）CTX chemotherapy group（continuous intraperitoneal injection of CTX from the 8th day to the 11th day,CTX injection scheme was the same as before+pure water gavage 0.3mL/time/d from day1 to day13）,CTX chemotherapy combined with AKQ treatment group（continuous intraperitoneal injection of CTX on 8-11 days and gavage of Aikeqing 2.6g/kg/d from from day1 to day13）.The experiment lasted for 14 days.Fresh feces were collected on the 12th day.Serum and heart were collected on the 14th day for follow-up detection.（3）Determination of myocardial enzymes and oxidation indexes:serum CK,CK-MB,SOD and MDA were detected.（4）Histopathological observation of heart:the heart tissues of mice in each group were HE-stained and observed.（5）Detection of apoptosis related proteins:Bax and Bcl-2 were detected by WB.（6）Detection of cardiac pathway proteins:WB and immunohistochemistry were used to detect the expression of cardiac pathway target proteins.（7）Tumor weight determination of tumor bearing mice:at the end of the experiment,the tumor of each group was weighed.（8）Serum metabonomics of tumor bearing mice:serum samples were collected from mice in each group for GC/MS non targeted metabonomics analysis.（9）Intestinal microflora analysis of tumor bearing mice:fresh unpolluted feces of mice were collected for 16S rRNA sequencing.Results:1.After intraperitoneal injection of cyclophosphamide（100 mg/kg/d x 2d,50 mg/kg/d x 2d）,Kunming mice showed obvious poisoning symptoms such as decreased activity,chilly curling,fluffy and erect hair,decreased food intake and weight loss.After addition of AKQ,the above poisoning symptoms were alleviated,and the mortality rate was significantly lower than that of model group（P=0.0028）.After cyclophosphamide injection,the white blood cells,platelets and lymphocytes of Kunming mice decreased significantly（P<0.001,P<0.001,P<0.01）,while the red blood cells,hemoglobin and monocytes did not change significantly;ast,ALT and cre did not change significantly,and CK increased significantly（P<0.05）.After oral administration of AKQ,the level of CK decreased significantly compared with the model group（P<0.05）.There was no significant difference in other blood biochemical indexes.HE staining showed that there was no significant difference in the pathological changes of liver,spleen,lung and kidney in each group;obvious myocardial damage was observed in the model group,and the degree of myocardial damage was reduced after taking AKQ.2.After intraperitoneal injection of cyclophosphamide（100 mg/kg/d × 2d,50 mg/kg/d ×2d）,the cardiac coefficient of Kunming mice was significantly higher than that of the normal group（P<0.05）,and the cardiac coefficient of low,medium and high dose AKQ groups was significantly lower than that of the model group（P<0.05,P<0.01,P<0.01）.Compared with the normal group,CK-MB and MDA in the model group were significantly increased（P<0.001,P<0.001）,GSH and SOD were significantly decreased（P<0.05,P<0.001）.After taking AKQ,the above indexes in each dose group of AKQ were improved in varying degrees,especially in the high-dose group.Compared with the model group,CK,CK-MB and MDA in the high-dose group were significantly decreased（P<0.05,P<0.001,P<0.001）.GSH and SOD increased significantly（P<0.05,P<0.001）.He staining showed obvious changes of myocardial injury in the model group,and the degree of myocardial lesion in the low,medium and high dose groups of Aikeqing was less than that in the model group in turn.Compared with the normal group,the expression of Bax and Bcl-2 in the heart tissue of the model group were increased,while the expression of Bax and Bcl-2 in each dose group of AKQ were decreased in a dose-dependent manner.3.The methanol extract of AKQ promoted the proliferation of H9c2 cells in a concentration dependent manner in the concentration range of 0-80 μ g/mL.the 24-hour IC50 of ACR on H9c2 cells was 20.37 μM.The damage of H9c2 cells induced by ACR for 24 hours involves morphological changes,decreased cell viability,increased number of hochester staining positive cells,increased proportion of flow cytometry annexin V-FITC/PI double positive cells,increased expression of apoptotic proteins Bax,Caspase3 and caspase9,and decreased expression of anti apoptotic protein bcl-2.Compared with the model group,AKQ treatment of H9c2 cells could improve the injury morphology,enhance cell viability,reduce the number of hochester staining positive and annexin V-FITC/PI double positive cells,inhibit the expression of Bax,Caspase3 and caspase9,and enhance the expression of Bcl-2 in a dose-dependent manner.The results of network pharmacology analysis showed that:192 targets were obtained by target screening and mapping,which were the main components of AKQ for the treatment of CTX cardiotoxicity;15 key compounds（including quercetin,luteolin,kaempferol,tanshinone IIA,naringenin,etc.）were obtained by constructing the network diagram of AKQ drugs active compounds therapeutic targets;30 by constructing PPI network Go analysis showed that the biological functions of the target mainly include the regulation of cell proliferation/apoptosis and oxidation/inflammation.KEGG pathway enrichment analysis showed that the top 10 signaling pathways involved PI3K-Akt,TNF and MAPK pathways.RT qPCR showed that the expression of PI3K and Akt in AKQ group was significantly higher than that in ACR group（P<0.001）.WB test also showed that the total protein and phosphorylated protein levels of PI3K and Akt in AKQ group were significantly higher than those in ACR group.In AKQ+ACR group,the up-regulated PI3K and Akt mRNA and protein levels were reversed when LY294002 was added,and the up-regulated Bax,Caspase3,caspase9 and down regulated Bcl-2 were also reversed in AKQ+ACR group.4.S180 sarcoma cell suspension with a density of 1 x 107 cells/mL was subcutaneously injected into the back neck of Kunming mice at a dose of 0.2mL,and the tumor model can be established in about 6 days.Compared with the tumor model control group,CK-MB and MDA in CTX chemotherapy group were significantly increased（P<0.001,P<0.001）,SOD was significantly decreased（P<0.001）,CK,CK-MB and MDA in CTX chemotherapy combined with AKQ group were significantly decreased（P<0.01,P<0.001,P<0.001）,while SOD was significantly increased（P<0.001）.He staining showed significant myocardial damage in CTX chemotherapy group,and the degree of myocardial damage in CTX chemotherapy combined with AKQ group was significantly less than that in CTX chemotherapy group.Compared with the tumor model control group,the expression of Bax was up-regulated and the expression of Bcl-2,PI3K and Akt was down regulated in CTX alone chemotherapy group.After adding AKQ,the expression of Bax was decreased and the expression of Bcl-2,PI3K and Akt was increased.After the end of the experiment,the tumor weight of CTX chemotherapy combined with AKQ treatment group was significantly lower than that of chemotherapy alone group（P<0.05）.Serum metabonomics analysis identified a total of 44 different metabolites among the three groups.AKQ intervention can make the profile of L-glutamic acid,L-aspartic acid,citric acid,glycine,ornithine and other metabolites changed by CTX chemotherapy approach to the tumor bearing control group;the metabolic pathways significantly affected by CTX combined with AKQ include:citric acid cycle,arginine biosynthesis,alanine aspartate and glutamic acid Metabolism,glyoxylic acid and dicarboxylic acid metabolism,arginine and proline metabolism,amino tRNA biosynthesis,glutathione metabolism,histidine metabolism,D-GLUTAMINE and D-glutamic acid metabolism.Intestinal microflora analysis showed that the relative abundance of Sphingomonas and Lysobacter was down regulated,and Prevotella9 was down regulated in CTX alone chemotherapy group.After CTX chemotherapy combined with AKQ,sphingomonadales and Lysobacter abundances were up-regulated,Prevotella9.In the CTX group,the down-regulated p53 signaling pathway,sesquiterpene biosynthesis,bile secretion and caffeine metabolism were up-regulated again after AKQ intervention.Conclusions:1.AKQ can improve the toxic symptoms of Kunming mice caused by CTX,significantly reduce the mortality;AKQ can reduce the abnormal elevation of myocardial enzymes caused by CTX,alleviate the imbalance of oxidative stress,reduce myocardial cell apoptosis,and improve the pathological changes of heart induced by CTX.2.AKQ can promote the proliferation of H9c2 cardiomyocytes,activate PI3K/Akt pathway and reduce ACR induced H9c2 apoptosis.3.CTX chemotherapy in tumor bearing mice can cause abnormal increase of cardiac kinase,imbalance of oxidative stress,increase of cardiomyocyte apoptosis and down-regulation of PI3K/Akt expression.Combination with AKQ can improve the above lesions and enhance the anti-tumor effect of CTX.4.AKQ can regulate the expression of 44 different metabolites,such as L-glutamic acid,L-aspartic acid,citric acid,glycine and ornithine,enhance the TCA cycle,arginine biosynthesis,alanine,aspartate and glutamic acid metabolism,up regulate the beneficial bacteria（sphingomonadales,Lysobacter）and down regulate the harmful bacteria（Prevotella9）It can enhance the function of p53 signaling pathway and bile secretion of intestinal bacteria,so as to play a role in reducing CTX cardiotoxicity. |
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