*3.4. PRHE Attenuates Diabetes-Induced Renal Oxidative Stress and Mitochondrial Dysfunction*

A significant increase in MDA but a decrease in GSH, SOD, and GPx were detected in the kidney tissues of diabetic rats (Figure 4). Treatment with PRHE and metformin and the combination regimen showed comparative efficacy to significantly attenuate these alterations. Diabetic rats also demonstrated a significant increase in mitochondrial ROS production, a decrease in mitochondrial membrane potential, and a swelling of the mitochondria (Figure 5). Supplementation of PRHE to the diabetic rats significantly restored all the changes. Metformin alone or in combination with PRHE showed similar results to PRHE treatment alone.

**Figure 4.** Effects of purple rice husk extract (PRHE) and metformin on renal oxidative stress indexes. (**a**) malondialdehyde; (**b**) reduced glutathione; (**c**) superoxide dismutase; (**d**) glutathione peroxidase. Values are the mean ± SD (*n* = 6). NDV: vehicle-treated normal group; DMV: vehicletreated diabetic group; DMM: metformin-treated diabetic group; DME: PRHE-treated diabetic group; DMME: metformin plus PRHE-treated diabetic group; KW: kidney weight. \* *p* < 0.05 vs. NDV, † *p* < 0.05 vs. DMV.

**Figure 5.** Effects of purple rice husk extract (PRHE) and metformin on kidney mitochondrial function. (**a**) ROS production; (**b**) membrane potential change; (**c**) mitochondrial swelling. Values are the mean ± SD (*n* = 6). NDV: vehicle-treated normal group; DMV: vehicle-treated diabetic group; DMM: metformin-treated diabetic group; DME: PRHE-treated diabetic group; DMME: metformin plus PRHE-treated diabetic group. \* *p* < 0.05 vs. NDV, † *p* < 0.05 vs. DMV.

#### *3.5. PRHE Modifies PGC-1α-SIRT3-Ac-SOD2-SOD2 Signaling Transduction*

To determine the signaling pathway involved in the benefits of PRHE on diabetic kidney injury, the protein expression of PGC-1α, SIRT3, Ac-SOD2, and SOD2 was determined. In the DMV group, the expression levels of PGC-1α, SIRT3, and SOD2 were significantly decreased compared to the NDV group, while the expression of Ac-SOD2

was significantly enhanced (Figure 6). These alterations were significantly reversed upon treated diabetic rats with PRHE. Similar results were observed in metformin monotherapy and in combination with PRHE.

**Figure 6.** Effects of purple rice husk extract (PRHE) and metformin on renal cortical expression of (**a**) Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α); (**b**) sirtuin 3 (SIRT3); (**c**) acetylated superoxide dismutase 2 (Ac-SOD2); (**d**) superoxide dismutase 2 (SOD2) and β-actin as a reference control. Values are the mean ± SD (*n* = 3). NDV: vehicle-treated normal group; DMV: vehicle-treated diabetic group; DMM: metformin-treated diabetic group; DME: PRHE-treated diabetic group; DMME: metformin plus PRHE-treated diabetic group. \* *p* < 0.05 vs. NDV, † *p* < 0.05 vs. DMV.

#### **4. Discussion**

This study reveals the protection against nephropathy in T2DM by PRHE through antioxidant abilities and mitochondrial protection, possibly via its major phytochemicals, particularly PCA and C3G. Results suggest that modifications of PGC-1α/SIRT3/SOD2 signaling are linked to this protection.

A combination of a high-fat diet and streptozotocin injection was used to develop a rat model of T2DM in the present study. Feeding rats with a high-fat diet promotes the development of obesity and insulin resistance, while injection of streptozotocin selectively destroys pancreatic β-cells and, thus, impairs insulin secretion [20]. This model has been shown to replicate the natural history and metabolic characteristics of humans and is also suitable for pharmacological screening [20]. However, the reliability and appropriateness of the model depend on the number of pancreatic β-cells remaining, which is largely regulated by the dose of streptozotocin injection. A high dose of streptozotocin has been shown to critically damage pancreatic β-cell function, leading to insulin deficiency, which is considered to resemble T1DM rather than T2DM [23]. Regarding our model, hyperglycemia, hyperinsulinemia, insulin resistance (presented by increased HOMA-IR), and lipid abnormalities were all detected in the DMV group. These metabolic alterations are compatible with the well-recognized characteristics of T2DM, thus confirming the successful T2DM induction in the current study. We did not quantify the number of functioning pancreatic β-cells. However, the observations of hyperglycemia together with hyperinsulinemia in our diabetic rats implied that the number of intact pancreatic β-cells was sufficient to increase insulin secretion in response to high glucose, even though it could not overcome a progressive decline in insulin action caused by diabetes. Additionally, the degree of hyperglycemia and the dose of streptozotocin (35 mg/kg) used in our study corresponded to a previous report showing that streptozotocin at the dose of 30–40 mg/kg has a moderate effect on plasma glucose and on the islets of Langerhans at a cellular level [24]. Taken together, all evidence points towards the development of T2DM in the

present study. Most importantly, our diabetic model also developed diabetic kidney injury, as indicated by accumulation of blood urea nitrogen and creatinine, reduced creatinine clearance, microalbuminuria, and histopathology that characterize diabetic nephropathy.

We found that diabetes-induced metabolic alterations were significantly diminished in the diabetic group given PRHE, suggesting the glucose-lowering ability of PRHE. To our knowledge, this is the first evidence showing antihyperglycemic potential in T2DM from the husk of purple rice, as a report is available only on its bran [25]. PCA and C3G, the main phytochemicals, may play a role in this action of PRHE. Previous publications showed the potential of PCA and C3G in reducing blood glucose, enhancing insulin sensitivity, and increasing glucose uptake, thereby improving glucose homeostasis in diabetes [26–28]. Studies also indicated that increased expression and translocation to the cell membrane of glucose transporters 1 (GLUT1) and 4 (GLUT4) contributed to the improvement of glucose tolerance by PCA and C3G [16–18]. The effectiveness on glycemic control by PCA and C3G was found to be comparable to metformin, a gold-standard antidiabetic drug, and was associated with the activation of AMPK [16]. Interestingly, our results also demonstrated a similar efficacy on metabolic improvement between PRHE, metformin, and PRHE+metformin. Metformin is an AMPK activator, and AMPK activation is recognized as the initial process for metabolic control by metformin [26]. It may be that PRHE (via PCA and C3G) stimulated AMPK and exerted its metabolic control through the same downstream signaling pathways as metformin; thus, no additional benefits were found in the combination therapy. Further study using cell culture experiments or the knockout mice model with an AMPK inhibitor or activator will be helpful to validate this possibility.

Our results showed that PRHE effectively protected against diabetic kidney injury. Because metabolic impairment was improved after PRHE treatment, it is likely that renal protection may result from glycemic control by PRHE. However, PRHE (particularly through PCA) may exert its benefits independent of glycemic control. This suggestion was based on a study using human mesangial cells exposed to high glucose in the presence or absence of PCA, which demonstrated that PCA was able to suppress mesangial cell proliferation in a dose-dependent manner by decreasing the levels of protein expression involved in mesangial expansion including type IV collagen, laminin, and fibronectin [29]. This in vitro evidence reinforces the potential direct action of PRHE at the kidney level in diabetes.

Studies have shown that oxidative stress plays an independent role in the development, progression, and severity of diabetic nephropathy [16,30]. ROS can damage renal cells by oxidizing membrane phospholipids, proteins, carbohydrates, and nucleic acids. ROS are also secondary messengers that activate many signaling cascades, leading to cell damage and deterioration of kidney function in diabetic kidney disease [16,30]. Mitochondria are well recognized as a major source of ROS production. There is evidence linking obesity and diabetes with oxidative stress and mitochondrial dysfunction [30]. The kidneys have high energy demand and are rich in mitochondria, thus mitochondrial dysfunction could contribute to renal oxidative cell injury and consequently renal functional and structural impairments. In line with this view, we detected mitochondrial damage in diabetic rats, as shown by increased ROS production, a dissipation of membrane potential, and a swollen and abnormal mitochondrial ultrastructure. Significant increases in the renal tissue levels of malondialdehyde (a lipid peroxidation product) and decreases in antioxidants (GSH, SOD, GPx) were also observed, indicating renal oxidative injury. These alterations were markedly diminished by PRHE, which is most likely mediated through the mitochondrial protective properties of PCA and C3G. PCA has been demonstrated to protect cardiac and brain mitochondrial dysfunction in streptozotocin-induced diabetic rats [31,32]. The protective role of C3G by modulating intracellular signaling and maintaining mitochondrial function, rather than acting solely as an antioxidant, has also been reported in cardiac ischemia-reperfusion injury [33]. Studies using HK-2 cells further demonstrated that C3G inhibited high glucose-induced ROS production, prevented mitochondrial membrane potential loss, and protected against mitochondrial dysfunction-

mediated cell death [34]. Taken together, it is proposed that treatment with PRHE preserves mitochondrial integrity, decreases oxidative stress, and, finally, leads to the protection of diabetic kidney injury. It is interesting to mention here that there was no greater production of mitochondrial ROS (including the kidney tissue levels of MDA) in the metformin-treated diabetic group, though blood glucose level was slightly higher than normal. Beyond its well-known blood glucose regulatory action, metformin has been confirmed to possess antioxidant properties [35]. This action of metformin is mediated through activation of AMPK secondary to its inhibition on the mitochondrial respiratory chain complex 1, the major site of ROS generation in mitochondria [35].

SIRT3, a major mitochondrial deacetylase, was recently highlighted as a novel regulator of mitochondrial function and redox homeostasis [13]. SIRT3 plays a key role in deacetylating and modifying the enzymatic activities of several mitochondrial proteins, including SOD2 [36]. SOD2 is the main antioxidant enzyme in the mitochondria that is responsible for scavenging the superoxide anion, a byproduct of the mitochondrial electron transport chain. This function allows SOD2 to clear mitochondrial ROS, maintain mitochondrial oxidative equilibrium, and confer protection against mitochondrial injury and death [37]. Evidence has suggested that the deacetylation of SOD2 by SIRT3 is necessary for the activation of SOD2, and this deacetylase activity of SIRT3 is further modulated by PGC-1α [13].

Herein, we detected a significant reduction in the expression of PGC-1α and SIRT3 in parallel with the increased Ac-SOD2 and decreased SOD2 expression in diabetic rats. Importantly, PRHE supplementation to the diabetes group was able to normalize these changes. This suggests that the protection against mitochondrial dysfunction and redox imbalance in diabetic nephropathy by PRHE may be a result of the modification of PGC-1α-SIRT3-SOD2 signaling transduction. Consistent with our results, an increased expression ratio of Ac-SOD2/SOD2 followed by increased mitochondrial oxidative stress and mitochondrial dysfunction were observed in Zucker diabetic fatty rats upon reduction of SIRT3 activity [38]. A study of SIRT3 knockout mice demonstrated similar results [36,39]. A recent publication also showed the protection against diabetic kidney injury in BTBR ob/ob mice by honokiol, a polyphenolic compound isolated from magnolia bark, through the maintenance of mitochondrial stability by activating SOD2 and restoring PGC-1α expression [40].

In this study, increased PGC-1α, SIRT3, and SOD2 expressions and decreased Ac-SOD2 expression in diabetic rats treated with a combination of metformin and PRHE were found to be very similar compared to the single therapy with metformin or PRHE. A previous study showed the upregulation of SIRT3 expression followed by a decrease in mitochondrial ROS formation after metformin treatment [41]. There is also a report that activation of PGC-1α is one of the therapeutic mechanisms of metformin in diabetes [42]. As PGC-1α and its subsequent downstream signals appear to be the same target for both metformin and PRHE, this may underlie the lack of additional therapeutic effects in diabetes treated with a combination regimen. However, this issue deserves future exploration.

#### **5. Conclusions**

This study provides convincing evidence indicating the promising role of PRHE in preventing the development and progression of diabetic nephropathy. The potential of PRHE is apparently associated with its ability to retain mitochondrial integrity and redox equilibrium within the kidney through the activation of the PGC-1α-SIRT3-SOD2 signaling pathway. The outcomes substantiate the worth of this agricultural waste and highlight the opportunity to develop purple rice husk as a dietary supplement or health product, which will be of great value in developing countries with limited resources and a high incidence of diabetes mellitus.

**Author Contributions:** Conceptualization, O.W. and N.L.; methodology, O.W., N.L., A.K. and W.P.; validation, O.W., A.K. and W.P.; formal analysis, O.W. and A.K.; investigation, O.W., N.L., A.K. and W.P.; data curation, O.W., N.L., A.K. and W.P.; writing manuscript, O.W.; project administration, O.W.; funding acquisition, O.W. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by Chiang Mai University (Grant Number: ĖĎ07/2561).

**Institutional Review Board Statement:** The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Institutional Animal Care and Use Committee at the Faculty of Medicine, Chiang Mai University (Project Number: 41/2559, approved on 3 October 2019).

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author.

**Acknowledgments:** Special thanks to the Faculty of Medicine, Chiang Mai University, for providing infrastructural support to carry out this research. We also extend sincere appreciation to Jannarong Intakhad and Parichat Tojing for their technical help during the experiment.

**Conflicts of Interest:** The authors declare no conflict of interest.
