Part2: 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

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3. General Overview of Biological Activities of Isorhamnetin

Oxidative stress is an endogenous and exogenous process that plays a role in aging. Oxidative stress occurs due to dysregulation in the antioxidant defenses resulting from the imbalanced production of ROS and nitrogen species [59]. Several researchers have shown that long-term exposure to free radicals contributes to the development of chronic diseases, such as cancer [60], diabetes [61], cardiovascular problems [62], and neurodegenerative disease [63]. Therefore, the study of the biological activities of isorhamnetin can be initiated by investigating the involvement of this molecule in the antioxidant phenomenon. Isorhamnetin belongs to the flavonol class known for its antioxidant potential[64,65]. Moreover, several studies have shown that this flavonol has remarkable antioxidant activity, scavenging DPPH radical and ABTS radical, and can inhibit lipid peroxidation [66-68]. Wu et al. showed that isorhamnetin and isorhamnetin-3-glucuronide could inhibit the human breast cancer MCF-7 cell proliferation [69]. Wei et al. proved that isorhamnetin inhibits the proliferation of a cervical cancer cell line HeLa [70]. The mechanism of the anti-proliferative effect of isorhamnetin was thoroughly related to cell cycle arrest at the G2/M stage by the activation of the ATM-Chk2 pathway. Li and colleagues revealed that isorhamnetin could also inhibit the growth of the gefitinib resistant PC9 cells(PC9-IR) by downregulating BCL-2 gene expression and PCNA protein expression, inhibiting DNA synthesis and upregulating P53, BAX, and CASP3 gene expression [22]. Along with this, Aonuma et al. [71]demonstrated that isorhamnetin might efficiently suppress hypertrophy and fibrosis induced by angiotensin IⅡ in cardiac tissue by regulating transforming growth factor(TGF-β)pathways. Furthermore, it might have appreciated properties on potential clinical consequences of cardiovascular diseases by the renin-angiotensin system regulation. Isorhamnetin exerted a neuroprotective effect against ischemic injury in mice by reducing infarct volume and Casp3 activity(a biomarker of apoptosis) and improving neurological function repossession. Likewise, the mice treated with this flavonol showed decreased cerebral edema, enhanced blood-brain barrier function, and upregulated gene expression of tight junction proteins, including Ocln, Zo-1, and Clin-5 [72]. Ishola et al. reported that isorhamnetin ameliorates scopolamine-induced spatial and non-spatial learning and memory impairment in vivo [73]. Likewise, it could reduce malondialdehyde(MDA) and nitrite production by increasing glutathione(GSH) level, SOD, and CAT activities in the prefrontal cortex and hippocampus. The anti-inflammatory effect of isorhamnetin and related mechanisms have been widely studied [21]. Yang et al. [32] demonstrated that isorhamnetin could alleviate LPS-induced acute lung injury by inhibiting Cox-2 expression in male BALB/c mice [74]. Similarly, the in vivo study of Dou et al.[75] showed that it could alleviate bowel disease by activating PXR and promoting the upregulation of PXR-mediated metabolism of probiotics and the downregulation of nuclear factor-kappa B (NF-kB) signal transduction [75].

Furthermore, isorhamnetin has been reported to prevent pulmonary tuberculosis [76]. Additionally, it may ameliorate acute kidney injury by inhibiting the NF-kB signaling pathway activation [31]. Additionally, isorhamnetin exhibits anti-thrombus[77], antihypertensive [78], anti-inflammatory [26], anti-osteoporosis [79], antiplatelet activity [80], hepatoprotective [81], and anti-hypoxia [82] effects. It has also been shown that isorhamnetin could boost innate immunity [83]. However, we have not found a wide range of publications that confirm the antimicrobial and antiviral activities of isorhamnetin. Still, some studies reported bactericidal effects of plant extracts containing isorhamnetin or its derivatives [84-86]. Antiviral activities of isorhamnetin were observed against influenza [87l, SARS-CoV-2 spike [24], and herpes simplex[88](see the summary in Figure 3). In summary, this interesting molecule is an immense source of biological activities. More than what we have mentioned, it is endowed with a critical anti-diabetic activity that we study in the next section of this review.

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4. Anti-Diabetic Effect of Isorhamnetin

Isorhamnetin has been reported to alleviate shared metabolic complications in type 1 diabetes mellitus (T1DM) and type 2 diabetes mellitus(T2DM), but until the present, it is more aligned with T2DM. For example, Matboli et al.showed that isorhamnetin used at three different doses(10, 20, 40 mg/kg for 3 weeks)possessed anti-diabetic action by regulating the insulin pathway at the microscopic, molecular, and protein levels in the streptozotocin/high fat diet-induced T2DM rat models [89]. Authors in this study concluded that isorhamnetin could be used as an alternative and/or potential complementary treatment in T2DM through modulation of insulin resistance signaling pathway-related gene expression. However, regarding T1DM, the effect of isorhamnetin is not yet well studied with in vitro and in vivo models.

4.1.General Overview of Diabetes and Its Link with Metabolic Syndrome

Metabolic syndrome is a constellation of cardiometabolic risk factors first described 90 years ago [90]. In 1988, the notion of syndrome X appeared [91]. It includes abdominal obesity, insulin resistance, glucose intolerance (or hyperglycemia), hypertension, and dyslipidemia. The combination of these risk factors is linked to an increased risk of developing T2DM and cardiovascular diseases. Diabetes mellitus (DM) is a prevalent metabolic disease characterized by abnormally elevated blood glucose levels. It is classified into type 1(T1DM) and type 2 (T2DM). The first category of diabetes(T1DM)is typically associated with failure in insulin production resulting from the destruction of pancreatic β-cells by T-cell-mediated autoimmunity. However, T2DM is more specific to insulin resistance and function [92]. The causes of metabolic syndrome are generally poorly understood but involve genetic factors linked to the environment. Among the genetic factors, one can quote those determining the corpulence, the distribution of the fatty mass, hyperinsulinemia, and various metabolisms(lipoproteins)93. The constitutive elements of these factors tend to derive from multivalent logic. The environmental factors, the sedentary lifestyle, smoking, excess calories provided in the form of lipids, and added sugars, in particular, are better known. Many other factors have been recently discovered, such as the presence of inflammatory cells in adipose tissue and alterations in the secretion of adipokines [93]. It is, therefore, necessary to find an adequate and safe treatment given the severe consequences of this disease on health, including diabetic nephropathy, retinopathy, neuropathy, cardiovascular diseases, and diabetic foot ulcer [92].

4.2. Effect of Isorhamnetin on Associated Metabolic Pathways

Several in vitro and in vivo studies showed that isorhamnetin might modulate carbohydrate metabolism and exhibit anti-diabetic activities by involving different mechanisms. Researchers have suggested that isorhamnetin promotes carbohydrate metabolism from the stages of digestion and intestinal absorption [94], improves glucose uptake by the liver and muscles [19,95], protects the pancreatic β-cells, and alleviates insulin secretion impairment [96].In addition, isorhamnetin regulates adipose tissue differentiation, growth, and maturation [29]. For anti-diabetic potential, researchers have investigated isorhamnetin isolated from different medicinal plants. In Table 1, we have listed the plant sources and extraction procedures of isorhamnetin that regulated diabetes-associated different intracellular signaling pathways. In the rest of this review, we will try to explain the impact of this molecule and its metabolic precursor (quercetin)[97] on the diabetes-associated mechanisms.

4.2.1. Effect of Isorhamnetin on Glucose Transporters

Glucose transporters (GLUT) are a large group of membrane proteins that transport glucose from epithelial cells to blood and from blood to cells passing the intestinal barrier in the gradient direction by passive transport. Some of them are insulin-dependent, notably GLUT4. It is a major insulin-regulated glucose transporter and coordinator of insulin action in adipose. In fact, it is well established that insulin, which enhances glucose uptake, also stimulates the recruitment of GLUT4 to the cell membrane of insulin-sensitive tissue. In a study underlying the anti-diabetic properties of quercetin and isorhamnetin, the authors proved that physiological concentrations of isorhamnetin promoted GLUT4 translocation to the plasma membrane in L6 myotubes through different mechanisms without altering GLUT4 expression [95]. In fact, isorhamnetin could activate the JAK-STAT signaling pathway at 1 nM and 10nM, allowing glucose transporter translocation induction. JAK-STAT is a signal transduction system composed of a transmembrane receptor, coupled to a Janus Kinase (JAK) enzyme and a STAT-type protein. When a ligand binds to the receptor, it changes its conformation, activating the enzyme JAK, which phosphorylates the STAT protein, inducing a transduction cascade and activating the transcription of specific genes [100]. In an in silico study, Selvaraj [101] showed that certain flavonoids, including isorhamnetin, can strongly interact with the active site of GLUT4 protein through H-bond interaction. This finding lets us look for the influence of this molecule on glucose transporters, particularly GLUT 4. By comparison, quercetin stimulates the insulin and AMPK-dependent pathway in the same L6 cells. In fact, Eid et al. have shown that an 18 h treatment of cultured rat L6 skeletal muscle cells with a dose of 50 uM quercetin could stimulate AMPK and increase GULT4 translocation [102]. Likewise, quercetin demonstrated divergent effects on insulin-mediated GLUT4 translocation in adipocytes under basal and insulin-resistant conditions related to its regulation of AMPKactivity[103]. Moreover, Eid et al. confirmed that quercetin aglycone, extracted from Vaccinium Viti's berries, could enhance muscle cell glucose uptake [98]. Guided fractionation of berry extract based on glucose uptake in muscle cells demonstrated that quercetin-3-O-glycosides were the main active compounds. At 50 μM， these compounds enhanced basal glucose uptake by up to 59% following an 18h treatment, an effect significantly greater than that of 100nM insulin.

4.2.2. Effect of Isorhamnetin on Peroxisome Proliferator-Activated Receptors (PPARs)

PPARs belong to the transcription nuclear receptor factors class II family with an important action in glucose and lipid metabolism regulation[104]. They are expressed in various cell types: pancreatic cells, hepatic cells, and adipose cells, thus controlling a wide range of biological processes by the modulation of related genes expressions [105]. In mammalian cells， three isoforms are present∶ PPARα， PPARβ/δand PPARy， which regulate genes implicated in glucose and lipid transport, synthesis, and fatty acid oxidation[106,107]. Many clinical studies are targeting PPARs for metabolic disorders and diabetes treatments. Thiazolidinediones (TZDs: pioglitazone and rosiglitazone) are a class of oral anti-diabetic drugs that reduce high blood sugar levels and improve the lipid profile of T2DM patients. A huge number of hypotheses suggest that the anti-diabetic effect of TZDs is exerted via PPARy. Fibrates that target PPARo have also been used to treat hyperlipidemia and diabetes. Isorhamnetin has been reported to exert similar effects as the mentioned drugs, mainly as PPARs antagonists or agonists [28], which ameliorates metabolic complications induced by a high-fat diet or leptin deficiency [33]. Pre-adipocyte cell line model 3T3-L1 cells are widely used in obesity and diabetes research [108,109]. Obesity-induced diabetes is characterized by hypertrophy and hyperplasia of adipocytes. Pretreatment of 3T3-L1 cells with different doses of isorhamnetin could inhibit adipocyte differentiation by decreasing triglyceride (TG) accumulation and glycerol-3-phosphate dehydrogenase (GPDH) activity[29]. At the molecular level, isorhamnetin could also regulate the mRNA expression of the major adipocyte markers, mainly the transcription factors PPARy and CCAAT/enhancer-binding protein-α(C/EBPa), the master co-working regulators of adipogenesis and cell differentiation [29,30]. These findings were also concordant with studies revealing that Nitraria retusa isorhamnetin-rich extract could reduce the fat accumulation in 3T3-L1 adipocytes in a dose-dependent manner [18]. The single-molecule isorhamnetin could also significantly inhibit the differentiation rate of 3T3-L1 cells, leading to reducing lipid droplet content by reducing cell size and number. In addition, oral administration of the Nitraria retusa extract could regulate PPARγ and lipogenic enzymes target genes LPL and FAS [17]. Importantly, a recent study revealed that isorhamnetin could significantly reduce the fat amount in the animal body via a PPARα-dependent pathway [27]. 

4.2.3. Effect of Isorhamnetin on Hepatic Enzymes

The liver is an organ that plays a key role in carbohydrate homeostasis [110]. Beyond the micro and macrovascular complications of diabetes, the T2DM patient presents more certain hepatic complications with, in the first place, hepatic steatosis. In T2DM, the risk of progression to fibrosis and hepatic inflammation is greater [111], exposing a certain number of diabetic patients to significant clinical consequences, particularly with hepatocarcinoma [112]. In parallel, hepatic complications in diabetic patients have been associated with a higher risk of cardiovascular events, which may have practical consequences in terms of optimization of cardiovascular prevention [113]. We focus on the most important liver complications related to diabetes: fatty liver diseases, hepatic fibrosis, and hepatocellular carcinoma (HCC).

Non-Alcoholic Steatohepatitis

Fatty liver disease or non-alcoholic steatohepatitis (NASH) represents a potential hepatic complication. It is characterized as an abnormal accumulation of TGs inside liver cells. In type 2 diabetes, insulin resistance leads to the accumulation of toxic fatty acids. When this accumulation occurs in liver cells it causes non-alcoholic fatty liver disease (NAFLD) and in severe forms steatosis or NASH[111,114]. Ganbold et al. have shown that isorhamnetin ameliorated hepatic steatosis and fibrosis in a NASH mouse model exhibits similar features to human NASH [19]. In fact, compared to the control mouse group, liver samples from the NASH-induced group showed acute fat accumulation overtaking 37% of the oil red O positive area. However, isorhamnetin treatment in the NASH group could reduce oil red Ostained area to 22%. In addition, isorhamnetin treatment alleviated NASH-induced gene expression alteration. The authors have shown that isorhamnetin efficiently improved the altered lipid metabolic process in NASH and inhibited de novo lipogenesis.

Hepatic Fibrosis

Hepatic fibrosis is excessive scarring resulting from the build-up of connective tissue in the liver. It is caused by excessive production and/or insufficient degradation of the extracellular matrix. There are many causes of liver fibrosis. Among them is NASHorigin. When there is resistance to insulin, fatty acids build up in the liver, causing toxicity and inflammation. This is accompanied by the production of cytokines and reactive oxygen species. In fibrosis, cytokines are responsible for the activation of hepatic stellate cells (HSCs) and will activate an excessive production of collagen fibers[115,116]. The trigger is chronic aggression, especially in the event of an inflammatory component [115]. TGF-βis a crucial mediator of HSC activation and extracellular matrix accumulation, leading to fibrosis [117]. Therefore, blocking this pathway could be a potential strategy for liver fibrosis [118]. In this context, Yang et al. demonstrated that isorhamnetin inhibited HSC activation and prevented TGF-β-induced expression of fibrogenic genes, including α-SMA, PAI-1, and COL1A1 [74]. In fact, the authors revealed that the inhibitory effect of this flavonol was a result of the canonical TGF-β/Smad signaling pathway inhibition. Additionally, isorhamnetin activated Nrf2-ARE signaling and suppressed TGF-β-mediated ROS production, which also contributed to the inhibition of fibrogenic gene expression. Furthermore, isorhamnetin significantly suppressed carbon tetrachloride (CCl4)-induced fibrosis in mice. Liu et al. also showed that isorhamnetin could alleviate CCl4 and bile duct ligation(BDL)-induced liver fibrosis by inhibiting TGF-β1 production and HSC activation [119]. Likewise, Lee et al. demonstrated that isorhamnetin, extracted from Oenanthe javanica, could reduce fibrosis in rat HSCs by blocking the extracellular signal-regulated kinase (ERK) signaling pathway and inhibiting the proliferation and collagen synthesis of HSC-T6 cells [99]. In fact, pretreatment of HSC-T6 cells with this flavonol repressed serum-induced ERK phosphorylation, in the same way as a MEK inhibitor(PD98059).

Hepatocellular Carcinoma

Hepatocellular carcinoma is often discovered late at the metastasis stage (in 64% of cases); the most frequent secondary locations are the lung, lymph nodes, kidneys, and adrenal glands [120]. As mentioned above, with the high accumulation of fatty acid in liver cells, physiological disturbances appear over time such as NASH, then inflammation (with the production of cytokines and ROS), then in more advanced cases cirrhosis and in severe forms carcinoma [121,122]. The role of isorhamnetin is deduced by its anti-tumor potential activities. Isorhamnetin, extracted from Hippophae rhamnoides L., showed potent anti-tumor activity in hepatocellular carcinoma BEL-7402 cells, exerting strong cytotoxicity with IC50 at the concentration of 74.4 ± 1.13 ug/mL and 72 h incubation [123]. Cytotoxicity of this flavonol on cancer cells depends on isorhamnetin cellular accumulation. Instead, the pibenzimol hydrochloride staining of isorhamnetin-treated cells displayed condensed and fragmented nuclei and apoptotic bodies. Furthermore, flow cytometry analysis used to evaluate cell cycle progression and differentiate between apoptosis and necrosis, revealed that 13.77% of isorhamnetin-treated BEL-7402 cells (with 50ug/mL for 48 h) appeared in the hypodiploid peak. Similar work by Wei et al. reported that isorhamnetin inhibited the cell cycle progression of the cervical cancer cell line HeLa, by arresting the G2/M phase via activating the ataxia-telangiectasia mutated Chk2 pathway [75]. In another work and by making a comparison, isorhamnetin-3-O-glucoside, quercetin 3-O-rhamnoside-7-O-glucoside, and kaempferol-3-O-glucoside-7-O-rhamnoside, extracted from Cleome droserifolia, reduced HepG2, a liver hepatocellular carcinoma cell, cell viability and anchorage-independent cell growth in a dose and time-dependent manner but with a high-est effect with the quercetin derivative. Furthermore, they exhibited a reduced migration capacity of HepG2 cells [124].

4.2.4. Effect of Isorhamnetin on Pancreaticβ-Cell Dysfunction

Insulin is a hormone naturally secreted by the body that helps our cells, particularly muscle cells, liver cells, adipose cells, and brain cells, absorb glucose from food to be converted into energy for all biological processes and functions[125,126]. In normal conditions, insulin is secreted by pancreatic β-cells, then released into the bloodstream. β-cell dysfunction is characterized by impaired insulin secretion, the critical feature of T2DM, both in its onset and progression [127]. Metabolic stress-induced β-cell dysfunction in T2DM can also be associated with high levels of saturated fats in obesity, leading to increased inflammation and oxidative stress within β-cells. Therefore, impairment of insulin secretion is more severe than insulin resistance [128]. Taken together, new anti-diabetic therapeutic strategies are needed to prevent or recover pancreaticβ-cell dysfunction. In this context, Wang et al. suggested that isorhamnetin, derived from Vernonia anthelmintic herb, could suppress the proliferation of pancreatic cancer cells (colorectal adenocarcinoma cell line)via arresting the cell cycle in the S phase, which may be an alternative way to prevent pan-creatic carcinoma and its metabolic complications [129]. Consistently, another report has shown that isorhamnetin and a glycosylated form of isorhamnetin (isolated from Salicornia herbacea plant) could promote glucose-stimulated insulin secretion in insulin-secreting rat insulinoma (INS-1)pancreatic β-cells without affecting cell viability [96]. Moreover, it could stimulate insulin secretion via phosphorylation of total ERK, insulin receptor substrate-2 (IRS-2), phosphatidylinositol 3-kinase (PI3K), Akt, and activated pancreatic and duodenal homeobox-1 (PDX-1)[96]. The enhancement of these markers may alleviate the pancreatic cell dysfunction causing diabetes.

Furthermore, Grdovic et al.reported that isorhamnetin originating from the Castanea sciatica plant species has a protective effect on streptozotocin-induced oxidative damage and β-cell apoptotic death[130]. This beneficial effect was correlated strongly to the antioxidant potential of this molecule by alleviating the cellular biomarkers of oxidative stress, i.e., MDA and intracellular GSHlevels, and enhancing the activities of antioxidant enzymes SOD and CAT. The effect was also confirmed at the molecular level by the decreased NF-kB transcription factor stimulated by cell oxidative stress, and thus suggestive of a protective effect within β-cells. Consistently, quercetin, which is metabolized in isorhamnetin, could reduce glucose levels by ameliorating insulin secretion in the streptozotocin-induced type 1 diabetes model in rats [131]. In the same animal model, quercetin showed a protective effect onβ-cell integrity by reducing oxidative stress markers [132]. It could also regenerate the islets in pancreatic cells [133].

4.2.5. Effect of Isorhamnetin on NF-kB

NF-kB is a protein of the superfamily of transcription factors involved in the immune and cellular stress responses and is considered the major mediator of the secretion of inflammatory markers such as cytokines, chemokines, and different key enzymes [130,134,135]. The role of NF-kβ in the pathophysiology of diabetes and its linked vascular complications has been investigated [136]. Hyperglycaemia activates this nu-clear factor that in turn induces the secretion and over-expression of pro-inflammatory cytokines, mainly TNF-α, interleukins, TGF-β, and Bcl2, causing vascular complications such as neuropathy, nephropathy, retinopathy, and cardiomyopathy [137]. Many experimental investigations on the preventive effect of flavonols on diabetes-induced vascular dysfunction have demonstrated that isorhamnetin could inhibit the activation of NF-kβthrough inducing the activity of pancreatic antioxidant enzymes [138]. Another study reported the inhibitory effect of isorhamnetin on iNOS expression and NO production in activated macrophages, leading to the blocking of NF-kβ [139]. Moreover, Qiu et al. have studied the renoprotective effect of the flavonol isorhamnetin in the T2DM rat model through the modulation of the NF-kβ signaling pathway [31]. Authors have shown that this molecule inhibited the NF-kB signaling activity by increasing the production of NF-kB p65, phospho-NF-kB p65, and phospho-IkBα and inflammatory mediators TNF-α, IL-1β, IL-6, ICAM-1, and TGF-β1, as well as decreasing the NF-kB p65 DNA-binding activity. In addition, it could alleviate oxidative cell stress by regulating MDA and SODlevels, leading to the recovery of renal damage in diabetic rats.