Xian Wang,Chang Liu,Mi Li,Yiqiang Xie＊,Kai Li＊

1School of Traditional Chinese Medicine,Hainan Medical University,Longhua District,Hainan,China.

#Xian Wang and Chang Liu made the equal contribution to this paper.

Abstract

Keywords：Alpinia oxyphylla,Diabetic nephropathy,Oxidative stress,p47phox

Background

Diabetic nephropathy (DN) is a common microvascular complication of diabetes,which is the main cause of end-stage kidney disease [1,2].The pathogenesis of DN is very complex.Although strict control of blood sugar can delay the progress of the disease,the current treatment is still not ideal [3].Many studies have confirmed that oxidative stress response is an important cause of DN [4].Under normal conditions,reactive oxygen species such as superoxide anion,hydroxyl free radical,hydrogen peroxide and so on have the function of stimulating cell growth.The production and scavenging of free radicals in the body are in equilibrium state.Once the balance is destroyed and the production is more than scavenging,there will be a lot of free radicals accumulation which will cause protein,fat,nucleic acid and other damages,that is oxidative stress damage[5].In previous studies,researchers found that there were abnormal increase in Reactive oxygen species (ROS) concentration,decrease in Superoxide dismutase (SOD) activity and significant increase Malondialdehyde (MDA) content in kidney tissues of DN rats [6].Among the many indicators of oxidative stress,ROS is an important one.Multiple intracellular and extracellular stimuli cause ROS to directly modify or indirectly damage intracellular nucleic acids,proteins,lipids,and then lead to cell damage.This cytotoxic effect of ROS plays an important role in the development of multiple acute and chronic kidney diseases [7].NADPH oxidases (Nox) is thought to be the leading producer of ROS in a variety of acute and chronic kidney diseases [8].To play a catalytic role,Nox needs to bind to a variety of regulatory subunits to form a stable complex [9].p47phoxis an important subunit of Nox family which can determine the catalysis of several Nox subtypes(Nox1,Nox2,Nox3,Nox4) [10].In a word,the expression of p47phoxplays an important role in the oxidative stress response of DN and could serve as an breakthrough point for the study of AO’s treatment mechanism.

According to the theory of Traditional Chinese Medicine (TCM),the AO has the effect of tonifying kidney and shrinking urine which is similar to the function of reducing urinary protein of Angiotensin Receptor Blocker (ARB).Previous studies have proved AO'seffects on DN [11-13].In this study,we put forward the hypothesis that AO could relieve oxidative stress of kidney and achieve the curative effect of DN by inhibiting the expression of p47phox.

Materials

Animals

Healthy male SD rats,weighed(180±20)g,SPF level,Changsha Tianqin Biotechnology CO.LTD,License:SCXK (Xiang) 2009 - 0012.After one-week’s adaptive feeding,the rats will be used for experiments which are in the absence of adverse reactions.The experiment followed the standards of the Ethics Committee of Hainan Medical University.

Drugs

AO,the powder of AO,was purchased from Beijing Kang Ren Tang Pharmaceutical Co.Ltd (Beijing,China).The drugs were dissolved with the distilled water to produce the suspension with required concentration.

Reagent

ROS,SOD,MDA and Nox2 kits were provided by Shanghai Xi Tang Biotechnology Co.Ltd (Shanghai,China).40% acrylamide solution,total protein extraction kit (containing Protease Inhibitor Cocktail)and BCA protein quantification kit were purchased from Guangzhou Beogene Biotechnology Co.Ltd(Guangzhou,China); PVDF Membrane (Millipore),anti-p47phoxantibody and reference β-actin were purchased from Santa Cruz (Cal.,USA); IgG-HRP in Sheep Anti-Mice,IgG-HRP in Sheep Anti-Rabbit(Pierce,USA),Super Signal?West Dura Extended Duration Sub-Strategy were purchased from Pierce,(Illinois,USA); X-ray Film (Kodak); TRlzol(Invitrogen)and RNase-Free DNase I(Qiagen);Prime Script TM II 1st Strand cDNA Synthesis Kit and SYBR?Prick Premix Ex TaqTM(Perfect Real Time)were purchased from Takara Biomedical Technology Co.Ltd.(Beijing,China)

Instrument

Protein ultraviolet spectrophotometer (Germany Eppendorf); Mini-PROTEAN Vertical electrophoresis and membrane rotating system (The United States Bio-Rad); Horizontal decoloring shaker (Shanghai);Pipetting device (Gilson);High speed refrigerated centrifuge (Sigma); Oscillator; Yue Zhun Glucose meter typeⅠ(Shanghai Yuyue Company);Ultraviolet spectrophotometer (Eppendorf company); iQTM5 Multiple real-time fluorescence quantification PCR instrument(The United States Bio-Rad company).

Methods

Grouping,Modeling and administration

The 32 rats were randomly divided into blank group,model group,AO group and IRB group according to random number table.In the light of modeling methods in references [14],24 SD rats in model,AO and IRB group received intraperitoneal injection with Streptozotocin (STZ) in the dose of 40 mg/kg one-time which was followed by high energy diet(high glucose and fat diet) feeding for 4 weeks.The other 8 rats in blank group were fed by normal diet without STZ injection.After the successful modeling,Rats drug dosage was converted according to method of human and animal surface area [14].Blank and model group were given distilled water by gavage of 10ml/kg.AO group was gavaged with suspension(dissolved from AO powder) in the dose of 20 g/(kg·d).IRB group was gavaged with IRB in the dose of 10mg/kg.Each group was given gavage once a day,and drinking and dieting freely.The intervention period was 4 weeks.Insulin and other hypoglycemic drugs were not used during the experiment.Keep room temperature between 18 and 20 ℃,humidity between 55% and 65%,with alternative lighting of 12h.

Pathological examination

After the rats were killed,the right kidney was re moved and cut along the sagittal median line.Hal fof the kidney was fixed,embedded,cut into slic esin continuous 20um and stained by HE staining.Then use a light mirror to observe.Renal histop athological changes were observed under light mic roscope.

Blood Glucose and 24h-urinary protein

Fasting blood glucose was collected from veins before dosing,at the end of 2ndand 4thweek.24h urine was collected from the metabolic cages and the urinary protein content was measured before dosing,at the end of 2ndand 4thweek.

Serum indicators

The values of serum ROS,SOD,MDA and Nox2 were determined by the ELISA method,which was performed according to the instructions in the kit.FiftyμL of PBS was added to the blank control wells.To each standard well,50μL of standards with different concentrations were added and the concentrations of each standard(S0-S5)are as follows:0,0.5,1,2,4,and 8 mmol/L.Then,10μL of samples were added to be tested and 40μL of sample diluent was added to sample wells.Then,100μL of detection antibody labeled with HRP was added to each well of the standard and sample wells,the reaction wells were sealed with a microplate sealer,and these were incubated in a water bath or incubator at 37 ℃ for 60min.The plate was washed five times with washing solution.50μL of substrate A and 50μL of substrate B were added to each well and incubated for 15 min at 37℃in the dark.Stop solution of 50μL was added to each well,and within 15 min,the OD value of each well was measured by a THERMO MK3 microplate(USA)at a wavelength of 450 nm.The standard curve was established according to the concentrations of the standards and the OD in the standard wells.The levels of serum lipids were calculated according to the standard curve.

Expression of p47phox by RT-PCR

Total RNA was extracted from kidney tissue using the Trizol RNA extraction kit and the concentration of sample RNA was measured.cDNA synthesis: The mixture of RT enzyme,oligo dT primer,and sample RNA was prepared in 0.2 mL of RNase free Eppendorf tubes and put on ice.The cDNA that was obtained at 37℃after 15 min,85℃after 5s,and 4℃after 60min were diluted by the addition of 180μL of distilled water and this was stored at -20 ℃.Quantitative PCR reaction: based on the published rat p47phoxand β-actin cDNA sequences from Genbank,using Primer Premier 5.0 software and Oligo6 software for homology correlation comparison,the regions with higher homology were selected to design amplification primers for p47phox.The primers were synthesized by Shanghai Biological Co.,Ltd.(China)with the following sequences(Table 1).

Table 1 Designed primer sequences

The PCR reaction mixture was prepared in a PCR tube and operated on ice.The mixture was fully mixed and three replicate controls were produced for each sample.The PCR program was progressed with Roche LightCycler?96 Fluorescence Quantitative PCR Instrument and was set: Stage 1: pre-denaturation at 95℃ for 30 s;Stage 2:PCR reaction at 95℃ for 5 s and 60 ℃for 34 s for 40-45 cycles; Stage 3:dissociation curve at 95℃ for 15 s,60℃ for 60 s,and 95℃ for 15 s.Onboard amplification was used for detection and the relative expression was calculated using the 2-ΔΔCt method.

The expression of p47phox protein were detected by western blotting

Cellular proteins were extracted using cell lysates containing PMSF with the final concentration of 1 mm/L and quantified using the BCA method.Proteins were transferred to a nitrocellulose membrane in SDS-PAGE at a concentration of 20%by switching on the power supply to 350 mA for 2 h.Ponceau red staining was used to observe the effect of protein imaging.The membranes were blocked by adding non-fat dry milk to a final concentration of 5% (w/v)in 1×TBST and shaken at room temperature for 1 h.The membranes were reacted with rabbit primary antibodies,including p47phox(1:1000),followed incubation with the goat anti-rabbit IgG/HRP secondary antibody (1:1000).The protein bands were detected by chemiluminescence using a Qinxiang ChemiQ4600 fluorescence chemiluminescence imager(China) and the results were scanned and analyzed using the automatic gel analysis and imaging software scanning system.The protein bands were analyzed using β-actin as an internal reference.

Statistical analysis

Results were analyzed by statistical software SPSS 17.0.Measurement data are expressed as mean±standard deviation (± s).Analysis of variance was used for comparison.A probability value ofP ＜0.05 was considered statistically significant.

Results

The effect of AO on renal pathological sections

In blank group,the volume and morphology of the glomeruli were normal,the basement membrane was not thickened and the mesangial region wasnot enlarged.In the model group,glomerular swelling and vacuolar changes in glomerular capillary epithelial cells could be observed,mesangial area was slightly widened.There were similar pathological changes in the AO and ARB group compared with the model group,but the degree of injury was reduced.(Figure 1).

The effect of AO on fasting blood glucose and 24h urinary protein

Compared with the blank group,the fasting blood glucose of the other groups was significantly increased (P ＜0.05),and there were no significant difference among the model,AO and IRB groups(P ＞0.05)(Table 2).The results indicates that both AO and IRB couldn't control blood glucose well.

Figure 1 Pathological sections of renal tissue under light microscope in each group (HE staining,400×)

Compared with the blank group,the 24h urinary protein in the model group was significantly increased after administration (P ＜0.05) which indicates that the occurrence of renal damage and the success of modeling.Compared with the model group,AO and IRB significantly reduced the amount of protein in the urine (P ＜0.05) and there was no significant difference between the two groups (P ＞0.05) which indicates that AO has certain curative effect on proteinuria in DN rats,which is similar to IRB (Table 3).

The effect of AO on oxidative stress indicators

SOD content:compared with the blank group,the model group significantly decreased (P ＜0.05).Compared with the model group,AO group and IRB group were significantly increased (P ＜0.05).There was no significant difference between AO group and IRB group(P ＞0.05).

ROS content:compared with the blank group,the ROS content in the model group was significantly increased(P ＜0.05).Compared with the model group,AO group and IRB group decreased significantly(P ＜0.05).There was no significant difference between AO group and IRB group(P ＞0.05).

MDA content:compared with the blank group,MDA content in the model group increased significantly (P＜0.05).Compared with the model group,AO group and IRB group decreased significantly (P ＜0.05).There was no significant difference between IRB group and AO group(P ＞0.05).

Nox2 content:compared with the blank group,Nox2 content in the model group increased significantly.Compared with the model group,AO group and IRB group decreased significantly (P ＜0.05).There was no significant difference between IRB group and AO group(P ＞0.05).(Table 4)

These oxidative stress indicators illustrate that the modeling methods induced oxidative stress in DN rats,AO and IRB significantly alleviated excessive oxidative stress and there was no significant difference between the two groups which indicates that AO could regulate oxidative stress response in DN rats,which is similar to IRB.

Effect of AO on p47phox mRNA expression in t he rat kidney

The expression levels of p47phoxmRNA in the kidney in model group are significantly higher than that in the blank control group (P ＜0.05).The expression levels of p47phoxmRNA in the kidney of AO and IRB group are significantly lower than those in the model group(P ＜0.05),which indicates that AO decreased the levels of p47phoxmRNA and there was no significant difference between IRB group and AO group (P ＞0.05)(Table 5).

The effect of AO on protein expression of p47phox

The band of p47phoxprotein in the model group is significantly wider and darker and the average protein gray value is higher than those in the blank group(P ＜0.05).The band of p47phoxprotein in the AO and IRB group are significantly thinner and lighter and the average protein gray value are lower than those in the model group (P ＜0.05),which indicates that the two drugs can decrease the expression levels of p47phox(Figure 2,Table 6).

Figure 2 Western blotting results of p47phox protein in rat kidney

Table 2 Effect of AO on fasting blood glucose(±s)(n=8,mmol/L)

Table 2 Effect of AO on fasting blood glucose(±s)(n=8,mmol/L)

Compared with the blank group,*P ＜0.05;Compared with model group,#P ＞0.05;Compared with AO group,△P ＞0.05.

Groups Before dosing 2nd week 4th week Blank 7.65±0.63 7.72±0.98 7.61±0.92 Model 24.45±2.31* 24.68±3.45* 24.94±3.81*AO 22.34±1.87# 23.65±2.14# 24.65±2.82#IRB 22.21±1.98#△ 23.29±2.38#△ 24.54±2.23#△

Table 3 Effect of AO on 24h urinary protein(±s)(n=8,mg/24h)

Table 3 Effect of AO on 24h urinary protein(±s)(n=8,mg/24h)

Compared with the blank group,*P ＜0.05;Compared with model group,#P ＜0.05;Compared with AO group,△P ＞0.05.

Groups Before dosing 2nd week 4th week Blank 0.27±0.08 0.28±0.04 0.27±0.05 Model 0.67±0.09* 1.93±0.15* 5.29±1.52*AO 0.42±0.05# 1.35±0.06# 2.01±0.36#IRB 0.40±0.06#△ 1.34±0.07#△ 1.99±0.21#△

Table 4 Effect of AO on oxidative stress indicators(n=8)

Table 5 Effect of AO on p47phox mRNA expression in the rat kidney(n=8）

Table 6 Comparison of mean gray values of p47phox protein in rat kidney

Discussion

Oxidative stress is an important mechanism of DN[15].Oxidative stress refers to the state of the body when the ROS and antioxidant defense system are out of balance when various harmful stimuli attack the body[16] which would cause the body of biological molecules such as nucleic acids,proteins,lipids suffer from oxidative damage [17,18].Studies have shown that oxidative stress plays a primary and independent role in the occurrence and development of DN [19].Oxidative stress of DN refers to ROS from kidney which has been associated with diabetic kidney.High glucose induces excessive production of ROS,which exceeds the body's clearance ability,resulting in the destruction of redox balance in patients with diabetic nephropathy,triggering a series of pathological processes [20].By affecting the renin-angiotensin system and TGF-βsignaling,chronic inflammation and hypertrophy of the glomerular and renal tubules are triggered,mesangial cell accumulation leads to renal fibrosis,extracellular matrix accumulation,thickening of renal tubules and glomerular membranes,dysfunctional podocytes and apoptosis,resulting in proteinuria,glomerulosclerosis and tubulointerstitial fibrosis.SOD is the main component of the antioxidant system and plays an important role in the antioxidant level.Studies have shown that SOD can remove ROS in mitochondria and reduce oxidative stress damage mediated by ROS.When SOD expression is changed or decreased,the body's antioxidant capacity is weakened,ROS cannot be removed in time,and the risk of oxidative stress injury in multiple organs would be increased [21].According to the research,superoxide anion free radical can directly damage the membrane structure of renal cells,thus causing the peroxidation of unsaturated fatty acids,causing cell degeneration,dissolution and death,SOD can promote superoxide anion free radical disproportionation reaction,blocking its damage to renal tissue.The decrease of antioxidant activity caused by oxidative stress is an important mechanism in the pathogenesis of DN,and SOD,as an indicator of antioxidant activity,can reflect the occurrence and development of DN[22].MDA is a reliable and stable indicator of oxidative stress in vivo.MDA content can reflect the degree of lipid peroxidation in the body,and indirectly reflect the degree of cellular damage [23].Nox is the only known exclusive source of ROS formation enzyme family.In animal models of type 1 and type 2 diabetic nephropathy,Nox plays an important role in the occurrence and development of renal injury and is a potential new target.Noxl,Nox2,Nox4 and Nox5 were found in glomerular cells (Mesangial cells,podocytes and capillaries),tubular cells and widely expressed in renal interstitial cells (tubular epithelial cells and interstitial fibroblasts) [24].Nox inhibitor oleander can improve renal blood flow,reduce renal vascular resistance and reduce renal fibrosis by reducing oxidative stress [25].In contrast-induced hypercholesterolemic rats in AKI model,hexone-diesterase phosphate inhibitor inhibits ROS production and thus protects renal tissue by inhibiting Nox activity [26].Nox proteins themselves have little catalytic activity and they need to be regulated by a variety of subunits bound to form a stable complex to play a catalytic role [9].When the cells are stimulated,the cytoplasmic subunits p67phox,p47phox,p40phoxand translocate to the cell membrane and bind to the membrane subunits gp91phoxand p22phoxto produce ROS [27].Further studies have shown that after knockout of the p47phoxgene,the glucose tolerance of the mice increased significantly,and the oxidative stress damage of podocytes,Mesangial cell proliferation and pathological changes such as glomerular hypertrophy were obviously reduced[28].

Conclusion

AO could significantly reduce the amount of 24h urinary protein and alleviate the excessive oxidative stress in the kidney of DN rats.The mechanism may be related to the inhibition of p47phoxexpression.