In this study, we established the DM（diabetes） rats model to investigate the effect of salidroside on the treatment of DM. The results showed that the learning and cognitive functions of rats in the DM and SAL groups was poorer than in the Sham group at 1d, 3 d, and 5 d after modeling, and the thickness of retinal pigment epithelial (RPE) was thinner in DM and Sham groups but thickened in SAL group. Additionally, three genes were screened out by 4D-DIA labeled quantitative proteomic analysis, including Nsmce1, Inpp5f and Vcan. The results showed Nsmce1, Inpp5f and Vcan genes were upregulated in DR(Diabetic retinal injury) group, Nsmce1 and Inpp5f formed stable molecules binding to salidroside but Vcan. The results of biological function enrichment analysis showed that the most important BP, CC, and MF was response to molecule of bacterial origin, raft, and cytokine receptor binding, respectively, and the most important signaling pathways were lipid and atherosclerosis.
DM is a metabolic disease mainly caused by abnormal insulin secretion and characterized by chronic hyperglycemia, in which long-term hyperglycemia can lead to DR and blindness, seriously affecting the quality of life of patients (Wang, Lan et al. 2019).
We sorted out the proteomic data and drew the Venny intersection map, obtaining three intersecting genes: Nsmce1, Inpp5f and Vcan. Nsmce1 gene is a nonstructural maintenance homolog of chromosomal element 1 (NSE1), which plays an important role in maintaining genomic integrity, DNA damage response and DNA repair (Gong, Wang et al. 2020). It has been found that Inpp5f expression is coordinated by insulin, glucose and lipid levels in STZ and high-fat diet (HFD) induced diabetic mouse models(Bai, Zhang et al. 2016). DR is also affected by insulin, blood sugar and lipid levels. Vcan, or Versican for short, interacts with inflammatory cells via hyaluronic acid either indirectly or directly via CD44, p-selectin glycoprotein ligand-1 (PSGL-1), and Toll-like receptors (TLRs) present on the surface of both immune and non-immune cells. These interactions activate signaling pathways that promote the synthesis and secretion of inflammatory cytokines such as TNFα, IL-6, and NFκB. Versican also influences inflammation by interacting with various growth factors and cytokines involved in regulating inflammation, thereby affecting its bioavailability and bioactivity. Versican is produced by a variety of cell types involved in inflammatory processes(Wight, Kang et al. 2020). In summary, Versican, as a component of ECM, affects immunity and inflammation by regulating the transport and activation of immune cells. Versican is emerging as a potential target for controlling inflammation in many different diseases(Islam and Watanabe 2020). The differential expression of heat map showed that Nsmce1, Inpp5f and Vcan genes were up-regulated in the diabetes group. In the SHAM group, Vcan was down-regulated, Nsmce1 was up-regulated, Inpp5F was down-regulated in C5-2, and the rest were up-regulated. In the salidroside treatment group, Nsmce1 and Inpp5f were down-regulated, Vcan was down-regulated in B2-2 and B1-2, and up-regulated in B5-2.
Molecular docking
Molecular docking of the three intersection genes with rhocidine found that Inpp5 and Nsmce1 were able to form a stable molecular binding pattern with rhocidine, while Vcan was not found in PDB.Inpp5 was found to be associated with retinal disease according to the Genecards query.The Nsmce1 gene is a non-structural maintenance homolog of chromosomal element 1 (NSE1), which is mainly involved in the maintenance of genome integrity, DNA damage response and DNA repair, and has a strong association with the treatment of retinal diseases
PPI interaction between proteomic genes and genes queried in PubMed
In this paper, PPI interaction was conducted on urine disease retinal injury and the protein relationship between salidroside and SHAM, which was found on PubMed. The top ten closely related genes were AGER and TLR4. AKT1, KEAP1; AKT1 and GAPDH; AKT1 and MAPK14; AKT1, MAPK3; AKT1 and PIK3CA; AKT1, TP53; AKT1, SOD1; AKT1, CASP3; AKT1, NOS2. These closely related genes may be the key genes in the treatment of diabetic retinal injury by salidroside.
Biological function enrichment analysis
In GO analysis, the most important BP is response to molecule of Bacterial Origin. Diabetes patients are often complicated with other complications, so the body resistance is low, bacterial infection is easy to occur, and the blood flow of the lesion site will be slow, the blood supply of organs and tissues will also be reduced, but also hypoproteinemia, malnutrition, bacteria are easy to invade, resulting in various infections. Once the infection occurs, the metabolic disorder will be aggravated, and all kinds of complications caused by the disease will become more complex, or even delayed, endangering the safety of patients. The most important CC process is membrane raft. Raft is a heterogeneous and dynamic domain in which lipids are tightly packed and rich in cholesterol, sphinolipids and other proteins such as various cell signaling proteins. Therefore, membrane rafts play an important role in cellular signaling pathways. And because they are rich in cholesterol, the raft domain is less mobile than other domains, which may stimulate signaling pathways(Li, Zhang et al. 2018). Sphingomyelin synthase (SMS) is the rate-limiting enzyme of sphingomyelin, and sphingomyelin is an indispensable component of lipid rafts. SMS2 is highly expressed in brain cells and is involved in the formation of sphingomyelin in the central nervous system. When SMS2 is deficient in organisms, sphingomyelin content in the plasma membrane is reduced, which may disrupt the formation of functional membrane rafts, which play an important role in cellular signaling pathways. More importantly, SMS2 deficiency results in a decrease in the abundance of LPS-stimulated TLR4 / MD2 complexes on the surface of macrophages to exert anti-inflammatory effects (Xue, Yu et al. 2019). Cytokine receptor binding is the most important MF process. Cytokines work by binding to specific extracellular receptors. In addition, cytokines play a crucial role in the regulation of immune homeostasis(Moraga, Spangler et al. 2014).
The first ten signaling pathways of KEGG were analyzed. The first pathway is Lipid and atherosclerosis. Both type 1 and type 2 diabetes have been proved to be accelerated the independent risk factors for the development of atherosclerosis, including diabetes and closely related to the pathogenesis of atherosclerosis, the connection of diabetes and atherosclerosis known pathological mechanism including dyslipidemia, associated with an increase in the AGE of high blood sugar, OS and inflammation (Poznyak, Grechko et al. 2020). The second channel is Salmonella infection. Diabetic retinal injury is associated with hyperglycemia, which promotes the growth and reproduction of bacteria, thus increasing the possibility of infection, which also leads to rebound hyperglycemia. Thus, the interaction between infection and hyperglycemia leads to the progression and aggravation of these diseases. Hyperglycemia impairs immune tolerance and accelerates the onset and progression of bacterial infections. Salmonella infection significantly increased the number of neutrophils in intestinal LP, with marked inflammation (Zhang, Wang et al. 2019). It has been reported that type 1 diabetes can be prevented and reversed by oral administration of msbB mutants of Salmonella (Cobb, Rawson et al. 2021). The third pathway is Fluid shear stress and atherosclerosis. Fluid shear stress (FSS) within the physiological range can stabilize the vessel, but prolonged low or high flow results in inward or outward remodeling of the vessel wall, respectively. Arterial regions with bifurcation, curvature, or valves have weak flow with complex directional changes that predispose them to atherosclerotic lesions. Occlusion due to atherosclerosis can also result in very high flow in the stenosis area and low flow downstream, thus affecting disease progression. Atherosclerosis may be considered as an ineffective remodeling of blood vessels that occurs in the interfered shear region in an inflammatory environment, while diabetic retinal injury leads to angiogenesis, neovascularization, and vascular inflammation (Baeyens, Bandyopadhyay et al. 2016). The fourth pathway is age-rage signaling pathway in diabetic complications. Age-rage signaling Pathway is one of the fibrosis signaling pathways. AGE/RAGE activation stimulates the secretion of many pro-fibrotic growth factors, promotes the increase of collagen deposition, leads to tissue fibrosis, and increases the expression of RAGE (Zhao, Randive et al. 2014). AGE/RAGE mediated vascular calcification as a key area of diabetes complications, AGE/RAGE signaling has been shown to increase OS by activating NOX-1 and decreasing SOD-1 expression, thereby promoting diabetes-mediated vascular calcification(Kay, Simpson et al. 2016). OS plays an important role in the pathogenesis of diabetic vascular complications. It is well known that the accumulation of advanced glycation end products (AGEs) and the activation of AGEs receptors (RAGE) induce sustained OS in vascular tissue (Wang, Zhang et al. 2019). Diabetic nephropathy is also a common complication of diabetes, and the binding of AGE receptor (RAGE) with its ligand causes OS and chronic inflammation in renal tissue, ultimately leading to renal function loss (Sanajou, Ghorbani Haghjo et al. 2018). The fifth pathway is nod-like receptor signaling pathway. Fibrosis is the ultimate common pathway for inflammatory diseases of various organs. Inflammasome as innate immune receptor plays an important role in the progression of fibrosis. There are four main members of inflammasome, such as NOd-like receptor protein 1 (NLRP1), Nod-like receptor protein 3 (NLRP3), and Nod-like receptor C4 (NLRC4). (Zhang, Chen et al. 2021). Nod-like receptors, NLRP3, have been reported to be STZ-induced DR in rats Increased vitreous expression in patients with retinopathy. These results suggest that Nod-like receptors are particularly important for the development of DR and may be an important factor in retinal cell death and retinal function loss. The sixth passage was Shigellosis. Shigellosis is a clinical syndrome caused by shigellosis invading the epithelium of the terminal ileum, colon and rectum. Shigella multiplies within the colon epithelial cells, causing cell death and lateral diffusion to infect and kill adjacent epithelial cells, causing mucosal ulcers, inflammation and bleeding. Treatment of shigellosis inflammatory response can alleviate the inflammatory response of DR (Niyogi 2005). The seventh pathway is Kaposi sarcoma-associated Herpesvirus infection. Kaposi's sarcoma-associated herpes virus is the cause of several malignant tumors of endothelial and B cell origin (Fröhlich and Grundhoff 2020). KSHV infection is associated with certain types of inflammation. In addition, KSHV hijacks and blocks the host's immune response by using, for example, extracellular vesicles (evs), which carry a variety of molecules that can be transferred from cell to cell and represent an important mode of cell-to-cell communication (Barrett, Dai et al. 2021). Studies have found that people with diabetes are often prone to pathogen infection and tumor progression. High glucose significantly increased the cleavage and reactivation of KSHV in latent infection cells, among which, high glucose activated transcription factor AP1 was the key to the cleavage and reactivation of KSHV. High glucose enhances the susceptibility of various target cells to KSHV infection, especially in endothelial and epithelial cells, where levels of specific cell receptors that KSHV enters, including integrin α3β1 and xCT/CD98, are elevated under high glucose, which is associated with increased cell susceptibility to infection (Chang, Yang et al. 2017). The eighth pathway is TNF Signaling Pathway. Tumor necrosis factor (TNF), also known as TNFα, is a tumor suppressor and pro-inflammatory mediator, and participates in systemic inflammatory response, which is one of the cytokines responsible for the acute phase response. By inhibiting TNF-α, DR can be reduced. The ninth pathway is toll-like receptor signaling pathway. Toll-like receptors (TLR), protective immune sentinels, are transmembrane pattern recognition receptors known for their role in innate immunity, used to detect and defend against microbial pathogens. TLRs guide immune and developmental programs by activating downstream signaling pathways, usually through activation of the NF-κB pathway. Toll-like receptor involvement in post-induced mitochondrial DNA (mtDNA) synthesis is critical for NLRP3 signaling, which has been reported to be an important factor leading to retinal cell death and loss of retinal function. The tenth pathway is Hepatitis B. In hbV-infected adults, diabetes is associated with progression of serious liver outcomes, including cirrhosis, HCC, and death, and diabetic retinal injury is a subset of diabetes. There is increasing evidence that adult populations, particularly those with DM, may benefit from HBV vaccination (Younossi, Kochems et al. 2017).