The Study On The Effect And Its Mechanism Of Oridonin On Proliferation,migration,Invasion And ECM Of Human Keloid Fibroblasts

Objective To investigate the effect of Oridonin（ORI）on the proliferation,migrat-ion,invasion,and protein expression related to the formation and deposition ofextracellular matrix（ECM）of human keloid fibroblasts（HKF）.Then explore poss-ible mechanism of it preliminarily.Methods Cell culture In vitro including thawing,subculture,cryopreservation etc,was performed on human keloid fibroblasts purchased from biotechnology company.Third-seventeen generation cells in the logarithmic growth phase were studied in this experiment.HKF was set as the control group and HKF+ORI was set as the experimental group:（1）The CCK-8 proliferation experiment was performed to detect the effect of proliferation ability on HKF with ORI intervention at different concentrations（10.0,12.5,15.0,17.5,20.0μM）at different action duration（24,48,72h）,and the half inhibitory concentration（IC50）of ORI acting on HKF for 24h,48,72h was measured,then the appropriate drug action concentration was screened for subsequent experiments.（2）Cell wounding healing assay could detect the effect of ORI on migration ability of HKF.（3）Cell Transwell invasion assay was performed to detect the effect of ORI on invasion ability of HKF.（4）Real-time quantitative reverse transcription polymerase chain reaction（RT-PCR）and western blot assay was conducted to explore the effect of ORI on the expressions level of HKF′s ECM-related proteins such as fibronectin 1（FN1）,α-smooth muscle actin（α-smooth muscle actin,α-SMA）,Collagen type I（COLI）,collagen type III COL III)and etc..In addition,the experiment was divided into control group（HKF）,model group（HKF+TGF-β1）,and experimental group（HKF+TGF-β1+ORI）.RT-PCR and Western blot assay was conducted to explore the effect of ORI on the expressions level of nucleotide-binding oligomerization domain（NOD）-like receptors Protein-3（NLRP3）,containing cysta Apoptosis-associated speck-like protein containing a C-terminal caspase-recruitment domain（ASC）,Smad2,Smad3 and their phosphorylation（p-Smad2,p-Smad3）in TGF-β1-induced HKF.Results（1）The results of CCK-8 showed that compared with the control group,the ORI experimental group had an inhibitory effect on the proliferation of HKF,and with the increase of drug concentration,the inhibitory effect became more and more significant,and it was dose-dependent（P<0.05 or P<0.01）.There was no significant difference in the inhibitory effect on the proliferation of HKF among different action duration（24,48,72h）with same concentration（P>0.05）.The IC₅₀of the drug action,measured for 24,48,72h,was 15.64,15.65,15.70μM.（2）Compared with the control group,the 24h migration ability of cells in the experimental group was significantly reduced,and the difference was statistically significant（P<0.05）.（3）Compared with the control group,the 24h invasion ability of cells in the experimental group was significantly reduced,and the difference was statistically significant（P<0.05）.（4）Compared with the control group,the expression levels of FN1,α-SMA,COL I.and COLIII in the experimental group were significantly downregulated（P<0.05）.（5）Compared with the control group,the model group′s expression levels of NLRP3,ASC,Smad2,Smad3 and p-Smad2/3（tested by WB）were significantly increased,and the difference was statistically significant（P<0.05）.Compared with model group,the experiment group′s expression levels of NLRP3,ASC,Smad2,Smad3 and p-Smad2/3（tested by WB）were significantly reduced,and the difference was statistically significant（P<0.05）.Conclusion ORI inhibited the proliferation,migration,invasion capacity and ECM formation or deposition of HKF.ORI reduces HKF formation of ECM by inhibiting the TGF-β1/Smad signaling pathway.The effect of ORI on HKF may be achieved by inhibiting the action of NLRP3.