Screening of key genes and pathways of microvascular complications in diabetes and prediction of traditional Chinese medicine by bioinformatics methods
In recent years, diabetes related epidemiological research shows that the incidence of hyperglycemia among people over 18 years old in China has reached 11.2%, and the incidence of diabetes continues to rise. Diabetes is a complex disorder of blood glucose metabolism. at a certain stage of the course of the disease, there may be a variety of acute and chronic complications of chronic hyperglycemia. Chronic hyperglycemia and genetic susceptibility ultimately affect microvessels, leading to complications primarily from the kidneys, eyes, and nervous system. The classic microvascular complications of diabetes include diabetes nephropathy (DKD), diabetes retinopathy (DR) and diabetes neuropathy (DPN). DKD is the leading cause of end-stage renal disease (ESRD), second only to various types of glomerulonephritis. DR is a common factor affecting vision and even blindness, while DPN is an important high-risk factor for foot ulcers and further amputations. These three microvascular complications seriously affect the health of patients with diabetes and cause serious economic burden to patients and society. However, modern medicine does not fully understand the specific pathogenesis of diabetes microvascular complications, and it is difficult to achieve good results with simple western medicine to treat diabetes microvascular complications with complex mechanisms. Traditional Chinese medicine has a long history and its clinical efficacy has been verified over a long period of time, especially in the treatment of some chronic diseases. Traditional Chinese medicine emphasizes a holistic concept and the unity of heaven and man. Clinical treatment emphasizes syndrome differentiation and treatment. Compared to single component Western medicine, traditional Chinese medicine can treat complex and varied human diseases through the integration and regulation of multiple links, levels, and targets. However, there is a lack of systematic mechanism explanation. Therefore, it is necessary to further study the microvascular complications of diabetes in order to obtain the pathophysiological mechanisms related to the development and progress of the disease, and to explore potential Chinese medicine therapeutic drugs.

With the rapid development of bioinformatics, effectively utilizing the abundant gene expression chips and high-throughput sequencing data to conduct big data mining and analysis of clinical samples can help explore the pathogenesis of diseases and provide more references for clinical practice. In this study, we used high-throughput sequencing to download gene expression profile chip data on microvascular complications of diabetes from GEO database for analysis, screened DEGs between long-term poor glycemic control with severe microvascular complications and long-term poor glycemic control without microvascular complications, analyzed the function and pathway enrichment of DEGs, and analyzed the interaction between proteins, further screened key target molecules and potential Chinese medicines involved in the pathogenesis of microvascular complications of diabetes, and provided new clues for clinical diagnosis, treatment and prevention.

 

Diabetes is a group of metabolic and heterogeneous diseases which can be caused by genetic, environmental and other factors. Most patients with diabetes will be accompanied by serious complications of chronic hyperglycemia at a certain stage of the disease course, including kidney disease, retinopathy, neuropathy and cardiovascular disease. There is currently no consensus on the pathogenesis of microvascular complications, leading to hypotheses such as hemodynamic abnormalities, non enzymatic protein glycosylation, increased activity of polyol channels, tissue self oxidation, and glycosylation. In clinical practice, we found that although the blood sugar was not well controlled for a long time, some patients with diabetes were still free from microvascular complications. To explore the pathogenesis of microvascular complications in diabetes and predict the related traditional Chinese medicine by analyzing the difference between long-term poor glycemic control with severe microvascular complications and long-term poor glycemic control without microvascular complications, so as to provide new clues for clinical prevention and treatment.

Using GEO2R to analyze and process the dataset GSE43950, 692 DEGs were obtained, including 121 upregulated genes and 571 downregulated genes. Through the enrichment analysis of GO function and KEGG signaling pathway in DGEs using the DAVID database, it was found that upregulated differentially expressed genes are mainly involved in the regulation of blood hemostasis and coagulation, and are associated with specific DNA binding to core promoter sequences and NF – κ B signaling pathway, AGE-RAGE signaling pathway, MAPK signaling pathway, etc. Downregulation of differentially expressed genes is mainly involved in neutrophil activation immune response, NOD like receptor signaling pathway, toll like receptor signaling pathway, endocytosis, and other related processes. Through research, we found that some enrichment functions and signal channels of differential genes are related to the disease process of microvascular complications in diabetes. For example, MAPK signaling pathway and AGE-RAGE signaling pathway in KEGG signaling pathway enrichment analysis that upregulates DEGs are closely related to the disease process of diabetes and its microvascular complications. The MAPK pathway has four main branching pathways: ERK, JNK, p38/MAPK, and ERK5. In diabetes and its late complications, the activity of MAPK in the body has increased, which has nothing to do with metabolic control. High blood sugar can activate the MAPK pathway and play an important role in mediating late stage complications. The MAPK pathway is closely involved in the progression of DR disease. By inhibiting the p38 and ERK signaling pathways, the damage caused by oxidative stress is alleviated, effectively delaying the further development of DR. P38/MAPK mediates the occurrence of diabetes nephropathy, which may promote the production of reactive oxygen species (ROS), mediate the release of inflammatory mediators, activate RAS system, increase glomerular internal pressure, and accelerate the disease progression of DKD. The role of advanced glycation end products (AGE) – AGE receptor (RAGE) signaling pathway has recently been identified in the pathogenesis of a variety of processes, such as inflammation, malignant tumors, diabetes and diabetes complications and neurodegeneration. Recent studies have shown that the interaction of AGE and RAGE receptors causes oxidative and pro apoptotic reactions in various types of cells and participates in the pathogenesis of vascular complications in diabetes. AGE leads to complications of diabetes through two mechanisms: one is that it is cross-linked in biomolecules to change their biological structure and function; the other is that it binds to related receptors on the cell surface, such as RAGE, to stimulate the signal pathway leading to oxidative stress deterioration. Compared with healthy individuals, patients with diabetes have higher concentrations of AGE and glucose oxidation products, and are believed to increase oxidative stress through interaction with their receptors (RAGE), thereby promoting vascular complications.

DEGs are mainly enriched in areas related to blood hemostasis, coagulation regulation, neutrophil activation immune response, and cell apoptosis. The protein interaction database STRING and the cytoHubba in Cytoscape software were used to analyze DEGs, and Top10 key genes were obtained. Key genes such as IL1B, TLR2, TLR4 and CYBB mediated the immune regulation of the body from multiple levels, suggesting that the immune regulation process is an important link in the occurrence and development of microvascular complications in diabetes, and regulating the immune function of the body may be an important target to prevent and treat microvascular complications in diabetes.

IL-1B, as a key pro-inflammatory factor in the interleukin-1 cytokine family, is the main regulator of inflammatory response and participates in a series of cellular activities such as proliferation, differentiation, and apoptosis. It is also associated with pain and autoimmune reactions in the body. Research has shown that IL-1B is a key upstream molecule that initiates pancreatic inflammation during the development of T2DM. A large number of experimental studies have shown that the activation of inflammatory bodies and the secretion of IL-1B are the key processes driving the formation of atherosclerosis. The pro-inflammatory cytokines IL-1B, interleukin-6 (IL-6), and interleukin-8 (IL-8) are mediators of acute phase response, typically associated with the induction of oxidative stress. Oxidative stress, as a common pathway to mediate the complications of diabetes, mediates many late complications of diabetes, including microvascular complications and macroangiopathy of diabetes.
TLRs belong to the pattern recognition receptor family and are closely related to the development of diabetes. TLRs can activate the innate immune system and are related to the pathogenesis of insulin resistance, diabetes and atherosclerosis, especially the expression of TLR2 and TLR4. TLR2 is mainly expressed in cells with immune related functions, such as dendritic cells, monocytes, and some endothelial cells. It participates in the pathogenesis of DN by regulating the cytokine signaling pathway in the body. Shao et al. studied the effect of paeoniflorin on the kidney of type 1 diabetes mice by using TLR2 knockout mice and wild type littermates. It was found that by inhibiting TLR2 signal pathway, albuminuria in diabetes mice was significantly reduced, and renal histopathology was weakened, which confirmed that TLR2 was closely related to DN. Meanwhile, TLR2 is a mediator of retinal degeneration in response to oxidative stress, serving as a bridge between oxidative damage and complement mediated retinal lesions, which may suggest a close relationship between TLR2 and the occurrence of diabetic retinopathy. In the TLRs family, TLR4, as a transmembrane protein receptor, is responsible for the activation of many signal transduction pathways. Chronic inflammation in diabetes is believed to be related to the activation of TLR4. Aly and other studies suggest that the increased expression of TLR2 and TLR4 may play an important role in DN progression and insulin resistance deterioration in type 2 diabetes patients. Therefore, TLR2 and TLR4 in the TLRs family may be a promising therapeutic target to prevent or delay type 2 diabetes and its complications.
CYBB encodes gp91phox, which is an important component of NADPH oxidase in phagocytic cells and a major source of oxidative stress in the kidneys. CYBB mediates the production of ROS and is involved in cell signaling related to differentiation, cell cycle, and apoptosis. Both diabetes patients and animal models have confirmed that the pathogenesis of DM may be mediated by the production of ROS in the body’s oxidative stress (OS). The excess of ROS is considered to be the main mechanism of diabetes complications. Increasing the production of mitochondrial ROS plays an important role in diabetes complications (including retinopathy).
By mapping key genes to the core medical database, and building a drug active ingredient target network, it was found that the four most frequently occurring traditional Chinese medicines (scutellaria baicalensis, salvia miltiorrhiza, achyranthes bidentata and yujin) and their active ingredients can target key genes that affect the microvascular complications of diabetes. All the four traditional Chinese medicines have a certain effect of promoting blood circulation and removing blood stasis. Salvia miltiorrhiza and yujin are more commonly used drugs for promoting blood circulation and removing blood stasis in clinical practice, and they perfectly match the pathogenesis of “diabetes goes into the collaterals after a long illness, and blood stasis occurs after a long illness”. The TCM name of diabetes is Xiaoke. Its pathogenesis is based on yin deficiency and marked by dryness and heat. Long term quenching of thirst can easily involve multiple organs, affecting the normal operation of qi and blood, causing yin deficiency, dryness and heat, and consuming body fluids, leading to poor blood circulation and blood stasis. Blood stasis is an important pathogenesis of diabetes, which is related to the progression of various chronic complications. In clinical practice, Chinese herbal medicines that promote blood circulation and remove blood stasis are often used to treat diabetes related complications. Modern pharmacology has also proved that some components of the above-mentioned traditional Chinese medicines play an important role in the treatment of diabetes and microvascular complications. Multiple bioactive drugs extracted from the root of Scutellaria baicalensis, such as baicalein and baicalin, have been found to increase glucose consumption and lower blood sugar levels in the body. Tanshinone IIA is a classic active ingredient of Danshen, which has anti-inflammatory, antioxidant, and anti fibrotic effects. Tanshinone IIA can improve painful diabetes neuropathy by inhibiting the expression and activity of voltage type sodium channels in the dorsal root ganglia of rats, suggesting that Tanshinone IIA may be a promising drug for DPN treatment. Yuan et al. found that tanshinone IIA can improve lipid metabolism, glucose metabolism and insulin resistance in type 2 diabetes rats through the AMPK signaling pathway induced by NF – κ B. Curcumin is the main active ingredient of Curcuma. A systematic review analysis showed that curcumin has the ability to inhibit oxidative stress and inflammatory process in the body. Nano curcumin based on nanotechnology is considered as a potential drug for drug treatment and management of diabetes patients. β – ecdysterone is mainly responsible as one of the active ingredients in Achyranthes chuanxiong, which can lower blood sugar, stabilize the level of NO metabolites in the body, and thus protect the kidneys.

Through information mining technology based on bioinformatics, gene difference analysis was carried out between long-term poor glycemic control with severe microvascular complications and long-term poor glycemic control without microvascular complications, and 10 genes with the largest difference were screened. Further analysis revealed that genes such as IL1B, TLR2, TLR4 and CYBB played an important role in the development of microvascular complications in diabetes, while traditional Chinese medicines such as salvia miltiorrhiza, scutellaria baicalensis, turmeric, and achyranthes bidentata and their active ingredients may become new drugs for the treatment of microvascular complications in diabetes, providing new ideas for further exploring the molecular mechanism of microvascular complications in diabetes and searching for new therapeutic drugs. However, this study still has the following shortcomings. First, the sample size in this research database is small, and diabetes types are not differentiated, so we need to further expand the sample size and refine diabetes classification; Secondly, there is a lack of precise experimental research and verification. Therefore, it is necessary to further explore the function of these key target molecules and potential drugs in the microvascular complications of diabetes.