Study On The Protective Effect Of AST-Ⅳ On Radioactive Cardiac Fibrosis Based On MiR-34c Mediated Radiation-induced Bystander Effect On Myocardial Fibroblasts

OBJECTⅣE: To predict the signaling pathways related to Astragaloside Ⅳ intervention on radiation-induced cardiac fibrosis by network pharmacology.Based on this,an experimental system of radiation-induced bystander effect was established by co-culturing X-ray irradiated CFs with non-irradiated CFs to observe the effect of miR-34 c regulating Notch1 signaling on phenotypic transdifferentiation and fibrogenic ability of bystander CFs.To investigate the molecular mechanism of miR-34 c mediated radiation-induced bystander effect in radiation-induced cardiac fibrosis and the protective mechanism of Astragaloside Ⅳ.METHODS: 1.Target genes related to "miR-34","fibrosis",and "Astragaloside Ⅳ" were collected from relevant databases,the PPI network was constructed by mining the STRING database while core targets were screened,then GO and KEGG signaling pathway enrichment analysis were performed.2.Firstly,miR-34 c inhibitor and miR-34 c inhibitor NC were transfected into CFs to construct miR-34 c inhibitor-CFs and NC-CFs,respectively,and AST-CFs were constructed with 4μg/m L Astragaloside Ⅳ pretreatment CFs.Subsequently,these CFs were irradiated with 2Gy X-ray,and co-cultured with normal CFs for 48 h to establish a radiation-side effect system.The experiment was randomly divided into five groups: Control group(Control group: normal CFs),irradiation co-culture group(X-co group:X-CFs+ normal CFs),miR-34 c inhibitor negative control co-culture group(X-NC-co group:X-(NC-CFs)+ normal CFs),miR-34 c inhibitor co-culture group(X-inhibitor-co group:X-(miR-34 c inhibitor-CFs)+ normal CFs)and Astragaloside Ⅳ intervention group(X-AST-co group: X-(AST-CFs)+ normal CFs).3.q RT-PCR and Western Blot were used to detect the expression of miR-34 c and Notch1 in irradiated CFs and non-irradiated bystander CFs.4.The expression of α-SMA molecule was detected by q RT-PCR and Western Blot,and α-SMA positive cells were detected by flow cytometry to observe the transdifferentiation of bystander CFs into myofibroblasts.5.q RT-PCR and Western Blot were used to detect the expression of TGF-β1 and Col I,CCK-8 assay was used to detect the cell proliferation,and wound healing assay was used to detect the cell migration,and the effect of radiation induced bystander effect on the fibrogenic ability of CFs was analyzed.RESULTS: 1.The pathways enriched by the core targets in the PPI network were mainly involved in MAPK signaling pathway,calcium signaling pathway,Erb B signaling pathway,gap junction,VEGF signaling pathway,Fc epsilon RI signaling pathway,Notch signaling pathway,NF-κB signaling pathway,etc.We focused on the relationship between Notch signaling pathway and myocardial fibers and carried out subsequent experimental studies.2.The expression of miR-34c: compared with the Control group,the expression level of miR-34 c in irradiated CFs group was gradually increased,and the expression reached the peak at 24h(P < 0.01),and the highest expression of miR-34 c was observed in non-irradiated bystander CFs at 48h(P < 0.01)and then began to decrease.3.Regulation of miR-34 c on Notch1 and intervention effect of AST in bystander CFs:compared with the Control group,the expression of miR-34 c was significantly increased in X-co group(P < 0.01),while Notch1 mRNA and protein expression were significantly decreased(all P < 0.01);Compared with X-co group,in the X-NC-co group,there was no statistically significant difference in the expression of miR-34 c and Notch1,while the expression of miR-34 c in X-inhibitor-co group and X-AST-co group was down-regulated(all P < 0.01),and the expression of Notch1 m RNA and protein was up-regulated(all P < 0.01).4.The effect of radiation-induced miR-34 c on the transdifferentiation of bystander CFs and the intervention effect of Astragaloside Ⅳ : Compared with the Control group,the expression of α-SMA at mRNA and protein levels was increased in X-co group(all P < 0.01),and the proportion of α-SMA positive cells also increased(P < 0.01);Compared with X-co group,there was no statistically significant difference in the expression of α-SMA and the proportion of α-SMA positive cells in X-NC-co group,while the expression of α-SMA in X-inhibitor-co group and X-AST-co group was decreased at the m RNA and protein levels(all P < 0.01),and the proportion of α-SMA positive cells also decreased(P < 0.01).5.The effect of radiation-induced miR-34 c on the fibrogenic ability of bystander CFs and the intervention effect of Astragaloside Ⅳ : compared with the Control group,the expression of fibrosis factors TGF-β1 and Col1 was increased at m RAN and protein levels in X-co group(all P < 0.01),and the proliferation activity and mobility of bystander CFs were increased(all P < 0.01);Compared with X-co group,the expression of fibrosis molecules and the proliferation and migration of CFs in X-NC-co group were not statistically significant different,while the expression of TGF-β1 and Col1 molecules in X-inhibitor-co group and X-AST-co group were decreased at m RAN and protein level(all P < 0.01),and the proliferation activity and mobility of bystander CFs were also decreased(all P < 0.01).CONCLUSION: 1.Network pharmacology studies predict that the pro-fibrotic effects of miR-34 and anti-fibrotic effects of AST may be related to MAPK,Erb B,VEGF,Fc epsilon RI,NF-κB,Notch and other signaling molecules.Literature studies have further shown that Notch signaling pathway plays an important role in protecting cardiac function.2.X-ray can increase the expression of miR-34 c in CFs and upregulate miR-34 c in bystander CFs,which further negatively regulating Notch1 signal to promote the transdifferentiation of CFs and enhance its fibrogenic ability.3.Astragaloside Ⅳ has an inhibitory effect on the transdifferentiation and pro-fibrosis of CFs by regulating miR-34c/Notch1 mediated radiation-induced bystander effect,thus playing a role in protecting radiation-induced cardiac fibrosis.