Avicularin Attenuates Lead-Induced Impairment of Hepatic Glucose Metabolism by Inhibiting the ER Stress-Mediated Inflammatory Pathway

Lead (Pb), an environmental hazard, causes several human diseases. Avicularin (Avi), a main dietary flavonoid found in several plants and fruits, exhibits potential protective properties on organs. However, the molecular mechanisms of Avi’s protective effects against Pb-induced damage are not clear. In our study, the effects of Avi on Pb-induced hepatotoxicity were evaluated using ICR mice. We have revealed for the first time that treatment with Avi significantly reduced hepatic inflammation, endoplasmic reticulum stress (ERS) and glucose metabolism disorder induced by Pb. Avi decreased the serum biochemical indicators of glucose metabolism. Avi increased the activities of glycogenolysis rate-limiting enzyme hexokinase (HK), pyruvate kinase (PK), glucokinase (GK) and glycogen phosphorylase (PYG) and inhibited the activities of gluconeogenesis rate-limiting enzyme phosphoenolpyruvate carboxy kinase (PEPCK) and glucose-6-phosphate dehydrogenase (G6PD). Avi decreased the protein expression levels of glucose-regulated protein 78 (GRP78), phosphorylated inositol requiring enzyme 1 (p-IRE1), phosphorylated RNA-dependent protein kinase-like ER kinase (p-PERK) and phosphorylated eukaryotic initiation factor 2α (p-eIF2α). The levels of tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) were decreased in the liver as a result of Avi suppression Pb-induced inflammation. These results indicated that Avi attenuated Pb-induced impairment of hepatic glucose metabolism by the ERS and inflammation pathway.


Introduction
Lead (Pb), an environmental hazard, causes severe diseases in the liver, kidney, cardiovascular system, hematopoietic system, reproductive systems and nervous system [1,2]. Pb can be obtained from the intake of food and drinking water [3][4][5]. It was reported that Pb exposure is closely associated with coronary artery disease [6]. Pb exposure can interfere with glucose metabolism and promotes diabetes in animals [2,3]. Pb exposure can impair brain glucose metabolism by affecting the expression levels of the key regulatory enzyme [7,8]. Pb exposure has also caused insulin resistance and metabolism disorder in the livers of experimental animals by regulating the activity of glycogenolysis and gluconeogenesis enzymes, including hexokinase (HK), pyruvate kinase (PK), glucokinase (GK), phosphoenolpyruvate carboxy kinase (PEPCK) and glucose-6-phosphate dehydrogenase (G6PD) [3,9,10]. Pb exposure also induces liver damage though the endoplasmic reticulum stress (ERS) pathway, which may cause glucose metabolism disorder [11,12]. However, the mechanisms of Pb-induced glucose metabolism disorder are still unclear.
Avicularin (Avi, quercetin-3-alpha-L-arabinofuranoside) ( Figure 1) is a dietary flavonoid found in several plants and fruits, which has displayed multiple pharmacological effects, including anti-tumor, anti-oxidative, anti-inflammatory, anti-depressant and hepatoprotective properties [13][14][15]. Avi supplementation improved insulin resistance by regulat- Therefore, Avi was evaluated for the first time for its hepatoprotective effects against Pb-induced hepatic inflammation and glucose metabolism disorder, and this study further clarified the role of the ERS pathway in Avi protection.

Animals and Ethics
Fifty male ICR mice (20 ± 1 g) were obtained from Beijing HFK Bioscience CO., LTD, (Beijing, China). All experiment processes were approved by Jiangsu Normal University Committees (approval No. 3/5/2018) and performed according to the relevant guidelines.

Experimental Design
The mice were positioned in the animal room, with a temperature of 23 ± 1 °C, a 12 h dark/light cycle and relative humidity (55 ± 5)%. After 1 week of adaptive rearing, the mice were randomly divided into five groups (10 mice/group): (1) Normal control group; Therefore, Avi was evaluated for the first time for its hepatoprotective effects against Pb-induced hepatic inflammation and glucose metabolism disorder, and this study further clarified the role of the ERS pathway in Avi protection.

Animals and Ethics
Fifty male ICR mice (20 ± 1 g) were obtained from Beijing HFK Bioscience Co., Ltd, (Beijing, China). All experiment processes were approved by Jiangsu Normal University Committees (approval No. 3/5/2018) and performed according to the relevant guidelines.
At the end of 3 months, blood and liver samples were collected immediately after the mice were decapitated. All the samples were frozen at −80 • C until they were assayed.

Statistical Analysis
The data was presented as mean ± standard error (SE). For multiple comparisons after the one-way variance (ANOVA) test, Tukey's test was applied.

Avi Rescues Pb-Induced Liver Dysfunction
Pb exposure resulted in a significant increase in serum ALT and AST activities compared to the control group (25.21 ± 1.07 and 37.42 ± 2.51) by 243.59% and 163.01%, respectively. The treatment with Avi (25 and 50 mg/kg) reduced the activities of ALT (by 15.61% and 34.09%, respectively) and AST (by 19.71% and 27.29%, respectively) compared to the Pb group (86.62 ± 1.76 and 98.42 ± 4.50), showing a dose-dependent relationship (R 2 ALT = 0.9976, R 2 AST = 0.9382) ( Table 1). Treatment with Avi only had no significant effect on the activities of these enzymes. Table 1. Effect of avicularin on the serum biochemical parameters of mice.

Effects of Avi on Pb-Induced Insulin Resistance
In order to examine the therapeutic potential of Avi on Pb-induced insulin resistance, serum FBG and IS levels were determined. Pb exposure increased the levels of serum FBG, IS and HOMA-IS compared to the normal control group by 30.50%, 153.03% and 218.18%, respectively. Avi (25 and 50 mg/kg) treatment decreased the serum FBG level (by 13.37% and 22.32%, respectively), IS (by 21.56% and 35.33%, respectively) and HOMA-IS (by 22.95% and 31.43%, respectively) compared to the Pb group (Table 1). Therefore, Avi only had no significant effect on the levels of serum FBG, IS and HOMA-IS.

Effects of Avi on the Abnormal Activities of Pb-Induced Glucose Metabolism in the Liver
To evaluate the effect of Avi on the glucose metabolism activities in the liver, we measure the activities of GK, PK, HK, PEPCK and G6PD in the liver. Pb exposure decreased the activities of the glycogenolysis enzymes GK, PK and HK compared to the normal control group by 65.75%, 43.16% and 63.55%, respectively. Pb exposure increased the activities of the gluconeogenesis enzymes PEPCK and G6PD compared to the normal control group by 96.23% and 184.75%, respectively. In contrast, Avi (25 and 50 mg/kg) treatment restored the activities of those glucose metabolism enzymes (Table 2). Therefore, Avi only had no significant effect on those enzyme activities in the liver. Table 2. Effect of avicularin on the activities of those glucose metabolism enzymes in the liver.

Avi Regulated the Expression Levels of Glucose Metabolism Enzymes in the Liver
Western blotting was used to determine liver glucose metabolism enzyme expression. As depicted in Figure 1, Pb exposure decreased the protein expression levels of GK and PYG and increased the levels of PEPCK and G6PC compared with the normal control group. However, Avi (25 and 50 mg/kg) restored the protein expression levels of those glucose metabolism enzymes ( Figure 2).

Avi Suppressed Hepatic Inflammation
Inflammation plays an important role in the pathogenesis of fatty liver disease. We evaluated the NF-κB nuclear translocation and the expression of pro-inflammatory cytokines TNF-α and IL-6 in hepatic tissue. As displayed in Figure 3, Pb exposure increased the levels of TNF-α, IL-6 and the NF-κB nuclear transcriptional activity compared to the normal control group. Avi (25 and 50 mg/kg) treatment decreased the expression of the

Avi Suppressed Hepatic Inflammation
Inflammation plays an important role in the pathogenesis of fatty liver disease. We evaluated the NF-κB nuclear translocation and the expression of pro-inflammatory cytokines TNF-α and IL-6 in hepatic tissue. As displayed in Figure 3, Pb exposure increased the levels of TNF-α, IL-6 and the NF-κB nuclear transcriptional activity compared to the normal control group. Avi (25 and 50 mg/kg) treatment decreased the expression of the inflammatory factor compared to the Pb group (p < 0.05).

Avi Suppresses the ERS Pathway in the Liver
ERS is involved in inflammation and glucose metabolism. We further examined the expression levels of GRP78, p-IRE1, p-PERK and p-eIF2α. As depicted in Figure 4, the expression levels of GRP78, p-IRE1, p-PERK and p-eIF2α were up-regulated in the Pb group. In contrast to the Pb group, treatment with different dosages of Avi significantly decreased the phosphorylation levels of these proteins.

Avi Suppresses the ERS Pathway in the Liver
ERS is involved in inflammation and glucose metabolism. We further examined the expression levels of GRP78, p-IRE1, p-PERK and p-eIF2α. As depicted in Figure 4, the expression levels of GRP78, p-IRE1, p-PERK and p-eIF2α were up-regulated in the Pb group. In contrast to the Pb group, treatment with different dosages of Avi significantly decreased the phosphorylation levels of these proteins.

Discussion
Pb is a ubiquitous, persistent and non-essential toxic heavy metal which can induce the disorder of glucose metabolism [2,3]. Pb exposure induces multiple liver injuries [9][10][11]. Our study found that Pb induced hepatic inflammation, ERS and glucose metabolism disorder (Table 1). Interestingly, we found that Avi supplementation mitigated Pb-induced liver injury.
Pb exposure could cause hyperglycemia and insulin resistance in many organs [3,5,19]. Pb exposure can interfere with glucose metabolism and promotes diabetes in animals [3,5]. Current research shows that Pb exposure increases the levels of serum FBG, IS and HOMA-IS compared with normal control groups, which indicates hyperglycemia [2,7]. Avi treatment showed a hypoglycemic effect in a diabetes model [13,16]. Our results revealed that Avi supplementation inhibited Pb-induced hyperglycemia and insulin resistance in mice (Table 1).
PYG is the rate-limiting enzyme in glycogenolysis [13,20]. PK and HK are the key glycolytic enzymes that control glycolysis rate, which are the key factors that administer glucose production [8,20]. GK is the rate-limiting enzyme modulating glucose metabo-

Discussion
Pb is a ubiquitous, persistent and non-essential toxic heavy metal which can induce the disorder of glucose metabolism [2,3]. Pb exposure induces multiple liver injuries [9][10][11]. Our study found that Pb induced hepatic inflammation, ERS and glucose metabolism disorder (Table 1). Interestingly, we found that Avi supplementation mitigated Pb-induced liver injury.
Pb exposure could cause hyperglycemia and insulin resistance in many organs [3,5,19]. Pb exposure can interfere with glucose metabolism and promotes diabetes in animals [3,5]. Current research shows that Pb exposure increases the levels of serum FBG, IS and HOMA-IS compared with normal control groups, which indicates hyperglycemia [2,7]. Avi treatment showed a hypoglycemic effect in a diabetes model [13,16]. Our results revealed that Avi supplementation inhibited Pb-induced hyperglycemia and insulin resistance in mice (Table 1).
PYG is the rate-limiting enzyme in glycogenolysis [13,20]. PK and HK are the key glycolytic enzymes that control glycolysis rate, which are the key factors that ad-Nutrients 2022, 14, 4806 7 of 9 minister glucose production [8,20]. GK is the rate-limiting enzyme modulating glucose metabolism, glycogen synthesis and insulin secretion [16,21]. PEPCK is a rate-limiting enzyme in gluconeogenesis, which also influences blood glucose levels and hepatic glucose production [3,10,13]. Research has shown that Pb decreased the activities of HK, PK and increased the activities of PEPCK, G6PC in brain and liver tissue [3,8]. Additionally, the expression of HK, PK, GK and PYG was significantly decreased [7,9] and the expression of PEPCK, G6PC was increased in the Pb group compared with controls [3,10]. Research found that Avi could up-regulate the expression of PK and PYG to alleviate glucose metabolism disorder [13]. In our current work, we found that Avi treatment recovered the activations of HK, PK, GK, PYG, PEPCK and G6PC in the Pb group ( Figure 2). Thus, the above results indicate that Avi ameliorated Pb-induced glucose metabolism disorder.
Inflammatory response is significantly associated with glucose metabolism [22]. Pb exposure reportedly stimulated inflammation and abnormal metabolism of gluconeogenesis and glycogenolysis in the liver [3]. Several studies revealed that Avi could inhibit the formation of inflammasome in the liver and brain of different experimental models, thus reducing toxin-induced tissue damage [15,17,23]. Avi could inhibit inflammation and fibrosis in osteoarthritis [24]. Avi could also prevent the release of IL-1β, IL-6 and TNF-α in various cells [24][25][26]. The results of this experiment showed that Pb stimulates NF-κB nuclear transcriptional activity and the release of IL-1β and TNF-α. Meanwhile, Avi could significantly prevent the secretion of these pro-inflammatory cytokines (Figure 3), indicating that Avi alleviates liver injury by inhibiting Pb-induced inflammation.
ERS is known as the "unfolded protein response (UPR)," which has three classical signaling pathways, IRE1α, PERK and ATF6 [5,27]. Excess ERS will not only induce apoptosis and inflammatory response but will also cause glucose metabolism disorder and other physiological diseases [27][28][29]. Hepatic ERS can induce gluconeogenesis by stimulating the activation of IRE1, PERK and eIF2α [30,31]. ERS disrupts insulin signaling and promotes hepatic insulin resistance and glucose production [31][32][33]. Pb was found to induce ERS in multiple tissues and cells, which further activated NF-κB and stimulated inflammation by the PERK and IRE1α signaling pathways [3,5,11]. As mentioned above, we found that Pb increased the expression of GRP78, p-IRE1, p-PERK and p-eIF2α, which indicated that Pb caused ERS in the liver [3]. However, Avi supplementation reduced the expression of GRP78, p-IRE1, p-PERK and p-eIF2α, and thus decreased Pb-induced ERS in the liver. We could claim that Avi was able to mitigate Pb-induced liver injury by inhibiting ERS (Figure 4).
In conclusion, Avi significantly alleviated Pb-induced hepatic inflammation and glucose metabolism disorder and inflammation by inhibiting the ERS pathway ( Figure 5). The hepatoprotection of Avi warrants further investigation in our future research. lism, glycogen synthesis and insulin secretion [16,21]. PEPCK is a rate-limiting enzyme in gluconeogenesis, which also influences blood glucose levels and hepatic glucose production [3,10,13]. Research has shown that Pb decreased the activities of HK, PK and increased the activities of PEPCK, G6PC in brain and liver tissue [3,8]. Additionally, the expression of HK, PK, GK and PYG was significantly decreased [7,9] and the expression of PEPCK, G6PC was increased in the Pb group compared with controls [3,10]. Research found that Avi could up-regulate the expression of PK and PYG to alleviate glucose metabolism disorder [13]. In our current work, we found that Avi treatment recovered the activations of HK, PK, GK, PYG, PEPCK and G6PC in the Pb group ( Figure 2). Thus, the above results indicate that Avi ameliorated Pb-induced glucose metabolism disorder. Inflammatory response is significantly associated with glucose metabolism [22]. Pb exposure reportedly stimulated inflammation and abnormal metabolism of gluconeogenesis and glycogenolysis in the liver [3]. Several studies revealed that Avi could inhibit the formation of inflammasome in the liver and brain of different experimental models, thus reducing toxin-induced tissue damage [15,17,23]. Avi could inhibit inflammation and fibrosis in osteoarthritis [24]. Avi could also prevent the release of IL-1β, IL-6 and TNF-α in various cells [24][25][26]. The results of this experiment showed that Pb stimulates NF-κB nuclear transcriptional activity and the release of IL-1β and TNF-α. Meanwhile, Avi could significantly prevent the secretion of these pro-inflammatory cytokines ( Figure  3), indicating that Avi alleviates liver injury by inhibiting Pb-induced inflammation.
ERS is known as the "unfolded protein response (UPR)," which has three classical signaling pathways, IRE1α, PERK and ATF6 [5,27]. Excess ERS will not only induce apoptosis and inflammatory response but will also cause glucose metabolism disorder and other physiological diseases [27][28][29]. Hepatic ERS can induce gluconeogenesis by stimulating the activation of IRE1, PERK and eIF2α [30,31]. ERS disrupts insulin signaling and promotes hepatic insulin resistance and glucose production [31][32][33]. Pb was found to induce ERS in multiple tissues and cells, which further activated NF-κB and stimulated inflammation by the PERK and IRE1α signaling pathways [3,5,11]. As mentioned above, we found that Pb increased the expression of GRP78, p-IRE1, p-PERK and p-eIF2α, which indicated that Pb caused ERS in the liver [3]. However, Avi supplementation reduced the expression of GRP78, p-IRE1, p-PERK and p-eIF2α, and thus decreased Pb-induced ERS in the liver. We could claim that Avi was able to mitigate Pb-induced liver injury by inhibiting ERS (Figure 4).
In conclusion, Avi significantly alleviated Pb-induced hepatic inflammation and glucose metabolism disorder and inflammation by inhibiting the ERS pathway ( Figure 5). The hepatoprotection of Avi warrants further investigation in our future research.