Evaluation of renoprotective effect of Chinese chive extracts on adenine-induced chronic renal failure

Qiang-Ming Li,Jian-Ping Luo,Li-Hua Pan,Xue-Qiang Zha

School of Food Science and Engineering,Hefei University of Technology,Hefei,230009,People’s Republic of China

Keywords:

ABSTRACT

1.Introduction

Chronic renal failure(CRF)is a progressive and potentially life-threatening renal injury disease that can induce anaemia,bone disease and cardiovascular disease[1,2].The basic clinical manifestation of CRF includes renal detoxification capacity degradation,acid-base equilibrium disturbance,water electrolyte balance disorder and endocrine imbalance[3].The onset of CRF exerts a serious impact on the patient’s survival[4,5].According to the statistics,the number of patients with CRF continues to enhance worldwide.In China,there are about 119.5 million patients with CRF in 2012,with an overall prevalence of 10.8%[6].Meanwhile,in USA,the prevalence rate of CRF was from 13.1% in 2004 up to 14.4% in 2010[7].However,due to the intricate pathogenic factors of CRF,the current therapeutic agents for CRF show poor efficiency and high side effects.Therefore,it is meaningful that finding an effective agent with little side effects to delay the development of CRF.Recently,to achieve this goal,more and more attention has been attracted to develop these agents from herbs or edible-medicinal materials[7,8].

Chinese chive(Allium tuberosum Rottl.Ex Spreng)is a famous daily edible green vegetable for Chinese and widely distributed in China[9,10].Additionally,it has long been used as a traditional Chinese medicine to treat impotence and nocturnal emissions[9].The traditional remedy indicates Chinese chive may have the ability to protect kidney.The previous researches demonstrated that some Allium plants extracts exhibited potential renoprotective effect[11–14].However,no experimental investigations have been carried out to evaluate the renoprotective effect of Chinese chive extracts.Thus,the aim of this study was to investigate the effects of Chinese chive ethanol and water extracts(CCEE and CCWE)on the adenine-induced CRF and its possible functional approach.

2.Material and methods

2.1.Preparation of extract

Chinese chive was purchased from local supermarket in Hefei city of China.The dried power of Chinese chive(1 kg)was extracted with 95% EtOH(10 L)at room temperature(3×1 d).The supernatant was concentrated under reduced pressure to obtain CCEE.The Chinese chive residue was further extracted with H2O(10 L)at 70°C(3×2 h),of which supernatant was concentrated to obtain CCWE.

2.2.Chemicals

Adenine was purchased from Biosharp(Hefei,China).Haikunshenxi capsules were obtained from Huinan Changlong Biochemical Pharmacy Co.,Ltd(Tonghua,China).The assay kits of superoxide

dismutase(SOD),glutathione reductase(GSH)and malonaldehyde(MDA)were purchased from Nanjing Jiancheng Bioengineering Institute(Nanjing,China).

Table 1 The effects of CCWE and CCEE on the body weight of adenine-induced CRF mice.

Table 2 Effects of CCWE and CCEE on renal pathological parameters in mice with adenine-induced CRF.

Fig.2.Effects of CCWE and CCEE on SCr(A)and BUN(B)contents of adenine-induced CRF mice.##P＜0.01(vs.normal group);*P＜0.05,**P＜0.01(vs.model group).

2.3.Study design

Kunming male mice(24±2 g)were purchased from the Laboratory Animal Center of Anhui Medical University.After an adaptation period of one week,mice were randomly divided into nine groups(12 mice per group),including normal group,model group,positive group,CCWE low-dose group(CCWEL),CCWE middle-dose group(CCWEM),CCWE high-dose group(CCWEH),CCEE low-dose group(CCEEL),CCEE middle-dose group(CCEEM),and CCEE highdose group(CCEEH).The normal group was fed a normal diet,while the others groups were fed the diet supplemented with adenine at the dose of 0.2%(w/w).The positive group was orally administered with Haikunshenxi capsule at the dosage of 150 mg/kg/day.The CCWEL,CCWEM and CCWEH groups were orally administered with CCWE by 50,100 and 200 mg/kg/day,respectively.The CCEEL,CCEEM and CCEEH groups were orally administered with CCEE by 50,100 and 200 mg/kg/day,respectively.All mice in all groups were weekly weighed during the experimental period.After five weeks administration cycle,all mice were euthanized with CO2.Serum and kidneys were collected for the analysis of blood biochemistry,pathology,and real-time quantitative polymerase chain reaction(RT-PCR).All animal handling procedures were performed strictly in accordance with the P.R.China Legislation on the Use and Care of Laboratory Animals.

2.4.Biochemical indicators measurements

The levels of serum creatinine(SCr)and blood urea nitrogen(BUN)in plasma were measured using automated analyzer(Accu-check Performa,Roche,Germany).The contents of SOD,GSH and MDA in kidney tissue were determined using commercial kit according to the manufacturer’s instructions.

2.5.Pathological analysis

After fixed in 4% paraformaldehyde,the kidneys were dehydrated in increasing concentrations of ethanol,cleared with xylene and embedded in paraffin.Then,five micrometer sections were prepared from the paraffin blocks and stained with hematoxylin and eosin(H&E)and Masson Trichome(MT)using standard procedures to assess inflammation and fibrosis as described earlier[15,16].

2.6.RT-PCR analysis

The total RNA of mice renal tissues was isolated using TRIZOL reagent(Gibco,BRL).The RNA was reverse transcribed into cDNA using iScriptTMcDNA Synthesis kit.Then,the RTPCR was performed on a BIO-RAD MyIQ2Real Time PCR system(California,USA)according to our reported protocol[17].All primer sequences were listed as follows:tumor necrosis factor alpha(TNF-α),5′-ACGGCATGGATCTCAAAGAC-3′(upper primer)and 5′-AGATAGCAAATCGGCTGACG-3′(lower primer);interleukinone (IL)-1β, 5′-GCAACTGTTCCTGAACTCAACT-3′(upper primer)and 5′-TCTTTTGGGGTCCGTCAACT-3′(lower primer);IL-6,5′-CCTTCCTACCCCAATTTCCAA-3′(upper primer)and 5′-AGATGAATTGGATGGTCTTGGTC-3′(lower primer);IL-10,5′-ACTGCACCCACTTCCCAGT-3′(upper primer)and 5′-TGTCCAGCTGGTCCTTTGTT-3′(lower primer); GAPDH,5′-GAAGGGTGGAGGCAAAAG-3′(upper primer) and 5′-ACCAGTGGTTGCAGGGAT-3′(lower primer).The mRNA levels of genes were normalized to that of GAPDH and presented as relative to the normal.

2.7.Statistical analysis

All experiments were carried out independently in triplicates and the data were expressed as mean±SD values.All data were analyzed statistically using one-way analysis of variance(ANOVA)followed by Tukey’s multiple-comparisons tests.Significant differences were set at P＜0.05.

3.Results and discussion

3.1.The effects of CCWE and CCEE on the body weight of CRF mice

The body weight of each experimental group was weekly recorded and shown in Table 1.In the first two weeks,the similar increase of mice body weight could be found in all groups.From the third week,the mice body weight of normal group was continuously increased.Meanwhile,the body weight of mice in model group was significantly reduced by the persistent treatment of adenine.The adenine-induced body weight reduction could be alleviated by the concomitant treatment of CCWE,although the mice body weight of CCWE groups was also decreased.In contrast,the body weight of CRF mice was slightly reduced by the concomitant treatment of CCEE.

3.2.The effects of CCWE and CCEE on the renal morphology and pathology of CRF mice

As shown in Fig.1A,the kidneys of mice in normal group were horse bean-shaped and henna-coloured with an obvious polish.Meanwhile,the kidneys of adenine-induced CRF mice were atrophic and pallid with a hoarfrost appearance.However,the hoarfrost appearance could be mitigated by CCWE in a dose-dependent manner.When the dose of CCWE reached 200 mg/kg/day,only subtle change could be found in renal morphology in comparison with that of normal group.But the renal morphology of CRF mice could not be improved by CCEE.These results indicated CCWE could alleviate the kidney damage of adenine-induced CRF mice,and was the main renoprotective fraction of Chinese chive.

In the present work,H&E and MT staining were further executed to analyze the effects of CCWE and CCEE on the renal pathology of CRF mice.As shown in Fig.1B,C,normal renal parenchyma with glomeruli and tubule could be observed in the kidney sections of normal group.Meanwhile,extensive inflammatory cell infiltration and interstitial fibrosis could be found in model group(Fig.1B,C and Table 2).However,the renal pathological damages of CRF mice could be significantly mitigate by CCWE in a dosedependent manner,but not CCEE.When the concentration of CCWE reached 200 mg/kg/day,the area of renal pathological damage was decreased to the 48.1% of model group.Thus,it was further confirmed that CCWE was the main renoprotective fraction of Chinese chive.

3.3.The effects of CCWE and CCEE on the blood biochemical index of CRF mice

The levels of serum SCr and BUN were considered as the important index to reflect the renal filtration function[18–20].To further evaluate their renoprotective function,the effects of CCWE and CCEE on the serum SCr and BUN levels of CRF mice were investigated.Compared to that of normal group,the serum SCr and BUN levels of model group were significantly increased by the treatment of adenine(Fig.2).However,it could be found that these enhancements were remarkably inhibited by CCWE in a dose-dependent manner,but not CCEE.Especially in the CCWEH group,the serum SCr and BUN were decreased to the 87.7% and 83.9% of model group,respectively.The results further confirmed that CCWE could improve the kidney function of the adenine-induced CRF mice,and was the main renoprotective fraction of Chinese chive.The previous study indicated that the main chemical constituents of CCWE are macromolecule polysaccharides and CCEE are small molecule compounds,including pyrazines,lignan and flavonoids[21,22].The chemical constituent difference of CCEE and CCWE might be the reason of these two extracts exhibiting different effects on the kidney function of CRF mice.

3.4.The effects of CCWE and CCEE on the antioxidant index of CRF mice

Fig.3.Effects of CCWE and CCEE on SOD(A),GSH(B)and MDA(C)contents in kidney tissue homogenate of adenine-induced CRF mice.##P＜0.01(vs.normal group);*p＜0.05,**P＜0.01(vs.model group).

Oxidative stress plays an important role in the pathogenesis of renal diseases,including CRF[23–25].To clarify the underlying mechanism of CCWE and CCEE,their effects on the antioxidant index of CRF mice,such as SOD,GSH and MDA,were researched.As shown in Fig.3,compared to those of normal group,the levels of SOD and GSH in model group were significantly raised by the treatment of adenine,and MDA was reduced.However,the alteration of SOD,GSH and MDA levels in CRF mice could be remarkably reversed by CCWE in a dose-dependent manner,but not CCEE.When CCWE dosage reached 200 mg/kg/day,comparing to those of model group,the levels of SOD and GSH were increased by 8.9%and 8.7%,and MDA was decreased by 12.1%.These results suggested that the antioxidant ability of CRF mice could be enhanced by CCWE,which might be one of the renoprotective mechanisms of CCWE.

Fig.4.Effects of CCWE and CCEE on mRNA level of inflammatory cytokines in kidney tissue homogenate of adenine-induced CRF mice.##P＜0.01(vs.normal group);*P＜0.05,**P＜0.01(vs.model group).

3.5.The effects of CCWE and CCEE on the inflammation of CRF mice

As a complicated self-defense process,inflammation also plays a key role in the pathogenesis of CRF[23,26,27].Thus,the effects of CCWE and CCEE on the inflammation of CRF mice were investigated.It could be found that the mRNA levels of pro-inflammatory cytokines TNF-α,IL-1β and IL-6 in model group were significantly increased by the treatment of adenine when compared to those of normal group,and the anti-inflammatory cytokine IL-10 was decreased(Fig.4).However,these changes of CRF mice could be remarkably reversed by CCWE in a dose-dependent manner,but not CCEE.When the concentration of CCWE reached 200 mg/kg/day,the mRNA levels of TNF-α,IL-1β and IL-6 were decreased to the 77.2%,72.8% and 73.4% of model group,respectively,and IL-10 was increased to 122.6%.These results were incorporated with the phenomenon observed in the H&E staining,which exhibited that the adenine-induced inflammatory cells infiltration was inhibited by CCWE(Fig.1B,C and Table 2),to confirm that the inflammation of CRF mice could be inhibited by CCWE.

4.Conclusion

In summary,the present work demonstrated that CCWE could improve the kidney function of the adenine-induced CRF mice via enhancing antioxidant ability and inhibiting inflammation,and was the main renoprotective fraction of Chinese chive.These results indicated that CCWE might be used to develop renoprotective functional food supplement to alleviate CRF in the future.

Conflicts of interest

The authors declare no conflicts of interest.

Acknowledgements

This work was financially supported by the National Natural Science Foundation of China(Grant No.31271814;No.21702040)and the Fundamental Research Funds for the Central Universities(Grant No.JZ2017HGPB0169;No.2014HFCH0011).