Effects of Manshenkangning Prescription on Adenine-induced Renal Interstitial Fi

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1. Guangdong Traditional Medical and Sports Injury Rehabilitation Research Institute, Guangdong Second Provincial General Hospital, Guangzhou 510317, China; 2. Center for Drug and Clinical Research, Southern Hospital, Southern Medical University, Guangzhou 510515, China; 3.Department of Traditional Chinese Medicine, the First Affiliated Hospital of Jinan University, Guangzhou 510632, China; 4. Department of Urology, Shanghai Zhongye Hospital, Shanghai 201900, China

Abstract [Objectives] To study the protective effects of Manshenkangning Prescription on adenine-induced renal interstitial fibrosis in rats, and explore the possible mechanism. [Methods] Sixty Wistar male rats were divided into normal group, model group, control group (administered with 10 mg/(kg·d) losartan) and high, medium and low dose experimental groups (30, 15, 7.5 mg/(kg·d) Manshenkangning). The rat models of renal interstitial fibrosis were induced by intragastric administration of adenine (250 mg/(kg·d)). After 2 h, the above drugs were administered intragastrically for 21 consecutive days and the administration time was 30 consecutive days. Serum creatinine (SCr), blood urea nitrogen (BUN), 24 h urinary protein (24 h MTP) and glomerular filtration rate (eGFR) were measured by biochemical method; renal histopathological changes were observed by hematoxylin-eosin (HE) staining. Renal collagen deposition in rats was observed by Masson staining. [Results] The SCr in model group and the high, medium and low dose experimental groups were (340.00±22.99), (176.80±18.60), (234.75±13.59), (266.11±14.78) μmol/L, and BUN were (23.74±2.51), (14.53±2.25), (18.78±0.88), (18.90±2.14) mmol/L; 24 h MTP were (675.86±74.58), (323.81±41.83), (438.84±34.69), (493.76±37.04) mg/d; eGFR were (19.30±2.48), (49.96±10.95), (32.61±10.75), (27.18±5.98) mL/min, and the difference was statistically significant compared with the normal group (all P<0.05). HE staining and Masson staining showed that compared with normal group, the renal interstitial lesions in model group were severe and the renal interstitial collagen material was deposited in a large amount. The renal interstitial tubule injury was relieved and the renal interstitial collagen deposition was reduced in experimental groups. And the difference was statistically significant (all P<0.01). [Conclusions] Manshenkangning can significantly protect the kidney against the progress of interstitial fibrosis in rats. Its possible mechanism is to regulate the activity of SIRT1 and inhibit the expression of COX-2 in order to resist the inflammatory reaction of kidney and improve the ability of anti-oxidative stress of kidney, thus delaying the occurrence and development of chronic renal failure.

Key words Manshenkangning, Renal interstitial fibrosis, Oxidative stress, Inflammatory reaction, Silent information regulator 1 (SIRT1), Cyclooxygenase-2 (COX-2)

1 Introduction

As the main pathological change of chronic kidney disease (CKD), renal interstitial fibrosis is also the common pathogenic outcome of the development of all kidney diseases[1]. Cyclooxygenase-2 (COX-2) is an induced enzyme that is closely related to the development of inflammation, while the silent information regulator (SIRT1) is a protective factor for renal immune oxidative stress injury[2-3]. In this experiment, it is intended to study the protective effects of Manshenkangning Prescription on adenine-induced renal interstitial fibrosis in rats, and explore the possible action mechanism.

2 Materials and methods

2.1 Materials

2.1.1Animals. Sixty male Wistar rats, SPF grade, weighing 160-200 g, were purchased from the Research Center for Laboratory Animal of Southern Medical University. Animal License Number: SCXK (Yue) 2006-0015.

2.1.2Drugs and reagents. Manshenkangning (main ingredients: Astragali Radix, Codonopsis Radix, and Poria,etc.) donated by Sanjiu Medical & Pharmaceutical Co. Ltd.; Losartan Potassium Tablets, specification: 100 mg/tablet, batch No.2010037, produced by Hangzhou MSD Pharmaceutical Co.,Ltd.; Adenine (C5H5N5), purity >98%, batch No.MC1025B0510J, produced by Sangon Biotech (Shanghai) Co., Ltd.; Trizol reagent kit, produced by American Invitrogen; reverse transcription reaction reagent and PCR reaction reagent, produced by TIANGEN Biotech (Beijing) Co., Ltd.

2.1.3Instruments. Chronmo4 full automatic fluorescent quantitative polymerase chain reaction (PCR) system, product of American MJ Research; 9600PCR amplification system, product of American PE.

2.2 Methods

2.2.1Animal modeling, grouping and drug administration. Rats (n=7) were randomly selected according to their weight using a random number table method as the normal group and the rest were model groups (n=53). According to previous studies, a model of adenine-induced renal interstitial fibrosis in rats was established[4]. After successful modeling,rats were randomly divided into model group, control group (losartan 10 mg/(kg·d), concentration 0.2%) and high, medium and low dose experimental groups (Manshenkangning 30 mg/(kg·d), concentration 300%; 15 mg/(kg·d), concentration 150%; 7.5 mg/(kg·d), concentration 75%), converted to the equivalent dose of high dose in rats as per the dose for human use. Within 2 h after animal modeling, rats were administered intragastrically. Rats in normal group and model groups were administered intragastrically with equivalent volume of distilled water (0.5 mL/100 g). The modeling period was 21 consecutive days and the administration time was 30 consecutive days. After the completion of intervention, the blood was collected and the serum was separated. After the rats were killed, the kidney tissue was taken 1/4 of the left kidney and fixed in 4% paraformaldehyde, and stored under normal temperature conditions. The remaining kidney tissue was wrapped with tin foil, marked and placed in a refrigerator at -80 ℃.

2.2.2Detection of serum creatinine (SCr), blood urea nitrogen (BUN), and 24 h urinary protein (24 h MTP) levels. SCr, BUN, and 24 MTP of rat serum were detected using automatic biochemical analyzer.

2.2.3Observation of pathological changes in renal tissue. With reference to literature[5], pathological changes in renal tissue were observed by HE staining method. In short, kidney tissue was fixed, dehydrated, waxed, embedded, dewaxed, dehydrated, stained, differentiated, washed, and re-stained. The stained sections were dehydrated again, cleared, and sealed and the pathological changes of renal tissues were observed under a light microscope (×200).

2.2.4Observation of renal tissue collagen deposition. With reference to literature[5], the renal tissue collagen deposition were observed by Masson staining method. The kidney tissue was fixed, dehydrated, waxed, embedded, sliced, mordanted 2, stained, ethanol differentiated, stained, weakly acid solution, dehydrated, cleared, and sealed, and the collagen deposition was observed under an optical microscope (×200).

2.2.5Detection of SIRT1 and COX-2 protein expression in rat kidney. With the reference to the literature[6], immunohistochemical staining (IHC staining) was applied to detect gray values with the aid of Motic Images Advanced 3.0 image analysis software, semi-quantitative analysis was carried out on the positive expression intensity of SIRT1 and COX-2 proteins in normal group, model group, control group and high, medium, and low dose experimental groups. The greater the gray value, the weaker the positive expression; otherwise, the positive expression will be stronger.

2.2.6Detection of SIRT1 mRNA and COX-2 mRNA expression. With the reference to the method in literature[6], the expression levels of SIRT1 mRNA and COX-2 mRNA in normal group, model group, control group and high, medium and low dose experimental groups were detected. The relative expression ofSIRT1andCOX-2was calculated by 2-ΔΔCTmethod.

3 Results and analysis

3.1 Effects on renal function of rats in each groupAfter the drug intervention, compared with the normal group, the levels of SCr, BUN, and 24 h MTP of the model group, the control group, and the high, medium, and low dose experimental groups showed an increasing trend, but the eGFR declined significantly (P<0.01). Compared with the model group, the levels of SCr, BUN and 24 h MTP in the control group and the low, medium and high dose experimental groups showed a significant decline (P<0.05), while eGFR was significantly increased (P<0.01). However, there were no significant differences in SCr, BUN, 24 h MTP and eGFR between the control group and the low, medium and high dose experimental groups (allP>0.05), as indicated in Table 1.

GroupnSCr ∥μmol/LBUN∥mmol/L24 h MTP ∥mg/deGFR∥mL/minNormal734.83±4.126.10±1.01115.67±5.7198.20±9.99Control6174.67±11.00##∗∗14.83±2.08##∗∗303.37±52.80##∗∗51.17±3.54##∗High dose experimental10176.80±18.60##∗∗14.53±2.25##∗∗323.81±41.83##∗∗49.96±10.95##∗∗Medium dose experimental9234.75±13.59##∗∗18.78±0.88##∗∗438.84±34.69##∗∗32.61±10.75##∗∗Low dose experimental9266.11±14.78##∗∗18.90±2.14##∗∗493.76±37.04##∗∗27.18±5.98##∗∗Model8340.00±22.99∗∗23.74±2.51∗∗675.86±74.58∗∗19.30±2.48∗∗

Note: SCr: Serum creatinine; BUN: Blood urea nitrogen; 24 h MTP: 24 h microalbumin; eGFR: Glomerular filtration rate; compared with normal group,*P< 0.05,**P<0.01; compared with model group,#P<0.05,##P<0.01.

3.2 Effects of renal pathological changes in rats with renal interstitial fibrosisNormal group: the morphological structure of glomeruli and renal tubules was normal, and the two were closely arranged and the ratio of renal interstitial was normal, and no obvious inflammatory cell infiltration was observed. Model group: the renal tubules were sparsely arranged, and even expansion and atrophy appeared alternately; a large number of inflammatory cells infiltrated in the renal interstitium, epithelial degeneration, necrosis and bundles, reticular and multifocal interstitial fibrosis, and there was much crystallization and deposition of many golden purine metabolite. In the high, medium and low dose experimental groups, the infiltration of inflammatory cells was less in the visual field, the renal tubules showed signs of improvement, and the crystallization of golden purine metabolites was gradually reduced. The control group and high dose experimental group showed similar pathological changes in the renal tissue, as shown in Fig.1.

Note: A: Normal; B: Model; C, D, E: Low, medium, high dose experimental; F: Control.

Fig.1 Comparison of each group of rat HE staining(×200)

3.3 Effects on collagen in rats with renal interstitial fibrosis

Normal group: no abnormal pathological changes were observed in the renal tissue field, in other words, tehre was no collagen in the renal interstitial or only a small amount of filament deposition; Model group: a large amount of collagen fibers accumulated in the glomeruli, renal interstitial and epithelial cells (collagen staining is blue), and the area was significantly increased, and at the same time, a markedly atrophic renal tubule was observed. In the other groups, the collagen deposition was improved by varying degrees after administration of different doses of the drug, as shown in Fig.2.

Note: A: Normal; B: Model; C, D, E: Low, medium, high dose experimental; F: Control.

Fig.2 Comparison of each group of rat Masson staining(×200)

3.4 Effects on oxidative stress and inflammatory reaction in rats with renal interstitial fibrosisThrough comparing the H2O2, GSSG and GSH indicators of the high, medium and low dose experimental groups and the control group and the model group, it showed that the difference was statistically significant (allP<0.01); through comparing the H2O2, GSSG and GSH indicators in Manshenkangning high, medium, and lose dose experimental groups and the control group, there were no significant differences (allP>0.05), but the high dose experimental group could significantly improve the levels of GSSG, GSH and H2O2, as shown in Table 2.

GroupnGSH ∥μmol/LGSSG∥μmol/LH2O2∥mmol/g/protNormal70.94±0.53∗0.46±0.36∗∗6.86±1.84∗∗Control60.43±0.040.31±0.017.29±2.49High dose experimental100.63±0.450.45±0.357.08±2.32Medium dose experimental90.53±0.310.43±0.258.21±3.16Low dose experimental90.55±0.570.31±0.078.43±3.62Model80.44±0.06#0.29±0.02##9.88±4.13##

Note: GSH: Glutathione; GSSG: Glutathione disulfide; H2O2: Hydrogen peroxide; compared with normal group,*P<0.05,**P<0.01; compared with model group,#P<0.05,##P<0.01.

3.5 Effects on renal tissue SIRT1 and COX-2 of rats with renal interstitial fibrosisRenal medulla interstitium is found in the distribution of SIRT1 and COX-2, and most of them are gathered in the cytoplasm of proximal tubular epithelial cells, and their brown/dark brown expression is positive expression. Results are listed in Table 3.

%

Note: compared with normal group,*P<0.05,**P<0.01; compared with model group,#P<0.05,##P<0.01.

3.6 Effects on renal tissue SIRT1 mRNA and COX-2 mRNA of rats with renal interstitial fibrosisCompared with the normal group, the expression level of SIRT1 mRNA in the renal tissue of the model group significantly declined (P<0.01). Compared with the model group, the expression of SIRT1 mRNA in the high, medium and low dose experimental groups and the control group showed a significant increase (P<0.05), but there was no significant difference between each group (P>0.05), as indicated in Table 4.

GroupnSIRT1 mRNACOX- 2 mRNANormal713.58±4.12∗0.24±0.16∗∗Control610.93±3.241.36±0.60High dose experimental1011.86±3.851.11±0.53Medium dose experimental97.23±3.141.78±0.62Low dose experimental97.01±3.2482.84±0.54Model86.35±2.98#3.15±0.62##

Note: compared with normal group,*P<0.05,**P<0.01; compared with model group,#P<0.05,##P<0.01.

4 Discussions

The progression and development of renal interstitial fibrosis is a common path of end-stage renal failure[7-8]. According to results of this experiment, Manshenkangning can significantly improve the renal function of renal interstitial fibrosis in rats, including SCr, BUN, 24 h MTP and eGFR levels, and the prescription has the functions of strengthening the body resistance to eliminate pathogenic factors, combining the dredging and tonifying, invigorating the spleen and benefiting the kidney, and clearing away the dampness and dispelling the turbidity. This experiment further explored the intrinsic mechanism of Manshenkangning Prescription on anti-oxidative stress and inflammatory reaction in rats with renal interstitial fibrosis. Compared with the model group, the high, medium and low dose experimental groups had mitigation of renal interstitial fibrosis injury and inflammatory cell infiltration, and the levels of SCr, BUN and 24 h MTP in rats were significantly decreased, while eGFR levels were increased, so the kidney functions were extended. Besides, immunohistochemistry and RT-PCR results indicated that the high, medium and low dose experimental groups could up-regulate SIRT1 protein expression and down-regulate COX-2 protein expression, and the high dose experimental group was more obvious in the improvement of the above expressions. However, the expression of COX-2 in the control group was lower than that in the model group, and the expression of SIRT1 was increased. However, the difference between the high-dose experimental group and the control group was not statistically significant. In summary, Manshenkangning can overcome the oxidative stress and inflammatory reaction through interfering with the SIRT1/COX-2 signaling pathway, thereby delaying the progression of renal interstitial fibrosis in chronic renal diseases.