Abstract This study aimed to apply transcriptomics to determine how Molor-Dabos-4 (MD-4) protects healthy rats against indomethacin (IND)-induced gastric ulcers and to identify the mechanism behind this protective effect. Rats were pretreated with MD-4 (0.3, 1.5, or 3 g/kg per day) for 21 days before inducing gastric ulcers by oral administration with indomethacin (30 mg/kg). Unulcerated and untreated healthy rats were used as controls. Effects of the treatment were assessed based on the ulcer index, histological and pathological examinations, and indicators of inflammation, which were determined by enzyme-linked immunosorbent assay. Transcriptomic analysis was performed for identifying potential pharmacological mechanisms. Eventually, after identifying potential target genes, the latter were validated by quantitative reverse-transcription polymerase chain reaction (qRT-PCR). After pretreatment with MD-4, gastric ulcers, along with other histopathological features, were reduced. MD-4 significantly (p < 0.05) increased the superoxide dismutase (SOD) levels in ulcers and reduced pepsin, TNF-α, and IL-6 levels. RNA-seq analysis identified a number of target genes on which MD-4 could potentially act. Many of these genes were involved in pathways that were linked to anti-inflammatory and antioxidant responses, and other protective mechanisms for the gastric mucosa. qRT-PCR showed that altered expression of the selected genes, such as Srm, Ryr-1, Eno3, Prkag3, and Eef1a2, was consistent with the transcriptome results. MD-4 exerts protective effects against IND-induced gastric ulcers by reducing inflammatory cytokines and pepsin and increasing the expression of SOD levels. Downregulation of Srm, Ryr-1, Eno3, Prkag3, and Eef1a2 genes involved in regulating arginine and proline metabolism, calcium signaling pathway, HIF-1 signaling pathway, oxytocin signaling pathway, and legionellosis are possibly involved in MD-4-mediated protection against gastric ulcers. Keywords: Molor-dabos-4, gastric ulcer, transcriptomic analysis, mechanism of action, Mongolian medicine 1. Introduction Gastric ulcers are one of the most common upper digestive disorders. An epidemiological survey on peptic ulcers showed that the prevalence of gastric ulcer disease increased from approximately 6 million in 1990 to 8 million in 2019 globally [[42]1]. In China, about 10% of people have suffered from gastric ulcers at some time in their life [[43]2]. An increase in aggressive agents (gastric acid, pepsin, and oxidative stress) and a decrease in protective factors (adhesive mucus, bicarbonate secretion, antioxidant defenses, and blood flow) causes ulcerations [[44]3]. Other etiological variables, including heavy alcohol consumption, sedentary lifestyle stress, and chronic nonsteroidal anti-inflammatory drug (NSAID) use can also contribute to gastric ulcers [[45]4]. In fact, in the latter case, even though NSAIDs are extensively used as analgesics, antipyretics and anti-inflammatory compounds, their use also promotes neutrophil adhesion, damage to the mucosal integrity and gastric ulcerations [[46]5,[47]6,[48]7]. Although proton-pump inhibitors and other synthetic medicines are available for treatment, their adverse effects may cause headache, dizziness, and gastrointestinal symptoms including abdominal pain, constipation, and diarrhea [[49]8,[50]9]. As a result, research has been focused on identifying medicines with greater effectiveness and safety and compounds that confer additional protection against stomach ulcers [[51]10,[52]11]. The use of herbal medications for treating gastric ulcers has been gaining popularity. This is not only because studies involving humans and animal models have shown that their efficacy was equivalent or even better than that of drugs, such as cimetidine and omeprazole [[53]12], but also because ulcer therapies based on herbal medicine tend to be less expensive than chemical-based ones [[54]13,[55]14]. For instance, the South Korean multiherbal formula SR-5 has been reported to considerably decrease the ulcer index in mice, with agarwood extracts producing similar effects, along with reduced inflammation, against stomach ulcers in rats [[56]15,[57]16]. Herbal medicines normally act by stimulating the proliferation of mucous cells, triggering antioxidation mechanisms, inhibiting the secretion of gastric acid, and increasing the activity of H(+)/K(+)-ATPases [[58]17], and thus their use can represent a valuable alternative for the effective treatment of gastric ulcers, while limiting the side effects. Molor-dabos-4 (MD-4) is a Mongolian folk medicinal prescription that is clinically used to treat gastric ulcers, gastroenteritis, and dyspepsia [[59]18]. The recommended clinical dosage for humans is 3 g per day for a patient weighing about 60 kg, and a therapy cycle lasts 21 consecutive days. MD-4 is composed of one mineral (halite) and three medicinal herbs, namely rhizome of Zingiber officinale Rosc. (ZOR), seed of Piper longum L. (PLL) and fruit of Terminalia chebula Retz. (TCR) with equal ratio combinations ([60]Table 1). Halite is composed of NaCl and trace elements including Br, Rb, Cs, Sr [[61]19]. The active substances of ZOR are volatile oil and gingerol; piperlongumine and piperine are proved to be effective components of PLL A number of glycosides and coumarin and phenolic acid have been isolated from TCR, including chebulosides, gallic acid, and chebulic acid. The research data on the chemical composition analysis of MD-4 are limited [[62]20,[63]21,[64]22,[65]23,[66]24]. Based on ethnopharmacological records, MD-4 is also effective for gastric protection, improving digestion and detoxification. However, its mode of action and antiulcer effects are yet to be determined. The current study examined how MD-4 affected indomethacin-induced gastric ulcers in healthy rats by measuring the extent of mucosal damage and the effects of gene expression on inflammation and gastrointestinal functions. Furthermore, using data mining and transcriptomic analyses, the underlying mechanism behind MD-4’s protective effects against indomethacin-induced ulcers were unraveled. Table 1. Components of MD-4. No Genus Species Common Names Plant Part Content (%) 1 Halite Salt - 25 2 ZOR Ginger Rhizome 25 3 PLL Piper longum Immature fruits 25 4 TCR Chebulae Fructus Fruits 25 Total content (%) 100 [67]Open in a new tab 2. Materials and Methods 2.1. Ethics Statements Six-week-old male Sprague Dawley rats (SPF grade, batch C-NMG2021012507) of weight 200 ± 20 g were obtained from Liaoning Chang-Sheng Biotechnology Co. (Shenyang, Liaoning, China) The Committee on the Ethics of Animal Experiments at Inner Mongolia Minzu University reviewed and approved the experimental protocols (approval NM-LL-2021-06-15-1), in compliance with the criteria and general principles of the Chinese Experimental Animals Administration Legislation. All surgical procedures were performed after the animals were euthanized using sodium pentobarbital, while minimizing suffering. 2.2. Animal Treatment Thirty-six male rats were allowed to acclimatize for seven days in a controlled environment (12 h light/dark cycle, 22 ± 2 °C, relative humidity of 50% ± 5%) with unrestricted access to water and food. Rats were randomly assigned to six groups: control group, IND group, 0.3, 1.5, and 3 g/kg MD-4 and ranitidine groups (a clinical antiulcer drug). They were then given a 21-day pretreatment as follows: sodium carboxymethylcellulose water solution (CMC-Na, Sigma; 0.5%, St. Louis, MO, USA) was given to the control and IND groups throughout the trial, while MD-4 groups at 0.3, 1.5, or 3 g/kg doses (provided by the Affiliated Hospital of Inner Mongolia Minzu University, Tongliao, China) and ranitidine (30 mg/kg) were suspended in 0.5% CMC-Na solution and provided once daily through gastric administration. 2.3. Gastric Ulcer Induction by Indomethacin The use of IND is a proven method for inducing gastric ulcerations in rats by a single orally administered dose. All rats were subjected to fasting for 24 h prior to the final drug administration. Three hours after providing the last treatment, IND (30 mg/kg) was orally administered to induce gastric ulcerations as previously described [[68]25]. Food and water were then withheld for six hours before eventually injecting pentobarbital intraperitoneally to euthanize the rats. To analyze the effects of treatments, gastric tissues were dissected and Guth’s method [[69]26] was subsequently applied to determine ulcer length and ulcer index. Percentage inhibition was then calculated as follows: inhibition rate = [(UImodel − UItreated)/UImodel] × 100%, where UI is ulcer index. 2.4. Pathological and Histopathological Observation Rats were euthanized as described above to obtain gastric tissues for pathological examinations. In this case, the tissues were fixed for 48 h in 4% paraformaldehyde prior to decalcification in EDTA (10%; Sigma-Aldrich, Darmstadt, Germany), processed and embedded in paraffin. Samples were then sliced into sections of 4 µm thickness and after dewaxing in xylene two times for 15 min and at 37 °C, ethanol of decreasing concentrations was used for rehydrating the tissues. This was followed by a 5 min washing step with distilled water at room temperature before eventually staining the tissues with hematoxylin and eosin (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). 2.5. Measurement of Cytokine Levels Blood collected from the rats was first centrifuged at 4 °C and 4500× g for 10 min. The resulting serum samples were then analyzed using ELISA kits to determine IL-6 and TNF-α levels. Washing and subsequent homogenization of tissues in ice-cold 10X Tris buffer (50 mM; pH = 7.4) was followed by centrifugation at 4 °C and 12,000× g for 10 min. Prostaglandin E2 (PGE2), superoxide dismutase (SOD), and malondialdehyde (MDA) levels were then determined for the resulting supernatant using commercially available assay kits (Jiangsu Jingmei Bioengineering Institute, Yancheng, China). 2.6. Transcriptome Analysis Six samples of gastric tissues were taken from the rats in the control, IND, and MD-4 treated (3 g/kg) groups and sent for sequencing in BioTree (BioTree, Shanghai, China). The integrity and purity of the extracted RNA were then measured using a NanoPhotometer^® UV/vis spectrophotometer (IMPLEN, Westlake Village, CA, USA), and a Bioanalyzer 2100 (Agilent, Santa Clara, CA, United States) respectively. With the help of poly(dT) oligos coupled to magnetic beads, mRNA was purified before being fragmented at high temperature using divalent cations. This was followed by the reverse transcription of fragments using random hexamer primers, with the resulting cDNA strands subsequently adenylated and ligated to NEBNext^® adapters with hairpin loop structures. After PCR amplification of the DNA fragments using universal PCR primers, Phusion High-Fidelity DNA polymerase and Index (X) Primer, the products were purified (AMPure XP system), and a library was prepared, with the latter’s quality assessed with an Agilent Bioanalyzer 2100 system. The TruSeq PE Cluster Kit v3-cBot-HS kit (Illumina) was then used for cluster amplification of the samples on a cBot Cluster Generation System prior to sequencing on an Illumina Novaseq platform to generate 150 bp paired-end reads. Eventually, KEGG pathway and GO functional analyses were used to identify genes that were differentially expressed (DEGs) between groups. In this case, an absolute fold change of ≥2 and a corrected p-value of 0.05 were selected as thresholds for considering a gene as being differentially expressed. 2.7. Validation by Real-Time Quantitative Reverse Transcription PCR (RT-qPCR) Based on the antiulcer results of MD-4, regulated cytokine levels, and transcriptomic analysis, six key DEGs related to inflammation and oxidative stress from six key KEGG pathways were validated by RT-qPCR to analyze the expression of Srm, Ryr1, Eno3, Prkag3, RPl3l and Eef1a2 for the MD-4, IND, and control groups. Using an oligo(dT) primer, a Quantiscript reverse transcriptase (Qiagen, Hilden, Germany) and the same RNA samples that were used for transcriptomics, cDNA strands were synthesized according to the manufacturer’s instructions. The sequences available in GeneBank were also used to design primers for the amplification of the selected genes ([70]Table 2). Eventually, qRT-PCR was carried out on a LightCycler 480 SW 1.5.1 (Roche LightCycler 480 II, Basel, Switzerland) with an initial 10 min denaturation step at 50 °C, followed by 40 amplification cycles, each with denaturation performed for 1 min at 95 °C, prior to a 1 min annealing and extension step at 60 °C. The process concluded with a melt curve obtained through an incremental increase in temperature from 72 °C to 95 °C before finally cooling to 40 °C. The β-actin gene was selected as an internal control and gene expression (ΔΔct) relative to the control was determined as a fold change for plotting. Table 2. Primers used for qRT-PCR to determine gene expression in gastric tissues. Gene Accession No. Forward Primer Reverse Primer Srm [71]NM_053464 ACTCTTGCCCACCAACCAAG TTGTTGGGTCACAGGGCATAG Ryr1 [72]XM_001078539 CTGAGCTGAATGAATACAACGC CCATGAGCCTTTCTAGCACTG Eno3 [73]NM_012949 CTGATGACTCTTCCAGCCTC ACACTTAGTTTCTTTTCCAGCA Prkag3 [74]NM_001106921 AGTCTGCAGGAAACATCGCT CTCTCTCTGCATTGGACCCC Rpl3l [75]NM_005061.3 GCTGGCACCAAGAAGAGAGT AGCATCCGTGGCCAAACTTA Eef1a2 [76]NM_012660 CGGTATCCTCCGTCCTGGTA CGGCGAATGTCCTTGACAGA β-actin [77]NM_031144.3 GGAGATTACTGCCCTGGCTCCTA GACTCATCGTACTCCTGCTTGCTG [78]Open in a new tab 2.8. HPLC-MS/MS Analysis of Chemical Constituents of MD-4 Extract The chemical composition of MD-4 was identified using liquid chromatography- tandem mass spectrometry (LC-MS/MS). MD-4 aqueous extract (145.54 mg) was poured into a 2 mL centrifuge tube, added 1 mL of 70% methanol and 3 mm steel balls, vibrated and crushed with a fully automatic sample rapid grinder (jxfstprp-48, 70 Hz) for 3 min, low-temperature ultrasound (40 KHz) for 10 min, and centrifuged at 4 °C 12,000 rmp for 10 min. The analysis was performed on a Thermo Ultimate 3000 LC-MS System (Waltham, MA, USA). The column is Zorbax eclipse C18 chromatographic column (1.8 μm × 2.1 × 100 mm) operated at 40 °C. The elution solvents were aqueous 0.1% formic acid (A) - acetonitrile (B). Samples were eluted using a linear gradient from 0–2.0 min, 5% B; 2.0–6.0 min, 30% B; 6.0–7.0 min, 30% B; 7–12 min, 78% B; 14–17 min, 95% B; 17–20 min, 95% B; 20–21 min, 5% B; and 21–25 min, 100% B. The flow rate was 0.3 mL/min. Mass spectrometry was performed using full scan (m/z 100~1500) and data-dependent secondary mass spectrometry scanning mode (dd-ms2, topN = 10). Primary mass spectrometry resolution was set at 120,000, secondary mass spectrometry resolution was 60,000. Collision mode was set as high-energy collision dissociation and ion heater temperature was 325 °C. Gas flow rate was 45 arb and auxiliary air velocity was 15 arb. Electric spray voltage was 3.5 kV and S-lens RF level was 55%. The chemical structures of MD-4 were characterized based on their retention behavior and MS information, and from reference to databases such as Scifinder and Chemspider, as well as the general literature. 2.9. Statistical Analysis All results were provided as means ± standard error of mean (SEM). Using the GraphPad Prism 5^® software (GraphPad software, San Diego, CA, USA), one-way analysis of variance (ANOVA) and LSD tests were then performed to analyze differences between means at 5% significance levels. 3. Results 3.1. Effects of MD-4 on IND-Induced Gastric Ulcers Macroscopic observations showed that, unlike the control for which hemorrhages were practically absent from gastric tissues, the IND-treated group displayed ulcerations and hemorrhagic lesions on the stomach’s mucosal layer. In contrast, both MD-4 and ranitidine reduced ulcerations in the treated groups, with the greatest change in terms of increased inhibition rate and decreased ulcer index being 53.27% after treatment with MD-4 at a dose of 3 g/kg. Histopathological observations indicated that, in addition to serious damage to epithelial tissues, the IND group also had necrotic lesions and extensive edematous submucosal layers, all of which were clear indications of ulcerations. MD-4- and ranitidine-treated groups showed less mucosal damage and milder inflammation in contrast to the IND group ([79]Figure 1A–C). Figure 1. [80]Figure 1 [81]Open in a new tab (A,B): Histopathological features after MD-4 treatment on IND-induced gastric ulcers in rats. In the IND group, rats showed severe injury and inflammation of the gastric epithelium and edema of submucosa. MD-4 improved these alterations dose-dependently and showed less mucosal damage and milder inflammation in contrast to the IND group. IND: indomethacin; MD-4: molor dabos-4 decoction. (C): Ulcer index. Data are reported as means ± SEM (n = 6). ** p < 0.01 vs. IND group; *** p < 0.001 vs. IND group. (D–I): Levels of TNF-α, IL-6 and pepsin in serum and SOD, MDA and PGE-2 in gastric tissues. Data are expressed as means ± SEM (n = 6). ^# p < 0.05 vs. control group; ^## p < 0.01 vs. control group; * p < 0.05 vs. IND group; ** p < 0.01 vs. IND group; *** p < 0.001 vs. IND group. 3.2. Modulation of Inflammatory and Oxidative Processes by MD-4 in IND-Induced Ulcers IND administration significantly increased the levels of IL-6, TNF-α and PP compared with the controls (p < 0.05), as shown in [82]Figure 1D–F, while treatment with MD-4 significantly decreased their concentrations (p < 0.05). In contrast, [83]Figure 1G shows that the IND- and MD-4-treated groups, respectively, showed a decrease and an increase in the SOD levels of gastric tissues (p < 0.05). Although IND did not influence MDA or PGE-2 levels in gastric tissue, MD-4 reduced MDA (at 0.3 mmol/L) and PGE-2 (at 1.5 and 3.0 mmol/L) at certain doses ([84]Figure 1H,I). 3.3. Altered Gene Expression by MD-4 in Ulcerated Rats The results of sequencing data quality showed that the base quality of sequences was above Q20 and there was no base shift ([85]Figure 2A). The sample correlation heat map showed the gene expression of the three groups showed intragroup correlation ([86]Figure 2B). A Venn diagram showed that 210 out of 821 DEGs responding to GU treatment were related to DEGs caused by MD-4 ([87]Figure 2C). Hierarchical clustering analysis showed that in each group DEGs expression was dramatically different ([88]Figure 2D). An analysis of the number of DEGs in ulcerated gastric tissues and subsequent volcano plots showed that 874 genes (403 downregulated and 471 upregulated) were differentially expressed between the control and the IND-treated groups while a comparison of the IND and MD-4 groups revealed 821 DEGs (388 downregulated and 433 upregulated) ([89]Figure 2E,F). Figure 2. [90]Figure 2 [91]Open in a new tab Transcriptomic analysis of MD-4-treated IND-induced gastric ulcerated rats. (A): Venn diagram shows the number of differentially expressed genes. Different colors represent different comparisons, adding all the numbers in one circle provides the number of differentially expressed genes for a particular comparisons, while overlapping areas represent common differentially expressed genes when two groups were compared. (B): Pearson correlation heat map between samples. The abscissa and ordinate are the square of the correlation coefficient of each sample. (C): Cluster heat map of genes that were differentially expressed. The abscissa indicates sample names, with the ordinate being the normalized FPKM vale. Red and green colors indicate upregulation and downregulation, respectively. (D,E): Volcano plots showing transcriptome data. (A,B): 874 and 821 genes were differentially expressed when comparing the control with IND treatment (A) and between IND- and MD-4-treated groups (B), respectively. Vertical lines indicate a log2 fold change, while the horizontal ones represent p-value of 0.05. Red and green colors indicate significant up- and downregulation of genes, respectively. Significant DEGs were identified based on a p-value of <0.05 and a log2 fold change of at least 2.0. Control: normal control, IND: indomethacin, MD-4: molor-dabos-4 decoction. 3.4. KEGG Pathway Analysis RNA-seq data were analyzed with R software, along with the KEGG database, to identify enriched pathways after treatment with IND and MD-4. Results of KEGG enrichment analysis indicated that genes that were differentially expressed between the control and the IND-treated groups ([92]Figure 3A) were mostly enriched for pertussis, fluid shear stress, atherosclerosis, rheumatoid arthritis, osteoclast differentiation, TNF, IL-17, glucagon, type C lectin receptor, and AGE-RAGE signaling pathways (padj < 0.05). On the other hand, when comparing the IND- and MD-4-treated groups ([93]Figure 3B), the DEGs were mainly enriched for ribosome, hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy (DCM), malaria, cardiac muscle contraction, vascular smooth-muscle contraction, and the IL-17, calcium, and oxytocin signaling pathways (padj < 0.05). Overall, six pathways involving 18 DEGs were common to the IND- and MD-4-treated groups when compared to the control and these included arginine and proline metabolism, ribosome, legionellosis, and the HIF-1, oxytocin, and calcium signaling pathways. Interestingly, patterns of gene expression followed a similar but opposite trend for the IND- and MD-4-treated groups, as the upregulation of one gene observed for one group was matched with the downregulation of the same gene in the second one ([94]Table 3). Figure 3. [95]Figure 3 [96]Open in a new tab KEGG pathway enrichment analysis. The abscissa is the ratio of the number of differential genes annotated to the KEGG pathway to the total number of differential genes, and the ordinate is the KEGG pathway. (A): control vs. IND, (B): IND vs. MD-4. Table 3. Main genes involved in the six major enriched Kyoto Encyclopedia of Genes and Genomes pathways common to IND- and MD-4-treated groups. KEGG Pathway Gene Symbol Official Full Name Log2 Fold Change GeneBank Accession No. Control vs. IND IND vs. MD-4 Arginine and proline metabolism Ckm creatine kinase, M-type +2.894 −3.376 [97]NM_012530 Srm spermidine synthase +2.146 −2.099 [98]NM_053464 Calcium signaling pathway Casq1 calsequestrin 1 +3.041 −2.955 [99]NM_001159594 Ryr1 ryanodine receptor 1 +3.152 −4.929 [100]XM_039100854 [101]AABR07005775.1 Rattus norvegicus strain mixed contig_5872, whole genome shotgun sequence +4.672 −8.455 [102]AABR07005775 Hrc histidine rich calcium binding protein +2.700 −2.963 [103]NM_181369 Mylk3 myosin light chain kinase 3 +3.985 −4.653 [104]NM_001110810 Tnnc2 troponin C2, fast skeletal type +2.653 −4.315 NM_0010373510 Trdn triadin +2.934 −3.886 [105]NM_021666 Mylk2 myosin light chain kinase 2 +4.898 −7.565 [106]NM_057209 HIF-1 signaling pathway Eno3 enolase 3 +2.209 −2.069 [107]NM_012949 Oxytocin signaling pathway Cacng6 calcium voltage-gated channel auxiliary subunit γ 6 +3.491 −2.382 [108]NM_080694 Prkag3 protein kinase AMP-activated non-catalytic subunit γ 3 +4.891 −4.264 [109]NM_001106921 Ryr1 ryanodine receptor 1 +3.152 −4.929 [110]XM_039100854 Mylk3 myosin light chain kinase 3 +3.985 −4.653 [111]NM_001110810 Mylk2 myosin light chain kinase 2 +4.898 −7.565 [112]NM_057209 Ribosome Rpl3l ribosomal protein L3 like +3.479 −4.562 [113]NM_001191589 Legionellosis Eef1a2 eukaryotic translation elongation factor 1 α 2 +2.396 −2.477 [114]NM_012660 [115]Open in a new tab 3.5. qRT-PCR Validation The MD-4 mediated changes in the expression of Srm, Ryr1, Eno3, Eef1a2, Rp131 and Prkag3 genes that could actually be related to anti-inflammatory or antioxidant responses were selected for further validation by qRT-PCR. Changes in the expression patterns of three out of five selected genes were consistent with the transcriptome results ([116]Figure 4). Expression of Srm, Ryr1, Eno3, Eef1a2 and Prkag3 mRNA were increased in the IND group and decreased by MD-4 treatment. No similar expression patterns were observed in the Rp131 gene. Figure 4. [117]Figure 4 [118]Open in a new tab Relative expression of Srm, Ryr1, Prkag3, Eno3, Rpl3l and Eef1a2 determined by qRT-PCR. β-actin was selected as an internal control. Values represent means ± standard error mean (n = 6). ^# p < 0.05 vs. control, ^##: p < 0.01 vs. control. * p < 0.05 vs. IND-treated, ** p < 0.01 vs. IND-treated. 3.6. Chemical Components of MD-4 Extract All compounds detected by HPLC-MS/MS and provided accurate relative molecular weight compared with the references. A total of 12 compounds