Expression of Nrf2/ARE pathway in diabetic nephropathy and renal protection of Qihuang Bushen Xiezhuo Formula

JIN Li-xia, ZHANG Xiao-dong, PAN Chao, LUAN Zhong-qiu, SONG Shu-juan, YIN Liying, WANG Wen-kai?

1. The First Affiliated Hospital of Heilongjiang University of Traditional Chinese Medicine, Harbin 150040, China

2. Heilongjiang University of Traditional Chinese Medicine, Harbin 150006, China

Keywords:Qihuang Bushen Xiezhuo Formula Oxidative stress High sugar Human glomerular mesangial cells Irbesartan

ABSTRACT Objective: To investigate the effect of Qihuang Bushen Xiezhuo Formula on the expression of the nuclear factor E2 related factor 2(Nrf2)/ antioxidant response element (ARE) signaling pathway of human glomerular mesangial cells (HMC) in high glucose medium and its protective effect on oxidative stress. Methods: The HMC were cultured in vitro to prepare normal rat serum. The rats were intragastrically administered with irbesartan and Qihuang Bushen Xiezhuo Formula. The serum containing the drugs was prepared after the blood concentration was reached. The rats were divided into the control group, the high glucose group, the irbesartan group and the Qihuang Bushen Xiezhuo Formula group. The HMC of the control group was cultured with normal rat serum (10% serum concentration) medium, while that of the high glucose group was cultured with high glucose medium of rat serum (10% serum concentration). The irbesartan group and the Qihuang Bushen Xiezhuo Formula group were respectively treated with 10% irbesartan and 10% Qihuang Bushen Xiezhuo Formula in serum high glucose medium. After 48 h of culture, the relevant indicators were collected and detected.The mRNA expression levels of Nrf2, gamma-glutamylcysteine synthetase(γ-GCS) and superoxide dismutase(SOD) in each group were detected by real-time PCR. The expressions of γ-GCS and SOD were detected by immunohistochemistry. The protein expressions of γ-GCS and SOD in HMC of each group were observed by Western Blot. Results: The expressions of Nrf2, γ-GCS, SOD mRNA and protein in experimental cells of each group were low in the control group, while those in the high glucose group were decreased as compared with the control group (P＜0.05). After interference of irbesartan and Qihuang Bushen Xiezhuo Formula the expressions were increased, and the increase in Qihuang Bushen Xiezhuo Formula group was more significant (P＜0.05). Conclusion: Qihuang Bushen Xiezhuo Formula can improve HMC oxidative stress injury in high glucose culture, and its mechanism may be achieved by activating Nrf2 and its related downstream proteins γ-GCS and SOD.

1. Introduction

Diabetic Nephropathy (DN) is the most important and common complication of diabetes, and the data show that DN occurs in about 45% of type 1 diabetic patients and about 30% of type 2 diabetic patients[1]. It is known as the leading cause of CKD and ESRD[2].The research on its pathogenesis in modern medicine is still unclear.Apart from various factors including glycolipid metabolism disorder,hemodynamic factors, genetic background, immune inflammatory reaction, etc., the significantly increased level of oxidative stress in patients with diabetes has been proved by a large number of studies.Kidney with rich vascular system is the most vulnerable to damage by oxidative stress, and it is one of the target organs of oxidative stress. Oxidative stress can directly damage podocytes, mesangial cells and endothelial cells, or indirectly activate other pathogenic pathways to cause damage, thereby promoting the occurrence and development of DN[3,4]. The main pathological characteristics of DN are glomerular hypertrophy and sclerosis, renal tubular injury and fibrosis formation[5]. Studies[6] have shown that there is a complex system in the body that can resist oxidative stress damage, and Nrf2-ARE is one of the most important signaling pathways. When the human body is stimulated by oxidative stress, a series of protective proteins are produced to protect cells. Under normal conditions,Kelch-like ECH-related protein 1 (Keap1), a cytoplasmic inhibitor,retains nuclear factor E2-related factor 2 (Nrf2) in the cytoplasm and promotes its ubiquitination, forming a dynamic balance with reactive oxygen species (ROS)[7]. In the high glucose state, as excessive ROS production would cause oxidative stress reaction[8],Nrf2 was separated from Keap1 and transferred to the nucleus,where it formed heterodimer with Maf protein and bound to the Antioxidant response element (ARE) in the promoter region of its target gene, genes encoding its downstream antioxidants, second detoxification enzyme, and related proteins, including superoxide dismutase (SOD), and γ-glutamylcysteine synthetase (γ-GCS),thereby improving the cell's ability to resist oxidative stress. Through collecting the latest literature both in China and abroad, we have found that the activation of the Nrf2-ARE signaling pathway can reduce glomerular oxidative stress damage in diabetic mice, delay renal fibrosis, and play a role in renal protection[9-13]. However, the molecular mechanism of traditional Chinese medicine compound prescription in regulating this pathway for the prevention and treatment of diabetic nephropathy remains to be further studied.

Qihuang Bushen Xiezhuo Formula mainly comprises Radix Astragali, wine-steamed Radix et Rhizoma Rhei, Semen Cuscutae,Dodder, Mao xucao, Salvia miltiorrhiza, Centella asiatica,Ostreae concha, epimedium, dandelion. It is an empirical formula summarized from years of clinical application and can effectively reduce proteinuria in DN patients and improve renal function. In our previous study[14], we found that Qihuang Bushen Xiezhuo Formula could interfere with the expression of Nrf2 and downstream related protein heme oxygenase-1(HO-1) to reduce the damage caused by oxidative stress. γ-GCS and SOD are also important antioxidant proteins in the downstream of Nrf2. It is reasonable to speculate that Qihuang Bushen Xiezhuo Formula can regulate Nrf2 and its downstream proteins γ-GCS and SOD to improve oxidative stress in DN, and has the potential to become a new target for clinical treatment of related diseases. The purpose of this study was to provide an experimental basis for early clinical intervention in the treatment of DN.

2. Materials and methods

2.1 Experimental cells, animals, and reagents

Human glomerular mesangial cells (HMC, Shanghai Fuheng Biotechnology Co., Ltd.), Wistar rats (SYXK (black) 2013-010, Drug Safety Evaluation Center, Heilongjiang University of Chinese Medicine). Irbesartan (Hangzhou Sanofi-Aventis Minsheng Pharmaceutical Co., Ltd, H20040494); Qihuang Bushen Xiezhuo Formula was prepared in the Preparation Room of the First Affiliated Hospital of Heilongjiang University of Chinese Medicine. SOD1(Wanleibio, China, WL01846), γ-GCS(Wanleibio,China, DF8550), biotinylated goat anti-rabbit IgG(Beyotime,China, A0277), DAB color solution (Solarbio, China, DA1010),hematoxylin (Solarbio, China, H8070), Goat serum (Solarbio, China,SL038), horseradish enzyme-labeled streptavidin (Beyotime, China,A0303).

2.2 Preparation of serum

After one week of feeding, 60 Wistar rats were randomly divided into a control group, a high glucose group, an irbesartan group, and a Qihuang Bushen Xiezhuo Formula group. Irbesartan group and Qihuang Bushen Chuzhuo Formula group were given irbesartan 1.35 mg/mL irbesartan solution prepared according to 13.5 mg·kg-1·d-1by intragastric administration, while Qihuang Bushen Chuzhuo Formula 0.324 mg·kg-1·d-1by intragastric administration. The rats in the control group and the high glucose group were intragastrically administered with equal volumes of normal saline. All rats were intragastrically administered once a day for 7 successive days. After anesthesia at the last 2 h of intragastric administration, blood was collected from the abdominal aorta to inactivate complement, which was then filtered, sterilized, subpackaged and stored at -80 ℃ for subsequent use.

2.3 HMC Cultivation and Grouping

The 4th to 5th generation HMC cell lines were passaged for 24 h in RPMI1640 medium containing 10% FBS and no FBS at 37 ℃and 5% CO2, respectively. The cells were randomly divided into four equal parts: the control group, the high glucose group, the irbesartan group, and the Qihuang Bushen Xiezhuo Formula group. The control group was cultured in the medium containing 10% normal rat serum,while the high glucose group was cultured in the high glucose medium containing 10% normal rat serum. The irbesartan group and the Qihuang Bushen Xiezhuo Formula group were cultured in the high glucose medium (30 mmol/L glucose) of rat serum containing 10% of thecorresponding drugs for 48 h, for the extraction of cellforming protein and the index detection after the experiment.

2.4 Real-time PCR assay was used to detect the mRNA expressions of Nrf2, γ-GCS and SOD in all groups

After extracting the total RNA from each group's HMC, the cDNA was obtained by reverse transcription according to the instructions and amplified on the PCR instrument. Next, we added reagents and put them into a fluorescence quantitative PCR instrument for reaction. Fluorescence quantitative data were collected and analyzed with ACTIN as the internal reference. See Table 1,2 for primer sequence design.

Tab 1 primer sequence of target genes

Tab 2 Total primer sequences

2.5 The expression levels of γ-GCS and SOD proteins in mesangial cells were determined by Western blot

After the cell protein was extracted, the protein extract was put into PBS buffer and mixed evenly at a ratio of 1:19 to prepare the protein test solution. BCA reaction: the volume ratio of solution A and solution B was 50:1, and they were added to each well. after they were fully mixed, they were placed at 37 ℃ for a reaction for 20 min. The solution changes from green to purple. Finally, we scanned the film and analyzed the optical density values using the Gel-Pro-Analyzer software.

2.6 Determination of the expressions of γ-GCS and SOD by immunocytochemistry

Determination of the expressions of γ-GCS and SOD by immunocytochemistry:0.1%TritonX-100. 3% hydrogen peroxide.Serum blocking. Primary antibody incubation. Secondary antibody incubation. Horseradish enzyme labelling. DAB colouration.Counterstaining of hematoxylin. Microscopic examination.

2.7 Statistical processing

All data were expressed as (±s). SPSS 25.0 was used to analyze the experimental data and Excel was used for mapping. One-way analysis of variance was used among groups. If the variances are not uniform, we will use the Tamhanes T2 test; If the variances are homogeneous, we will choose the LSD test.

3. Results

3.1 The mRNA expression levels of Nrf2, γ-GCS and SOD in mesangial cells of each group were detected by quantitative real-time PCR analysis

The results of Real-time PCR showed that after HMC culture for 48 hours, the mRNA of Nrf2, γ-GCS and SOD were expressed in the control group. Intracellular Nrf2, γ-GCS, and SOD mRNA expressions were decreased in the high glucose group as compared with the control group(P＜0.05). Compared with the high glucose group, the expressions of Nrf2, γ-GCS, and SOD mRNA in cells of the irbesartan group and the Qihuang Bushen Xiezhuo Formula group were increased(P＜0.05). Moreover, it was more obvious in the Qihuang Bushen Xiezhuo Formula group(P＜0.05). The results are presented in Table 3, Figure 1.

Tab 3 Comparison of relative values of Nrf2, γ-GCS and SOD mRNA expression after 48 h of HMC culture among different groups(n=8, ±s)

Tab 3 Comparison of relative values of Nrf2, γ-GCS and SOD mRNA expression after 48 h of HMC culture among different groups(n=8, ±s)

Note: Compared with the control group at the same time, #P＜ 0.05; the ratio of the same time to the high glucose group *P＜ 0.05.

Group Nrf2 γ-GCS SOD control group 0.99±0.08 0.97±0.10 0.99±0.06 High glucose group 0.52±0.05# 0.48±0.04# 0.60±0.04#Irbesartan group 2.55±0.21* 1.63±0.15* 1.37±0.09*Qihuang Bushen Xiezhuo Formula Group 3.51±0.31* 2.73±0.25* 2.42±0.20*F 394.166 300.249 344.401 P 0.000 0.000 0.000

3.2 The protein expressions of-GCS and SOD in mesangial cells were determined by Western blot

After 48 h culture of HMC, the relative expression levels of γ-GCS and SOD in the high glucose group were significantly lower than those in the control group(P＜0.05). As for the prognosis of irbesartan and Qihuang Bushen Xiezhuo Formula, compared with the high glucose group, the relative expressions of γ-GCS and SOD in the irbesartan group and Qihuang Bushen XieZhuo Formula group were increased(P＜0.05). And the up-regulation in the Qihuang Bushen Xiezhuo Formula group was the most significant(P＜0.05). Results are shown in Figures 2,3 and Table 4.

3.3 The expressions of γ-GCS and SOD were detected by immunocytochemistry

The results of immunohistochemistry showed that after 48 h of HMC culture in each group, the expressions of γ-GCS and SOD in the high glucose group were significantly lower than those in the control group (P＜0.05). As for the prognosis of irbesartan and Qihuang Bushen XieZhuo Formula, compared with the high glucose group, the expression levels of γ-GCS and SOD in the irbesartan group and Qihuang Bushen XieZhuo Formula group were increased(P＜0.05), and the increase in the Qihuang Bushen XieZhuo Formula group was the most significant (P＜0.05). Results are presented in Figure 4.

Fig 1 Comparison of relative values of Nrf2, γ-GCS and SOD mRNA expression after 48 h of HMC culture among different groups

Fig 2 The expressions of γ-GCS and SOD proteins in HMC of all groups at the end of 48 h were determined by Western blot

Tab 4 The relative values of γ-GCS and SOD protein expressions of HMC in each group at 48 h (n=8, ±s)

Tab 4 The relative values of γ-GCS and SOD protein expressions of HMC in each group at 48 h (n=8, ±s)

Note: Compared with the control group at the same time, #P＜ 0.05; the ratio of the same time to the high glucose group *P＜ 0.05.

Group γ-GCS/β-actin SOD/β-actin control group 0.26±0.02 0.40±0.04 High glucose group 0.17±0.02# 0.20±0.01#Irbesartan group 0.41±0.03* 0.75±0.05*Qihuang Bushen Xiezhuo Formula Group 0.65±0.06* 1.10±0.13*F 249.450 258.476 P 0.000 0.000

Fig 3 The expressions of γ-GCS and SOD proteins in HMC of all groups at 48 h

Fig 4 The expression levels of HMC γ-GCS and SOD at 48 h by immunohistochemistry

4. Discussion

Diabetic nephropathy is a common clinical disease with hidden symptoms, which can be manifested as increased urinary albumin excretion rate in the early stage. The symptoms are atypical and easy to be ignored. If it is not found in the early stage and is not effectively controlled, the condition will progress rapidly. Once persistent proteinuria occurs, it will inevitably progress to endstage renal disease[15]. Although the pathogenesis of DN has not been clearly understood in modern medicine, many related studies in recent years have shown that oxidative stress and inflammatory response play very important roles in the development of DN[16].Oxidative stress refers to the imbalance between oxidants and antioxidants in the body. Under normal physiological conditions,ROS production and elimination in the body are in dynamic balance[17]. In diabetes, ROS is overproduced, and the antioxidant capacity of the body decreases[18]. Oxidative stress not only directly attacks DNA, protein or lipids for oxidative modification, but also activates various signaling pathways to destroy the ability of kidney tissue to remove free radicals[19]. These alter the morphology and function of the renal tissue, eventually forming tubulointerstitial fibrosis and proteinuria that develop into DN. There is a set of defence mechanisms in the man-machine body to deal with the damage of free radicals and toxic substances, and its principle is a set of complex antioxidant response mechanisms. The most important pathway in this anti-oxidative response mechanism is Nrf2-ARE.Its effects on anti-oxidative stress and protecting renal function have been proved by a large number of experimental studies. ARE, as a promoter binding sequence on a cell protection protein gene, can be activated by the transcription factor Nrf2. The process is relatively stable under physiological conditions, but under stress, Nrf2 is transferred to the nucleus for recognition and combines with ARE to form the Nrf2-ARE pathway, further producing antioxidant proteins such as SOD, γ-GCS and the like to exert antioxidant stress effects,improve kidney pathological changes and glucolipid metabolism disorders, and inhibit kidney inflammatory reaction caused by ROS accumulation[21-22]. SOD is a major antioxidant enzyme in the body and also an important indicator for the evaluation of oxidative stress. SOD can play an antioxidant role by promoting the disproportionation reaction of superoxide anion radical to block its damage to the renal cell membrane structure[23]. γ-GCS is a rate-limiting enzyme for the synthesis of reduced glutathione(GSH) in vivo, and it has attracted the most attention among many downstream proteins in the Nrf2-ARE pathway. Its content and activity directly determine the speed and quantity of GSH synthesis.The increase in the quantity of GSH can further enhance the body's ability to resist oxidative stress[24]. Therefore, regulating the Nrf2-

ARE signalling pathway and promoting the transcription of SOD and γ-GCS in response to oxidative stress may become a new target for the treatment of DN.

In this study, the expression of Nrf2 and downstream antioxidant proteins γ-GCS, SOD mRNA and protein of Nrf2-ARE signal pathway in human Mesangial cells cultured with high glucose were observed to improve the molecular mechanism of Qihuang Bushen Xiezhuo Formula in reducing oxidative stress and preventing the progression of diabetic nephropathy. The results of this study showed that: the results of Western blot and immunocytochemistry showed that human Mesangial cells cultured with normal rat serum expressed a small amount of γ-GCS and SOD. After the stimulation of high glucose, its expression decreased significantly, and the addition of irbesartan and Qihuang Bushen Xiezhuo drug serum could increase the expression of γ-GCS and SOD to varying degrees.The results of Real-time PCR showed that the expressions of γ-GCS and SOD mRNA of irbesartan and Qihuang Bushen Xiezhuo recipe were higher than those of the high glucose group. No matter from the gene level or protein level, the expression of SOD and γ-GCS increased after drug intervention, especially in the Qihuang Bushen Xiezhuo Formula group.

To sum up, Oxidative stress can promote the occurrence and development of DN. Qihuang Bushen Xiezhuo Formula can regulate the expression of Nrf2, γ-GCS, SOD mRNA and protein in HMC cultured with high glucose, and reduce the damage of cells caused by oxidative stress. Therefore, we speculate that Qihuang Bushen Xiezhuo Formula can interfere with the Nrf2-ARE signal pathway,promote the production of antioxidant proteins, reduce oxidative stress, and thus protect the kidney and delay the progress of DN.The purpose of this study is to provide the experimental basis for the clinical application of Qihuang Bushen Xiezhuo Formula and early intervention of DN.

The pathogenesis of diabetic nephropathy is complex. In this study,we preliminarily discussed the mechanism of traditional Chinese medicine compound intervention on the Nrf2-ARE signal pathway and its related protein antioxidant stress. Next, we will further study diabetic nephropathy and its related signal pathway, and intervene with traditional Chinese medicine compound prescription. To provide a more theoretical basis for the prevention and treatment of diabetic nephropathy with traditional Chinese medicine.

Author’s contributions

Jin Li-xia and Wang Wen-kai participated in the whole process, instructed the experimental operation and wrote the paper. Zhang Xiao-dong and Pan Chao raised animals, tested the experimental related indicators, data statistical analysis, participated in paper writing, Luan Zhongqiu, Song Shu-juan, Yin Li-ying for immunohistochemical, Western Blot, Real-time PCR detection of related indicators and data statistics.

All authors declare that there is no conflict of interest.