Study On The Intervention Effect Of Individual/Combinatorial Bioactive Parts From Corni Fructus And Radix Rehmanniae On Diabetic Nephropathy

Object:Diabetic nephropathy(DN)acts as the major microangiopathy in diabetes,results in disability and death.Non-enzymatic glycosylation plays an important role in the pathological process of DN.Studies indicated that AGEs/RAGE pathway was important in structure and function changes of renal inherent cells.AGEs induced human renal mesangial cells(HRMCs)damage mainly cause the DN.Radix rehmanniae and Cornus officinalis were used to treat diabetes mellitus and its complications for long in traditional Chinese medicine(TCM).In our study,db/db mice and AGEs-induced HRMCs were used to study the effect of Radix rehmanniae and Cornus officinalis in DN by modern biomedicine.Moreover,it would provide scientific basis for the clinical TCM in DN.Method:(1)In vivo:db/db mice(FBG≥15 mmol/L)with high-microalbuminuria were selected as the DN mice.Model mice were divided into related groups:DM,Met(200 mg/kg),IGCO(59 mg/kg),TACO(15 mg/kg),IGRR(121 mg/kg)and CBP(195 mg/kg),the mice were gavaged with related compounds for 8 weeks with indicators detected.①Body weights,24 h food consumption,24 h water intake,24 h urine volume and fasting blood glucose levels were measured every 4 weeks.②Glycated serum protein(GSP),secrum insulin,total cholesterol(TC),triglyceride(TG),24 h urinary proteins,urea nitrogen and creatinine were determined.③Pancreas and renal lesion were observed by HE,Masson and PAS methods.RAGE,SphKl,NF-κB in renal cortex were measured by immunohistochemical method.Microstructure changes in renal were also checked using TEM.④RAGE,SphKl,P38MAPK,PTK,NOX4,TGF-βand NF-κB expression levels in renal cortex were measured using western blot.(2)In vitro:Different concentrations of AGEs(50,100,200 and 400 mg/L)were used in inducing HRMCs damage.The suited concentration of AGEs was decided after cell proliferation and microstructure changes of HRMCs evaluated.Furthermore,different concentrations(10、1、0.1 μmol/L)of loganin,morroniside,catalpol and acteoside were used to intervene the effect of AGEs in HRMCs.①Cell proliferation was measured using MTT method,and microstructure changes were checked using TEM.②Cell injury were detected using AO/EB method.ROS、LDH、FN and COL-IV in cell upernate were measured.③Phosphorylation of SphK1,P38MAPK,PTKs,MEK1/2,ERK1/2 and NF-κB,protein levels of RAGE,Ras,S1P2;TGF-β,ROS,NOX4 and nuclear-NF-κB were detected using western blot.(3)Targets exploring:①Based on the RAGE-silenced,loganin,morroniside,catalpol and acteoside were used to intervene the effect of AGEs in HRMCs.S1P2,NOX4,TGF-β,p-SphK1,p-P38MAPK,p-PTK,p-ERK1/2,p-MEK1/2 and p-NF-kB expression levels were measured using western blot.② Inhibitors of target proteins were added into AGEs/RAGE-sthenic HRMCs,loganin,morroniside,catalpol and acteoside were used to intervene the effect of inhibitors.Western blot was used to detected the levels of FN and COL-Ⅳ under the inhibiting effect of inhibitors.Results:(1)IGCO,TACO,IGRR and CBP could improve the DN.And the CBP expressed the advantages in DN:①24 h food consumption,24 h water intake,24 h urine volume,GSP,secrum insulin,TC,TG and fasting blood glucose levels were deceased(P<0.05,P<0.01).②Urea nitrogen,serum creatinine and 24 h urinary proteins were reduced(P<0.05,P<0.01).③Pancreas and renal lesions were improved,collagen and glycogen deposition were remittenced(P<0.05,P<0.01).④Serum and renal AGEs levels reduced.RAGE,SphK1,P38MAPK,PTK,NOX4,TGF-βand NF-κB expression levels in renal cortex were decreased.⑤Damages in HRMCs and podocyte were improved.(2)Loganin,morroniside,catalpol and acteoside could improve the AGEs-induced HRMCs damage:①AGEs(200 mg/L)was used to establish the cell model.The Cell proliferation and microstructure changes of HRMCs were severe in HRMCs(P<0.01).②Phosphorylation of SphK1,P38MAPK,PTKs,MEK1/2,ERK1/2 and NF-κB,protein levels of RAGE,S1P25 TGF-β,ROS,NOX4 and nuclear-NF-κB were reduced(P<0.05,P<0.01).③Fluorescence intensity of ROS,RAGE and NF-κB decreased(P<0.05,P<0.01).Moreover,the microstructure changes in HRMCs were improved。(3)The potential targets of loganin,morroniside,catalpol were Sphk1,ERK1/2 and NF-κB respectively:①After RAGE silenced,only the expression s of S1P2,p-SphK1,p-P38MAPK,p-ERK1/2,p-MEK1/2 and p-NF-kB reduced except the NOX4,TGF-β and p-PTK(P<0.05,P<0.01).②In the AGEs/RAGE-sthenic HRMCs with targets-inhibited,COL-Ⅳ and FN secretion decreased in HRMCs(P<0.05,P<0.01).Particularly,PF543,VX-11e,PDTC respectively eliminated the effect of loganin,morroniside and catalpol.Conclusions:In vivo study indicated that bioactive parts in Radix rehmanniae and Cornus officinalis impove the diabetic symptoms,aberrant structure and function of renal,by intervening the AGEs/RAGE pathway.Morever,combination of bioactive parts in Radix rehmanniae and Cornus officinalis could represent its advantage in treating DN.In vitro study demonstrated typical compounds of iridoid glycosides in Radix rehmanniae and Cornus officinalis suppress the AGEs induced HRMCs proliferation,ECM secretion,AGEs/RAGE pathway activation.Furthermore,SphK1,ERK1/2 and NF-κB were the potential targets of loganin,morroniside,catalpol respectively.Above all,combination of effective parts in Radix rehmanniae and Cornus officinalis could represent its advantage in treating DN via synergistic effect in AGEs/RAGE pathway of different effective constituents.These datas expound the dominance and scientificity of the compatibly using of TCM in clinical.