Mechanism Study Of AGEs-induced Renal Pathology Interfered By Catalpol,Loganin And Their Compatibility Based On The Function Of AGEs Receptors

Objective:The accumulation of Advanced Glycation Endproducts(AGEs)is one of the core mechanisms that mediates the onset of Diabetic Nephropathy(DN).Although AGEs can bind to the matrix membrane and change its properties via binding to the receptor for Advanced Glycation Endproduct(RAGE),they promote the release and generation of fibrogenic factors.It is acknowlegable that AGEs play a pivotal role in the development of the glomerulus and renal interstitium injury and amplification of the inflammatory response in partial renal tissues.The contiuous accumulation of AGEs result in the circulation and kidney injuries during the pathogenic procedures of DN.Galectin-3 is a 32-35kDa multifunctional lectin protein,which expresses in many organs,including renal tissues.The renal injuries and fibrosis are highly associated with the expression of Galectin-3.Meanwhile,Galectin-3 is a member of the receptors of AGEs.Research show that Galectin-3 is a scavenger receptor,which plays a protective role under specific conditions.However,it has been reported that the inhibition of Galectin-3 ameliorate the renal fibrosis induced by various pathological factors.It is worth intensive studies based on the controversial role of Galectin-3 in AGEs-induced renal injuries.Endothelial dysfunction is a key pathogenic mechanism of DN when AGEs-RAGE triggers.AGEs can disrupt the cross-linked reactions of the sub-endothelial matrix molecules,for instance,it can destroy the generation of collagens and the interaction between matrix-matrix and intercellular communications thus make damge to the intergrities of vessal wall and besement membrane.Another important mechanism is that AGEs can disturb the synthesis mode of Nitrc Oxide(NO)in endothelial cells,thus negatively affect the function of endothelium.Catalpol and Loganin are the main medicinal components extracted from Rehmannia glutinosa Libosch and Cornus officinalis.The Rehmannia glutinosa Libosch and Cornus officinalis drug pair was presented in the ancient books Synopsis of Golden Chamber "Shenqi Wan" and Intergrating Chinese and Western Mediclne "Zicui Yin" and other formulae etc,which are mainly used to treat diabetes.Rehmannia glutinosa Libosch can nourish yin and excrete heat by bitter and cold properties,nourishing body fluid,downbear fire and protect kidney and improve kidney function.Cornus officinalis has the effect of invigorating kidney yin and yang,astringent essence and solid removing.It can strengthen the function of invigorating liver and kidney,nourish liver and kidney,clear deficiency and heat,nourish kidney water Yin and cold deficiency,nourish yin fluid,moisten dryness and reduce fire.In clinic,Shuangxu Zishen Decoction and Qingzao Ziyin Decoction are used in many prescriptions to treat Xiaxiao,which is the main manifestation of "nourishing Yin and clearing heat".In preclinic study,it has been demonstrated that the irdoid glycosides from Rehmannia glutinosa Libosch and Cornus officinalis ameliorate the renal function via inhibiting AGEs-RAGE activation in DN animal models.Therefore,this paper mainly discusses the two major medicinal ingredients to improve the glomerular endothelial dysfunction caused directly by AGEs-RAGE axis hyperactivityMethods:①Establishment of an animal model of AGEs-RAGE renal hyperactivation in vivoWe tried to explore whether tail vein injection(i.v.)of AGEs(100mg/kg)could damage the kidney by unilateral renal vein hydrodynamic retrograde injection of empty vector adenovirus Ad-GFP into renal veins..AGEs(100mg/kg)were continuously injected into the tail veins for 8 days and urine was collected on the 7th day.On the 8th day,the renal cortex was taken to observe the transfection.The 2×1010PFU Ad-RAGE-GFP was diluted to 3×107,3×106 and 3×105PFU(Plaque-Forming Unit)according to the concentration gradient.AGEs were injected unilaterally into the left kidney by renal vein hydrodynamic retrograde injection.AGEs were injected continuously into the tail vein for 8 days after operation.On the 7th day,urine was collected 24 hours a day,animals were sacrificed and transcardially perfused on the 8th day,and the renal cortex was taken to measure the changes of renal function.②The effects of catalpol,loganin and their compatibility on renal pathological changes and inflammation in rats with AGEs-RAGE overactivation in vivo.After intravenous injection of Ad-RAGE-GFP,AGEs were administered continuously by retrograde hydrodynamic method.Catalpol,loganin and compatibility were administered simultaneously by gavage for 8 days.Urine samples were collected on the 8th day.To observe whether Catalpol low dose group(20mg/kg),Catalpol high dose group(100mg/kg),loganin low dose group(20mg/kg),loganin high dose group(100mg/kg),Catalpol+loganin(6.6+3.3)mg/kg,Catalpol+loganin(5+5)mg/kg,Catalpol+loganin(66+33)mg/kg,Catalpol+loganin(50+50)mg/kg can improve abnormal renal function by inhibiting the hyperactivation of AGEs-RAGE axis.③Catalpol,loganin and their compatibility inhibit the inflammation of AGEs on glomerular endothelial cells and its mechanismCCK-8 method and LDH method were used to observe the effects of catalpol,loganin and compatibility on the proliferation and toxicity of mGECs in glomerular endothelial cells.WB method was used to observe the effects of NF-κB/iNOS and PI3K/Akt/eNOS pathways stimulated by 200 μg/mLAGEs.Transwell method was used to observe the migration of macrophages interferred by single administration and compatibility.FITC-Dextran method was used to observe 200 μg/mLAGEs-induced vascular endothelial hyperpermeability interferred by single and compatibility.WB method was used to observe the expression of VE-cadherin and occludin.④Mechanisms of Catalpol on Inflammatory Infiltration in Rats with Overactivation of AGEs-RAGE KidneyImmunofluorescence assay(IF)was used to observe whether Catalpol inhibited the infiltration of F4/80(macrophage marker)inflammation cells induced by overexpression of AGEs-RAGE axis,the early proliferation of glomerular vasculitis CD34(endothelial marker),and the deposition of AGEs in renal cortex.At the same time,the expression of MCP-1 and ICAM-1 in renal cortex was observed.Western Blotting and IF methods were used to observe whether Catalpol could stimulate the expression of eNOS,iNOS,NF-kappa B,PI3K and Akt proteins in vascular endothelial dysfunction induced by AGEs.At the same time,pull down method was used to observe whether Catalpol could inhibit AGEs-induced vascular inflammation through RhoA/ROCK pathway.⑤Preliminary study of catalpol,loganin and compatibility on AGEs-galectin-3 mediated endothelial dysfunctionThe effects of AGEs on the fate of mGECs at low doses were observed by real-time dynamic cell imaging and CCK-8.The contents of Galectin-3,VEGFR-1,VEGFR-2 and VEGF after AGEs stimulation were observed by ELISA and WB.In order to further observe their effects,ELISA was used to detect the secretion of proangiogenic factors Ang-1,Ang-2 and Tie-2 by AGEs stimulation.WB and IF were used to detect the secretion and expression of Ang-1,Ang-2 and Tie-2 and HIF-1α in mGECs stimulated by the use of drugs alone and in combination.Meanwhile,for macrophages,ELISA and WB methods were used to detect the expression of Galectin-3/HIF-1 alpha in macrophages stimulated by AGEs at low doses.The changes of Galectin-3/HIF-1 alpha expression in macrophages were observed after catalpol,loganin,catalpol and loganin treatment.Results:①Establishment of an animal model of AGEs-RAGE renal hyperactivation in vivoThe experimental results showed that retrograde hydrodynamic intravenous injection of empty virus vector Ad-GFP and continuous intravenous injection of AGEs(100mg/kg)for 8 days had no significant effect on BUN,UPr,sCr,uCr and other renal function indicators in rats,and had no significant change in renal morphology.However,when Ad-RAGE-GFP was injected intravenously and AGEs(100mg/kg)was injected intravenously for 8 consecutive days,the injected kidney was atrophied obviously,and a large number of necrotic foci appeared in the cortex.The renal function indexes of BUN,UPr,sCr and uCr in rats were significantly different from saline group and Ad-GFP group,and positively correlated with the titer of transfected adenovirus(**p<0.05,**p<0.001).Masson staining of renal cortical tissue sections showed that local overactivation of AGEs-RAGE axis could disrupt the glomerular and tubular structure of the kidney and induce abnormal collagen proliferation.The expression of iNOS and F4/80 in renal tubules and glomerular cells was observed by immunofluorescence assay,suggesting that the activation of AGEs-RAGE axis is closely related to NO production and macrophage infiltration(**p<0.05,***p<0.001).②The effects of catalpol,loganin and their compatibility on renal pathological changes and inflammation in rats with AGEs-RAGE overactivation in vivoCatalpol 100 mg/kg and Catalpol 66 mg/kg+loganin 33 mg/kg in compatible group were the best for improving renal function(**p<0.05,***p<0.001).Catalpol 100 mg/kg,loganin 100 mg/kg and compatible high dose groups could significantly reduce the number of inflammatory cells in whole blood and kidney tissue,Catalpol 100 mg/kg and compatible high dose group could significantly improve collagen deposition,tube formation and glomerular swelling caused by renal injury.High dose of 1:1 combination group and Cat.100mg/kg could improve the ultrastructural changes of kidney such as vascular lumen swelling,endothelial space enlargement,inflammatory infiltration and mesangial dilatation(**p<0.05,***p<0.001).③Catalpol,Loganin and their compatibility inhibit the inflammation of glomerular endothelial cells induced by AGEs and its mechanismsCatalpol,loganin and compatible drugs had no significant effect on the proliferation or obvious toxicity of mGECs.The drugs and their compatibilities could inhibit the activation of NF-κB/iNOS.In addition,the drug could reverse the inhibitory effect of PI3K/Akt/eNOS pathway,decrease the secretion of proinflammatory cytokines stimulated by 200 μg/mLAGEs and exert anti-inflamatory effect.Both single administration and compatibility could inhibit the migration of macrophages.Both single and combined administration could inhibit the increase of vascular endothelial permeability induced by 200μg/mLAGEs stimulation in vitro.Catalpol 10μM and Catalpol+loganin(5+5)μM had the best effect in single and compatibilities respectively(**p<0.05,***p<0.001).Drugs could increase the expression of VE-cadherin and occludin and protect vascular endothelial function(**p<0.05,***p<0.001).④The mechanism of Catalpol on inflammatory infiltration in rats with AGEs-RAGE renal hyperactivationCatalpol could inhibit the infiltration of inflammatory cells and early proliferation of glomerular vasculitis caused by overexpression of AGEs-RAGE axis in renal cortex,and inhibit the deposition of AGEs in renal cortex(**p<0.05,***p<0.001).At the same time,it inhibited the release of MCP-1 and ICAM-1 in renal cortex.Catalpol repaired vascular dysfunction and inhibited the secretion of chemokines and adhesion molecules by regulating the expression of eNOS/iNOS in vascular endothelium stimulated by AGEs.At the same time,Catalpol partly inhibited the inflammation induced by AGEs through RhoA/ROCK pathway(**p<0.05).⑤Preliminary study of catalpol,loganin and compatibility on AGEs-Galectin-3 mediated endothelial dysfunctionWhen AGEs were deposited in mGECs at low doses(100μg/mL),the proliferation of mGECs was observed(**p<0.05,***p<0.001).At the molecular level,AGEs activated Galectin-3(###p<0.001),a large amount of vascular endothelial growth factor,further up-regulate the expression of vascular endothelial growth factor-1 and vascular endothelial growth factor-2 were released from mGECs.At the same time,AGEs stimulated mGECs to release a large number of proangiogenic factors Ang-1,Ang-2 and Tie-2(**p<0.05,***p<0.001),the expression of HIF-1αwas up-regulated(**p<0.05,***p<0.001),which stimulated the proliferation of mGECs.Catalpol,loganin and combined administration inhibited the proliferation of mGECs at low dosage by tail vein injection of AGEs in vivo.Judging from the results of inhibiting proliferation effect,Catalpol+loganin(5+5)μM in vivo and Catalpol+loganin(50+50)mg/kg in vitro were the best(**p<0.05,***p<0.001).In the molecular mechanism of galectin-3/VEGFR expression inhibition on mGECs,the inhibitory effect of the compatible group Cat.+Log.(5+5)μM was better than that of the single group(**p<0.05,***p<0.001).Loganin alone group had some inhibitory effect on proliferation,but the effect of single group was weaker than other groups.In terms of macrophages,AGEs at low doses promoted the proliferation of macrophages by up-regulating Galectin-3/HIF-1α.Catalpol,catalpol and loganin(5+5)μM inhibited the proliferation of macrophages by down-regulating the expression of Galectin-3/HIF-1α(**p<0.05,***p<0.001).In conclusion,it is the best group in single and compatible group respectively that catalpol and catalpol-loganin 1:1 combination group inhibited the proliferation of mGECs and inflammatory macrophages through Galectin-3.Conclusion:①Renal vein injection of Ad-RAGE-GFP and continuous tail vein injection of AGEs(100mg/kg/d)for 8 days causes significant local renal injury accompanied by inflammatory cell infiltration,suggesting that activation of AGEs-RAGE axis is one of the direct factors of renal injury.From the morphological changed,renal vein injection of Ad-GFP with empty carrier and tail vein injection of AGEs(100mg/kg/d)are used for continuous tail vein injection show no significant difference after 8 days of intravenous injection.②Catalpol(20mg/kg,100mg/kg),loganin(20mg/kg,100mg/kg)and Catalpol+loganin(10+10,13.3+6.6,50+50,66.6+33.3mg/kg)were administered orally to animals with unilateral hypernephrosis of AGEs+Ad-RAGE-GFP.The changes of renal function and morphology among groups were observed and compared.From the experimental results,each group has the effect of improving renal function and pathological morphology,and the effect of compatibility group is better than that of single drug group.Catalpol(100 mg/kg)and Catalpol+loganin(50+50 mg/kg)had the best renal protection in vivo.③mGECs stimulated by 200 ug/mLAGEs in vitro,Catalpol(1 μM,10 μM,loganin(1μM,10 μM)and Catalpol+ loganin(0.5+0.5,5+5,0.66+0.33,6.6+3.3 μM)were administered to the cells simultaneously.To clarify the relationship between activation of AGEs-RAGE axis and regulation of NO,we observe that catalpol,loganin and compatible drugs had no significant effect on the proliferation of mGECs and had no significant toxicity when administered alone,but the activation of NF-κB/iNOS and its inflammatory effect stimulated by 200 μg/mLAGEs were inhibited.10 μM Catalpol had the best overall effect.Catalpol+Loganin(5+5)μM had the most significant effect in the compatibility group.In addition,the drugs can reverse the inhibitory effect on PI3K/Akt/eNOS pathway stimulated by 200μg/mLAGEs and protect endothelial function.Both single administration and compatibilities can inhibit the migration of macrophages,of which Cat.10um has the best effect.Both single and combined administration inhibit the increase of vascular endothelial permeability induced by 200 μg/mLAGEs stimulation in vitro.Cat.10μm had the best effect.Drugs can increase the expression of VE-cadherin and occludin and protect vascular endothelial function.Catalpol can also improve AGEs-RAGE activation by iNOS/eNOS,which leads to endothelial dysfunction.④Catalpol,Catalpol+loganin 1:1 combination group can inhibit the proliferation of endothelial cells by inhibiting the high expression of Galectin-3 induced by low dose of AGEs stimulation,and ameliorate the proliferation and infiltration of macrophages by inhibiting Galectin-3.