Material Basis And Action Mechanisms Of Eucommia Ulmoides Oliv.lignans Against Diabetic Kidney Injury

Diabetic kidney injury(DKI)is a chronic kidney disease caused by hyperglycemia,leading to pathophysiological changes in the glomeruli,renal tubules,renal interstitium,tiny blood vessels and so on.In recent years,the incidence of DKI has increased rapidly,but effective drugs for it are still lacking.Based on the high growth rate of incidence and numbers,it is urgent to develop a new effective drug against DKI.Eucommia ulmoides Oliv.is a unique traditional medicine in China,which is called the gold in plant.Based on its traditional efficacy,medicine theory and previous pharmacological research results,we can known that Eucommia ulmoides Oliv.lignans(DZ-Ligs)has a good protective effect on kidney injury.Therefore,aiming to explore a adaptive drug theoretical basis for DKI treating,we screen some activated molecule chemical entities from DZ-Ligs being extracted for renal protective,and explore their active components and possible mechanisms against DKI.Purposes:1.To investigate the protective activity and the effect of DZ-Ligs on diabetic kidney injury.2.To analyze the chemical components of DZ-Ligs,including the isolation and identification of the structure of chemical compounds.3.To investigate the protective activity and the action mechanisms of monomeric chemical compounds obtained from DZ-Ligs on diabetic kidney injury.Methods:1.The renal protective activity and the action mechanism of DZ-Ligs were evaluated in experimental rats.After 1 week of adaptive feeding,diabetic rat models were established via intraperitoneal injection of STZ and induced by high-fat feeding.Successfully induced model rats were then randomly categorized into control group,positive drug group(metformin 200 mg/kg/day),and three different concentration groups of high,medium,low groups(50 mg/kg/day,100 mg/kg/day,200 mg/kg/day,respectively)of lignans obtained from E.ulmoides Oliv(DZ-Ligs).The rats in each group were administered via gavage for 4 weeks,and their body weight,fasting blood glucose level,serum insulin level,and urinary protein levels were regularly analyzed.The plasma sample was assayed by measuring the molecular levels of serum creatinine(SCR),blood urea nitrogen(BUN),malonaldehyde(MDA),and estimating the enzymatic activities of superoxide dismutase(SOD)and glutathionidase(GSH)after the last administration.The renal tissues were prepared for staining with hematoxylin and eosin(HE),as well as periodic acid-Schiff(PAS)stains to observe the morphological changes in the samples.The expressions of nephrin,Nrf2,and HO-1 in the tissues were measured by immunohistochemical method,while the expressions of Nrf2,HO-1,and TGF-β1-related proteins in the renal tissues were quantified by Western blotting.2.The original material was extracted by using the water reflux method.The total compositions of water extract were enriched through porous resin column chromatography.Tracking analysis of the distribution of each fraction of the compound was conducted through ultraviolet(UV)spectrophotometry and high-purity liquid chromatography(HPLC)combined with mass spectrometry(MS).Subsequently,the lignans constituents were enriched,isolated,and purified via chromatographic techniques such as silicone column chromatography,Sephadex LH-20,Prep-HPLC,and LC-MS/MS.The planar structure and absolute configuration of the compounds were confirmed through the comprehensive application of UV spectrophotometry,infra-red spectrum(IR),1 and 2 dimensional-NMR spectra(1/2D-NMR),high-resolution mass spectrum(HRMS),optical rotatory dispersion(ORD),circular dichroism(CD)combined with electronic circle dichromatography(ECD),and chemical communication.3.Potential action targets to the components of E.ulmoides Oliv.were predicted with reference to a public network database to screen the related targets for diabetic kidney injury therapy.The core components and the targets were confirmed by using network tools such as String.The pathway annotation analysis of GO and KEGG was conducted by using the DAVID database.The network relationship of"components-targets-pathway" was constructed through Cyto scape to predict the potential mechanism of the target compounds.The components with higher binding capacity were virtual screened based on the potential antioxidant targets of Nrf2 by using the Modulus of Elastic(MOE)software.In an HRMC cell model of high glucose-induced abnormal proliferation,we further verified whether isoeucommin A could interfere with the Nrf2/HO-1 signaling pathway in high-glucose-stimulated HRMCs.Next,we detected the expression levels of antioxidant SOD and oxidative stress factor MDA in each group of HRMCs.Moreover,to explore the effect of isoeucommin A on the RTECs injured by H2O2,we first examined the expression of Nrf2/HO-1 signal pathway-related proteins.The compound isoeucommin A may inhibit the killing effect of H2O2 on RTECs to a certain extent.Then,the role of isoeucommin A on oxidative stress of H2O2-stimulated RTECs was analyzed by determining the content of SOD,MDA,and GSH.Results:1.For diabetes renal injury with high-fat feeding and the STZ injection model,the fasting blood glucose levels of rats were significantly increased,while their insulin levels were significantly decreased.After the DZ-Ligs administration intervention,the blood fasting glucose levels were decreased significantly relative to those of the model group,while the insulin levels were significantly increased in the high-dose administration group.In addition.the levels of Scr,BUN,and MDA in the blood and the urine protein level were decreased,while the serum SOD activity was increased.As for the histomorphology results,the increased glomerular volume,swelled endotheliocyte and vacuolar degeneration,and the significantly increased interstitial inflammatory cell infiltration played an essential role for the pathological traits in the model group,while the traits in the intervention groups after DZ-Ligs administration apparently presented with an improved outcome in the renal tissues,including vacuolar degeneration in the endothelial cells in a mitigated degree,reduced inflammatory cell infiltration,and a declining number of glomerular cells.When compared with the model group,the groups with the DZ-Ligs intervention recorded an improvement in the expression levels of Nrf2 and HO-lin the rat renal tissues,while the levels of Smad3 and TGF-β1 protein evidently reduced.2.The total lignin content of the present batch of E.ulmoides Oliv.in this experiment was 10.89%as measured by UV spectrophotometry.The UPLCQ-TOF-MS analysis results suggested 89 absorption peaks of the chemical components in DZ-Ligs and the prediction of 37 lignans based on the corresponding mass spectrum.Column chromatography and pre-HPLC methods helped isolate and identify 27 compounds from DZ-Ligs,in which compounds 1 and 2 were new compounds identified spectroscopically.These compounds were respectively identified as noreucol A(1);(+)-epicycloolivil(2);(+)-cycloolivil(3);caruilignan D(4);(+)-medioresinol(5);(+)-episyringaresinol(6);(+)-syringaresinol(7);(+)-epipinoresinol(8);(-)-epipinoresinol(9);(+)-pinoresinol 4-O-β-Dglucopyranoside(10);(-)-pinoresinol 4-O-β-D-glucopyranoside(11);(+)-isoeucommin A(12);(+)-medioresinol 4-O-β-D-glucopyranoside(13);(+)-pinoresinol 4,4’-O-bis-β-D-glucopyranoside(14);(-)-olivil(15);coniferin(16);caffeic acid(17);chlorogenic acid(18);(+)-genipin(19);geniposidic acid(20);geniposide(21);aucubin(22);(-)-β-sitosterol(23);kaempferol(24);rutin(25);3-methoxy-4-hydroxybenzaldehyde(26);3,5-dimethoxy-4-hydroxybenzaldehydeby(27).3.The predicted renal protective mechanisms of the target components involved cell proliferation and differentiation,oxidative stress,inflammatory response,and fibrosis.The lignans screened,such as(5),(9),(10),(11),(12),and(14),were confirmed to show a good affinity with Nrf2 protein.In an HRMC cell model of high glucose-induced abnormal proliferation,we next further verified whether(+)-isoeucommin A interfered with the Nrf2/HO-1 signaling pathway in high-glucose-stimulated HRMCs.When compared with that in the control group,the expressions of Nrf2 and HO-1 were elevated with increasing concentrations of(+)-isoeucommin A.The p-GSK-3β level was significantly elevated after(+)-isoeucommin A treatment,which decreased the protein stability of GSK-3 β and reduced its inhibitory effect of Nrf2.We also found that the SOD level in the HG group was reduced relative to that of the control group,albeit the MDA level was increased.In addition,the SOD content in the(+)-isoeucommin A groups was increased when compared with that in the HG group,albeit the MDA content was decreased.The results revealed that(+)-isoeucommin A could reduce the oxidation effect induced by high glucose levels and thereby protect HRMCs.To explore the effect of(+)-isoeucommin A on the RTECs injured by H2O2,we first examined the expression of Nrf2/HO-1 signal pathway-related proteins.With the stimulation of H2O2 in RTECs,the expressions of HO-1 and Nrf2 were downregulated.When the(+)-isoeucommin A was added into H2O2-stimulated RTECs,both the expressions were improved robustly in a concentration-dependent manner.In contrast with that in the control group,H2O2 treatment reduced the cell viability with significant differences.The cell viability was found to advance with the addition of(+)-isoeucommin A,particularly,in the medium-and high-concentration groups.Then,the role of(+)-isoeucommin A on oxidative stress of H2O2-stimulated RTECs was analyzed.Statistical analysis indicated that H2O2 reduced the levels of SOD and GSH slightly,but apparently hiked the MDA level.After the addition of(+)-isoeucommin A,the situation of the three enzymes was reversed in H2O2-stimulated RTECs.Instead,the MDA level was reduced by isoeucommin A treatment,and the effect was most obvious in the high-concentration group.Conclusion:1.DZ-Ligs evidently demonstrated protective effects on kidney injury in a diabetic rat model through the inhibition of oxidative stress for renal protection.2.A total of 27 compounds were isolated and identified from the E.ulmoides Oliv.,among which compounds 1 and 2 are new compounds.3.(+)-Isoeucommin A could activate the Nrf2/HO-1 signaling pathway in high-glucose-stimulated HRMCs as well as effectively activated the Nrf2/HO-1 signaling pathway to counteract the oxidative stress induced by H2O2 in RTECs.