**1. Introduction**

Diabetes is a critical metabolic syndrome associated with aberrant glucose metabolism, and cause chronic damage and dysfunction of various organs, such as blood vessels, heart, nerves, eyes, liver, and kidneys. In particular, hyperglycemia-induced renal inflammation can develop chronic lesions with histological and functional defects in kidney [1,2].

Hyperglycemia results in overproduction of reactive oxygen species (ROS), which cause oxidative stress in various organs in cases of diabetes [3,4]. Oxidative stress due to uncontrolled blood glucose leads to activation of the nucleotide-binding oligomerization domain-like pyrin domain containing receptor 3 (NLRP3) [5]. Recent studies have shown that activation of NLRP3 inflammasome in renal cells promotes renal inflammation and contributes to chronic kidney damage [6,7]. NLRP3 inflammasome

acts as the molecular sensor that responds to dangers such as pathogen-associated molecular patterns (PAMPs) and danger-associated molecular patterns (DAMPs) [6]. In sensing those dangers, NLRP3 can recruit the apoptosis-associated speck-like proteins including caspase recruitment domain (ASC) and pro-caspase-1 [6]. Activated NLRP3 inflammasome allows the activation of interleukin (IL)-1β by cleaved caspase-1, and is involved in inflammatory response [6].

Pro-inflammatory cytokines include tumor necrosis factor-α (TNF-α), which is activated by free radicals involved in proinflammatory signals by binding to TNF-α receptors on tubular cell surfaces [8]. These responses trigger activation of nuclear factor kappa B (NF-κB) by particularly encouraging the phosphorylated IκB (p-IκB), which allows nuclear translocation of NF-κB [9]. NF-κB activation induces inflammatory cytokines including IL-6 and IL-1β, and maximizes inflammatory response [10]. Chemokines including monocyte chemoattractant protein-1 (MCP-1) are also involved in inflammatory response by recruiting active components of inflammatory cells and adhesion molecules including intercellular adhesion molecule 1 (ICAM-1) [11]. The inflammatory mediators are involved in the attachment of leukocytes, which can release proteolytic enzymes leading to renal damage [11,12]. The various changes promote the loss of function, viability, and harmful mutations. Eventually, excessive oxidative stress and chronic inflammation accelerate radical-mediated damage, resulting in cell degradation and renal damage [13,14]. Hence, suppressing the activation of NLRP3 inflammasome and subsequent hyper-inflammatory response would be a therapeutic target strategy for ameliorating renal damage [15–18].

The adenosine monophosphatekinase (AMPK)/Sirtuin 1 (SIRT1)/ peroxisome proliferator-activated receptor gamma coactivator α (PGC-1α) signaling pathway is also related to renal damage [19]. SIRT1 is known to protect pathogenesis of diabetic nephropathy (DN) along with regulation of mitochondrial biogenesis [19]. In diabetes, p65 acetylation accelerates the transcription activity of the NF-κB complex [10]. However, SIRT1 interacts with the p65 subunit of the NF-κB complex, deacetylates p65, and consequently suppresses NF-κB activation [20]. Previous studies have focused on the fact that SIRT1 suppresses NLRP3 inflammasome activation [20,21]. Moreover, SIRT1 was found to ameliorate podocyte loss and albuminuria by suppressing the expression of claudin-1 in podocytes [20]. SIRT1 also prevented hyperglycema-induced mesangial expansion by intensifying the AMPK-mammalian target of rapamycin (mTOR) signaling pathway [22]. In addition, PGC-1<sup>α</sup>, a downstream molecule of AMPK/SIRT1 signaling pathway, suppressed ROS overexpression and renal hyper-inflammation [23]. Hence, activation of the AMPK/SIRT1/PGC-1α pathway could be a possible mechanism associated with the therapeutic approach for hyperglycemia-induced renal damage.

*Lespedeza bicolor* (LB), named by American botanist Asa Gray, is a species of warm-season perennial deciduous shrub, which belongs to the genus *Lespedeza* (Leguminosae), and widely grows in the United States, Asia, and Australia [24]. LB has been used traditionally for the treatment of inflammation of the urinary tract, nephritis, and diabetes [25,26]. Recently, some studies have reported that natural compounds have therapeutic effects on various organ damages [27]. LB also contains many compounds such as genistein, quercetin, daidzein, catechin, rutin, luteolin, and naringin [28]. These natural phytochemicals in *Lespedeza bicolor* extract (LBE) have been determined for their antioxidant and anti-inflammatory activities, as well as their blood glucose lowering effect in hyperglycemia [27,28]. In particular, various polyphenols such as genistein, quercetin, and naringin have an antioxidant function—electron donating and ROS scavenging activity. Our previous study showed that LBE attenuated advanced glycation end product (AGE) formation and breakage in addition to endothelial dysfunction, which was triggered by methylglyoxal-induced glucotoxicity in vitro [29,30]. Furthermore, LB attenuated methylglyoxal (MGO)-induced diabetic renal damage in vitro and in vivo [31]. However, effects of LBE on NLRP3 inflammasome-associated hyperinflammation and AMPK/SIRT1/PGC-1α signaling under hyperglycemic condition have not ye<sup>t</sup> been revealed. Therefore, we investigated whether LBE has ameliorating effects on renal damage by suppressing NLRP3 inflammasome-related hyperinflammation and activating AMPK/SIRT1/PGC-1α signaling in type 2 diabetes mellitus (T2DM) mice.
