| Purpose: The propose of this experience is to assess the effects of FZLFR on the treatment of cancer cachexia and the alleviation of skeletal muscle atrophy caused by cancer cachexia through establishing the mice model of cancer cachexia.Based on the network pharmacology analysis,we probe the key targets and pathway of FZLFR against cancer cachexia,and verify targets by in vivo experiment,further,to clear the mechanism of FZLFR against cancer cachexia.Methods: 1.The study of anti-tumor cachexia by FZLFR(1)Establishing the mice model of cancer cachexia 40 male C57BL/6 mice that were randomly divided into 4 groups,each group contains 10 mice.The Lewis Lung Carcinoma(LLC)cells subcutaneously injected into the right forelimb axillary back of Group Cachexia,Group Cachexia+MA,and Group Cachexia+FZLFR of mice.Mice of Group Control were injected with the same amount of PBS buffer at the same location.(2)Detection of experimental indicators After inoculated the LLC cells,we observed the generally status of mice daily.Each group of mice were treated on the 8th day,continuous administration for 14 days,food intake and body weight of mice were recorded each day,tumor sizes were measured each 2 days.The experiment ended on the 22 st day.The solid tumor tissue,bilateral gastrocnemius muscle,tibial anterior muscle,epididymal fat and other organs were surgically removed and weighed.Detected the proliferative activity of spleen cells by MTT,observed the changes of cross-sectional area of gastrocnemius muscle and tibial anterior muscle by HE staining,examined the protein expression levels of gastrocnemius muscle of MAFBx,MURF1,NF-κB and TWEAK by Western Blot,calculated the serum levels of TNF-ɑ and IL-6 by ELISA.2.The anti-mechanism of cancer cachexia by FZLFR and verification of experiment based on network pharmacology analysis(1)To probe the mechanism of FZLFR anti-cancer cachexia through network pharmacology analysis Acquired the ingredients and targets of FZLFR by the TCMSP platform and the Drug Bank database,obtained the targets of cancer cachexia through Gene Card and OMIM platforms,took intersection of differential genes of disease and target genes of FZLFR.Originated “FZLFR-ingredients-targets-cancer cachexia” by using the cytoscape.Formed the PPI interaction network and sequenced key targets from higher score to lower score via STRING database.Attained GO function and KEGG pathway enrichment analyses of all targets from Metascape.(2)Verification of molecular mechanism Ordering by protein interaction targets from high to low,we applied Western Blot to analyze the expression levels of MAPK8、VEGFA、EGFR and ESR1 in tumor tissue of each group.Results: 1.The study of anti-tumor cachexia by FZLFR(1)General status of mice On the 8th day of subcutaneous inoculation of LLC cells in mice,the tumor growth can be touched,on the 18 th day,the tumor mass was increased significantly,the body weight and food intake of mice decreased obviously(P < 0.05),the tumor bearing mice initiated the cachexia state.During the treatment of disease,the free body weight of Group Cachexia and Group Cachexia+MA were decreased by the time,while the free body weight of Group Cachexia+FZLFR was increased.The food intake of mice of Group Cachexia+FZLFR was elevated compared with Group Cachexia and Group Cachexia+MA(P<0.05).(2)Spleen Index and the proliferative activity of spleen cells of mice Compared with the Group Control,the SI and the proliferative activity of spleen cells of Group Cachexia was strikingly increased(P<0.05).The SI of Group Cachexia+FZLFR was comparable contrast to Group Cachexia,while the proliferation activity of spleen cells of Group Cachexia+FZLFR was higher compared with Group Cachexia and Group Cachexia+MA(P<0.05).(3)The cross-sectional area of gastrocnemius and anterior tibial muscles of mice The weight of gastrocnemius and anterior tibial muscles of Group Cachexia and Group Cachexia+MA were lowered compared with Group Control(P<0.05).The weight of gastrocnemius and anterior tibial muscles of Group Cachexia+FZLFR was higher compared with Group Cachexia(P<0.05)and Group Cachexia+MA.Compared with Group Control the cross-sectional area of gastrocnemius and anterior tibial muscles of Group Cachexia were reduced obviously(P<0.05).The cross-sectional area of gastrocnemius of Group Cachexia+FZLFR was improved compared with Group Cachexia(P<0.05).(4)The expression levels of MAFBx and MURF1,NF-κB,TWEAK proteins in gastrocnemius of mice.The expression levels of MAFBx and MURF1 proteins in the gastrocnemius of Group Cachexia were higher compared with Group Control(P<0.05);Compared with Group Cachexia,the Group Cachexia+FZLFR significantly reduced the protein expression levels of MAFBx and MURF1(P<0.05).Besides,there was significant differences between Group Cachexia and Group Control in the expression level of NF-κB(P<0.05);Compared with Group Cachexia,the expression level of NF-κB of Group Cachexia+FZLFR and Group Cachexia+MA were notable declined(P<0.05).The expression levels of TWEAK in the gastrocnemius of Group Cachexia was enhanced compared with Group Control;Compared with Group Cachexia and Group Cachexia+MA,the Group Cachexia + FZLFR appreciably reduced the protein expression levels of TWEAK(P<0.05).(5)The serum levels of TNF–α and IL–6 Compared with Group Control,the serum levels of TNF–α and IL-6 were arose markedly(P<0.05).Apparently,Group Cachexia+FZLFR had significantly declined the serum levels of TNF–α and IL-6 compared with Group Cachexia(P<0.05).2.The anti-mechanism of cancer cachexia by FZLFR and verification of experiment based on network pharmacology analysis(1)The mechanism of FZLFR anti-cancer cachexia through network pharmacology analysis The TCMSP database screened FZLFR compounds that met the conditions of OB≥30% and DL≥0.18,and obtained 138 drug targets through the Drug Bank platform,obtained 2118 tumor cachexia-related genes from OMIM and Gene Cards databases.FZLFR corresponded to the target protein and the intersection of tumor cachexia-related genes.Obtained 60 FZLFR anti-tumor cachexia core target genes.Imported core targets into Cytoscape and STRING databases to construct a network of "FZLFR-ingredientstargets-cancer cachexia" of which quercetin,kaempferol,beta-sitosterol,luteolin and Stigmasterol are the main active ingredients of FZLFR,and to set up protein interaction network of related targets,among which MAPK8,IL-6,VEGFA,EGFR and ESR1 were related 57 core genes.The GO and KEGG enrichment pathways of core targets showed that FZLFR anti-tumor cachexia involved tumor-related signaling pathways such as TNF signaling pathway,Erb B signaling pathway,and VEGF signaling pathway.(2)Verification of molecular mechanism Compared with Group Cachexia,the expression levels of MAPK8 and VEGFA of Group Cachexia+FZLFR was appreciably reduced(P<0.05).Likewise,the expression levels of EGFR of Group Cachexia+FZLFR was considerably reduced compared with Group Cachexia(P<0.05).Moreover,the expression levels of ESR1 in tumor of Group Cachexia+FZLFR was noticeably amplified compared with Group Cachexia(P<0.05).Conclusions: 1.C57BL/6 mice were transplanted subcutaneously with LLC cells to successfully establish cancer cachexia model.FZLFR increased the diet of mice,enhanced the free body weight of mice,and reduced skeletal muscle atrophy.FZLFR can decrease the protein expression levels of MAFBx,MURF1,NF-κB and TWEAK,decline the serum levels of TNF-ɑ and IL-6,regulate the activity of the UPP signaling pathway,and uniformly correct the skeletal muscle atrophy caused by cancer cachexia.2.In this study,based on network pharmacology analysis,the targets and pathways of FZLFR intervention in tumor cachexia were obtained.The mechanism may be the main ingredients such as quercetin,kaempferol,beta-sitosterol,luteolin and Stigmasterol of FZLFR through the TNF signaling pathway,Erb B signaling pathway and VEGF signaling pathway mediated by the related core targets of MAPK8,IL-6,VEGFA,EGFR and ESR1 which inhibits tumor proliferation,regulates inflammatory response,and achieves the effect of anti-tumor cachexia.3.FZLFR reduced the serum levels of IL-6 in cachexia mice,declined the expression levels of MAPK8,VEGFA and EGFR in tumor tissues,and improved the expression level of ESR1.It is suggested that FZLFR can inhibit tumor cell proliferation and reduce inflammatory reaction by inhibiting MAPK8,IL-6 mediated TNF signaling pathway,EGFR intervened Erb B signaling pathway and VEGFA intermeddled VEGF signaling pathway,thereby alleviating the rapid progress of tumor cachexia and achieving the effect of anti-cancer cachexia. |
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