Part3: Effects Of Isorhamnetin On Diabetes And Its Associated Complications: A Review Of In Vitro And In Vivo Studies And A Post Hoc Transcriptome Analysis Of Involved Molecular Pathway
Mar 29, 2022
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5. A Post Hoc Transcriptome Analysis Predicts the Potential Effect of the Isorhamnetin on Diabetes in a Stem Cell-Based Tool
We have established a stem cell-based tool using a perinatal stem cell, human amniotic epithelial cells(hAECs), to evaluate the bioactivities of natural compounds employing whole-genome microarray analysis [71,140-144]. In recent years, an increasingly large number of bioactive compounds in medicinal plants have been screened for their potential therapeutic and preventive effects. In this context, stem cell-based approaches using human pluripotent stem cells(hPSCs) receive great attention as physiologically more relevant in vitro human models for drug screening and validation of thousands of compounds in both academic research and the pharmaceutical industry[145-147]. However, hPSCs, including embryonic stem cells(hESCs) and induced pluripotent stem cells (hiPSCs), have limited cell resources, require invasive extraction procedures, expensive cell reprogram-ming, and critical maintenance procedures, as well as pose ethical constraints, and therefore are less favorable as a practical source for drug screening. On the other hand, hAECs are de-rived from discarded term placenta, a medical waste product. They do not require invasive harvesting procedures and have minimum ethical concerns. Furthermore, hAECs are de-rived from the pluripotent epiblasts and thus maintain ESC-like multilineage differentiation potential and can be differentiated into cells from all three germ layers [148-151].
It is worth noting that upon appropriate differentiation protocol, hAECs can be differentiated into hepatocyte-like cells [152-155], cholangiocytes [156], and, most importantly, pancreatic β-like insulin-producing cells[157-160]. Transplantation of hAEC-induced pan-creatic cells into streptozotocin-induced diabetic mice could normalize the blood glucose level[161]. hAECs[162], as well as exosomes derived from hAECs [163], could accelerate diabetic wound healing via promoting angiogenesis and fibroblast function and reducing inflammation. Incorporating hAECs into islet organoids [164] and shielding native islets with a layer of hAECs [165] could enhance islet engraftment and revascularization in diabetic mice models. Additionally, hAEC-derived hepatocyte-like cells, as well as hAEC itself, have been reported to have therapeutic efficacy in liver diseases, including hepatic fibrosis [166,167], cirrhosis [168], and hepatic failure [169].
Considering the complex pathophysiology of DM, hAEC may not be an ideal in vitro model to study the ant-diabetic effects of compounds. However, because of its stem cell-like properties, it can be used for the initial screening of target compounds. We have previously explored the antifibrotic[71] and hepatic differentiation-inducing [170]potential of isorhamnetin in hAECs. In the present study, we have performed a targeted secondary analysis of our previously published data [71] to explore the potential functionalities of isorhamnetin in diabetes(Figure 4). Data analysis was conducted for three biological replicates of day 10 control (n = 3) and isorhamnetin-treated (n =3) hAECs. The cells were grown in 3D cell culture. Control cells were maintained in placental basal epithelial cell medium(Promo Cell, Cat.#C-26140)in absence of any differentiation medium or growth factors, whereas treatment cells were supplemented with 20 mM of isorhamnetin (Sigma-Aldrich, Japan) for 10 days. Differentially expressed genes (DEGs) are referred to as genes with a linear fold change>2 and p-value<0.05(one-way between-subjects ANOVA). A total of 303 DEGs were identified; among them, 60 were upregulated and 243 were downregulated. Details of methodology have been explained elsewhere [71,170]. All microarray data are available at Gene Expression Omnibus(GEO) under accession number: GSE153149 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE153149,accessed on 24 November 2021).
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5.1. Cell Type Signature Gene Sets
In our previous studies on hAECs, we found that different types of compounds could direct the differentiation of hAECs towards different cell lineages, such as a caffeic acid ester, rosmarinic acid [142] and a caffeoylquinic acid derivative3,4,5-Tri-O-Caffeoylquinic Acid (TCQA)[140] which could enhance neural cell differentiation, whereas an anthocyanin, cyanidin-3-O-glucoside(Cy3G), induced adipocyte differentiation [143] in hAECs. We observed that the bioactivities or functionalities of natural compounds could generally be predicted from the enriched cell types by the DEGs.
We examined the significantly enriched cell type signature data sets using the Molecular Signatures Database (MSigDB)ver. 7.4 of GSEA online software (https://software. broadinstitute.org/gsea/index.jsp; accessed on 26 November 2021)[171]. These gene sets contain cluster marker signature genes for cell types identified in human tissue single-cell sequencing studies and facilitate the cell type assignments in data sets, such as experiments of developing organoid models.
We found that the most significantly enriched cell type gene set was pancreatic mesenchymal stromal cells [172](Figure 4A). Additionally, pancreatic ductal and endothelial cell types were significantly enriched [172]. Pancreatic signature genes in isorhamnetin-treated hAECs are involved in epithelial-mesenchymal transition, TGF-β signaling, TNF-αsignaling via NF-kB, KRAS signaling, and fatty acid metabolism. Several hepatic signature gene sets were also significantly enriched, such as HSCs, kupffer cells, bile duct cells [173], and fetal liver mesothelial cells [174]. There were also several significantly enriched skeletal muscle signature gene sets, including fibrillin1+fibro-adipogenic progenitor (FBN1+FAP)cells, fibro-adipogenic progenitor(FAP) cells, and skeletal muscle pericytes [175]. The biological functions of hepatic signature genes in isorhamnetin-treated hAECs include several inflammatory response pathways, whereas skeletal muscle signature genes regulate wound healing, collagen fibril organization, and MAPK cascade. Significance was measured as the false discovery rate, an analog of hypergeometric p-value after Benjamini and Hochberg correction for multiple hypothesis testing (FDR q-value <0.05).
5.2.Significantly Enriched Hallmark Gene Sets
Next, we examined the significantly enriched hallmark gene sets on MSigDB (retrieved on 26 November 2021). Hallmark gene sets represent specific, well-defined biological states or processes generated based on identifying gene set overlaps and retaining genes that display coherent expression. The hallmarks have a collection of 50 gene sets condensed from over 4000 overlapping gene sets and thus have reduced noise and redundancy [176].
Significantly enriched hallmark gene sets include genes defining epithelial-mesenchymal transition, genes upregulated in response to hypoxia, genes regulated by NF-kB in response to TNF, genes up and downregulated by KRAS activation, genes mediating apoptosis by activation of caspases, genes involved in myogenesis, genes upregulated in response to TGF-β1, genes upregulated by STAT5 in response to IL-2 stimulation, genes defining inflammatory response, genes involved in p53 pathways and networks, and genes encoding proteins involved in glycolysis and gluconeogenesis (Figure 4B). Significance was considered at FDR q-value <0.05.
An interesting finding is the enrichment of KRAS activation by the DEGs of the isorhamnetin-treated hAECs. Several KRAS-induced gene expressions were found to be significantly downregulated by isorhamnetin, such as MMP9, TSPAN1, and ITGBL1. Hyperglycemia triggers genomic instability leading to KRAS mutations in pancreatic cells [177] and has also been associated with increased risk and invasiveness of pancreatic [178] and colon cancers [179]. Wang et al.reported that isorhamnetin suppresses the proliferation of pancreatic adenocarcinoma cell line PANC-1 through downregulating Ras/MAPK signaling pathway activity [134]. Therefore, as mentioned in Section 4.2.4, the effect of isorhamnetin on the KRAS-induced risk of cancers in diabetes, especially pancreatic cancers, is worth further exploration.
5.3. Significantly Enriched Pathways
Further pathway analysis of the DEGs was conducted using the Comparative Toxicogenomics Database(CTD)(http://ctdbase.org/; accessed on 29 November 2021)[180]. CTD represents the Kyoto Encyclopedia of Genes and Genomes (KEGG) and REACTOME pathways. We found that several inflammatory pathways, collagen formation and assembly, PI3K-Akt signaling pathway, and AGE-RAGE signaling pathway in diabetic complications were significantly enriched (Figure 4C).
Advanced glycation end products (AGEs) are produced through the non-enzymatic glycation and oxidation of proteins, lipids, and nucleic acids. The receptors for advanced glycation end products (RAGE)belong to the immunoglobulin superfamily. AGE/RAGE signaling is a complex and intricate cascade that activates multiple intracellular signal pathways involving protein kinase C, NADPH oxidase, and MAPKs, resulting in NF-kB -induced expression of IL-1, IL-6, TNF-α, VCAM-1, and VEGF. Particularly, AGE/RAGE signaling has been implicated in diabetes-mediated vascular calcification through activation of TGF-β mediated fibrosis, NFkB, and ERK1/2 pathways [181-184]. We found that isorhamnetin significantly decreased AGE/RAGE signaling-related gene expression, such as COL1A1, COL1A2, COL4A6, FN1, MMP2, and SERPINE1. As discussed in the earlier section, isorhamnetin's antifibrotic effects have been well documented [71,74,99,119], and therefore, it can be asserted that isorhamnetin may also have beneficial effects in diabetes-induced vascular pathology.
5.4.Signifiantly Enriched Metabolic Diseases and Related Gene Expressions
Curated gene-disease association data were retrieved from the CTD (retrieved on 29 November 2021). We curated only significantly enriched metabolic diseases. The significance of enrichment was calculated by the hypergeometric distribution adjusted by the Bonferroni method. Significantly enriched metabolic diseases included DM, glucose, and lipid metabolism disorders, hyperglycemia, and obesity (Figure 4D). Heatmap shows that PPARs, TGFs, TNFs, ILs, collagen, and apoptosis-inducing gene expressions were significantly downregulated in isorhamnetin-treated hAECs(Figure 4E). On the other hand, insulin receptors, lipoprotein lipases, and apoptosis inhibitors were significantly upregulated in isorhamnetin-treated hAECs.
Our targeted microarray data analysis of isorhamnetin-treated hAECs also confirmed the potential of isorhamnetin in regulating biological functions related to DM and its associated complications.
6. Bioavailability and Intestinal Absorption of Isorhamnetin Aglycone and Its Glycosylated Derivatives
With the presence of various categories of flavonoids in nature, it is interesting to analyze the presence of these compounds in the body. When we talk about the metabolism of isorhamnetin and its availability in the human body after consumption, we are talking about the origin, the metabolism, and the transport. This is based on an observation made by chromatography of some flavonoids such as flavonols in human serum. Liquid chromatography-mass spectrometry provides insight into the bioavailability of certain flavonoids in their aglycone and glycoside forms in the body [49]. Mass spectrometry can be used to determine flavonoids in biological samples [185]. A study reported 23 mixed sulfate, methyl, glucuronide, and glucose derivatives of quercetin in both urine and plasma of human volunteers 1 h after ingestion of lightly fried red onions. This study detected glycosides of both quercetin and isorhamnetin in plasma [186].
Several factors play a role in the entry of nutrients through the digestive tract. For example, enzymes from the intestinal microbiota affect the entry of phenolic compounds.
A study carried out on the ginkgo leaf extracts in a mice model showed the importance of gut microbiota on the bioavailability and absorption from the gastrointestinal tract of some bioactive molecules, especially isorhamnetin [187]. In this step, enzymes of gut microbiota produce flavonoid aglycones and a variety of ring fission products. Analyses of the whole blood samples indicated that the uptake of isorhamnetin was increased by antibacterial treatment, suggesting that gut microbiota enzymes have a negative effect on the pharmacokinetics of natural molecules, such as isorhamnetin. Antibacterial or probiotic consumption may increase the bioavailability of the glycoside form of isorhamnetin. In addition, the in vitro biotransformation rates and residence times of bioactive molecules differed between normal, diabetic, and diabetic nephropathy rats [188].
On the other hand, different membrane transporters control the transport of flavonoids, such as the sodium-dependent glucose transporter 1(SGLT1)and the multidrug resistance-associated proteins 2 and 3 (MRP2 and MRP3)[189]. In this context, MRP transporters regulate the transcellular and the paracellular transport pathways of isorhamnetin [190]. Inside cells, the transport of isorhamnetin from the apical to the basal side was 6.8-9.3-fold higher. In Figure 5, the anti-diabetic effects of isorhamnetin are summarized.
7. Conclusions
Isorhamnetin is a phenolic compound of the flavonoid family, more precisely of the flavonols. Originally it is a quercetin molecule but has undergone methylation. Isorham-netin is distributed in the plant kingdom in many wild and cosmetic plants. In addition, several medicinal plants produce this molecule and several studies have attested to its anti-diabetic effect among other biological activities. All these data thus show the interest of isorhamnetin in the therapeutic industry. From this perspective, it would be very interesting to explore the effect of isorhamnetin and its derivatives, isolated especially from natural resources, on metabolic disorders. It is also necessary to highlight and review the clinical studies performed in this context using flavonoid-rich fractions and natural products in order to avoid the impact of side effects caused by synthetic and chemical drugs.
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