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Original Article Protective effects of She Jing Xiao Bai capsule on diabetic nephropathy in a diabetic rat model

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Original Article

Protective effects of She Jing Xiao Bai capsule on

diabetic nephropathy in a diabetic rat model

Ouyang Jin, Leiping Gao, Xiao Chen, Ying Zhu, Yufeng Zhou, Weiming Wu, Mei Dai, Chunhua Wang, Lili Xie, Xiuping Xiong

Department of Internal Medicine, Changshu Hospital of Traditional Chinese Medcine (New Area Hospital), Changshu 215500, Jiangsu, China

Received August 14, 2015; Accepted January 23, 2016; Epub February 15, 2016; Published February 29, 2016

Abstract: Objective: To investigate the protective effects and mechanism of She Jing Xiao Bai capsule, a traditional Chinese medicine, on diabetic nephropathy (DN) in a rat diabetes model. Methods: Diabetes was induced in 32 male Sprague-Dawley rats by intraperitoneal injection of streptozotocin (STZ). Rats were randomly allocated to a control group, and groups given She Jing Xiao Bai capsule, irbesartan, or She Jing Xiao Bai capsule plus irbesartan. Serum creatinine and 24 h urine protein were assayed, and renal pathology was evaluated at 2, 4, 6 and 8 weeks after treatment. Transforming growth factor-β (TGF-β) and connective tissue growth factor (CTGF) were assayed by immunohistochemistry. Spleen CD4+Foxp3+T cells were assayed by flow cytometry. Results: Urine protein levels were significantly lower in rats treated with She Jing Xiao Bai capsule, irbesartan, or She Jing Xiao Bai capsule plus irbesartan than in control rats. Renal TGF-β and CTGF expression decreased, and splenic CD4+Foxp3+T cells in-creased, with treatment. The strongest effect was observed following treatment with She Jing Xiao Bai capsule plus irbesartan. Conclusion: She Jing Xiao Bai capsule significantly reduced renal injury in this STZ-induced DN model. Treatment was associated with decreased TGF-β and CTGF expression and increased numbers of CD4+Foxp3+T cells.

Keywords: Diabetic nephropathy, She Jing Xiao Bai capsule, urine protein, CD4+Foxp3+T cells

Introduction

The worldwide prevalence of type 2 diabetes mellitus (T2DM) is expected to rise along with increasing urbanization and aging of the popu-lation. Diabetic nephropathy (DN) is a common microvascular complication of T2DM, and is estimated to occur in 40% of diabetes pati- ents. The pathogenesis of DN is not fully under-stood, but may include innate immune respons-es [1]. The formation of immune complexrespons-es such as NLRP3 inflammasomes in patients with hyperuricemia leads to hyperplasia and thickening of the glomerular basement mem-brane and mesangial matrix [2, 3]. Regulatory T cells (Tregs), a subpopulation of T cells previ-ously called suppressor T cells, modulate the immune system, maintaining tolerance to self-antigens, and preventing autoimmune disease [4]. Tregs suppress or downregulate the stimu-lation and proliferation of effector T cells. Lower Treg counts have been observed in T2DM pati- ents than in healthy controls, with significant reductions associated with disease

progres-She Jing Xiao Bai capsule used in this study were prepared at our hospital from purified Chinese medicines and contained black soy-bean, atractylodes, astragalus, Chinese yam, ant powder, and hirudo powder. We previously reported that treatment with She Jing Xiao Bai capsule plus irbesartan significantly reduced urine protein levels in a diabetes model [6]. However, the efficacy of She Jing Xiao Bai cap-sule monotherapy in a DN model, especially the effect on immune function is largely unknown. In this study, the protective effect of She Jing Xiao Bai capsules was investigated in an animal T2DM model with the objective of providing experimental data supporting clinical applica-tion in DN patients.

Materials and methods

Animal model and treatments

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strep-sodium citrate buffer, pH 4.2). The rats weighed approximately 220 g and were fasted for 12 h before treatment, but had free access to water. After 72 h, blood was collected from the tail vein to measure fasting blood glucose (FBG). FBG concentrations >16.7 mM for 10 consecu-tive days indicated successful development of the diabetes model. A 24 h urine protein con-centration >30 mg indicated the presence of DN. Thirty-two rats developed DN and were ran-domly allocated in equal numbers (n=8) to a model control group and experimental groups given She Jing Xiao Bai capsule, irbesartan, or She Jing Xiao Bai capsule plus Irbesartan. All treatments were administered intragastrically. She Jing Xiao Bai was given at a dose of 2 g/kg/d; each capsule contained black soybean (0.04 g), atractylodes (0.04 g), astragalus (0.04 g), Chinese yam (0.04 g), ant powder (0.04 g), and hirudo powder (0.02 g). Irbesartan was administered at 90 mg/kg/d as monotherapy, and the rats given combined therapy received 2 g/kg/d She Jing Xiao Bai capsule plus 90 mg/kg/d Irbesartan. The model control rats were given saline. Treatment continued for 8 weeks. Urine samples were collected at 2, 4, 6, and 8 weeks, and blood was collected from the tail vein at 2, 4, 6 weeks. At 8 weeks, animals were sacrificed by decapitation. At that time, blood was collected from the heart, and kidney tissue was obtained and either fixed in 10% formalin for hematoxylin-eosin (HE) staining and immunohistochemistry or frozen in liquid nitrogen for western blotting and quantitative real-time polymerase chain reaction (qRT-PCR). Rats were not given insulin to control the blood glucose level. This study was approved by Hospital of Changshou City New District.

Biochemical assays

A metabolic cage (Taichang Co, Ltd, China) was used to collect urine. Urine protein level was determined by the biuret method and serum

creatinine (SCr) was assayed by the saturated picric acid method following the kit manufac-turer’s instructions (Sichuang Maike Techno- logy, China).

Immunohistochemistry

Fixed renal tissue was embedded in optimum cutting temperature compound (OCT) and sec-tioned at 5 μm for staining. The immunohisto-chemistry procedure was conducted following the staining kit manufacturer’s instructions (Beijing Zhongshan Biotech, Co, Ltd, China). Western blotting

A 100 mg sample of renal column tissue taken from frozen kidney tissue was homogenized in proteolysis buffer and the total protein was iso-lated by centrifugation. The proteins in aliquots (50 μL) prepared from tissue of rats in each experimental group were separated by sodium dodecyl sulfate polyacrylamide gel electropho-resis (SDS-PAGE) and transferred to nitrocellu-lose membranes for staining. The membranes were incubated overnight with anti-transform-ing growth factor beta (TGF-β, 1:1000; Beijanti-transform-ing Zhongshan Biotech, Co, Ltd, China) and anti-connective tissue growth factor (CTGF, 1:1000; Beijing Zhongshan Biotech, Co, Ltd, China). After washing, secondary antibody (1:5000) was applied for 1 h. The optical density of the blots was normalized against actin.

Electron microscope

Fresh tissue was fixated in 2.5% glutaralde-hyde. After washed with 0.01 mol/L PBS buffer, the tissues were dehydrated in alcohol, embed-ded in EPOR812, microtomed, stained with acetic acid uranium and citromalic acid and observed by an electron microscope (Hitachi, Japan) with the magnification of 10000×. qRT-PCR

mRNA was isolated from kidney tissue of each group using a trizol agent kit (Beijing Zhong shan Biotech, Co, Ltd, China) and the RNA con-tent was assayed by ultraviolet spectrophotom-etry (Puruisi Instrument Co, Ltd, China) (A260/ A230>1.8). Reverse transcription was carried out using random primer and Moloney murine leukemia virus reverse transcriptase (Promega, Madison, USA). The PCR assay of TGF-β and CTGF was performed with a quantitative

ther-Table 1. Effects of treatment on FBG level FBG (mmol/l)

Group n Before treatment At 8 weeks

G1 8 15.35±4.73 16.45±2.34

G2 8 16.05±2.68 16.04±2.29

G3 8 16.76±3.80 14.87±2.80

G4 8 15.76±2.24 14.65±2.60

[image:2.612.91.290.84.164.2]
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mal cycler (Mastercycler ep realplex, Eppen- dorf, Germany) using the following procedure: 50°C 20 min (×1), 95°C 10 min (×1), 94°C 15 s, 46.4°C 20 s, 72°C 30 s (×45). Relative ex- pression was calculated as the ratio of target cDNA to GAPDH. The primers used were TGF-β (449 bp) sense 5’-TGAACCAAGGAGACGGAA- TA-3’ and antisense 5’-CACGCAGCACGGTGATG- 3’; CTGF (533 bp) sense 5-’CCGCCAACCGCAA- GATT-3’ and antisense 5’-CTTGGCAATTTTAGG- CGTCC-3’; and GAPDH (115 bp) sense 5’-AT- CGTGGAAGGGCTAATG-3’ and antisense 5’-ATC- GTGGAAGGGCTAATG-3’.

CD4+Foxp3+T in spleen

Fresh spleen cell suspensions were prepared, and red blood cells were hemolyzed using lysis buffer. The CD4+Foxp3+T cells were counted in suspensions containing at least 106 cells labeled with fluorescein isothiocyanate (FITC)-conjugated anti-CD4, allophycocyanin (APC)-conjugated anti-CD25, anti-CD16/23 and phy-coerythrin (PE)-conjugated anti-Foxp3 antibodi- es.

Statistical analysis

All data were reported as means ± standard deviation (SD). The statistical analysis was

car-saline) the urine protein level increased gradu-ally, and was significantly higher at weeks 4, 6, and 8 than it was at week 2. Urine protein levels increased in rats treated with She Jing Xiao Bai capsule, but the rate of increase was much slower than that observed in the model control group. At week 8, urine protein levels in the She Jing Xiao Bai capsule, irbesartan, and She Jing Xiao Bai capsule plus irbesartan groups were significantly lower than that in the model con-trol group (Table 2).

She Jing Xiao Bai capsule decreased SCr in DN rats

SCr levels were measured in each group at 2, 4, 6 and 8 weeks. In the model control group, SCr levels increased gradually, and were significant-ly higher at weeks 4, 6, and 8 than at week 2. At week 8, SCr levels in the She Jing Xiao Bai cap-sule, irbesartan, and She Jing Xiao Bai capsule plus irbesartan groups were significantly lower than that in model control group (Table 3). Kidney pathology after She Jing Xiao Bai cap-sule treatment

[image:3.612.89.342.98.176.2]

Morphological changes in kidney tissue were monitored by evaluation of HE-stained sec-tions. Compared with normal tissue, glomeruli

Table 2. The effects of treatment on 24 h urine protein levels in DN rats

24 h urine protein (mg)

Week G1 G2 G3 G4

2 63.4±17.5 59.4±15.2# 43.4±12.5# 39.2±11.4# 4 73.4±16.7* 62.3±16.3# 52.1±13.5#,* 44.3±14.4#,* 6 123.2±20.2* 64.4±19.2# 63.1±13.3#,* 60.4±13.3#,* 8 145.2±12.3* 73.3±15.4#,* 70.4±14.3#,* 62.7±19.5#,* #P<0.05 vs. G1 at the same time; *P<0.05 vs. the same group at week

2. G1, model control; G2, She Jing Xiao Bai capsule; G3, irbesartan; G4, She Jing Xiao Bai capsule plus irbesartan.

Table 3. The effects of treatment on Scr level (mg) in DN rats

Week G1 G2 G3 G4

2 173.4±32.4 129.3±25.5# 143.3±22.5# 139.3±31.0# 4 214.3±36.2* 132.1±29.2# 132.2±33.3# 128.3±23.5# 6 227.3±19.4* 140.3±39.4# 149.1±19.3# 145.2±33.9# 8 240.3±27.4* 137.4±35.3# 150.3±24.5# 142.4±21.4# #P<0.05 vs. G1 at the same time; *P<0.05 vs. the same group at week

2. G1, model control; G2, She Jing Xiao Bai capsule; G3, irbesartan; G4, She Jing Xiao Bai capsule plus irbesartan.

ried out by ANOVA with post hoc Turkey test using SPSS 12 (SPSS, Chicago, USA) P<0.05 was considered statisti-cally significant.

Results

FBG level was not affected by the ex-perimental treatments

FBG levels were determined in each group before and at 8 weeks after treatment. There were no significant differences in FBG levels among the groups of rats with DN before the start of treatment or after 8 weeks of treatment Moreover, the experimental treatments did not affect the FBG lev-els within each group (Table 1). She Jing Xiao Bai capsule decreases urine protein in DN rats

[image:3.612.92.341.254.322.2]
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of DN rats had an increased diameter with expansion of capillary loops, increased cellular-ity and expansion of the matrix in the glomeru-lar mesangial area, and lymphocyte and mono-nuclear cell infiltration. The epithelium of the renal tubules had begun to degenerate, and glomerular sclerosis was observed. In contrast, the pathological changes in the She Jing Xiao Bai capsule, irbesartan group, and She Jing Xiao Bai capsule plus irbesartan group were less frequent and less severe (Figure 1A). Electron microscopy showed that in DN model control rats, the glomerular basement mem-brane was thickened and matrix was expanded. Other changes consistent with DN included fusion of epithelium podocyte feet, amplifica-tion of the mesangial region, and deposiamplifica-tion of highly electron-dense substance. In contrast, those characteristics were attenuated by treat-ment with She Jing Xiao Bai capsule, tan, and She Jing Xiao Bai capsule plus irbesar-tan (Figure 1B).

She Jing Xiao Bai capsule decreased TGF-β

and CTGF mRNA expression in DN rats

The expression of TGF-β and CTGF mRNA was assayed by qRT-PCR. As shown in Figure 2A, She Jing Xiao Bai capsule, Irbesartan, and She Jing Xiao Bai capsule plus Irbesartan signifi-cantly down-regulated TGF-β and CTGF mRNA expression.

Immunohistochemical staining revealed that TGF-β was expressed primarily in the cytoplasm

and matrix of hypertrophic glomeruli and rarely in renal tubules. Compared with the model con-trol group, treatment with She Jing Xiao Bai cap-sule, irbesartan, and She Jing Xiao Bai capsule plus irbesartan significantly decreased TGF-β and CTGF expression (Figure 2B). These stain-ing results were consistent with the results of Western blotting (Figure 2C).

She Jing Xiao Bai capsule increased the num-ber of CD4+Foxp3+T cells in DN rats

Flow cytometric analysis of spleen cell popula-tions labeled with fluorescent antibodies re- vealed that the numbers of Treg cells in each of the three experimental groups was significantly higher than in the model control group (Figure 3A, 3B).

Discussion

[image:4.612.94.525.76.246.2]

DN is one of the most severe complications of DM, and is also an important step in the patho-genesis of more advanced renal disease [7]. Because damage to glomerular vessels is a major contributor to death in end-stage renal disease, effective treatments are urgently needed. The clinical manifestations of DN include proteinuria, edema, hypertension and decreased renal function. Although the patho-genesis of DN is not fully known, recent studies point to metabolic and endocrine disorders together with microvascular hemodynamic and structural changes [8]. Glomerular hypertrophy and sclerosis that occur in DN lead to alteration of glomerular function, increased synthesis and decreased degradation of ECM [9].

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There is evidence for a role of cytokines and growth factors in the development of DN, [10], and one of the candidates is TGF-β [11]. Released by autocrine or paracrine secretion, TGF-β induces cell hypertrophy or stimulates the accumulation of extracellular matrix (ECM). The binding of TGF-β to its receptor is followed

by a number of effects including: (1) regulation of cell proliferation and differentiation; (2) pro-motion of protein synthesis and cell hypertro-phy; (3) stimulation of ECM and platelet-derived growth factor (PDGF) by glomerular and mesen-chymal cells; and (4) induction of the transfor-mation of renal tubular epithelial cells to

[image:5.612.91.516.76.568.2]
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enchymal cells [12]. In this study, the potential role of TGF-β in DN was investigated in an experimental T2DM rat model. We found that TGF-β was upregulated in model control DN rats, and that the increase in TGF-β was attenu-ated by treatment with She Jing Xiao Bai cap-sule, irbesartan, and a combination of the two agents.

In a mouse model, glomerular CTGF expression was found to have increased at 7 days after development of DM and to increase 28-fold after 3.5 months [13]. Concurrent decreases observed in matrix metalloproteinase (MMP) might contribute to strong expression of proteo-glycan, collagen type I and collagen type IV [14]. The increased synthesis and decreased degra-dation of ECM expected under those conditions would be expected to result in glomerular scle-rosis and fibscle-rosis [15]. CTGF has thus emerged as a key mediator of renal fibrosis. The increase of CTGF expression observed in our DN rat model is thus consistent with the findings of previous studies, and it was attenuated by She Jing Xiao Bai capsule and/or irbesartan treatment.

Tregs are a subtype of T cells that can suppress activation of the immune system and inhibit

inappropriate reactions leading to autoimmune diseases. They also restrict the area, extent and reaction time of immune responses. Treg cells thus have a key role in regulating the sta-bility of immune reactions including those rele-vant to type 1 diabetes mellitus (T1DM) [16]. Indeed, Treg cell counts were found to decre- ase before the appearance of T1DM and non-obese diabetes (NOD) [16]. Dysfunctional Tregs lose expression of CD25during cell prolifera-tion [17], and in T2DM, Tregs counts were found to decrease [17]. Moreover, a continuing Treg decrease has been related to disease progres-sion. Thus development of DN is related to immune dysfunction. In this study, She Jing Xiao Bai capsule with irbesartan, and alone increased Treg cell populations.

[image:6.612.95.519.71.366.2]

In traditional Chinese medicine theory, symp-toms of DN are “Xiao ke”, “hydroncus”, and “asthenia”, and the pathogenesis of DN is due to long-duration “Xiao ke”, which leads to con-sumption of “Jing” and “Qi”, and weakness of “Qi” and “Yin”. Cumulative damage of the neph-ron leads to blood stasis. She Jing Xiao Bai cap-sule consists of black soybean, atractylodes, astragalus, Chinese yam, ant powder, and hiru-do powder. Based on its prescription, She Jing Xiao Bai capsule acts to supplement Qi and

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nourish Yin, invigorate the spleen, prevent blood stasis, and alleviate water retention. It should therefore be beneficial for the treatment of DN.

In this study, She Jing Xiao Bai capsule signifi-cantly decreased SCr and urine protein levels and mitigated renal pathology. She Jing Xiao Bai treatment increased the Treg count in this DN model, but this experimental effect was seen in a context that differs from clinical prac-tice. In clinical practice, most patients are in an early stage of DN, whereas here the treatment was at a later stage [6]. Finally, She Jing Xiao Bai capsule reduced the extent of renal pathol-ogy possibly through multiple pathways includ-ing TGF-β, CTGF and Tregs. Although the active chemical compounds are not known, this cap-sule has been used in our hospital for more than 20 years. Future studies should be con-ducted to identify the active compounds. In conclusion, She Jing Xiao Bai capsule clearly reduced renal injury in this STZ-induced DN model. The mechanism of action might involve decrease of TGF-β and CTGF expression and increase in the numbers of CD4+Foxp3+T cells. Inflammatory factors such as IL-10 might be also involved. The study results warrant further study of its effectiveness and mechanism.

Disclosure of conflict of interest

None.

Address correspondence to: Xiuping Xiong, De- partment of Internal Medicine, Changshu Hospital of Traditional Chinese Medcine (New Area Hospital), The Yellow River Road, Changshu 215500, Jiangsu, China. Tel: +8613862257981; Fax: 0512-523422- 73; E-mail: 964359553@qq.com; xiongxp1028@ 126.com

References

[1] Mao Y, Ootaka T, Saito T, Sato H, Sato T and Ito S. The involvement of advanced glycation end-products (AGEs) in renal injury of diabetic glo-merulosclerosis: association with phenotypic change in renal cells and infiltration of immune cells. Clin Exp Nephrol 2003; 7: 201-209. [2] Kim SM, Lee SH, Kim YG, Kim SY, Seo JW,

Choi YW, Kim DJ, Jeong KH, Lee TW, Ihm CG, Won KY and Moon JY. Hyperuricemia-induced NLRP3 activation of macrophage contributes to the progression of diabetic nephropathy. Am

J Physiol Renal Physiol 2015; 308: F993- F1003.

[3] Gao P, Meng XF, Su H, He FF, Chen S, Tang H, Tian XJ, Fan D, Wang YM, Liu JS, Zhu ZH and Zhang C. Thioredoxin-interacting protein medi-ates NALP3 inflammasome activation in podo-cytes during diabetic nephropathy. Biochim Biophys Acta 2014; 1843: 2448-2460. [4] Vignali DA, Collison LW and Workman CJ.

How regulatory T cells work. Nat Rev Immunol 2008; 8: 523-532.

[5] Schneider A, Rieck M, Sanda S, Pihoker C, Greenbaum C and Buckner JH. The effector T cells of diabetic subjects are resistant to regu-lation via CD4+ FOXP3+ regulatory T cells. J Immunol 2008; 181: 7350-7355.

[6] Gao L, Zhu Y and O J. The effects of She Jing Xiao Bai capsule associated with irbesartan on urine protein level in diabetic nephropathy pa-tients. Jiangsu J Tradit Chin Med 2010; 42: 2. [7] Kumawat M, Sharma TK, Singh I, Singh N,

Ghalaut VS, Vardey SK and Shankar V. Anti- oxidant Enzymes and Lipid Peroxidation in Type 2 Diabetes Mellitus Patients with and without Nephropathy. N Am J Med Sci 2013; 5: 213-219.

[8] Mason RM and Wahab NA. Extracellular matrix metabolism in diabetic nephropathy. J Am Soc Nephrol 2003; 14: 1358-1373.

[9] Lenz O, Elliot SJ and Stetler-Stevenson WG. Matrix metalloproteinases in renal develop-ment and disease. J Am Soc Nephrol 2000; 11: 574-581.

[10] Chen S, Jim B and Ziyadeh FN. Diabetic ne-phropathy and transforming growth factor-be-ta: transforming our view of glomerulosclerosis and fibrosis build-up. Semin Nephrol 2003; 23: 532-543.

[11] Langham RG, Kelly DJ, Gow RM, Zhang Y, Cordonnier DJ, Pinel N, Zaoui P and Gilbert RE. Transforming growth factor-beta in human dia-betic nephropathy: effects of ACE inhibition. Diabetes Care 2006; 29: 2670-2675.

[12] Lan HY. Transforming growth factor-beta/Smad signalling in diabetic nephropathy. Clin Exp Pharmacol Physiol 2012; 39: 731-738. [13] Crean JK, Lappin D, Godson C and Brady HR.

Connective tissue growth factor: an attractive therapeutic target in fibrotic renal disease. Expert Opin Ther Targets 2001; 5: 519-530. [14] Conway BR, Betz B, Sheldrake TA, Manning JR,

Dunbar DR, Dobyns A, Hughes J and Mullins JJ. Tight blood glycaemic and blood pressure control in experimental diabetic nephropathy reduces extracellular matrix production with-out regression of fibrosis. Nephrology (Carlton) 2014; 19: 802-813.

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[16] Zhang Y, Bandala-Sanchez E and Harrison LC. Revisiting regulatory T cells in type 1 diabetes. Curr Opin Endocrinol Diabetes Obes 2012; 19: 271-278.

[17] Chapoval S, Dasgupta P, Dorsey NJ and Kee- gan AD. Regulation of the T helper cell type 2

Figure

Table 1. Effects of treatment on FBG level
Table 2. The effects of treatment on 24 h urine protein levels in DN rats
Figure 1. Morphological changes in renal tissue from DN rats following treatment. A. HE staining; B
Figure 2. The effects of treatment on TGF-β and CTGF expression in DN rats. A. mRNA expression; B
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References

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