Protective Effects of Artemisia selengensis Turcz. Leaf Extract on Caenorhabditis elegans under High Glucose Diet

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Introduction
During the last decades, the dietary habit of people has gradually shifted to high sugar and high lipid.Diets enriched in glucose, one of the most extensively studied sugar, have been reported to cause several metabolic-related diseases such as diabetes mellitus and obesity [1].Recently, growing evidence has emerged that high glucose diet could also lead to accelerated aging in diferent organisms, thus resulting in reduced longevity and locomotion, and increased neuronal defects [2][3][4].Nutritional intervention has been considered to be an efective approach to attenuate the detrimental efects induced by high glucose diet and promote healthy aging [5,6].
Artemisia selengensis Turcz.(AST), as a widely edible vegetable in the central region of China, has been reported to possess many bioactivities such as antioxidation, antidiabetic, and anticancer activities [7][8][9].Recent study also reveals that ASTE is a potential α-glucosidase inhibitor, which may be active in regulating glucose metabolism [10].Moreover, cafeoylquinic acids, the major components of ASTE, have been suggested to mediate glucose metabolism and alleviate the impact of diabetes complications [11].Emerging evidence demonstrates that sustained elevated plasma glucose level afects organismal aging process [3].Several natural ingredients that regulate glucose metabolism have been found to prevent the negative impact of glucose on longevity [12,13].However, little is known about the efects of ASTE in alleviating the detrimental impacts and delaying the accelerated aging induced by high glucose diet.
Due to the short lifespan, easy cultivation, and facile gene manipulation, C. elegans has been utilized as a powerful tool to explore the metabolic and phenotypic efects of high glucose diet [14].In addition, nutrient-sensing pathways involving IIS, AAK-2/AMP-activated protein kinase (AMPK), and SKN-1/nuclear factor erythroid 2-related factor 2 (Nrf2) can regulate glucose level and aging process by sensing nutrient or metabolites, and these pathways are highly conserved between nematodes and mammals [15].Tus, natural ingredients identifed, with protective efects against glucotoxicity on C. elegans, may have the potential to promote human health in high sugar environments.
In the present study, the efects and underlying mechanisms of ASTE on alleviating high glucose-induced damages were investigated by employing the C. elegans model.Tis research reveals the potential of ASTE in preventing glucose toxicity and promoting healthy aging.

Preparation of ASTE.
ASTE was obtained from our laboratory according to our previous study [16].In brief, Artemisia selengensis Turcz.leaves were ground to powder and extracted with 50% ethanol three times, then partitioned with ethyl acetate to obtain the crude extracts.Once removing the solvents, the extract was loaded onto a resin HP-20 column to purify.Afterwards, the eluted fractions with 50% ethanol were concentrated and freeze-dried to yield ASTE. Te ASTE was diluted in dimethyl sulfoxide (DMSO) to prepare 60 mg/mL stock solution.

Lifespan Assay.
Te late L4-stage synchronized worms were transferred into a Transwell-96 well plate containing liquid S-complete medium with/without glucose [18], and treated with 0.6% DMSO (control) or various concentrations of ASTE (60, 240, and 360 μg/mL).Te FUDR and heatkilled E. coli OP50 were also added.For glucose addition, sterilized stock solution of glucose was added to the liquid Scomplete medium to the desired concentration (1%, 2%, and 4% glucose, respectively).Te living number of worms was recorded until 100% mortality.

Motility Assays.
Worms were cultured as outlined in the lifespan assay.On the 3rd, 6th, and 9th day of cultivation, worms were moved onto new NGM plates, and the number of body bending and pharyngeal pumping rates in 20 s was counted using an anatomic microscope according to a previous report [19].Each trial was repeated 3 times with at least 30 worms.
2.6.Intracellular ROS Assay.Te nematodes were removed into a black 96-well plate (30 worms per well) after culturing for 7 days as described in Section 2.4 and then treated with 50 μM fresh H 2 DCF-DA for 2 h at 37 °C [20].Te fuorescence intensity was then recorded every 20 min at an excitation/emission wavelength of 485/530 nm.

GSH Contents and SOD Activities Determination.
Worms were prepared as outlined in Section 2.4.After treated for 3 days, the nematodes (approximately 2000 worms per group) were collected with M9 bufer, ground in a tissue grinder, and successively centrifuged to obtain the supernatant [21].Te GSH contents and SOD activities were analyzed by commercial kits (Nanjing Jiancheng Bioengineering Inst., Jiangsu, China) based on the manufacturers' instructions.Te results were normalized to the protein content, which was analyzed by a BCA kit (Nanjing Jiancheng Bioengineering Inst., Jiangsu, China).

Glucose and Triglyceride
Quantifcation.On the 3rd day of adulthood, the nematodes (approximately 2000 worms per group) were collected and washed with M9 bufer for 3 times, then homogenized and centrifuged to obtain the samples for subsequent assays.Te glucose and triglyceride contents were then quantifed by the commercial kits (Nanjing Jiancheng Bioengineering Inst., Jiangsu, China).
2.9.Oil-Red O Staining.Based on the previous report, the lipid droplets of nematodes were detected by Oil-Red O staining [22].In brief, after treatment for 3 days, the worms were collected and fxed with 2% paraformaldehyde for 30 min.Afterwards, the working solution of Oil-Red O was added and incubated overnight for staining.Te photographs of stained worms were then recorded under an inverted microscope.Te intensity of the stained fat of each worm was quantifed using ImageJ software.

2.10.
Quantitative RT-PCR Analysis.Worms were collected on the 3rd day of adulthood treated as for lifespan assay.RNA in each group was extracted and converted to cDNA (Takara), as described previously [16].By using SYBR Green PCR Master Mix (Takara), the qRT-PCR reaction was carried out on a qTOWER 2.0 PCR system (Analytik JenaAG, Germany).Te mRNA expression level of gene was quantifed by 2 −ΔΔCT method, and act-1 was chosen as 2 Journal of Food Biochemistry endogenous control.Te primer sequences of abovementioned genes are presented in Supporting Information Table S1.

High Glucose Diet Decreased the Lifespan of C. elegans.
To evaluate the efect of glucose on the lifespan of C. elegans, we exposed wildtype worms to glucose at a range of concentrations.Te lifespan assay was then carried out.In comparison with the control, worms fed with 1%, 2%, and 4% glucose markedly decreased the mean lifespan by 14.58%, 22.22%, and 31.50%,respectively (Figure 1(a), Table 1, p < 0.01).Since the epidermis of C. elegans treated with 4% glucose was slightly wrinkled during the experiment, we used 2% glucose for the subsequent experiments.

ASTE Extended the Lifespan of C. elegans on High Glucose
Diet.Te major phytochemical compounds in ASTE were chlorogenic acid, 1,4-dicafeoylquinic acid, 3,4-dicafeoylquinic acid, 1,5-dicafeoylquinic acid, 3,5-dicafeoylquinic acid, and 4,5-dicafeoylquinic acid, as we have previously reported [16].Te HPLC profle of ASTE is shown in supplementary information Figure S1.Based on our previous study, these plant-derived polyphenols have great benefts on antiaging activity, therefore, we further examined whether ASTE could protect worms from the negative impact of a high-glucose diet on lifespan.Te results showed that high glucose diet (2%) feeding decreased the lifespan by 28.42%, while ASTE at 240 μg/mL and 360 μg/mL notably prolonged the mean lifespan of worms by 28.70% and 27.93%, respectively, (p < 0.001 and p < 0.01, Figure 1(b), Table 2).Tese fndings confrmed that ASTE can alleviate the adverse impacts of high glucose on longevity.In addition, the dose of 240 μg/mL ASTE was applied for the subsequent experiments due to its remarkable efect.

ASTE Attenuated the Negative Efects of High Glucose Diet on Healthspan.
In addition to a reduction in lifespan, a high glucose diet also had adverse efects on healthspan [23,24].With increasing age, the locomotion ability of nematodes decreased in both the normal diet and high glucose diet group; however, the pharyngeal pumping rates and bodybending rates of worms grown on high glucose diet were further decreased compared with the normal diet group (Figures 2(a) and 2(b), p < 0.001).In addition, the results also revealed that the pharyngeal pumping and body bending rates obviously recovered to a certain extent in worms fed with 2% glucose after the addition of ASTE, but did not return to the level of the normal diet group.Taken together, ASTE, as a natural plant-derived ingredient, partially mitigated the damage to the healthspan of C. elegans caused by high glucose diet.

ASTE Enhanced the Antioxidant Activity in C. elegans.
On the basis of previous reports, excessive glucose intake may lead to an enhanced oxidative stress by increasing the level of cellular reactive oxygen species (ROS), thereby accelerating the aging process [14,25].To verify whether ASTE alleviated the detrimental efects of high glucose diet via decreasing the oxidative stress, we examined the accumulation levels of ROS and the activity of some antioxidant enzymes.As displayed in Figure 3 4(e).In C. elegans, oga-1 has been reported to encode an active O-GlcNAcase, which was related to glucose uptake [26].Te results showed that the expression of oga-1 was obviously increased by 2.82-fold in worms on high glucose diet, whereas ASTE decreased the expression of oga-1 by 0.73-fold (Figure 4(e), p < 0.001).Meanwhile, the expressions of lbp-5 (intracellular lipidbinding protein) and acs-2 (acyl-CoA synthetase) were signifcantly reduced by 0.53-fold and 0.57-fold, respectively, (Figure 4(e), p < 0.001 and p < 0.01), while the expression of fat-6 (stearoyl-CoA desaturase) was increased by 2.77-fold in worms on high glucose diet.On the contrary, ASTE notably upregulated the expressions of lbp-5 (1.99-fold) and acs-2 (3.75-fold), and inhibited the expression of fat-6 (0.11fold) in worms on high glucose diet.Above all, these results indicated that ASTE played an important role in reducing glucose and fat contents in C. elegans.

ASTE Extended the Lifespan of C. elegans on High Glucose
Diet by Regulating the IIS Pathway.Te IIS pathway was reported to regulate aging, stress resistance, metabolism, and development, while inhibiting IIS pathway was an efective approach to promote the longevity [27,28].Terefore, we examined whether ASTE-induced lifespan extension on high glucose diet relied on the IIS pathway.Te results showed that daf-2 and daf-16, which were two critical regulatory factors of the IIS pathway, abolished ASTEinduced lifespan extension (Figures 5(a) and 5(b), Table S2), demonstrating that daf-2 and daf-16 were indispensable for the ASTE-mediated longevity promotion efect on high glucose diet.Moreover, compared with the normal diet group, the expressions of daf-2, age-1, and akt-2 were signifcantly increased in the 2% glucose diet group (Figure 5(c), p < 0.05).Meanwhile, the expressions of critical transcription factor daf-16 and its downstream   stress-response genes sod-3 (superoxide dismutase), mtl-1 (metallothionein), and hsp-12.3(heat shock proteins) were markedly inhibited in the 2% glucose diet group (p < 0.05).However, the supplementation of ASTE on 2% glucose diet obviously decreased the expressions of daf-2, age-1, and akt-2, and elevated the expressions of daf-16, sod-3, mtl-1, and hsp-12.3(Figure 5(c), p < 0.01).Taken together, these data indicated that ASTE prolonged the lifespan of C. elegans on high glucose diet by inhibiting the IIS pathway.

ASTE-Induced Lifespan Extension on High Glucose Diet
Was Dependent on skn-1 and aak-2.SKN-1, a homologue of mammalian Nrf2, was found to activate phase II detoxifcation and antioxidative stress response pathway in C. elegans [29].In addition, aak-2, a homologue of α-catalytic subunits of mammalian AMPK, was reported to regulate oxidative stress resistance and energy metabolism in nematodes [30].Considering that ASTE could decrease the ROS level in worms on high glucose diet, we further examined whether the lifespan extension efect of ASTE was relied on skn-1 and aak-2.Te results showed that ASTE failed to prolong the lifespan of skn-1 and aak-2 null mutants (Figures 6(a) and 6(c), Table S2), indicating that skn-1 and aak-2 were required for ASTE-induced lifespan extension on high glucose diet.In addition, the expressions of skn-1 and aak-2 were signifcantly inhibited in the high glucose diet group in comparison to the normal one, whereas ASTE intervention obviously upregulated the expressions of skn-1 and aak-2 (Figures 6(b) and 6(d), p < 0.01).Tese fndings suggest that ASTE might prolong the lifespan of worms grown on high glucose diet by activating skn-1 and aak-2.

Discussion
Tere is growing evidence that excess glucose intake afects the lifespan of C. elegans [14,31].One previous study has demonstrated that ASTE is a potential resource of α-glucosidase inhibitor, which might help to reduce the postprandial blood glucose level [10].However, little is known about the efects of ASTE on the lifespan of C. elegans on high glucose diet.Terefore, by employing the C. elegans model, we investigated the benefts of ASTE in face of high glucose diet.Herein, we found that ASTE, rich in caffeoylquinic acids, was able to partially rescue the shortened lifespan and mitigate the damage to the healthspan caused by high glucose diet.Moreover, ASTE decreased the oxidative stress and the glucose and fat contents in worms grown on high glucose diet.Te protective efects of ASTE were mediated by inhibiting the IIS pathway and activating skn-1 and aak-2.
It is well known that a consistently large amount of dietary sugar can lead to the increase in obesity, diabetes, and cardiovascular diseases [32,33].However, there is limited information about the direct efects of a high sugar diet on aging, especially on longevity.Our results indicated that addition of glucose shortened the lifespan and induced damage to the healthspan of C. elegans.ASTE, as a natural plant-derived ingredient, was found to extend the lifespan and improve the locomotion ability of C. elegans, thus delaying aging induced by a high glucose diet.Besides, ASTE has been reported to inhibit α-glucosidase [10], suggesting that ASTE has potential in regulating glucose metabolism.Tis was supported by the current observation that ASTE obviously decreased the glucose content in C. elegans.Moreover, ASTE was found to remarkably reduce the fat level in C. elegans, which was consistent with a previous report that Lonicera japonica extracts rich in cafeoylquinic acids could decrease fat accumulation in worms [34].Taken together, our fndings demonstrated that ASTE not only promoted longevity and ftness, but also regulated the glucose and fat metabolism of C. elegans on high glucose diet.
Excessive glucose intake may lead to an enhanced oxidative stress by increasing the level of cellular reactive oxygen species (ROS), thereby accelerating the aging process.Specifcally, sustained hyperglycemia induces the formation of ROS via increasing the formation of advanced glycation end products, activating protein kinase C and mitochondrial electron transport chain, thus causing damages to various macromolecules including protein, DNA, and lipids in the long run [35,36].Terefore, removing the excessive ROS induced by hyperglycemia was considered to be an efective strategy to alleviate diabetes complications [37].In our study, high glucose feeding increased the oxidative stress in C. elegans through accumulating ROS and reducing the SOD activity and GSH content, while ASTE treatment signifcantly decreased the oxidative stress induced by high glucose.Combined with the fact that ASTE could inhibit the activity of α-glucosidase [10], our results were in line with a previous study that inhibition of α-glucosidase was responsible for decreased oxidative stress [36].Above all, it is conceivable that the reduction of oxidative stress may contribute to the longevity extension efect of ASTE in C. elegans on high glucose diet.
A few previous studies have demonstrated that an increased dietary glucose and lipid intake are closely associated SOD activities, and (c) GSH contents in worms on normal diet or high glucose diet.Statistical analysis was performed by two-tailed Student's t test.* p < 0.05, * * p < 0.01, and * * * p < 0.001 represented the comparison between the 2% glucose group and the normal diet group, while ## p < 0.01 and ### p < 0.001 represented the comparison between the ASTE group and the 2% glucose group.6 Journal of Food Biochemistry with metabolic dysregulation, which ultimately decreases the lifespan of C. elegans [3,14].Based on the fact that some genes involved in regulating glycolipid metabolism also mediated longevity, we further investigated whether ASTE extended the lifespan of C. elegans on high glucose diet through modulating the metabolic-related genes.Te glycosylation gene oga-1 has been reported to afect glucose uptake in C. elegans, and inactivation of oga-1 leads to reduced glucose uptake and prolonged lifespan [38,39].In the current study, ASTE could signifcantly inhibit the  Journal of Food Biochemistry expression of oga-1 in worms grown on high glucose diet, which contributed to the decrease of glucose content as well as the lifespan extension in C. elegans.Furthermore, defciency of lbp-5, an intracellular lipid chaperone gene, to increased ROS production and fat accumulation [40].Our results demonstrated that the expression of lbp-5 in worms fed with high glucose was remarkably elevated by ASTE, thus leading to reduced fat accumulation and enhanced antioxidant capacity.Collectively, these fndings confrmed that the lifespan promotion efect of ASTE was tightly correlated to its regulation of glycolipid metabolism.
In C. elegans, the IIS pathway has been well known to regulate growth, reproduction, metabolism, and aging, while inhibiting the IIS pathway is an efective strategy to prolong lifespan [41,42].Tus, we examined whether the longevity promotion efect of ASTE on high glucose diet was dependent on the IIS pathway.In the current study, the lifespan extension was completely abolished in the daf-2 and daf-16 null mutants, which were two critical regulatory factors of the IIS pathway, indicating that daf-2 and daf-16 were required for ASTE to prolong the lifespan on high glucose diet.Besides, a previous study has reported that glucose-enriched diets shorten the lifespan of C. elegans by downregulating the DAF-16/FOXO, but the mechanism by which high glucose inhibiting DAF-16/FOXO remains unclear [43].Our results revealed that high glucose feeding signifcantly upregulated the expressions of daf-2, age-1, and akt-2, and downregulated the expressions of daf-16, sod-3, mtl-1, and hsp-12.3,indicating that high glucose inhibited the activity of DAF-16/FOXO by activating the IIS pathway, and ultimately reduced the lifespan of worms.However, ASTE addition was proved to upregulate DAF-16/FOXO and its downstream target genes by downregulating the IIS pathway.Tis was in line with a previous report that chlorogenic acid, one of the main bioactive ingredients of ASTE, extended the lifespan of C. elegans by modulating the IIS pathway [44].
It has been well documented that overconsumption of glucose may result in increased cellular oxidative stress, which negatively impacts glucose metabolism and apoptosis * p < 0.05, * * p < 0.01, and * * * p < 0.001 represented the comparison between the 2% glucose group and the normal diet group, while ## p < 0.01 and ### p < 0.001 represented the comparison between the ASTE group and the 2% glucose group.[25].Te transcription factor SKN-1, ortholog of the mammalian Nrf2 in C. elegans, plays a key role in regulating oxidative stress response pathway and longevity [45].Considering that ASTE could reduce the oxidative stress in worms on high glucose diet, we investigated the role of SKNin ASTE-mediated lifespan extension.Our results indicated that high glucose treatment obviously inhibited the expression of skn-1, which was consistent with a previous report that high glucose intervention shortened the lifespan of worms by inactivating SKN-1 [46].Nevertheless, ASTE treatment considerably upregulated the skn-1 and failed to extend the lifespan of skn-1 deletion mutants, indicating that skn-1 was required for the lifespan extension efect of ASTE on high glucose diet.Besides, another study suggested that constitutive activation of SKN-1 could protect the worms from fat accumulation grown on a high glucose diet [47].Tus, future experiments are needed to investigate the role of SKN-1 in ASTE-regulated fat metabolism.
AMPK, as a crucial cellular energy sensor, has been reported to regulate energy homeostasis and metabolism, and high cellular energy levels caused by a glucose-rich diet will lower the AMP/ATP ratio and inactive AMPK [3,48].Tis was supported by the fact that high glucose uptake disrupted aak-2, a homologue of AMPK, and led to survival reduction in C. elegans [49].Consistently, our results showed that high glucose treatment downregulated the aak-2, while ASTE addition enhanced the expression of aak-2.Combining with the fact that ASTE failed to prolong the lifespan of aak-2 null mutants on high glucose diet, we speculated that ASTE extended the lifespan of C. elegans by activating akk-2.Tese fndings were consistent with the previous studies showing that the main compounds of ASTE, such as chlorogenic acid, 3,4-dicafeoylquinic acid, 3,5-dicafeoylquinic acid, and 4,5-dicafeoylquinic acid were reported to activate AMPK signaling pathway and regulate lipid metabolism [50,51].Taken together, it is conceivable that in addition to extending the lifespan, the activation of aak-2 might contribute to the lipid-lowering efect of ASTE.

Conclusion
In summary, this demonstrates that ASTE protects C. elegans from glucotoxicity by extending the lifespan, strengthening the locomotive abilities, decreasing the oxidative stress, and lowering the accumulation of glucose and lipid.Meanwhile, the mechanistic study reveals that ASTE can reverse survival reduction by regulating skn-1, aak-2, and IIS pathway.Our fndings reveal the benefcial efects of ASTE in the face of unhealthy high-sugar diet, thus broadening the application of ASTE for health promotion.

Figure 1 :
Figure 1: ASTE extended the lifespan of C. elegans on high glucose diet: (a) lifespan curves of N2 treated with DMSO (control) or 1%, 2%, and 4% glucose and (b) lifespan curves of N2 treated with 0.6% DMSO (control) or diferent doses of ASTE on a high glucose diet (2%).

Figure 2 :
Figure2: ASTE promoted the healthspan of C. elegans on high glucose diet.Te efect of ASTE on the rates of (a) pharyngeal pumping and (b) body bending of worms on the days 3, 6, and 9 of adulthood.Statistical signifcance was performed by two-tailed Student's t test.* * * p < 0.001 represented the comparison between the 2% glucose group and the normal diet group, and ### p < 0.001 represented the comparison between the ASTE group and the 2% glucose group.

Figure 3 :
Figure3: Efects of ASTE on ROS levels and antioxidant enzyme activities of nematodes on high glucose diet: (a) intracellular ROS levels, (b) SOD activities, and (c) GSH contents in worms on normal diet or high glucose diet.Statistical analysis was performed by two-tailed Student's t test.* p < 0.05, * * p < 0.01, and * * * p < 0.001 represented the comparison between the 2% glucose group and the normal diet group, while ## p < 0.01 and ### p < 0.001 represented the comparison between the ASTE group and the 2% glucose group.

Figure 4 :
Figure 4: Efects of ASTE on glucose and fat metabolism of C. elegans on high glucose diet.Te glucose content (a) and triglyceride contents (b) Oil-Red-O staining pictures of worms (c) and relative quantifcation of fat content (d) by Oil-Red-O staining.(e)Te mRNA expression levels of oga-1, lbp-5, fat-6, and acs-2.Statistical signifcance was performed by two-tailed Student's t test.* * p < 0.01, and * * * p < 0.001 meant the comparison between the 2% glucose group and the control group, while # p < 0.05, ## p < 0.01, and ### p < 0.001 meant the comparison between the ASTE group and the 2% glucose group.
(a), the intracellular ROS level of worms on high glucose diet was signifcantly higher than that of worms on normal diet (p < 0.05).However, supplementation with ASTE considerably reduced the ROS accumulation in the worms on high glucose diet.Besides, the SOD activity and GSH content of worms on high glucose diet signifcantly decreased by 39.30% and 40.93%, re- [10]tively, compared with that in worms on normal diet (Figures3(b) and 3(c), p < 0.001 and p < 0.01).With the addition of ASTE, the SOD activity and GSH content of worms on high glucose diet considerably increased by 39.83% and 74.39% (p < 0.01).Abovementioned data demonstrated that ASTE enhanced the antioxidant activity of C. elegans by activating the antioxidant enzymes and lowering ROS accumulation.3.5.ASTE Regulated the Glucose and Fat Metabolism of C. elegans.It has been reported that six cafeoylquinic acid compounds identifed from Artemisia selengensis Turcz.(AST) could inhibit α-glucosidase activity[10].To assess whether ASTE could actually mediate the glucose metabolism in vivo, we checked the glucose content in worms on high glucose diet.As expected, the glucose content of nematodes incubated with 2% glucose was obviously increased by 5.6-fold (p < 0.001), compared with that in the normal diet group, whereas ASTE remarkably declined the glucose content in worms on high glucose diet (Figure4(a), p < 0.01).Meanwhile, the triglyceride contents of worms treated with high glucose signifcantly increased by 2.26fold, suggesting that the dietary glucose was indeed absorbed by C. elegans and stored as fat (Figure4(b), p < 0.01).

Table 1 :
Efects of diferent doses of glucose on the lifespan of C. elegans.
Diferent letters (a, b, c, and d) indicated a signifcant diference among diferent groups.p values were analyzed by the log-rank test.

Table 2 :
Efects of ASTE on the lifespan of C. elegans on 2% glucose diet.Letter a meant the comparison between the 2% glucose group and the control group.Letters (b, c, and d) meant the comparison between the 60 μg/mL, 240 μg/ mL, and 360 μg/mL ASTE group and the 2% glucose group, respectively.p values were analyzed by the log-rank test.