Role And Molecular Mechanism Of BCL6 In Hypertensive Renal Inflammation And Vascular Remodeling

Proto-oncogene B-cell lymphoma 6(BCL6)is a sequence-specific transcriptional repressor with a structure consisting of an N-terminal hydrophobic BTB/POZ region and a C-terminal six kruppel-type zinc-finger motifs.BCL6 inhibits transcription by specific binding to the DNA sequence in the promoter region of the target gene.Target genes regulated by BCL6 are mainly related to cell activation,differentiation,apoptosis,and cell cycle arrest.BCL6 plays a broader role in macrophage quiescence and the termination of the inflammatory response.BCL6 recruits con-repressor SMRT/NCoR cistrome to repress inflammation and attenuate atherosclerosis.The thesis is divided into two parts.(1)Effects of BCL6 on renal inflammation in spontaneously hypertensive rats.(2)Effects of BCL6 on vascular remodeling in hypertension.Part 1 BCL6 attenuates renal inflammation via negative regulation of NLRP3 transcription1.BackgroundRenal inflammation has an important role in the development of hypertension and may be a target for attenuating hypertension.Multiple models of inflammatory diseases showed activation of NACHT,LRR and PYD domains-containing protein 3(NLRP3)inflammasome,however,studies on NLRP3 inflammasome in renal injury associated with hypertension are very limited.The transcriptional repressor BCL6 negatively regulates NF-?B expression and thereby inhibits NF-KB-mediated inflammation.BCL6 is considered as a therapeutic target for autoimmune diseases and cancer treatment.The role of BCL6 in hypertensive renal inflammation remains unclear.The present study was designed to determine the effect of BCL6 on hypertensive renal inflammation and its molecular mechanism.2.ObjectiveThe study was to designed to investigate the role and molecular mechanism of BCL6 on NLRP3 inflammasome activation and explore the therapeutic effect of BCL6 overexpression on renal inflammation in spontaneously hypertensive rats.3.MethodsHK-2 cells were cultured in Dulbecco's Modified Eagle Media:Nutrient Mixture F-12(DMEM/F12)with 10%FBS,penicillin(100 units/ml)and streptomycin(100 mg/ml)at 37°C in a humidified atmosphere of 95%air and 5%C02.The HEK293 cells were maintained in DMEM containing 10%FBS,with penicillin and streptomycin.Male spontaneously hypertensive rats(SHR)and WKY aged 13 weeks were randomly divided into two groups.BCL6 overexpression lentivirus vector and enhanced red fluorescent protein(ERFP)lentivirus vector were performed through intraparenchymal injection.After 2 weeks,intravenous injection of recombinant lentivirus-expressing BCL6 or ERFP was carried out via tail vein to ensure the adequate upregulation of BCL6 in kidneys.2 weeks later,blood pressure of tail artery was measured in conscious state using a noninvasive computerized tail-cuff system.Acute experiments were carried out.Serum blood urea nitrogen and creatinine concentrations were examined with the Urea Assay Kits and Creatinine Assay Kit.BCL6,NLRP3,apoptosis-associated speck-like protein containing a caspase recruitment domain(ASC),pro-cysteinyl aspartate specific proteinase 1(pro-caspase-1),neutropil gelatinase-associated lipocalin(NGAL),Kidney injury molecule-1(kim-1),Cystatin C,Interleukin-18(IL-18),Interleukin-1?(IL-1?),glyceraldehyde phosphate dehydrogenase(GAPDH)were measured by Western blot.Expressions of BCL6 and NLRP3 were also measured by Immunohistochemistry.Real-time quantitative PCR were used to dected the mRNA levels of BCL6,NLRP3,IL-1?,Interleukin-6(IL-6),tumor necrosis factor-?(TNF-a),chemokine CCL2 and CXCL2.Double luciferase reporter gene was used to detect the binding activity of BCL6 and NLRP3 promoter.The electrophoretic mobility shift assay(EMSA)and Chromatin Immunoprecipitation(ChIP)were performed to verify the binding sites of BCL6 binding in the NLRP3 promoter.4.Results(1)BCL6 mRNA and protein levels were downregulated,while NLRP3 inflammasome was activated in the kidneys of SHR compared with those of WKY.(2)Ang ? or LPS reduced BCL6 expression in HK-2 cells.Overexpression of BCL6 not only attenuated Ang II-induced up-regulation of NLRP3 expression,inflammation,and cell injury,but also attenuated LPS-induced upregulation of NLRP3 and inflammation in HK-2 cells.(3)Double luciferase reporter assays showed that BCL6 inhibited NLRP3 transcription by binding to the NLRP3 promoter.EMSA showed BCL6 binding sites at +283/+296 in the promoter region and the result was consistent with ChIP results.(4)The BCL6 knockdown increased the NLRP3 expression at both protein and mRNA levels in PBS-or Ang II-treated HK-2 cells but had no significant effects on ASC,pro-caspase-1 and pro-IL-1? protein expression levels.Furthermore,mature IL-1? protein was upregulated by the BCL6 knockdown in both PBS and Ang?-treated HK-2 cells.(5)The BCL6 overexpression caused a persistent reduction in SBP and MAP in SHR,prevented the upregulation of NLRP3 and mature IL-1? expression levels but had no significant effects on the upregulation of ASC,pro-caspase-1 and pro-IL-1?expression levels in the renal cortex of WKY and SHR.5.ConclusionBCL6 attenuates Ang ? or LPS-induced inflammation in HK-2 cells by inhibiting NLRP3 transcription.BCL6 was down-regulated in SHR rats.Overexpression of BCL6 in SHR rats inhibited NLRP3 expression and inflammation in the renal cortex.Part 2 BCL6 attenuates proliferation and oxidative stress of vascular smooth muscle cells in hypertension1.BackgroundVascular smooth muscle cells(VSMCs)are the major components of the middle vascular wall in blood vessels.VSMCs proliferation plays an important role in cardiovascular diseases including hypertension,atherosclerosis and vascular stenosis.VSMCs are intrinsically contractile in nature and exhibit very low rates of proliferation.However,in pathological conditions associated with vascular injury,such as in hypertension,VSMCs proliferate,undergo hypertrophy.VSMCs proliferation is a main feature of vascular remodeling.Vascular remodeling is one of the important causes of the pathogenesis of hypertension.Therefore,intervention of hypertensive vascular remodeling has become an important entry point for preventing and treating hypertension.However,it is unclear whether BCL6 plays a role in VSMCs proliferation in hypertension.This study was to investigate the effect of BCL6 on VSMC proliferation and its mechanism.2.ObjectiveThis study was designed to determine the effects and molecular mechanisms of BCL6 on VSMCs proliferation and to explore the therapeutic effect of BCL6 on vascular remodeling and blood pressure in SHR.3.MethodsHuman VSMCs were cultured in F12-K medium with 10%FBS,penicillin(100 units/ml)and streptomycin(100 mg/ml)at 37? in a humidified atmosphere of 95%air and 5%C02.Male SHR and WKY were randomly divided into two groups.BCL6 overexpression lentivirus vector and enhanced red fluorescent protein(ERFP)lentivirus vector were performed.blood pressure of tail artery was measured in conscious state using a noninvasive computerized tail-cuff system.The efficiency of the BCL6 overexpression was measured by Western blot.The descending thoracic aorta were stained with Masson's trichromestaining to analyse vascular remodeling.The effect of BCL6 on the proliferation of VSMCs was detected by CCK-8 kit.DNA synthesis of VSMCs was detected by EdU incorporation assay.The protein expressions of Proliferating cell nuclear antigen(PCNA)?Nicotinamide adenine dinucleotide phosphate oxidase2(NOX2)?Nicotinamide adenine dinucleotide phosphate oxidase4(NOX4)?GADPH were measured by Western blot.Real-time PCR was used to detect the mRNA levels of NOX2 and NOX4.Dihydroethidium(DHE)was used to evaluate the production of reactive oxygen species.4.Results(1)Overexpression of BCL6 attenuated the VSMCs proliferation induced by Ang ?,and decreased the number of EdU-positive cells and the high protein expression of PCNA in VSMCs induced by Ang ?.(2)Overexpression of BCL6 attenuated the high protein expression of NOX4 in VSMCs induced by Ang II and reduced the production of ROS induced by Ang ?.(3)BCL6 shRNA aggravated VSMCs proliferation and oxidative stress induced by Ang ?.(4)Lentivirus-mediated overexpression of BCL6 reduced blood pressure in SHR,and significantly inhibited vascular remodeling and oxidative stress in SHR.5.ConclusionBCL6 attenuates Ang ?-induced VSMC proliferation and oxidative stress.Overexpression of BCL6 in SHR rats lowers blood pressure,reduces vascular remodeling and oxidative stress.