**3. Results**

#### *3.1. E*ff*ect of LBE Supplementation on Body Weight, Food Intake, Fasting Blood Glucose (FBG), and Glycated Hemoglobin (HbA1c) in T2DM Mice*

The body weight of the DMC group was significantly increased compared to those of the NC group (Table 1). Simultaneously, there was no e ffect on body weight change among the DM groups. FBG of the DMC group showed significant elevation compared to that of the NC group, but the LL group showed lower FBG compared to the DMC group (Table 1). Levels of HbA1c in the DMC group

were significantly higher than those in the NC group. However, the LL and the HL groups showed lower levels of HbA1c compared to the DMC group.


**Table 1.** Effect of *Lespedeza bicolor* extract (LBE) supplementation on body weight, food intake, kidney weight, fasting blood glucose level (FBG), and hemoglobin A1c (HbA1c) in T2DM mice.

All values are means ± SD. \* *p* < 0.05 compared with the normal control (NC) group; # *p* < 0.05 compared with the diabetic control (DMC) group. NC, normal mice (negative control); DMC, T2DM mice (positive control); LL, T2DM mice supplemented with low dose (100 mg/kg/day) of LBE; HL, T2DM mice supplemented with high dose (250 mg/kg/day) of LBE.

#### *3.2. E*ff*ect of LBE Supplementation on Renal Function and Renal Morphology in T2DM Mice*

The ACR in the DMC group was significantly higher than that in the NC group over the whole experiment period (Figure 1A). However, the ACR in the LL group was significantly lower than that in the DMC group at the mid and late stages of the experiment. Plasma creatinine and BUN were significantly higher in the DMC group compared to the NC group. At the same time, a high dose of LBE supplementation significantly decreased the level of plasma creatinine and BUN in the diabetic mice.

In the NC group, capsular space was observed as a thin white line (Figure 1C). Capsular space of the DMC group was thickened compared to that of the NC group. However, a high dose of LBE supplementation improved corpuscular architecture and tubular necrosis compared to the DMC group.

**Figure 1.** Effect of LBE supplementation on renal function and morphology in T2DM mice. (**A**) Urine albumin/creatinine ratio (ACR) during experiment period, (**B**) plasma creatinine and blood urea nitrogen (BUN), (**C**) kidney morphology (magnification ×400), and glomeruli size. \* *p* < 0.05 compared with NC group; # *p* < 0.05 compared with the DMC group.

*3.3. E*ff*ect of LBE Supplementation on Renal Receptor for Advanced Glycation end Products (RAGE) Formation in T2DM Mice*

The protein level of RAGE was significantly elevated in the DMC group compared to that of the NC group (Figure 2). The protein level of RAGE was significantly reduced in the LL group compared to that of the DMC group.

**Figure 2.** Effect of LBE supplementation on receptor for advanced glycation end products (RAGE) in T2DM mice. A representative band image of repeated experiments is shown in the left panel. \* *p* < 0.05 compared with NC group; # *p* < 0.05 compared with the DMC group.

#### *3.4. E*ff*ect of LBE Supplementation on Renal Inflammation in T2DM Mice*

The protein levels of NLRP3, ASC, procaspase-1, caspase-1, pro IL-1β, and mature IL-1β were significantly elevated in the DMC group compared to those of the NC group (Figure 3A). However, the protein levels of NLRP3, procaspase-1, caspase-1, pro IL-1β, and mature IL-1β showed significant reduction in the HL group compared to those of the DMC group. There was no significant difference of the protein level of ASC among the DMC group and LBE-supplemented groups.

**Figure 3.** Effect of LBE supplementation on renal NLRP3 inflammasome and inflammation in T2DM mice. Protein levels of (**A**) nucleotide-binding oligomerization domain-like pyrin domain containing receptor 3 (NLRP3) inflammasome: nucleotide-binding oligomerization domain-like pyrin domain containing receptor 3 (NLRP-3); apoptosis-associated speck-like proteins including caspase recruitment domain (ASC), caspase-1, and interleukin (IL)-1β; and (**B**) markers of pro-inflammatory response: monocyte chemoattractant protein-1 (MCP-1) and intercellular adhesion molecule 1 (ICAM-1); and nuclear factor kappa B (NF-κB)-related inflammatory response: nuclear factor kappa B (NF-κB), phosphorylated IκB (p-IκB), tumor necrosis factor-α (TNF-α), interleukin (IL)-6, and inducible nitric oxide synthase (iNOS); representative band images of each marker are shown in the left panel. \* *p* < 0.05 compared with NC group; # *p* < 0.05 compared with the DMC group.

The DMC group showed greater levels of the protein related to inflammation including MCP-1 and CRP than the NC group (Figure 3B). The DMC group also showed higher protein levels of nuclear NF-κB, phosphorylated IκB, ICAM-1, TNF-<sup>α</sup>, IL-6, and inducible nitric oxide synthase (iNOS) than the NC group. Simultaneously, the protein levels of MCP-1, nuclear NF-κB, phosphorylated IκB, ICAM-1, and iNOS significantly decreased in both LBE-supplemented groups compared to the DMC group. The protein levels of TNF-α and IL-6 were significantly lowered in the LL group compared to the DMC group.

#### *3.5. E*ff*ect of LBE Supplementation on Renal Oxidative Stress in T2DM Mice*

The renal protein level of 4-hydroxynonenal (4-HNE) was significantly higher in the DMC group than that of the NC group (Figure 4A). Simultaneously, the protein level of 4-HNE was significantly reduced in the LBE-supplemented groups compared with that in the DMC group. The level of renal protein carbonyls was significantly increased in the DMC group compared to that in the NC group. However, the level of renal protein carbonyls was significantly decreased by LBE supplementation in the DM group. The protein levels of nuclear Nrf2 (nuclear factor erythroid 2-related factor 2) and cytosolic heme oxygenase-1 (HO-1), NAD(P)H dinucleotide phosphate dehydrogenase quinone 1 (NQO1), catalase, and manganese superoxide dismutase (MnSOD) were significantly higher in the DMC group compared to those in the NC group (Figure 4B). The protein levels of Nrf2 and NQO1 in the LBE supplementation groups were significantly lowered compared to those in the DMC group. The protein levels of HO-1 and catalase in the high dose of LBE supplementation groups were significantly lowered compared to those in the DMC group. The protein level of MnSOD was significantly reduced in the LL group compared to the DMC group.

**Figure 4.** Effect of LBE supplementation on renal oxidative stress in T2DM mice. Representative band images of (**A**) 4-hydroxynonenal (4-HNE) and protein carbonyl groups and (**B**) nuclear factor erythroid 2-related factor 2 (Nrf2)-associated antioxidant defense markers: heme oxygenase-1 (HO-1), NAD(P)H dehydrogenase quinone 1 (NQO1), catalase, and manganese superoxide dismutase (SOD) are shown. \* *p* < 0.05 compared with NC group; # *p* < 0.05 compared with the DMC group.

#### *3.6. E*ff*ect of LBE Supplementation on AMPK Phosphorylation and SIRT1 in T2DM Mice*

The protein level of AMPK was significantly decreased in the DMC group compared to the NC group and was increased in the LL and the HL groups compared to that of the DMC group (Figure 5A). At the same time, the protein level of phospho adenosine monophosphate kinase (pAMPK) was significantly higher in the LL group compared to that of the DMC group. Consequently, the AMPK phosphorylation ratio (pAMPK/AMPK) was significantly increased in the LL group compared to that of the DMC group.

The renal protein levels of SIRT1 and peroxisome proliferator-activated receptor gamma -coactivator α (PGC1α) were significantly lower in the DMC group than those of the NC group (Figure 5B). Simultaneously, the protein level of SIRT1 was significantly increased in the LL group compared to that of the DMC group. The protein levels of PGC1α were significantly decreased in the DMC group compared to those of the NC group. However, there was no significant difference of the protein levels of PGC1α in the LBE supplementation groups compared to the DMC group.

**Figure 5.** Effect of LBE supplementation on renal adenosine monophosphate kinase (AMPK) phosphorylation and Sirtuin (SIRT)-1/ peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α) signaling in T2DM mice. (**A**) Renal AMPK phosphorylation and (**B**) SIRT1-PGC1 activation. Representative band images of each marker are shown in the left panel. \* *p* < 0.05 compared with NC group; # *p* < 0.05 compared with the DMC group.
