The endocrine function of the pancreas is performed by the islets, which are the center of controlling the dynamic balance of blood glucose in the body and are an essential endocrine organ in the pancreatic tissue. Among them, β-cell is the primary cell of the islets and mainly secrete insulin. If insulin production is insufficient, or if IR occurs, it may lead to elevated blood glucose. Puerarin acts on the following molecular mechanisms of the pancreas to lower blood glucose.

The skeletal muscle is the significant tissue of glucose metabolism, accounting for nearly 75% of the whole-body insulin-stimulated glucose uptake. IR in skeletal muscle is a critical component of the etiology of diabetes (Chen et al., 2018a). As the largest endocrine organ in the body, adipose tissue secretes various protein substances that regulate blood glucose metabolism and are essential regulators of human glucose homeostasis. Puerarin regulates blood glucose metabolism in skeletal muscle and adipose tissue through the following molecular mechanisms.

The pathophysiological mechanism of elevated blood glucose involves a variety of tissues and cells, of which the liver has the most closed relationship with type 2 diabetes. The liver plays a crucial role in glucose metabolism. The regulation of glucose production and storage by the liver is crucial for gluconeogenesis and glycogen synthesis. The most important source of endogenous glucose production in hepatic gluconeogenesis, a hallmark in type 2 diabetes patients (Liu et al., 2021).

The PI3K/Akt signaling pathway is considered the foremost signal transduction pathway and plays a significant regulatory role in gluconeogenesis. Glucose-6-phosphatase (G6pase) and phosphoenolpyruvate carboxykinase (PEPCK) are the pivotal rate-limiting enzymes in gluconeogenesis, and Foxo1 modulates insulin sensitivity. Under the condition of insulin resistance, PI3K/Akt activity is decreased, and hepatic gluconeogenesis is promoted due to increased PEPCK and G6pase expression driven by Foxo1 (Liu et al., 2021).

In vitro and in vivo experiments confirm, Puerarin could enhance the phosphorylation of Foxo1 by activating PI3K/Akt signaling pathway in liver tissues of type 2 diabetes rates and elevating the pFoxo1/Foxo1 protein and mRNA expressions, and further inhibiting the expression of G6pase and PEPCK. Thus hepatic gluconeogenesis and endogenous glucose production in the liver are suppressed. Furthermore, Puerarin improved IR (Shen et al., 2019; Liu et al., 2021). (Figure 3)

Moreover, in vitro, the protein kinase Bα2 (Akt1) is predicted to be a target protein of Puerarin. Puerarin targeted the PH domain of Akt1, inhibited Akt1’s transmembrane effect, and activated Akt1 to improve the phosphorylation or activity of downstream proteins, such as glycogen synthase kinase-3β (GSK-3β) and PDE3, and ultimately regulated glucose and lipid metabolism (Shen et al., 2019). (Figure 3)

Consequently, Puerarin inhibits hepatic gluconeogenesis by activating PI3K/Akt signaling pathway. The hypoglycemic mechanism of Pueraria acting on the liver is shown in Table 3.

Finally, Puerarin regulates systemic functions to lower blood sugar. In vitro and in vivo studies prove that the antidiabetic effects of Puerarin might be related to the inhibition of protein tyrosine phosphatase-1B.

(PTP1B), therefore, better the insulin signaling pathway and increased insulin receptor’s, achieving the purpose of moderating blood glucose levels by markedly boosting glucose uptake and escalating glucose tolerance (Sun et al., 2019). Moreover, Puerarin significantly increased plasma β-endorphin (β-EP) concentrations and reduced blood glucose levels in diabetic rats via within α1-adrenergic receptors (α1-AR) and adrenal medulla 1-adrenaline (1-A) activation (Chen et al., 2004). In addition, Puerarin decreased the level of blood glucose and aldose reductase activity in red blood cells, accordingly inhibiting the formation of glycation products and expression of AGE mRNA (Kim et al., 2006; Zhang et al., 2006; Zhang et al., 2009; Liu et al., 2018). See Table 4 for details.

Some studies discovered that reduction of NAD + profile was induced by hyperglycemia in the podocytes. Following the condition, the expression levels of SIRT1 and peroxisome proliferator-activated receptorγcoactivator-1α (PGC-1α) were decreased, and mitochondrial damage occurred (Xu et al., 2016). Puerarin reduced oxidative stress by activating SIRT1. SIRT1 serves as a regulator of Foxo1, Puerarin activated downstream pathway of Foxo1 through activating SIRT1, accordingly stimulated the synthesis of PGC-1α, subsequently elevated the anti-oxidant target gene MnSOD and catalase (CAT) by SIRT1/Foxo1 pathway, and driven the anti-oxidant effect, may lead to a reduction of ROS, meanwhile, down-regulation of IL-6, TNF-α in the kidney (Xu et al., 2016). On the other hand, Puerarin regulated NADPH oxidase 4 (NOX4) expression through the SIRT1-NF-κB pathway in podocytes. Puerarin decreased nuclear factor kappa-light-chain-enhancer of activated β-cells (NF-κB) activation through activating SIRT1, subsequently restrained release to proinflammatory cytokines, and decreased NOX4 expression, the primary enzyme contributing to the increased oxidative stress in podocytes among the different NADPH oxidase (NOX) isoforms, thus reduced oxidative stress (Xu et al., 2016; Li et al., 2017). It also acted directly on nuclear factor-kBp65 (NF-kBp65) and reduced tumor necrosis factor-α (TNF-α) expression by down-regulating NF-kBp65 expression in kidney tissue (Cui and Wang, 2010). Alternatively, it acted directly on intercellular cell adhesion molecule-1 (ICAM-1) and TNF-α and inhibited expressions of ICAM-1 and TNF-α, inhibited non-enzymatic glycosylation of proteins, and relieved oxidative stress inflammatory reaction damage (Pan et al., 2015).

On the other hand, transforming growth factor-β1 (TGF-β1) is one of the probiotic growth factors. A high blood glucose level can induce broad expression of the TGF-β1 gene and protein in the kidney (She et al., 2014). Excessive activation of the TGF-β1/Smad2 signal pathway results in the kidney’s extracellular matrix (ECM) accumulation. Therefore Puerarin exerted its anti-diabetic effect by inhibiting the TGF-β1/Smad2 signaling pathway and reducing the accumulation of extracellular matrix in the kidney (She et al., 2014). In addition, Puerarin could restrain the protein expressions of TGF-β1 and TGF-β1 receptors (TGF-β-RI) in the kidney tissue of KKAy mice via reducing the expression of α-smooth muscle actin (α-SMA) (Yi et al., 2013). It also inhibited the TLR4/MyD88/NF-kBp65 pathway by up-regulating miRNA-140-5p, thus reducing expression levels of TNF-α, interleukin-1β (IL-1β), interleukin-6 (IL-6), interferon-γ (INF-γ), and TGF-β1 in renal tissues of diabetic rats (Xu X et al., 2020). These changes may inhibit and reverse the epithelial-mesenchymal transition process, thus delaying the occurrence, preventing diabetes-induced renal damage and the development of DN.

Moreover, Puerarin can promote autophagy mechanisms through the following two signaling pathways. Puerarin modulated ERS/autophagy crosstalk by regulating activated extracellular signal-regulated kinase (p-ERK), persistent ERS activated the p-ERK-eukaryotic translation initiation factor 2α (eIF2α) signaling pathway, activating transcription factor 4 (ATF4) is then up-regulated in response to eIF2α phosphorylation. CHOP and Beclin-1 were activated through regulating of the PERK/eIF2α/ATF4 signaling pathway, up-regulated the levels of autophagy markers Beclin-1, microtubule-associated protein light chain 3 II (LC3II), and autophagy-related 5 homolog (Atg5), and down-regulated the level of p62, thus resulted in the autophagy response (Xu Y et al., 2020). It also protected against podocyte injury by restoring the autophagic activity via the AMPK/SIRT1 pathway. In vitro and in vivo experiments demonstrate, Puerarin stimulated SIRT1 expression in podocytes to deacetylate liver kinase B1 (LKB1) and then phosphorylated AMPK-mTOR pathway to induce autophagy (Li et al., 2020).

Advanced glycation end products (AGEs) and RAGE play an essential role in developing diabetic nephropathy. Puerarin could protect the renal tissue from the impairment of hyperglycemia and AGE by decreasing AGEs contents and inhibiting the expression of RAGE mRNA in the kidney, which may due to decrease blood glucose directly, or reduce AGEs formation by inhibiting oxidative stress, aldose reductase activity, and, so on (Shen et al., 2009). Furthermore, Puerarin also attenuates eNOS expression in glomerular endothelial cells (Zhang et al., 2015).

Together, Puerarin can achieve inhibitory non-enzymatic glycosylation of the protein, alleviate oxidative stress or inflammatory response damage, and promote autophagy response through the above pathways, thus protecting diabetes-induced kidney damage and delaying the occurrence and development of DN.

Hyperglycemia could cause a non-classic inflammation response in the vascular endothelium and contributes to the inflammation response through the aggregation of intracellular ROS (Lian et al., 2019). So there is a correlation between DM and the occurrence of oxidative stress products and inflammatory factors in a high-glucose environment. Puerarin inhibits the occurrence of oxidative stress and inflammatory response mainly through the following signaling pathways.

ROS in vascular the primary source is the NADPH oxidase (NOX family). ROS is a critical upstream activator of the NF-kB pathway (Li et al., 2016), which consequently increases the expressions of lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1), ICAM-1, and E-selectin, and Nlrp3 inflammasome activation (Li et al., 2016). High mobility group box 1 (HMGB1) release is a downstream product of Nlrp3 inflammasome activation (Lian et al., 2019). Thus, NF-kB, the critical transcription factor in regulating molecular adhesion expression, plays an essential role in DM. Puerarin inhibited NADPH oxidase 2 (NOX2) and NOX4 expressions in vascular smooth muscle cells (VSMCs) through regulating NF-kBp65 to suppress oxidative stress and expressions of cell adhesion molecules (Li et al., 2016). It also attenuated IKKb phosphorylation and effectively blocked NF-kB activation by inhibiting NF-kBp65 phosphorylation and decreased TNF-α and IL-6 production by inhibiting IKKb/NF-kB activation (Huang et al., 2012). In addition, this is a new protection mechanism of Puerarin that inhibited NF-kB activation by suppressing oxidative stress, subsequently inhibited Nlrp3 inflammasome activation, attenuated TXNIP-NLRP3 binding, decreased subsequent Caspase-1 activation, and decreased the release of HMGB1 (Lian et al., 2019).

Moreover, Puerarin also stimulated endothelial nitric oxide synthase (eNOS) phosphorylation and nitric oxide (NO) production via activation of an estrogen receptor-mediated PI3K/Akt and CaMKII/AMPK dependent pathway. It attenuated phosphorylation of IRS-1 at S307 and effectively ameliorated the tyrosine phosphorylation of IRS-1, which activated PI3K, subsequently activated PI3K phosphorylates and activated downstream target Akt which directly phosphorylated eNOS at Ser1177, leading to increased production of NO(Hwang et al., 2011). (Figure 3)

In vitro, Puerarin disrupted the phosphorylation and membrane translocation of PKCβ2 as well as Rac1, p47phox, and p67phox subunits and NADPH oxidase activation in VSMCs may exert inhibitory effects on high-glucose-induced VSMC proliferation via interfering with PKCβ2/Rac1-dependent ROS pathways (Zhu et al., 2010).

In summary, Puerarin inhibited inflammatory response and oxidative stress via inhibiting NF-kB activation, and it inhibited oxidative stress via ameliorating PI3K/Akt, CaMKII/AMPK, and PKCβ2/Rac1 pathway. Accordingly, Puerarin ameliorated IR-associated endothelial dysfunction.

DR is an alteration of diabetic microangiopathy in the specific environment of the fundus. Long-term chronic hyperglycemia causes oxidative stress, inflammatory response, and non-enzymatic glycosylation of proteins, which promotes apoptosis and accelerates the onset of DR. Puerarin protects retinal function through the following pathways.

In response to high glucose toxicity, the activity of ROS is increased, causing phosphorylation of retinal capillary endothelial cells in JAK2/STAT3, thereby increasing the expression of vascular endothelial growth factor (VEGF). Thus, the mechanism of the Puerarin effect is hypothesized to be due to inhibition of the phosphorylation of JAK2/STAT3, thereby reducing the expression of VEGF and the inflammation of the retina, and that results in preventing the occurrence of DR (Cai et al., 2017). Furthermore, Puerarin decreased the expression levels of retinal vascular endothelial growth factor (VEGF) and IL-1β through activating nuclear factor-E2 related factor2 (Nrf2)/HO-1 signaling pathway (Zhang and Li, 2019) and inhibiting the Nrf2/ERK signaling pathway (Zhang and Wang, 2019), thus, reduced the inflammatory response and inhibiting oxidative stress of retinal tissue.

The Fas/FasL system is considered the primary signal transduction pathway to mediate apoptosis, and it may affect and strengthen the apoptosis process mediated by ONOO- (Hao et al., 2010; Hao et al., 2012). Thus, Puerarin could protect retinal pigment epithelial cells (RPE) of diabetic rats through inhibiting inducible nitric oxide synthase (iNOS) up-regulation and ONOO-generation mediated through Fas/FasL signal pathway (Hao et al., 2010; Hao et al., 2012).

The prolonged hyperglycemic state can induce the production of AGEs. Puerarin inhibited the productions of AGEs-modified proteins and their accumulation in the retina by inhibiting the non-enzymatic glycosylation of proteins to improve DR. Components of flavonoid structures can capture dicarbonyl compounds (key intermediates in AGEs formation) and form adducts, thus exerting an inhibitory effect on AGEs formation, and it is speculated that the mechanism of action of total flavonoid extracts of Pueraria to inhibit AGEs levels may be related to this (Liu et al., 2018; Deng et al., 2021).

In addition, Puerarin raised the expression of insulin-like growth factor-1 (IGF-1) and decreased the expressions of TNF-α, ICAM-1, IL-1β, and IL-6 in the retina to protect the function of the retina (Li, 2007; Yang et al., 2016b; Deng et al., 2021).

In conclusion, Puerarin could protect RPE cells and the function of the retina by reducing the inflammatory response, inhibiting oxidative stress and apoptosis of retinal tissue, inhibiting nonenzymatic glycosylation reactions of the proteins.

Chronic hyperglycemic environments promote oxidative stress and the release of inflammatory factors through processes such as glycosylation, thus damaging the myocardium. Puerarin may reduce myocardial damage through the following pathways.

On the one hand, Puerarin increased Bcl-2 expression in the myocardium, afterward suppressed permeabilization of the mitochondrial inner membrane to Cyt-C, subsequently reduced the release of Cyt-C, inhibited the formation of apoptotic bodies, subsequently regulated activation of the Caspase-3, are suggested to be the mechanisms responsible for puerarin’s anti-apoptotic effect against diabetic cardiomyopathy in STZ induced SD rats (Ling et al., 2011). (Figure 2)

Moreover, it also reduced RAGE expression at the mRNA level by reducing blood sugar, subsequently reduced AGE-RAGE binding, reduced the myocardial oxidative stress and the inflammatory response. Thus injury was relieved by AGEs in diabetic rats (Ye et al., 2013).

For another, AT1R mediates almost all cardiovascular effects of angiotensin-II (Ang-II). Puerarin decreased myocardial AT1R protein expression and reduced cardiac Ang-II levels in diabetic rats. On the one hand, it inhibited renin-angiotensin system (RAS) activation and suppressed cardiomyocyte hypertrophy and fibrosis, thus improving cardiac systolic and diastolic functions. On the one hand, it inhibited TNF-α and IL-1β expressions, suppressed Caspase-9 activation, inhibited inflammatory factor release and cardiomyocyte apoptosis, thereby protecting the myocardium. In addition, Puerarin could improve IR by decreasing Ang-II levels (Zhao et al., 2008; Chen et al., 2013; Gao et al., 2017).

DPN is one of the most prevalent and debilitating microvascular complications of diabetes, affecting at least 50% of people with diabetes (Xue et al., 2017). Schwann cells (SCs) are the most critical myelinating cells of the peripheral nervous system. Hyperglycemia-induced abnormalities of SCs could cause a cause of demyelination of nerve fibers, reduction of regeneration capability in peripheral nerves, and axonal atrophy, resulting in the development of DPN(Xue et al., 2017). Therefore, to SCs, Puerarin significantly inhibited glucose fluctuation-stimulated ROS production and mitochondrial depolarization in SCs, subsequently suppressed mitochondrial dysfunction, down-regulated the expression of proapoptotic factors (e.g., Bax), and up-regulated the expression of anti-apoptotic factors (e.g., Bcl-2), which subsequently suppressed Caspase-3 activation and PARP cleavage in SCs, thus inhibited SCs apoptosis (Xue et al., 2017). In addition, Puerarin activated T-type Ca2+ channel and mitogen-activated protein kinases (MAPK) signaling by up-regulating NO level, which up-regulated the expression of the calcitonin gene-related peptide (CGRP) gene to enhance the antagonistic effect of CGRP on endothelin-1 (ET-1), improved endure blood supply (Chen et al., 2010b). The anti-inflammation effect of Puerarin might be related to the suppression of spinal NF-kB activation or cytokines upregulation (Liu M et al., 2014).

Moreover, Puerarin significantly inhibited the proinflammatory response and oxidative stress in the cerebral cortex and hippocampus by activating PI3K/Akt signaling pathway (Hao et al., 2019) and inhibiting NF-kB activation (Liu et al., 2016), which determines its cognitive protection in diabetes (Yang et al., 2015).