**4. Discussion**

Various medical plants and natural products have been found to be antidiabetic agents, including banaba, fenugreek, and gymnema, among others. Among these plants, LB is a perennial deciduous shrub belonging to the Leguminosae family and has been used for treatment of inflammation throughout Asia. LB as a legume family contains phenolic compounds including many different types of flavonoid derivatives. The previous study reported by our group found that LBE contained polyphenolic compounds such as quercetin (0.853 mg/g), genistein (0.053 mg/g), daidzein (0.165 mg/g), and naringenin (0.08 mg/g) [24,30,31]. These four components known as major active compounds in Leguminosae have been shown to exert antioxidant effects by inhibiting AGEs formation [24]. Furthermore, quercetin and genistein in LB increased free amine contents, resulting in the increased breakage of AGEs and inhibition of AGE formation [24]. The findings support that the ameliorative effect of LBE against diabetes might be due to synergistic or additive effects of these active ingredients. In the current study, we hypothesized that LBE supplementation could ameliorate hyperglycemia-induced renal inflammation by regulation of NLRP3-inflammasome associated hyperinflammation in an in vivo diabetic model.

First of all, it is well-known that chronic hyperglycemia contributes to renal malfunction such as progression of elevated urinary albumin excretion [34,35]. HFDs induce insulin resistance, and potentially moderate pancreatic beta-cell dysfunction [36]. HbA1c is known to reflect average blood glucose within 3 months. A recent study reported that HbA1c level is a better indicator of hyperglycemia with fewer variables compared to overnight fasting blood glucose [37]. The current study demonstrated that LBE supplementation, regardless of dose, decreased the level of HbA1c in the diabetic mice. Previous studies have shown that decreased glucotoxicity caused by hyperglycemia improved renal function [31,38,39]. In the present study, a high dose of LBE supplementation declined the level of BUN and serum creatinine, and a low dose of LBE supplementation lowered the ACR. Elevated BUN, serum creatinine, and urine ACR level are well-known markers of renal malfunction with albuminuria [40]. Furthermore, the low dose of LBE supplementation attenuated albuminuria and the high dose of LBE supplementation minimized basement membrane thickening in diabetic kidney morphology. It can be inferred that LBE attenuated renal malfunction by regulating hyperglycemia accompanied by morphological alteration of diabetic kidney.

Hyperglycemia-induced oxidative stress results in the formation of AGEs and activation of protein kinase C (PKC). These changes are accelerated by mitochondrial overproduction of ROS in prolonged exposure to hyperglycemia. Activated renal RAGE stimulated production of cytosolic ROS, leading to mitochondrial ROS [41,42]. A previous study reported that LB acts as an AGE modulator by suppressing oxidative stress [30]. Furthermore, LBE ameliorated oxidative stress by reducing RAGE expression, which subsequently reduced AGE–RAGE interaction in MGO-induced renal glucotoxicity [31]. The current study demonstrated that the high dose of LBE supplementation reduced the renal protein level of RAGE in diabetic mice. Both LBE supplementations also suppressed the protein levels of 4-HNE and protein carbonyls. 4-HNE is a representative biomarker of lipid peroxidation, and has cytotoxic and mutagenic activities in various tissues [43,44]. ROS overproduction also leads to oxidative modification of proteins and produces protein carbonyls via lipid peroxidation or glycation/glycoxidation [45]. These data sugges<sup>t</sup> that LBE supplementation ameliorated AGE formation and ROS production by remarkably decreasing lipid peroxidation and protein oxidation. Furthermore, activation of Nrf2 corresponding with 4-HNE and protein carbonyls could serve as an illustration that oxidative stress induces the stimulation of Nrf2 and its related antioxidant defense systems via the compensatory activation against overproduction of ROS. Our results demonstrated that LBE supplementation regulated increased Nrf2 and its related antioxidant defense enzymes in the type 2 diabetic mice.

Moreover, ROS overproduction activates inflammatory response including NLRP3 inflammasome. NLRP3 inflammasome activates various pro-inflammatory cytokines including IL-Iβ, TNF-a, and IL-6. The pro-inflammatory cytokines can critically trigger inflammatory response in diabetic kidney [4,5]. Therefore, suppression of NLRP3 inflammasome could be a potential target of hyperglycemia-induced renal damage [4,5]. A previous study demonstrated that administration of polyphenols inhibited TNFα-associated pro-inflammatory cytokines [6]. Genistein, quercetin, daidzein, and naringenin in LBE are known to inhibit nuclear NF-κB translocation, along with decreases in iNOS and NO productions in vitro and in vivo [27]. In particular, inhibition of NLRP3 inflammasome could prevent facilitation of the progression of hyperglycemia-induced renal failure [5]. For the first time, the current study showed that the high dose of LBE supplementation suppressed NLRP3 inflammasome along with nuclear NF-κB activation. Indeed, LBE supplementation ameliorated inflammatory responses via suppression of NLRP3 inflammasome.

AMPK has some physiological e ffects on mitochondrial biogenesis and glucose metabolism. AMPK also ameliorates the NF-κB related inflammatory response through activation of SIRT1 and PGC-1 α [9,10]. SIRT1 suppresses NF-κB by deacetylation of p65 and decreases priming of NLRP3 protein [10]. One previous study suggested that SIRT1 decreased activation of NLRP3 inflammasome in vitro [10]. Furthermore, SIRT1 was found to preserve podocyte function by regulating claudin-1, which induces podocyte e ffacement and albuminuria [46,47]. Hence, the AMPK/SIRT1 signaling pathway could be a therapeutic target in diabetic kidney damage. Other studies sugges<sup>t</sup> that anthocyanin and resveratrol reduce albuminuria, glomerulosclerosis, and tubulointerstitial fibrosis in diabetic nephropathy through the activation of AMPK/SIRT1 signaling [20,21]. The current study showed that LBE supplementation activated SIRT1 and AMPK in diabetic kidney. It would be inferred that LBE supplementation has protective e ffects on hyperglycemia-induced renal damage by activation of AMPK/SIRT1 along with reduced NLRP3 inflammasome-associated hyper-inflammation in diabetes.

Various pathways accelerate transcription of nuclear NF-κB. Oxidative stress and downregulated AMPK/SIRT1 signaling pathway can directly and indirectly activate NF-κB in hyperglycemia. As mentioned before, our results showed that LBE supplementation has protective action against oxidative stress demonstrated by RAGE, protein carbonyls, and 4-HNE. In particular, the high dose of LBE supplementation was more e ffective on suppression of NLRP3 inflammasome in our study. However, the low dose of LBE supplementation accelerated the activation of AMPK/SIRT1 more than did the high dose of LBE supplementation in the diabetic condition. The lower protein level of NF-κB in both doses of LBE supplementation might be influenced by various pathways, including AMPK/SIRT1 and oxidative stress as well as NLRP3 inflammasome [48]. In our previous study, LBE supplementation alleviated hepatic inflammation by suppressing the activation of NF-κB. Collectively, LBE supplementation, particularly at a high dose, ameliorates renal inflammation in diabetes according to these findings.
