Network pharmacology is a rapidly emerging discipline. It has also transformed the concept of drug discovery from "one target, one drug" to "network target, multi-component therapy". Because of the advantage of network pharmacology research strategy, it can open up a new and innovative way for the development of traditional Chinese medicine. The purpose of this study is to analyze the active components, target and related signal pathway of Milkvetch Root in improving glycolipid metabolism of diabetic nephropathy by network pharmacology, and to explore the possible mechanism of action.
Using network pharmacological analysis, we found that there are 20 active components and predicted 180 potential targets in Milkvetch Root. The results of T-D network analysis show that there are 360 edges in the network, which represent the interaction between active component and target. Among them, quercetin has the most potential targets with a total of 136, followed by kaempferol with 51 potential targets. Others, such as 7-O-methylisomucronulatol, formononetin, isorhamnetin and(6aR,11aR)-9,10-dimethoxy-6a,11a-dihydro-6H-benzofurano[3,2-c] chromen-3-ol also have more corresponding targets, which are 33, 28, 25, and 19 respectively. The corresponding targets of active ingredients are AKT1, VEGFA, IL6, PPARG, NOS3, etc.
Quercetin is a potent antioxidant flavonoid found in many common medicinal herbs, and possesses a wide spectrum of biologic activities . It also has anti-oxidant, hypoglycemic, hypolipidemic, tumor suppression, and anti-inflammatory effects [28.29.30]. One study showed that quercetin liposome or free quercetin can prevent weight loss, decrease kidney hypertrophy index, decrease blood glucose level and 24-hour urine protein in diabetic nephropathy model rat . Kaempferol is a kind of natural peroxisome proliferator-activated receptor-γ(PPARγ) agonist, and PPARγ agonist has also become a common drug in the treatment of diabetes and its complications . Kaempferol has similar hypoglycemic effect with Rosiglitazone, but its adverse reactions are significantly lower than the latter. It can improve the glucose uptake of 3T3-L1 cells, control blood glucose, and improve the oxidative stress damage of kidney caused by glucose metabolism disorder . There are also studies that suggest that kaempferol can work as RhoA/Rho Kinase inhibitor and may attenuate progression of diabetic complications with specific emphasis on DN . Formononetin is a polyphenolic compound. Formononetin is a potential molecule which increases the expression of SIRT1 in kidney tissue of diabetic, which is an effective molecule to control nephropathy in type 2 diabetes mellitus .7-O-methylisomucronulatol has the similar pharmacological effect with formononetin. It can prevent and treat DN by inhibiting the proliferation of mesangial cells and the production of nitric oxide .
Isorhamnetin can inhibit the NF-κB signaling activity, decreased the production of inflammatory mediators and attenuated oxidative stress in diabetic rats and glomerular mesangial cells(GMCs), thus reducing urinary albumin filtration, reducing renal damage, improving renal pathological changes and so on . This fully shows the complex network relationship between drugs and targets, and verifies that Milkvetch Root plays the role of improving DN in a multi-component and multi-target way.
In addition, quercetin, kaempferol, formononetin and isorhamnetin are all flavonoids. Studies on the mechanism of action suggested that flavonoids can improve the metabolism of sugar and lipid, enhance insulin resistance, inhibit the activity of relevant glucose metabolic enzymes, and avoid oxidative damage of DM [38.39]. Therefore, it is point out that these components may be the main components of Milkvetch Root. It can be seen that flavonoids of TCM may be a novel drug for diabetic nephropathy, which has a broad prospect of development.
In terms of target, there are three isoforms of AkT: AKT1 (PKBα), AKT2 (PKBβ), and AKT3 (PKBγ), which each have their own physiologic functions . The protein kinase AkT, also known as protein kinase B (PKB), has been shown to regulate a variety of cell functions, and is particularly important for glucose metabolism, cell growth, and cell survival. Therefore, changes in its expression or activity are thought to be involved in the pathogenesis of diabetes and DN .
In humans, there are five secreted glycoproteins that make up the VEGF family member: VEGF-A, VEGF-B, VEGF-C, VEGF-D and placental growth factor (PlGF) . Previous studies have demonstrated that angiotensin type 1 receptor blocker (ARB) can inhibit the synthesis of VEGF mediated by Ang‑II and can effectively treat diabetic nephropathy . VEGF-A is an important regulator of angiogenesis and vascular permeability with a possible pathogenic role in diabetic nephropathy [44.45]. VEGF-A is essential for the normal growth of podocytes. When the expression of VEGF-A was lower than the normal level, the podocytes were damaged . In conclusion, blockade of VEGF-A can effectively restore renal function in diabetic nephropathy.
IL-6 in the pathogenesis of DN and its association with insulin resistance. Studies suggested that IL-6 affect the dynamics of ECM surrounding cells and may increase GBM and endothelial permeability . Current evidence suggests that IL-6 responses are mediated via gp130-STAT3 dependent mechanisms which, on one hand, trigger globally the transition from innate to adaptive immune response, and on the other hand act locally for tissue remodeling and immune cell infiltration . Therefore, the regulation of IL6 target is of great significance in the treatment of DN.
PPARG is a kind of transcription factor activated by ligands. At present, there are three subtypes: PPARα, PPARβ, PPARγ . Some studies have found PPARG is the risk of progression of diabetic nephropathy in China .
Nitric oxide (NO) is closely related to kidney by regulating renal hemodynamics, renin secretion, inhibiting renal tubular sodium reabsorption, renal tubular glomerular feedback (TGF) and renal sympathetic nerve activity [51.52]. The synthesis of NO in vivo is closely related to nitric oxide synthetase (NOS2/3). The active components act on NOS related targets, enhance the biological activity of NOS, restore no pathway can down regulate the expression of inflammatory factors, thereby reducing creatinine level, protein filtration rate, and play the role of renal protection .
In this study, according to the result of molecular docking and network analysis, all protein-pathway pairs were distributed among oxidative stress, inflammation, metabolism, immune system, apoptosis, and multiple pathways. For instance, Oxidative stress and inflammation prompted by hyperglycemia are the key initiators finally leading to renal damage and nephropathy [54.55]. AGE-RAGE signaling pathway in diabetic complications, HIF-1 signaling pathway, PI3K-Akt signaling pathway and TNF signaling pathway are responsible for imparting therapeutic effects on DN, and these pathways have been widely studied. Some studies have confirmed that AGE-RAGE signaling pathway is a signaling mechanism in the pathogenesis of diabetes and its complications . It can aggravate the vascular damage of diabetes through oxidative stress , increase the risk of renal function deterioration and cardiovascular events, leading to an increase in all-cause mortality . HIF can activate in the early stage of DN under hypoxia, and stimulate the proliferation and aggregation of inflammatory factors in the injured kidney, which paves the way for renal fibroblast scar [59.60]. Secondly, HIF can be combined with fibrosis-promoting genes such as collagen 1, connective tissue growth factor, and plasminogen activator inhibitor 1, to generate interstitial collagen and reduce degradation of the extracellular matrix (ECM), eventually leading to renal fibrosis . PI3K-Akt signaling pathway has been indicated as the source of glomerular hypertrophy and ECM accumulation . PI3K can activate its downstream molecule Akt, and Akt further phosphorylates fox OS, GSK-3, Bad, mTOR and other proteins to cause the cascade reaction of signal pathway, which plays a key role in the accumulation of extracellular matrix, mesangial cell proliferation, epithelial mesenchymal transformation and other aspects of diabetic nephropathy[63.64]. TNF-α can stimulate the aggregation and adhesion of inflammatory cells, increase the permeability of small blood vessels, and damage the glomeruli through inflammatory reactions .