Protective Effects Of Echinacoside On Renal Function，Renal Tissue And Mesangial Cell Injury in Rats With Diabetic Nephropathy

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HE Qin， LIU Fan， WANG Hongli， LI Jianping， DUAN Jun（Department of Cadre Medicine， Caotang Ward， Sichuan People’s Hospital， Sichuan Academy of Medical Sciences， Chengdu 610072 Sichuan， China）

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 group（high， medium and low doses ECH， 100， 50， 20 mg·kg- 1 ·d- 1）and losartan potassium group（losartan potassium， 16 mg·kg- 1 ·d- 1）， 10 rats in each group. The model group， ECH group, and losartan potassium group were fed with high- fat and high-sugar diet， and intraperitoneally injected streptozotocin（40 mg ·kg- 1）to establish DN rat models. After 2 weeks of treatment， the 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 the expression of B-cell lymphoma（Bcl-2）， Bcl2-associated X protein（Bax）， Caspase- 3 mRNA. Western 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， the body weight of rats was significantly increased in the medium-dose ECH group， high-dose ECH 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 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

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Diabetic nephropathy (Diabetic nephropathy, DN) is a common diabetic microvascular complication, which can not only cause kidney damage but also affect other organs. Renal tubulointerstitial fibrosis is the main pathological feature in clinical [1]. The pathogenesis of diabetic nephropathy is complicated. Previous studies [2-3] believed that the disease was mainly caused by hyperglycemia in diabetic patients, which caused renal hemodynamic changes and abnormal metabolism. The clinical manifestations were proteinuria and decreased glomerular filtration rate. Wait. At present, patients with diabetic nephropathy 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, which has the effect of nourishing the kidney and strengthening the yang [4]. Modern pharmacological research [5] shows that echinacoside has anti-oxidation, anti-apoptosis, anti-inflammatory, and pharmacological effects such as improving blood flow and microcirculation. It can also regulate the body's glucose metabolism and improve glucose tolerance. In recent years, studies [6] have shown that echinacoside may inhibit the renal fibrosis of diabetic nephropathy rats by inhibiting the transforming growth factor (TGF-β) signaling pathway and protecting rat kidney tissue. However, there are few reports in the literature on the treatment of diabetic nephropathy with echinacea, and its specific mechanism for protecting diabetic nephropathy in rats is still unclear. Therefore, this study further explored the effects and possible mechanisms of echinacoside on renal fibrosis and renal tissue cell apoptosis in diabetic nephropathy rats and aimed to provide a theoretical basis for the clinical application of echinacoside.

Causes of diabetes and traditional treatment methods

The onset of diabetic nephropathy is hidden, and the pathogenesis is complex and diverse. Once formed, it is difficult to cure. It is a chronic disease with great harm and an important cause of end-stage renal disease [8]. Patients with diabetic nephropathy are often treated with angiotensin-converting enzyme inhibitors and angiotensin receptor antagonists, but the clinical treatment effect is not very satisfactory [9]. Losartan potassium is an angiotensin Ⅱ receptor antagonist, which can be used to treat essential hypertension. A number of studies [10-11] show that Losartan potassium can also inhibit the renal interstitium of rats with diabetic nephropathy Fibrosis, protect kidney function. Therefore, this study chose Losartan potassium as a positive control to compare the protective effects of echinacoside on the renal tissue of rats with diabetic nephropathy.

Clinical studies [12-13] have shown that the content of echinacoside in Changui Yishen Capsules is about 3.104 mg · kg-1. After conversion based on the human and mouse administration dose, the lowest dose of Changui Yishen Capsule used for diabetic nephropathy in rats is 0.648 g·kg-1, which is approximately equivalent to echinacoside 2 mg·kg-1. Due to the current clinical concerns about echinacoside in the treatment of diabetes

There are few reports in the literature of kidney disease. Therefore, this study chose 10 times the lowest clinical dose, that is, 20 mg·kg-1 echinacoside as a low dose, and gradually increased, setting 50 mg·kg-1 echinacoside Is a medium dose,

100 mg·kg-1 Echinacoside is a high dose.

Demonstration of Echinacoside in Treating Diabetes and Improving Renal Function

In this study, low-dose echinacoside has no obvious effect on the kidney tissue of diabetic nephropathy rats, while medium and high-dose echinacoside can significantly improve the pathological changes of kidney tissue and improve renal tissue damage in diabetic nephropathy rats, Suggesting that medium and high doses of echinacoside can protect the kidney tissue damage of diabetic nephropathy rats in a concentration-dependent manner, and it is expected to be used in the treatment of clinical diabetic nephropathy.

medium and high doses of echinacoside can significantly increase the body mass of rats with diabetic nephropathy and reduce their water intake, urine output, and fasting blood glucose. Clinical studies [14] have shown that insulin in patients with diabetic nephropathy is in absolute or relative deficiency, and blood glucose levels are elevated. Excessive blood glucose concentration will exceed the glomerular reabsorption capacity. It is excreted in the urine, resulting in an increase in the frequency of urination and increased drinking water. The decrease in body mass is basically consistent with the results of this study, suggesting that echinacoside may increase the body mass of diabetic nephropathy rats by lowering blood sugar and reducing their drinking and urine output.

medium and high doses of echinacoside can significantly reduce urine microalbumin, creatinine, and urea nitrogen in rats with diabetic nephropathy. Creatinine and urea nitrogen are mainly excreted through glomerular filtration, which can more accurately reflect kidney function. When renal dysfunction, the glomerular filtration rate decreases, the concentration of blood creatinine and urea nitrogen will increase significantly [15]. The urine protein level is one of the most commonly used indicators to reflect the degree of glomerular damage, which can directly damage mesangial cells and can also aggravate kidney damage and renal dysfunction [16]. Research by Zhang Yun et al. [17] showed that hyperglycemia in rats with diabetic nephropathy can cause a decrease in glomerular filtration rate, leading to an increase in blood creatinine, urea nitrogen, and urine microalbumin levels. The results of this study suggest that echinacoside may reduce blood sugar, improve renal function, reduce blood creatinine, urea nitrogen, and urine microalbumin levels, and improve renal tissue cell damage.

medium and high doses of echinacoside can significantly down-regulate the expression of Bax and Caspase-3 mRNA in diabetic nephropathy rats, up-regulating the expression of Bcl-2 mRNA, and inhibit the apoptosis of renal tissue cells. Bax gene is a pro-apoptotic member of the Bcl-2 family. Under physiological conditions, Bax and Bcl-2 form a heterodimer, which reduces cell membrane permeability and cytochrome C release. Once the expression of Bcl-2 is down-regulated, It will cause the increase of cell permeability, the release of cytochrome C, activate the downstream Caspase cascade, and induce cell apoptosis [18].

Caspase-3 is an apoptosis executive molecule, which can degrade a variety of important proteins in cells and participate in cell apoptosis [19]. The study of Dong et al. [20] showed that echinacoside can inhibit the apoptosis of a variety of cells, which is basically consistent with the results of this study, suggesting that echinacoside can inhibit apoptosis of renal tissue cells, thereby protecting renal tissue damage.

medium and high doses of echinacoside can significantly down-regulate the expression of VCAM-1 protein in diabetic nephropathy rats. VCAM-1 is an important vascular cell adhesion molecule, which is closely related to the occurrence and development of diabetic nephropathy and can be used for the clinical evaluation of patients with diabetic nephropathy [21]. The study of Zhang et al. [22] showed that VCAM-1 can induce neuronal apoptosis after intracerebral hemorrhage in rats. The results of this study suggest that medium and high doses of echinacoside may inhibit the expression of VCAM-1 and inhibit renal cell apoptosis in rats with diabetic nephropathy.

In this study, medium and high doses of echinacoside can significantly reduce the levels of HA, TIMP-1, and LN in rats with diabetic nephropathy. HA is the main component of the extracellular matrix, and LN is an important structural glycoprotein, which is a commonly used clinical indicator of tissue fibrosis [23]. TIMP-1 is an inhibitor of matrix metalloproteinases (MMP), which can inhibit the expression of MMP, inhibit the degradation of extracellular matrix, and promote renal tissue fibrosis [24]. The study by Bieniaa et al. [25] showed that TIMP-1 can inhibit the expression of MMP, promote the expression of HA and LN, and promote the occurrence and development of renal tissue fibrosis. This is basically consistent with the results of this study, suggesting that echinacoside may inhibit HA, TIMP-1, and LN levels, inhibit the occurrence and development of renal fibrosis.

medium and high doses of echinacoside can significantly down-regulate the expression of TGF-β and α-SMA proteins in diabetic nephropathy rats. A large number of studies [26-27] have shown that the TGF-β/Smad signaling pathway plays an important role in the occurrence and development of renal interstitial fibrosis. When TGF-β binds to its receptor in the cell membrane, it can phosphorylate Smad2 and Smad3, and the activated Smad forms a trimer, which is transferred to the nucleus to regulate the expression of downstream effector molecules such as α-SMA and matrix metalloproteinases. Induce the production of renal interstitial fibrosis [28]. α-SMA is a sign of the transformation of fibroblasts into myofibroblasts, which can promote the secretion and deposition of extracellular matrix, and promote the occurrence of renal interstitial fibrosis [29]. Research by Li Zhong et al. [30] showed that TGF-β/Smad plays an important role in regulating renal tissue damage in diabetic nephropathy. The results of this study suggest that echinacoside may inhibit the expression of TGF-β, inhibit the expression of α-SMA, and inhibit renal tissue fibrosis.

Conclusion

In summary, medium and high doses of echinacoside can reduce blood sugar in diabetic nephropathy rats, and may inhibit the renal interstitial fibrosis and renal interstitial fibrosis in diabetic nephropathy rats by inhibiting the protein expression of TGF-β and VCAM-1. Tissue cell apoptosis improves the renal function of rats.