Shanna phenol regulates AMPK/NOX4 pathway to inhibit high glucose induced oxidative stress and extracellular matrix accumulation in glomerular mesangial cells
Diabetes nephropathy (DN) is a microvascular complication of diabetes and an important cause of end-stage renal failure. Abnormal function of glomerular mesangial cells (GMCs) can promote pathological and physiological changes in diabetic nephropathy. Under renal pathological conditions, long-term hyperglycemia leads to the accumulation of a large amount of reactive oxygen species (ROS) in the body, which activates downstream glucose signaling responses, promotes extracellular matrix (ECM) accumulation, and directly stimulates abnormal proliferation of GMCs. This suggests that intervening in oxidative stress (OS) and ECM accumulation in GMCs may be an effective strategy for preventing diabetic nephropathy.

DN has multiple pathogenesis mechanisms, among which OS is a key pathogenic factor in DN, caused by elevated ROS levels. The reduced nicotinamide adenine dinucleotide phosphate (NADPH) family (NOX 1-5 and Duox 1-2) transfers electrons to oxygen molecules through the plasma membrane to generate ROS, which is one of the main sources of ROS in DN. NADPH oxidase 4 (NOX4) is a highly expressed subtype of NADPH in the kidneys of DN patients and is the main cause of high glucose induced OS production in GMCs. Elevated levels of NOX4 can directly cause glomerular fibrosis. Silencing NOX4 expression will weaken glomerular membrane dilation, glomerulosclerosis, and ECM accumulation. Revealing the molecular mechanism of high glucose induced upregulation of NOX4 expression and the upstream and downstream effector factors involved is of great significance.

Phosphorylation of 5 ‘- adenosine monophosphate activated protein kinases (AMPK) can block high glucose induced NOX4 expression and inhibit abnormal proliferation and activation of renal fibroblasts. Sestrins is an important stress-induced protein family that plays a crucial role in maintaining cellular environmental stability. Sestrin2 is one of the most important members of the family and plays a critical role in protecting cells from oxidative damage by activating AMPK phosphorylation. In addition, AMPK may be a negative regulator of NOX activation, as Sestrin2 and AMPK activation can inhibit ROS and protect GMCs from OS invasion, indicating that activation of Sestrin2 and AMPK phosphorylation may have a preventive effect on DN.

The accumulation of OS and ECM has been proven to play an important role in the pathogenesis of diabetic nephropathy, so it is particularly urgent to search for traditional Chinese medicine and natural products with antioxidant effects and weakened DN effects. Flavonoids have attracted increasing attention from scholars due to their excellent efficacy. Kaempferol (KAE) is a flavonoid compound that has been proven to have multiple pharmacological activities. KAE reduces heart damage caused by high blood sugar by inhibiting inflammatory response and OS; By inhibiting RhoA/Rho kinase mediated inflammatory signaling, DN can be improved. At present, there is relatively little research on the intervention of KAE in high glucose induced GMCs OS and ECM accumulation by regulating the AMPK/NOX4 pathway. This study used an in vitro high glucose model of GMCs to investigate the effects of KAE on the accumulation of OS and ECM in high glucose induced GMCs and its protective mechanism against DN.

Elevated blood glucose level is considered to be the key factor of renal function and pathological changes in diabetes nephropathy. Due to the similarity in phenotype between mesangial cells cultured in HG environment and DN patients, in this experiment, we investigated the protective effect of KAE on HG induced GMC damage in vitro. The results showed that KAE inhibited HG induced GMC cell proliferation, OS, and ECM accumulation by regulating the AMPK/NOX4 pathway.

The early characteristics of DN are abnormal proliferation of mesangial cells and excessive deposition of ECM, which usually leads to mesangial proliferation, renal fibrosis, and end-stage renal damage. Collagen, fibronectin (FN), and laminin are the main components of ECM, which are mainly synthesized under high blood glucose stimulation. In DN, TGF – β 1 is a key cytokine involved in the accumulation of renal ECM. Under normal physiological conditions, various cells in the body can secrete inactive TGF – β 1; In a high glucose environment, the inactive TGF – β 1 can be transformed into an active state, promoting the accumulation of glomerular ECM by increasing the expression of ECM genes (such as Col IV) and reducing ECM degradation. Inhibiting TGF – β 1 can weaken the high glucose induced changes in ECM gene expression, thereby reducing ECM accumulation in DN. The results showed that in high glucose stimulated GMCs cells, the mRNA and protein levels of TGF – β 1 and Col IV were significantly increased, while KAE treatment could reduce their expression, indicating that KAE could play an anti diabetes role by reducing the accumulation of ECM and promoting its degradation.

The pathogenesis of DN is related to the occurrence of OS, which can activate multiple signaling pathways within cells and stimulate transcription factors, leading to increased ECM accumulation and reduced matrix degradation. HG induced ROS generation can disrupt the balance between oxidants and antioxidants, interfere with antioxidant defense systems, and lead to damage to GMCs. Based on this, the excessive production of ROS plays an important role in the pathogenesis of diabetic nephropathy. Under pathological conditions, ROS clearance requires the joint involvement of enzymatic and non enzymatic systems, such as SOD and MDA. SOD is an antioxidant metalloenzyme that plays an important role in maintaining the balance between oxidation and antioxidation by catalyzing the dismutation of superoxide anions and hydrogen peroxide. MDA has an impact on the antioxidant defense system, as MDA levels are directly correlated with membrane lipid peroxidation levels in GMCs. The results showed that KAE may increase SOD activity and reduce MDA content in HG induced GMCs, indicating that KAE has potential antioxidant properties.

ROS transduction and enhanced glucose signaling are considered important pathogenic factors that promote the development of diabetic nephropathy. There are many sources of ROS in the kidney, and the NADPH oxidase subtype of NOX family is mainly related to diabetes nephropathy. So far, studies have found seven subtypes of the NOX family, among which NOX4 is highly expressed in rat and mouse glomeruli, podocytes, and tubular cells. Normally, NOX4 binds to the transmembrane subunit (p22phox) to form an active complex, which is responsible for its biological function. The upregulation of NOX4 is associated with the increase of FN and TGF – β 1 in mesangial cells and tubular cells exposed to HG. NOX4 is associated with TGF – β 1-induced ECM accumulation in fibroblasts, which contributes to their differentiation into fibrotic fibroblast phenotypes. NOX4 has been shown to mediate an increase in mesangial hypertrophy and FN elevation under angiotensin II treatment. Excessive accumulation of OS and ECM may lead to kidney damage and is considered an important cause of DN. To investigate the relationship between OS, ECM accumulation, and NOX, Western blot and qRT PCR were used to measure the protein and mRNA levels of NOX4 subtypes highly expressed in DN. Research has shown that eliminating NOX4 activity leads to a decrease in OS and ECM in HG induced GMCs. The results of this experiment are consistent with previous studies. HG upregulates the mRNA and protein expression of NOX4 and p22phox, stimulating the generation of ROS, TGF – β 1, and Col IV in GMCs. The use of KAE and silencing NOX4 and p22phox can inhibit the accumulation of OS and ECM, indicating that the antioxidant effect of KAE is regulated through the NOX4/p22phox pathway.

The Sestrins family is composed of a group of stress-induced proteins involved in intracellular stability regulation, which is one of the antioxidant defense mechanisms and has two different biological activity functions. Firstly, inhibiting ROS accumulation to exert antioxidant effects may involve regulating antioxidant transcription factors. Secondly, Sestrins act as feedback inhibitors of mTORC1 by activating AMPK or inhibiting Rag GTPases. AMPK is an important metabolic sensor widely expressed in almost all eukaryotic cells. Research has shown that under HG conditions, Sestrin2 and AMPK activation inhibit NOX4 induced ROS accumulation in GMCs. AMPK phosphorylation inhibits ROS production and ECM accumulation in human mesangial cells. In addition, AMPK phosphorylation can inhibit the accumulation of TGF – β 1 in urine, suppress mesangial matrix expansion, and reduce collagen deposition in DN mouse models. More and more studies suggest that AMPK is closely related to NOX activity in DN. In this experiment, it was found that KAE significantly inhibited HG induced cell proliferation, ROS production, NOX4, TGF – β 1, and Col IV expression by upregulating AMPK. In addition, compound C reversed the protective effect of KAE on HG induced OS and ECM deposition in GMCs.

In summary, this study demonstrates that KAE exerts a protective effect on HG induced accumulation of GMCs OS and ECM by regulating the AMPK/NOX4 pathway. It is expected that KAE can serve as a potential candidate drug or lead compound for the prevention and treatment of DN.