| Objective:Hepatic ischemia/reperfusion injury(IRI),one of the most common complications in liver transplantation,is an almost unavoidable liver injury process,which can seriously affect the prognosis of patients by initiating a series of molecular events such as an inflammatory cascade reaction.However,there is still an enormous lack of effective clinical treatment for hepatic IRI up to now.Therefore,it is urgent to search for potential therapeutic targets in hepatic IRI.Micro RNA(mi R)-450b-5p is a non-coding micro RNA that has emerged recently in solid organ immunity.Although mi R-450b-5p was significantly up-regulated in liver IRI model in line with our preliminary experiment,there is little relevant studies on mi R-450b-5p in liver IRI.Furthermore,through bioinformatics methods,we found that CRYAB,an anti-inflammatory protein widely presented in human body,might be a predicted target of mi R-450b-5p.In particular,our previous experiments showed that CRYAB was significantly down-regulated in liver IRI model and negatively correlated with mi R-450b-5p.Based on this,we speculated that mi R-450b-5p might promote the occurrence of hepatic IRI by targeting CRYAB.The purpose of this study was to detect the expression of mi R-450b-5p and CRYAB in animal and cell models with hepatic IRI.By further clarifying the targeting effect of mi R-450b-5p on CRYAB,the immune regulatory functions of mi R-450b-5p and CRYAB in liver IRI animal model and cell H/R model were verified.Finally,the regulatory mechanism of mi R-450b-5p and CRYAB was explored in the cell model,which might help to provide some potential research basis and transformation ideas for the treatment of hepatic IRI.Method:1.The RAW 264.7 cell line was used as the classical surrogate cell of Kupffer cells,and the classic cell model of liver IRI was established through tri-gas chambers,namely the H/R model of cell hypoxia/reoxygenation.Hypoxia/reoxygenation time gradients were set,and q RT-PCR was applied for determination of mi R-450b-5p,CRYAB and TNF-α levels.Western Blot was applied for determination of CRYAB,IκBα,p-IκBα,NF-κB p65,p-p65,NIK and NF-κB p52 levels.Results from both q RT-PCR and Western Blot were taken into consideration to screen the optimal combination of hypoxia/reoxygenation time points.2.The protein expression of CRYAB,IκBα,p-IκBα,p65,p-p65,NIK and NF-κB p52 were detected by Western Blot.The nuclear translocation of p65 was detected by immunofluorescence.The levels of inflammatory cytokines including representative TNF-α,IL-1β and IL-6 in cell supernatant were determined by ELISA.The targeting effect of mi R-450b-5p on CRYAB was determined by the dual luciferase reporter gene assay.3.Hepatic IRI animal model was established using C57BL/6J mice.The levels of inflammatory factors in mouse serum were detected by ELISA,whilst mi R-450b-5p expression in hepatic IRI were further monitored.Both of CRYAB and mi R-450b-5p were interfered in vivo,and the pathological changes of mouse liver were observed by HE staining(according to Suzuki score).TUNEL was used to detect the apoptosis of mouse hepatic cells.ELISA was used to detect the release of inflammatory cytokines in serum of mice after interference.Serum AST and ALT levels were assessed using liver enzyme detection kits.4.The specific IKK pathway inhibitor,IMD 0354,was used in vitro,and the expression of CRYAB was interfered.The activation of both IKKαand IKKβ subunits were observed by IF.The protein expression of CRYAB,IκBα,p-IκBα,IKKα,p-IKKα,NIK,IKK,p-IKK,p65,p-p65,NF-κB p52,IKKβ,p-IKKβ,and IL-1β were detected by WB.Same as above,to detect the levels of inflammatory factors in cell supernatant,ELISA was performed.5.IMD 0354 was used in vivo and the expression of CRYAB was interfered.Liver pathological changes of mice were observed by HE staining.TUNEL was used to evaluate the apoptosis of liver cells in mice.Serum inflammatory factors were detected by ELISA.Serum ALT and AST levels were detected by liver enzyme detection kits.6.The expression of mi R-450b-5p and CRYAB was interfered in vitro,and the protein expression of M2-type polarization marker including Arg-1and TGFβ1,within pathway protein comprising AKT1,activation form as p-AKT1,m TOR,activation form as p-m TOR,downstream S6K/4E-BP pathway protein in macrophages was detected by WB.m RNA levels of M2 markers were detected by q RT-PCR.The activation of M2 marker protein was detected by immunofluorescence.7.The expression of Arg-1,TGFβ1,AKT1,p-AKT1,m TOR,p-m TOR,S6 K,P-S6 K,4E-BP and p-4E-BP was detected by Western Blot after in vitro useage of m TOR activator,MHY 1485,within simultaneous interference of CRYAB.m RNA levels of M2 markers were detected by q RT-PCR.The activation of M2 related protein was detected by immunofluorescence.8.CRYAB was interfered in vivo,while IMD 0354 and MHY 1485 were used at the same time.Same as above,to determine the pathological alterations in mouse liver in hepatic IRI,HE staining was applied.Meanwhile,to evaluate the apoptosis of mouse liver cells in hepatic IRI,TUNEL was applied.Serum liver enzyme levels of mice were detected by liver enzyme kit.Results:1.mi R-450b-5p,IκBα/NF-κB p65 and NIK/NF-κB p52 as inflammatory signaling pathway protein were significantly up-regulated in the cell H/R model,while CRYAB was significantly down-regulated.Combining q RT-PCR and WB results,we finally decided to choose 6/12 as the ideal H/R time for subsequent cell experiments.2.Inhibition of mi R-450b-5p in the cell H/R model could increase the expression of CRYAB and inhibit the activation of IκBα/NF-κB p65 and IKKβ,while further inhibition of CRYAB significantly reversed the effects of the mi R-450b-5p inhibitor.mi R-450b-5p/CRYAB did not significantly affect the activation of NIK/NF-κB p52 or IKKα.3.mi R-450b-5p was significantly upregulated in mouse IRI model,and inhibition of mi R-450b-5p in vivo significantly alleviated the liver pathological injury,apoptosis,release of inflammatory factors and liver enzymes in mice.Further inhibition of CRYAB significantly reversed the effect of mi R-450b-5p in vivo.4.The use of the IKKβ inhibitor,IMD 0354,in a cell H/R model significantly reversed the effects of CRYAB deficiency on IκBα/NF-κB p65 and IKKβ inflammatory signaling.The application of IMD 0354 in mouse hepatic IRI model significantly reversed the effects of CRYAB down-regulation on liver pathological injury,apoptosis,serum inflammatory factors and liver enzyme release in mice.5.In the cell H/R model,inhibition of mi R-450b-5p significantly enhanced AKT1/m TOR signaling protein and M2 polarization marker,and further inhibition of CRYAB could reverse the effects derived from mi R-450b-5p inhibitor.6.The use of m TOR agonist,MHY 1485,in cell H/R model significantly reversed the effects of CRYAB shortage on M2 polarization markers and AKT1/m TOR signaling protein.7.Simultaneous use of IKKβ inhibitor IMD 0354 and m TOR agonist MHY 1485 in mouse IRI model significantly reversed the effects of CRYAB deficiency on liver pathological injury,apoptosis,and serum liver enzymes in mice.Conclusion:1.mi R-450b-5p was significantly up-regulated in hepatic IRI mouse model and cell H/R model,while CRYAB was significantly down-regulated.2.mi R-450b-5p can enhance the occurrence of hepatic IRI by directly targeting CRYAB.3.Regulation of hepatic IRI by mi R-450B-5p/CRYAB axis is realized through controlling IKK/NF-κB p65 and AKT/m TOR signaling. |
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