Application And Mechanism Of Drug Repositioning In Osteoarthritis Vitro Model

Several chronic inflammatory diseases have significant overlap at the molecular level.However,the phenotype in different cells and tissues suggests some individual pathological features contribute to these molecular mechanisms.Based on this theory,"Drug Repositioning" has recently been developed.Drug Repositioning is used to apply a large number of known drugs which treat other diseases,to both identify new treatments and advance our understanding of the molecular basis within chronic inflammatory diseases,particularly at the molecular/ biochemical level.Thus,Drug Repositioning in concept may greatly shorten the time for drug safety testing and reduce research costs,bringing better patient outcomes.Osteoarthritis(OA)is one of the most common degenerative diseases.Although there has been many studies reported,there is a key limitation in almost all therapies for OA: drug therapy is only targeted to the inflammatory response caused by OA,but not to regenerate the cartilage tissue.To integrate the above issues,our research screened 80 kinds of chemicals with research background,to develop novel drugs for OA treatment.OBJECTIVE: To combine research of the molecular basis with OA biomarkers selected as targets,and use this to identify potential mechanisms of applying Drug Repositioning.METHODS: 80 drugs were selected based on a literature review.Then cytotoxic drugs were screened out.10 n M Interleukin-1 ?(IL-1?)was used in combination with Tumor Necrosis Factor-?(TNF-?)to establish a model for the OA-like cell damage;morphological changes of human chondrosarcoma cells OUMS27 within 24 hours after adding drugs were observed by light microscopy.Moreover,key proteins in OA was detected by Western blotting at 6,12,24,48,and 72 hours after modeling,and the most obvious changes was selected,used as biomarkers;q RT-PCR was used todetect the effect of previously selected drugs on target protein expression;Western blotting was used to detect the activation/ phosphorylation levels of three classical pathways;different concentrations of phosphorylation inhibitors were used to detect target m RNA levels.The regulation followed by the use of large doses of upstream and downstream inhibitors in the primary pathway further confirmed the details of pathways that regulate the target protein.RESULTS: 1.Observed by light microscope,there were 5 levels of cell condition for the groups of the candidate drugs;from the fullness of the cell morphology with the close connection to died cells in a spherical shape: the state deteriorated in turn.The OA group stimulated by IL-1? and TNF-? was in grade III,and to ensure the safety of the drug in determined dose,the cell condition was screened in the non-grade I drug group within 24 hours;17 drugs remained after this screening.MTT cell activity assay to determine whether 17 drugs can improve the in vitro model of OA established by IL-1? and TNF-?.Among them,four drugs,represented by ?-glucosylrudin,significantly enhanced cell viability(P< 0.001).2.The expression of key proteins at different time points in the OA model was detected by Western blot.Aggrecan,fragmented Aggrecan(Nitege)and ADAMTS 4 accordingly have a time-dependent trend,but the change is not obvious;the expression of ADAMTS 5 has no change at OA group;Hyaluronan-Binding Protein Involved Expression in Hyaluronan Depolymerization(HYBID)peaked at 12 hours.q RT-PCR analysis of its m RNA further verified that HYBID expression was 30-fold higher than the control group at 12 hours(P < 0.001),HYBID was selected as the target protein for subsequent experiments and detected at 12 hours after model establishment.3.After 6 hours stimulation of OUMS27 cells with IL-1? and TNF-?,four selected drugs were added to OA group for 6 hours.After total 12 hours,HYBID m RNA level was detected by q RT-PCR.Except for indomethacin,?-glucosylrutin,praziquantel,and aspirin all had significant negative effects on HYBID expression(P<0.001).4.The three classical signal pathways AKT,ERK,and p38 can be triggered by inflammatory signals and activated in the short term.AKT,ERK,and p38 all have affect on HYBID expression by using 5 ?M and 50 ?M inhibitors,respectively.50?M of ERK inhibitor had a significant inhibitory effect on the m RNA expression of HYBID(P<0.001),and further investigations on the upstream and downstream of ERK revealed that JAK and p90,which are signaling proteins on the ERK pathway,were activated on OA.Inhibiting their activity can also reduce the expression of HYBID.Thus,JAK/ERK/p90 might be the dominant signaling pathway regulating HYBID(P<0.001).CONCLUSION: The theory of Drug Repositioning has a wide range of application in many diseases.This study found that 1 ?M ?-glucosylrudin may inhibit the expression of the key protein HYBID through JAK/ ERK/ p90 signaling pathway,which caused beneficial effects in the OA vitro model.Further study may shed more detailed mechanisms which may benefit patient outcome if used to develop new drug targets and could lead to a treatment which promotes tissue regeneration as well as anti-inflammatory effects.