AR is a traditional Chinese medicine for invigorating Qi that is widely used in clinical Chinese medicine, and its clinical efficacy has been universally recognized by modern medicine . Qi is a relatively abstract concept in traditional Chinese medicine. The theory of traditional Chinese medicine believes that Qi is the driving force of human life activities and one of the basic substances that maintain the normal life activities of the human body from different aspects. Studies have shown that AR extract has complex components, including Astragaloside, Astragalus flavonoids, APS and amino acids, which play a role in immune regulation, multi-organ protection, hypoglycemia, anti-virus, and anti-tumor [13, 14]. As so far, the cellular biological mechanism of AR curative effect is not clear.
In order to understand the AR effects on cells through modern technology, we used AR extract to treat cells for different times and concentrations. With the help of transcriptomics technology, we analyzed the changes in cell gene transcription levels and reveal the significance of AR in cell life activities. The results showed that treatment of cells with a low concentration of AR extract can screen out prolific DEGs, and the co-DEGs of different treatment times were mainly enriched in cell growth, metabolic processes, immune response and other related pathways, which were consistent with the multiple clinical function of AR in Chinese medicine . After a high dose of AR acts on cells, its signal pathways are mainly concentrated on disease-related pathways such as systemic lupus erythematosus, viral carcinogenesis, and Shigellosis. The results well explained that the high dose of AR extract may cause some side-effect in healthy people.
The growth and development of cells is usually a complex process involving various regulatory molecules. Cell growth depends on the comprehensive effects of biochemical and molecular processes that synthesize its various components inside the cell. In our previous experimental results, it was found that low dose of AR increased the content of ATP and GSH in cells . ATP is the carrier of energy released by cells during respiration and directly provides energy for various life activities. Mitochondrial homeostasis is important for maintaining mitochondrial function and cell stability . Low concentration of AR acted on the differential genes screened by cells, and multiple genes become potential target genes, such as ceramide synthase 1(Cers1), aquaporin 3 (Aqp3), Interleukin-2 (Il2) and so on (Result 3.1). Cers1 is a transduction stress signal that regulates lipid metabolism from the endoplasmic reticulum to the outer mitochondrial membrane . The transmembrane transport of H2O2 requires the participation of Aqp. H2O2 crosses the cytoplasmic membrane from outside the cell into the cytoplasm with the assistance of Aqp3 . Aqp3 can also inhibit cell apoptosis by reducing the genes expression related Wnt/GSK-3β/β-catenin pathway. Toll-like receptor 4(Tlr4) participates in a variety of autoimmune diseases by acting as a bridge between innate immunity and acquired immunity. The activation of Tlr4 induces mitochondrial dynamic imbalance and promotes the occurrence of diseases . NIMA-related kinase 7 (Nek7) is a multifunctional kinase that affects centrosome replication, mitochondrial regulation, intracellular protein transport, DNA repair and mitotic spindle assembly , and participates in NF-κB signaling. (Il2) is a pro-inflammatory cytokine that can regulate cell growth, proliferation, and apoptosis. After Il2 stimulation, the quality of mitochondria changes, or the production of ROS in cells increases [21, 22]. In addition, there are some differential gene functions related to mitochondria, such as the perredoxin (Prx) family that controls the level of H2O2 in the cell and regulates signal transduction . Glutathione S-transferase (GST) family members 7 (Gstm7) exerts the detoxification effect of electrophilic compounds by binding to reduced glutathione . The transcription of E3 ligase proteins Fbxo15 and Ring finger protein 26 (Rnf26) is involved in the immune regulation function of cells [25, 26]. These genes are directly and indirectly related to the function of mitochondria. And it can be seen from Fig. 1, E that in the BP of co-DEGs, protein modification related genes had the most enrichment. It showed that low concentrations of AR may play a role in changing the function of mitochondria by modifying the protein after expression of mitochondrial-related genes, thereby improving cellular immune function and cell growth.
The decrease of mitochondrial membrane potential can cause a series of biochemical changes in the mitochondrial membrane, which is an early event in the process of cell apoptosis . High concentration of AR reduces the production of ATP and lowers the membrane potential of mitochondria . Excessive mitochondrial fission can cause mitochondrial ROS overload and inhibit the production of mitochondrial ATP . As it showed in Table 1, the high concentration of AR extract produced drug toxicity for cell growth and caused cellular immune response. Among the co-DEGs shared with the low-concentration AR extract, the expression levels of multiple genes showed opposite results (Table 2). The expression patterns of these genes under the action of high concentrations of AR were mostly related to the occurrence of diseases. Studies have found that human neutrophils treated with Interleukin-27 (Il27) swelled the cells, ruptured mitochondria and condensed the nucleus . Chemokine (C-C motif) ligand 20(CCL20) is expressed in various human tissues and immune cells, and involved in local immune cell recruitment . CCL20 is significantly up-regulated in a variety of cancer tissues. CCL20 produced by liver cancer cells recruits CCR6 + CD5 + B cells and induces blood vessels generated to promote tumor growth . In our experiments, it was also found that Ccl20 expression was highly expressed under the action of high concentrations of AR. The mutation of Clcn1 is related to myotonia . GPIb-IX-V complex is expressed on megakaryocytes and platelets, which acts as a receptor for von Willebrand factor and mediates platelet-subendothelial interactions, and is a major participant in hemostasis. The glycoprotein Ib (GPIb) -IX-V complex contains four different transmembrane subunits: GPIbα, GPIbβ, GPIX and GPV. Mutations in GPIbβ caused GPIb-IX-V defects . Some experiments have found that high concentrations of AR can shorten the lifespan of bees and also have an inhibitory effect on the growth of cancer cells [34, 35]. It was obvious that high concentrations of drugs had adverse effects on cells and may promote the occurrence of diseases. In addition, the high dose of AR extract also changes the expression of multiple histones, which can cause some genes to change their expression patterns at the DNA level. This also shows that the high concentration of AR extract may have drug toxicity on cells, resulting in changes in gene expression patterns related to diseases.
Based on the RNA-Seq analysis, we gained further understanding of the molecular mechanism of AR, and also learned the preliminary functional mechanism for the side effects of the high drug concentration AR. Several genes were identified as potential targets of AR, which were expected to become treatment strategies for diseases caused by abnormal expression of the them. Our future research will focus on animal model to verify these hypotheses.