The protective effect of echinacoside on diabetic nephropathy

Contact: Audrey Hu audrey.hu@wecistanche.com

Protective Effects of Echinacoside on Renal Function, Renal Tissue and Mesangial Cell Injury in Rats with Diabetic Nephropathy

HE Qin, LIU Fan, WANG Hongli, LI Jianping, DUAN Jun

Abstract:

Objective To explore the protective effect of echinacoside (ECH) on renal dysfunction, histopathology, and mesangial cell injury in diabetic nephropathy (DN) rats and its mechanism. Methods Sixty SD rats were randomly divided into control group,model group,ECH (echinacoside) group(high,medium and low doses ECH (echinacoside),100,50, 20 mg·kg'·d') and losartan potassium group(losartan potassium,16 mg·kg'·d'),10 rats in each group. The model group, ECH (echinacoside) group, and losartan potassium group were fed with a high-fat and high-sugar diet, and intraperitoneally injected streptozotocin(40 mg·kg"')to establish DN rat models. After 2 weeks of treatment, body weight,24h water intake, and urine volume of rats were recorded. The contents of fasting blood glucose, serum creatinine, urea nitrogen, urine microprotein, hyaluronic acid(HA), tissue metalloproteinase inhibitor-1(TIMP-1), and laminin(LN) were determined. HE staining and TUNEL staining was performed to observe pathological changes in kidney tissue. RT-PCR was performed to detect expression of B-cell lymphoma(Bcl-2),Bcl2-associated X protein(Bax), Caspase-3 mRNA. Wester Blot was performed to determine expression levels of vascular cell adhesion molecule-1(VCAM-1), Transforming growth factor(TGF-β),α-Smooth muscle actin(α-SMA). Results Compared with the control group, the bodyweight of the model group was significantly decreased(P< 0.05), water intake, urine volume, fasting blood glucose, urine microalbumin, urea nitrogen, creatinine, HA, TIMP-1 and LN were significantly increased (P<0.05). Bax, Caspase-3 mRNA, and expression of VCAM-1, TGF-β and α-SMA protein were significantly up-regulated(P< 0.05), expression of Bcl-2 mRNA was significantly down-regulated (P< 0.05). Compared with the model group, bodyweight of rats was significantly increased in the medium-dose ECH (echinacoside) group, high-dose ECH (echinacoside) group, and losartan potassium group(P<0.05), water intake, urine volume, fasting blood glucose, urine microalbumin, urea nitrogen, creatinine, HA, TIMP-1 and LN were significantly decreased(P<0.05).Bax,Caspase-3 mRNA, expression of VCAM-1,TGF-β and α-SMA protein were significantly down-regulated(P< 0.05); and expression of Bcl-2 mRNA was significantly up-regulated(P<0.05).HE and TUNEL staining showed that pathological changes in renal tissue were significantly decreased. Conclusion Medium and high doses of ECH (echinacoside)can protect renal tissue damage, and improve renal function in DN rats by inhibiting protein expression of TGF-β and VCAM-1 and inhibiting renal interstitial fibrosis and renal cell apoptosis.

Keywords: Cistanches herba; Echinacoside; diabetic nephropathy; renal interstitial fibrosis; apoptosis; rats

cistanche echinacoside treat diabetic nephropathy

Introduction

Diabetic nephropathy (Diabetic nephropathy, DN) is a common diabetic microvascular complication, which can not only cause kidney damage but also affect other organs. The clinical pathological feature of renal tubulointerstitial fibrosis is the complicated pathogenesis of diabetic nephropathy. Previous studies [P2-5 believe that the disease is mainly due to hyperglycemia in diabetic patients, which causes renal hemodynamic changes and abnormal metabolism. The clinical manifestations are mainly proteinuria and decreased glomerular filtration rate. At present, diabetic nephropathy Patients still lack effective clinical treatment methods, and finding new effective drugs is the key to the treatment of diabetic nephropathy.

echinacoside (ECH) is one of the extracts of Cistanche Tubulosa. It has the effect of invigorating the kidney and strengthening yang." Modern pharmacological studies have shown that echinacoside has antioxidant, anti-apoptotic, anti-inflammatory, and improved blood flow Microcirculation and other pharmacological effects can also regulate the body's glucose metabolism and improve glucose tolerance. In recent years, studies have shown that echinacoside may inhibit the transformation growth factor (Transforming growth factor, TGF-β) signaling pathway and inhibit diabetic nephropathy. Interstitial fibrosis of rat kidneys protects rat kidney tissue. However, there are few reports in the literature about the treatment of diabetic nephropathy with echinacoside, and its specific mechanism for protecting diabetic nephropathy in rats is not clear. Therefore, this study further explored The effect of echinacoside on renal fibrosis and renal cell apoptosis in diabetic nephropathy rats and its possible mechanism is intended to provide a theoretical basis for the clinical application of echinacoside.

Benefit of cistanche: treat kidney disease and improve kidney function

1 Material and methods

1.1 Animals:

60 SD rats, male, SPF grade, 8 weeks old, body weight 240~280 g, provided by Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd., animal license number: SCXK (Beijing) 2015-0001, animal quality is qualified Certificate number: 11400700106210, raised in the animal center laboratory of the hospital, keeping the room temperature constant at 25°C, simulating the day and night, changing the lighting conditions once every 12 hours, and the animals are free to eat and drink.

1.2 Drugs and reagents:

Echinacoside, China Institute for Food and Drug Control, batch number: 111670-201706; Losartan potassium, Hangzhou Merck Pharmaceutical Co., Ltd., National Medicine Standard: H20030654; Streptozotocin, American Sigma Company, batch number: 040103; Urine Protein content detection kit, Ningbo Meikang Biotechnology Co., Ltd., batch number: 20170612; TUNEL detection kit, Amicage Technology Co., Ltd., batch number: 20180406; RNA extraction kit, reverse transcription kit, Shanghai Hengfei Bio-Technology Co., Ltd., batch number: 20180705, 20180904; Serum hyaluronic acid (HA), tissue metalloproteinase inhibitor-1 (TIMP-1) and laminin (laminin, LN) detection kits, Shanghai Yuanmu Biotechnology Co., Ltd., the batch numbers are 20180612, 20180624, 20180706.

1.3 Apparatus

7600 type automatic biochemical analyzer, Japan HITACHI company; portable blood glucose meter, Beijing Yicheng Bioelectronics Technology Co., Ltd.; B X60 optical microscope, Japan OLYMPUS company; SN-695B radioimmunoassay analyzer, Shanghai Rihuan Instrument Shenbei Co., Ltd. company

1.4 Grouping, model replication and administration method

After one week of pre-feeding, 60 rats were randomly divided into the control group, the model group, the echinacoside high, medium and low dose groups and the Losartan potassium group, with 10 rats in each group. The model group, echinacoside group and losartan potassium group were treated with high-fat and high-sugar diet combined with low-dose streptozotocin to induce diabetic nephropathy in rats. Before the model was replicated, the rats fasted with water for 12 h. The rats in the control group were fed with ordinary feed and the other groups were fed with high-fat and high-sugar feed. After 4 weeks of continuous feeding, the rats in the control group were given a single intraperitoneal injection of an equal volume of citrate buffer, and the remaining groups were given a single intraperitoneal injection of streptozotocin Bacteriocin (40 mg·kg') to replicate the rat model of diabetic nephropathy. 72 hours after the rats were injected with streptozotocin, the blood glucose was ≥16.7mmol·L, and the urine protein excretion rate of 24 hours after 3 weeks was higher than that of the control group. The quantitative urine protein of 24 hours was greater than 50% before the model replication, indicating that the model was successfully replicated. Four weeks after the injection of streptozotocin, the low, medium and high dose groups of echinacoside were given 20 mg·kg', 50 mg·kg, and 100 mg·kg' echinacoside (according to 5mL·kg in rats). kg"’ bodyweight is dissolved in 37°C normal salines), rats in the Losartan potassium group are intragastrically administered with Losartan potassium 16mg·kg', and the model group and control group are intragastrically administered with equal volumes of normal saline, once a day. Gavage was continued for 2 weeks.

1.5 Specimen collection

After the last administration, the rats fasted without food and water. The 24 hours of drinking water of the rats were recorded, and the 24 hours of urine were collected. In the early morning of the second day, the rats were weighed. After collecting urine, intraperitoneal injection of 2% pentobarbital sodium solution 2mL·kg~ for anesthesia, separation of rat abdominal aortic blood, centrifugation (3 500 r·min²', 15 min), take the serum, and place in the refrigerator- Temporarily store at 20 ℃; separate rat kidney tissue and divide into two parts, one part is fixed in 4% paraformaldehyde, the other part is prepared as 10% homogenate, and placed in -80 ℃ refrigerator for later use.

1.6 Determination of glucose and lipid metabolism in rats

The serum of each group of rats was taken, fasting blood glucose was measured with a blood glucose meter, and triglyceride, cholesterol, and low-density lipoprotein levels were detected by an automatic biochemical analyzer.

1.7 24 h urine protein, renal function and renal fibrosis-related indicators

The 24-hour urine of rats in each group was collected, and the 24-hour urine microprotein content was determined by radioimmunoassay. The operation was carried out in strict accordance with the instructions. The serum of each group of rats was collected, and the automatic biochemical analyzer was used to detect the levels of blood creatinine and urea nitrogen, which are related to renal function, the level of serum TIMP-1 was detected by ELISA, and the levels of HA and LN were detected by radioimmunoassay. The operation was carried out in strict accordance with the instructions.

1.8 HE staining to observe the pathological changes of kidney tissue and TNUEL staining to observe the apoptosis of kidney tissue cells

The fixed kidney tissues of each group were taken, routinely embedded in paraffin, and sectioned. One section is used for HE staining to observe the morphological changes of renal tubules, glomeruli and renal interstitium under a light microscope; the other section is used for TUNEL staining, operating according to the kit instructions, and observing the number and positivity of renal tissue cells under the microscope The number of cells is calculated as the ratio of the number of positive cells to the number of renal tissue cells, which is the rate of apoptosis.

1.9 RT-PCR method to detect the expression of apoptosis-related genes in renal tissues

Take rat kidney tissue homogenate, extract total RNA by Trizol method, determine the concentration by ultraviolet spectrophotometer, and synthesize cDNA with reverse transcription kit.

1.10 Western Blot method to determine the expression of renal tissue VCAM-1, TGF-β and α-SMA protein.

Take about 50 mg of rat kidney tissue homogenate, dissolve it with cell lysate containing protease inhibitors, and extract total protein with cell lysate, Using the BCA method for protein quantification. The total protein sample extracted by 50 μmol·L" was transferred to a nitrocellulose membrane by polyacrylamide gel electrophoresis and sealed with 5% skimmed milk, and the primary antibody (1∶1000) and β-actin protein were added respectively. Antibody (1:300), incubate overnight at 4°C. Finally, add the secondary antibody and incubate for 2h at 37°C, monitored by electrochemiluminescence. Use Photoshop image analysis software to analyze the gray value of protein bands.

1.11 Statistical processing methods

SPSS 17.0 software was used to analyze the obtained data, and the measurement data that satisfies the normal distribution are expressed as mean ± standard deviation ((x±s), and single-factor analysis of variance is used to compare the differences between groups. Pairwise comparison between groups uses SNK- With q test, P<0.05 indicates that the difference is statistically significant.

2 Results

2.1 The effects of echinacoside on the body mass, drinking water and urine output of rats with diabetic nephropathy are shown in Table 1.

Table 1 Effects of ECH on body weight， water intake and urine volume in DN rats（x ± s，n=10）

Compared with the control group, the bodyweight of the model group rats was significantly reduced, and the 24-hour drinking water and urine output were significantly increased. The difference was statistically significant (P<0.05); compared with the model group, the middle and high doses of echinacoside The bodyweight of the rats in the Losartan potassium group increased significantly, and the 24-hour drinking and urine output decreased significantly, and the difference was statistically significant (P<0.05).

2.2 The effects of echinacoside on glucose and lipid metabolism in diabetic nephropathy rats are shown in Table 2.

Table 2 Effects of ECH on glucose and lipid metabolism in DN rats

Note: Compared with the control group,' P<0.05; compared with the model group, *P<0.05

Compared with the control group, the fasting blood glucose of the model group rats was significantly increased, and the difference was statistically significant (P<0.05); triglycerides, cholesterol, and low-density lipoprotein increased, but the difference was not statistically significant (P>0.05) ). Compared with the model group, the fasting blood glucose of rats in the middle and high dose groups of echinacoside and the losartan potassium group was significantly reduced, and the difference was statistically significant (P<0.05); triglycerides, cholesterol, and low-density lipoprotein decreased, But the difference was not statistically significant (P>0.05).

2.3 The effects of echinacoside on 24-hour urine protein and renal function of diabetic nephropathy rats are shown in Table 3.

Compared with the control group, the urine microalbumin, urea nitrogen and creatinine levels of the model group were significantly increased, and the differences were statistically significant (P<0.05); compared with the model group, the middle and high dose echinacoside groups The levels of urinary microalbumin, urea nitrogen and creatinine of rats in the and losartan potassium group were significantly reduced, and the differences were statistically significant (P<0.05).

Table 3 Effects of echinacoside on 24h urine protein and renal function of rats with diabetic nephropathy (x±s, n=10)

2.4 The effects of echinacoside on renal fibrosis in diabetic nephropathy rats are shown in Table 4.

Compared with the control group, the serum levels of HA, TIMP-1 and LN in the model group were significantly increased, and the differences were statistically significant (P<0.05); The levels of HA, TIMP-1 and LN of rats in the sartan potassium group were significantly reduced, and the differences were statistically significant (P<0.05).

Table 4 Effects of ECH on renal fibrosis in DN rats

2.5 HE staining to observe the pathological changes of kidney tissue in diabetic nephropathy rats (see Figure 1).

HE staining of rats in the control group showed that the structure of renal tissue cells was intact, and no obvious pathological changes were seen. The HE staining of rats in the model group and the low-dose echinacoside group showed: glomerulus swelled and enlarged in size, renal tubular epithelial cells swelled or fell off, renal interstitium was accompanied by inflammatory cell infiltration, and some mesangial hyperplasia and interstitial proliferation were also visible. Qualitative fibrosis. Echinacoside medium and high dose groups and Losartan potassium groups. The staining showed that the pathological changes of the kidney tissue were significantly reduced, accompanied by a small amount of inflammatory cell infiltration, and the degree of mesangial hyperplasia and interstitial fibrosis was also lighter than that of the model group.

Figure 1 Pathological changes of renal tissues of rats in each group

2.6 The effect of echinacoside on the apoptosis of kidney tissue in diabetic nephropathy rats is shown in Figure 2.

TUNEL staining showed that compared with the control group, the apoptosis rate of rats in the model group was significantly increased, and the difference was statistically significant (P＜0.05); compared with the model group, the middle and high doses of echinacoside and Corsa The apoptotic rate of rats in the potassium group was significantly reduced, and the difference was statistically significant (P<0.05).

Figure 2 Apoptosis of renal tissue cells in DN rats

2.7 The effect of echinacoside on the expression of apoptosis-related genes in renal tissues of diabetic nephropathy rats is shown in Table 6.

RT-PCR results showed that compared with the control group, the expression of Bax and Caspase-3 mRNA in the model group was significantly up-regulated, and the expression of Bcl-2 mRNA was significantly down-regulated, and the difference was statistically significant (P<0.05); compared with the model group In comparison, the expression of Bax and Caspase-3 mRNA was significantly down-regulated in the middle and high-dose echinacoside groups and the Losartan potassium group, and the expression of Bcl-2 mRNA was significantly up-regulated, and the differences were statistically significant (P<0.05).

Table 6 Effects of ECH on the expression of the apoptosis-related gene in renal tissue of DN rats

2.8 The effects of echinacoside on the expression of VCAM-1, TGF-β and α-SMA proteins in diabetic nephropathy rats are shown in Figure 3 and Table 7. Compared with the control group, the expression of VCAM-1, TGF-β and α-SMA protein in the model group was significantly up-regulated, and the difference was statistically significant (P＜0.05); The expressions of VCAM-1, TGF-β and α-SMA proteins in rats in the dose group and the Losartan potassium group were significantly down-regulated, and the differences were statistically significant (P<0.05).

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