Effect Of Ulinastatin On The Expression Of MiR-21 In LPS-induced Mouse Macrophages And Its Preliminary Mechanism

Objective: To investigate the protective effect of ulinastatin on lipopolysaccharide-induced RAW264.7 cells and its effect on miR-21 expression.Methods: The expression of miR-21 and tumor necrosis factor-?(TNF-?)mRNA,interleukin-6(IL-6)mRNA were detected by RT-PCR at different concentrations(100,250,500 ng / mL)mRNA;RAW264.7 cells were stimulated with 500 ng / mL LPS for 6h,12 h,24h,and TNF-? mRNA was detected by RT-PCR;The effects of different concentrations(10,100,1000 U / mL)UTI on the activity of RAW264.7 cells were detected by MTT assay.Set blank group,model group(LPS 500ng/mL)?LPS +UTI(1000 U / mL)group?UTI 1000 U / mL group,and observe the RAW264.7 cell growth status.The expression of tumor necrosis factor TNF-? mRNA,IL-6 mRNA and miR-21 were detected by RT-PCR after pretreatment with different concentrations(10,100,1000 U / mL)for 2 h,then stimulated mouse RAW264.7 cells with LPS for 12 h;The expression of PTEN in UTI(1000 U / mL)was detected by Western Blot.Set blank group,model group(LPS 500ng/mL)?LPS +mi R-21 mimic group?LPS +miR-21 inhibitor group:The expression of mi R-21 was detected by RT-PCR.The expression of PTEN was detected by Western Blot.Results: 1.UTI protective effect on cells: 1 UTI on the RAW264.7 cells toxicity: UTI concentration of less than 1000 U / mL when RAW264.7 cells no toxic effects,the group OD value of the difference was not statistically significant(P = 0.117)2 UTI could effectively decrease the expression of TNF-? mRNA and IL-6 mRNA at different concentrations(10,100,1000 U / mL)(P1 <0.05,P2<0.05).2.The expression of miR-21 in different inflammatory response: compared with the normal group,the secretion of TNF-?mRNA and IL-6 mRNA was significantly increased compared with the basal secretion(P < 0.05)after LPS(100,250,500 ng / mL)stimulated RAW264.7 cells,and miR-21 expression was significantly increased,and increased with the LPS concentration increased(P <0.05).3.Effects of UTI on mi R-21 expression:UTI could decrease the expression of miR-21 at different concentrations(10,100,1000 U / mL),the difference was statistically significant(P = 0.000).4.Effects of miR-21 on PTEN protein: Compared with the blank group,the expression of miR-21 was up-regulated and the expression of PTEN protein was down-regulated in LPS group.The expression of miR-21 was significantly decreased and the expression of PTEN protein was significantly increased in the miR-21 inhibitor group(P <0.05).The expression of miR-21 was significantly increased in the miR-21 simulant group and the expression of PTEN protein was significantly decreased(P <0.05).5.Effects of UTI on the expression of PTEN protein: The effect of UTI on miR-21 target gene PTEN protein was further observed based on the experimental results of miR-21 expression level.Compared with the blank group,the expression level of PTEN protein in LPS group was significantly decreased,and the expression level of PTEN protein was significantly increased in UTI group(P <0.05).Conclusion:1.UTI can reduce the expression of TNF-? and IL-6 mRNA in LPS-induced RAW264.7 cells.2.LPS could induce the high expression of miR-21 in RAW264.7 cells in a concentration-dependent manner.The high expression of miR-21 could inhibit the expression of PTEN protein.3.UTI significantly down-regulated the expression of mi R-21 and up-regulated the expression of target gene PTEN protein.Therefore,UTI can inhibit the inflammatory response of LPS-induced RAW264.7 cells,which plays a protective role on the cells.The mechanism may be related to the down-regulation of miR-21 expression induced by UTI.