Investigation Of The Effect And Mechanism Of Ferrostatin-1 On Abnormal Fluid Shear-induced Synovitis

Objective:(1)To detect the difference in the expression of ACSL4,GPX4 in the synovial fluid of patients with tmporalandibular joint internal derangement(TMJID)and Temporomandibular joint osteoarthritis(TMJOA).(2)To investigate the effects of Fluid flow shear stress(FFSS)intervention at different intensities on the morphology of Fibroblast-like synoviocytes(FLSs)as well as indicators related to inflammation,catabolism,and iron death.(3)To investigate the role of Ferrostatin-1(Fer-1)on abnormal FFSS-induced synovial inflammation and related mechanisms.Methods:(1)Joint synovial fluid of patients diagnosed with temporomandibular joint disorder(TMD)was collected and divided into TMJID group and TMJOA group according to medical history,clinical and imaging examination.The TMJOA group was divided into stage I,stage II and stage III.Baseline data were collected,synovial fluid was collected,and the expressions of ACSL4 and GPX4 in the joint synovial fluid of the two groups of patients were detected by ELISA.(2)FFSS intervention with 0dyn/cm~2,1dyn/cm~2,3dyn/cm~2,5dyn/cm~2,10dyn/cm~2of FLSs for 1 h.The changes of cell morphology in each group were observed under inverted microscope.The expression levels of intracellular ferrous ions and Reactive oxygen species(ROS)were detected in each group by different kits.The expression levels of IL-1β,IL-18,MMP3,MMP13,GPX4,ACSL4,Nrf2 and XC-were detected by Western blot.(3)cck-8 assay to detect the effect of Fer-1 on cell viability and determine the optimal intervention concentration.The effect of Fer-1 on intracellular divalent iron and reactive oxygen species in each group was examined by different kits.Western blot was performed to detect the effect of Fer-1 on the protein expression levels of IL-1β,IL-18,GPX4,ACSL4 and Nrf2 in each group.Results:(1)There was no statistically significant difference between the two groups in age,gender,ethnicity,pain,misalignment and restricted opening(P<0.05).There were statistically significant differences in the expression of ACSL4,GPX4 between the two groups.The expression of ACSL4 was lower(P<0.05)and the expression of GPX4 was relatively higher(P<0.05)in the TMJOA group compared with the TMJID group.(2)In terms of cell morphology,after1 h of FFSS intervention with different force values,compared with the control group(0dyn/cm~2),the number of cells was significantly reduced,the cavity gradually increased,the cytosol around the cavity was stretched,the nucleus was shifted,and some cells showed vacuoles and envelope rupture.The intracellular ferrous ion and ROS content increased step by step with the increase of force value,and the difference was statistically significant(P<0.05).The results of cellular immunofluorescence showed that the fluorescence intensity of GPX4 gradually decreased with the increase of force value,while that of ACSL4 gradually increased,and the difference was statistically significant(P<0.05).WB results showed that the protein expression of IL-1β,IL-18,MMP3,MMP13,ACSL4,and Nrf2 tended to increase with increasing force values(0,1,3,5,and10dyn/cm~2),while the protein expression of GPX4,Xc-tended to decrease and the differences were statistically significant(P<0.05).(3)The cck-8 results showed that different concentrations of Fer-1 had no effect on the cell viability of FLSs.No significant differences were observed in the number of cells,morphology,and the cavities that appeared after the addition of force in the Fer-1 group compared to the addition of force(5dyn/cm~2)under inverted microscopy,but the phenomenon of cell membrane rupture was reduced.The levels of intracellular ferrous ions and ROS were decreased in the Fer-1intervention group compared with the adjuvant group alone(5dyn/cm~2),and the difference was statistically significant(P<0.05).The cellular immunofluorescence results showed an increase in the fluorescence intensity of GPX4 and a decrease in the fluorescence intensity of ACSL4 in the Fer-1 group compared to the adjuvant group(5dyn/cm~2),and the difference was statistically significant(P<0.05).WB results showed that the expression of IL-1β(P<0.05),IL-18(P>0.05)and ACSL4(P<0.05)was reduced and the expression of GPX4(P<0.05)and Nrf2(P<0.05)was elevated in the Fer-1 intervention group relative to the adjuvant group(5dyn/cm~2).Conclusion:(1)This experiment was the first to detect the difference in the expression of iron death markers ACSL4 and GPX4 in the synovial fluid of TMD patients,and it was hypothesized that iron death might be associated with the progression of early TMD.(2)In this part of the experiment,FFSS with different force values were used to intervene on FLSs,and the many effects of mechanical factors on FLSs were identified by observing cell morphology and detecting inflammation,catabolism,and iron death-related indicators.(3)This experiment is the first to find that mechanical signals can cause changes in iron death-related indicators in FLSs.By investigating the role of Fer-1,the relationship between cellular iron death and abnormal FFSS-induced synovitis was indirectly confirmed,laying the foundation for more in-depth studies in the future.