| Part I:Construction of a mouse model of sepsis-induced acute kidney injury and construction of an LPS-induced cellular inflammation model and temporal expression of miR-21-5p and YAP1 in vitro and in vivoObjective: A mouse septic acute kidney injury model was constructed,and the success of the model was judged by molecular biology methods such as renal histopathology scoring,enzyme-linked immunosorbent assay(ELISA),and biochemical assays to detect serum inflammatory factors and serum creatinine urea nitrogen.Though the quantitative real-time fluorescence assay(q PCR),protein blotting(Western Blot,WB),and immunofluorescence to detect the expression of miR-21-5p and YAP1 in kidney tissues.A model of inflammatory damage in HK-2cells was constructed,and the success of the model was judged by detecting serum inflammatory factors.The expression levels of miR-21-5p and YAP1 in the cells were detected by q PCR,WB,and immunofluorescence.Methods:Animal experiment: 42 SPF-grade male C57BL/6 mice,the weight is 20-25 g,the age about 8 weeks,which were selected,and randomly divided into cecum ligation and perforation(CLP)group,Sham operation(Sham)group,,and the CLP group was further divided into different time point groups,namely,2 h,4 h,6 h,8 h,12 h and 24 h.The Sham group underwent only post-open abdominal closure,and the CLP group underwent cecum ligation and perforation.Blood was taken from the tip of the snack at a predetermined time for each group as well as removal of both kidneys.Cellular experiments: HK-2 cells were modeled using LPS,divided into ctrl and LPS groups,and LPS groups were divided into different time point groups,2h,4h,6h,8h,12 h and 24 h groups.Though ELISA to detect TNF-α molecular expression levels in plasma and culture medium supernatant,biochemical assays to measure creatinine and urea nitrogen in plasma,and histopathological scoring of the kidney.Though WB and q PCR to detect the expression of miR-21-5p and YAP1 in plasma and culture medium supernatants,and the entry of YAP1 into the nucleus was detected by immunofluorescence in kidney tissues and cells.Results: 1.Comparison of serum inflammatory factors and creatinine urea nitrogen in mice compared with the Sham group,TNF-α,Scr,Bun started to increase 6 h after CLP(P<0.01)and reached the highest level 12 h after surgery(P<0.0001).2.In the Sham group the renal pathology and pathological scores of renal tissue were no obvious pathological changes,and 6 h after CLP started to show changes in renal histopathology,mainly swelling of renal tubular epithelium,absence of brush border,vacuolar degeneration,and inflammatory infiltration.renal histopathology score started to increase in the mice in the 6h postoperative group of CLP(P<0.0001)with increasing time after CLP.3.In mice,compared with the Sham group,miR-21-5p increased with time in the CLP group after surgery in the 2h,4h,(P>0.05),decreased and reached the lowest level in the 12 h group(P<0.001),and slightly increased in the24 h group(P<0.001).YAP1 did not change significantly in the 2h,4h,6h,and 8h groups(P>0.05),increased and reached the highest peak in the 12 h group(P<0.0001),and slightly decreased in the 24 h group(P<0.0001).4,In the medium of HK-2 cells with LPS concentration of 200 μg/ml for 6 h compared to the ctrl group,the expression level of TNF-α in was significantly increased.(P<0.05).5.Compared to the ctrl group,in HK-2 cells,the expression level of miR-21-5p did not change significantly in the 2h and 4h groups(P>0.05).YAP1 expression did not change significantly in the 2h and 4h groups(P>0.05),YAP1 expression in the 6h group decreased and reached the lowest level(P<0.001),and increased in the 8h,12 h,and24h groups compared with the 6h group,but was still lower than that in the ctrl group(P<0.0001).,but still higher than the ctrl group(P<0.05).Conclusions: 1.cecum ligation perforation(CLP)can well replicate the model of septic acute kidney injury.2.miR-21-5p expression level decreased to the lowest level and YAP1 expression level increased to the highest level in kidney tissues of mice at 12 h and 24 h after CLP;the expression levels of the two molecules were not yet significantly different between 2h and 8h after CLP.3.HK-2 cells were The expression of miR-21-5p decreased to the lowest level and the expression of YAP1 increased to the highest level 6h after LPS treatment;between 6h and 24 h after LPS treatment,the expression of the two molecules still differed and showed opposite trends.It is speculated that the expression level of YAP1 correlates with the expression level of miR-21-5p.Part II: Occurrence of mitophagy in CLP-induced septic acute kidney injury in mice and in an LPS-induced inflammatory model of HK-2 cells.Objective: Detection of mitophagy in mouse S-AKI model and HK-2 cellular inflammation model.Methods: Animal experiment: 12 SPF-grade male C57BL/6 mice,whose age about 8weeks old,weighing 20-25 g were selected and randomly divided into cecum ligation perforation 12 h group(CLP 12h)and Sham-operated(Sham)group,the expression levels of mitophagy-related proteins TIM23,PINK1 and LC3 in mouse kidney tissues were detected by WB and by electron microscopy to detect the increase of vesicles.Cell experiment: HK-2 cells were divided into ctrl group and LPS 6h group,and to model HK-2 cells by LPS concentration of 200ug/ml,and though the WB to detect the expression levels of mitophagy-related proteins TIM23,TOM20,PINK1 and LC3 II,and the fusion of mitochondria and autophagosomes was detected by immunofluorescence.Results: 1.At the cellular level,compared with the ctrl group,the proteins PINK1 and LC3 which expression levels of mitophagy-related were increased(P<0.01,P<0.01)and the expression levels of TOM20 and TIM23 were decreased(P<0.0001,P<0.0001)in the cells of the 6h group;immunofluorescence showed an increase in fusion light;2.At the renal tissue level,compared with the Sham group,the CLP12 h group showed an increase in the expression levels of mitophagy-related proteins PINK1 and LC3 expression levels were increased(P<0.01,P<0.0001)and TIM23 expression levels were decreased(P<0.01);electron microscopy showed increased mitochondrial autophagic vesicles.Conclusion: Mitophagy occurred 6h after LPS treatment and 12 h after CLP treatment.Part III: miR-21-5p inhibits mitophagy of HK-2 cells by acting on YAP1,thereby aggravating the inflammatory damage of HK-2 cellsObjective: To detect the targeting effect of miR-21-5p and YAP1 in 293 T cells.The expression levels of miR-21-5p and YAP1 and the effects on mitophagy were examined in HK-2 cells by transfection with miR-21-5p mimics as well as the oeYAP1 plasmid.Methods: 1.HK-2 cells were transfected with miR-21-5p mimics,and detecting the occurrence of mitophagy in HK-2 cells.by WB and Qt-PCR to dectect the expression levels of mitophagy-related proteins(TIM23,TOM20,PINK1,LC3).;mitophagy vesicles were detected by immunofluorescence as well as electron microscopy.2. Targetscan,miRDB and miRWalk that are databases predict the downstream target genes of miR-21-5p.The intersection of the three databases was taken to identify downstream targets,and then YAP1 was finally selected as the downstream study target by enrichment analysis of the intersecting targets.Though the Double luciferase reporter gene assay to verify the binding of YAP1 m RNA 3’UTR and miR-21-5p.3.HK-2 cells were transfected with the plasmid of YAP1 overexpression and miR-21-5p mimics,and the mitophagy in HK-2 cells was detected.4.The expression level of TNF-α in the supernatant of HK-2 cell culture medium after transfection of miR-21-5p mimics and YAP1 plasmid was detected by ELISA.By WB and Qt-PCR to detect the proteins(TIM23,TOM20,PINK1,LC3),mitophagic vesicles were detected by immunofluorescence as well as electron microscopy.The expression level of TNF-αis detected by ELISA.Results: 1.Effect of overexpression of miR-21-5p on mitophagy-related proteins in HK-2 cells: compared to the ctrl group,miR-21-5p expression levels were reduced in the LPS group,LPS+miR-21-5p NC group(P<0.001).,compared with the LPS+miR-21-5p NC group,the expression level of miR-21-5p was increased in HK-2 cells in the LPS+miR-21-5p mimics group after transfection with miR-21-5p mimics(P<0.05),TIM23 and TOM20 protein expression levels of the LPS group,compared to the LPS+miR-21-5p NC group,the LPS+LPS+miR-21-5p NC group were decreased(P<0.001)and LC3 II,PINK1,and YAP1 protein expression levels were increased(P<0.05)in the LPS+miR-21-5p mimics group(P<0.05),and LC3 II,PINK1,and YAP1 expression was decreased(P<0.05).2.Effect of overexpression of miR-21-5p on YAP1 expression in HK-2 cells: YAP1 protein as well as m RNA expression levels were elevated in the LPS group,LPS+miR-21-5p NC compared to the ctrl group(P<0.05);LPS+miR-21-5p mimics group had reduced YAP1 protein as well as m RNA expression levels compared to the LPS+miR-21-5p NC group(P<0.05);3.YAP1 is a direct downstream target gene of miR-21-5p: though the dual luciferase reporter gene experiments that the WT plasmid+miR-21-5p mimics group had fluorescence ratio was significantly lower than that of the WT plasmid + miR-21-5p NC group(P<0.05);between the fluorescence ratio of the MUT plasmid + miR-21-5p mimics group and the fluorescence ratio of the MUT plasmid + miR-21-5p NC group,there was no significant difference(P>0.05);4.In HK-2 cells,miR-21-5p effect on mitophagy was achieved by targeting YAP1: the m RNA expression level of YAP1 was elevated in the LPS+miR-21-5p mimics+oe-YAP1 group compared with the LPS+miR-21-5p mimics group,(P<0.05),compared with the LPS+miR-21-5p mimics group,the expression of TIM23 and TOM20 protein levels were decreased(P<0.05)and the expression of PINK1,LC3 and YAP1 protein levels were increased(P<0.05)in the LPS+miR-21-5p mimics+oe-YAP1 group compared with the LPS+miR-21-5p mimics mimics+oe YAP1 group had increased mitochondrial autophagosome fusion yellow light.5.In HK-2 cells,overexpression of miR-21-5p promoted inflammatory damage in HK-2 cells: Compared with LPS+miR-21-5p NC group,the expression level of TNF-α in LPS+miR-21-5p mimics group was increased(P<0.05).6.In HK-2cells,overexpression of YAP1 can reduce the inflammatory damage of HK-2 cells:compared with LPS+miR-21-5p mimics group,the expression level of TNF-α in LPS+miR-21-5p mimics+oe-YAP1 group is reduced(P<0.0001)Conclusions: 1.YAP1 is the direct downstream target gene of miR-21-5p;2.Overexpression of miR-21-5p can inhibit mitophagy of HK-2 cells;3.The effect of miR-21-5p on mitophagy is achieved by inhibiting YAP1;4.Overexpression of miR-21-5p aggravates the inflammatory damage of HK-2 cells;5.Overexpression of YAP1 can reverse the inflammatory damage effect of overexpression miR-21-5p on HK-2 cells. |
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