| Rheumatoid arthritis(RA)is an autoimmune disease characterized by synovial inflammation.At present,nearly 1% of the global population is suffered from RA,which greatly affects people’s quality of life.Traditional drugs for the treatment of RA mainly include non-steroidal anti-inflammatory drugs,glucocorticoids,and diseaseimproving anti-rheumatic drugs.Although these drugs can alleviate the disease process of RA to a certain extent,they could not fundamentally achieve the RA treatment.In addition,these drugs are likely to cause serious side effects such as gastrointestinal diseases,cardiovascular diseases and infections.Therefore,it is urgent to develop new treatment strategies to achieve the fundamental treatment of RA.During the occurrence and development of RA,the abnormal expression of genes breaks the body’s immune dynamic balance,thereby intensifying local tissue inflammation and bone and joint destruction.Therefore,using gene therapy strategies to regulate gene expression and achieve RA treatment is expected to become a new direction for future research.In therapeutic genes,micro RNA plays an important role in regulating gene expression.It combines with the untranslated region(UTR)of m RNA to degrade m RNA to achieve the purpose of inhibiting protein translation.Previous studies have shown that mi R-30 a is under-expressed in autoimmune diseases such as RA.Therefore,in-depth exploration of the key role of mi R-30 a in the occurrence and development of RA will provide an important theoretical basis for mi RNA-based RA gene therapy.At the same time,the in vivo application of mi RNA needs to overcome multiple biological barriers,such as the degradation of RNase in the blood,the activation of the immune system,barriers across cell membranes,and lysosome escape.Therefore,the design of a suitable gene delivery vector will enable the efficient and stable delivery of mi RNA and effectively exert its biological functions in vivo.Based on the above considerations,this research system explored the mechanism of mi R-30 a in the occurrence and development of RA,and constructed an efficient and safe delivery system-fluorinated modified PAMAM(FP)to achieve the in vivo delivery of mi R-30 a,and evaluate the above system The therapeutic effect and mechanism of RA.The main research contents and results of this paper are as follows:1.Through GEO Data Set database analysis,the expression level of mi R-30 a in RA patients and their disease models is significantly reduced.Taking RAW264.7macrophages as a model and Lipofectamine2000 liposomes as a carrier to mediate mi R-30 a delivery,it can effectively inhibit the expression of inflammatory factors such as IL-6,IL-1β and TNF-α.The target genes of mi R-30 a were screened through the three databases of Target Scan,mi RDB and mi RWalk,and six inflammation-related targets were obtained.Molecular biology experiment analysis showed that mi R-30 a can significantly down-regulate the expression levels of Snail and Prlr genes.The expression of Snail in the synovial tissue of rheumatoid arthritis mice is significantly increased,which is closely related to the occurrence and development of RA.Therefore,this study chose Snail as the key target of mi R-30 a to regulate the development of the disease.STRING analysis showed that Snail interacts directly with Cadherin-11 to regulate inflammatory signal-related pathways.Up-regulating the level of mi R-30 a can significantly reduce the expression level of Snail,inhibit the expression of Cadherin-11,the key downstream protein,and significantly reduce p65,p65 and Cadherin-11.The phosphorylation of p-ERK1/2 and p-JNK realizes the inhibition of NF-κB and MAPK signal pathways,and finally exerts anti-inflammatory biological functions,which provides a theoretical basis for mi R-30 a as a RA gene therapy drug.2.We use PAMAM as the parent and heptafluorobutyric anhydride as the fluorination reagent,the fluorinated modified dendrimer FP was constructed to mediate the efficient and stable delivery of mi R-30 a.The nanocomposite formed by the two can effectively protect mi R-30 a from nuclease degradation,promote mi R-30 a to enter the cell and realize the escape of endosomes,and exert its biological effects.After the FP/mi R-30 a nanocomposites enter the cells,it can significantly reduce the expression of Snail and Cadherin-11,thereby inhibiting the expression of inflammatory factors such as IL-6,IL-1β and TNF-α,and has a good ability to inhibit inflammation in vitro.Using DBA/1J mice to construct a collagen-induced rheumatoid arthritis(CIA)mouse model,the nanocomposite was injected through the tail vein,after 7 consecutive administrations,the results showed that the FP/mi R-30 a nanocomposite can Effectively inhibit the pathogenesis of mice with rheumatoid arthritis,reduce the swelling of the feet,and restore the exercise ability of the mice.Through micro-CT analysis of the integrity of the bone tissue and bone parameters of the mouse’s hind limb paw,FP/mi R-30 a can protect the integrity of the cortical bone and cancellous bone of the mouse paw.The analysis of H&E staining and Safranin Fast Green staining of the ankle joint showed that FP/mi R-30 a has a good protective effect on cartilage,and at the same time,the inflammatory cell infiltration and synovial hyperplasia of joint synovial tissue are significantly reduced.The immunohistochemical analysis of the synovial tissues of the knee and ankle joints showed that FP/mi R-30 a effectively reduced the expression of Snail and Cadherin-11,and inhibited the inflammatory factors(IL-6,IL-1β and TNF-α)in the joints and serum Secretion to achieve inflammation suppression.In addition,compared with the free mi R-30 a,the FP/mi R-30 a nanocomposite can accumulate in the joints of rheumatoid arthritis mice through ELVIS(Extravasation through leaky vasculature and subsequent inflammatory cell-mediated sequestration)effects,and improve joints.Inflammation treatment effect,at the same time has good biological safety.The above studies show that FP can effectively mediate the efficient and stable delivery of mi R-30 a,inhibit the expression of Snail and Cad-11 proteins,regulate the inflammatory factor network,and achieve the treatment of rheumatoid arthritis.In summary,this study provides a new idea to reveal the key role mi R-30 a in the development of rheumatoid arthritis.Based on this,the stability of mi R-30 a can be achieved based on the fluorinated modified polymer carrier.Delivery,obtained good therapeutic effects of RA,and provided important theoretical support and technical guidance for the development of rheumatoid arthritis mechanism and related gene therapy strategies. |
PDF Full Text Request