Mechanism Of Eicosapentaenoic Acid In Inhibiting Mesenchymal Transformation And Fibrosis Of Renal Tubular Epithelial Cells

Part I Mechanism of eicosapentaenoic acid in inhibiting mesenchymal transformation and fibrosis of renal tubular epithelial cells in vitroBackgrounds: Epithelial to mesenchymal transformation(EMT)is an important process in the development of renal diseases.Transforming growth factorβ1(TGF-β1)is a kind of common fibrogenic cytokines.TGF-β1 /ILK and integrin-β1 /ILK signaling pathway plays an important role in the chronic inflammatory changes of interstitial and the accumulation of extracellular matrix in the process of renal fibrosis.PPARy can down regulate the activity of ILK and delay EMT.Several studies have confirmed that mi RNAs are involved in tubular level cell development,such as mir-4756,mi R-34 a and mir-210.Microphthalmia transcription factor(MITF)is the target of mir-541,and the MITF / mir-541 axis is a new regulatory pathway of cardiac hypertrophy.Mi RNAs,an important regulator of tumor metastasis and EMT,participate in the development of various tumors by regulating EMT related genes.Therefore,it is not clear whether mir-541 is involved in tubuloepithelial interstitial transformation and fibrosis.EPA can reduce proteinuria,reduce renal damage and delay renal progression.However,it is not clear whether the renal protective effect of EPA is mediated by TGF-β1 /ILK signaling pathway or integrin-β1 /ILK signaling pathway,and whether mir-541 is involved.Objective : In this study,HK-2 model was stimulated by human albumin,and the pathological process of proteinuria was simulated.Aim to analyze the effect of EPA on the expression of EMT,fibrosis and signal pathway related proteins,to evaluate the role of mi R-541 in this process,and to explore its potential mechanism.Based on this,this vitro experimental study will explore the mechanism of EPA’s inhibition of renal tubular epithelial cell mesenchymal transition and fibrosis in order to provide a theoretical basis for clinicaltreatment.Methods:In the logarithmic growth phase of HK-2,the cells were cultured for 24 hrs,and then placed in serum-free culture for 24 hrs.The cells were cultured in the GO phase.(1)5mg / ml albumin was added as the albumin group(model group);EPA10μmol / l + 5mg / ml albumin(low dose EPA intervention group),EPA10μmol / l + 5mg / ml albumin(high dose EPA intervention group),and a normal control group was set.The above groups were cultured for 24 hrs,48hrs,and 72 hrs,and their specific indexes were measured.(2)After transfection of mi R-541 inhibitor or mi R-nc inhibitor for 24 h,then cells were washed and then treated with or without 5 mg/ml Alb,with or without 30 μmol/l EPA for another 24 h,the changes of specific indexes were observed.Six repeat holes were set in each experimental group.(3)The effect of alb or EPA on the activity of HK-2 cells was determined by MTT method.(4)The m RNA expression of ILK,integrin β1,PPAR γ,mi R-541,a-SMA,E-cadherin,type I collagen and FN were detected by q PCR or RT-PCR.The levels of TGF-β 1,Smad2 / 3,p Smad7 and ILK,integrin β 1,PPAR γ,mi R-541,a-SMA,E-cadherin and type I collagen were measured by Western blot.Enzyme-linked immunosorbent assay(ELISA)was performed to detect FN expression.(5)The target relationshipbetween mi R-541 and TGF-β1 was confirmed by bioinformatics,luciferase reporter assay and western blot assay.Results:(1)Low doses of Alb had no effect on the cell viability of HK-2 cells,while EPA repressed the cell viability of HK-2 cells in a concentration-dependent manner.(2)HK-2 under the action of albumin,the expression of ILK m RNA and protein was up-regulated,while that of integrinβ1 and PPARγwas down regulated,and EPA inhibited the process to some extent,which was concentration and time-dependent.(3)EPA could inhibit EMT and fibrosis and increase the mi R-541 expression of HK-2 cells exposed to Alb.Interestingly,introduction of mi R-541 effectively abolished the EMT and fibrosis of HK-2 cells stimulated by Alb.(4)Bioinformatics analysis predicted TGF-β1 as a target gene of mi R-541,and subsequent luciferase reporter assay and western blot assay further supported the prediction.mi R-541 counter-regulated TGF-β1 expression,and inhibited TGF-β1/Smad3/ILK pathway.Alb treatment activated TGF-β1/Smad3/ILK pathway,while EPA inhibited the activation of the pathway.(5)mi R-541 inhibitors reversed the effects of EPA on EMT,fibrosis and TGF-β1/Smad3/ILK pathway-related proteins expression induced by Alb.Conclusions:EPA can reduce EMT and renal fibrosis by activating directly nuclear receptors through PPARγ mediated ILK/ integrinβl signaling pathway or by targeting mi R-541 through TGF-β1 / Smad3 / ILK pathway.Part II Mechanism of eicosapentaenoic acid in inhibiting mesenchymal transformation and fibrosis of renal tubular epithelial cells in vivoBackgrounds:Supplementing n-3 polyunsaturated fatty acids such as docosahexaenoic acid(DHA)and eicosapentaenoic acid(EPA)in chronic kidney disease may benefit patients,such as reduce proteinuria,reduce renal damage,delay renal progress,etc.However,no specific mechanism is still unclear.Objective:In this study,we intend to use EPA to intervene KK-Ay/Ta in type 2 diabetic nephropathy mice at the early stage,to explore the mechanism of EPA’s inhibition of renal tubular epithelial interstitial transformation and fibrosis in vivo by detecting the phenotypic changes of mice and analyzing the pathological changes of kidney.This study will provide a theoretical basis for EPA to treat chronic kidney disease,especially early diabetic nephropathy.Methods:In this experiment,in the body will be 20 KK-Ay/Ta male mice group is divided into two groups by random number table method in each group are 10 male mice,in which the treatment group from mice weeks since 12 th week to the EPA,EPA1g/kg/d each intraperitoneal injection,injection of eight weeks in a row,the treatment group in the 12 weeks intraperitoneal injection of physiological saline was given 1 g/kg/d,injection of eight weeks in a row,The control group consisted of 10 Balb/c A male mice.Phenotypic changes such as blood pressure,blood glucose,urine protein / creatinine ratio were detected at 12 weeks,16 weeks and 20 weeks,and serum fatty acid,triglycerides,cholesterol,advanced oxidation protein products(AOPP),malondialdehyde(MDA),insulin,glucose tolerance levels were detected at 20 weeks.The KK-Ay/Ta male mice were killed after 8 weeks of treatment,the renal tissue was taken out,and the renal tissue was observed and detected by morphology.The expression of TGF β 1,α-SMA,type I collagen,monocyte chemoattractant protein-1(MCP-1)and macrophage infiltration were detected by image analysis software and immunohistochemistry.At the same time,the m RNA expression of MCP-1,TGF β 1,α-SMA and type I collagen was detected by RT-PCR.Results:after EPA treatment on KK-Ay/Ta male mice,compared with the control group,the level of EPA in serum phospholipids increased significantly.The results of this study suggested that EPA injected intraperitoneal was fully absorbed by KK-Ay/Ta male mice.After EPA treatment,the AOPP and MDA level of KK-Ay/Ta male mice was significantly decreased,and the serum triglyceride of KK-Ay/Ta male mice was significantly decreased,which significantly reduced the excretion rate of urinary albumin,and significantly improved the glucose intolerance.Immunohistochemical test and morphological examination confirmed that EPA treatment on KK-Ay/Ta male mice could significantly reduce the accumulation of glomerular extracellular matrix,reduce the degree of EMT and tubulointerstitial fibrosis,and effectively weaken the expression of MCP-1 and macrophages infiltration in the kidney.Real-time PCR indicated that after EPA treatment on KK-Ay/Ta male mice,compared with the untreated group,the protein and expression levels of MCP-1,TGF-β 1,α-SMA and collagen I m RNA were all decreased.Conclusion:EPAcan effectively alleviate the accumulation of extracellular matrix,reduce EMT and renal interstitial fibrosis by inhibiting local inflammation of kidney,correcting abnormal metabolism in vivo,regulating TGF-β1 level and oxidative stress.