Therapeutic Effect Of Responsive And Targeted Anti-inflammatory Peptides On Rheumatoid Arthritis

Objective: The multi-functional anti-inflammatory peptides that integrate multiple functions such as targeting,enzymtic sensitivity and cell penetrating were designed and synthesized by solid phase synthesis,and the anti-rheumatoid arthritis efficacy of the peptides was evaluated in vivo and in vitro.Methods: Solid phase synthesis was carried out to prepare the peptides,namely RMTQ,TATQ,QAW and RMT.The purity of the synthesized peptides and the molecular weight of the peptides were analyzed by High performance liquid phase(HPLC)and Mass spectrometry(MS)respectively.The circular dichroism spectroscopy was used to examine the secondary structure of each peptide.the enzyme digestion of RMTQ was analyzed using Tricine-SDS-PAGE in vitro.The toxicity of peptides to mononuclear macrophage Raw264.7 was investigated by CCK-8 method.Raw264.7 cells were stimulated by Lipopolysaccharide(LPS)to establish the inflamed cell model in vitro.ELISA was used to detect the cellular inflammatory factors IL-6 and TNF-?.The content of nitric oxide(NO)was quantitatively detected by a commercial kit,and the reactive oxygen species(ROS)content was qualitatively detected by fluorescence microscopy.The cellular uptake of the peptides was quantitatively and qualitatively examined separately by flow cytometry and confocal microscopy.The lysosomal escape of the peptides was determined by laser confocal microscopy.Adjuvant was used to establish inflamed animal model.randomly grouped mice were continuously administrated for 14 days with anti-inflammatory drug-dexamethasone,free peptide-QAW,TATQ,RMT and RMTQ.The indexes of body weight,paw volume,clinical arthritis score,serum concentration of IL-6 and TNF-?,and observation of ankle joint tissue sections were examined.The study demonstrated a comprehesive evaluation of the anti-inflammatory activity of multifunctional peptide in vitro and in vivo.Results: The MS and HPLC results showed that the synthesized peptides possessed expected molecular weight and high purity.The results of CD spectrashowed that except for the peptide QAW,the other three peptides were randomly conformated in aqueous solution at room temperature.Tricine-SDS-PAGE results indicated that the enzyme-sensitive peptide chain could be cleaved by type IV collagenase.In the cell studies,CCK-8 results showed that peptides were non-toxic to Raw264.7 cells when the concentration of each peptide was less than 30 ?M.Compared with other groups,RMTQ could inhibit the secretion of inflammatory factors IL-6 and TNF-? and reduce the content of NO and ROS in inflammatory cells more efficiently.In the cellular uptake study,the fluorescence intensity of the RMTQ was similar to the TATQ group at 1 h after administration.At 2 h and 4 h,the fluorescence intensity of the RMTQ group was slightly higher than that of the TATQ group,both of RMTQ and TATQ group showed much higher fluorescent intensity than the free anti-inflammatory peptide QAW.Confocal microscopy results were consistent with the flow cytometry results.In animal studies,compared with the free anti-inflammatory peptide QAW,targeting anti-inflammatory peptides can reduce the volume of the inflammatory paws,clinical scores,and levels of inflammatory factors in serum.Compared with the mice in the model group,each administration group can increase the body weight of the mice and reduce the content of inflammatory cells in the blood.Conclusion: The multifunctional anti-inflammatory peptides can increase the cellular uptake rate,increase the anti-inflammatory activity of QAW in vitro and in vivo.Therefore,the targeted peptides hold a great potential for clinical use.