Exploring the potential mechanism of Morus alba in the treatment of diabetes peripheral neuropathy based on network pharmacology and molecular docking
Diabetes peripheral neuropathy (DPN) is one of the most common and easily neglected diseases in the chronic complications of diabetes. It is a chronic, synchronous and progressive distal symmetric polyneuropathy characterized by chronic hyperglycemia and microangiopathy. Epidemiological survey found that about 26% of adolescents and 46% of adults with diabetes will have peripheral neuropathy, which to some extent increases the risk of pain, foot ulcer and amputation; It exacerbates the occurrence of depression, reduces the quality of life of patients, and increases the economic burden on society. The pathogenesis of this disease is complex and may be closely related to factors such as activation of the polyol pathway under chronic high glucose conditions, accumulation of advanced glycation end products (AGEs), activation of protein kinase C (PKC), activation of the hexosamine biosynthesis pathway, overexpression of oxidative stress, immune damage, activation of related factors and pathways in inflammatory responses, deficiency of neurotrophic factors, vascular damage and activation of poly ADP ribose polymerase (PARP), activation of MAPK pathway, inhibition of the Hedgehog (Hh) pathway, and inhibition of the Wnt pathway. At present, there is no recognized treatment method to prevent or reverse diabetes peripheral neuropathy. Western medicine mostly chooses mecobalamin (nerve repair), vitamin B1, B6, B12 (nutrient nerve) and other therapeutic drugs for the cause of disease on the basis of improving lifestyle and blood sugar control, or epasta, lipoic acid and other drugs for the pathogenesis to prevent the occurrence and development of diabetes peripheral neuropathy, but the effect is not ideal. Traditional Chinese medicine has a long history, extensive and profound, and has rich clinical experience in the treatment of diabetes peripheral neuropathy. The role of mulberry resources in the prevention and treatment of type 2 diabetes and chronic complications has been recognized. The white bark of mulberry plants, which removes the cork and retains the root bark, was first recorded in the “Shennong Bencao Jing”. It has a sweet taste and a cold nature, and is mainly used for treating injuries and can supplement deficiency. The “Biography of Famous Doctors” believes that mulberry bark can remove water vapor from the lungs, promote diuresis, relieve heat, and quench thirst; The “Treatise on Medicinal Properties” believes that it can treat lung asthma. Mulberry white bark has many active ingredients, mainly including quercetin, kaempferol, sitosterol, flavonoids, etc. Pharmacological studies have confirmed that Mulberry white bark and its active ingredients have antitussive, expectorant, antiasthmatic, diuretic, anti-inflammatory, hypoglycemic, lipid-lowering, peripheral analgesia, nerve repair, immune regulation, antioxidant and other effects, and can treat diabetes peripheral neuropathy by affecting the expression of nerve factors, protecting the structure and function of the sciatic nerve.

Traditional Chinese medicine excels in treating complex diseases from the perspective of holistic view and dialectical analysis. However, due to the complex composition and unclear mechanism of action of traditional Chinese medicine, the modernization development of traditional Chinese medicine has encountered difficulties. Network pharmacology is a new discipline based on the theory of systems biology that conducts network analysis of biological systems, selects specific signal nodes for multi-target drug molecule design, and effectively connects traditional Chinese medicine characterized by a holistic view with modern cutting-edge technologies oriented towards systems, providing new opportunities and directions for the modernization of traditional Chinese medicine. In view of the complexity of the mechanism of diabetes peripheral neuropathy, the application of network pharmacology molecular docking technology reveals the intervention effect of the traditional Chinese medicine Morus alba on the disease from different perspectives, predicts the targets and pathways of Morus alba in the treatment of diabetes peripheral neuropathy, and provides scientific basis and data reference for the development and clinical application of Morus alba resources, especially Morus alba.

There is no corresponding name of diabetes peripheral neuropathy in the ancient books of Chinese medicine. According to the principle of symptom similarity, it is more appropriate to classify diabetes peripheral neuropathy into the category of “thirst dissipating arthralgia syndrome” of Chinese medicine. The pathogenesis is related to the duration of thirst dissipating, deficiency of both qi and yin, and blood stasis of the veins. The traditional Chinese medicine mulberry bark is the dried root bark of mulberry trees in the mulberry family. It can clear the heat in the lungs and stomach, and its efficacy is comparable to that of “Little White Tiger Decoction”; It can also tonify deficiency and invigorate qi (Shennong Bencao Jing and Bencao Jing Shu), promote blood circulation and remove blood stasis (Compendium of Materia Medica and Compendium of Materia Medica), generate fluids and quench thirst (Mingyi Bielu and New Edition of Materia Medica), and relieve pain (Treatise on Medicinal Properties and Dietary Therapy Materia Medica). Pharmacological studies have confirmed that mulberry bark can enhance immune regulation and reduce the production of B cell antibodies; Can lower blood lipids and regulate glucose and lipid metabolism disorders; Can dilate blood vessels, increase the number of capillaries, and improve blood flow status; It has a protective effect on the neuronal damage induced by high glucose in PC12 cells. Ma Songtao confirmed through a series of experimental studies that Mulberry white extract can increase the content of cGMP and cAMP in the sciatic nerve of diabetes rats, increase the activity of Na+⁃ K+⁃ ATPase in red blood cells, and improve the nerve conduction speed; Increase the activity of serum superoxide dismutase, increase the expression of myelin protein, promote the expression of nerve growth factor, reduce the content of AGE in nerve tissue, and alleviate and repair the damage of diabetes peripheral nerve to a certain extent. We found that the current research lacks the complex research on the multi target and multi approach of the effective components in Mulberry white bark acting on the body to treat diabetes peripheral neuropathy, which is precisely the network pharmacology to make up for this defect.

This study used network pharmacology methods to screen 31 active ingredients and 312 active ingredient related targets from the traditional Chinese medicine mulberry bark; 4661 genes related to diabetes peripheral neuropathy were found by GeneCards database and OMIM database; 120 common targets of Morus alba – diabetes peripheral neuropathy were obtained by intersection of Morus alba and related genes of disease diabetes peripheral neuropathy. The related targets of the active ingredients of Morus alba were intersected with the common targets of Morus alba – diabetes peripheral neuropathy, and 23 effective ingredients of Morus alba were obtained for the treatment of diabetes peripheral neuropathy, and a Chinese medicine regulation network diagram of “Chinese medicine – effective ingredients – target genes – disease” was constructed. The common targets of Sangbaibai diabetes peripheral neuropathy were recorded into STRING database for analysis and PPI network diagram was obtained. It was found that the core genes of PPI were transcription factor AP ⁃ 1 (JUN), mitogen activated protein kinase 1 (MAPK1), transcription factor p65 (RELA), serine threonine protein kinase 1 (AKT1), interleukin 6 (IL-6), etc. Some studies have confirmed that Schwann cells, the glial cells of the peripheral nervous system, can promote the demyelination of the peripheral nerves by attenuating the transcription factor AP-1 (JUN), and improve the demyelination pathology of diabetes peripheral neuropathy; MAPK1 can delay the occurrence and development of diabetes peripheral neuropathy by inhibiting the activation of the nervous system should activate protein kinase; RELA, IL-6 are associated with inflammatory regulatory response; AKT1 affects the proliferation, apoptosis, and regeneration of pancreatic beta cells and nerve cells.

The GO enrichment analysis results showed that the most enriched Chinese herb Mulberry White in the treatment of diabetes peripheral neuropathy was DNA binding transcriptional activator activity, RNA polymerase II specific, followed by cytokine receptor binding, receptor ligand activity, cytokine activity, ubiquitin like protein ligase binding, protein serine/threonine kinase activity, etc. It is involved in gene transcription, cytokine activity, protein kinase and other biological processes, indicating that the Chinese medicine Mulberry bark plays an intervention role in the treatment of diabetes peripheral neuropathy by participating in a variety of biological processes. KEGG pathway enrichment analysis showed that the most significant of the Chinese herbal medicine Sangbai in treating diabetes peripheral neuropathy was AGE-RAGEsignaling pathway in diabetic complications, fluid shear stress and atherosclerosis, followed by Kaposi sarcoma associated herpesvirus infection, MAPK signaling pathway, and human cytomegalovirus infection. and tumor necrosis factor signaling pathway. The late glycation end products and their receptor signaling pathways in the complications of diabetes are closely related to oxidative stress. The accumulation of AGE and the activation of RAGE can continuously induce oxidative stress, and can also cause immune imbalance in diabetes patients, which is an important link in the occurrence of diabetes peripheral neuropathy. Research has found that the accumulation of AGEs can activate p38MAPK, leading to neuronal apoptosis. The active ingredient kaempferol has been confirmed to reduce the formation and accumulation of AGE, alleviate the neuroinflammatory reaction in STZ induced diabetes rats, and reduce the pain sensitivity of diabetes neuropathy. The MAPK signaling pathway consists of four pathways: p38MAPK, extracellular signal regulated kinase 1/2 (REK1/2), c-Jun amino (N) terminal kinase 1/2/3 (JUK1/2/3), and ERK5. MAPK signaling pathway is closely related to inflammation, oxidative stress, genetic engineering, etc. Ren et al. found that long non coding RNAs (IncRNAs) in MAPK signaling pathway mediate the occurrence of neuropathy in diabetes by enhancing purine receptor mediated neuropathic pain and releasing the expression of inflammatory factors. Therefore, we can protect diabetes peripheral neuropathy by interfering with MAPK signaling pathway. In KEGG analysis, MAPK signal pathway is a signal pathway with more abundant genes. We can speculate that MAPK pathway may be one of the key pathways in the treatment of diabetes peripheral neuropathy with Morus alba. The representative factor in the tumor necrosis factor signaling pathway is TNF – α, which is an important inflammatory factor produced by activated macrophages, lymphocytes, natural killer cells, mast cells, and eosinophils. It has toxic effects on neurons and glial cells, leading to damage to nerve cells and demyelination of nerve fibers; It also stimulates the secretion of IL-1 β and IL-6 by endothelial cells and monocytes, exacerbating the inflammatory response of endothelial cells and causing damage to endothelial cells. The study found that the level of TNF – α in diabetes and diabetes peripheral neuropathy patients increased, and the expression of myelin basic protein in nerve tissue decreased; After inhibiting the expression of TNF – α with recombinant human TNF – α receptor antibody fusion protein (rhTNFR: Fc), the phenomena of nerve conduction velocity, fiber demyelination and axonal structure disorder were improved, and the expression of myelin basic protein was increased, which indicated that TNF – α played an important role in the pathogenesis of diabetes peripheral neuropathy. This study successfully docked all the core ingredients selected from Mulberry White Paper with key targets. Among them, three key effective ingredients had the best binding energy with the target MAPK1. Among the three key effective molecules, the binding energy of quercetin with the target MAPK1 and RELA was better than the other two key effective ingredients, both of which were less than 8.0kJ/mol, suggesting that the core ingredients in Mulberry White Paper had good binding activity with the key targets, indicating that they might be potential active ingredients for the treatment of diabetes peripheral neuropathy. This shows that the potential active ingredients and key targets of Sangbaibai in the treatment of diabetes peripheral neuropathy have certain scientific basis and theoretical support through network pharmacology molecular docking technology. The results of enzyme-linked immunosorbent assay and fluorescence spectrum experiment showed that the levels of IL-6, TNF – α and AGEs in the model group were increased. Mulberry white bark could reduce the levels of IL-6, TNF – α and AGES expression, indicating that it could inhibit the expression of IL-6, TNF – α and AGEs and protect diabetes peripheral nerves from damage.

To sum up, from the perspective of network pharmacology and molecular docking, this study shows that the prevention and treatment of diabetes peripheral neuropathy by Morus alba is through quercetin, sitosterol, kaempferol and other effective ingredients acting on multiple gene targets such as MAPK1, JUN, RELA, thereby affecting AGE-RAGE signal pathway, fluid shear force and atherosclerosis, Kaposi sarcoma related herpesvirus infection, MAPK signal pathway, human cytomegalovirus infection, tumor necrosis factor signal pathway and other multiple pathways. It also verifies the results of some network pharmacology from the experimental aspect, and confirms that Morus alba alba has the characteristics of multi-component, multi target, and multi pathway in the treatment of diabetes peripheral neuropathy. It provides data support for traditional Chinese medicine, especially Sangke resources, to prevent and treat diseases around diabetes, contributes to the inheritance and innovation of Sangke resources, and provides a theoretical basis for clinical application.