H. erythrostictumextracts enhance protection against toxic compounds

To determine whether H. erythrostictum water extract (HEWE) and butanol extract (HEBE) have protective effects against toxic substances in the intestine, adult flies were fed DSS or SDS, respectively. These chemicals are toxic compounds that disrupt normal intestinal barrier function and cause intestinal inflammation. To determine the survival rate, adult flies were fed multiple concentrations of HEWE (4, 6, and 8 mg/ml) or HEBE (1, 2, and 4 mg/ml). After 7 days, the survival rates of the flies in the 4% DSS and 0.6% SDS groups substantially decreased by 71% and 96.8%, respectively (Fig. 1A-D). However, compared to those in the control groups, the survival rates of the experimental groups administered HEWE or HEBE were significantly greater, with 8 mg/ml HEWE and 1 mg/ml HEBE having the greatest protective effects. After exposure to 4% DSS, supplementation with 8 mg/ml HEWE or 1 mg/ml HEBE increased survival by 55.8% (P < 0.0001) and 54.2% (P < 0.0001), respectively (Fig. 1A, B). The survival rates increased substantially by 51.6% (P < 0.0001) and 35.5% (P < 0.0001), respectively, when 0.6% SDS was utilized as an inflammatory agent. (Fig. 1C, D). The toxicity of H. erythrostictum extracts to Drosophila was subsequently evaluated. As shown in Fig. 1E and F, the survival rates of the group fed HEWE or HEBE did not differ from the survival rate of the group fed sucrose alone, indicating that the H. erythrostictum extracts were not toxic to Drosophila. Overall, 8 mg/ml HEWE and 1 mg/ml HEBE significantly enhanced the survival rates of flies exposed to the inflammatory factors DSS and SDS. Next, we used only DSS to induce intestinal dysfunction.

Fig. 1

Both HEWE and HEBE enhance the survival of flies exposed to DSS or SDS. (A-D) Wild type w1118 flies were reared on various media. Suc, 5% sucrose medium; DSS, 5% sucrose containing 4% DSS; SDS, 5% sucrose containing 0.6% SDS; experimental group, DSS or SDS medium supplemented with HEWE (4, 6, and 8 mg/ml) or HEBE (1, 2, and 4 mg/ml), respectively. Supplementation with HEWE or HEBE at each concentration significantly increased the survival rate of flies exposed to toxic compounds. (E, F) Flies were reared in 5% sucrose medium supplemented with HEWE (E) or HEBE (F) without toxic compounds. Neither HEWE nor HEBE at any concentration was harmful to fruit flies. Differences in survival were analyzed using the log-rank test. nsP > 0.05,**P < 0.01,***P < 0.001,****P < 0.0001

H. erythrostictum extracts protect against the degradation and dysfunction of intestinal epithelial cells caused by DSS

As H. erythrostictum extracts improved Drosophila survival after ingestion of toxic compounds, we hypothesized that they may protect intestinal function. 7-AAD staining was used to evaluate DSS-induced intestinal epithelial cell death. The fluorescence intensity of 7-AAD was substantially greater in DSS-treated flies than in sucrose-fed flies (P < 0.0001, Fig. 2A, B, N). However, the fluorescence signal decreased by more than 76% after supplementation with H. erythrostictum extracts (HEWE:P < 0.001, HEBE: P < 0.001, Fig. 2C, D, N). In addition, adherens junctions regulate cell adhesion and cytoskeletal organization in a variety of cell types. Armadillo (Arm) is a crucial protein that binds to other proteins to form a cell‒cell junction complex [27]. Previous research has demonstrated that DSS stimulation disrupts and disorganizes the adhesion of intestinal epithelial cells [28]. To assess cell adhesion against intestinal damage, we utilized anti-Arm antibodies. In the sucrose-fed group, clear small progenitor cells and large ECs, which make up the majority of the intestinal epithelium, were observed, whereas in the DSS-treated group, cell boundaries were disorganized and cell types could not be distinguished (Fig. 2E, F). After supplementation with HEWE or HEBE, the cell adhesions resembled those of the sucrose-fed group in terms of clarity and orderliness (Fig. 2G-H).

Fig. 2

Both HEWE and HEBE can prevent DSS-induced intestinal epithelial cell death and dysfunction. (A-D) w1118 flies were exposed to 6% DSS for 72 h, and 7-AAD was used to detect dead cells. The addition of HEWE or HEBE significantly reduced DSS-induced cell death. (E-H) The cell membranes were stained with anti-Arm antibodies. Following the administration of DSS, the intestinal cell arrangement was disrupted; however, supplementation with HEWE or HEBE restored the aberrant cellular arrangement. (I-L) Images of ‘Smurf’ flies on different foods. ‘Smurf’ flies discharge blue dye from the intestine into other tissues. (M) Illustration of intestinal acid‒base homeostasis indicated by the bromophenol blue pH indicator. ‘Homeostasis’ indicates that the CCR is yellow, ‘Perturbed A’ indicates that the CCR is blue, and ‘Perturbed B’ indicates that the flies were not administered bromophenol blue and that the intestinal tract was not stained with a pH indicator. (N) Fluorescence intensity of 7-AAD as measured in A-D (n = 12–16). Three separate experimental repeats were performed. (O) Quantification of the percentage of Smurf flies in I-L. Sixty flies were included in each group. Three separate experimental repeats were performed. (P) The percentage of intestinal acid‒base homeostasis in M. Each group consisted of 60 flies. Three experimental repeats were performed. Quantification results represent mean ± SEM.ns P > 0.05, *P < 0.05, **P < 0.01,***P < 0.001,****P < 0.0001. Scale bars: 50 μm

Cell mortality and disordered cell arrangement may induce disruption of the epithelial barrier, thereby increasing intestinal permeability. Next, we determined the integrity of the intestinal epithelial barrier in a Smurf assay. In the Smurf experiment, a nonabsorbable blue food dye (FD&C Blue #1) was used, and flies whose entire bodies exhibited blue color after ingesting the dye were referred to as ‘Smurf’ flies [29]. In the DSS-fed group, 42% of flies exhibited the ‘Smurf’ phenotype (P < 0.0001, Fig. 2I, J, O). In contrast, supplementation with 8 mg/ml HEWE or 1 mg/ml HEBE significantly reduced the Smurf phenotype to 20% (P < 0.01) and 18% (P < 0.001), respectively (Fig. 2K, L, O).

Previous research has suggested that intestinal acid‒base homeostasis plays an essential role in metabolic regulation [30], and instability of intestinal epithelial cells results in a loss of acid‒base homeostasis [31]. The copper cell region (CCR) of fruit flies is located in the middle midgut and is composed of a group of copper cells implicated in gastric acid secretion [32]. Bromophenol blue (BPB) is a pH sensitive dye that transforms from yellow at pH 3.0 to blue at pH 4.6 or higher. Therefore, when Drosophila consumes BPB, the CCR under intestinal homeostasis appears yellow. We used BPB to determine whether DSS administration could disrupt the acid‒base homeostasis of CCR in the intestine. In the DSS-fed group, acid-base homeostasis was significantly reduced by 74.5% (P < 0.0001) compared to that in the sucrose-fed group; however, in the HEWE- and HEBE-supplemented groups, acid-base homeostasis was significantly improved by 25% (P < 0.001) and 23.4% (P < 0.001), respectively, compared to that in the DSS-fed group (Fig. 2M, P). These results suggested that H. erythrostictum extracts could protect against the disruption of intestinal morphology and function caused by DSS administration.

H. erythrostictum extracts reduce excessive AMP levels and steatosis caused by Ecc15

Antimicrobial peptides (AMPs) are small, functional proteins with potent antiviral, antibacterial, and antifungal activities [33]. AMPs for host defense against microbial infection in Drosophila are produced essentially in adipocytes, but also in gut epithelial cells [34]. According to previous studies, AMP can protect intestinal homeostasis in Drosophila by responding to gram-negative bacterial infection [34], but excessive expression of AMP is detrimental to epithelial cells [35,36,37]. In this study, we examined the expression levels of the reporter genes defencin (Def)-GFP, drosomycin (Drs)-GFP, and diptericin (Dipt)-GFP following infection with the gram-negative bacterium Ecc15. The AMP levels were substantially greater in the Ecc15-infected group than in the sucrose-fed group (Def: P < 0.0001, Drs: P < 0.001, Dipt: P < 0.0001); however, after the addition of 8 mg/ml HEWE or 1 mg/ml HEBE, the AMP levels decreased to levels comparable to those in the uninfected group (Def: HEWE P < 0.0001, HEBE P < 0.0001; Drs: HEWE P < 0.05, HEBE P < 0.05; Ditp: HEWE P < 0.0001, HEBE P < 0.0001, Fig. 3A-L, Q-S). To further confirm these results, we performed qRT-PCR analysis of AMP gene expression. As anticipated, the Def, Drs, and Dipt transcript levels were 5.8-, 1.9-, and 6.5-fold greater, respectively, in the infected group than in the uninfected group (Def: P < 0.0001, Drs: P < 0.001, Ditp: P < 0.001). In contrast, supplementation with H. erythrostictum extracts significantly decreased AMP gene transcription (Def: HEWE P < 0.01, HEBE P < 0.01; Drs: HEWE P < 0.001, HEBE P < 0.001; Ditp: HEWE P < 0.01, HEBE P < 0.01, Fig. 3T-V).

Fig. 3

Both HEWE and HEBE can reduce excessive AMP levels and steatosis during Ecc15 infection. (A-L) Using anti-GFP antibodies, immunofluorescence images of the posterior midgut were obtained. The transgenic fly lines Def-GFP (A-D), Drs-GFP (E-H), and Dipt-GFP (I-L) were infected with Ecc15 for 16, 16, and 24 h, respectively. Infection with Ecc15 substantially increased Def, Drs, and Dipt levels. However, supplementation with HEWE or HEBE significantly reduced the increase in AMP levels caused by Ecc15 infection. (M-P) Bodipy was used to obtain representative images of midgut lipid droplets (LDs). Ecc15 infection causes LD accumulation in the midgut, but supplementation with HEWE or HEBE significantly decreases LD accumulation. (Q, R, S) Fluorescence intensities of Def-GFP (Q), Drs-GFP (R), and Dipt-GFP (S) in A-D, E-H, and I-L, respectively (n = 12–17). (T-V) After Ecc15 infection for 6 h, qRT-PCR analysis of Def (T), Drs (U), and Dipt (V) was performed. All mRNA levels were normalized to rp49 expression. (W) Quantification of the fluorescence intensity of LDs in M-P (n = 12–14). Quantification results represent mean ± SEM.*P < 0.05, **P < 0.01,***P < 0.001,****P < 0.0001. Scale bars: 50 μm

Adult fly bacterial infection induces intestinal steatosis due to lipid accumulation, and intestinal steatosis is an antibacterial immune response marker and modulator [38]. BODIPY staining revealed significant lipid accumulation in response to Ecc15 infection (P < 0.0001); however, supplementation with HEWE or HEBE reduced lipid droplet numbers in the intestine (HEWE: P < 0.0001, HEBE: P < 0.001, Fig. 3M-P, W). Taken together, these findings showed that H. erythrostictum extracts have intestinal-protective effects by regulating excessive AMP levels and lipogenesis in the Drosophila gut caused by Ecc15 infection.

H. erythrostictum extracts eliminate the excessive accumulation of ROS in the intestine

In clearing microbes from the gut, reactive oxygen species (ROS), which are highly effective immune effector molecules, exert broad-spectrum microbicidal effects [39]. However, ROS are destructive to intestinal epithelial cells; therefore, their effects must be transient and at low levels [40]. Excessive amounts of ROS cause tissue damage by oxidizing proteins, lipids, and DNA [41]. In the present study, for the first time, the ROS-scavenging activity of H. erythrostictum extracts was examined. The levels of ROS in the intestine were measured using dihydroethidium (DHE) and 5-(6)-chloromethyl-2’,7’-dichlorodihydrofluorescein diacetate (CM-H2DCFDA). After exposure to DSS, flies had a much stronger fluorescence intensity in the posterior midgut than did those fed sucrose (DHE: P < 0.0001, CM-H2DCFDA: P < 0.0001). However, 8 mg/ml HEWE or 1 mg/ml HEBE supplementation decreased DHE (HEWE: 41.7%, P < 0.0001; HEBE: 32.5%, P < 0.0001) or CM-H2DCFDA (HEWE: 22.2%, P < 0.001; HEBE: 22.1%, P < 0.0001) fluorescence intensity (Fig. 4A-H, M, N). Next, we evaluated intestinal ROS levels in ROS-sensitive GstD1-GFP transgenic flies [42]. In DSS-stimulated flies, the GFP intensity was substantially greater than that in sucrose-fed flies (P < 0.0001, Fig. 4I, J, O), corroborating the findings of a previous study indicating that GstD1 activity is increased in differentiated cells in response to stress [43]. As expected, supplementation with HEWE or HEBE significantly decreased GstD1-GFP levels (HEWE: P < 0.0001, HEBE: P < 0.0001, Fig. 4K, L, O).

Fig. 4

Both HEWE and HEBE can decrease ROS levels in the gut of Drosophila fed DSS. (A-H) ROS levels were monitored with DHE and CM-H2CFDA. Wild-type w1118 flies were exposed for 48 h to 6% DSS for DHE staining and for 96 h to 3% DSS for CM-H2DCFDA staining. After ingesting DSS, ROS levels in the gut were significantly elevated (A, B, E, F). Both HEWE and HEBE ameliorated the DSS-induced increase in ROS levels (C, D, G, H). (I-L) Photographs of the midguts of gstD1-GFP transgenic flies. After treatment with 6% DSS for 48 h, the GFP levels increased significantly (I, J). Supplementation with HEWE or HEBE significantly reduced the expression of gstD1-GFP (K, L). (M) Quantification of DHE intensity in A-D (n = 10–16). (N) Quantification of CM-H2DCFDA intensity in E-H (n = 10–16). Quantification of the GFP intensity in I-L (n = 12–27). Quantification results represent mean ± SEM.***P < 0.001,****P < 0.0001. Scale bars: 50 μm

Through enzymatic and nonenzymatic antioxidant defenses, living cells have evolved a balanced system to counteract excessive ROS levels [44]. Catalase is one of the oldest known enzymes and has been extensively characterized as a critical defense system against reactive oxygen species and free radicals [45]. This enzyme decomposes H2O2 into oxygen and water and plays a crucial role in the metabolism of H2O2 [46]. In addition, superoxide dismutase (SOD) enzymes catalyze the breakdown of superoxide into hydrogen peroxide and water and are central regulators of ROS levels, the first line of defense against free radicals [47, 48]. In a previous study, when excessive ROS were induced with paraquat, ISC proliferation and an increase in progenitor cells were observed [49]. Therefore, we questioned whether Catalase and Sod2 inhibition would result in an increase in progenitor cells. The GFP signal fromesg-Gal4 UAS-GFP flies can be used to identify both ISCs and EBs in the intestinal tract of Drosophila. When Sod2 or Catalase was knocked down in ISCs/EBs using the esg > GFP driver, we showed that the numbers of progenitors and mitotic cells were significantly greater than those inesg > GFP/+ flies (Sod2 RNAi: esg+ cells P < 0.001, PH3+ cells P < 0.0001; Catalase RNAi: esg+ cells P < 0.0001, PH3+ cells P < 0.0001, Figure S1A, D, G, J, M-P). However, supplementation with H. erythrostictum extracts reduced the numbers of esg+ (HEWE: 27.6%, P < 0.0001, 41.4%, P < 0.001; HEBE: 33.4%, P < 0.001, 41.1%, P < 0.0001) and PH3+ (HEWE: 49.6%, P < 0.01, 50.4%, P < 0.0001; HEBE, 36.6%, P < 0.01, 55.9%, P < 0.01) cells compared to those in cells with low levels of antioxidant enzyme-induced increases in cell numbers (Figure S1B, C, E, F, H, I, K, L, M-P). These results indicated that both HEWE and HEBE possess ROS-scavenging activity, which, by reducing ROS accumulation, can reduce progenitor overproliferation and differentiation.

H. erythrostictum extracts inhibit the proliferation and differentiation of stem cells induced by DSS

To preserve the epithelial barrier, microbial or chemical damage to epithelial surfaces activates a number of signal transduction pathways that accelerate progenitor proliferation and differentiation [40]. In a previous experiment, DSS-exposed Drosophila intestinal epithelial cells died, and intestinal integrity was compromised. Therefore, we examined whether H. erythrostictum extracts could maintain intestinal epithelial homeostasis to protect intestinal integrity. In the midgut epithelium of the sucrose-fed group, modest and dispersed subpopulations of esg+ cells were observed (Fig. 5A). Many large and concentrated esg+ cells were observed after 72 h of feeding with 3% DSS, and the number of progenitor cells increased by more than 82% (P < 0.0001, Fig. 5B, Q). Compared to those in DSS-fed group, supplementation with 8 mg/ml HEWE or 1 mg/ml HEBE significantly reduced the numbers of esg+ cells by 29.6% (P < 0.001) and 31.6% (P < 0.01), respectively (Fig. 5C, D, Q). To further confirm the presence of mitotic stem cells, we analyzed cell proliferation with an anti-phospho-histone H3 (anti-PH3) antibody. After DSS stimulation, the number of PH3+ cells in the entire midgut significantly increased (P < 0.0001); however, supplementation with HEWE or HEBE inhibited the number of PH3+ cells by 35.35% (P < 0.001) and 43.6% (P < 0.0001), respectively (Fig. 5E-H, R).

Fig. 5

Both HEWE and HEBE prevent excessive proliferation and differentiation of ISCs induced by DSS. (A-P) Representative images of the posterior midguts of esg > GFP/Cyo(ISC/EB marker, A-H), Dl-lacZ (ISC marker, I-L) and Su(H)-lacZ (EB marker, M-P) transgenic flies. Guts were stained using anti-GFP (green), anti-PH3 (red), and anti-β-gal antibodies (green). After treatment with 3% DSS for 72 h, the numbers of esg+ cells, PH3+ cells, ISCs, and EBs significantly increased. Both HEWE and HEBE supplementation significantly reduced the increases in all types of targeted cells. However, HEBE did not reduce the increase in EBs. (Q-T) Quantification of the numbers of esg+ cells in A-D (n = 13–30), the numbers of PH3+ cells in E-H (n = 25–32), the numbers of ISCs in I-L (n = 12–14), and the numbers of EBs in M-P (n = 12–14). Quantification results represent mean ± SEM. nsP > 0.05, *P < 0.05, **P < 0.01,***P < 0.001,****P < 0.0001. Scale bars: 50 μm

Next, we utilized transgenic flies expressing the reporter genes Delta-lacZ (an ISC-specific marker) and Su(H)GBE-lacZ (an EB-specific marker) to analyze the numbers of ISCs and EBs, respectively. As expected, the numbers of Dl+ and Su(H)+ cells increased substantially after three days of treatment with 3% DSS (Dl+ cells: P < 0.0001, Su(H)+ cells: P < 0.0001). However, both the Dl+ and Su(H)+ cell numbers decreased by 29.2% (P < 0.05) and 52.0% (P < 0.0001), respectively, in the HEWE-supplemented group, while only the Dl+ cell number decreased by 28.9% (P < 0.05) in the HEBE-supplemented group (Fig. 5I-P, S, T). Our results demonstrated that H. erythrostictum extracts have a protective effect against DSS-induced abnormal proliferation and differentiation of progenitors.

H. erythrostictum extracts modulate progenitor overproliferation and differentiation by inhibiting the ROS-associated JNK signaling pathway

ROS-induced cellular damage and mortality, as well as bacterial or chemical ingestion, can activate JNK stress signaling pathways in ECs [50]. Activation of these pathways leads to the nuclear translocation and activation of the transcription factor AP1 (downstream of the JNK pathway), which induces cytokine and growth factor expression. Activation of JNK signaling pathways was observed by monitoring target gene expression. Using puc-lacZflies, we first measured the expression level of a reporter of JNK signaling. After 3 days of treatment with 3% DSS, the number of lacZ+ cells significantly increased compared to that in the sucrose-fed group (P < 0.0001). However, this increase was completely reversed by supplementation with 8 mg/ml HEWE or 1 mg/ml HEBE (HEWE: P < 0.0001, HEBE: P < 0.001, Fig. 6A-D, O). An anti-p-JNK antibody was subsequently used to assess JNK pathway activation. H. erythrostictum extracts significantly attenuated increase in the fluorescence intensity of p-JNK (HEWE: P < 0.0001, HEBE: P < 0.0001, Fig. 6E-H, P).To confirm the inhibitory effect of H. erythrostictum extracts on the JNK signaling pathway, we specifically overexpressed the JNK kinase hemipterous (Hep) in progenitor cells and counted the numbers of progenitor and mitotic cells. As a controls, the esgts> GFP transgene was crossed with wild-type w1118. In esgts> GFP; HepWT transgenic flies, the numbers of esg+ and PH3+ cells dramatically increased (P < 0.0001, P < 0.0001); however, after feeding with 8 mg/ml HEWE or 1 mg/ml HEBE, the numbers of progenitor and mitotic cells decreased by more than 25.4% (HEWE: P < 0.0001, HEBE: P < 0.0001) and 51.1% (HEWE: P < 0.001, HEBE: P < 0.0001), respectively (Fig. 6I-N, Q, R). These findings demonstrated that H. erythrostictum extracts can inhibit excessive progenitor proliferation and differentiation by decreasing JNK pathway activity.

Fig. 6

Both HEWE and HEBE inhibited the activation of the JNK pathway by DSS. (A-H) Representative images of the posterior midguts of puc-lacZ (A-D) and w1118 (E-H) flies. Guts were stained with anti-β-gal antibodies (green) and anti-p-JNK antibodies (red). After treatment with 3% DSS for 72 h, the number of β-gal+ cells and the p-JNK level increased. Both HEWE and HEBE supplementation effectively decreased the increase in the number of β-gal+ cells and the level of p-JNK. (I-N) Representative images of the posterior midgut of esgts> GFP; HepWT transgenic flies stained with anti-GFP (green, I-K) and anti-PH3 (red, L-N) antibodies. In flies supplemented with HEWE or HEBE, the numbers of esg+ and PH3+ cells were significantly lower than those in the sucrose group. (O-R) Quantification of the numbers of β-gal+cells in A-D (n = 12–15), p-JNK intensities in E-H (n = 12–13), the numbers of esg+ cells in I-K, and the numbers of PH3+ cells in L-N (n = 12–16).Quantification results represent mean ± SEM. ***P < 0.001, ****P < 0.0001. Scale bars: 50 μm

H. erythrostictumextracts regulate intestinal homeostasis by inhibiting the JAK-STAT pathway

Increased JNK activity in ECs is sufficient to promote ISC proliferation by stimulating the expression of the cytokines Upd2 and Upd3. These cytokines promote regeneration by stimulating JAK/STAT signaling in ISCs to promote their proliferation and in EBs to promote their differentiation [40, 51]. Upd3 is one of the main cytokines that acts as a signal to elicit antibacterial and reparative host responses during oral infection with entomopathogenic bacteria such as Ecc15 [20, 51]. Therefore, we analyzed Upd3 expression in upd3 > GFP/CyO flies. As expected, after oral infection with Ecc15, GFP levels increased by more than 71.6% (P < 0.0001); however, after supplementation with HEWE or HEBE, GFP signals decreased almost to control levels (HEWE: P < 0.0001, HEBE: P < 0.0001, Fig. 7A-D, O). Next, the activity of the JAK/STAT pathway was evaluated using the 10XSTAT-GFP reporter, a common JAK/STAT signaling reporter containing 10 Stat92E-binding sites derived from the suppressor of cytokine signaling at 36E (Soc-s36E) gene [52]. As expected, supplementation with 8 mg/ml HEWE or 1 mg/ml HEBE reduced the number of GFP+ cells induced by Ecc15 (HEWE: P < 0.0001, HEBE: P < 0.0001, Fig. 7E-H, P).

Fig. 7

Both HEWE and HEBE inhibited the activation of the JAK–STAT pathway byEcc15. (A-H) Representative images of the posterior midguts of upd3 > GFP/CyO (A-D) and 10×STAT-GFP (E-H) flies. Guts were stained with anti-GFP antibodies (green). After infection with Ecc15 for 16 h, the intensity of GFP and the number of GFP+ cells increased. Both HEWE and HEBE supplementation effectively reduced the increase in the intensity of GFP and the number of GFP+ cells. (I-N) Representative images of the posterior midgut of esgts> GFP; hopCA transgenic flies. Guts were stained with anti-GFP (green, I-K) and anti-PH3 (red, L-N) antibodies. In flies supplemented with HEWE or HEBE, the numbers of esg+ and PH3+ cells were significantly lower than those in the sucrose group. (O-R) Quantification of GFP intensities in A-D (n = 11–15), GFP+ cell numbers in E-H, the numbers of esg+ cells in I-K, and the numbers of PH3+ cells in L-N (12–19) (n = 13–15). Quantification results represent mean ± SEM. ***P < 0.001, ****P < 0.0001. Scale bars: 50 μm

We overexpressed hopscotch (hop) of Drosophila JAK kinase in ISCs/EBs using the esgts> GFP driver to confirm the inhibitory effect of H. erythrostictum extracts on the JAK/STAT signaling pathway. The numbers of progenitor and mitotic cells increased by approximately 1.7- and 5.6-fold, respectively (esg+ cells: P < 0.0001, PH3+ cells: P < 0.0001). However, supplementation with HEWE or HEBE significantly suppressed the JAK/STAT pathway-induced proliferation and differentiation of cells. The numbers of progenitor and mitotic cells drastically decreased, with the progenitor number resembling that in the sucrose group (esg+ cells: HEWE P < 0.0001, HEBE P < 0.0001; PH3+ cells: HEWE P < 0.001, HEBE P < 0.0001, Fig. 7I-N, Q, R). In conclusion, these findings demonstrated that H. erythrostictum extracts inhibit the JAK-STAT pathway to maintain intestinal homeostasis.

H. erythrostictum extracts inhibit intestinal injury induced by DSS via the EGFR pathway

The EGFR/Ras/MAPK pathway is crucial for ISC proliferation, and JNK and JAK/STAT signaling induce ISC proliferation by activating EGFR signaling [7, 16]. Therefore, we examined whether H. erythrostictum extracts could protect against intestinal injury induced by DSS via the EGFR pathway. We initially stained the midgut with an anti-p-Erk antibody that recognizes the activated form of MAPK [53]. After 3 days of stimulation with 3% DSS, the p-Erk intensity in the midgut increased by approximately 92.5% compared to that in the sucrose-fed group (P < 0.0001); however, supplementation with 8 mg/ml HEWE or 1 mg/ml HEBE significantly decreased the p-Erk intensity (P < 0.0001, P < 0.0001, Fig. 8A-D, O). Three EGFR ligands, Vein, Spitz, and Keren, are expressed in the intestine of Drosophila. Vein is expressed in the visceral mesoderm (VM) surrounding the intestinal epithelium in a stable state and is the most essential ligand for stimulating ISC division [7, 54, 55]. The expression level of the EGFR signaling pathway ligand was subsequently detected using the Vein (Vn)-lacZ reporter. The fluorescence intensity of Vn-lacZ+ cells in the midgut was significantly greater in the DSS-fed group than in the sucrose-fed group (P < 0.001). Supplementation with 8 mg/ml HEWE or 1 mg/ml HEBE significantly decreased Vein expression in response to DSS stimulation (HEWE: P < 0.0001, HEBE:P < 0.0001, Fig. 8E-H, P).

Fig. 8

Both HEWE and HEBE inhibited the activation of theEGFR pathway by DSS. (A-H) Representative images of the posterior midguts of w1118 (A-D) and Vn-lacZ (E-H) flies. Guts were stained with anti-p-ERK (red) and anti-β-gal antibodies (red). After treatment with 3% DSS for 72 h, the levels of p-ERK and β-gal increased. Both HEWE and HEBE supplementation effectively decreased the elevated p-ERK and β-gal levels. (I-N) Representative images of the posterior midgut of esgts> GFP; EGFRCA transgenic flies. Guts were stained with anti-GFP (green, I-K) and anti-PH3 (red, L-N) antibodies. In flies supplemented with HEWE or HEBE, the numbers of esg+ and PH3+ cells were significantly those