The GO terms enriched with CXC and HXMMT targets were similar and focused mainly on the response to reactive oxygen species and oxidative stress, regulation of blood vessels and blood coagulation.
Radix salviae is an important component that is closely related to the response to reactive oxygen species and oxidative stress. In an in vitro model of hypoxia and reoxygenation, Hu’s [10] study showed that Radix salviae obviously alleviated cardiomyocyte apoptosis and protected mitochondrial function and cell membrane skeleton integrity in H9c2 cells. In addition, Zhang’s experiment in rodents [11] revealed that Danshen (Radix salviae) dripping pill inhibited apoptosis and exerted neuroprotective effects in the retinas of diabetic rats by increasing the expression of Bcl-2, Bcl-2 associated X and caspase-3 in diabetic rats. Moreover, according to the I-T networks of CXC and HXMMT, Radix salviae contained a greater number of active ingredients related to potential therapeutic targets in PDR than did the other herbal components, suggesting that Radix salviae may play a crucial role in PDR treatment. A randomized controlled trial (RCT) performed by Lian and colleagues [5] showed that a Radix salviae-containing Chinese herbal product was effective in treating DR and in delaying the progression from non-PDR to PDR by reducing the area of capillary nonperfusion and degree of vascular leakage. Our study identified tanshinone as one of the most important active ingredients of Radix salviae. According to previous studies, tanshinone exerts protective effects on retinal pigment epithelium and retinal endothelial cells [12–14].
Regarding circulation-related effects, some studies [7, 15] have shown that CXC contributes to the attenuation of streptozotocin (STZ)-induced retinal lesions, including ameliorating increases in erythrocyte aggregation, plasma viscosity, and acellular vessel and pericyte loss, by reversing the hyperexpression of vascular endothelial growth factor (VEGF) and intercellular adhesion molecule-1 and endothelin-1 and the hypoexpression of pigment epithelium-derived factor and occludin in the retinas of STZ-induced rats. In addition, Liu’s study [16] showed that different core bioactive ingredients in CXC had novel therapeutic uses in managing blood circulation. Panaxytriol and ginsenoside Rb1 were related to red blood cell aggregation, while angoroside C was involved in platelet aggregation. Protocatechualdehyde was related to intrinsic clotting activity, while calycosin-7-O-beta-D-glucoside was related to extrinsic clotting activity. However, the effect of HXMMT has seldom been evaluated in DR; indeed, the only study is one by Long [6] conducted in rat models of branch retinal vein occlusion (BRVO), which indicated that HXMMT may alleviate retinal edema by regulating the expression of VEGF-α and improving microcirculation. Further studies should be performed to clarify the mechanism of HXMMT.
Utilizing a network pharmacology approach, Piao [17] found that MMP9 and IGF-1 (an IGF family member contained in Radix salviae) may be key therapeutic targets in DR. Consistent with Piao’s result, we found that MMP9 was included in both the CXC and HXMMT I-T networks. Hyperglycemia may increase the activity of MMP9 and therefore provides growth space and nutrients for neovascularization by degrading the basement membrane and relaxing the cell structure [18]. In addition, IGF-2, another IGF family member, was included in our networks but is not targeted by Radix salviae. IGF is expressed in many tissues, including the retina, where it is found in cells such as retinal endothelial cells and retinal pigment epithelial cells. IGF is a crucial regulator of cell differentiation and is closely related to blood-retinal barrier breakdown and retinal neovascularization [19, 20]. However, IGF-2 was related to 2 herbal components of CXC (Huangqi and Sanqi) and 9 herbal components of HXMMT (Cheqianzi, Chishao, Huangqin, Mohanlian, Mudanpi, Muzei, Nvzhenzi, Puhuang and Xiakucao), implying that different active ingredients may share common therapeutic targets. Combined and stronger therapeutic effects may be exerted on a therapeutic target shared by a greater number of active ingredients. HXMMT contains more components than CXC; therefore, HXMMT may have more therapeutic targets. In addition, many of the genes targeted only by HXMMT but not by CXC were related to circulation and blood coagulation. For instance, CYCS was shown to be involved in blood platelet formation and regulatory processes [21, 22]. APOD, a crucial component of lipoproteins that transports lipids and stabilizes the structure of lipoproteins, was found to also be closely related to angiogenesis, a critical pathophysiological process in PDR [23]. PECAM1 was suggested to play an important role in the maintenance of human vascular endothelial barrier integrity and function [24]. Similarly, our topological analysis showed that some genes targeted only by HXMMT had many other functions. For example, YWHAB may perform specific functions in rod photoreceptors [25]. RACK1 may promote the expression of VEGF in endothelial cells and subsequently facilitate angiogenesis [26]. PARP1, activated by reactive oxygen species, was proven to be involved in inflammation, cell death, and retinal disease progression [27, 28]. In summary, its stronger effects at a given dose and more numerous gene targets may be two major reasons that HXMMT is more strongly recommended than CXC by TCM doctors for treating fresh VH secondary to PDR.
These two CPMs shared many pathways in our study, and the AGE-RAGE signaling pathway in diabetic complications was the most significantly enriched pathway. RAGE is expressed in almost all retinal cells. Retinal Müller cells, the major glial cells in the retina, play a critical role in maintaining the structure and normal functions of the retina, and these cells express high levels of RAGE [29]. In addition, Zong’s study [30] demonstrated that RAGE plays an essential role in retinal neurodegeneration induced by diabetes and that early induction of RAGE expression by hyperglycemia in retinal Müller cells contributes to the increased levels of proinflammatory cytokines, including VEGF (a crucial downstream growth factor in angiogenesis) and monocyte chemoattractant protein-1 (MCP-1), in vivo and in vitro. Moreover, Hirata [31] found that increased production of VEGF secondary to retinal Müller cell activation may account for neovascularization in PDR. Therefore, the AGE-RAGE signaling pathway may not only provide neuroprotection in DR but also participate in crosstalk between neuroprotection and vascular protection. The difference in the enriched genes between CXC and HXMMT was that one additional gene (COL1A2) was included among the HXMMT targets. COL1A2 has seldom been studied in DR; Zou’s research [32] is the only DR study involving COL1A2 to date. Zou revealed that silencing of circular RNA COL1A2 (circCOL1A2) suppresses angiogenesis during PDR progression by regulating the miR-29b/VEGF axis, suggesting that circCOL1A2 and its related genes may be therapeutic targets in DR.
According to the pathway map, the AGE-RAGE signaling pathway in diabetic complications pathway is also closely connected with the PI3K-Akt signaling pathway and VEGF. The PI3K-Akt signaling pathway is one of the most frequently studied pathways in DR [33–35]. The proliferation, migration and invasion of retinal vascular endothelial cells, retinal pericytes, retinal pigment epithelial cells and microglial cells can be regulated through this pathway [36–39]. A series of pathophysiological processes, including oxidative stress regulation, inflammatory response regulation, angiogenesis and neuroprotective regulation, are also involved.