| Introduction:Diabetic nephropathy(DN)is a major complication and cause of death in type 1 diabetes(T1D)and type 2 diabetes(T2D),as well as a major cause of chronic kidney disease(CKD).With the progress of the disease,DN may eventually develop into end-stage renal disease(ESRD).DN has become a public health problem,which seriously endangers human health.Conventional treatment of DN does not completely halt or delay the development of ESRD.Therefore,it is extremely necessary to actively search for therapeutic targets for DN and develop new therapeutic strategies.The pathogenesis of DN is complex,and the pathways involved are also diverse.DN has been considered as a non-inflammatory disease caused by metabolic abnormalities in the past,but with the deepening of research,more and more evidence shows that inflammation is a key factor in its occurrence and development,and is also involved in cardiovascular complications and all-cause poor prognosis of diabetes.Circular RNAs(circ RNAs)are a new class of RNA molecules discovered in recent years,which are backspliced from exons or introns.They are characterized by covalent closed-loop structure and have become a research hotspot in the field of RNA.A large body of evidence suggests that circ RNAs are involved in the pathogenesis of kidney diseases,and an increasing number of regulatory roles of circ RNAs in DN have been reported.In a previous study,our research group found that silencing circ ACTR2(circ Base ID:hsa＿circ＿0008529)inhibited high glucose-induced human renal tubular epithelial cell line(HK-2)cells pyroptosis.However,its specific mechanism of action is still unclear and needs to be clarified in the next step.Macrophage migration inhibitory factor(MIF)is a pleiotropic cytokine that is widely expressed and involved in the occurrence of many immune and inflammatory diseases.It is a potential therapeutic target for many diseases.Recently,MIF has been found to mediate the activation of Nucleotide binding and oligomerization domain-like receptor family pyrin domain-containing protein 3 inflammasome.In DN,the role of NLRP3 inflammasome in pyroptosis has been extensively explored.Chicago sky blue 6B(CSB6B)is a small-molecule inhibitor of MIF.Recent studies have shown that CSB6B treats a variety of inflammatory reactive diseases,such as osteolytic bone disease,spinal cord injury,neuropathic inflammation,inflammatory pain,and Alzheimer’s disease,but whether it has a therapeutic effect on DN has not been studied.Whether inhibition of MIF exerts its renoprotective effect on DN by regulating NLRP3-mediated pyroptosis is currently unknown.The aim of this study is to investigate whether CSB6B can be used to treat diabetic nephropathy and improve renal injury and prognosis of DN by regulating pyroptosis.Methods:Part Ⅰ:At the cellular level,to determine whether pyroptosis and changes in the expression levels of hsa＿circ＿0008529,miR-451a and MIF occurred in human renal tubular epithelial HK-2 cells treated with high glucose.The relationship between hsa＿circ＿0008529 and miR-451a,miR-451a and MIF was verified by bioinformatics analysis,Quantitative Real-time PCR(q-PCR)experiment and dual luciferase gene reporter experiment.The downstream NLRP3 signaling pathway was detected by western blot.Lactate dehydrogenase release assay was used to evaluate the cell damage and death in each group.Immunofluorescence assay was used to detect the expression of GSDMD.The ratio of pyroptosis cells(FAM-YVAD-FMK~+/PI~+cells)in each group was analyzed by flow cytometry.The protein expressions of extracellular matrix Collagen I(Col I)and Fibronectin(FN)were detected by Western blot.In addition,the levels of MIF,IL-1βand TGF-β1 in cell supernatant were detected by enzyme-linked immunosorbent assay(ELISA).Part Ⅱ:To determine whether MIF affects diabetic intrinsic cell pyroptosis and inflammatory injury by mediating the activation of NLRP3 in human renal proximal tubular epithelial cells under high glucose environment.In this study,HK-2 cells were cultured with 5.5m M,30m M D-glucose and different concentrations of MIF inhibitor CSB6B.CCK-8 assay was used to detect the toxicity of CSB6B on the cells.The expression of MIF mRNA was detected by q-PCR.The effects of CSB6B on the expression of NLRP3/Caspase-1/GSDMD signaling pathway in HK-2 cells were detected by Western blot analysis of MIF,NLRP3,Caspase-1 p20 and pyroptosis marker GSDMD-NT.Western blot was used to detect the expression of fibrosis markers FN and Col I protein in the total protein of each group.ELISA kits were used to detect the expression of MIF,IL-1βand TGF-β1 in the cell culture supernatant of each group.Cell membrane integrity was assessed by measuring cell LDH release by lactate dehydrogenase(LDH)assay kit(Promega).Part Ⅲ:To further clarify whether CSB6B can be used as an effective treatment for DN,all experimental animals were acclimated to the SPF barrier environment for1 week.Eight-week-old male db/m mice were used as the normal control group.Eight-week-old male db/db mice with type 2 diabetic nephropathy were randomly divided into model group,low-dose CSB6B treatment group(db/db+CSB6B 2mg/kg)and high-dose CSB6B treatment group(db/db+CSB6B 8mg/kg).The body weight and blood glucose of mice were monitored,and serum and urine samples were collected to detect serum creatinine,urea nitrogen,urinary albumin/creatinine ratio and urinary NGAL.HE,PAS and Masson staining were used to analyze the pathological changes of the kidney in each group.The expressions of MIF and NLRP3 in renal tissues were analyzed by immunohistochemistry.The expression of NLRP3 and IL-1βmRNA in the renal cortex of mice in each group was detected by real-time quantitative PCR.The expressions of MIF,NLRP3,Caspase-1,GSDMD and IL-1βin the renal cortex of each group were detected by Western blot.The expression of Col I and FN in renal tissue was detected by Q-PCR and immunohistochemistry.Results:Part Ⅰ:High glucose induces the activation of NLRP3/Caspase-1/GSDMD pyroptosis pathway in human renal tubular epithelial HK-2 cells.The expression levels of hsa＿circ＿0008529 and MIF mRNA and protein in HK-2 cells in the high glucose group were significantly higher than those in the normal glucose group,and MIF secretion was significantly increased(P<0.05),while the expression level of miR-451a was significantly lower than that in the normal glucose group(P<0.05).Bioinformatics analysis,dual luciferase gene reporter assay and q PCR confirmed that there was a direct interaction between hsa＿circ＿0008529 and miR-451a,miR-451a and MIF.hsa＿circ＿0008529 promotes HG-induced pyroptosis and the production of inflammatory factors in HK-2 cells by regulating the NLRP3 inflammasome axis through miR-451a/MIF.In addition,hsa＿circ＿0008529 regulated extracellular matrix synthesis and TGF-β1 secretion of HK-2 cells under high glucose through miR-451a.Part Ⅱ:In this study,we found that CSB6B could effectively inhibit the secretion of inflammatory cytokines such as interleukin-1βand TGF-β1 by HK-2 cells stimulated by high glucose without affecting the viability of HK-2 cells.In addition,CSB6B effectively inhibited the expression and secretion of MIF in HK-2 cells stimulated by high glucose,down-regulated the expression of NLRP3,inhibited the activation of NLRP3 inflammasome,reduced the production of pyroptosis-related proteins,and significantly reduced the expression of extracellular matrix collagen I and FN in HK-2 cells induced by high glucose.Part Ⅲ:CSB6B treatment significantly reduced body weight,blood glucose,serum creatinine,urine ACR and NGAL,and improved renal function in db/db mice.In addition,CSB6B could significantly improve diabetic renal pathological damage,such as reducing mesangial matrix proliferation,reducing uneven thickening of glomerular basement membrane,and reducing podocyte injury.CSB6B can effectively reduce the protein expression of MIF,down-regulate the high expression of NLRP3,Caspase-1,GSDMD and IL-1βin the renal cortex of diabetic nephropathy mice,and reduce the transcription and translation of Col I and FN in the extracellular matrix.Conclusions:Part Ⅰ:hsa＿circ＿0008529 targets MIF to regulate NLRP3/Caspase-1/GSDMD axis through miR-451a,which promotes pyroptosis,inflammation and fibrosis in human renal tubular epithelial cells under high glucose.Our findings provide new insights into the development and progression of DN,and hsa＿circ＿0008529 may be a target to alleviate the progression of DN.Part Ⅱ:CSB6B,an inhibitor of MIF,effectively reduced the expression and secretion of MIF protein in HK-2 cells stimulated by high glucose,and significantly inhibited the expression of NLRP3.CSB6B inhibits pyroptosis by inhibiting the activation of the NLRP3/Caspase-1/GSDMD pyroptosis signaling pathway,reducing the secretion of cytokines such as IL-1βand TGF-β1,and reducing the accumulation of extracellular matrix Collagen I and FN.Part Ⅲ:CSB6B may inhibit the activation of NLRP3 inflammasome through MIF,thereby inhibiting the expression of NLRP3/Caspase-1/GSDMD/IL-1βsignaling pathway,inhibiting cell pyroptosis,reducing inflammatory response in renal tissue,and reducing extracellular matrix proliferation,thus protecting the kidney.CSB6B may be a potential drug for the treatment of diabetic nephropathy. |
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