Protective Effects of Astragaloside IV on Uric Acid-Induced Pancreatic β-Cell Injury through PI3K/AKT Pathway Activation

Background Elevated uric acid (UA) has been found to damage pancreatic β-cell, promote oxidative stress, and cause insulin resistance in type 2 diabetes (T2D). Astragaloside IV (AS-IV), a major active monomer extracted from Astragalus membranaceus (Fisch.) Bunge. which belongs to TRIB. Galegeae (Br.) Torrey et Gray, Papilionaceae, exhibits various activities in a pathophysiological environment and has been widely employed to treat diseases. However, the effects of AS-IV on UA-induced pancreatic β-cell damage need to be investigated and the associating mechanism needs to be elucidated. This study was designed to determine the protective effects and underlying mechanism of AS-IV on UA-induced pancreatic β-cell dysfunction in T2D. Methods UA-treated Min6 cells were exposed to AS-IV or wortmannin. Thereafter, the 3-(45)-dimethylthiahiazo(-z-y1)-35-di-phenytetrazoliumromide (MTT) assay and flow cytometry were employed to determine the effect of AS-IV on cell proliferation and apoptosis, respectively. Insulin secretion was evaluated using the glucose-stimulated insulin secretion (GSIS) assay. Finally, western blot and quantitative real-time polymerase chain reaction (qRT-PCR) were performed to determine the effect of AS-IV on the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) pathway in UA-treated cells. Results AS-IV had no cytotoxic effects on Min6 cells. UA significantly suppressed Min6 cell growth, promoted cell apoptosis, and enhanced caspase-3 activity; however, AS-IV abolished these effects in a dose-dependent manner. Further, decreased insulin secretion was found in UA-treated Min6 cells compared to control cells, and the production of insulin was enhanced by AS-IV in a dose-dependent manner. AS-IV significantly increased phosphorylated (p)-AKT expression and the ratio of p-AKT/AKT in Min6 cells exposed to UA. No evident change in AKT mRNA level was found in the different groups. However, the effects of AS-IV on UA-stimulated Min6 cells were reversed by 100 nM wortmannin. Conclusion Collectively, our data suggest that AS-IV protected pancreatic β-cells from UA-treated dysfunction by activating the PI3K/AKT pathway. Such findings suggest that AS-IV may be an efficient natural agent against T2D.


Introduction
Diabetes mellitus (DM), a frequent chronic disease, is known to have a remarkable burden on public health [1]. According to the US diabetes epidemiological statistics, 8% of people had diabetes in 2011, and 80 million adults were diagnosed with prediabetes [2]. Type 2 diabetes (T2D), a type of DM, is characterized by insulin tolerance and β-cell damage [3].
Many reports have revealed that β-cell dysfunction will appear when the pancreas cannot excrete adequate insulin [4][5][6]. us, protecting pancreatic β-cell functions may be an approach for T2D development and therapy. Based on several reports, uric acid (UA) level is associated with β-cell functions, insulin secretion, and the risk of T2D [7][8][9][10]. Several studies also revealed that increased UA could abduct insulin resistance and inhibit pancreatic β-cell survival and insulin product [11][12][13]. However, the latent mechanism of UA-induced β-cell dysfunction in T2D has not been fully elucidated.
Astragaloside IV (AS-IV) is a bioactive saponin purified from Astragalus membranaceus which belongs to TRIB. Galegeae (Br.) Torrey et Gray, Papilionaceae [14]. Based on accumulating evidence, AS-IV exerts multipotent activities in many diseases, such as anti-inflammation [15] and antioxidant [16], with no toxicity. Zhang et al. revealed that AS-IV extends the lifespan of Caenorhabditis elegans by promoting age-related functional declines and inducing antioxidant responses [17]. Some of these pharmacological activities resulted in the regulation of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) pathway signaling pathways [18][19][20]. e PI3K/AKT pathway has been found to be involved in the prevention of UA-induced dysfunction of β-cells [8]. For instance, Zhang and Qui revealed that the PI3K/AKT inhibitor LY294002 promotes UA-induced dysfunction of β-cells [21]. We speculated that the activation of PI3K/AKT may be involved in the protective effect of AS-IV on UA-induced β-cell injury in diabetes.
e aim of this study was to illustrate the potential therapeutic effects of AS-IV on UA-stimulated β-cell injury and elucidate the underlying molecular mechanism of AS-IV in T2D.

Glucose-Stimulated Insulin Secretion (GSIS) Assay.
Min6 cells (5 × 10 4 cells per well in 6-well plates) were cultured in DMEM at 37°C for 24 h. ereafter, the cells were pretreated with various concentrations of AS-IV for 2 h followed by 5 mg/dl UA for another 24 h. e Krebs-Ringer Buffer (Sigma-Aldrich, St. Louis, Mo, USA) supplemented with 0.1% bovine serum albumin (BSA; Sigma-Aldrich, St. Louis, MO, USA) was applied to the cells, which were then incubated for 1 h. e cells were then stimulated with Krebs-Ringer Buffer containing low or high concentrations of glucose for another 1 h. Finally, the insulin content in samples was assessed by radioimmunoassay, as described previously.
2.6. Caspase-3 Activity Detection. Min6 cells were pretreated with AS-IV or/and wortmannin for 2 h followed by treatment with UA (5 mg/dl) for another 24 h. To determine the activity of caspase-3 in Min6 cells (2 × 10 7 cells) from different groups, a caspase-3 activity detection kit (Beyotime, Shanghai, China) was used according to the manufacturer's instructions.

qRT-PCR Analysis.
Min6 cells were exposed to AS-IV, UA, or wortmannin for the indicated time. TRIzol reagent (Invitrogen, Carlsbad, CA, USA) was used to extract total RNA from Min6 cells, according to the manufacturer's instructions. Total RNA was then reverse-transcribed into complementary (c) DNA with the Reverse Transcription Kit ( ermo, San Jose, CA, USA). e expression of AKT was detected using a SYBR Green PCR Master Mix Kit (TaKaRa, Japan). e primers for qPCR were obtained from Sangon Biotech (Shanghai, China). e reactions were run in triplicate on the ABI PRISM 7900 sequence detection system (Applied Biosystems, USA). Target gene expression was calculated using the 2 −ΔΔCt method.

Statistical Analysis.
We used the Kolmogorov-Smirnov test to determine the normality of the data in SPSS. Statistical analysis was performed using Statistical Package for the Social Sciences (SPSS) 20.0 (IBM Corp.). All experiments were conducted more than 3 times. e data are expressed as mean ± standard deviation (SD) of three independent experiments. Differences between the two groups were estimated using an unpaired Student's t-test. Statistical differences among multiple groups were analyzed by one-way analysis of variance (ANOVA) followed by Tukey's post hoc tests. P < 0.05 was considered to indicate significant difference.  ereafter, Min6 cells were stimulated with 5 mg/dl UA for another 24 h. UA was found to prominently suppress Min6 cell viability (Figure 2(a)), enhance caspase-3 activity (Figure 2(b)), promote cell apoptosis, and markedly enhance the ratio of apoptotic cells (Figures 2(c) and 2(d)) in UA-treated Min6 cells. However, AS-IV abolished these results in a dose-dependent manner.

Effects of AS-IV on Insulin Secretion in Glucose-Stimulated Min6 Cells.
en, this study revealed the roles of AS-IV in Min6 cell dysfunction and adopted AS-IV (12.5, 25, and 50 μmol/l) and UA (5 mg/dl) to treat Min6 cells. Different concentrations of glucose were then used to stimulate Min6 cells for another 1 h. As displayed in Figure 3, UA remarkably reduced insulin secretion under 16.7 mM glucose treatment. Compared with UA, AS-IV evidently promoted high glucose-stimulated insulin production in a dose-dependent manner.

AS-IV Protects UA-Stimulated Cell Damage by Regulating the PI3K/AKT Signal Pathway.
To conduct an in-depth exploration of the related mechanism for the effect of AS-IV on UA-induced Min6 cells, the expression of proteins in the PI3K/AKT pathway was detected. Based on western blot and qRT-PCR, UA notably inhibited p-AKT protein levels and the ratio of p-AKT/AKT in Min6 cells. Nevertheless, after AS-IV treatment, the protein expression levels of p-AKT and the p-AKT/AKT ratio were amplified in a dose-dependent manner (Figures 4(a) and 4(b)). However, there were no significant changes in AKTmRNA levels among the different groups (Figure 4(c)).

Effect of Wortmannin on Pancreatic β-Cell Growth and Apoptosis in Min6 Cells after UA and AS-IV Treatment.
To further confirm whether AS-IV can resist UA-induced pancreatic β-cell damage and dysfunction by activating the  PI3K/AKT pathway, wortmannin, a PI3K/AKT inhibitor, was applied. Min6 cells were treated with 25 μmol/l AS-IV or/and 100 nM wortmannin for 2 h. ereafter, the cells were treated with 5 mg/dl UA for another 24 h. As shown in Figure 5, 100 nM wortmannin obviously restrained Min6 cell viability (Figure 5(a)), enhanced caspase-3 activity ( Figure 5(b)), and induced Min6 cell apoptosis (Figures 5(c) and 5(d)) when compared to the UA + 25 μmol/l AS-IV group.

Effect of Wortmannin on Insulin Production in Min6
Cells after UA and AS-IV Treatment. As shown in Figure 6, 100 nM wortmannin markedly decreased insulin production in glucose-induced Min6 cells (Figure 6), suggesting that AS-IV exhibited protective effects on UA-induced Min6 cells via the PI3K/AKT pathway.

AS-IV Protects Pancreatic β-Cell Functions by Activating the PI3K/AKT Pathway in Min6 Cells after UA Treatment.
Finally, this study explored whether AS-IV protects pancreatic β-cell functions by activating the PI3K/AKT pathway in Min6 cells after UA treatment. Based on western blot and qRT-PCR, wortmannin significantly suppressed the protein expression of p-AKT (Figure 7(a)) and the ratio of p-AKT/ AKT (Figure 7(b)) in Min6 cells after UA + 25 μmol/l AS-IV stimulation. However, the mRNA levels of AKT were not significantly altered among various groups (Figure 7(c)).

Discussion
DM is a familiar chronic disease that leads to cell injury, increases serum UA, and accelerates the progression of DM [22]. T2D, which is a type of DM, is characterized by insulin disorder or β-cell injury [23]. In recent years, the potential   (d) Quantification of apoptotic cells. Data are expressed as mean ± SD. Statistical differences among groups were analyzed by one-way ANOVA followed by Tukey's post hoc tests. * * P < 0.01 compared to the control group; # , ## P < 0.05, 0.01 vs. UA group; and P < 0.05. relationship between UA level and the risk of T2D has been revealed in various clinical studies. For example, Guarda et al. revealed that high serum UA is associated with tubular damage and kidney inflammation in T2D patients [24]. Moreover, Wang et al. reported the relationship between serum UA and ischemic stroke in a large T2D population in China [25]. Currently, drug therapy is the main treatment for DM, including the dipeptidyl peptidase-4 inhibitor, sitagliptin, which is approved in more than 130 countries worldwide as monotherapy and in combination with other antihyperglycemic drugs for the treatment of adult patients with T2D. e pathogenesis of DM and the development of treatment strategies have always been a hot spot in this research field. AS-IV, an active saponin, is extracted from Astragalus membranaceus. A previous report revealed that AS-IV displayed various activities in diseases, including T2D [26]. Based on these assessments, this study opted to determine whether AS-IV could be a useful agent to relieve pancreatic β-cell dysfunction and evaluate the underlying mechanisms.
Pancreatic β-cell dysfunction is widely recognized as a significant feature of T2D [27]. erefore, this investigation assessed the roles of AS-IV in pancreatic β-cells by employing different concentrations of AS-IV (12.5, 25, 50, 10, and 200 μmol/l) to stimulate Min6 cells for 24 h. Firstly, the data showed that AS-IV had no cytotoxic effect on pancreatic β-cells. Based on previous studies, excess UA may induce insulin resistance and pancreatic β-cell injury, which was associated with the progression of T2D [7][8][9][10][11][12][13]  Data are expressed as mean ± SD. Statistical differences among groups were analyzed by one-way ANOVA followed by Tukey's post hoc tests. * * P < 0.01 compared to the control group; # , ## P < 0.05, 0.01 vs. UA group; and P < 0.05. [28]. e present study further explained the functions of AS-IV in UA-stimulated pancreatic β-cells. In partial accordance with other reports, this study revealed that UA evidently suppressed Min6 cell viability and promoted cell apoptosis compared to the control. Caspase-3 is a significant mediator of the apoptosis-related pathway [29]. Accordingly, caspase-3 activity was determined in this study. Based on the findings, caspase-3 activity was significantly enhanced in Min6 cells after UA stimulation. Nevertheless, UAstimulated Min6 cell viability and apoptosis were abolished by AS-IV in a dose-dependent manner, suggesting that AS-IV alleviated pancreatic β-cell injury caused by UA.
Several reports have revealed that insulin resistance is the principal cause of T2D development. Owing to UA treatment, insulin secretion in pancreatic β-cells was obviously inhibited [30]. Hence, in the current study, insulin content in glucose-stimulated Min6 cells was determined. e results of the GSIS assay revealed that AS-IV significantly altered insulin production in a dose-dependent manner, indicating that AS-IV inhibited UA-induced Min6 cell dysfunction by suppressing apoptosis and stimulating insulin release.
Some pharmacological activities of AS-IV are closely related to the regulation of the PI3K/AKT signaling pathway [18][19][20]. e activation of the PI3K/AKT pathway has been reported to be responsible for the antiapoptotic    [32]. To further verify our speculation, the PI3K/AKT pathway was determined. Findings revealed that the PI3K/ AKT pathway was substantially inhibited in UA-treated Min6 cells. However, the effects of UA on the PI3K/AKT pathway were found to be reversed by AS-IV. ese findings suggest that AS-IV prevented pancreatic β-cell from UA-induced damage by regulating the PI3K/AKT pathway. To further analyze the roles of the PI3K/AKT pathway in the regulation of pancreatic β-cell functions, the PI3K inhibitor wortmannin was employed. Wortmannin (100 nM) was found to remarkably suppress the protective effect of AS-IV on UA-treated Min6 cells, as demonstrated by decreased cell viability, insulin production, and enhanced apoptotic cells and caspase-3 activity.
ese findings further demonstrated that AS-IV protected pancreatic β-cell damage induced by UA by activating the PI3K/AKT pathway.
is study also had some limitations. For example, the effect of more doses of AS-IV on UA-treated Min6 cells was not assessed. Further, the effect of AS-IV on pancreatic β-cell function in T2D was not evaluated in vivo. ese issues will be addressed in future studies.
In conclusion, this study revealed that AS-IV protected UA-induced pancreatic β-cell dysfunction by agitating the PI3K/AKT pathway. Altogether, the findings of the current study provided a better understanding of the potential mechanism of AS-IV on T2D and revealed that AS-IV may serve as a novel agent for the treatment of T2D.

Data Availability
All datasets used and/or generated during the current study are available from the corresponding author on reasonable request.

Conflicts of Interest
e authors declare that they have no conflicts of interest. Evidence-Based Complementary and Alternative Medicine 7