The Lingqihuangban Granule (LQHBG) is a classic prescription for treating diabetic retinopathy disease with blood stasis syndrome, consisting of 9 herbs of Cistanche, Lucid ganoderma, Lycium barbarum, Angelica sinensis, Semen Cuscutae, Rhizoma atractylodis, Ligusticum wallichii, Salvia miltiorrhiza and Codonopsis pilosula, altogether playing roles in treating DR. To determine the relationship between the bioactive ingredients of LQHBG and DR and the potential mechanism, this study constructed a biological network of interactions between bioactive components and relative targets. In this study, 357 bioactive components in the Lingqihuangban Granule were screened, acting on a total of 86 DR targets and involving 851 interaction relationships. As seen from the active components–targets network diagram, for single herbs, active ingredients such as sitosterol, quercetin, and fucosterol of Lycium barbarum can both regulate CYP2C19 and PPARA targets, while ALB, TNF, and MAPK3 can be influenced by more than one active ingredient, showing that the multiple targets can be mediated by multiple bioactive ingredients. Among the 9 diverse herbs, sitosterol is the common bioactive ingredient of Lycium barbarum, Lucid ganoderma, Angelica sinensis and Ligusticum wallichii with the same targets, and quercetin is the common bioactive ingredient of Lycium barbarum, Lucid ganoderma, Cistanche and Semen Cuscutae, indicating that there are synergistic effects between drugs. All of the above findings reflected the mechanisms of coordination and joint regulation of LQHBG in the treatment of DR and show that there were a variety of interactions among targets, which together constituted a complex biological network system.
In the PPI network diagram, the higher the degree of connectivity was, the greater the possibility of LQHBG treating DR through these targets was. The results showed that INS, ALB, GAPDH, TNF, IL6 and MAPK3 were the six core targets selected according to the degree ranking. Some of the effective genes have been identified by animal experiments and clinical tests. For example, INS is a gene that encodes insulin and plays a vital role in the regulation of carbohydrate and lipid metabolism and stimulates glucose uptake with the binding of the insulin receptor (INSR). Previous studies illustrated that INSC94Y transgenic pigs developed a persistent diabetic phenotype, such as abnormalities of intraretinal microvasculature similar to human beings, confirmed by regulating the functional proteins for Müller glial cell metabolism (e.g., GS) and the vital retinal water channel protein AQP4 . Moore et al. reported that retinal microvascular endothelial cells (RMECs) treated with glycated albumin (AGE-Alb) gave rise to increases in the regulation of the NF-κB signaling pathway and leukocyte adhesion, accompanied by a dysfunctional blood–retinal barrier, while Treins et al. stated that AGE-Alb stimulated VEGF expression through an ERK-dependent pathway, playing an important role in the pathogenesis of proliferative diabetic retinopathy [39, 40]. Literature studies and network pharmacology results suggested that INS and ALB may be the key targets for the LQHBG treatment of DR, directly indicating the accuracy of network pharmacology in target prediction.
The results of DAVID (Version 6.8) Pathway and Reactome Pathway annotation analyses showed that the potential mechanisms of the LQHBG in the treatment of DR was mainly focused on the signaling pathways of biological metabolism, circulatory transport and anti-inflammatory and antioxidant functions. The diagram of the main effective pathways revealing the potential mechanisms of LQHBG for the treatment of DR is shown in Fig. 6. Based on the integration of pathway analysis and the theory of compatibility in traditional Chinese medicine, the Chinese herbal compound LQHBG played a hypoglycemic role of improving insulin resistance, reducing fat load and promoting the synthesis of liver glycogen. In addition, the components possess anti-inflammatory and antioxidant properties that regulate the synthesis of inflammatory factors and further prevent cell apoptosis and functional damage. At the same time, the herbs can effectively regulate blood circulation through reducing the oxidative stress response and preventing the proliferation of vascular endothelial cells, strengthening the adhesion of cells and the blood–retinal barrier (BRB). The above corresponded respectively with the theories of traditional Chinese medicine of invigorating spleen and benefiting digestion, tonifying kidney and warming Yang, nourishing blood and generating essence, with the result of clearing collaterals and clarifying eyes. To be specific, the pathways related to microvascular circulation include the AGE-RAGE pathway, the HIF-1α pathway and the VEGF pathway, which correspond to herbs containing the functions of strengthening Qi and blood and regulating the balance of Yin and Yang in the kidney, such as Lucid ganoderma, Salvia miltiorrhiza, Semen Cuscutae and Rhizoma atractylodis. All of the herbs acted on the AGE-RAGE signaling pathway, especially Lucid ganoderma and Salvia miltiorrhiza, reflecting the particularity and commonness of drug actions. The accumulation of AGEs plays a significant role in the progression of DR through exerting an inflammatory reaction and oxidative stress and increasing the expression of pro-apoptotic cytokines, which induce neurodegeneration, apoptosis of retinal pericytes and retinal microvascular dysfunction . Brunelle et al. illustrated that AGEs activated the mitochondria-dependent apoptosis pathway by upregulating Bax expression and inhibiting Bcl-2 expression, participating in the release of pro-apoptotic cytochrome c from the mitochondria through the pores in the outer mitochondrial membrane, thus leading to the activation of caspase 9 and caspase 3 . Moreover, the AGE-RAGE axis was linked to the blood-retinal barrier (BRB) breakdown through the mediation of leukocyte adherence barrier dysfunction, leading to the production of VEGF and angiogenic/vasopermeability growth factor in the retinal Müller glia  and to the activation of transcriptional factor nuclear factor-κB (NF-κB) and p38 MAPK expression, regulating pro-inflammatory cytokine release [41, 44]. The quercetin of Lucid ganoderma, Lycium barbarum, and Semen Cuscutae and the luteolin of Salvia miltiorrhiza and Codonopsis pilosula mainly enriched the AGE-RAGE pathway and induced the apoptosis of retinal pericytes and neurons and mediated microvasculature dysfunction. Under hypoxia conditions, the production of hypoxia-inducible factor (HIF)-1α induces body glycolysis and erythrocytosis, giving rise to blood thickening and mitochondrial metabolism dysfunction . Various clinical studies have confirmed that HIF-1α upregulates the transcription of the VEGF gene, stimulating angiogenesis and thus facilitating disease progression [46, 47]. The Ergosta-7,22-dien-3beta-yl palmitate in Lucid ganoderma, the sesamin in Semen Cuscutae and the epidanshenspiroketallactone in Salvia miltiorrhiza are mainly enriched in the HIF-1α pathway and prevent the occurrence of proliferative diabetic retinopathy (PDR) by reducing VEGF expression and suppressing angiogenesis.
The pathways related to biological metabolism include the sphingolipid signaling pathway and the PI3K-Akt signaling pathway, which corresponded to herbs containing the function of spleen’s hypoglycemic effect through improving insulin resistance, reducing fat load and promoting the synthesis of liver glycogen, such as Codonopsis pilosula, Rhizoma atractylodis, Ligusticum wallichii and Lucid ganoderma. Evidence exists that the sphingolipid pathway is associated with inflammation and apoptosis through metabolic regulation . A variety of sphingolipid metabolites, such as ceramide (Cer), ceramide-1-phosphate (C1P), sphingosine-1-phosphate (S1P), and lactosylceramide (LacCer), serve as the biologically active lipids, mediating important functions in biological metabolism and signal transduction . Yaribeygi et al. illustrated that the upregulation of ceramides increases DR risk through inducing β-cell apoptosis and pancreatic inflammation , while Arai et al. reported that the accumulation of LacCer and S1P accelerate the proliferation of retinal endothelial cells and the eventual production of new blood vessels [51, 52]. The stigmasterone of Codonopsis pilosula, the NSC63551 of Rhizoma atractylodis, and the myricanone of Ligusticum wallichii mainly enrich the sphingolipid pathway and inhibit the synthesis and secretion of adipokines and proinflammatory cytokines. Recent studies have shown that the PI3K/Akt/VEGF signaling pathway gives rise to enhanced viability of retinal vascular endothelial cells (RVECs) and inhibition of angiogenesis and apoptosis, stimulated by miR-21 overexpression and phosphatase and tensin homolog (PTEN) suppression . In addition, glycogen synthase kinase-3b (GSK3b) is a critical enzyme that reduces the synthesis of liver glycogen and increases the blood sugar concentration. Evidence has shown that the PI3K/Akt/GSK3 signaling pathway is involved in the metabolism of glycogen, playing a crucial role in the glucose output responsible for insulin resistance . The stimulation of PI3K is essential for the insulin-stimulated glucose uptake, while phosphorylated Akt induces the prevention of GSK3 expression, which promotes glucose intake and inhibits the inflammatory response [55, 56]. The myricanone of Ligusticum wallichii and the lauric acid of Codonopsis pilosula mainly act on the PI3K/Akt pathway and regulate glucose metabolism and fat metabolism to resist the progression of insulin resistance. The pathways related to anti-inflammatory and antioxidant functions include the MAPK signaling pathway, the NF-kappa B signaling pathway and the NOD-like receptor signaling pathway, which correspond to herbs containing the functions of strengthening Qi and blood and regulating the balance of Yin and Yang in the kidney, such as Lycium barbarum, Angelica sinensis and Cistanche. The mitogen-activated protein kinase (MAPK) signaling pathway is an important signal transduction pathway, consisting of p38 MAPK, JNK, and ERK and correlating to certain pathological processes, such as cell proliferation, apoptosis, inflammation, and necrosis . Under the condition of injury, MAPK aggravates the inflammation in a vicious circle, with the downstream P-p38 MAPK entering the nucleus to release inflammatory factors, such as IL-1 and IL-18, and regulating the expression levels of Bax and Bcl-2 and the apoptosis effector protein Caspase-3, which is closely associated with apoptosis [58, 59]. The mandenol of Lycium barbarum and the quercetin of Cistanche may inhibit the MAPK signaling pathway and alleviate the inflammatory reaction and the progression of apoptosis. Nod-like receptor family pyrin domain-containing protein (NLRP) is closely related to the chronic inflammatory reaction and innate immune response, accompanied by high expression levels of proinflammatory molecules such as IL-1β and TNF-α in DR . It has been reported that increased levels of NLRP3 inflammasomes exert downstream inflammatory cytokines and signal transduction of the NF-κB pathway, which induce a cascade of inflammatory response in the aggravation of DR . The inosine of Lycium barbarum and the suchilactone of Roucourong mainly act on the NF-kappa B signaling pathway and the NOD-like receptor signaling pathway, which block the activation of the inflammatory response and the oxidation reaction in preventing abnormal biological processes.
In short, DR is characterized by structural and functional changes that are affected by multiple factors. The current clinical studies identified new targets for the treatment of DR and confirmed that the traditional Chinese medicine components acting on these potential targets play important roles in the prevention and treatment of DR. In this study, the bioactive components and targets of the LQHBG in treating DR were analyzed and predicted through a network pharmacology method, while the mechanisms of action of key genes and pathways were analyzed through bioinformatics methods. It can be seen from the network diagram of bioactive components–targets and pathway analysis that quercetin is most the common component of the four single drugs in the LQHBG, which act together on the core gene of ALB, reflecting the synergistic effects of compound Chinese medicine components. Each herb consists of different bioactive ingredients, acting on diverse drug targets, referred to as the AGE-RAGE, HIF-1a, PI3K Akt, MAPK, and NF-kappa B signaling pathways, treating DR from different therapeutic aspects of the circulation, metabolism, inflammation, and aging, embodying the complementary effects of compound Chinese medicine in the treatment of DR with multiple components, multiple targets and multiple approaches, providing a new idea for the treatment of DR with complex pathogenesis. Because the results of this study were only obtained from available databases and the obtained pathways were relatively wide, failing to fully elucidate the molecular regulation and interaction relationships, the next step will be undertaking in vitro experiments and performing analysis of the biological technology, further verifying the accuracy of the targets of the Chinese herbal compound LQHBG and will be focusing on each single pathway to study the molecular regulation and interaction relationships, further exploring the specific mechanisms of action of this Chinese herbal compound in treating DR.