Studies On The Role Of Ribosomal Protein S5 In The Treatment Of Hepatic Fibrosis With Luteolin

Cirrhosis which is the main liver disease can cause death in some case and the hepatic fibrosis plays an important role in the cirrhosis.The hepatic fibrosis is reversible thus the treatment of liver fibrosis becomes an important means for the prevention of liver cirrhosis.The main features of hepatic fibrosis are extracellular matrix multiplication and precipitation,which derive from the hepatic stellate cell activation and multiplication,and the epithelial mesenchymal transition that forms fibroblasts.Alpha smooth muscle actin??-SMA?and type I collagen are the major biological indexes for hepatic stellate cell activation and epithelial-mesenchymal transition.Both of their expression levels can be detected to verify the occurrence of hepatic fibrosis.As one kind of natural flavonoids,luteolin?Lut?can effectively reduce the levels of?-SMA and type I collagen mRNA in the liver tissue and subsequently inhibit the activation of hepatic stellate cells and the synthesis of collagen.This can effectively lessen the degree of hepatic fibrosis.Recent studies have shown that the ribosome protein S5?RPS5?can inhibit the activation of hepatic stellate cells and consequently involves in the treatment process of hepatic fibrosis by matrine and its derivatives.This result presumed that RPS5 is the potential target protein for the treatment of hepatic fibrosis.However,whether the action mechanism of luteolin theraping the hepatic fibrosis is also associated with RPS5 is unclear.The previous research had found that His₆-Tag-RPS5 expressed in vitro can combine with Lut,but the binding sites are so far unknown.This thesis mainly focuses on the studies of the role played by RPS5 in the therapy process of Lut for hepatic fibrosis.RPS5 and its mutants were expressed in vitro and the binding regions and the binding sites of RPS5 to Lut were explored,which can provide clues to the mechanism of Lut to treat the fibrosis in which the RPS5 participates from the perspective of molecular structure.The expression level of RPS5 mRNA in the hepatic stellate cells were interfered with RPS5 siRNA and the expression level of RPS5 was determined by means of RNA extraction,reverse transcription PCR and real-time fluorescent quantitative PCR?Real-time PCR?technologies.The?-SMA and type I collagen mRNA expression levels are significantly increased when the RPS5 expression level is suppressed,while this phenomenon can be conversely reversed by the addition of Lut.Thus,RPS5 is assumed to participate in the treatment progress of hepatic fibrosis with Lut.To research the binding sites of RPS5 to Lut,the recombinant plasmid pET-28a-RPS5was constructed by cloning cDNA of RPS5 and connecting it to pET-28a vector.The plasmid pET-28a-RPS5 was transformed into E.coli BL?DE3?to express RPS5 under optimizing expression conditions.The purity RPS5 was then separated and purified through SP and DEAE ion exchange columns and G-75 column.The potential binding regions and binding sites of RPS5 to Lut were analyzed by using molecular simulation software DS3.5 to simulate the combination of Lut and RPS5.RPS5 mutants were expressed and purified,and the binding constants of Lut with RPS5 and its mutants were measured by OCTET and Biacore technologies to conform the binding sites of RPS5 to Lut.The preliminary results show that the residues of Lys63,Arg81,Lys85,Lys193 and Arg145 in region I?MET76^LYS85 and ARG159?and region II?ARG60^GLN65 and ARG145?could probably be the binding sites.In conclusion,RPS5 was experimental confirmed involving in the treatment progress of hepatic fibrosis with Lut from the view of molecular biology.The purity RPS5 was expressed,separated and purified,and the binding regions and binding sites in the protein to Lut were verified by computer simulation,protein mutation,OCTET and Biacore technologies.It is significant for clarifying the mechanism of Lut to treat the hepatic fibrosis and designing the new drugs for anti-hepatic fibrosis.