Identification of chemical compounds from QWS aqueous extract
As shown in Fig. 1, the result of total ion chromatogram of mass spectroscopy illustrated that 183 compounds in QWS aqueous extract were identified through chromatogram matching. Additional file 1: Table S1 shows retention time, experimental and calculated molecular weight, molecular formulas, errors in parts per million (ppm), and major MS/MS fragments, etc. The mass error of all identified compounds was less than 5 ppm.
Prediction of targets and pathways by network pharmacology technology
Firstly, 98 compounds were screened out by SwissADME based on DL and GA, and a total of 725 targets were obtained from these compounds, and 1356 OU related targets were gathered in corresponding databases after removing duplicate values. Then, a total of 269 overlapping genes were screened out through Venn analysis to be potential targets related to OU (Additional file 2: Fig. S1A), which corresponded to 79 of 98 compounds of QWS. PPI analysis was performed on above 269 targets through STRING database and used to build network further by Cytoscape software (Fig. 2A). The PPI network consisted of 218 nodes and 2241 edges and 30 nodes with degree value ≥ 11, betweenness centrality ≥ 0.01045, closeness centrality ≥ 0.3412 and average shortest path length ≤ 3.1944 were selected as major genes, including SRC, STAT3, HSP90AA1, MAPK3, PIK3CA, MAPK1, AKT1, RELA, EP300 and VEGFA. These genes were likely to play an important pharmacological role in OU process.
To further investigate the mechanisms of QWS on OU at a systematic level, 269 overlapping targets were uploaded into DAVID. The results of GO analysis showed that 246 biological processes (BP), 34 cell components (CC), and 57 molecular functions (MF) enriched for these targets were recognized (P < 0.01, FDR < 0.01). Top 20 terms in BP, CC and MF were presented in Fig.S1B-D based on gene count. Enriched BPs included signal transduction, positive regulation of transcription from RNA polymerase II promoter, response to drug, positive regulation of cell proliferation, inflammatory response. Enriched CCs included plasma membrane, cytoplasm, cytosol, extracellular exosome, nucleoplasm. Enriched MFs included protein binding, ATP binding, protein homodimerization activity, identical protein binding, enzyme binding. According to results of pathway enrichment, 59 target-related pathways had been found (P < 0.01, FDR < 0.01). Top 20 KEGG pathways were presented in Fig. 2B based on gene count. The KEGG enrichment analysis provided insight that QWS might act on PI3K-Akt signaling pathway, Neuroactive ligand-receptor interaction, Rap1 signaling pathway, Ras signaling pathway, MAPK signaling pathway, HIF-1 signaling pathway, TNF signaling pathway, Toll-like receptor signaling pathway, NF-kappa B signaling pathway.
In order to further explore the relationship between active ingredients of QWS and therapeutic effect on OU, a component-target-pathway network was constructed (Fig. 2C), which includes 79 compounds, 269 targets and 59 pathways. A total of 26 components (degree ≥ 16, betweenness centrality ≥ 0.01149, closeness centrality ≥ 0.3187 and average shortest path length ≤ 3.2080) were screened out as vital pharmacological chemicals of QWS on OU, such as 13(S)-HOTrE, 13S-hydroxyoctadecadienoic acid, 3,4-Dimethoxycinnamic acid, Ferulaldehyde, 4-Methylumbelliferone, Ferulic acid, etc. Multiple targets were associated with multiple compounds, indicating that different components in QWS had synergistic effects in the therapeutic process. And the targets were distributed in different pathways and were coordinated with each other. The results revealed that QWS treatment may exert therapeutic effects on OU through multiple pathways and multiple targets.
The changes of water and food intake, weight body and fasting blood glucose level in mice after QWS treatment
The mice in normal control and T2DM groups exhibited shiny hair, a certain level of movement vigor, and regular dietary, stool and urine. The mice in T2DM + SH group showed lusterless hair and hoarseness. They were more irritable and aggressive. Their daily urine volumes were less and more yellow, and their stool presented dry and hard. Compared with T2DM group, water and food intake of mice were notably increased (P < 0.01, Fig. 3A, B) and body weight was significantly decreased (P < 0.05, Fig. 3E) in T2DM + SH group, which were consistent with common signs or symptoms of stomach heat syndrome in humans with T2DM .
After 7 days of QWS administration (D22), mice suffered with OU showed lower food intake than that of normal control group, which might be associated with ulcers pain. Mice in T2DM + SH + OU group exhibited significantly increased water intake (P < 0.05) and decreased body weight (P < 0.01), and food intake showed an upward trend, compared with T2DM+OU group. The amount of food and water intake in QWS-L and QWS-H groups still showed a downward trend, and body weight in QWS-L group suggested an upward trend compared with T2DM + SH + OU group (Fig. 3C–E). These results illustrated that QWS treatment was able to ameliorate some stomach heat symptoms. However, there was no obvious effect of QWS on fasting blood glucose level (Fig. 3F). Considering short administration time of QWS (7 days), whether it has a hypoglycemic efficacy remains to be further studied. Compared with normal control group, body weight and fasting blood glucose level of db/db mice were significantly increased (P < 0.01, Fig. 3E, F), which was consistent with symptoms of T2DM.
The characteristics of gastric and oral mucosa in T2DM + SH + OU mice model
Under observation of microscope, HE staining illustrated gastric mucosa edema and congestion, epithelial cell necrosis and shedding, accompanied by gland deformation and disordered arrangement in T2DM + SH + OU mice model, fulfilling the pathological criteria of stomach heat syndrome in humans  (Fig. 4). After inducing oral ulcer to db/db mice administered by the decoction of dried Zingiber officinale Rosc. rhizome for 24 h, a round-like ulcers appeared in left buccal mucosa. Ulcers surface was covered with gray-white or yellow pseudomembrane with a central depression and surrounding hyperemia edema, appearing obvious OU. Under observation of microscopy, oral mucosa exhibited severe injuries with massive infiltration of lymphocytes and neutrophils, and blood capillaries were dilated and congested, as well as collagen fibers were irregularly arranged, thin and broken (Fig. 4). It indicates that the establishment of stomach heat syndrome’s OU model in db/db mice was successful.
QWS treatment ameliorated ulcer healing
As shown in Fig. 5A, in normal control group, oral mucosa of mice was pink, intact, smooth and no ulceration. While in other groups, it showed an obvious round-like OU on left cheek after cauterized by NaOH for 24 h, and no difference was observed in ulcers area among groups (P > 0.05). After low and high doses of QWS administration for four days (D19), ulcer area of mice was decreased in varying degrees, and ulcers became shallower with less congestion and edema. After seven days of treatment (D22), OU of mice in T2DM + SH + OU group and T2DM + OU group were still visible and mucosal hyperemia and edema around ulcer were decreased, and parts of pseudomembrane fell off from surface, but in QWS-treated mice, mucosa in injured area was similar to normal mucosa without obvious hyperemia and edema. No significant differences in ulcers area and wound healing rate were found between T2DM + OU group and T2DM + SH + OU group at D19 and D22 (P > 0.05). Compared with T2DM + SH + OU group at the same time point, ulcers area in QWS-L and QWS-H groups was notably reduced, and wound healing rate was definitely increased (P < 0.01). Although there was no significant difference in ulcers healing between KQKYS group and QWS-H group, high dose of QWS treatment showed better therapeutic effects on reducing ulcers area and improving ulcers healing rate (Fig. 5B, C).
QWS treatment improved pathological morphology of gastric and oral mucosa
Gastric wall of mice in normal control and T2DM + OU groups showed distinct structure, and glands of lamina propria were clear, well-arranged, neither atrophy, shedding nor defect was observed. Gastric mucosa of T2DM + SH + OU mice was mildly damaged and a small number of epithelial cells shed off. Gastric mucosal structure of mice in QWS-L and QWS-H groups were intact, and epithelial cells and glands were arranged neatly, and mucosal hyperemia was obviously attenuated (Fig. 6).
In normal control group, oral mucosa tissue structure was distinct, and cell structure was intact with neatly arranged cells without inflammatory cells infiltration. In T2DM + SH + OU group, the integrity of oral mucosa was broken including shed epithelial cells and irregular and pyknotic nuclei and hyperchromatic change, and its surface was covered with necrotic tissue, as well as was infiltrated by a large number of neutrophils and lymphocytes, even presented focal pyogenic or hemorrhagic lesion in some cases. Histological lesions in oral mucosa were milder in T2DM + OU group than those in T2DM + SH + OU group. Pathologic degree of oral mucosa in QWS-L and QWS-H groups were significantly improved compared with that in T2DM + SH + OU group, and inflammatory cell infiltration was obviously relieved, with visible part of new epithelial proliferation and intact cell morphology (Fig. 7A1–F1).
Collagen fiber of oral mucosa was stained in blue. In normal control group, collagen fibers were abundant, evenly distributed and orderly. In T2DM + OU and T2DM + SH + OU groups, it showed broken, thin, disordered and scattered collagen fiber bundles. In QWS-L and QWS-H groups, collagen fibers were thick and intact, uniformly colored, and arranged neatly and regularly (Fig. 7A2–F2).
QWS treatment decreased the levels of TXB2, inflammatory cytokines and 5-HT
No significant differences in the levels of TXB2 and 6-keto-PGF1α in gastric tissue were observed between normal control and T2DM + OU groups (P > 0.05). In comparison with normal control group, the levels of TXB2 and the ratio of TXB2/6-keto-PGF1α were increased in T2DM + SH + OU group, although there was no statistic difference. Compared with T2DM + SH + OU group, low and high doses of QWS and KQKYS treatments significantly reduced TXB2 level (P < 0.01), and high dose of QWS treatment markedly decreased the ratio of TXB2/6-keto-PGF1α (P < 0.05) (Fig. 8A–C).
Compared with normal control group, the levels of TNF-α and IL-2 in serum were increased significantly in T2DM + OU group (P < 0.01), and the levels of IL-1β, IL-2, IL-6 and TNF-α presented obviously high in T2DM + SH + OU group (P < 0.05). There were no significant differences in the levels of IL-1β, IL-2, IL-6 and TNF-α between T2DM + OU group and T2DM + SH + OU group, although the levels of IL-1β, IL-6 and TNF-α in T2DM + SH + OU group were higher than those in T2DM + OU group. Compared with T2DM + SH + OU group, low and high doses of QWS treatment significantly reduced IL-2, IL-6 and TNF-α levels (P < 0.01), and high dose of QWS treatment markedly decreased IL-1β level (P < 0.01). Although there was no significant difference in serum inflammatory factors between KQKYS and QWS-H groups, the suppression effect on inflammatory factors was more pronounced in QWS-H group (Fig. 8D–G).
Compared with serum 5-HT level in normal control group, it was significantly increased in T2DM + OU and T2DM + SH + OU groups (P < 0.01), while serum β-EP concentration showed no statistical significance. Additionally, there were no significant differences in the levels of 5-HT and β-EP between T2DM + OU and T2DM + SH + OU groups. Compared with T2DM + SH + OU group, treatments of QWS and KQKYS notably reduced 5-HT level, respectively (P < 0.01), and QWS treatment failed to influence β-EP level (P > 0.05) (Fig. 8H, I).
QWS treatment downregulated TLR4/MyD88/NF-κB pathway
As shown in Fig. 9, the protein expressions of TLR4, TRAF6, MyD88, p-IκΒα, NF-κB p65 and the ratio of p-IκBα/IκBα in oral mucosa in T2DM + OU and T2DM + SH + OU groups were obviously increased compared with normal control group (P < 0.05). There were no marked differences of protein expressions mentioned above between T2DM + OU and T2DM + SH + OU groups. High dose of QWS treatment decreased the expressions of TLR4, TRAF6, MyD88, p-IκΒα and NF-κB p65, as well as downregulated the ratio of p-IκBα/IκBα compared with T2DM + SH + OU group (P < 0.01).
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