Qingwei San treats oral ulcer subjected to stomach heat syndrome in db/db mice by targeting TLR4/MyD88/NF-κB pathway

Abstract

Background: Qingwei San (QWS), one of classic Chinese Medicine prescripts, has been widely used to treat stomach heat syndrome which manifests oral ulcer (OU), periodontitis and upper gastrointestinal bleeding for seven hundred years. However, the therapeutic effects of QWS on diabetic OU subjected to stomach heat syndrome are still ambiguous. In the study, we investigated the pharmacological mechanisms.

Methods: The main components of QWS aqueous extract were analyzed by LC-MS, and potential pathways of QWS targeting OU were predicted by network pharmacology. The db/db mice were administered with the decoction of dried Zingiber officinale Rosc. rhizome combined with NaOH cauterization to establish the model of diabetic OU subjected to stomach heat syndrome. Subsequently, the model mice were treated with QWS, and OU wound healing status were recorded. The pathological changes of gastric tissue and oral mucosa were evaluated using hematoxylin-eosin staining, and the morphology of collagen fibers in oral mucosa was assessed by Masson staining. The levels of thromboxane B₂ (TXB₂), 6-Keto-prostaglandin F1α (6-keto-PGF1α), interleukin-1 β (IL-1β), IL-2, IL-6, tumor necrosis factor-α (TNF-α), β-endorphin (β-EP) and 5-Hydroxytryptamine (5-HT) were determined by ELISA assay. The protein expressions of Toll-like receptor 4 (TLR4), TNF receptor associated factor 6 (TRAF6), myeloid differentiation factor 88 (MyD88), inhibitor of NF-κB alpha (IκΒα), p-IκΒα and nuclear factor kappa-B (NF-κB) p65 were measured by Western Blotting.

Results: A total of 183 compounds in QWS were identified by LC-MS, and identified 79 bioactive compounds corresponded to 269 targets and 59 pathways. QWS high-dose treatment significantly reduced the level of TXB₂ and the ratio of TXB₂/6-keto-PGF1α. Meanwhile, it improved mucosal pathological morphology, and reduced the area of OU and local edema. Simultaneously, the levels of TNF-α, IL-1β, IL-6, IL-2 and 5-HT, and the expressions of TLR4, TRAF6, MyD88, p-IκΒα and NF-κB p65 were decreased.

Conclusion: QWS treatment facilitates the healing of OU, ameliorates pathological morphologies of gastric and oral mucosa and decreases the levels of pro-inflammatory cytokines in db/db mice subjected to stomach heat syndrome, whose mechanism may be associated with the inhibition of TLR4/MyD88/NF-κB signaling pathway to exert anti-inflammatory effects.

Keywords: Diabetes mellitus; Oral ulcer; Qingwei San; Stomach heat syndrome; TLR4/MyD88/NF-κB.

Fig. 1

Total ion chromatogram of QWS aqueous extract

Fig. 2

Network pharmacology analysis for screening targets and pathways of QWS. A PPI network of common targets. The color of node indicated the size of degree value; The greater the degree value corresponding to node color from green to red; The thickness of edge and combine score value have a positive correlation. B KEGG pathway analysis for common targets. C Component-target-pathway network

Fig. 3

The changes of water and food intake, weight body and fasting blood glucose level in db/db mice subjected to stomach heat syndrome. A Comparison of food intake in mice between T2DM group and T2DM + SH group. B Comparison of water intake in mice between T2DM group and T2DM + SH group. C The changes of food intake after QWS treatment. D The changes of water intake after QWS treatment. E The changes of body weight after QWS treatment. F The changes of fasting blood glucose after QWS treatment. T2DM, type 2 diabetes mellitus; OU, oral ulcer; SH, stomach heat syndrome; QWS-L, Qingwei San aqueous extract (1.32 g/kg); QWS-H, Qingwei San aqueous extract (5.14 g/kg). Data were shown as mean ± SD. *P < 0.05, **P < 0.01 vs. normal control group; ^#P < 0.05, ^##P < 0.01 vs. T2DM + OU group; ^&P < 0.05, ^&&P < 0.01 vs. T2DM + SH + OU group

Fig.4

Histopathology of oral and gastric mucosa in stomach heat syndrome’s OU db/db mice (×200, scale bar = 100 μm)

Fig.5

Therapeutic effect of QWS on ulcers healing. A Images of ulcers in six groups were presented. B Quantification of ulcers area. C Quantification of ulcer healing rate. T2DM, type 2 diabetes mellitus; OU, oral ulcer; SH, stomach heat syndrome; QWS-L, Qingwei San extract (1.32 g/kg); QWS-H, Qingwei San extract (5.14 g/kg). Data were shown as mean ± SD. ^&P < 0.05, ^&&P < 0.01 vs. T2DM + SH + OU group

Fig. 6

Histopathological examination of gastric mucosa. A normal control, B T2DM + OU, C T2DM + SH + OU, D KQKYS, E QWS-L, F QWS-H (HE, ×200, scale bar = 100 μm)

Fig. 7

Histopathological examination of oral mucosa. A normal control, B T2DM + OU, C T2DM + SH + OU, D KQKYS, E QWS-L, F QWS-H (×200, scale bar = 100 μm)

Fig. 8

Effects of QWS on the levels of TXB₂, 6-keto-PGF1α and inflammatory cytokines. A TXB₂ level was quantified, B 6-keto-PGF1α level was quantified, C. the ratio of TXB₂/6-keto-PGF1α was quantified. D–G the levels of IL-1β, IL-2, IL-6, TNF-α level were quantified. H, I 5-HT and β-EP levels were quantified. T2DM, type 2 diabetes mellitus; OU, oral ulcers; SH, stomach heat syndrome; QWS-H, Qingwei San extract (5.14 g/kg). Data were shown as mean ± SD (n = 10). *P < 0.05, **P < 0.01 vs. normal control group; ^&P < 0.05, ^&&P < 0.01 vs. T2DM + SH + OU group

Fig. 9

QWS treatment suppressed TLR4/MyD88/NF-κB pathway. A Western blotting assay was performed to measure the expressions of TLR4, MyD88, TRAF6, IκΒα, p-IκΒα and NF-κB p65 in oral mucosa. B–F Relative expressions of TLR4, MyD88, TRAF6, p-IκBα/IκBα and NF-κB p65 were quantified. T2DM, type 2 diabetes mellitus; OU, oral ulcers; SH, stomach heat syndrome; QWS-H, Qingwei San extract (5.14 g/kg). Data were shown as mean ± SD (n = 3). *P < 0.05, **P < 0.01 vs. normal control group; ^&P < 0.05, ^&&P < 0.01 vs. T2DM + SH + OU group