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Protective effect of tetramethylpyrazine isolated from Ligusticum chuanxiong on nephropathy in rats with streptozotocin-induced diabetes.

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Keywords: Diabetes Tetramethylpyrazine Lignsticum chuanxiong Diabetic nephropathy Vascular endothelial growth factor

ABSTRACT

Purpose: This study was designed to investigate the protective effect of tetramethylpyrazine isolated from Ligusticum chuanxiong, a traditional Chinese medicine, on diabetic nephropathy in a rat model, and to explore the possible mechanism involved in a protective function.

Materials: Diabetes was induced in male Sprague-Dawley rats by a single intraperitoneal injection of 70mg/kg of streptozotocin. One week later, 200mg/kg/day of tetramethylpyrazine was administered intragastric gavage daily for 8 weeks. Renal functions and expression of vascular endothelial growth factor were examined at 4 and 8 weeks after tetramethylpyrazine administration. Results: Blood glucose and renal function were significantly improved in the tetrametfty/pyrazine-treated group compared to the untreated diabetic rats. Diabetic nephropathy resulted in an increase in the expression of vascular endothelial growth factor, while tetramethylpyrazine administration greatly decreased the expression.

Conclusions: Our results suggest that administration of tetramethylpyrazine may reduce kidney damage caused by diabetes. This protective effect may be mediated, in part, by downregulated expression of vascular endothelial growth factor in the kidney.

[c] 2011 Elsevier GmbH. All rights reserved.

Introduction

Diabetic nephropathy is one of the most serious microvascular complications and a leading cause of mortality and morbidity in diabetic patients [Sung et al. 2010]. Although many therapies have been tested in animal models, and have yielded significant efficacy for diabetic nephropathy, translating these experimental therapies to humans is enormously challenging [Mahnensmith et al. 2010; O'Loughlin et al. 2010].

Tetramethylpyrazine (TMP) is a compound, originally isolated from the rhizome of Ligusticum chuanxiong, a well-known traditional Chinese medicine. Ligusticum chuanxiong's active ingredients include an alkaloid, TMP, ferulic acid, chrysophanol, sedanoic acid, and essential oils such as ligustilide and butylphthalide [Hong 1986]. TMP is one of the most important active ingredients of Ligusticum chuanxiong [Lu et al. 1978; Liu et al. 2002], and has been applied in the treatment of stroke and cardiovascular diseases for a long time in Oriental medicine. TMP has increasingly gained attention due to its significant vascular protective properties. A number of studies on the effects of TMP on ischemic neural disorders and cardiovascular diseases have been documented. However, reports of the use of TMP for diabetic nephropathy are extremely limited, with only one study showing a protective effect of TMP on diabetic nephropathy published at this time [Huang et al. 2004]. Therefore, the effects of TMP treatment on diabetic nephropathy, and the possible mechanisms by which TMP protects against renal damage resulting from diabetes need to be explored.

Some previous studies have shown that TMP has vasodilatory and antihypertensive effects [Kwan 1994]. It may also have antioxidant effects and create endothelial protection [Kang et al. 2009]. In traditional Chinese clinical practice, TMP has been found to improve microcirculation, correct hypercoagulability, and promote vascular recanalization. All these findings suggest that protective effects of TMP may include multiple mechanisms.

One of the critical mechanisms of kidney damage resulting from diabetes involves the vascular endothelial growth factor (VEGF) [Long et al. 2010]. VEGF, also known as vascular permeability factor, is a dimeric glycoprotein which plays a crucial role in the microvascular complications of diabetes, including diabetic nephropathy [Long et al. 2010]. It has been demonstrated that the pathogenesis of a variety of inflammatory diseases, including the microvascular complications of diabetes, are linked with high levels of VEGF [Eremina et al. 2008; Chen and Ziyadeh 2008]. VEGF is indeed a major mediator of diabetic nephropathy [Aiello et al. 1994].

These factors led to the hypothesis that an effect on the level of VEGF expression could, in part, explain a protective effect of TMPon diabetic renal damage.Therefore, the present study was designed to confirm the protective effects of TMP, using a rat model of diabetic nephropathy, and to elucidate the role of VEGF in the protective mechanism of TMP.

Materials and methods

The study was considered, and approved by the Ethics Committee of the Third Military Medical University, China.

Animals and induction of diabetes

Forty male Sprague-Dawley rats, weighting 200-220 g, were randomly divided into three main groups with eight rats in each group: Control, Saline and TMP groups.

Diabetes was induced in the Saline and TMP groups with a single intraperitoneal injection of 70mg/kg of streptozotocin (STZ) (Sigma, USA). One week after the STZ injection, a blood sample was obtained, and blood glucose was determined with a glucometer (Johnson & Johnson, USA). Rats with a blood glucose level more than 200mg/dl were considered diabetic. The animals serving as the controls had free access to tap water and rat chow throughout the experiment.

All animals were individually housed in plastic metabolic cages under conditions of constant temperature and humidity and with a 12-h dark/light cycle. The duration of the experiment from the first day when TMP was administered was eight weeks, half of the rats in TMP- and Saline-groups were killed at 4 weeks and half at 8 weeks, respectively.

Effect of TMP on blood glucose and renal functions

One week after the STZ injection, rats in the Saline group were administered 2 ml of saline, and rats in the TMPgroups were administrated 200mg/kg of TMP by intragastric gavage. The same dose was infused every 24 h for 8 weeks. Blood glucose levels from a tail vein blood sample were evaluated at 4 and at 8 weeks after treatment. Twenty-four hour urine collection was used for assessment of urinary protein excretion and creatinine clearance at 4 and at 8 weeks.

Determination of VEGF expression

After serum glucose and 24-h urinary protein and creatinine clearance rates were evaluated at 4 and 8 weeks, the rats were scarified, and their kidneys were removed for determination of the expression of VEGF mRNA and immunohistochemical staining for VEGF protein. The quantitation of renal VEGF mRNA was performed by real time RT-PCR using a Syber Green RT-PCR kit (Qiagen, Valencia, USA). The quantitation of rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a control. All assays were performed in triplicate.

For histological examination and immunohistochemical staining of VEGF, paraffin embedded tissues were cut into 5[micro] m-thick slices, and were placed on slides. The slides were processed with a biotinylated secondary antibody (Santa Cruz Biotechnology, USA) and incubated with a peroxidase substrate.The stained tissues were examined by light microscopy and the optical density of the stained VEGF was evaluated.

Statistical analysis

Data were presented as mean [+ or -] standard error of the mean (SEM). Comparisons of levels of blood glucose, urine volume, urinary protein excretion and creatinine clearance at different time points were performed with two-way ANOVA and Scheffe's post-hoc comparison. Values were considered significant with P<0.05. All experimental data were analyzed using a statistical software package, SPSS 11.0.

Results

Effects of TMP on body weight and blood glucose

All animals survived and completed the study. There was a significant difference in body weight between the Control and Saline groups: body weights were significantly lower in the Saline Group. Compared with the Control Group, body weight was lower in the TMP Group, but was significantly higher than in the Saline Group. Blood glucose levels increased approximately 3- to 4-fold in the Saline group, compared with the Control group. There were no statistically significant differences between the Control and TMP groups in blood glucose level, even if blood glucose level in the TMP group was higher than that in the Control group (Table 1).
Table 1
Body weights (g) and blood glucose levels (mg/dl) at 4 and 8 weeks.

Groups   N        Body weight (g)

                       Week 4                       Week 8

Control  8             312 [+ or -] 20.6            481 [+ or -] 45.3
Saline   8           234 [+ or -] 19.4 *          252 [+ or -] 31.2 *
TMP      8      291 [+ or -] 34.2 (#/NS)      477 [+ or -] 7.8 (#/NS)

Groups       Blood glucose (mg/dl)

                     Week 4                         Week 8

Control              97.2 [+ or -] 9.1            98.6 [+ or -] 8.4
Saline            320 [+ or -] 43.1 **       408 [+ or -] 52.2 (**)
TMP           110 [+ or -] 11.2 (#/NS)      102 [+ or -] 9.8 (#/NS)

The body weights of diabetic rats (Saline group) were significantly
reduced compared to the control rats (Control group). Blood glucose
was also markedly increased in the Saline group compared to the
Control group. TMP administration has effects on the reduction of
blood glucose, and the increase of body weight.
Data are mean [+ or -] SD.
Comparations in TMP Group: TMP-Control/TMP-Saline.

NS: No significance, compared to the Control group.

TMP: Tetramethylpyrazine.

* p < 0.05, compared to the Control group.

** p < 0.01, compared to the Control group.

# p < 0.05, compared to the Saline group.


Effects of TMP on renal function

Renal functions were evaluated by assessment of urine protein excretion and creatinine clearance. Renal functions in the Control animals were normal. A clear difference was observed between TMP and Saline groups in the renal functions. STZ-induced diabetes resulted in a significant renal dysfunction in the Saline group, but TMP administration greatly reduced this dysfunction (Table 2).
Table 2
Changes of 24-h urinary protein excretion (mg/day) and creatinine
clearance (ml/min).

Groups   N        Urinary protein (mg/day)

                      Week 4              Week 8

Control  8            3.2 [+ or -] 0.6         3.4 [+ or -] 0.3
Saline   8  11.4 [+ or -] [0.4.sup.**]     15.2 [+ or -] 0.3 **
TMP      8  5.1 [+ or -] [0.3.sup.*/*]  4.7 [+ or -] 0.6 (NS/*)

Groups            Creatinine clearance (ml/min)

                   Week 4                   Week 8

Control           3.7 [+ or -] 0.1          3.6 [+ or -] 0.4
Saline          7.0 [+ or -] 0.3 *       8.2 [+ or -] 0.2 **
TMP        4.6 [+ or -] 0.2 (NS/*)  3.9 [+ or -] 0.8 (NS/**)

24-h urinary protein excretion in the TMP rats (TMP group) were
significantly reduced as compared to the diabetic rats (Saline
group). Creatinine clearance was also markedly reduced in the TMP
group compared to the Saline group. TMP administration has
effects on the reduction of 24-h urinary protein excretion and
creatinine clearance. Data are mean [+ or -] SD.
Comparations in TMP Group: TMP-Control/TMP-Saline.

NS: No significance.

TMP: tetramethylpyrazine.

* p < 0.05, compared to the Control group.

** p < 0.01, compared to the Control group.


Effects of TMP on the expression of VEGF mRNA

STZ-induced diabetes elevated the expression of VEGF mRNA in the Saline group, while TMP administration significantly decreased the expression of VEGF mRNA (Fig. 1), compared to the Control group.

Effects of TMP on VEGF protein expression

Immunohistochemical staining for VEGF was visibly increased in the Saline group compared to the Control group, and decreased in the TMP group (Fig. 2a-cc). The intensity of the immunohistochemical staining for VEGF was evaluated by an optical density. The optical density of the immunohistochemical staining for VEGF was significantly increased in the Saline group, and significantly decreased in the TMPgroup compared to the Control group (Fig. 2d). There was no significant difference seen in the optical density of VEGF staining between week 4 and week 8 of TMP treatment

Discussion

Diabetic nephropathy is the leading cause of chronic renal disease and a major contributor to cardiovascular mortality [Zelmanovitz et al, 2009]. Abnormal renal hemodynamics occurs in the early stages of diabetic nephropathy, and progresses to proteinuria, glomerulosclerosis, and renal dysfunction. Therefore, management of renal hemodynamic abnormality and reduction of proteinuria are important to prevent the deterioration of kidney function. In the present study, we demonstrated that TMP administration protected renal functions.

[FIGURE 1 OMITTED]

STZ-induced diabetes resulted in extensive damage to renal functions, but TMP treatment significantly reduced the damage. The improvement in urine protein excretion and creatinine clearance with TMP treatment was accompanied by a significant decrease in VEGF expression. This result suggests that the protective effect of TMP treatment may occur by the suppression of VEGF expression.

The effect of TMP is related to its antioxidative function [Kang et al. 2009; Lee et al. 2002 ], and the inhibition of hydrogen peroxide-induced apoptosis in endothelial cells [Ou et al. 2010]. In addition, the crucial role of VEGF in microvascular complications of diabetes has been recently examined [Long et al. 2010]. Pharmacological and genetic disruptions of VEGF have been shown to result in significant proteinuria and glomerular endotheliosis [Eremina et al. 2003; Sugimoto et al. 2003]. VEGF is known to have a central role in angiogenesis, vascular homeostasis, and the maintenance of capillary integrity, particularly in the kidney glomerulus [Long et al. 2010]. It has been suggested that precise regulation of glomerular VEGF expression in the kidney is required for the proper development and function of glomeruli [Long et al. 2010; Eremina et al. 2008; Chen and Ziyadeh 2008]. Moreover, the use of anti-VEGF in STZ-induced diabetic animals has been shown to result in improvement in kidney function in animal models of diabetic nephropathy [De Vriese et al. 2001; Flyvbjerg et al- 2002].

A single published study [Huang et al. 2004], using a combination of TMP with aminoguanidine, has shown an effect of TMP on diabetic nephropathy. However, this study did not explore the mechanism behind the protective effect of TMP. In our present study, we found that the expression of VEGF was elevated in the animals with diabetic nephropathy, while the expression was significantly suppressed by administration of TMP. Thus, one of the mechanisms of TMP protection against renal dysfunction may be the depressed transcription of VEGF. Proper expression of VEGF provides protection against microvascular complications of diabetes, including diabetic nephropathy [Long et al. 2010].

In addition to the protection against renal dysfunction, the decreased blood glucose level was seen in the diabetic rats following TMP treatment. Even though the glucose level in the TMP-treated group was slightly higher than in the Control group, there were no statistically significant differences between the Control and TMP groups. Thus, the mechanism of TMP protection against renal dysfunction may involve the depressed transcription of VEGF on the one hand, and the protective effects may also involve the depressed glucose level in the diabetic rats on the other hand. The present study did not examine how the blood glucose level was reduced by TMP treatment in the diabetic rats.

Interestingly, both Ligusticum chuanxiong and Cnidium belong to Umbelliferae, and are both being used to treat the same conditions [Baek et al. 2003; Chung 2004]. In a study of the inter-genomic relationships between these two medical herbs, it was proposed that either Ligustkum chuanxiong originated from Cnidium or Ligustkum chuanxiong and Cnidium diverged from the same parental plant [Lee et al. 2010], but further evidence has shown that the origin of Ligustkum chuanxiong and Cnidium is identical [Liu et al. 2002].

[FIGURE 2 OMITTED]

The analysis of single constituents of Ligusticum chuanxiong could allow us to better understand their individual toxicities. Symptoms of vomiting and dizziness caused by an overdose of Ligusticum chuanxiong have been reported [Bensky and Gamble 1993]. Prescriptions in traditional Chinese medical practice, which usually include five to ten or even more herbs per formula, make it difficult to research the efficacy and toxicity of Chinese medicinal herbs. However, the medicinal effect of Iigusticum and its well established traditional use have many potential clinical and therapeutic applications [Sinclair 1998].

Conclusions

STZ-induced diabetes resulted in significant renal damage with abnormal 24-h urinary protein and creatinine clearance rates. TMP treatment had a protective effect on the kidney in this diabetes model. Our results suggest that the expression of VEGF may be involved in the protective mechanism of TMP against diabetic nephropathy, and that TMP may be beneficial for the clinical treatment of diabetic nephropathy.

doi: 10.1016/j.phymed.2011.05.003

0944-7113/$ - see front matter [c] 2011 Elsevier GmbH. All rights reserved. doi: 10.1016/j.phymed.2011.05.003

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Qi-Hong Yang (a), YongLiang (b), QiangXu (a), Yi Zhang (a), Li Xiao (a), Liang-Yi Si (a), *

(a) Department of Geriatrics, Southwest Hospital, Third Military Medical University, Chongqing, China

(b) Department of Orthopedics, First Affiliated Hospital of Chongqing Medical University, Chongqing, China

* Corresponding author. Tel.: +86 23 68754151.

E-mail address: siliangyi@163.com (L.-Y.Si).
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Author:Yang, Qi-Hong; Liang, Yong; Xu, Qiang; Zhang, Yi; Xiao, Li; Si, Liang-Yi
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:9CHIN
Date:Oct 15, 2011
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