Abstract: In order to study the molecular mechanism of rosemary extract (RE) on female Drosophila melanogaster life extension, 2-d-old female Drosophila melanogaster were selected and randomly divided into four groups, namely the control group, the low-dose group (0.2 mg/mL RE), the medium-dose group (0.5 mg/mL RE), and the high-dose group (1.5 mg/mL RE), and the culture medium was changed every 3 days. The medium was changed once every 3 d. Life span, body weight, food intake, activity of antioxidant enzymes, staining of intestinal anatomy, and quantitative real-time polymerase chain reaction (RT-qPCR) were performed at different time points of feeding.
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The results showed that the addition of RE increased the lifespan by 13.10% and protected against oxidative stress damage, and the RE treatment enhanced the activity of endogenous antioxidant enzymes and reduced the malondialdehyde (MDA) content. In addition, RE inhibited the mRNA expression of mTOR and activated autophagy to maintain intestinal homeostasis. At the same time, RE improved the intestinal barrier function, reduced the abnormal proliferation rate of intestinal stem cells (ISCs) by 35.89%, and improved the intestinal integrity of aged Drosophila. In conclusion, RE can maintain intestinal homeostasis through PI3K, a key gene of insulin pathway, and mTOR, a key gene of rapamycin pathway, and improve antioxidant and stress resistance, thus prolonging life span.
Aging is an unavoidable physiological phenomenon that leads to a gradual decline in the function of human organs. The "free radical theory of aging" is supported by many studies [1]. Reactive oxygen species (ROS) have been reported to be one of the major factors contributing to aging [2]. In living organisms, the maintenance of appropriate levels of ROS is essential for the maintenance of redox homeostasis. In a healthy state, free radicals are used to scavenge senescent cells, clean up or control mutated cells, and keep the body in a harmonious and organized state[3] . However, oxidative stress caused by excess peroxides and free radicals can lead to apoptosis or necrosis, resulting in the development of many diseases that accelerate aging. As we age, the activity of endogenous antioxidant enzymes decreases, leading to a reduction in the function of the antioxidant defense system, an increase in free radical damage, and aging [4].
Rosemary is a plant of the genus Rosmarinus officinalis in the family Labiatae, with a long history of development along the Mediterranean Sea[5] . Rosemary extract (RE) has a variety of biological functions such as antioxidant, anti-inflammatory, antimicrobial, immunomodulatory and lipid metabolism, and is a potential alternative to antibiotics[6] . Many reports have shown that there are many active substances in RE, such as rhamnoside, rhamnol, and rosmarinic acid[7] . The previous experiments also found that 1.5 mg/mL RE has certain antioxidant effects on both male and female Drosophila, and can effectively delay oxidative damage caused by high fat, thus further delaying aging[8-10] . Drosophila melanogaster, as a classical model organism, has aging genes and metabolic pathways that are very similar to those of humans[11] . It was found that female Drosophila has a high estrogen content and is more sensitive to intestinal development than male Drosophila[12] , so female Drosophila was chosen as a model to study the intestinal tract.
At the molecular level, human aging is closely related to the insulin pathway, a highly conserved transduction pathway that regulates the life cycle [13]. Akt, as a pivot between the insulin pathway and the rapamycin pathway, is activated by insulin growth factor, which inhibits the activity of the mTOR-negative regulators, TSC2 and TSC1, and thus reduces autophagy regulation and accelerates aging [14]. In recent years, it has been found that autophagy can be activated by mTOR. In recent years, autophagy has been found to regulate the proliferation of intestinal stem cells (ISCs) and intestinal barrier dysfunction, in order to maintain intestinal homeostasis and reduce diseases caused by intestinal imbalance [15].
In recent years, the antioxidant activity of RE has been widely reported, but its mechanism of regulating lifespan in mTOR and autophagy pathways has been less studied. Therefore, in the present work, we investigated the antioxidant effects of RE on female Drosophila, its regulation of intestinal function during aging in female Drosophila, and the potential molecular mechanisms, so as to provide a reference for the development and further exploration of RE products.
1 Materials and Methods
1.1 Materials and reagents
RE: Tianjin Jianfeng Natural Products Research and Development Co., Ltd; wild-type Drosophila W1118: provided by the Research Laboratory of Food Additives and Nutritional Regulation, Tianjin University of Science and Technology; Esg-Gal4 UAS-GFP transgenic Drosophila, which carries the marker green fluorescent protein of Drosophila intestinal precursor cells, was provided by the School of Life Sciences, Northeast Forestry University; hydrogen peroxide, non-digestible dye (FD&C blue NO 1), Trizol reagent, SYBR Green dye, Lyso-Tracker Red fluorescent probe: Sigma Corporation, USA. Hydrogen peroxide, non-digestible dye (FD&C blue NO.1), Trizol reagent, SYBR Green dye, Lyso-Tracker Red fluorescent probe: Sigma Co.
cDNA (complementary DNA) reverse transcription kit: Sigma, USA; total superoxide dismutase (SOD) kit, copper/zinc superoxide dismutase (Cu/Zn-SOD), manganese superoxide dismutase (Mn-super- oxidedismutase, Mn-SOD) kit, catalase (CAT) kit, malondialdehyde (malondialdehyde) kit, and manganese superoxide dismutase (Mn-super- oxidedismutase, Mn-SOD) kit. Cu/Zn-superoxide dismutase (Cu/Zn-SOD), manganese superoxide dismutase (Mn-super- oxidedismutase, Mn-SOD) kit, catalase (CAT) kit, malondialdehyde (MDA) kit: Nanjing Jianjian Reagent Co. Lactobacillus agar, tryptone, yeast powder, NaCl, glucose, agar, mannitol, nutrient agar (all analytical pure): Beijing Dingguo Changsheng Biotechnology Company. The primers were designed by Beijing Dingguo Changsheng Biotechnology Company.
1.2 Instruments and equipment
OLYMPUSU-RFLT50 fluorescence microscope: Olympus, Japan; G:box gel image acquisition and analysis system: Syngene, U.K.; BIO-RAD real-time quantitative polymerase chain reaction (PCR) instrument: Beijing Yuanye Bole Technology Development Co; Thermo Finnigan Surveyor liquid chromatograph mass spectrometer (LC-MS): Thermo Finnigan Company, USA; SMZ-140 enzyme labeling instrument: McAudi (Xiamen) Electric Co: Glass homogenizer: Anhui Weiss Experimental Equipment Co.
1.3 Component analysis of RE
A Thermo Finnigan Surveyor LC-MS system equipped with a photodiode array detector and a Venusil XBP C18 column (2.1 mm×150 mm) was used for the determination of the components in the rosemary extract. The flow rate was 0.2 mL/min, the detection wavelength was 280 nm, and the oven temperature was 30 ℃. The mobile phases were 0.1% A phase (formic acid) and B phase (acetonitrile). The gradient elution program was as follows: 10% B: 0-5 min; 20% B: 40 min-45 min; 90% B: 45 min-55 min; and finally 10% B for 55.1 min-60.0 min. The mass spectrometry was performed in positive ionization mode using an electron spray ionization (ESI) source with a spray voltage of 4.5 kV, a capillary voltage of -10 V, a capillary temperature of 275 ℃, a nitrogen (N2) flow rate of 30 arb, and an auxiliary gas flow rate of 5 arb. The molecular weights were in the range of 100~500 m/z in the full scan mode. The molecular weight range was 100~500 m/z in full scan mode.
1.4 Preparation of Drosophila culture media
Drosophila basal medium (750 mL): distilled water (750 mL), cornstarch (72 g), anhydrous dextrose (72 g), yeast powder (10 g), agar powder (6 g), and preservative (40 mL) (1% ethyl para-hydroxybenzoate) [16]. Drosophila experimental medium was prepared by adding RE (0.2, 0.5, 1.5 mg/mL) to the basal medium.
1.5 Lifetime experiment
Wild-type Drosophila W1118, which had not been mated for 2 d, was divided into four groups of 200 individuals each. One group was a control group on basal diet, and the other three groups were 0.2, 0.5, and 1.5 mg/mL RE experimental groups, respectively. The medium was changed every 3 d and the number of survivors was recorded. Maximum lifespan was calculated as 10% maximum survival time[17] .
1.6 Food intake and body weight experiments
Wild-type Drosophila W1118 was collected 2d after plumage, and randomly divided into 4 groups, 200 flies in each group, and cultured as in 1.5 Drosophila Life Span Cultivation Methods. Drosophila were cultured as in 1.5 Drosophila life span culture method. Food intake was measured: after 4 days of culture, the flies were first transferred to empty tubes containing filter paper strips moistened with distilled water and starved for 2 h, and then transferred to diets containing 0.2% rhodamine B sodium sulfonate for 2 h. The degree of red abdomen was measured using a double-blind subjective scale of 0 (colorless abdomen) to 5 (completely red abdomen), and the degree of redness was used as an indicator of food intake for each Drosophila [17]. Body weight experiment: The change in body weight was also considered as an indicator of food intake. On day 20 of rearing, Drosophila were anesthetized with CO2 and then weighed and the mean body weight was calculated [18].
1.7 Climbing experiments
Drosophila melanogaster treated with RE (0.2, 0.5, 1.5 mg/mL) or control were transferred to empty tubes at 0, 15, 30, and 45 d. The number of Drosophila that climbed more than 7 cm in 20 s was counted. The experiments were conducted three times in parallel [18].
1.8 Stress injury experiments
Wild-type Drosophila W1118 were collected at 2 d post-feathering and randomly divided into four groups of 200 flies each. flies were starved for 2 h at 25 d in RE (0.2, 0.5, 1.5 mg/mL) or control treatments, and then transferred to new vials containing strips of filter paper impregnated with 1 mL of saturated 30% hydrogen peroxide (H2O2) or 6% dextrose solution containing 20 mmol/L paraquat. The vials contained 1 mL of filter paper strips saturated with 30% hydrogen peroxide (H2O2) or 6% glucose solution containing 20 mmol/L paraquat. The number of survivors was counted every 2 h until all Drosophila died, and each independent experiment was repeated three times [9].
1.9 "Smurf" experiments
Wild-type Drosophila W1118 were collected 2 d after plumage and randomly divided into 4 groups of 200 flies each. Drosophila were cultured as in 1.5 Drosophila Lifespan Culture Method. On the 20th and 50th day, the control group was fed in medium without RE and containing 2.5% blue dye, while the experimental group was fed in medium with different concentrations of RE and containing 2.5% blue dye. After 2 h of starvation, Drosophila were fed with blue dye for 9 h, then stunned with CO2, and their abdomens were observed under a microscope to count the number of "Smurfs" [17]. Each independent experiment was repeated three times.
1.10 Determination of total intestinal colonization
Lactobacilli (LMRS) selective medium: 70 g/L Lactobacilli agar; Enterobacteria (ENT) selective medium: 10 g/L peptone, 1.5 g/L yeast extract, 10 g/L glucose, 5 g/L NaCl, 12 g/L agar; Acetobac- teria (ACE) selective medium: 25 g/L mannitol, 5 g/L yeast extract, 3 g/L peptone, 15 g/L agar; Nutrient culture medium: 25 g/L mannitol, 5 g/L yeast extract, 3 g/L peptone, 15 g/L agar. Acetobac- teria (ACE) selective medium: 25 g/L mannitol, 5 g/L yeast extract, 3 g/L tryptone, 15 g/L agar; nutrient rich medium (NR): 23 g/L nutrient agar.
Wild-type Drosophila W1118 was collected 2 days after plumage and randomly divided into 4 groups of 200 flies each, and cultured as in 1.5 Drosophila Lifespan Cultivation Methods. When the flies were reared for 20 and 50 d, they were fasted for 2 h. After anesthetized with CO2, the midgut (n=10) was taken out with a dissecting needle under a microscope and placed in 1 mL of phosphate buffer (pH 6), and then grinded and diluted with a glass homogenizer. 0.1 mL of the diluted solution was coated on the selective medium of Enterobacteriaceae (ENT), Lactobacillus lactis (LMRS), Acetococcus acetosus (ACE), and nutrient medium (NR), and the colonies were counted by plate counting. The total number of intestinal colonies was calculated by plate colony counting[16] . Each independent experiment was repeated three times.
1.11 Fluorescent Staining and Fluorescence Microscopic Observation
Esg-Gal UAS-GFP Drosophila were cultured with RE (0.2, 0.5, 1.5 mg/mL) or control diet for 20 or 50 d. The midgut was dissected out and the fluorescent spots were observed under an inverted fluorescence microscope. In the fluorescence staining experiments, Drosophila were fed with RE or normal diet for 20 d. The intestines were dissected in phosphate buffer and stained with 1 μmol/L Lyso-Tracker Red Fluorescent Staining Solution for 3 min; washed with phosphate buffer (pH 6) and fixed in 4% formaldehyde for 30 min, and then sealed with 70% glycerol. The slices were visualized using an inverted fluorescence microscope[19] . Each independent experiment was repeated three times.
1.12 Determination of antioxidant enzyme activity and lipid peroxidation content
Wild-type Drosophila W1118 (n=200) was collected 2 d after plumage and cultured according to the Drosophila lifespan culture method in 1.5. Drosophila were continuously cultured for 45 d, starved for 2 h, weighed, homogenized and diluted at 1:9 (mg/mL) body weight in saline, and the supernatant was taken to determine the enzyme activities of SOD, CAT, MDA and MDA according to SOD, CAT, MDA kit method [20].
1.13 Real-time fluorescence quantitative polymerase chain reaction experiments
Drosophila were fed with RE (0.2, 0.5, 1.5 mg/mL) or control diet for 45 d and ground in liquid nitrogen . Total RNA was extracted using TRizol and cDNA was constructed using a cDNA reverse transcription kit. gene expression was calculated based on cycle threshold (CT) values. The primer information for each gene amplification is shown in Table 1 (RP49 was used as the internal reference gene).
1.14 Statistical analysis
Statistical analysis was performed using Origin 2021 and the results are expressed as mean ± standard deviation. Survival experiments were analyzed by Kaplan- Meier analysis using SPSS 17.0 software and significant differences between groups were analyzed by log-rank test. One-way analysis of variance (ANOVA) was used to assess the significance of differences between means. Fluorescence levels were analyzed using ImageJ software. p<0.05 indicates significant differences, while p<0.01 indicates highly significant differences.
2 Results and analysis
2.1 Components of RE
LC-MS analysis was used to identify the active ingredients in RE and the results are shown in Table 2.
A total of five different compounds were identified by liquid chromatography-mass spectrometry (LC-MS/MS), as shown in Table 2, which were sage phenol, sage acid, ligustral phenol, ligustral acid, and methyl ligustralate, respectively.
2.2 Effect of RE on Drosophila lifespan
The effect of RE on the longevity of female Drosophila is shown in Table 3.
As the body's ability to self-regulate and function decreases [21], the aging process is often accompanied by a decrease in the body's adaptability or resistance to external stress [3]. As shown in Table 3, compared with the control group, the average lifespan of the 1.5 mg/mL RE group increased from 60 d to 70 d, which was 16.67% (P＜0.05); the maximum lifespan of the 1.5 mg/mL RE group increased from 84 d to 95 d, which was 13.10% (P＜0.05); the lifespan of the 1.5 mg/mL RE group increased from 84 d to 95 d, which was 13.10% (P＜0.05); and the lifespan of the 1.5 mg/mL RE group increased from 84 d to 95 d, which was a dose-dependent effect. Therefore, it is believed that RE has the effect of delaying aging.
2.3 Effect of RE on Drosophila feeding and body weight
The effects of RE on food intake and body weight of female Drosophila are shown in Fig. 1 and Table 4.
Dietary restriction has been previously reported to have a lifespan-extending effect [22], but in the present study, in order to exclude the effect of dietary restriction on lifespan, Drosophila body weight and food intake were measured according to the method described in 1.6. As shown in Table 4, the average body weight of female Drosophila after RE feeding was not significantly different from that of the control group, and no significant difference was found between the experimental group and the control group by measuring the amount of food intake of the female Drosophila (Fig. 1).
2.4 Effect of RE on Drosophila crawling ability
With the aging of the organism, physiological functions and self-regulation ability gradually weakened, and the crawling ability indirectly reflected the health status and aging of the organism[19] .The effects of RE on the crawling ability of female Drosophila are shown in Fig. 2.It can be seen in Fig. 2 that the crawling ability of Drosophila increased in a dose-dependent manner with the addition of RE, and the average percentage of crawling of the 1.5 mg/mL RE-treated group increased significantly by 18.25% (P < 0.05) compared with the control group. The average percentage of crawling in the 1.5 mg/mL RE-treated group significantly increased by 18.25% (P<0.05) compared with the control group. The crawling ability of Drosophila was significantly improved after the administration of RE, and the mean percentage of crawling in the 1.5 mg/mL RE group increased by 33% and 28% at day 30 and day 45, respectively. The above results showed that RE could improve the crawling ability of Drosophila and enhance the regulation of the body.
2.5 Effect of RE on Drosophila resistance to oxidative stress
The effects of RE on paraquat and hydrogen peroxide stress in female Drosophila are shown in Figure 3.
In different biological models, lifespan is closely related to increased stress tolerance[23] . Redox reactions under normal metabolism or environmental stress generate ROS, which disrupt the intracellular redox balance and accelerate aging. Endogenous cellular antioxidant enzymes, such as SOD and CAT, can maintain the balance of ROS. As shown in Fig. 3 A, 0.5 and 1.5 mg/mL RE can prolong the maximum life span of Drosophila. Compared with the control group, the average lifespan of 1.5 mg/mL RE was significantly increased by 26.32% (P<0.05). As shown in Figure 3B, the maximum lifespan of Drosophila melanogaster was increased by 20.01% and 50.20% (P<0.05) in the 0.5 mg/mL and 1.5 mg/mL RE-treated groups, respectively, compared with that of the control group. The above results indicated that RE had a protective effect on the oxidative damage induced by H2O2 and paraquat.
2.6 Effect of RE on gut microorganisms
The effect of RE on the gut microbiota of female Drosophila is shown in Figure 4.As the organism ages, the regulatory capacity decreases, the intestinal microbiota structure becomes unbalanced, intestinal barrier dysfunction occurs, and free radicals accumulate [24]. In recent years, intestinal homeostasis has been shown to play an important role in determining the lifespan of Drosophila [25]. As shown in Figure 4, after 20 d of RE feeding, there was no significant difference in LMRS, ENT, ACE and NR in the RE group compared with the control group. After 50 d of RE administration, the number of microorganisms in the intestinal tract was significantly reduced compared with that of the control group (P < 0.05). In female Drosophila, when the concentration of RE was 1.5 mg/mL, the LMRS, ENT, ACE, and NR were down-regulated by 15.56%, 14.62%, 28.31%, and 13.27%, respectively, when compared with the control group (P<0.05). The results showed that RE was able to maintain the balance of intestinal microorganisms in aged female Drosophila and inhibit the abnormal proliferation of intestinal microorganisms caused by aging.
The effect of RE on the intestinal barrier function of Drosophila is shown in Figure 5.
During aging, a decline in physiological regulation leads to a decrease in barrier function, reduced intestinal integrity and increased permeability. Gut homeostasis is one of the determinants of lifespan in Drosophila. A non-digestible dye (FD&C blue NO.1) was fed to Drosophila to observe the aging of intestinal tissue. As shown in Fig. 5 A, when Drosophila were fed the blue dye at 20 d of age, the dye was only present in the prostate and digestive tract. However, at 50 d of age, the blue dye was clearly visible throughout the body. These blue-colored Drosophila are known as "Smurfs", and the proportion of "Smurfs" in the overall sample was counted to reflect the degree of intestinal aging in Drosophila, and furthermore, the degree of aging in Drosophila. In Fig. 5 B, the percentage of Smurfs in the control group increased with age, but at 50 d, the number of Smurfs in the RE-treated group decreased by 22.86% in the 1.5 mg/mL RE group compared with the control group (P < 0.05). Thus, RE may protect intestinal integrity by improving intestinal permeability in Drosophila.
2.7 Effect of RE on the proliferation of ISCs
The effects of RE on the proliferation of intestinal stem cells in female Drosophila are shown in Figures 6 and 7.
The loss of intestinal barrier function in aging Drosophila has been found to be related to Drosophila genotype and environmental conditions. In Drosophila, intestinal tissue homeostasis is maintained by pluripotent ISCs distributed along the basement membrane[26] . As Drosophila ages, the intestinal epithelial cells in Drosophila activate regenerative mechanisms that accelerate stem cell proliferation to maintain damaged cells, but further lead to abnormal stem cell proliferation[27] . GFP-positive cells show a proliferative rate of ISCs, as esg is specifically expressed in ISCs and enterocytes[28] .
Therefore, the expression of esg in the midgut of Drosophila was examined in this paper. Figure 7 shows that the proliferation of control group and RE group remained normal at 20d, with no significant difference (P>0.05). The proliferation of ISCs increased significantly with age. As shown in Figure 7, the number of esg-positive cells in the 1.5 mg/mL RE-treated group was significantly reduced by 35.89% (P<0.05) compared with the control group in aged Drosophila (50 d).
2.8 Effect of RE on antioxidant parameters in Drosophila
The effects of RE on total SOD, Cu-Zn-SOD, CAT activity and MDA content are shown in Table 5, and the effects of RE on the expression of antioxidant genes in Drosophila are shown in Figure 8.
2.9 Molecular mechanisms
The effect of RE on lysosomal fluorescence intensity and quantitative results in female Drosophila are shown in Fig. 9, and the effect of RE on insulin pathway and autophagy genes are shown in Fig. 10.
Autophagy is a powerful initiator of metabolic homeostasis, maintaining homeostasis and preventing degenerative diseases by degrading lipid peroxidation, damaged organelles and pathogens[29] . Autophagy regulates intestinal barrier defense, prevents intestinal flora imbalance, and plays a specific role in maintaining intestinal homeostasis [30]. In order to investigate whether the lifespan extension effect of RE in Drosophila is related to the activation of autophagy, RE treatment was analyzed by lysosomal staining to determine whether it leads to an increase in autophagosomes and lysosomes. It was found that autophagosomes in the intestine increased in a dose-dependent manner after RE treatment (Fig. 9), and the real-time PCR results showed that the autophagy-related genes of Atg-1, Atg-5, Atg-8a, and Atg-8b were increased by 67.21%, 43.16%, 53.23% (P＜0.01), and 45.07% (P＜0.01), respectively, after 1.5 mg/mL RE treatment. and 45.07% (P < 0.05), respectively (Figure 10). Therefore, the above results indicated that RE activated the autophagy pathway.
In nematodes, Drosophila and mammals, inhibition of key genes or proteins in the insulin pathway, which are closely related to aging, can effectively prolong life [31]. As shown in Figure 10, after treating Drosophila with 1.5 mg/mL RE, the mRNA expression levels of PI3k, Akt-1 and mTOR were reduced by 16.04%, 52.14% and 40.07%, respectively, suggesting that RE may regulate lifespan through insulin signaling pathway.
3 Conclusion
Addition of a certain dose of RE significantly prolonged the life span of female Drosophila, slowed down the decline of locomotor activity due to aging, enhanced stress resistance, reduced aging-induced intestinal barrier dysfunction and intestinal flora imbalance, inhibited the abnormal proliferation of intestinal precursor cells, and maintained intestinal ecology The probable mechanism of life prolongation by RE treatment is through the inhibition of insulin pathway and the activation of the rapamycin pathway factor, mTOR, to activate autophagy pathway and improve antioxidant capacity and intestinal imbalance. The possible mechanism of life extension by RE therapy is the activation of the autophagy pathway through inhibition of the insulin pathway and activation of the rapamycin pathway factor mTOR, which improves antioxidant capacity and intestinal imbalance. The results of this study provide a theoretical basis for the subsequent development and application of rosemary functional products, but the exact mechanism remains unclear and needs to be further explored.
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