I. cicadae was collected from a suburb of Ya’an, Sichuan province, China. DSS (40 KD) was purchased from Seebio Biotechnology Co., Ltd (Shanghai, China). TLR4 protein (purity ≥ 87%) was purchased from Sino biological. Bovine serum albumin (BSA) was purchased from Gemini. Animal Total RNA Isolation and Cell Total RNA Isolation Kits (RE-03014 and RE-03014113, respectively), RT EasyTM II (Master Premix for first-strand cDNA synthesis for Real-Time PCR RT-01022), and Real-Time PCR EasyTM-SYBR Green I (QP-01012) were purchased from Foregene (Chengdu, China). RNA Isolater Total RNA Extraction Reagent was purchased from Vazyme (R401-01, Nanjing, China). The Immobilon Western Chemiluminescent HRP Substrate was from Millipore (WBKLS0500, United States).

Air-dried powder of I. cicadae (10 Kg) was extracted using 95% aqueous MeOH (3 × 100 l) under reflux. A crude extract was obtained after removing the solvent under reduced pressure. This extract was then suspended in distilled water (10 l) and placed on a macro-porous resin D101 column (D × L: 20 × 120 cm). The column was eluted with MeOH-H₂O (0:100, 30:70, 60:40, 100:0, V/V) to yield four fractions (A-D). Fraction C (150 g) was placed on a silica gel column (100–200 mesh, D × L: 15 × 80 cm) and eluted with a PE-acetone gradient (100:0→1:1, V/V) to afford eight subfractions (C1-C8). Fractions C2 (4.1 g), C3 (1.9 g), and C4 (3.9 g) were combined, guided by TLC, and passed through a Sephadex LH-20 column (D × L: 2 × 180 cm, CHCl₃-MeOH, 1:1). Finally, the combined fractions were further purified by preparative HPLC (5 μm, 120 A, D × L: 10 × 250 mm) with MeOH-H₂O (30:70, 10 ml/min) to yield NADD (1.26 g, purity ≥98%, (Supplementary Figures S1, S2).

All animal experiments were conducted as per protocols accredited by the Animal Welfare Committee of Chengdu University of Traditional Chinese Medicine, and in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Imprinting control region male mice (7 weeks old, 30–35 g) were supplied by Specific Pathogen Free (SPF) Biotechnology Co., Ltd (Beijing, China) and housed in the animal facility of the Laboratory Animal Center of Chengdu University of Traditional Chinese Medicine, under standard conditions. All mice were provided food and distilled water. Animal experiments were designed following reported procedures (Ma et al., 2018; Shi et al., 2020; Wang et al., 2021; Zhang et al., 2021). After one week of acclimatization, mice were randomly divided into six groups (n = 8 per group): normal group (normal), NADD administration group (NADD (20 mg/kg)), UC model group (DSS), rescue UC model groups administrated different concentrations of NADD—DSS + NADD (5 mg/kg), DSS + NADD (10 mg/kg), DSS + NADD (20 mg/kg). NADD was dissolved in normal saline.

Mice administered DSS or DSS combined with NADD were supplied with freely accessible 2.5% DSS in drinking water for first 7 days. DSS was discontinued in favor of distilled water for the last 3 days (days 8–10). Normal mice and mice administered NADD (20 mg/kg) were provided with only distilled water for all 10 days. From the third day, mice in DSS + NADD (5 mg/kg), DSS + NADD (10 mg/kg), DSS + NADD (20 mg/kg) groups were administrated low, middle, and high concentrations of NADD by gavage. Mice in normal and DSS groups were administrated the same volume of normal saline by gavage as parallel controls. Body weight and DAI were recorded daily during the experiment. DAI was evaluated on the last day as the sum of body weight loss, diarrhea, and fecal bleeding (Wang et al., 2021). All groups of mice were sacrificed and colon tissues were obtained; colon length of each mouse was measured. Colon tissues were divided into five parts for different analyses.

Colon tissues were fixed with 4% paraformaldehyde for 24 h and dehydrated with 30% sucrose at 4°C for 72 h. Tissues were then embedded in paraffin. Embedded tissues were cut into 5 μm- thick sections and stained with hematoxylin-eosin (HE) or Periodic Acid-Schiff (PAS) (Naeem et al., 2020). Stained sections were visualized and images were captured by light microscopy.

Total RNA of colon tissues was isolated using an Animal Total RNA Isolation Kit, and mRNA was reverse transcribed into cDNA using an RT EasyTM II Kit following the manufacture’s protocol. Expression of target genes was assessed with RT-PCR using Analytikjena (qTOWER3 G, Germany) and Real-Time PCR EasyTM-SYBR Green I. Sequences of primers TNF-α, IL-1β, iNOS, IL-6 and β-actin were previously reported (Wang et al., 2014). The primer sequences of Ikbkb, CCL2, Rela (p65), Nfkb1 (p50) and Nfkb2 (p52) were designed as previously described (Listwak et al., 2013).

Colon tissues were lysed in PBS using a tissuelyzer (Servicebio, KZ-III F, China), then centrifuged and supernatants collected. TNF-α, IL-1β and IL-6 in supernatants were assessed using enzyme-linked immunosorbent assay (ELISA) kits (Multi Science, China), following the manufactures instruction. Absorbance was measured at 450 nm.

Murine leukemic monocyte macrophage cells (RAW264.7 macrophages) were cultured in a DMEM complete medium with 10% fetal bovine serum and 1% penicillin-streptomycin at 37°C in a humidified atmosphere with 5% CO_2. In cell culture experiments, both NADD and LPS were dissolved in DMSO, therefore, DMSO was used as control.

RAW 264.7 macrophages were seeded into 96-well plates at a density of 8 × 10³ cells/well. Cells achieved a density of 60–70% by the next day. Cells were then pre-treated with different concentrations of NADD (0 μM, 10 μM, 30 μM, 60 μM, 100 μM, 200 μM) for 1 h, followed by co-treating with or without 1 ug/mL LPS for 24 h. Subsequently, 10 μl of Cell Counting Kit-8 reagent (CCK8, MCE, HY-K0301) was added to each well and incubation continued for 3 h. Finally, optical density of formazan was measured at 450 nm using a microplate reader to assess cell viability (Thermo, Varioskan, MA, United States).

RAW264.7 macrophages were seeded into 10 cm plates. When cells achieved a density of 60–70%, they were pre-treated with NADD at the concentration of 60 μM or without NADD for 1 h, followed by co-treatment with or without 1 μg/ml LPS for 24 h and generated three groups (DMSO group, LPS group, NADD + LPS group). A total of 5 × 10⁶ cells were collected and lysed with RNA Isolater Total RNA Extraction Reagent. Lysates or fresh colon tissues were sent for transcriptome sequencing at Annoroad Biotechnology Co., Ltd (Zhejiang, China): Purity, concentration, and integrity of total RNA samples were assessed before further analysis. A cDNA library was obtained after amplification and purification. Finally, the cDNA library was sequenced using the Illumina NovaSeq 6000 platform with a 150-bp pair-end sequencing strategy to generate raw reads. Clean reads were obtained after removing adapter, poly-N sequences, and inferior quality reads. These reads were mapped to the Mus musculus (GRCm39 (Howe et al., 2021)) reference genome sequence using HISAT2 (Kim D. et al., 2019) tools. We assembled transcripts using HTSeq (Anders et al., 2015) for each sample to obtain gene read counts. Differential gene expression was assessed using DESeq2. Only genes with p-value < 0.05 and fold change value ≥ 1.5 were considered as significantly differentially expressed. We use cluster profiler (Wu et al., 2021) to assess KEGG pathway enrichment of DEGs. Volcano and heatmap plots were drawn in R. We used the upregulated genes and downregulated genes after LPS treatment as the background gene sets datasets, respectively. The upregulated and downregulated genes after LPS + NADD treatment were sorted according to the fold changes and used as input data for GSEA analysis. The related results showed whether the upregulated and downregulated genes were enriched in the term of background gene sets.

RAW264.7 macrophages were seeded into 6-well plates at a density of 5 × 10⁵ cells/well. When the cells achieved a density of 60–70%, they were pre-treated with different concentrations of NADD (10 μM, 30 μM, 60 μM) for 1 h, followed by co-treatment with or without 1 ug/mL LPS for 24 h. Subsequently, cells were collected and lysed with RIPA lysis buffer at 4°C for 30 min, centrifuged, and supernatants collected. The protein concentrations were then analyzed using a BCA protein assay kit (Beyotime, P0012, China) following the manufacturer’s instructions. Aliquots with equal amounts of protein were loaded into wells of SDS-PAGE gels, subjected to electrophoresis and protein transferred to PVDF membranes (Ju et al., 2021). After blocking with 10% BSA, membranes were incubated with primary antibodies at 4°C overnight. Primary antibodies were: TLR4 (1:4000, Proteintech, 66350-1-Ig, China), IKK-α (1:1,000, CST, 11930 s, United States), p-IKK-α (1:1,000, CST, 2,697 s, United States), NF-κB-P65 (1:1,000, CST, 8,242 s, United States), p-NF-ΚB-P65 (1:1,000, CST, 3,033 s, United States), IκB-α (1:1,000, CST, 4812 s, United States), p-IκB-α (1:1,000, CST, 5,209 s, United States), P38 (1:1,000, CST, 8,690 s, UnitedStates), p-P38 (1:1,000, CST, 4511s, United States), R-iNOS (1:1,000, NOVCCS, NB300-605SS, United States), COX-2 (1:1,000, abcam, ab179800, United States), α-tubulin (1:20,000, proteintech, 66031-1-Ig, China). Membranes were washed three times, and incubated with species-specific secondary antibodies at room temperature for 2 h: HRP-Anti-mouse-IgG (1:5,000, servicebio, GB23301, China), and HRP-Anti-rabbit-IgG (1:5,000, servicebio, GB23303, China). Finally, targeted proteins were visualized using HRP substrate for immunoblotting and detected using a Chemiluminescent Imaging System (MiniChemi 610, China). The immunoblots of targeted proteins were analyzed using ImageJ.

All inter-group comparisons were assessed using one-way analysis of variance by SPSS 26 and reported as mean ± standard deviation (SD). p < 0.05 was considered statistically significant, and a post-hoc analysis was done to determine which comparisons were significant.

The UC model was specifically constructed by receiving 2.5% DSS in drinking water on days 0–7, followed by normal drinking water for the last 3 days before sacrifice. Subsequently, different concentrations of NADD were administered orally from the fourth day to assess their impact on colitis. Parallel control groups (normal and DSS) were administrated with normal saline (Figure 1B). The UC model was constructed successfully as indicated by findings in normal and DSS group mice (Figures 1C–E). Further, mice administered NADD were compared to UC model mice to assess the therapeutic effect of NADD. The colon in DSS group mice appeared red, bleeding and shortened. In contrast, the colon for animals administered NADD gradually improved and became similar to normal mice. No differences were observed between colons from normal and NADD treated mice. Colon length shortened significantly in mice exposed to DSS when compared with normal animals (Figure 1C, ^## p < 0.01). However, NADD administration gradually reversed this shortening increasing oral dose (Figure 1D, ^* p < 0.05, ^** p < 0.01). No changes in DAI were noted between colons from normal and NADD treated mice. Compared with normal and NADD treated mice, all the DAI increased with time in DSS and DSS + NADD treated animals. However, the increase of DAI in DSS group was most notable over time, because NADD administration reversed this increase of DAI induced by DSS in a dose-dependent manner (Figure 1E, ^* p < 0.05, ^** p < 0.01). The body weight of DSS-exposed mice decreased over time, while the body weight of normal and NADD treated animals increased gradually. Compared with DSS group mice, the body weight of DSS + NADD treated animals increased significantly each day: In the low concentration group [DSS + NADD (5 mg/kg)], there were a trend of body weight increasing on days 5–10, but only on day 7, the increase was significantly; In the middle concentration group [DSS + NADD (10 mg/kg)], the trends of body weight increasing were more notable on days 4–10, and there were significantly increasing of body weight on days 5, 6, 7, and 9; In the highest concentration group [DSS + NADD (20 mg/kg)], the trends of body weight increasing were most notable, and there were significantly increasing of body weight on days 4, 5, 6, 7, 8, 9, and 10. To sum up, increases were most significant for mice administered NADD+20 (mg/kg) (Figure 1F, ^* p < 0.05, ^** p < 0.01). NADD thus relieved the symptoms of DSS-induced UC in a dose-dependent manner.

Histological damage in colon tissues from DSS-induced UC reflected injury seen in human UC. Pathology included the disappearance of colonocytes, mucin depletion, decreases in goblet cells and crypts, neutrophil infiltration of lamina propria, and submucosa, and edematous submucosa (Perse and Cerar, 2012). Normal mucosa, thin submucosa and muscularis was found in the colon of normal and NADD (20 mg/kg) treated mice, and columnar colonocytes, goblet cells and long crypts were found in the mucosa (Figure 2A). The mucosa layer in DSS-exposed mice was disrupted, colonocytes and goblet cells disappeared, long crypts loosened, submucosa was edematous, and a large number of neutrophils infiltrated into the mucosa and submucosa, accompanied by crypt abscesses. However, NADD administration protected colon tissues from damage induced by DSS. Colonocytes, goblet cells and long crypts increased, the submucosa became thinner, and neutrophil infiltration was less prominent. These protective effects of NADD became more obvious with increasing concentration. Conversely, PAS was used to identify goblet cells by staining secreted mucin glycoproteins. Mucin layers are considered protective factors against colitis (Chen et al., 2019). Colons from normal and NADD group mice showed mucin glycoprotein (Figure 2B). NADD increased mucin glycoproteins in a concentration-dependent manner. The grading of histological score was refer to references (Cooper et al., 1993; Kim S. E. et al., 2019). The pathohistological scores increased and the number of goblet cells lost significantly in the DSS only group than Normal and NADD only groups. However, the administration of NADD significantly decreased the pathohistological scores and increased the number of goblet cells in a concentration-dependent manner (Figures 2C,D). NADD thus protected against morphological changes induced by DSS exposure.

UC is typically accompanied by the secretion of various proinflammatory factors, including TNF-α, IL-6, IL-1β, and NF-κB. For example, NF-κB induces an increase in NO concentrations by activating inducible nitric oxide synthase (iNOS) (Kimura et al., 2000). In this study, transcription levels of NF-κB, TNF-α, IL-6, IL-1β and iNOS were increased significantly in the colon tissues of DSS group mice compared with that of the normal and NADD only group animals ( ^** p < 0.01). Thus, DSS exposure successfully induced inflammation of colon tissues in mice (Figure 3A). Transcription levels were downregulated in a concentration-dependent manner by NADD ( ^** p < 0.01). Similarly, protein levels of TNF-α, IL-6, and IL-1β were also highest in DSS group mice, and became lower following NADD treatment ( ^** p < 0.01). IL-6 levels of DSS + NADD (10 mg/ml) and DSS + NADD (20 mg/ml) groups also decreased significantly ( ^# p < 0.01, ^## p < 0.01) compared with mice treated with the low concentration of NADD (DSS + NADD (5 mg/ml)) (Figure 3B). NADD thus shows anti-inflammation activity for DSS-induced UC.

RAW264.7 macrophages are a monocyte/macrophage-like cell linage (Kong et al., 2019) and LPS stimulates RAW264.7 to induce inflammation. This activity is accompanied by the expression of proinflammatory cytokines and iNOS (Plociennikowska et al., 2015; Kong et al., 2019). RAW264.7 macrophages activated by LPS change morphology from round to irregular and pseudopodia are expanded (Figure 4A). Cells treated with DMSO and 60 μM NADD were round and small, basically with no pseudopodia. After LPS exposure, most cells became irregular, cell bodies enlarged, and pseudopodia expanded. RAW264.7 macrophages treated with NADD showed reduced numbers of activated cells, and greater numbers of normal cells in a concentration-dependent manner. The statistical analysis showed that the morphological changes were significant as compared with the LPS group (Figure 4B). Further, NADD significantly decreased the generation of NO. This effect was also concentration-dependent (Figure 4D, ^* p < 0.05, ^** p < 0.01). Compared with 10 μM and 30 μM NADD, the higher concentration of NADD (60 μM) induced a better anti-inflammation (Figure 4D) effect without adverse impacts on cell phenotype and viability with or without LPS stimulation (Figures 4A,C). NADD thus shows anti-inflammatory activity in vitro.

Transcriptome analysis was used to assess anti-inflammatory mechanisms of NADD. First, RAW264.7 macrophages were treated with LPS for 24 h to induce inflammation in vitro. Cells treated with DMSO served as a parallel control. Another group of cells were pre-treated with NADD (60 μM) for 1 h followed by LPS treatment for the next 24 h. Genome-wide profiles of differentially expressed genes (DEGs) were obtained by RNA-seq from all groups of cells. Cut-off criteria for volcano plots was fold change≥1.5 and p < 0.05 to identify DEGs. Many more DEGs were observed when comparing LPS and DMSO treated cells than when comparing LPS + NADD and LPS treated cells. Almost all genes downregulated by LPS treatment were upregulated after LPS treatment plus NADD treatment (Figures 5A,B), for example, Nrros, which downregulates ROS production (Bonini and Malik, 2014). Also, almost all genes upregulated by LPS treatment were downregulated after LPS treatment plus NADD treatment (Figures 5A,B), for example, Saa3, which is an inflammatory ligand that stimulates the production of IL-6 and TNF-α (Zhuang et al., 2017). The Venn diagrams (Figure 5C) showed 7,206 DEGs between LPS and DMSO treated cells, and 284 DEGs between LPS + NADD and LPS treated cells. Notably, almost 72% of the LPS + NADD affected genes (204/284) were also DEGs affected by LPS treatment alone. 93 DEGs that were down-regulated by LPS could be up-regulated by treatment of LPS + NADD, and 45 DEGs that were up-regulated by LPS could be down-regulated by treatment of LPS + NADD. The heat-map (Figure 5D) also shows that expression changes after LPS treatment are reversed by NADD treatment (fold change≥1.5, p < 0.05): Downregulated genes after LPS treatment were upregulated by NADD and vice-versa. Furthermore, gene set enrichment analysis (GSEA) illustrated the gene sets of up-regulated and down-regulated genes following LPS + NADD treatment (Figure 5E). As expected, NADD could reprogram the expressions of LPS regulated gene sets (Figure 5E). The intersection pathways enriched by both conditions were showed in Figure 5F, and the results showed that DEGs under both conditions were enriched in NF-kB and MAPK pathways. These findings suggest that NADD reverses LPS-induced inflammation at a transcriptional level. NADD inhibited LPS-induced Inflammation through TLR4/NF-kB and MAPK pathways.

RAW264.7 macrophages were treated with LPS and different concentrations of NADD to further explore anti-inflammatory effects of NADD in vitro. Western blotting was used to detect representative proteins involved in inflammatory signaling. Protein levels of p-IKK/IKK, p-NF-κB p65/ NF-κB p65, and p-IκBα/ IκBα in the NF-κB pathway were significantly elevated in LPS treated cells compared with DMSO and 60-μM treated cells. However, this effect was reversed significantly by NADD in a concentration-dependent manner (^* p < 0.05, ^** p < 0.01) (Figures 6A–D). LPS treatment also significantly upregulated protein levels of p-JNK/JNK, p-ERK/ERK and p-P38/P38 compared with DMSO and 60 μM treated cells, and NADD reduced the levels of these proteins in a concentration-dependent manner (^* p < 0.05, ^** p < 0.01) (Figures 6E–H). NADD thus suppress inflammation through modulation of NF-κB and MAPK pathways.