Mechanism Study Of Catalpol,Loganin And Their Compatibility On Diabetic Renal Lesions Caused By Excessive Activation Of AGEs-RAGE Axis Via Regulating Endothelial-Macrophage Interaction

Aim:Diabetic nephropathy(DN)is the most common microvascular complication of diabetes mellitus,and inflammatory response plays an essential role in the progression of DN.The metabolic inflammatory response is accompanied by the infiltration and activation of monocyte-macrophages,which further aggravates the inflammatory damage of glomerular endothelial cells.The RAGE/RhoA/ROCK signaling pathway is a central link in the interaction between macrophages and renal endothelial cells,and the resulting inflammatory mediators are an important material basis for promoting diabetic kidney injury.In this thesis,the molecular mechanism of macrophage-endothelial cell interaction response was elucidated in depth through a mouse model of spontaneous diabetes mellitus with renal RAGE overexpression and a model of endothelial-macrophage cell co-culture injury induced by AGEs.The effects of catalpol,loganin and their combinations on DN were investigated,providing new ideas and methods for DN prevention and treatment.Methods:1.In vivo:Eight-week-old male KK/Ay mice were fed high-fat chow for 2 weeks,and mice with blood glucose values≥11.1 mmol/L were used as DM mice.The adeno-associated virus(rAAV-DJ-mRAGE-IRES-ZsGreen)was injected into the tail vein of DM mice,and those with more than 10-fold elevated urinary protein and successful enrichment of adeno-associated virus in the kidney were used as RAGE overexpressing DN mice after 2 weeks.The mice that were successfully molded were randomly divided into:model group,aminoguanidine group(100 mg/kg/d),catalpol low-dose group(50 mg/kg/d),catalpol high-dose group(100 mg/kg/d),loganin low-dose group(50 mg/kg/d),loganin high-dose group(100 mg/kg/d)and catalpol-loganin combination group(catalpol 50 mg/kg/d+loganin 50 mg/kg/d).In addition,KK-Ay mice without adeno-associated virus injection were taken as DN control group and healthy C 5 7BL/6J mice were taken as blank control group,6 mice in each group.The mice were administered by gavage according to the above groups for 8 weeks,and equal amounts of saline were given by gavage to the blank control group.DN control group and model group,and the indexes were determined as follows:①Mice were executed by decortication,and the cortical parts of the kidney were fixed in 10%neutral formalin and 2.5%Gluta respectively to HE、Masson staining and transmission electron microscopy to observe the glomerular structure;②Blood collected from the orbit was used to detect serum levels of MCP-1,M-CSF.IL-12,IL-10;③ Immunohistochemistry and Western blot were used to detect the protein expression of Occludin and VE-Cadherin in the renal cortex;④Immunohistochemistry was employed to detect macrophage marker protein EDA1 expression in kidneys,and Western blot was added to detect expression of macrophage M1/M2 type polarization marker proteins iNOS and Arg-1;⑤Western blot was employed to detect the expression of AGEs-RAGE/RhoA/ROCK pathway proteins in renal cortex.2.In vitro:(1)Construction of endothelial-macrophage co-culture injury model caused by AGEs:Mouse glomerular endothelial cells and macrophages were cultured in vitro at 37℃ in a 5%CO2 incubator.①Endothelial cells and macrophages were stimulated with different concentrations of AGEs(50,100,200,400 mg/L),and a blank control group and a negative control group with 0-BSA(200 mg/L)were set up to detect the effects of 0-BSA and AGEs(50,100,200,400 mg/L)on cell viability by CCK-8 assay;②The effects of AGEs on apoptotic damage of endothelial cells was detected by AO/EB staining;③Different concentrations of AGEs stimulated endothelial cells in Transwell culture plates.The effect of different concentrations of AGEs on RAGE protein expression in endothelial cells was detected by Western blot,and the morphology of co-cultured macrophages was observed under light microscopy;④ Different concentrations of AGEs stimulated macrophage in Transwell culture plates.The effect of different concentrations of AGEs on RAGE protein expression in macrophages was detected by Western blot,and the morphology of endothelial cells in co-culture was observed under light microscope.(2)Effect of catalpol,loganin and their combination on endothelial cell mediating macrophage migration and polarization stimulated by AGEs:Based on AGEs-stimulated renal endothelial cell-mediated macrophage co-culture model,different concentrations(1 and 10 μM)of catalpol and loganin and their combinations(1:1 mixture of catalpol and loganin)were added.①ELISA and Western blot were employed to detect the levels of AGEs-stimulated endothelial cells secreting MCP-1,M-CSF,and ICAM-1 levels;② Crystal violet staining was used to observe the migration rate of macrophages mediated by AGEs-stimulated endothelial cell;③Immunofluorescence and Western blot were employed to test the expression of the M1-type polarization marker protein iNOS,CD 16/32 and the M2-type polarization marker protein CD206,Arg-1 in macrophages mediated by AGEs-stimulated endothelial cell.(3)Effect of catalpol,loganin and their combination on macrophage mediating endothelial cell injury stimulated by AGEs:Based on AGEs-stimulated macrophage-mediated endothelial cell co-culture model,different concentrations(1 and 10 μM)of catalpol and loganin and their combinations(1:1 mixture of catalpol and loganin)were added.① The levels of TNF-α,IL-6,IL-12 and IL-10 secreted by AGEs-stimulated macrophages were measured by ELISA.②The proteins expression of Occludin and VE-Cadherin of endothelial cells mediated by AGEs-stimulated macrophage were measured by Western blot and immunofluorescence.(4)Study on the mechanism of endothelial-macrophage interaction regulated by catalpol,loganin and their combination:① Based on the endothelial-macrophage co-culture model,different concentrations(1 and 10 μM)of catalpol,loganin and their combinations(1:1 mixture of catalpol and strychnine)were added.RAGE.RhoA and ROCK protein expression were detected by Western blot;② Endothelial cells were transfected with RAGE,RhoA,ROCK lentivirus and divided into blank group,model group,RAGE overexpression group,RAGE overexpression+catalpol-loganin combination group,RhoA overexpression group,RhoA overexpression+catalpol-loganin combination group.ROCK overexpression group,ROCK overexpression+catalpol-loganin combination group.Western blot was employed to detect RAGE,RhoA and ROCK protein expression in endothelial cells and iNOS protein expression in co-cultured macrophages;Macrophages were transfected with RAGE.RhoA and ROCK lentivirus,then RAGE,RhoA,ROCK protein expression in macrophages and Occludin protein expression in co-cultured endothelial cells were detected by Western blot;③ Endothelial cells were treated with FPS-ZM1,Rhosin hydrochloride and Y27632,which were inhibitors of RAGE,RhoA and ROCK,and divided into blank group,model group,catalpol-loganin combination group,FPS-ZM1+catalpol-loganin combination group,Rhosin hydrochloride+catalpol-loganin combination group,Y27632+catalpol-loganin combination group.Western blot assay was added to detect RAGE,RhoA and ROCK proteins expression in endothelial cells and iNOS protein expression in co-cultured macrophages;macrophages were treated with FPS-ZM1,Rhosin hydrochloride and Y27632,then the expression of RAGE,RhoA and ROCK proteins in macrophages and Occludin protein in co-cultured endothelial cells were measured by Western blot.Results:1.In vivo:① RAGE overexpression significantly aggravated the renal pathological changes in DN mice.Catalpol,loganin and their combination reduced glomerular thylakoid matrix hyperplasia,collagen deposition,interstitial fibrosis and basement membrane endothelial space enlargement(P<0.05,P<0.01),with the best improvement effect in the combination group;②Serum MCP-1,M-CSF and IL-12 levels were obviously increased and IL-10 levels were clearly decreased in RAGE overexpression DN mice(P<0.01).Catalpol,loganin and their combination could decrease MCP-1,M-CSF,IL-12 levels and increase IL-10 levels(P<0.05,P<0.01),among which the combination group had the best improvement effect.③ RAGE overexpression significantly decreased the levels of Occludin and VE-Cadherin in kidneys of DN mice(P<0.01).Catalpol,loganin and their combination restored the protein expression of Occludin and VE-Cadherin(P<0.05,P<0.01),with the best improvement in the combination group;④ RAGE overexpression significantly upregulated the positive staining of macrophage marker protein EDA1 in DN mouse kidney,and upregulated the expression of M1-type macrophages polarization marker protein iNOS and downregulated the expression of M2-type macrophages polarization marker protein Arg-1(P<0.01).Catalpol,loganin and their combination reduced the positive staining of EDA1,downregulated iNOS expression and upregulated Arg-1 expression(P<0.05,P<0.01),with the best improvement in the combination group.Catalpol was better than loganin in downregulating iNOS,while loganin was better than catalpol in upregulating Arg-1.⑤ The over-activation of AGEs-RAGE axis significantly increased RhoA and ROCK protein expression in DN mice kidneys.Catalpol,loganin and their combinations reduced AGEs,RAGE,RhoA and ROCK protein expression,with the best effect in the combinations group.Catalpol down-regulated RhoA protein expression better than loganin,while the down-regulation of ROCK protein expression was better by loganin than catalpol.2.In vitro:(1)Construction of endothelial-macrophage co-culture injury model caused by AGEs:①Endothelial cells and macrophages were stimulated with different concentrations of AGEs for 24h and 48h,respectively.The CCK-8 assay detected that the proliferation inhibition rate of endothelial cells reached 51.4%and the proliferation rate of macrophages reached 93.7%when stimulated with 200 mg/L AGEs for 48h;②The highest apoptosis rate of endothelial cells was observed when endothelial cells were stimulated with 200 mg/L AGEs for 48h;③ Stimulation of endothelial cells with different concentrations of AGEs for 48h,we found that RAGE protein expression in endothelial cell was concentration-dependent with AGEs(P<0.05,P<0.01),and light microscopy observed that 200 mg/L AGEs-stimulated endothelial cell-mediated macrophages protruded pseudopods and changed from round to polygonal shape;④ Different concentrations of AGEs stimulated macrophages for 48h,we found that upregulation of macrophage RAGE protein expression was optimal at 200 mg/L AGEs stimulation(P<0.01),and crinkling of co-cultured endothelial cell morphology and reduction in the number of adherent cells were observed under light microscopy.(2)Effect of catalpol,loganin and their combination on endothelial cell mediating macrophage migration and polarization stimulated by AGEs:AGEs stimulated endothelial cell secretion of MCP-1,M-CSF,and ICAM-1(P<0.01),inducing migration of co-cultured macrophages to endothelial cells(P<0.01),upregulating iNOS and CD16/32 proteins expression(P<0.01),and downregulating CD206 and Arg-1 proteins expression(P<0.01).Catalpol,loganin and their combinations reduced MCP-1,M-CSF and ICAM-1 secretion from endothelial cells stimulated by AGEs(P<0.05,P<0.01),inhibited macrophage migration(P<0.01),down-regulated iNOS and CD16/32 proteins expression(P<0.05,P<0.01),and up-regulated CD206 and Arg-1 proteins expression in macrophages(P<0.05,P<0.01).The effect of catalpol-loganin combination was optimal,with catalpol down-regulating M1-type polarization marker protein in macrophages better than loganin,while loganin up-regulating M2-type polarization marker protein was better than catalpol.(3)Effect of catalpol,loganin and their combination on macrophage mediating endothelial cell injury stimulated by AGEs:AGEs stimulation promoted macrophage TNF-α,IL-6,IL-12 secretion,inhibited IL-10 secretion(P<0.01),decreasing Occludin,VE-Cadherin protein expression in co-cultured endothelial cells(P<0.01).Catalpol,loganin and their combination reduced TNF-α,IL-6 and IL-12 secretion and increased IL-10 secretion in macrophages(P<0.05,P<0.01),restoring Occludin and VE-Cadherin protein expression in endothelial cells(P<0.05,P<0.01),with the best effect in the combination group.(4)Study on the mechanism of endothelial-macrophage interaction regulated by catalpol.loganin and their combination:① AGEs stimulation obviously increased RAGE,RhoA and ROCK protein expression in endothelial cells and macrophages(P<0.01).Catalpol,loganin and their combination down-regulated the expression of RAGE,RhoA and ROCK proteins(P<0.05,P<0.01).The down-regulation of RhoA protein by catalpol was superior to loganin,while the down-regulation of ROCK protein expression by loganin was superior to catalpol.② Lentiviral transfection of endothelial cells revealed that iNOS protein expression was elevated in co-cultured macrophages(P<0.01),and RAGE overexpression in endothelial cells significantly upregulated protein expression of RhoA and ROCK(P<0.01),while RhoA and ROCK overexpression had no significant effect on RAGE protein expression,suggesting that RAGE is upstream of RhoA and ROCK.RhoA overexpression significantly upregulated the protein expression of ROCK(P<0.01),while ROCK overexpression had no significant effect on the protein expression of RAGE and RhoA,suggesting that ROCK is downstream of RAGE and RhoA.Catalpol-loganin combination downregulated RAGE,RhoA and ROCK expression in endothelial cells(P<0.01),and downregulated co-cultured macrophages iNOS protein expression(P<0.01);Occludin protein expression in co-cultured endothelial cells was reduced after lentiviral transfection of macrophages(P<0.01).Catalpol-loganin combination restored Occludin protein expression in endothelial cells(P<0.01),and downregulated RAGE,RhoA,and ROCK protein expression in macrophages(P<0.01).③FPS-ZM1,Rhosin hydrochloride,and Y27632,which the inhibitors of RAGE,RhoA,and ROCK,in combination with catalpol-loganin,respectively,had better down-regulation of AGEs-stimulated endothelial cell-mediated macrophage iNOS protein than catalpol-loganin alone(P<0.01);FPS-ZM1,Rhosin hydrochloride,and Y27632,in combination with catalpol-loganin,respectively,had better up-regulation of AGEs-stimulated macrophage-mediated endothelial cell Occludin protein than catalpol-loganin alone(P<0.01).Conclusion:Catalpol.loganin and their combination interfere with the interaction response between macrophages and endothelial cells by regulating AGEs-RAGE/RhoA/ROCK signaling pathway,reducing endothelial cell injury and chemokine secretion,inhibiting macrophage migration and regulating M1/M2 phenotypic transition balance.Catalpol inhibits the polarization of macrophages to M1 type better than loganin,while loganin promotes their polarization to M2 type better than catalpol,Thus the combination of catalpol and loganin was found to be synergistic in improving diabetic inflammatory damage of kidney.Notably,catalpol showed stronger inhibition of RhoA downstream of RAGE,while loganin showed stronger inhibition of ROCK downstream of RAGE,revealing the advantage of catalpol-loganin combination in delaying DN.