Emodin Regulates MiR-21 Mediated Autophagy to Alleviate Renal Oxidative Damage in diabetes Nephropathy Mice
Diabetes nephropathy (DN) is the most common serious secondary glomerular disease of diabetes and the common cause of chronic renal failure. Excessive blood sugar can trigger oxidative stress in the kidneys, leading to the accumulation of reactive oxygen species (ROS) in the intrinsic cells of the kidneys. This can cause oxidative damage to podocytes, endothelial cells, tubular epithelial cells, and mesangial cells, exacerbating the occurrence and development of diabetic nephropathy. Autophagy is an important internal degradation mechanism that maintains the stability of the intracellular environment in eukaryotic cells. It acts by degrading lysosomal proteins, clearing damaged structures or overexpressed proteins, and participating in maintaining cell renewal and homeostasis. The pathological and physiological processes of DN are closely related to endoplasmic reticulum stress, mitochondrial dysfunction, and inflammatory response. Autophagy clears misfolded proteins and damaged organelles, promotes autophagosome formation and autophagy lysosome fusion, and alleviates endoplasmic reticulum stress and cell apoptosis. ROS generated under oxidative stress can induce autophagy, which can clear free radicals and alleviate oxidative stress damage to kidney tissue and cells. Autophagy is an important survival promoting mechanism for cells. Effectively inducing autophagy and controlling the inflammatory process are effective strategies for treating DN.

In recent years, studies have shown that microRNAs (miRNAs) are abnormally expressed in DN patients and participate in pathological processes such as mesangial extracellular matrix (ECM) aggregation and podocyte injury, especially microRNA-21 (miR-21), which has become a hot research topic. MiR-21 is widely present and regulates cell differentiation, proliferation, and apoptosis. Multiple studies at home and abroad have found that the serum miR-21 concentration in DN patients is significantly higher than that in normal individuals, and miR-21 is positively correlated with renal tubulointerstitial fibrosis in DN patients’ renal tissue biopsies. Therefore, silencing miR-21 or inhibiting its expression is of great significance in inhibiting long-term or short-term complications of diabetes. MiR-21 has been reported to regulate cellular autophagy by modulating signaling pathways such as Akt/mTOR and PTEN. However, there is no report on reducing renal oxidative damage in diabetes nephropathy by regulating miR-21 mediated autophagy.

Emodin (EM) is an effective active ingredient in the Polygonaceae plants Rheum palmatum L., Rheum tanguticum Maxim. ex Balf., and Rheum officinale Baill. It belongs to the anthraquinone class of compounds and has pharmacological effects such as antibacterial, anti-inflammatory, immune regulation, anti-tumor, and renal function improvement. Previous studies have found that emodin can delay the progression of diabetic nephropathy by inhibiting high glucose induced proliferation of glomerular membrane cells and promoting apoptosis of mesangial cells. The preliminary experimental results also showed that 25 mmol/L glucose can significantly promote the expression of miR-21 in glomerular mesangial cells. This study aims to explore the mechanism of emodin in alleviating renal oxidative damage in diabetes nephropathy mice by regulating miR-21 mediated autophagy.

The model of diabetes nephropathy in mice was induced by intraperitoneal injection of high sugar and high fat diet plus streptozotocin. The successful mice were randomly divided into model group, glimepiride group (0.6 mg/kg/d, ig), emodin high-dose group (50 mg/kg/d, ig), emodin low-dose group (25 mg/kg/d, ig), and normal group (10 in each group). After one week of treatment, blood glucose, kidney weight coefficient, and pathological morphology were measured in each experimental group of mice; The fluorescence probe method was used to measure the ROS content in the kidneys, while the Elisa method was used to measure the BUN, Cr, and uAE content in the kidneys; Transmission electron microscopy was used to observe the degree of autophagy in renal cells, and Western blot was used to measure the expression of P62, Atg7, and LC3 proteins. The representative results are as follows.

Oxidative stress and apoptosis have been confirmed in the kidney tissues of patients with diabetes nephropathy and animal models. In vitro experiments have also found that high glucose environment can induce podocytes to undergo oxidative stress, apoptosis, inflammation and other events. Renal podocytes play an important role in maintaining the integrity of the glomerular filtration barrier and regulating glomerular permeability. In this process, glomerular podocyte autophagy is closely related to the pathogenesis of diabetes nephropathy. Once the autophagy activity of podocytes is altered, overactivated, or inhibited, it can cause inflammatory damage to podocytes and damage to the filtration barrier. In this experiment, we also observed that the podocyte autophagy in the kidney of DN mice is seriously insufficient, and ROS is significantly increased. ROS is a signal molecule in the mitochondria of podocytes, and is the starting link for the occurrence and development of diabetes and its complications. Reducing the production of ROS is a potential method to prevent the progress of DN. After oral administration of emodin to mice, we observed a significant decrease in renal ROS and an increase in autophagy, leading to an improvement in a series of cascade reactions, such as a significant reduction in renal inflammatory pathological damage and a significant decrease in BUN, Cr, and uAE levels. Therefore, promoting autophagy to clear high glucose induced ROS in mouse kidneys may be an important mechanism for emodin to improve DN mice. Meanwhile, we also observed that Glim can significantly reduce the levels of BUN, Cr, and uAE in DN mice. Glim is a sulfonylurea based hypoglycemic drug, which acts by binding to sulfonylurea receptors on the surface of pancreatic beta cells and releasing it uniformly and slowly in vivo. At the same time, Glim can enhance the sensitivity of surrounding tissues to insulin and improve insulin resistance. The utilization of glucose by surrounding tissues increases, and blood glucose levels decrease significantly. After reaching the blood sugar standard, the production of glycation end products decreases, and the damage to the kidneys is reduced.

MiR-21, as a broad-spectrum gene, has been proven to be positively correlated with the onset of diabetic nephropathy. Previous literature both domestically and internationally has shown that miR-21 can participate in the occurrence and development of diabetic nephropathy by mediating signaling pathways such as TGF – β/Smad, PTEN/Akt, and MMP-S/TIMPS. In terms of autophagy, there are also literature reports that miR-21 can intervene in the Akt/mTOR signaling pathway to inhibit autophagy in renal podocytes and exacerbate the pathogenesis of diabetic nephropathy. These pieces of evidence indicate that inhibiting miR-21 and regulating its downstream pathways is an effective strategy for improving DN. This study also observed that the expression of miR-21 in the kidneys of DN mice was significantly upregulated compared to the normal group, which may be related to the weakened autophagy in the kidneys of DN mice. In non DN diseases, miR-21 has also been shown to participate in vital activities such as pulmonary fibrosis, macrophages, and hypoxic oval cells through negative regulation of autophagy. After administration of emodin, the expression of miR-21 in the kidneys of DN mice was significantly downregulated, while autophagy inhibition was relieved. However, whether emodin directly mediates autophagy or regulates miR-21 mediated cellular autophagy needs to be confirmed through target gene prediction analysis, as some researchers have reported that emodin can regulate autophagy through lysosomal and mitochondrial pathways, but whether it is related to regulating miR-21 requires further research.

In terms of exploring autophagy mechanisms, we detected the expression of three classic autophagy marker proteins, P62, Atg7, and LC3. These three proteins are key members of the autophagosomal pathway, which plays an important role in eliminating and degrading damaged organelles, denatured proteins, and other biomolecules, and is an important repair mechanism for cells. Therefore, we speculate that the enhanced autophagy and recovery of renal pathological and physiological functions in DN mice by emodin may be related to the autophagosomal pathway. The results showed that the expression of P62, Atg7, and LC3 proteins in the kidneys of DN mice was upregulated after administration of emodin, suggesting that emodin may act on the autophagy lysosome pathway of renal podocytes to protect the kidneys of DN mice.