| ObjectivesPediatric anorexia(PA)is a chronic digestive disorder syndrome in children,manifested as long-term loss of appetite or disappearance,reduction or even refusal to eat,which can cause malnutrition,delay development,and then affect the cognitive ability and immunity of patients.Xiao’er Kaiwei Zengshi Mixture is a traditional Chinese medicine prescription clinically used in the treatment of PA,and it has been proved to be effective.However,the mechanism behind its efficacy remains unexplored.The purpose of this study was to elucidate the bioactive components of Xiaoer Kaiwei Zengshi Mixture in the treatment of infantile anorexia and its possible mechanism of action.Methods(1)The SD rat model of anorexia was induced by special feed,and then these rats were randomly divided into Control group(n=10),Model group(n=10),western medicine group(WM,n=10),and Xiao’er Kaiwei Zengshi Heji low-dose group(Low-XKZH,n=10),medium-dose group(Middle-XKZH,n=10)and high-dose group(High-XKZH,n=10).The food intake and body weight of rats were measured during the modeling period and the intervention period.HE staining was selected to observe the effect of Xiao’ er Kaiwei Zengshi Heji on pathological changes of stomach tissue,so as to evaluate its therapeutic efficacy.(2)Based on TMT quantitative proteomics technology to detect differential proteins in rat jejunal and gastric tissues.The differential proteins were selected and their function enrichment,kyptp encyclopedia of genes and genomes(KEGG)pathways,and their interactions were analyzed.The expressionn level of the differential proteins regulated by Xiao’ er Kaiwei Zengshi Heji was verified by western blot.(3)Based on the network pharmacological method,TCMSP,Dis Ge NET,TTD,and OMIM databases were used to screen and analyze the effective chemical components and their corresponding targets of Xiao’er Kaiwei Zengshi Heji.Enrichment analysis of Gene ontology(GO)function and KEGG pathway was performed on the potential targets screend out.(4)Comprehensive analysis of proteomics data and network pharmacology data,screening of the core targets of Xiao’er Kaiwei Zengshi Heji.The protein expression of screened core targets was tested by western blot,and the m RNA expression of core targets-related pathway factors was tested by Real time polymerase chain reaction(RT-PCR).Results(1)A rat model of anorexia was successfully established.The body weight and feed intake of rats in each group were counted,and it was found that compared with the control group,the body weight and feed intakes were significantly lower in model group(P<0.01).After the intervention of the Xiao’er Kaiwei Zengshi Heji in anorexia rats,the body weight and feed intake of rats in the low-XKZH group,middle-XKZH group and high-XKZH group were significantly higher than those in the model group(P<0.01),and showed a dose dependence.(2)HE results showed that there were obvious pathological changes in jejunum and stomach in model group,which were characterized by disordered arrangement of villi in jejunum,reduced atrophy of gastric glands,destruction of both structures,and obvious inflammatory cell infiltration in the tissue;Compared with the model group,the pathological changes of jejunum and stomach in low-XZKH,middle-XZKH and high-XZKH groups were significantly improved in a dose-dependent manner.(3)Proteomics results showed that a total of 4116 proteins were quantified in this study.Compared with the rrats in the control group,there were 32 differential proteins(20up-regulated and 12 down-regulated proteins)in the gastric tissue of rats in model group.Compared with model group,there were 77 proteins expresssiom differences(65 up-regulated and 12 down-regulated proteins)in the gastric antral tissue of the XKZM group rats.GO enrichment analysis showed that these differential proteins were mainly concentrated in cellular processes,adenosne triphosphate(ATP)binding,antioxidant activity,protein binding and signal transduction.KEGG pathway analysis showed that differential proteins were enriched in glycolysis and glycometabolism,biosynthesis,insulin-related signaling,ligand-receptor interaction in nerve tissue,metabolism,Rat sarcoma(RAS)signaling,and Mitogen a ctivated protein kinase(MAPK)signal.The results of protein interaction analysis showed that helicobacter pylori(HP),catalase(CAT),albumin(ALB),UDP-glucuronyl transferase 1A1(UGT1A1),NADH dehydrogenase,quinone 1(NQO1),apolipoprotein E(APOE),glutathione S transferase mu 3(GSTM3)and transthyretin(TTR)proteins were interacted with other proteins as pivotal proteins.Western blot results showed that compared with the control group,the protein expression of sulfotransferase 1C2A(SULT1C2)and acid binding protein 2(CRABP2)in gastric antrum tissue of rats in model group were significantly lower(P<0.05),and the expression of Enolase 3(ENO3)and Keratin 5(KRT5)protein were increased(P<0.05).Compared with the Model group,the XKZM group SULT1C2 And CRABP2 protein expression increased(P<0.05),ENO3 and KRT5 protein expression decreased(P<0.05).The expression trend of these differential proteins is consistent with the results of proteomics.(4)Proteomic results showed that a total of 3575 proteins were quantified in gastric antrum tissue.Compared with control group,34 differential proteins(11 up-regulated proteins and 23 down-regulated proteins)were found in gastric tissue of model group;Compared with model group,71 differential proteins(29 up-regulated proteins and 42 down regulated proteins)were found in XKZM group.These differential proteins can be enriched into many biological processes related to gastric tissue secretion,gastric motility and gastrointestinal protection,including positive regulation of constitutive secretion pathway,amide transport,regulation of constitutive secretion pathway,ceramide transport from endoplasmic reticulum to Golgi,drug transmembrane transport,biosynthesis of glutamine family amino acids and so on;The enriched biological functions include electron transporter-electron transfer within COQH2-Cyt c reductase complex activity,triphosphate monoester hydrolase activity,d GTPase activity,ceramide transporter activity,serine endopeptidase activity,oxidoreductase activity and estradiol 17 β Dehydrogenase activity,cytoplasmic dipeptidase activity,amino acid binding,phosphatidylserine binding,etc.Further pathway analysis showed that the differential proteins were related to steroid biosynthesis,ABC transporter,bile secretion,pyruvate metabolism,amino acid metabolism and so on.Protein interactions revealed that microtubule multispecific organic anion transporter 1(ABCC2),epidermal growth factor receptor(EGFR),cytochrome P450 family 3 subfamily a polypeptide 62(Cyp3a62)Cadherin-17(Cdh17),cytosolic non-specific dipeptidase(Cddp2),cytochrome P450 family 2subfamily B polypeptide 4(cyp2d4),ezrin(Ezr),granzyme A(gzma),ATP binding cassette subfamily G member 2(ABCG2)occupy the center of the network diagram and interact with other proteins as a hub.The results of Western blot showed that compared with the blank control group,the relative expression of Scamp3,Tmem86 a and Akr1b7 protein in the model group increased significantly(P < 0.05),while the relative expression of Smarc1,Nudt13,Rpp30,Rps4y2,Art2 b and Glmp protein decreased significantly(P < 0.05);Compared with the model group,the relative expression levels of Scamp3,Tmem86 a and Akr1b7 protein in Xiaoer Kaiwei Zeng Shi mixture group decreased significantly(P < 0.05),and the relative expression levels of Smarc1,Nudt13,Rpp30,Rps4y2,Art2 b and Glmp protein increased significantly(P < 0.05).The expression trend of these differential proteins was consistent with the results of proteomics.(5)Network pharmacology,according to the results of screening in 142 compounds as infantile appetizing food mixture of potential effective components,their role in the 82 target of anorexia,involving 26 pathological pathways,including nerve activity ligand receptor interaction in neural tissue(27 targets),calcium ion signaling(18 targets),retinol metabolism(6 targets),arachidonic acid metabolism(6 targets),drug metabolism(5 targets),and fat cytokine signaling pathways(5 targets).(6)Comprehensive analysis of proteomics data and network pharmacology data,it was found that ATP binding cassette subfamily G member 2(ABCG2)could be used as the core therapeutic target of Xiao’er Kaiwei Zengshi Heji.ABCG2 was related to biological processes such as drug response,protein dimerization activity and plasma membrane regions,and the ABC transporter pathway.The results of western blot illustrated that ABCG2 protein level in the stomach tissue of the model group was significantly up-regulated compaerd with that of the control group(P<0.05).Compared with the model group,the expression of ABCG2 protein in the gastric tissue of the XKZH group was decreased(P<0.05).This result is consistent with the proteomics result.The RT-PCR test results showed that ABCG2,p-glycoprotein(p-GP)and multidrug resistance associated protein 2(MRP2)m RNA expression in the stomach tissue of the model group were significantly higher than thos of the control group(P<0.05).And the levels of ABCG2,p-GP,and MRP2 m RNA in the stomach tissue in the XKZH group were significantly down-regulated compared with those in the model group(P<0.05).Conclusion(1)Xiaoer Kaiwei Zeng Shi mixture has obvious therapeutic effect on juvenile anorexia rats with simulated etiology.(2)Xiaoer Kaiwei Zeng Shi mixture can increase the expression of apo E,ndhl,SCP2 and DHCR24 proteins related to blood lipid metabolism in jejunum of PA model,regulate the expression of eno3,krt5,sult1c2 a and crabp2 proteins in PA model,promote blood lipid metabolism,and affect glycogen decomposition and glycometabolism,RNA degradation,amino acid biosynthesis,carbon metabolism ATP production and HIF-1 signal pathway can achieve the effect of treating PA,which may be the main therapeutic target of kaiweizengshi mixture for children.(3)Xiaoer Kaiwei Zeng Shi mixture can affect the glutamine transport and synthesis process,amino acid metabolism pathway,energy metabolism pathway,bile secretion process and drug excretion pathway in PA gastric antrum,which provides a certain theoretical support for the clinical application of Xiaoer Kaiwei Zeng Shi mixture in the treatment of anorexia.(4)Based on the method of network pharmacology,it is found that there are 142 kinds of effective active components in Xiaoer kaiweizengshi mixture,which act on 82 targets of anorexia.The main effective components include laxin,Medicago sativa,stigmasterol,Poria acid,mangprocyanidin,visfatin,pentenoic acid,isoflavones,methoxyphenol,hydrazine carboxyamide hexenoic acid and di-n-octyl phthalate,etc.PTGS2,ESR1,AR,ESR2,PTGS1,ADRB2,RXRA,ADRA1 B,CHRM1 and KCNH2 may be the potential therapeutic targets of kaiweizengshi mixture for anorexia in children.Neuroactive ligand receptor interaction signaling,calcium ion signaling,retinol metabolism,arachidonic acid metabolism,gap junction,cytochrome P450 metabolism to xenobiotics,drug metabolism,steroid hormone biosynthesis,linoleic acid metabolism,tryptophan metabolism and vascular smooth muscle contraction may be the potential treatment of anti anorexia with kaiweizengshi mixture in children.(5)Based on the comprehensive analysis of proteomics and network pharmacology,it is found that ABCG2,as the target of laxin and Glycyrrhiza chalcone B,is one of the core targets of kaiweizengshi mixture for children.ABCG2 protein participates in its therapeutic process through ABC transporter pathway.Xiaoer Kaiwei Zeng Shi mixture inhibits the expression of ABCG2 in tissues,thus inhibiting the excretion of effective active ingredients and enhancing its sensitivity to drugs,so as to achieve the purpose of treating diseases. |
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