A Network Pharmacology Approach to Understanding the Mechanisms of Action of Traditional Medicine: Rheum L. for Diabetic Kidney Disease

Abstract


Background
Diabetic kidney disease (DKD) is one of the most common and serious complications of diabetes mellitus.Pathological changes in renal cells of DKD patients include glomerular hypertrophy, mesangial expansion, and tubulointerstitial brosis due to the accumulation of extracellular matrix (ECM) proteins, thickening of basement membrane, and podocyte dysfunction 1 .DKD is now the main cause of CKD worldwide and the leading cause of end-stage-renal disease (ESRD) requiring renal replacement therapy.
Approximately 20-40% of diabetic patients suffer from DKD in China, which is the main cause of CKD and end-stage renal failure in patients with diabetes 2 .DKD is a major but under-recognized contributor to the global burden of disease.Between 1990 and 2012, the number of deaths attributed to DKD rose by 94% 3 .This dramatic rise is one of the highest observed for all reported chronic diseases 4 .Early medical research has shown that angiotensin-converting enzyme inhibitors (ACEIs) and angiotensin II receptor blockers (ARBs), the rst-line treatment for DKD, can reduce proteinuria and have a certain effect on delaying the progress of renal dysfunction 5 .However, the side effects of ACEI/ARB, such as dry cough, hypotension, hyperkalemia, and angioedema, limit the application of these drugs.Evidence-based medical studies have shown that these agents have not signi cantly reduced DKD vascular event rate and mortality 6 .Therefore, it is urgent to discover potential therapeutic targets and develop new therapeutic strategies for the treatment of DKD.
Traditional Chinese Medicine (TCM) is a branch of traditional oriental medicine, characterized by its systematic and holistic philosophy, which has been widely used in Asian countries 7 .TCM have multiple ingredients that contain a variety of advantages, such as synergy, minor side effects, and improvement of adaptive resistance 8 .TCM network pharmacology not only con rms and optimizes multiple targets interventions by modeling signaling pathways and precise processes, but also estimates the e cacy of drugs, especially multi-target drugs 9,10 .Based on the theory of traditional Chinese herbal medical science, TCM can offer a treatment for the prevention and treatment of DKD in a systematic way.As a traditional medicinal plant, Rheum L. has satisfactory drug e cacy for the alleviation of DKD, which indicates the existence of certain pharmacological components in Rheum L. 11 .In clinical practice, we discovered that some formulas, which contained Rheum L., could effectively decrease 24hurinary protein level, increase glomerular ltration rate in type 2 DKD patients with macroalbuminuria, and ameliorate diabetic kidney injury 12,13 .However, the pharmacological mechanisms of Rheum L. are still vague.
The aim of our study was to screen the related ingredients of Rheum L. using multiple databases and acquire the potential targets by target shing.Then, we aimed to screen the related targets of DKD by consolidation of a multi-source database.Based on the matching results between Rheum L. potential targets and DKD targets, we aimed to build a protein-protein interaction (PPI) network to analyze the interactions among these targets and screen the hub targets based on topology.Moreover, using The DAVID bioinformatics resources, we aimed to obtain the enrichment analysis of the GO-BP and KEGG.This study is necessary to investigate how Rheum L. alleviates DKD via the molecular mechanisms predicted by network pharmacology and how the network pharmacology approach can be an effective tool to reveal the mechanisms of TCM.The owchart of the experimental procedures of our study is shown in Fig. 1.

Ingredients in Rheum L. and its targets
To collect the ingredients in Rheum L., we used the Traditional Chinese Medicine System Pharmacology Database 14 (TCMSP, http://lsp.nwu.edu.cn/tcmsp.php,2020.2.28).Ninety-two herbal ingredients were recorded in this process.Retrieving the small molecular structure information of the active ingredients in Rheum L. on the PubChem database (https://pubchem.ncbi.nlm.nih.gov/) and the Swiss Target Prediction webserver (http://www.http://www.swisstargetprediction.ch/index.php).Twenty three targets that matched the related targets of Rheum L. on DKD were collected as related targets for the effect of Rheum L. on DKD (Fig. 2 and Fig. 3).

GO biological process and KEGG pathway enrichment analysis
In the String database, the PPI network of the 23 targets was established.The details are shown in Fig. 4.
represent the degree, as the scale indicates.The size of the circle also indicates the degree.

GO biological process enrichment analysis
DAVID v6.8 was used for enrichment analysis of the 23 targets.The screening threshold was P < 0.01 and 11 GO items were retrieved.We selected 18 KEGG pathways for analysis.The details are shown in Fig. 6.
. Figure 6.The main 18 pathways enriched by major hubs from the DAVID database.

KEGG pathway enrichment analysis
The 11 biological processes were mainly involved in in ammatory response, apoptosis, brosis, and peripheral circulation.The details are shown in Fig. 7.The processes were, in the aspect of cell proliferation: positive regulation of transcription from RNA polymerase II promoter (GO:0045944), positive regulation of cell proliferation (GO:0008284), transmembrane receptor protein tyrosine kinase signaling pathway (GO:0007169), and transcription, DNA-templated (GO:0006351);in the aspect of protein metabolism: negative regulation of protein binding (GO:0032091) and positive regulation of protein binding (GO:0032092); in the aspect of in ammatory response: response to oxidative stress (GO:0006979); in the aspect of apoptosis: negative regulation of extrinsic apoptotic signaling pathway (GO:2001237); and in the aspect of peripheral circulation regulation: glucose homeostasis(GO:0042593), regulation of blood pressure(GO:0008217), and blood coagulation(GO:0007596).Based on these ve main aspects, a complex multi-path synergetic effect may be the cause of the effect of Rheum L. on DKD.

In ammatory response
It is known that PTGS2 is often called cyclooxygenase 2 (COX-2), several in ammatory cytokines contribute to DKD pathogenesis, including COX-2, in ammatory parameters in patients with type 2 diabetes at an early stage of nephropathy are independently associated with urinary albumin excretion (UAE) 19 .High glucose activates protein kinase C (PKC) to increase the permeability of Glomerular endothelial cells (GECs),which up-regulates the COX-2 in endothelial cells and COX-2 up-regulation is associated with an imbalance in eicosanoids pro le, with an increase in vasoconstricting thromboxane B2 and a decrease in vasodilatory 6-keto-prostaglandin Fα 20 .These changes lead to renal hemodynamic changes, which may participate in glomerular hyper ltration observed in early DKD.In addition, increased ROS production upregulates COX-2 gene transcription and modi es the production of prostanoids 21,22 which control vascular reactivity of the renal glomerular arterioles and deteriorate the glomerular endothelial surface layer 23 .In mice, diabetes-induced endothelial injury, albuminuria, podocyte loss, and glomerulosclerosis are ameliorated by scavenging of mitochondrial ROS prevent endothelial mitochondrial oxidative stress 24 .
Adiponectin exerts favorable effects in anti-in ammatory, anti brotic, antioxidant effects, and potently stimulates ceramidase activity.Low circulating adiponectin levels in obese patients who have a risk of insulin resistance, type 2 diabetes, and cardiovascular diseases, and increased adiponectin expression in the state of albuminuria suggest a protective and compensatory role for adiponectin in mitigating further renal injury during the development of overt DKD.Enhancing the AMPK/PPARα pathway and ceramidase activity alleviate renoprotective role against lipotoxicity and oxidative stress 25 .Moreover, a study demonstrated that peroxisome proliferator-activated receptor γ (PPARγ) coactivator-1α (PGC-1α) participates in mitochondrial biogenesis in tissues with high energy consumption, as a key nuclear factor in energy and oxidative stress 26 .PGC-1α is highly expressed in the kidney 27 , and is reported to protect against several kidney diseases.PGC-1α upregulation has been demonstrated to alleviate mitochondrial dysfunction in acute kidney injury and an adriamycin nephrosis model 28,29 .
IGF1 is a powerful regulatory factor in various cell types, including glomerular and tubular cells 30 .It is an important growth factor for keeping the nephritic structure and function.It also plays a key role in the pathological process of DKD 31 .Inhibition of IGF1R could alleviate in ammation in DKD more e ciently 32 .Li J el al 33 found that inhibition of IGF1R was a more effective choice for in ammation treatment than insulin in diabetic kidney disease (DKD).The IGF1R inhibitor blocked pathological changes induced by the over-expression of IGF1 in DKD without up-regulating SOCS2 protein levels.

Apoptosis
Apoptosis, which programmed cell death, plays an essential role in the development and homeostasis of metazoans.Deregulation of apoptosis leads to a variety of pathologic disorders, including cancer, autoimmune diseases, and neurodegenerative disorders.Caspases, part of the cysteine protease family, are central to the initiation and execution of apoptosis, acting to speci cally cleave the C-terminal side of an aspartate residue in substrates.The apoptotic caspases are generally classi ed into initiator and executioner caspases,and one of the principal executioner caspases is Caspase-3 34,35 .Researchers have found that apoptosis had an indispensable role in renal ischemia-reperfusion injury and contrast-induced acute kidney injury 36,37 .Markers of apoptosis have repeatedly been linked with DKD 38 .Wang et al 39 showed that GSDME could switch Caspase-3-dependent non-in ammatory and immunologically silent apoptosis to a terminal phase, namely secondary necrosis.Wen et al 40 found that Caspase-3 could promote renal injury and brosis in DKD through gasdermin E-mediated progression to secondary necrosis during apoptosis.
IGF-I is synthesized in renal glomeruli and distal tubules.IGF-I raises GFR through reducing arteriolar resistance and increasing LpA.IGF-I has been associated with the initiation of hypertrophy in models of compensatory renal growth and may contribute to the accumulation of extracellular matrix proteins in the nephron in chronic renal diseases.IGF-1 can promote sclerosis by either increasing synthesis, or decreasing the degradation of glomerular extracellular matrix (ECM), suggesting that IGF-1 could be a major contributor to the development of diabetic glomerulosclerosis 41,42 .In addition, insulin and IGF-1 are major anti-apoptotic hormones, as well as metabolic and growth hormones.Their effects are mediated through activation of the insulin and IGF-1R and the consequent activation of the PI3K-Akt (phosphatidylinositol 3-kinase to Akt) and the Ras-Raf-MEK-ERK (Ras to Raf to mitogen-activated or extracellular signal-regulated protein kinase to extracellular signal-regulated protein kinase) pathways 43,44 .In brown preadipocytes in the basal state, the presence of insulin or IGF-1 receptors plays a permissive role in apoptosis, which is blocked either by ligand binding or by deletion of both receptors, revealing the apoptosis that is independent of their kinase activity 45 .

Fibrosis
PPARγ belongs to the ligand-activated type II nuclear receptor superfamily and predominantly expressed in adipose tissues 46 .In the kidney, PPARγ is mainly located in the medullary collecting duct, and glomeruli and proximal tubules were also expressed in small amounts 47 .Activated PPARγ delayed the progress of DKD by improving insulin resistance 48 , lowering blood pressure 49 , ameliorating in ammation 50 , reversing cell cycle arrest 51 , increasing adiponectin 52 , improving oxidative stress 53 , and other mechanisms.On the other hand, the study has shown that PPARγ single nucleotide polymorphism (SNP) is associated with the risk of diabetic kidney disease 54 .All of these indicated that PPARγ signal pathway plays a protective role in DKD 55 .Moreover, The PPARγ agonist may induce PGC-1α expression to maintain mitochondrial function and to reduce ROS generation, and it may ameliorate podocyte impairment, GBM thickening and kidney brosis to aid in the prevention of DKD occurrence and progression 56 .
IGF-1 participates in multiple biological metabolic pathways of DKD.Dong et al 57 discovered that IGF-1R inhibitor could ameliorate urinary albumin excretion and kidney histological changes due to diabetes, including amelioration of glomerulomegaly, in ammatory in ltration, and tubulointerstitial brosis.
Activation of IGF-1 in diabetic kidneys induces brogenesis through Snail1 upregulation.The diabetesrelated histological and functional changes, as well as brogenesis, can be attenuated by IGF-1/IGF-1R inhibition.

Peripheral circulation regulation
In type 1 diabetes (T1D), adjuvant treatment with inhibitors of the renin-angiotensin-aldosterone system (RAAS), which dilate the efferent arteriole, is associated with prevention of progressive albuminuria and renal dysfunction.In the setting of chronic hyperglycemia, overactivation of the RAAS is strongly implicated in the initiation and progression of DKD 58,59 .Angiotensin II (Ang II) is the major peptide of RAS, and is formed under the action of the angiotensin converting enzyme (ACE).Ang II shows increased activity under high glucose conditions that causes hypertrophy of various renal cells and has a pressor effect on arteriolar smooth muscles, thereby resulting in increased vascular pressure 60 .The ACE gene is composed of 26 exons and 25 introns, with a length of 21 kb.Recently, many researchers have demonstrated that the ACE I/D polymorphism is related to diabetic microangiopathy, and that the D allele might be a susceptibility factor for patients with DKD [61][62][63] .The ACE I/D polymorphism was suggested to be strongly associated with the development of DKD, and using valsartan in DKD patients could signi cantly decrease the excretion of urinary albumin 64 .
Rheum L., one of the most popular traditional Chinese medicines used to control various diseases for thousands of years, there are several studies that demonstrate that Rheum L. can effectively reduce in ammation, apoptosis, antioxidant stress, and blood pressure.

Glycolipid metabolism
Cheng et al. 65 found that the fasting blood glucose (FBG), total cholesterol (TC), and triglyceride (TG) levels in the serum of rats in the puri ed anthraquinone-Glycoside from Rheum palmatum L. treatment groups were signi cantly decreased.The expression of Fas ligand (FasL), cytochrome C (Cyt-c), and Caspase-3 in pancreatic tissue was obviously decreased, and the pathological damage to the liver, kidney, and pancreas was improved, which indicated that PAGR can reduce oxidative stress in rats with diabetes mellitus by improving blood lipid metabolism and enhancing their antioxidant capacity, thereby regulating the mitochondrial apoptotic pathway to inhibitβ-cell apoptosis and improve β-cell function.Furthermore, PAGR could activate the GLP-1/cAMP pathway to decrease glycated serum protein (GSP), insulin concentration and HOMA-IR index, increase GLP-1 concentrations, ameliorate insulin resistance, and restore the gut microbiota 66 .

Improving renal function
The changes in mean blood glucose levels, normalized kidney weight, urinary albumin excretion, serum creatinine levels and tubulointerstitial injury index (TII) scores of the rats with DN were signi cantly attenuated by emodin.Furthermore, treatment with emodin signi cantly inhibited in ammation-related factors and oxidative stress, suppressed the expression of intercellular adhesion molecule 1 (ICAM-1) and B-cell lymphoma 2-associated X protein (Bax), increased phosphorylated Akt and phosphorylatedglycogen synthase kinase 3 (p-GSK-3β) expression and inhibited caspase-3 activity in diabetic rats 67 .Li et al 68 discovered that using Rheum L. anthraquinones extract decreased the serum creatinine (SCr), blood urea nitrogen (BUN) and urine protein (UP) values in diabetic nephropathy rats, which suggested that it displayed certain therapeutic and preventive effects against the diabetic nephropathy.Moreover, administration of Rheum L. inhibited increase of BUN and serum creatinine in cases with chronic renal failure and was effective for the reduction of uremic substances, which can retard the introduction-period of hemodialysis and inhibit deterioration of the disease 69 .Gentamicin increased the level of urinary glucose and protein, and increased malondialdehyde while it decreased thiol in kidney tissue, and increased the concentration of urea and creatinine in the serum.However, hydroalcoholic extract of Rheum turkestanicum was able to improve gentamicin toxicity, it has positive effects in the attenuation of gentamicin-induced nephrotoxicity 70 .

Delaying renal brosis
Shui et al 71 found that Dahuang Fuzi Decoction could attenuate renal brosis and ameliorates mitochondrial dysfunction in chronic aristolochic acid nephropathy.Besides, Dahuang Fuzi Decoction could alleviate adenine-induced tubular epithelial apoptosis and renal damage in vivo, presumably through the suppression of TGF-β1-JNK pathway activation 72 .Chinese researchers believed that Niaoduqing particles safely and effectively delayed CKD progression in patients with stage 3b-4 CKD.This traditional Chinese medicine may be a promising alternative medication for patients with moderateto-severe renal dysfunction 73 .As the main active ingredient of Rheum L., emodin regulates lipopolysaccharide-induced toll-like receptor 4 and reduces the expression of tumor necrosis factor alpha and interleukin 6, all three being synthesized by renal tubular epithelial cells 74 .Emodin acts as an antiin ammatory agent by inhibiting the differentiation and maturation of dendritic cells and increasing the number of regulatory T-cells 75 .It also can bring high blood pressure down, decrease blood lipids and improve microcirculation, protect kidney 76 .Tu et al 77 discovered that rhein could inhibit autophagy in rat renal tubular cells by the regulation of the AMPK/mTOR, p38/Erk MAPKs and Akt-independent signaling pathways, which may explain the therapeutic mechanisms of Rheum L. and rhein in treating CKD patients.
Advances in traditional herbal medicine suggest Chinese herbal remedies can be considered promising agents in the prevention and treatment of DKD.However, the clinical application of Chinese herbal medications is pretty limited, due to the following concerns and barriers (1) Lacking of high-quality animal studies,focusing on the role of herbal medicine on DKD.(2) The potential side effects associated with herbal medicine may be underrecognized and underreported.(3) The screened active ingredients may be inconsistent with the actual absorbed components in the blood of patients with DKD.(4) The interaction relationships between the nodes in the network construction methods are still unclear.Therefore, further experimental veri cation of the potential active ingredients is needed to verify this theoretical prediction.

Conclusion
This study used a scienti c approach to decipher the pharmacological mechanisms of Rheum L. in the treatment of DKD.We discovered that the effects may be associated with in ammatory response, apoptosis, brosis, and peripheral circulation.Among these crucial biological functions, twelve targets were identi ed as key active factors involved in the related pathways.This research suggests that Rheum L. can alleviate DKD via the molecular mechanisms predicted by network pharmacology and that the network pharmacology approach can be an effective tool to reveal the mechanisms of TCM.However, to improve the reliability of the results, further experimental experiments are needed to validate these results.
Institution of Guang'anmen Hospital, China Academy of Chinese Medical Science, Beijing, 100000, China.

Figures
Page 20/    The C-T network that consists of 6 nodes and 271 targets.Yellow and blue nodes denote the compounds and targets, respectively.
The C-T network that consists of 6 nodes and 271 targets.Yellow and blue nodes denote the compounds and targets, respectively.
Page 23/30  The 23 matching targets of related targets in Rheum L. on DKD.
The 23 matching targets of the related targets in Rheum L. on DKD.The main 18 pathways enriched by major hubs from the DAVID database.
Page 29/30 The main 18 pathways enriched by major hubs from the DAVID database.

Figure 1 The
Figure 1

Figure 1 The
Figure 1

Figure 3 The
Figure 3

Table 1
The list of active ingredients of Rheum L.