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

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.

Qingwei San Treats Oral Ulcer Subjected to Stomach Heat Syndrome in db/db Mice by Targeting TLR4/MyD88/NF-κB Pathway

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 investigate 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 B 2 (TXB 2 ), 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 TXB 2 and the ratio of TXB 2 /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.

LncRNA- and circRNA-associated competing endogenous RNA (ceRNA) networks in leukocytes in traditional Chinese medicine (TCM)-defined Pi-qi-deficiency syndrome and Pi-wei damp-heat syndrome resulting from chronic atrophic gastritis

Background To investigate lncRNA/circRNA-associated competing endogenous RNA (ceRNA)-gene regulation underlying leukocyte functions and characteristics, especially the potential ceRNA biomarkers, implicated in traditional Chinese medicine (TCM)-defined Pi-qi-deficiency syndrome (PQDS) and Pi-wei damp-heat syndrome (PDHS) resulting from chronic atrophic gastritis (CAG). Methods Based on RNA-sequencing approach, comparing with healthy control population, we identified the PDHS- or PQDS-specific lncRNAs/circRNAs in leukocytes, especially the Zheng (syndrome)-specific ceRNAs and their corresponding ceRNA regulatory networks, further decoding their potential functions and pathways. Results Despite being the TCM-defined Zhengs resulting from the same disease of CAG, the Zheng-specific lncRNAs/circRNAs in leukocytes were not same in PQDS and PDHS. There were the Zheng-specific lncRNA/circRNA-associated ceRNAs identified in the leukocytes, and their corresponding ceRNA regulatory networks were generated, including the ceRNA-gene binary relationship networks and the miRNA-centered ceRNA-miRNA-gene triple relationship networks. In the generated Zheng-specific ceRNA networks, the PQDS-specific ceRNA-governed genes in leukocytes, keeping more complex interactions with each other, were enriched in pathways related to MAPK signaling, receptor tyrosine kinases signaling as well as complement and coagulation cascades. Notably, the enriched pathways associated with adherens junction, focal adhesion, ECM-receptor interaction, ECM-organization and cell surface interactions, were implicated in cell-to-cell adhesion/junction and communication, probably contributing to the characteristics and functions of leukocytes. The PDHS-specific ceRNA-regulated genes, seemed to have no more interactions with each other, were enriched in the biological processes related to regulation of cell morphogenesis, neutrophil activation and degranulation, and lymphocyte mediated immunity such as B-cell receptor signaling, complement and coagulation cascades, and regulation of NK/T-cell mediated cytotoxicity. Importantly, the five exosome-encapsuled ceRNAs, containing ZFAS1 (NR_036658.2), AL353719.1 (ENST00000566847), LOH12CR2 (NR_024061.1) and two new lncRNAs (TCONS_00038035 and TCONS_00027600), particularly higher expression in the leukocytes in PQDS rather than PDHS, could be the potential ceRNA biomarkers for differentiation of the TCM-defined PQDS and PDHS among CAG patients. Conclusions These results may provide new insights into the characteristic and functional changes of leukocytes in the two TCM Zhengs, especially the Zheng-specific ceRNA-mediated gene regulation underlying leukocyte characteristics and functions, with potential leukocyte biomarkers for future application in integrative medicine.

Network Pharmacology-Based Analysis of Gegenqinlian Decoction Regulating Intestinal Microbial Activity for the Treatment of Diarrhea

Gegenqinlian decoction (GD) has been extensively used for the treatment of diarrhea with intestinal dampness-heat syndrome (IDHS) with a satisfying therapeutic effect. The purpose of this study is to clarify the active ingredients and mechanism of GD in the treatment of diarrhea with IDHS. The TCMSP database was used to screen out the active ingredients of the four Chinese herbal medicines in GD, and the targets of the active ingredients were predicted. We selected the targets related to diarrhea through the DisGeNET database, then used the NCBI database to screen out related targets of lactase and sucrase, and constructed the visual network to search for the active ingredients of GD in the treatment of diarrhea and related mechanisms of the targets. Combined with network pharmacology, we screened out 146 active ingredients in GD corresponding to 252 ingredient targets, combined with 328 disease targets in diarrhea, and obtained 12 lactase targets and 11 sucrase targets. The key active ingredients involved quercetin, formononetin, β-sitosterol kaempferol, and wogonin. Furthermore, molecular docking showed that these five potential active ingredients had good affinities with the core targets PTGS2. The active ingredients in GD (such as quercetin, formononetin, and β-sitosterol) may increase the microbial activity of the intestinal mucosa of mice and reduce the microbial activity of the intestinal contents through multiple targets, thereby achieving the effect of treating diarrhea.