Identification and Validation of Molecular Mechanisms of Salvia Miltiorrhiza in Treating Diabetic Retinopathy: A Network Pharmacology and Molecular Docking Study

    

    

    Figure 8: Binding affinities (kcal/mol) of key targets and their targeted active constituents of salvia miltiorrhiza. The constituents and target proteins(X-axis) plotted against the binding affinities (Y-axis).
    

Discussion

Diabetic retinopathy is an eye condition caused by damage to the blood vessels of the light-sensitive tissue at the back of the eye (retina) [27]. DR is clinically characterized by retinal microvascular pathologic changes, such as microaneurysms, hemorrhages, capillary occlusion, and neovascularization, finally leading to severe vision loss and irreversible blindness [28]. Salvia Miltiorrhiza constituents such as luteolin and tanshinone IIA, a Chinese Traditional Medicine (TCM) extracts, have been demonstrated to have in vivo and in vitro medicinal benefits on DR. Still, their mechanism is yet to be well defined. The mechanisms of action of TCM treatments are intricate, with numerous constituents and targets. When the pathophysiology of pathogens is yet to be explicated, it becomes more problematic to scrutinize the mechanical action of a TCMs treatment effects [22]. Network pharmacology and Molecular docking analysis enable us to organize the investigation of the targets, active constituents, and pathways of medications at the molecular level, thereby enhancing our understanding of the links between constituents-targets and pathways in treating disease.

Employing network pharmacology and molecular docking techniques, we discovered 59 active constituents and 123 targets of SM, and 1271 targets of DR; 40 targets overlapped with the therapeutic targets of DR. An investigation of the SM active constituent-target network revealed that luteolin and tanshinone IIA two of the 49 corresponding SM active components, may act strongly on many network targets. This finding suggests that these two constituents may be essential for the efficiency of the therapy of SM in DR. Luteolin prevails as the greatest potential target, accompanied by tanshinone IIA in the 49-constituents-40 shared targets network.

One of the key elements in the pathophysiology of metabolic syndrome and diabetes is inflammation. The primary cause of type 1 diabetes and a common sign of type 2 diabetes is chronic inflammation. TCM extracts have been reported to have pharmacological effects in disease treatment by mitigating factors or biomarkers associated with diseases. Reports have shown the antioxidant ability of luteolin. A study on active components SM, mainly Asiatic acid and Salvianolic acid j, reported that the SM components (Asiatic acid and Salvianolic acid j) might regulate cell proliferation and response to inflammation and hypoxia [29]. Previous research shows that Luteolin augments insulin sensitivity and raises insulin-mediated glucose uptake [30]. It has also been demonstrated to inhibit increased glucose-induced VEGF synthesis, lowering ROS manufacture and fat buildup [31,32]. In an animal model of diabetes-induced retinal neurodegeneration, Luteolin reduced inflammatory markers, including oxidative stress and cytokines levels [31,33,34]. Heme-Oxygenase-1 (HO-1) is an antioxidant and a cytoprotectant. Luteolin increases HO-1 expression and phosphorylation of AKT and prevents morphological destruction in diabetic-associated complication tissues.

Tanshinone IIA has also been reported to enhance the morphological functioning of the retina in diabetic mice and inhibit diabetic retinopathy through its anti-inflammatory, anti-angiogenic, and antioxidant properties [35-37]. Excessive production of VEGF can cause retinal neovascularization. Findings have shown that Tanshinone IIA significantly reduced VEGF expression in both in vivo and in vitro studies [35,38]. Tanshinone IIA has shown its anti-inflammatory ability in TNF (a pro-inflammatory cytokine that leads to increased vascular permeability, breakdown of the Blood-Retinal Barrier (BRB), and loss of capillary pericytes) by reducing the contents of TNF in the retina of diabetic mouse [35]. The inflammatory cytokines reduce beta cell function and induce insulin resistance. Luteolin and Tanshinone IIA attenuate inflammation-induced diabetes and diabetes-induced inflammation. The TCM extracts might exert their protective effects by ameliorating the hyperglycemia-induced increase in inflammatory markers. In conformity with the above reports, these constituents may serve as therapeutic effects on DR.

AKT1, VEGFA, TP53, TNF, EGFR, SRC, ESR1, JUN, HRAS, MMP9, CXCL8, and FOS were the top 12 targets acting on DR in the PPI network among the 40 shared targets in the SM-DR network, according to the degree of the node. The study selected the first four genes (AKT1, VEGFA, TP53, and TNF mainly connected to the core components of SM Luteolin and Tanshinone IIA base on the order of high-degree nodes for docking analysis. These genes are regarded as hub genes in the study's network and may play essential roles in the therapeutic efficacy of SM in DR. These target genes are implicated in oxidative stress, inflammation, vascular permeability, and immune system modulation [22,29], which are hallmark for DR progression.

For instance, the AKT1 protein targeted by this study as a hub gene is abundantly present in insulin-responsive tissues. It initiates a mechanism that increases glucose absorption by cells by activating glucose transport proteins, thereby improving glucose metabolism [39,40]. The PI3K/AKT pathway, which controls cell proliferation and survival, is among the major mechanisms involving AKT1 in DR. This system can be dysregulated in diabetes, resulting in increased inflammation, oxidative stress, and aberrant blood vessel formation in the eye, which are defining characteristics of DR. AKT1 controls these activities and targeting AKT1 or the PI3K/AKT pathway has been suggested as a promising diabetic retinopathy treatment [41]. Preclinical studies using animal models indicate that modulating AKT1 and VEGFA activities through various means, such as pharmacological inhibitors or gene silencing techniques, can reduce retinal inflammation, abnormal blood vessel growth, and oxidative stress leading to improved outcomes in diabetic retinopathy [42].

The hub target, VEGFA, is present in microvessels of arteries, veins, and lymphatic vessels and in the endothelial cells of larger vessels [43]. The VEGFA gene is also implicated in the aberrant development of retinal blood vessels. Hyperglycemia, as observed in diabetes, can destroy retinal blood vessels [44] , triggering the release of VEGF-A, a gene-encoded protein. VEGF-A stimulates neovascularization in the retina, which can disturb normal retinal function and potentially lead to vision loss. In DR, the overexpression of VEGF-A due to an increase in VEGFA gene activity contributes to the progress of aberrant blood vessels in the retina, which can leak fluid and blood, resulting in retinal edema, hemorrhages, and other severe complications. Therefore, VEGFA and its protein product VEGF-A are targeted in treating DR to inhibit abnormal blood vessel growth and reduce vision-threatening complications.

According to research, TP53, one of our 12 hub genes, is connected with cancer and may also play a role in the growth of DR by inducing inflammation, oxidative stress, retinal neovascularization, and vascular endothelial dysfunction. In a study conducted by Zafer et al., it was observed that there is a notable augmentation in the expression levels of the p53 protein in retinal pericytes subjected to elevated glucose concentrations. The investigation revealed a concomitant escalation in the O-linked Β-N-acetylglucosamine (O-GlcNAc) modification levels of p53 within retinal pericytes exposed to high glucose conditions. The outcomes of the study strongly propose that the post-translational O-GlcNAc modification of p53, coupled with its heightened expression, potentially plays a contributory role in the selective early demise of pericytes during diabetic conditions [45].

TNF is a pro-inflammatory cytokine that is vital to the pathophysiology of DR. Chronic hyperglycemia in diabetes releases TNF and other pro-inflammatory cytokines, which cause retinal blood vessel inflammation and damage. This injury causes the flow of fluid and blood into the retina and the formation of aberrant blood vessels, which can result in vision loss and blindness. In addition, TNF promotes blood-retinal barrier degradation, which is essential for maintaining the integrity of the retina. It also stimulates the creation of other inflammatory molecules and apoptosis in retinal cells. Our investigation revealed that the Fluid shear stress and atherosclerosis pathway contains a notable concentration of the four major targets AKT1, VEGFA, TP53, and TNF. Additionally, AKT1, TP53, and VEGFA were revealed to be considerably enriched in diabetes complication-associated PI3K-Akt signaling and Lipid and atherosclerosis pathways. The antioxidant, anti-inflammatory, and anti-neovascularization activities of SM extracts such as Luteolin and Tan-IIA have been reported by previous studies. Our study showed Luteolin and Tanshinone-IIA are mainly connected to the core genes associated with diabetic retinopathy and may serve as targeted components in DR treatment.

KEGG enrichment findings indicate numerous signaling pathways contributing to DR progression and initiation. We chose three of the top 20 pathways enriched with our four core genes. These pathways consist of the PI3K-Akt, Lipid and Atherosclerosis, and Fluid Shear Stress and Atherosclerosis pathways. Thus, it is believed that the constituents of SM may possess anti-diabetic potential by acting on these signaling pathways. PI3K-AKT, a pathway that plays a significant role in DR formation and growth [46], has exhibited a crucial role in forming new blood vessels in DR. When activated, PI3K/AKT can prolong the lifespan of endothelial cells and work in tandem with VEGF to facilitate cell survival and movement, culminating in the initiation of neovascularization [47,48].

Moreover, PI3K signaling may be triggered in diabetic conditions by hypoglycemia and hypoxia, and activation of AKT may facilitate vascular dilation, remodeling, and vascularization [49]. Once activated, AKT may perform diverse functions by acting upon various downstream target proteins. For example, it can promote cell growth and proliferation by phosphorylating mTOR or impede cell apoptosis by inhibiting the Low-Density Lipoprotein (LDL) cholesterol expression [50]. PI3K/AKT signaling pathway has been reported that its activation cause the pathogenesis of DR and stimulates the propagation and movement of retina endothelial cells [51].

Lipids, a form of fat, can potentially influence the progress of DR in atherosclerosis. Increasing Low-Density Lipoprotein (LDL) cholesterol, commonly regarded as "bad" cholesterol, can lead to atherosclerosis, a condition characterized by the accumulation of fatty deposits inside the arterial walls. The progression of DR may be aided by the restriction of blood flow to the retina caused by atherosclerosis, which narrows the blood vessels. In addition, the atherosclerosis pathway is associated with inflammation, which contributes to the development of diabetic retinopathy [52,53]. Atherosclerosis can also produce blood clots, which can restrict blood flow to the retina and contribute to improving DR. Therefore, targeting lipids and atherosclerosis by inhibiting the atherosclerosis pathway might serve as a mechanism for treating DR.

Fluid shear stress and atherosclerosis, which have demonstrated significant roles in the formation and progression of DR, are other essential pathways reported in this work. Blood flow exerts a physical force known as fluid shear stress on the inner walls of blood vessels, including the retina. Under certain conditions, fluid shear stress can protect blood arteries by stimulating the production of anti-inflammatory chemicals. However, it can also contribute to the advancement of atherosclerosis. For instance, aberrant blood flow patterns, such as those that occur at branching points or bends in blood vessels, can result in areas of lower shear stress [54], which can stimulate the release of pro-inflammatory genes and promote the formation of atherosclerotic plaques [52]. Furthermore, blood flow alterations and elevated glucose and lipid levels can create excessive shear stress on the retinal blood vessels in diabetic retinopathy. This can result in oxidative stress, inflammation, and endothelial dysfunction [55], which are crucial factors in the growth of DR. Luteolin and Tanshinone-IIA have exhibited anti-atherosclerosis efficacy in preclinical studies. A study conducted in a mouse model showed luteolin to remarkably attenuated atherosclerosis in high-fat diet-induced ApoE-/- mouse via alleviating inflammation [56]; Tan IIA, on the other hand, has been shown to ameliorate endothelial cell dysfunction and prevent oxidative stress and inflammatory damage to vascular endothelial cells [57]. Based on the mechanisms mentioned earlier, SM constituents are hypothesised to reduce DR's progression and maintain retinal function by inhibiting oxidative stress, inflammation, neovascularization and endothelial dysfunction through PI3K/Akt signaling and Lipid and Fluid shear stress-induced atherosclerosis pathways. The reported data propose that the Salvia Miltiorrhiza targets identified via network pharmacology may have a therapeutic role against DR.

To further identify the mechanism behind the therapeutic impact of SM in DR, GO analysis was done on the 40 shared targets of Salvia Miltiorrhiza. In this study, we chose the top 20 most significantly enriched BP, CC, and MF terms. The data indicate that Salvia Miltiorrhiza has intricate and multifaceted effects on DR. The GO analyses demonstrated that regulation of kinase activity and positive regulation of protein phosphorylation significantly impact the advancement of DR. Kinases are enzymes crucial in numerous biological activities, such as cell signaling and the expression gene regulation. Kinases have been connected to multiple features of diabetic retinopathy, including the onset of retinal neovascularization and inflammation [6,58]. In diabetic retinopathy, positive regulation of protein phosphorylation is an essential biological process that plays a significant role in the onset and progression of the disease. Protein phosphorylation is a process by which phosphate groups are added to specific amino acid residues of proteins. This modification is vital for regulating various cellular processes involving metabolism, cell signaling, and gene expression. High blood glucose levels in DR can cause oxidative stress and retinal inflammation. This can activate various signaling pathways that result in the abnormal phosphorylation of proteins involved in angiogenesis, inflammation, and apoptosis. For instance, the phosphorylation of VEGF receptor-2 can promote angiogenesis, forming new blood vessels that can be leaky and dysfunctional [59]. The investigation revealed that the molecular functions of our target genes were primarily associated with kinase binding and kinase regulation. For the examination of cellular components, it was also observed that the targets were abundant in the membrane side and membrane raft. Collectively, these results demonstrate the intricacy of the pathogenic mechanism underlying DR. The GO annotation and KEGG signaling pathways showed that the hub constituents of Salvia Miltiorrhiza, including Luteolin and Tanshinone-IIA, may play a role in inhibiting oxidative stress, inflammation, neovascularization and endothelial dysfunction by regulating the binding and recognition of intracellular (such as AKT1, TNF and TP53) and extracellular (such as VEGF) related proteins, via PI3K/Akt signaling and Lipid and Fluid shear stress-induced atherosclerosis pathways.

Docking technology efficiently determines how traditional medicine constituents bind with essential target proteins related to diseases. This strategy can assist researchers in selecting which herbal compounds to examine in the wet lab [60]. To authenticate the predictions derived from network pharmacology assessment, docking was done on four hub genes linked with DR. This study used Luteolin and Tanshinone IIA as ligands to dock the DR-associated hub genes. The results proved that Luteolin binds to all 4 hub genes, while Tanshinone IIA binds with AKT1 and TP53. The ingredients bind to their target genes with affinities scores more significant than -5kcal/mol. Docking results confirmed stable interactions between SM constituents (tanshinone IIA and luteolin) and DR targets with the highest binding affinities of -10.3 and -8.9 kcal/mol. These findings demonstrate a persistent connection between the receptors and ligands.

Extracts from Salvia Miltiorrhiza (SM), such as tanshinone IIA and Luteolin, have been utilized in clinical interventions for metabolic and other diseases [35,61,62]. However, comprehensive clinical studies are imperative to ascertain the efficacy and safety of these compounds in Diabetic Retinopathy (DR). Presently, pharmaceutical strategies for diabetic retina predominantly involve blood lipid regulation, blood sugar reduction, blood pressure management, and anti-VEGF drugs. Despite the prevalent use of anti-VEGF drugs, their limited half-life necessitates repeated administration, prompting the need for rigorous, long-term efficacy and safety validation [63]. This investigation, consistent with prior research, suggests that tanshinone IIA and Luteolin, acting as multi-targeted drugs, hold promise in impeding neovascularization, mitigating oxidative stress, and inhibiting inflammation.

These compounds can be employed individually or in synergy as an SM-hybrid or potentially in combination with other pharmaceutical agents for treating diabetic retinopathy. Their observed effects on shared targets such as TNF, ATK1, and VEGF, among others, signify potential biomarkers or therapeutic targets for diabetic retinopathy interventions. The demonstrated efficacy of tanshinone IIA and Luteolin in targeting neovascularization, oxidative stress, and inflammation proposes a more sustained and effective treatment avenue compared to current anti-VEGF drugs.

Understanding the intricate biological processes influenced by SM aids in elucidating the mechanisms underlying its reparative effects on diabetic retinopathy. These pathways emerge as potential targets for drug development or interventions aimed at modulating the progression of diabetic retinopathy retinopathy.

Conclusion

The investigation uncovered that potent pharmacological mechanisms are strictly linked to endothelial dysfunction, neovascularization, inflammation, and oxidative stress. Based on the results, luteolin and tanshinone-IIA may inhibit oxidative stress, inflammation, neovascularization, and endothelial dysfunction by controlling the binding and recognition of related extracellular (VEGF) and intracellular (AKT1, TNF, and TP53) proteins. The biological characteristics are mainly controlled via the PI3K/Akt signaling and Lipid and Fluid shear stress-induced atherosclerosis pathways. The outcomes of molecular docking suggested that the active constituents of SM bind tightly with essential targets, including AKT1, VEGFA, TP53, and TNF, in DR treatment. The identified targets, pathways, and constituents from SM provide a robust foundation for further research and the development of drugs or interventions for diabetic retinopathy. Additionally, this knowledge furnishes a roadmap for translational research, holding the potential to unveil novel therapeutic agents in the ongoing pursuit of effective treatments for diabetics. However, this study has limitations, like the possibility that some compounds or targets may not have been involved in the analysis and the absence of experimental authentication for the results obtained employing network pharmacology and docking. Therefore, we suggest that our group conduct further wet lab experimental studies to confirm these findings.

Author Statements

Data Availability Statement

The original data presented in the study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding author.

Author Contributions

Conceptualization KL; MY; Formal analysis, KL, SLG, and QZ; Investigation, KL; Resources, MY; Data curation, K. L, and MY; Writing-original draft preparation, KL, and SLG; Writing-review & editing, KL, ZL, and ML, Funding acquisition, ZL All authors have read and agreed to the published version of the manuscript.

Acknowledgements

This project was supported by Zhejiang Province "leading goose" research and development project (number: 2023C02017).

Conflicts of Interest

The authors have no conflicts of interest to declare relevant to this article's content.

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