All flies were reared on standard medium at 25 C and approximately 65% humidity under a 12 h light/12 h dark cycle. Flies lines used in this study were as follows: esg-Gal4, UAS-GFP was kindly provided by Dr. Lihua Jin, Northeast Forestry University; 10×STAT92E-GFP and gstD1-GFP lines were gifts from Dr Fengwei Yu, Temasek Life Sciences Laboratory, National University of Singapore. w ¹¹¹⁸ (#5905) were obtained from the Bloomington Drosophila stock center (BDSC; Indiana, United States). 3–5 day old adult female or male were collected using light CO₂ anesthesia and allowed to recover for 2 days before further experimentation.

Flos Puerariae extract (FPE) was purchased from Shaanxi Ruilin parnell biotechnology Co. Ltd. The dried Flos Puerariae was mixed with 50% (V/V) methanol solution with the solid-to-liquid ratio at 1∶30. The mixture was extracted with ultrasound for 2 h at 70°C. The extracted products were then purified sequentially by petroleum ether, ethanol and chloroform-butyl alcohol, and eluted gradually with mixed mobile phase of methanol-chloroform solution in the silica gel column system. In the end, the isolated ingredients were further analyzed by color reaction, ultraviolet spectrophotometry, high performance liquid chromatography, infrared spectrum and mass spectrum. FPE was diluted with a standard cornmeal-molasses medium to different concentrations (0, 1, 5 and 10 mg/ml). w ¹¹¹⁸ flies were supplied with different concentrations of FPE in their standard diet from the 1st instar larvae until experiments. 0.4 mm ML385 (SML 1,833, Sigma, China) and 0.6% SDS together were provided to adult females during experiments.

Liquid chromatography coupled to mass spectrometry (LC-MS) was used as a reference standard for the quality control of FPE. LC-MS analysis was performed as previously described (Wang et al., 2020). Separation was achieved by utilizing a Ascentis Express C18 (100 mm × 2.1 mm i.d., 1.8 µM). Formic acid (0.05%, V/V) with water (eluent A) and acetonitrile (eluent B) were used for gradient elution with the following time program (%B in A): from 0 to 20 (1 min), from 20 to 70 (8 min), from 70 to 95 (3.5 min), from 95 to 0 (1 min), and finally isocratic reconditioning for 1 min. The flow rate was set at 0.35 ml/min and the temperature was set at 40°C. The injection volume is 2 μL. Data acquisitions were performed using Analyst 1.6.3 software (Sciex). Multiquant 3.0.3 software (Sciex) was used to quantify all metabolites.

3–5 days old males or females of the 4 groups (including the control groups and the experimental groups) fed the corresponding mediums were cultured for 2 days, and then removed to empty vials for 2 h. 20 flies per vial were transferred into vials containing five layers of filter paper infiltrated by 0.6% SDS and 5% sucrose solution, or 5% H₂O₂ and 5% sucrose solution, respectively. Filter papers were replaced every 2 days and the number of dead flies was recorded. Four treatments were performed, and each with at least three replicate vials.

The “Smurfs” assay, as previously described, refers primarily to the use of a non-absorbent blue dye (FD&C Blue #1) to analyse the intestinal barrier integrity of Drosophila (Rera et al., 2011). Briefly, after fed in the medium containing 5% sucrose with or without 0.6% SDS for 72 h, females were transferred to the medium with blue dye (2.5% w/v) for 18 h. The number of “Smurfs” was then calculated.

3–5 days old females were starved in empty vials for 2 h at 25°C, then 20 females per vial were transferred to vials containing 5 layers of filter paper on the bottom. Filter papers were wetted with 5% sucrose solution (control) or 0.6% SDS and 5% sucrose solution (experiment). After 16 h, guts were dissected and examined.

After fed in the medium containing 5% sucrose with or without 0.6% SDS for 16 h, 15–20 females per group were used to dissect intestines in cold PBS, the isolated guts were fixed with 3.7% formaldehyde for 30 min at room temperature. Following three washes with PBST for 10 min each, the samples were stained with 4’ 6-diamidino-2-phenylindole (DAPI) for 10 min. The tissues were then mounted in cedar wood oil (Beijing Solarbio Science & Technology Co., Ltd, China) and observed under an Olympus FV1000 confocal microscope (Olympus, Japan). ISCs were identified by their esg-GFP expression, their size, and their basal location within the intestinal epithelium. The experiments were independently repeated at least three times.

Adult females were incubated with 0.6% SDS and 5% sucrose medium or individual 5% sucrose for 16 h, then total RNA of 60 females was extracted with TRIzol reagent (Invitrogen, Carlsbad, CA, UAS) according to manufacturer’s instructions. Total RNA was reverse transcribed using Hieff® reverse transcriptase (Shanghai YEASEN, China). Quantitative PCR was performed with a GFX 96 Connect^TM Optics Module (Bio-Rad Laboratories) using Multiplex PCR Master Mix (Shanghai YEASEN, China). Ribosomal gene RP49 was used as the internal control. The primer sequences were listed in Table 1. The gene expression was calculated using the comparative threshold cycle (Ct) method. The levels of gene expression in all groups were shown as a ratio to the SDS treated control group value. At least three replicates were established for each group, and all experiments were repeated at least three times.

Statistical analysis was performed using GraphPad Prism software. Comparisons among groups were made by analysis of variance (ANOVA) followed by Dunnett’s t-test. Survivorships among groups were compared and tested for significance with a Log-rank test. Differences between treatment groups were considered to be statistically significant at *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001.

The primary constituent of Flos Puerariae is isoflavonoids, including puerarin, daidzein, daidzin, genistein and apigenin (Eom et al., 2018; Lertpatipanpong et al., 2020). To identify these bioactive compounds in FPE, LC-MS analysis was used. The chromatogram of the standard was eluted at a retention time of 30 min, and it was determined that FPE contained puerarin, daidzein, daidzin, rutin, apigenin and genistein (Figure 1).

To determine the protective effect of FPE, adult females or males were orally treated with inflammatory reagent or ROS-producing agents, SDS or H₂O₂ respectively. SDS interferes with the normal function of the intestinal barrier and stimulates local and systemic inflammation (Song et al., 2021). Survival rates were remarkably increased in FPE treated females or males compared to the control flies, following exposure to SDS (Figures 2A–D). FPE had stronger protective function in females than in males, in which 10 mg/ml FPE treated males did not enhance the survival rates under SDS stimulation. FPE treated flies did not exhibit significantly extended survival rates compared to the control flies under H₂O₂ stimulation (Figures 2E,G). However, females fed with 5 mg/ml and 10 mg/ml FPE remarkably increased the maximal life span (Figures 2F,H), indicating FPE had weak antioxidant ability in vivo. Thus, these findings indicate that FPE has protection against chemical-induced inflammatory injury in flies, and the protective function of FPE is stronger in females than in males.

Drosophila development is considered to be a high-throughput method for assessing drug safety (Abnoos et al., 2013; Gao et al., 2020). To determine the effect of FPE on development, we analyzed the effect of FPE on growth phase and hatchability. FPE supplementation did not regulate the growth phase from egg to pupa (Figures 3A,B) and also did not affect hatchability (Figure 3C). It indicates that FPE has no role to mediate development. In addition, the body weight was determined in adult flies after hatching (Figure 3D). Flies fed with FPE at 1, 5 and 10 mg/ml had the similar body weight with control flies. Next, we attempted to determine the possible effect of FPE (1, 5 and 10 mg/ml) on food intake (Figures 3E,F). As a control, the total amount of food consumption was not altered when flies fed with dose-dependent FPE. Thus, these results indicate that FPE has no effect on the development or metabolism in flies.

The ingestion of SDS could induce melanotic tumors and morphological changes in the Drosophila intestine (Liu et al., 2016). To evaluate whether FPE maintains intestinal morphological integrity under SDS stimulation, the “Smurfs”, intestinal length and melanotic tumors were tested in females after 0.6% SDS treatment for 3 days. The “Smurfs” experiment was used to determine the integrity of the intestinal epithelial barrier (Rera et al., 2011). Approximately 35% of SDS-treated flies had “Smurfs” in the gut, and the proportion of “Smurfs” decreased to 25% after 5 mg/ml FPE treatment (Figures 4A,B). SDS supplementation significantly shorted the intestinal length (Figure 4C). Following SDS treatment, female flies fed with 5 mg/ml FPE had longer intestinal length than control flies. About 30% “melanotic tumors” were observed in SDS treated flies, and it was decreased to about 12% after flies fed with FPE (Figures 4D,E). Together, these results suggest that SDS ingestion disrupts the intestinal barrier and morphology, and FPE has protective function in flies.

Intestinal morphology damage leads to the activation of intestinal stem cells (ISCs) proliferation to regenerate the damage intestinal epithelium (Wu et al., 2020). We next investigate the effect of FPE on ISCs proliferation induced by SDS (Figure 5A). First, we characterized the number of dividing cells using Escargot (Esg), a specific marker of stem cells and enteroblasts (Korzelius et al., 2014). Esg-Gal4; UAS-GFP reporter gene was only rarely expressed in a small, dispersed subset of rounded cells in the intestinal epithelium (Figure 5B). SDS stimulation led to the significant increase in the area and number of GFP-positive cells along the gut, which indicates an extensive increase in stem cell-derived cells (Figure 5C). 5 mg/ml FPE supplementation reduced the increased area and number of GFP-positive cells in the gut (Figures 5D–I). To extend this finding, we used an anti-phosphohistone H3 (anti-PH3) antibody that marks dividing cells to stain gut. A high number of PH3-positive cells were detected in the intestinal epithelium of SDS treated flies, while a very low number of PH3-positive cells in control flies gut (Figures 5E,F). 5 mg/ml FPE supplementation significantly decreased the number of PH3-positive cells in the gut (Figures 5D–J). Thus, FPE can inhibit the ISCs abnormal proliferation induced by SDS.

Extensive ROS is produced in the gut under injury, and can stimulate ISCs proliferation and damage the intestinal epithelium (Zhang et al., 2020). To assess the effect of FPE on ROS levels during SDS stimulation, the gstD1 (gstD1-GFP) transgenic flies sensitive to ROS were used (Zemanová et al., 2016; Tan et al., 2018). The activity of gstD1 is increased in differentiated cells and tissues of flies in response to stress (Tan et al., 2018). We determined the role of FPE on regulating ROS levels in intestinal epithelium under SDS stimulation. We found that under SDS stimulation, gstD1-GFP was expressed highly in intestinal epithelium (Figures 6A,B). 5 mg/ml FPE supplementation significantly inhibited the gstD1-GFP expression in SDS-treated flies (Figures 6C,G). Next we detected ROS level in the gut using dihydroethidium (DHE). After exposed to SDS for 72 h, flies had a robust intensity of fluorescence probe in the midgut compared to control flies (Figures 6D,E,H). 5 mg/ml FPE supplementation remarkably reversed the SDS-induced ROS accumulation (Figures 6F,H). These results suggest that FPE has function to protect the intestinal epithelium against SDS-induced oxidative damage.

To further certificate the role of FPE on regulating Nrf2/Keap1 signaling mediated by SDS, we detected the mRNA expression of CncC, dKeap1, gstD1, gstD2, Sod1, Sod2 and Cat in gut. As depicted in Figure 6I, exposure of females to SDS for 16 h initiated the increased mRNA expression of CncC, Keap1, gstD1, gstD2, Sod1 and Cat. After 5 mg/ml FPE treatment, CncC, dKeap1 and gstD1 expressions were recovered in SDS-treated flies (Figure 6I). To extend this studying, we fed Nrf2 inhibitor ML385 to inhibit the activity of Nrf2 in flies. 0.4 mm ML385 supplementation did not affect the survival rate under SDS stimulation in control flies, while it shorted the survival rate in FPE treated females (Figure 6J). 5 mg/ml FPE supplementation still extended the survival rate in ML385 treated females, following exposure to SDS. It indicates that FPE protect against SDS-induced injury via activating Nrf2/Keap1 signaling and other signaling.

To further examine the molecular mechanism of FPE on protecting the intestinal injury, firstly network pharmacology analysis was used. JAK-STAT signaling pathway were defined as one of core pathways (Supplementary Figure S1). The JAK-STAT pathway can be activated by cytokines, such as the Unpaired family (UPD, UPD2 and UPD3) in the midgut (Zhou et al., 2013). We then investigated the activity of JAK-STAT pathway in the gut of flies. A fly line carrying a reporter construct composed of ten tandem Stat92E DNA binding sites inserted upstream of a minimal promoter and a GFP reporter (referred to as 10×STAT92E-GFP) (Bach et al., 2007). We found a weak GFP signal was present in intestinal epithelium of control flies (Figure 7A). The increased number of GFP-positive cells was detected in the gut after SDS stimulation for 16 h (Figure 7B). However, 5 mg/ml FPE supplementation remarkably inhibited the increased levels of GFP expression along the gut in SDS stimulated flies (Figures 7C–D). It indicates that SDS can activate JAK-STAT pathway in the gut, which is inhibited by FPE. Then, we monitored the mRNA levels of Upds (Upd, Upd2 and Upd3) and JAK-STAT pathway (Hop and Socs36E) (Bach et al., 2007). SDS stimulation increased expression of Upd, Upd2, Upd3, Hop and Socs36E in the gut (Figure 7E). mRNA expression levels of Upd, Upd2, Upd3 and Hop was decreased, but Socs36E expression was increased in SDS stimulated flies fed with 5 mg/ml FPE compared to flies without FPE treatment (Figure 7E). It indicates that FPE has function to inhibit the increased JAK-STAT pathway induced by SDS in the gut. The activation of Wnt signaling pathway is a classic feature of chronic IBD and its animal models (Bradford et al., 2017; Lee et al., 2021). We measured mRNA levels of Wg, the homologue of wnt-1, and Arm as a homology of β-catenin in the gut (Clevers, 2006). SDS ingestion resulted in a significant increase in Wg and Arm gene expression in the intestine, which was reduced by FPE intervention (Figure 7F). It suggests that FPE can inhibit the activation of the Wnt signaling pathway. Together, these results indicate that FPE has function to protect intestinal homeostasis and ISCs proliferation by inhibiting JAK-STAT pathway and Wnt signaling pathway.