A Review of Experimental Studies on Natural Chalcone-Based Therapeutic Targeting of Genes and Signaling Pathways in Type 2 Diabetes Complications

Diabetes mellitus type 2 (T2DM) is a common chronic condition that presents as unsettled hyperglycemia (HG) and results from insulin resistance (IR) and β-cell dysfunction. T2DM is marked by an increased risk of microvascular and macrovascular complications, all of which can be the cause of increasing mortality. Diabetic nephropathy (DNE), neuropathy (DNU), and retinopathy (DR) are the most common complications of diabetic microangiopathy, while diabetic cardiomyopathy (DCM) and peripheral vascular diseases are the major diabetic macroangiopathy complications. Chalcones (CHs) are in the flavonoid family and are commonly found in certain plant species as intermediate metabolites in the biosynthesis of flavonoids and their derivatives. Natural CHs with different substituents exert diverse therapeutic activities, including antidiabetic ones. However, the therapeutic mechanisms of natural CHs through influencing genes and/or signaling pathways in T2DM complications remain unknown. Therefore, this review summarizes the existing results from experimental models which highlight the mechanisms of natural CHs as therapeutic agents for T2DM complications.

Despite several experiments on CHs as potential targets for T2DM treatment [25][26][27][28], there is no review of experimental studies exploring the mechanisms of natural CHs for the treatment of T2DM complications.Therefore, this review highlights the existing experiments focusing on the therapeutic mechanisms of all natural CHs in T2DM complications.

Diabetic Nephropathy
Several experiments have shown that natural CHs, including ILTG, XN, LicoA, PH, HSYA, ISO, CAD, and BU, alleviate DNE via altering the genes/signaling pathways of renal fibrosis, proliferation, angiogenesis, apoptosis, inflammation, and oxidative stress.The treatment of human renal mesangial cells under conditions of high glucose with nontoxic ILTG (≥10 µM) inhibited glomerulosclerosis/mesangial fibrosis and mesangial cell proliferation by reducing type IV collagen production and the expression of tissue inhibitor of matrix metalloproteinases-2 (TIMP-2) and cellular connective tissue growth factor (CTGF) through repressing the transforming growth factor β (TGF-β)-mothers against decapentaplegic homolog (SMAD) signaling pathway [29].ILTG treatment at 100 and 200 nM concentrations showed a significant inhibition of advanced glycation end product (AGE)-induced tubular epithelial-myofibroblast transdifferentiation and collagen secretion in renal proximal tubule cells through downregulating vimentin levels, signal transducer and activator of transcription 3 (STAT3) phosphorylation, and the TGF-β/SMAD/STAT3 signaling pathway [30].Oral administration of ILTG reduces renal damage in diabetic rats by downregulating oxidative stress markers and renal inflammatory cytokines [31].ILTG was reported to inhibit high-glucose-induced proliferation and inflammation and the accumulation of the extracellular matrix (ECM) in mouse glomerular mesangial cells when treated at a concentration of 20 µM.This could be related to a decrease in the mRNA levels of pro-fibrotic markers and proinflammatory cytokines through suppressing the Janus kinase 2 (JAK2)/STAT3 signaling pathway [32].ILTG effectively inhibited the viability of high-glucose-induced renal cells when treated at a dose of 20-80 µg/mL through ameliorating oxidative stress markers.ILTG also improved renal morphology and oxidation stress markers while suppressing fibrosis-related factors and the JAK2/STAT3 signaling pathway in streptozotocin (STZ)-induced diabetic rats [33].
An experiment showed that XN reduces blood glucose levels and modulates the vascular endothelial growth factor (VEGF) angiogenic pathway in the kidneys of diabetic mice by reducing phospho-vascular endothelial growth factor receptor-1/2 (p-VEGFR-1/2) expression and its downstream effectors [34].It has been shown that oxidative stress reduces in the kidneys of diabetic mice supplemented with XN by decreasing the accumulation of AGE and galectin-3 (Gal3) expression [35].XN treatment at a dose of 50 µM reduced high-glucose-induced oxidative stress in human kidney tissues and STZ-induced kidney injury in diabetic mice.This is mediated by decreasing ROS levels and increasing catalase (CAT)/SOD activity, as well as by the mRNA levels of nuclear factor erythroid 2-related factor 2 (Nrf2) and its downstream genes [36].
LicoA treatment reduces DNE in diabetic mice through downregulating and/or upregulating of proteins expression and oxidative stress parameters [37].Treatment with PH at a low concentration (20 mg kg −1 ) significantly alleviated podocyte injury in diabetic rats through restoring podocin and nephrin expression [38].An experiment carried out on STZ and high-fat-diet-induced DNE in mice showed that HSYA treatment at a concentration of 120 mg/kg attenuates renal cell apoptosis, oxidative stress, and inflammation, as evidenced by increased and/or decreased apoptotic protein expression and by inflammatory and oxidative stress markers in the kidneys [39].HSYA treatment at a dose of 100-200 µM was shown to reduce HG-induced inflammation and podocyte apoptosis in mouse macrophage cells by decreasing the protein and mRNA levels of inflammatory and oxidative stress genes while increasing mannose receptor 206 (CD206) and arginase-1 (Arg-1) protein expression [40].In vitro and in vivo experiments showed that HYSA treatment at a concentration of 10 mg/kg decreases renal fibrosis in human renal glomerular endothelial cells and STZ-induced diabetic rats by ameliorating oxidative stress/inflammatory markers and downregulating the Toll-like receptor 4/nuclear factor-κB (p65) (TLR4/NF-κB p65) signaling pathway [41].
ISO treatment attenuates high-glucose-induced endothelial damage in vitro renal glomerular endothelial cells model, as well as suppresses renal inflammation and glomeru-lar endothelial cell apoptosis in STZ-induced DNE rats by reducing the expression of inflammatory cytokines through blocking the activation of NF-κB signaling pathway [42].It has been reported that CAD treatment at different concentrations reduces kidney damage in methylglyoxal (MGO)-treated rat kidney tubular epithelial cells through altering DNE-mediated gene expression and signaling pathways [43].Treatment with BU showed anti-diabetic activity and normal morphology of glomerulus and renal tubular in STZinduced diabetic rats without any side effects through the downregulation of PPARγ expression [44].

Diabetic Neuropathy
A few in vivo experiments have shown that ILTG and PH modulate gene expression of autophagy, inflammation, and oxidative stress in a rat model of DNU.ILTG administration to diabetic rats and neuro2a cells promoted Sirtuin (SIRT1) activity and NAD+/NADH ratio in peripheral sciatic nerves, resulting in alleviated neuropathic pain, decreased ROS production, and increased Nrf2 activity through upregulating Forkhead box O3 (FOXO3) signaling, peroxisome proliferator-activated receptor-γ coactivator 1 α (PGC-1α)-mediated mitochondrial biogenesis, and AMPK-mediated autophagy, along with the suppressing of mammalian target of rapamycin (mTOR) phosphorylation [45].The combinational treatment of sciatic nerve tissue with PH and duloxetine (an antidepressant drug) was shown to decrease inflammatory and oxidative stress markers in diabetic rats [46].

Diabetic Retinopathy
The few experiments that have been performed suggest that XN, HSYA, PH, and BU attenuate DR by altering the gene expression and/or signaling pathways of oxidative stress inflammation, autophagy, apoptosis, and angiogenesis.XN treatment was shown to inhibit angiogenesis and regulate autophagy dysregulation in hypoxia and high-glucose-induced human retinal microvascular endothelial cells by downregulating and/or upregulating the protein and mRNA levels of angiogenesis and oxidative stress markers through the inhibition of PI3K/AKT/mTOR signaling pathway [47].HSYA treatment was found to improve DR and attenuate retinal ganglion cell apoptosis in diabetic rats due to a mechanism involving increased and/or decreased inflammatory and oxidative stress markers [48].PH treatment at a concentration of 20 mg/kg significantly improved DR in diabetic mice by upregulating and/or downregulating the expression of proteins involved in energy metabolism, apoptosis, and oxidative stress [49].PH treatment improves human retinal pigment epithelial cells through reducing glucose uptake, inhibiting JNK phosphorylation and lipopolysaccharide (LPS)-induced proinflammatory cytokine production, and upregulating Nrf2 activity [50].The findings of an in vitro experiment showed that BU treatment at different concentrations inhibits fluorescent AGEs and α-crystallin-induced lens proteins aggregation [51].

The Therapeutic Mechanisms of Natural CHs in T2DM Macrovascular Complications
Results from a few experiments demonstrated that ILTG, HSYA, and PH reduce DCM, diabetic atherosclerosis (DAS), and diabetic vascular injury (DVI) through the modulation of multiple genes.

Diabetic Cardiomyopathy
An experiment using embryonic rat heart cells demonstrated the inhibition of highglucose-induced apoptosis, fibrosis, and hypertrophy following treatment with ILTG at different concentrations via ameliorating inflammatory and oxidative stress markers at the mRNA and protein levels.ILTG treatment was also found to attenuate apoptosis/heart fibrosis and myocardial inflammation and oxidative stress in diabetic rats [52].HSYA administration to diabetic mice supplemented with STZ and a fat diet demonstrated antioxidant effects against DCM by regulating the balance between ROS and antioxidant enzymes [53].Oral administration of PH to db/db mice protected against DCM by altering proteins involved in cardiac lipid metabolism and myocardial mitochondria [54].PH was demonstrated to exhibit inhibitory effects on HG-induced fibrosis injury, cardiomyocyte oxidation, and hypertrophy in cardiac cells when treated at different concentrations, with no toxic effect observed.PH treatment also effectively prevented cardiomyocyte injury and suppressed cardiac oxidative stress/fibrosis in diabetic mice via the downregulation of Kelch-like ECH-associated protein 1/Nrf2 (Keap1/Nrf2) signaling pathways [55].Treatment with PH at different concentrations showed effective in vitro results in attenuating HG-induced inflammation, apoptotic death, and fibrotic response in cardiomyocytes and preventing cardiomyocyte injury, as well as inhibiting cardiomyocyte inflammatory response in STZ-induced diabetic mice.These effects were mediated by the amelioration of inflammation, fibrosis, and hypertrophy-related genes at the mRNA and protein levels via upregulating SIRT1 expression [56].

Diabetic Atherosclerosis
HSYA has exerted antioxidant and anti-ferroptosis effects in diabetic mice and human umbilical vein endothelial cells by reducing atherosclerotic plaque formation via the regulation of ferroptosis and oxidative stress-related markers [57].PH exerts a therapeutic effect against HG-induced endothelial cell dysfunction and decreases the acceleration of atherosclerosis in diabetic mice treated at a dose of 20 mg/kg through the upregulation of nitric oxide synthase (eNOS) and kruppel-like factor 2 (KLF2) expression [58].

Diabetic Vascular Injury
An in vitro experiment showed that HSYA at a concentration of 50 µM reduces highglucose-induced vascular injury by decreasing endothelial cell apoptosis, monocyte adhesion, vascular permeability, oxidative stress markers, and cell adhesion molecule (CAM) levels [59].PH has demonstrated the effects of alleviating high-glucose/AGE-induced vascular injury and the EndMT process in human umbilical vein endothelial cells following treatment at a nontoxic concentration of 40 µM through regulating the protein and mRNA levels of mesenchymal/endothelial markers.Furthermore, PH has the potential to reduce vascular fibrosis and endothelial damage in diabetic mice by downregulating the mRNA levels of TGFβ, intercellular adhesion molecule (ICAM), bone morphogenetic protein-2 (BMP2), and monocyte chemoattractant protein-1 (MCP1) in aorta tissues [60].
The mechanisms of natural CH treatment for T2DM microvascular complications is presented in Table 2.

Conclusions
T2DM is a disease characterized by HG and resulting from increased pancreatic β-cell dysfunction and IR.The literature has demonstrated that T2DM causes several micro-and macrovascular complications via multiple gene expression and signaling pathways.However, the specific mechanisms of natural CHs through modulating gene expression/signaling pathways and the protective role thereof in T2DM complications remain unclear and mostly unexplained.
Natural CHs, and ILTG, HSYA, and PH, in particular, act on multiple targets of macrovascular T2DM complications and regulate signaling pathways/genes, resulting in protection against DCM, DAS, and DVI.A few experiments in rat embryonic heart cells and diabetic rats have confirmed the potential of ILTG, HSYA, and PH in ameliorating HG-induced DCM through reducing oxidative stress, inflammatory/fibrotic responses, and apoptotic death [52][53][54][55][56].The limited evidence available in diabetic rats and human umbilical vein endothelial cells suggests the efficacy of HSYA and PH in ameliorating DAS and DVI-associated oxidative stress and ferroptosis [57][58][59][60].

Future Directions
Further experiments are warranted to explore the role of natural CHs in the treatment of T2DM complications through the regulation of multiple genes and/or signaling pathways involved in inflammation, oxidative stress, angiogenesis, apoptosis, and autophagy.Both in vivo and in vitro experiments demonstrated antidiabetic effects, but the ideal concentration in treatment still needs more investigation.
A few experiments indicate that ILTG and PH protect against DNE, DCM, and DVI, with no toxic effect observed.More experiments are needed to determine if natural CHs have any toxic effects on human/rat cells at concentrations that have shown efficacy in treating T2DM complications.The effects of natural CH-rich extracts from plants on T2DM complications need to be further analyzed in human and animal models.Proteomics, transcriptomics, and metabolomics are among the omics technologies targeting T2DM complications and are used as single techniques or are combined in experiments.There is a need for further experiments utilizing multi-omics technologies to determine the underlying cellular pathways and molecular genes of T2DM complications at different dimensions.Only a few human experiments suggest that natural CHs alleviate T2DM complications.Further human experimental and clinical trials are needed to explore the role of natural CHs as therapeutic agents in T2DM complications.

Table 1 .
Summary of experimental models focusing on natural CHs for the treatment of microvascular T2DM complications.

Table 2 .
Summary of experimental models focusing on natural CHs for the treatment of macrovascular T2DM complications.