Identify the Key Active Ingredients and Pharmacological Mechanisms of Compound XiongShao Capsule in Treating Diabetic Peripheral Neuropathy by Network Pharmacology Approach

Compound XiongShao Capsule (CXSC), a traditional herb mixture, has shown significant clinical efficacy against diabetic peripheral neuropathy (DPN). However, its multicomponent and multitarget features cause difficulty in deciphering its molecular mechanisms. Our study aimed to identify the key active ingredients and potential pharmacological mechanisms of CXSC in treating DPN by network pharmacology and provide scientific evidence of its clinical efficacy. CXSC active ingredients were identified from both the Traditional Chinese Medicine Systems Pharmacology database, with parameters of oral bioavailability ≥ 30% and drug-likeness ≥ 0.18, and the Herbal Ingredients' Targets (HIT) database. The targets of those active ingredients were identified using ChemMapper based on 3D-structure similarity and using HIT database. DPN-related genes were acquired from microarray dataset GSE95849 and five widely used databases (TTD, Drugbank, KEGG, DisGeNET, and OMIM). Next, we obtained candidate targets with therapeutic effects against DPN by mapping active ingredient targets and DPN-related genes and identifying the proteins interacting with those candidate targets using STITCH 5.0. We constructed an “active ingredients-candidate targets-proteins” network using Cytoscape 3.61 and identified key active ingredients and key targets in the network. We identified 172 active ingredients in CXSC, 898 targets of the active ingredients, 110 DPN-related genes, and 38 candidate targets with therapeutic effects against DPN. Three key active ingredients, namely, quercetin, kaempferol, and baicalein, and 25 key targets were identified. Next, we input all key targets into ClueGO plugin for KEGG enrichment and molecular function analyses. The AGE-RAGE signaling pathway in diabetic complications and MAP kinase activity were determined as the main KEGG pathway and molecular function involved, respectively. We determined quercetin, kaempferol, and baicalein as the key active ingredients of CXSC and the AGE-RAGE signaling pathway and MAP kinase activity as the main pharmacological mechanisms of CXSC against DPN, proving the clinical efficacy of CXSC against DPN.


Introduction
Diabetic peripheral neuropathy (DPN) is one of the most common complications of diabetes, affecting approximately 50% of people with diabetes [1][2][3]. Characterized by numbness, pain, paresthesia, and sensory loss, it becomes a major cause of disability and mortality [4][5][6]. Given the estimated global prevalence of diabetes of 693 million by 2045 [4], DPN is likely to affect as many as 346.5 million people worldwide, at a tremendous cost.
The pathogenesis of DPN is complex and multifactorial [7,8]. It is generally accepted that chronic hyperglycemia induces the generation of advanced glycation end products (AGEs) in peripheral nervous tissue, and AGEs frequently result in neurological dysfunction mainly by modifying nervous structural proteins or overexpressing their receptors (RAGE) [9][10][11]. In addition, high glucose level activates mitogen-activated protein kinase (MAPK) in sensory neurons [8]. Moreover, elevated p38 and ERK activities may  cause neuron apoptosis, affecting neuronal functions and accelerating the progress of DPN [8,12]. Despite advancements in the understanding of DPN pathogenesis, prevention and therapy of DPN mainly focus on glucose control and lifestyle modification [13]. There is still a lack of therapies against DPN pathogenesis other than aldose reductase inhibitors, i.e., epalrestat, and antioxidants, i.e., alpha-lipoic acid [14,15]. In addition, these therapies show modest efficacy in clinical practice and exhibit certain adverse reactions, such as nausea, vomiting, and dizziness [14,16]. Thus, safe and effective treatments of DPN that act on its mechanisms are urgently needed [13].
However, the active ingredients of CXSC and their potential pharmacological mechanisms have not been fully studied. In a previous study, network pharmacology was used to discover the active ingredients and elucidate the mechanisms of herbal formulae [28]. Thus, our study aimed to use the network pharmacology approach to identify the key active ingredients of CXSC and their pharmacological mechanisms in treating DPN. A flowchart of the network pharmacology approach is presented in Figure 1.
The TCMSP database is a unique TCM platform for identifying the relationships between drugs, targets, and diseases [29]. HIT is a database containing herbal ingredients and validated protein targets derived from more than 3250 literatures [31]. . . Targets of Active Ingredients. The validated targets of the active ingredients of CXSC were collected from the HIT database [32,33]. In addition, the predicted targets of these active ingredients were obtained by using ChemMapper (http://www.lilab-ecust.cn/chemmapper/index.html), a web server for predicting potential drug targets based on 3D structure similarity [34], with 3D structure similarity of above 1.0 and prediction score of above 0 [32,33]. Duplicates of the validated and predicted targets were eliminated, and the targets of the active ingredients of CXSC were screened.
. . Genes Related to DPN Obtained. Known genes of DPN were identified from five currently available databases using "diabetic peripheral neuropathy" as the keyword. In addition, the main differentially expressed genes (DEGs) between DPN patients and diabetic patients were extracted from microarray data GSE95849 [35] in the Gene Expression Omnibus (GEO; http://www.ncbi.nlm.nih.gov/geo/) database [36], with a cutoff value of P < 0.05 and fold change |FC| of ≥ 1.5 [37]. DPNrelated genes were identified after removal of duplicates.
The five databases were: the Therapeutic Target

. . Candidate Targets and Proteins Interacting with Candidate
Targets. Candidate targets with therapeutic effects against DPN were identified by mapping the targets of the active ingredients of CXSC and the genes related to DPN [33]. The proteins that interacted with these candidate targets were screened by using STITCH 5.0 (http://stitch.embl.de/) [43], a database of known and predicted interactions between chemicals and proteins, with the species limited to "Homo Sapiens" and a confidence score of > 0.9.

. . Network Construction and Enrichment Analysis.
A network of "active ingredients-candidate targets-proteins" was constructed using the Cytoscape 3.61 software [44]. The key active ingredients of CXSC and the key targets in this network were simultaneously identified by Network Analyzer plugin [45] using the following criterion: nodes with degree values exceeding twice the average value of all nodes in the network [46]. The degree value is the number of edges a node has in a network, which indicates how many active ingredients/targets/proteins one active ingredient/target/protein is related with. The larger the degree value, the more critical a role the node (active ingredient, target, or protein) is believed to play in the network [46]. A widely used visualization software, ClueGO [47], was used to explore the potential KEGG pathways and molecular functions of the key targets.

Results
. . Active Ingredients of CXSC. A total of 172 active ingredients (Supplementary Table 1) were identified in this study, of which 133 were collected from the TCMSP database and 39 were obtained from the HIT database. The numbers of active ingredients in RP, AgR, RCX, RS, CL, CT, PGS, SM, RM, RC, OF, and SP were 35,22,22,21,21,13,13,13,12,7,5,5, and 4, respectively. The active ingredients that overlapped between herbs are shown in Table 1; for example, beta-sitosterol was identified in seven herbs, sitosterol was identified in six herbs, and quercetin was identified in five herbs. Table 2) were identified in our study, including 229 validated targets and 669 predicted targets. Our analysis results showed that quercetin, baicalein, and kaempferol were the top three active ingredients targeting 308, 296, and 224 targets, respectively. Furthermore, there were 140 common targets of 12 herbs, 72 common targets of 11 herbs, 30 common targets of 10 herbs, 26 common targets of 9 herbs, 33 common targets . . Genes Related to DPN. A total of 51 DEGs were extracted from microarray data GSE95849, including 23 upregulated genes and 28 downregulated genes, as shown in Figure 3. After combination with the 59 known targets from the other five databases, a total of 110 DPN-related genes were identified (Supplementary Table 3).  Figure 4: "Active ingredients-candidate targets-proteins" network. The triangle nodes represent active ingredients, the rectangle nodes represent validated targets, the diamond nodes represent predicted targets, and the circular nodes represent interacting proteins. The color of the nodes is shown in a gradient from purple to transparent according to descending order of the degree value. The three key active ingredients and 25 key targets are listed in the center circle.

. . Key Active Ingredients and Key Targets in the Network.
An "active ingredients-candidate targets-proteins" network of 38 candidate targets and their interacting proteins was constructed using the Cytoscape 3.61 software (Figure 4). There were 328 nodes and 1396 edges in the network, with an average degree value of 8.767, as calculated by using the NetworkAnalyzer plugin. There were 28 nodes with degree values exceeding 18 (twice the average value of the degree), comprising of three active ingredients (quercetin, kaempferol, and baicalein) representing the key active ingredients ( Figure 5) and 25 targets representing the key targets ( Table 2).

. . KEGG Pathways and Molecular Functions Enriched.
The enrichment analysis results showed enrichment in the KEGG signaling pathways and molecular functions. As shown in Figure 6, the following three KEGG pathways were enriched: the AGE-RAGE signaling pathway in diabetic complications, B cell receptor signaling pathway, and cocaine addiction pathway, with the AGE-RAGE signaling pathway accounting for 88% of all the pathways enriched. The enriched molecular functions were divided into three groups: MAP kinase activity, phosphatase binding, and neurotransmitter: sodium symporter activity (Figure 7).

Discussion
TCM pharmacological mechanisms have always been associated with multiple components and multiple targets that are difficult to explain. However, the holistic ideas of TCM have been revealed by emerging network pharmacology approaches studying the relationships between drugs, targets, and diseases [28]. In our study, the active ingredients of CXSC, targets of these active ingredients, and genes related to DPN were identified comprehensively from the databases. Next, an "active ingredient-target-protein" network with 328 nodes and 1396 edges was constructed by the Cytoscape software. After analyzing the network, three key active ingredients and 25 key targets of CXSC were determined, and the pharmacological mechanisms of CXSC in treating DPN were elucidated by enrichment analysis of the 25 key targets.
. . Quercetin, Kaempferol, and Baicalein as the Key Active Ingredients of CXSC. The "active ingredients-targets-proteins" network showed quercetin, kaempferol, and baicalein as the key active ingredients of CXSC that exhibit therapeutic effects against DPN. Moreover, these key ingredients targeted 308 (34.3%), 296 (33.0%), and 244 (27.2%) targets, respectively. Furthermore, these three key ingredients targeted 22 of the 38 candidate targets with therapeutic effects against  Ad Ad A A Ad Ad dip dip Ad A Ad Ad Ad Ad ocyt ocytok ok ki ki ki kin in in ki ki k ki ki ki ok e si e si e signal gnal gnaling ing ing pat path pat path path path path ath path path pa path path at a athway way way way way ay ay ay ay y ay y ay y mia mia mia a a a i i i RIG RIG RIG RIG G G IG G-G-G-IG G G-G I-li I-li I-like r ke r ke recep ecep eceptor tor tor sign sign ign g g g g g g g g g alin alin aling g g pa pa pa g g g g g g g g thwa thwa thway y y g g g g g g g g g g rog rogest est est ) ) ) ) ) ) ) ) ) ) ) ) ) T T T ll ll ll t t t i i i li li li g pa g pa g p g pa p thw thw thw thwa thwa hwa thw thw thw hw thw thw y y y y y y y y y y y y y y y y Epit Epit Epithe he e eli eli e e he e e eli e e e al c al c al cell ell l l l l l l l l ell l l sign sign sign sig si si si si s s si s si s alin alin l n n n n n n n n ng g in g in g in g g g g g Hel Hel Helicob icob icobacte acte acter py r py r pylori lori lori inf inf infecti ecti ection on on Salm Salm Salmon on on on on onel onel onel n on on n on n la i alin n n n n n n n n ng g g g g g g g g g g g g p p eceptor tor tor or sig sign signalin alin n n n n n n n n n ng g g ing ing g pat pat pathway hway hway ing ng ng ng pat pat pat p hway hway hway hway ing ing ing ng pat pat p p hway hway hway hway ng n ng g ng pat pat pathway hway way wa    DPN, as well as 21 of the 25 key targets of CXSC, as shown in Figure 8. Interestingly, several studies have previously shown that quercetin may be helpful against diabetic neuropathy by attenuating cold allodynia and hyperalgesia [48,49], maintaining the density of the general neuronal population [50], and restoring sciatic nerves injuries [51] in streptozotocin-(STZ-) induced diabetic rats. Kaempferol [52] and baicalein [53] also showed neuroprotective effect against STZ-induced diabetic neuropathy. Thus, we speculated that CXSC exhibited significant therapeutic effect against DPN mainly through the synergistic actions of quercetin, kaempferol, and baicalein.
. . Regulations of the AGE-RAGE Pathway and MAPK Activity as the Pharmacological Mechanisms of CXSC erapeutic Effect against DPN. Kaempferol and baicalein, the key active ingredients of CXSC, were previously confirmed to reduce the formation of AGEs, thereby reducing inflammatory responses in diabetic rat nerves [52,54]; thus, these two compounds may exert therapeutic effect against DPN. Quercetin, another key active ingredient of CXSC, protected rat dorsal root ganglion neurons against high glucoseinduced injury in vitro through dose-dependent inhibition of the NF-B signaling pathway; thus, this compound may be beneficial as a treatment of diabetic neuropathy [55]. Furthermore, activation and perpetuation of the AGE-RAGE signaling pathway have been reported in diabetic neuropathy [9]. AGEs act on RAGEs to allow sustained activation of NF-B, which aggravates inflammatory reactions and leads to neuronal dysfunction [9,11]. Lukic I.K [10] identified the AGE-RAGE signaling pathway as a new therapeutic target of neuronal dysfunction. In our study, the AGE-RAGE signaling pathway of diabetic complications accounted for 88% of all the enriched pathways, suggesting that the therapeutic effect of CXSC against DPN was mainly related to reduction in AGE formation and inhibition of NF-B.
In addition, AGEs can activate p38 MAPK, leading to neuron apoptosis [56]. MAPK may play important roles in neuronal cell death or regeneration [57,58]. For example, p38 MAPK was reported to aggravate oxidative stress and inflammation through induction of nitric oxide synthase, thereby resulting in NO production, as well as through regulation of the production of cytokines, such as TNF and IL-10, leading to neuronal cell death [59,60]. Notably, increased phosphorylation of MAPK has been detected in diabetic animal models [61][62][63] and in the peripheral nerves of patients with DPN [12]. p38 MAPK, especially has been proved to be associated with DPN pathogenesis, such as increased mechanical hyperalgesia [64][65][66] and reduced nerve conduction velocity [67]. Thus, adjustment in the activity of MAPK may be the basis of preventive treatments of diabetic neuropathy [9]. Fortunately, tetramethylpyrazine, a main ingredient of RCX in CXSC, was found to block MAPK and suppress reactive oxygen species in N9 microglial cells, which may shed light on future treatments of neurodegenerative diseases [68].
Baicalein, one of the three key active ingredients identified in the current study, counteracts diabetes-associated p38 MAPK phosphorylation and oxidative-nitrosative stress, targeting several mechanisms implicated in DPN [53]. Therefore, the effects of CXSC on MAPK regulation against DPN may be attributed to tetramethylpyrazine and baicalein. Our molecular function analysis also indicated that the therapeutic effect of CXSC against DPN was related to MAPK activity. Our results were consistent with those of previous studies, which indicated that p38 MAPK activation mediates RAGEinduced, NF-B-dependent secretion of proinflammatory cytokines, leading to accelerated inflammation. Activation of the AGE-RAGE pathway and MAPK appeared to be present not only in DPN but also in diabetic nephropathy [69] and diabetic keratopathy [56]. Thus, further studies are needed to investigate whether CXSC is effective against other diabetic complications.

Conclusions
In conclusion, the multicomponent and multitarget features of the therapeutic effects of CXSC against DPN were effectively elucidated through network pharmacology approach. Quercetin, kaempferol, and baicalein were determined as the key active ingredients of CXSC. In addition, the AGE-RAGE signaling pathway and regulation of MAPK activity were shown as the main pharmacological mechanisms of the therapeutic effects of CXSC against DPN, thereby providing scientific evidence of the clinical efficacy of CXSC against DPN.

Data Availability
The data of our research can be acquired from the Supplementary Materials uploaded with this article.
Evidence-Based Complementary and Alternative Medicine 9

Conflicts of Interest
The authors declare no conflicts of interest.