In TCM theory, RS is supposed to be the most responsible herb for CDDP’s effects. According to our putative active compounds of CDDP for DR, RS has 48 among a total of 54 active compounds with 27 targets. 45 of 48 active compounds are specific in the herb, indicating that RS may play a vital role in the formula consistent with TCM theory. RS have shown some anti-diabetic properties by regulating multiple pathways and biological processes related to the development of diabetes.27 Thus anti-diabetic compounds are expected to be identified from it. In our study, Tanshinones were identified as the major effective compounds of RS demonstrating multiple bioactivities. It is suggested that tanshinones could be therapeutic agents for diabetes as insulin sensitizer and AMPK pathway activators.28–30 Early application of tanshinones may prevent the progress of DR. Besides, many tanshinones have shown anti-inflammatory effect,31 and total tanshinones exhibit a stronger effect than a single one.32 Since inflammation is supposed to play a vital role in DR, the application of tanshinones could also be beneficial for DR in this respect. Among the tanshinones, tanshinone IIA stood out with the highest node degree. It has been suggested exerting anti-angiogenesis, anti-oxidative stress, anti-inflammatory effects in the retina through suppressing VEGF, AGE, NF-κB pathway.33–35 There were still some non-specific compounds of CDDP at the top of the list (Table 1), which could not be ignored. Polyphenols like quercetin and luteolin have been suggested to be therapeutic for DR.36 Luteolin from RS is believed to have effects on activating anti-inflammatory, antioxidant responses, neuroprotective effects and regulating relaxation, lipid profile via acting on multiple signaling pathways.37–42 Taken together, tanshinones and luteolin may be responsible for the effectiveness of CDDP against DR.
For PN, although 5 compounds have 43 targets, most of them like quercetin could be found in plenty of Chinese herbs, and PN is less dosage in the formula, the compounds identified were not suggested as the representative compounds for CDDP. The situation of BS is almost the same as PN. But their compatibility with RS is necessary according to TCM theory. Pharmacokinetics showed that the bioavailability of PN extract significantly increased when co-administered with BS through oral administration in rats.43 It suggests that BS might work as a potential absorption enhancer, and an upper ushering to brain drug.44 Although there is no significant difference in pharmacokinetic parameters between single RS or PN extract and combination treatment through oral administration,43 the combination treatment enhanced RS compounds in the brain and cerebrospinal fluid while decreasing the distribution of PN compounds via inner ear administration in guinea pigs.45 These researches indicate that herb-herb interactions may improve pharmacokinetic behaviors of CDDP’s active compounds.
Our results indicated that the predominant targets of CDDP against DR were ESR1, AR, CASP3, IL6 and VEGFA. ESR and AR are steroid hormone receptor. ESR signaling has shown its importance in ischemia-induced angiogenesis46,47 and neural injury gradually48–50. But the effects of sex hormones in DR are not fully understood and quite controversial at present.51 Estrogen could be neuroprotective52,53 or useless54 or harmful55. Its neuroprotective effect may be related to anti-apoptosis and anti-autophagy effects, while androgen is more pro-apoptotic56. From our results, CDDP could have a potential influence on both ESR and AR signaling. Furthermore, CDDP showed a potential influence on CASP-3, a downstream executioner of apoptosis in DR,57–60 in our study and its inhibitory effect on CASP-3 expression has been reported.61 Combining with the GO enrichment analysis, CDDP was strongly indicated involved in the regulation of steroid hormone response and apoptosis in DR.
Chronic inflammation has been suggested by growing evidence playing a prominent role in the pathogenesis of DR.62–64 IL-6, one of the inflammatory mediators, is found elevated in diabetic retinopathy.65–67 IL-6 is a pleiotropic cytokine, and its effects could be quite different through activating different pathways. Binding to membrane-bound IL-6 receptor and gp130, IL-6 activates classical IL-6 signaling pathway, which is supposed to be regenerative and protective; while binding to the soluble IL-6 receptor, IL-6 activates IL-6 trans-signaling pathway which is chronic pro-inflammatory and resulting in endothelial cell dysfunction and vascular leakage.68–70 both pathways could be activated in the retina under high glucose.71,72 Meanwhile, IL-6 is highly correlated with the aqueous level of VEGF in DR.73 The correlation between VEGF and the pathogenesis of DR is uncontroversial, especially for proliferative DR.21,74,75 Downstream events of VEGF include survival, proliferation, and permeability.76 It is supposed to improve the survival of retinal cells in the first place. But over time, it disrupts the outer blood-retinal barriers (BRB) and acts as a pathologic angiogenic stimulus.77,78 These studies indicate the feasibility of CDDP against DR by targeting these predicted targets and their biological processes. Our results showed that CDDP might regulate multiple targets of DR, including inflammatory cytokines and growth factors et al.
At present, several biological processes and pathways have been proposed to participate in the development of DR including oxidative stress, AGE, protein kinase C (PKC) activation, inflammation, VEGF, sorbitol, erythropoietin, the renin-angiotensin system (RAS), peroxisome proliferator-activated receptor α (PPAR-α).79,80 These processes or pathways could be the potential therapeutic targets for DR. As our results indicated, the mechanisms underlying the effect of CDDP for DR might involve in multiple pathways. A total of 97 KEGG pathways were enriched out including the AGE-RAGE and VEGF pathway, fluid shear stress and atherosclerosis, apoptosis et al. AGE-RAGE and VEGF pathways play critical roles in DR and has been discussed a lot.19–21, 81-87 Except for the biochemical changes, physiological changes also play a role in the process of DR. Hemodynamic dysfunction has been found in DR and may precede clinical DR.88–92 Hemodynamic parameters, wall shear rate and wall shear stress are suggested reduced in the early stage of DR, as well as retinal blood velocity and flow, which could be markers of micro-vasculopathy.88,93,94 Atherosclerosis has also been considered associated with the severity of DR, manifesting increased macrovascular artery intima-media wall thickness and accompanied by wider retinal vessel diameter in DR.95–97 These studies emphasize the vital role of hemodynamic changes in the early stage of DR. CDDP in TCM theory is characterized by improving blood circulation (Pharmacopoeia of the People's Republic of China). The early application of CDDP in NPDR patients can significantly shorten the retinal circulation time and reduce capillary hemangioma, hard exudates, retinal hemorrhaging.8 And CDDP may also have an anti-atherosclerosis property.98 The potential regulation on hemodynamics through fluid shear stress and atherosclerosis pathway may make CDDP a suitable medicine for an early intervention of DR. Besides, fluid shear stress is related to the release of vasodilatory factors nitric oxide (NO).99–101 As we all know, blood pressure has been suggested as one of the risk factors of DR.102 The regulation of fluid shear stress and atherosclerosis pathway by CDDP may affect blood pressure control. And anti-hypertension of RS has been reported by some researches.103–105 In summary, CDDP may be beneficial in the early stage of DR by acting on AGE-RAGE, VEGF, fluid shear stress and atherosclerosis, and apoptosis pathway.
Except for vascular dysfunction, retinal neurodegeneration has been recognized in DR with growing evidence. New insights suggest that vascular dysfunction and neural degeneration may be parallel in this disease. Thus physiological changes of DR should be viewed as changes of retinal neurovascular unit.79,106 The unit includes the interaction of vascular cells, retinal glial cells and neural cells. Because of the interaction, elevated pro-inflammatory or pro-angiogenesis cytokines in DR may contribute to both the blood-retina barrier (BRB) disruption and synaptic or neuronal degeneration.79 Cholinergic signaling may be blunted in diabetic retina, which may associate with synaptic degeneration, resulting in endothelium-dependent vasodilatation dysfunction and ischemia.107,108 And CHRM has been suggested to play an important role in retinal neuron survival109,110 and insulin release stimulation.111–113 It is rational to hypothesize that active cholinergic signaling could be protective in DR. In the compound-target network of CDDP, it showed that CDDP might have a potential regulation on cholinergic signaling through CHRM1-5 and ACHE (Fig. 1). Although it is not clear what exact role of cholinergic signaling plays in DR, therapies targeting cholinergic signaling could be worth trying considering the neuroprotective effects of its agonists towards other neural degenerative diseases like Alzheimer’s and Parkinson’s diseases.114