Therapeutic Effect Of FAPI-siRNA Targeting FAP Positive Fibroblasts In Pulmonary Fibrosis

Background:Idiopathic pulmonary fibrosis(IPF)is a chronic,irreversible and fatal interstitial lung disease(ILD)that is characterized by an unrestrained accumulation of extracellular matrix(ECM),resulting in healthy lung tissue being replaced by ECM and alveolar structures being destroyed,thereby reducing lung compliance,oxygenation damage,and eventually respiratory failure.At present,treatment options for patients with IPF remain limited.Lung transplantation remains the only treatment that clearly improves survival in carefully selected patients.myofibroblasts are the key effectors cells in the IPF.Many studies have shown that IPF patients have high expression of fibroblast activation protein-α(FAP),while normal tissue cells hardly express FAP.Therefore,small molecule nucleic acid drugs that specifically target FAP can theoretically effectively inhibit the progression of pulmonary fibrosis,but further research and demonstration are still needed.Objective:To explore whether FAPI-siRNA can specifically inhibit and clear activated fibroblasts to effectively inhibit the progression of pulmonary fibrosis,and provide an effective treatment strategy and theoretical basis for the clinical treatment of idiopathic pulmonary fibrosis.Methods:Part Ⅰ:(1)Western blot and Pathological evaluation such as HE,Masson,immunohistochemistry and immunofluorescence was used to detect the difference of FAP in the lung tissues of IPF patients and healthy people.Single-cell sequencing verifies the difference in FAP expression between healthy people and IPF patients.(2)The primary fibroblasts were stimulated with TGF-β1,and changes in FAP levels were detected by Western blot.Part Ⅱ:(1)Pathological evaluation,cell model,and single-cell sequencing verification are the same as those in Part Ⅰ.(2)RNA extraction was used to detect the differences in the expression of EIF3B and BIRC5 between normal individuals and IPF patients at the mRNA molecular level,and to identify the main sources of these two proteins.(3)The expression of EIF3B and BIRC5 genes is interfered in human primary fibroblasts derived from patients with IPF,cell proliferation and apoptosis are detected,fibrosis indicators(α-SMA、Collagen Ⅰ),proliferative markers(PCNA),and apoptotic pathways(Bax,Bcl2)were detected by Western blot and qRT-PCR;α-SMA and Collagen Ⅰ are detected by immunofluorescence to determine whether cells are transformed into activated fibroblasts;detection of apoptosis ratio by flow cytometry(Annexin V);(4)Collagen Ⅰ is detected by qRT-PCR and immunofluorescence as a marker of inhibited fibroblast activation.Part Ⅲ:(1)FAPI-siRNA coupling compounds were synthesized and tested for their ability to specifically recognize binding activated fibroblasts.(2)The interference efficiency of FAPI-siRNA conjugates was verified by in vitro interference experiments.(3)Male NCG mice were selected and grouped according to siEIF3B,siBIRC5,FAPI-siEIF3B,and FAPI-siBIRC5.Each mouse was subcutaneously injected with 200μl of activated fibroblasts containing a high concentration of matrix.On the day 6,the siRNA compound was injected into the tail vein,and FAP-sc Fv-ICG in vivo imaging was taken at the same time,and then euthanized on day 20 for analysis of fibrosis.Results:Part Ⅰ:(1)Compared with normal people,FAP is abundantly expressed in patients with idiopathic pulmonary fibrosis and localized in myofibroblasts.(2)The expression levels of FAP were evaluated based on the protein levels in fibroblasts treated with TGF-β1.Part Ⅱ:(1)Compared with normal people,EIF3B and BIRC5 are highly expressed in patients with idiopathic pulmonary fibrosis and localized in myofibroblasts.(2)The expression levels of EIF3B and BIRC5 were evaluated based on the protein levels in fibroblasts treated with TGF-β1.(3)Knock down the BIRC5and EIF3B promotes apoptosis and inhibit proliferation in myofibroblast,and apoptosis was promoted through endogenous mitochondrial pathways.Part Ⅲ:(1)FAPI-siRNA conjμgated compounds can specifically recognize and bind to myofibroblasts.(2)FAPI-siEIF3B and FAPI-siBIRC5 can significantly knock down the target gene and effectively interfere with the synthesis of collagen in humanized mice.Conclusion:Compared with normal people,FAP~+BIBC5~+/FAP~+EIF3B~+double positive cells are abundantly expressed in IPF fibrotic lungs and predominantly localized in myofibroblasts.Meanwhile,knock down the BIRC5 and e IF3B promotes apoptosis and inhibit proliferation in myofibroblast,thereby effectively inhibiting the progression of pulmonary fibrosis.Furthermore,FAPI-siRNA conjμgated compounds can specifically recognize myofibroblasts and inhibit the progression of pulmonary fibrosis in vitro.In summary,our results show that FAPI-siRNA targeted therapy could be an attractive strategy for attenuating pulmonary fibrosis in vivo.