ABSTRACT
ETHNOPHARMACOLOGICAL RELEVANCE: Chinese herbal medicine Gegen Qinlian Decoction (GQD) has been clinically shown to be an effective treatment of ulcerative colitis (UC) in China. However, the underlying mechanism of GQD's anti-ulcerative colitis properties and its effect on gut microbiota still deserve further exploration. AIM OF THE STUDY: This study observed the regulatory effects of GQD on Th2/Th1 and Tregs/Th17 cells balance, the NOD-like receptor family pyrin domain containing 3 (NLRP3) infammasome and gut microbiota in TNBS-induced UC in BALB/c mice. MATERIALS AND METHODS: 61 main chemical compounds in the GQD were determined by UPLC-Q-TOF/MS. The UC BALB/c model was established by intrarectal administration of trinitrobenzene sulfonic acid (TNBS), and GQD was orally administered at low and high dosages of 2.96 and 11.83 g/kg/day, respectively. The anti-inflammatory effects of GQD for ulcerative colitis were evaluated by survival rate, body weight, disease activity index (DAI) score, colonic weight and index, spleen index, hematoxylin-eosin (HE) staining and histopathological scores. Flow cytometry was used to detect the percentage of CD4, Th1, Th2, Th17 and Tregs cells. The levels of Th1-/Th2-/Th17-/Tregs-related inflammatory cytokines and additional proinflammatory cytokines (IL-1ß, IL-18) were detected by CBA, ELISA, and RT-PCR. The expressions of GATA3, T-bet, NLRP3, Caspase-1, IL-Iß, Occludin and Zonula occludens-1 (ZO-1) on colon tissues were detected by Western blot and RT-PCR. Transcriptome sequencing was performed using colon tissue and 16S rRNA gene sequencing was performed on intestinal contents. Fecal microbiota transplantation (FMT) was employed to assess the contribution of intestinal microbiota and its correlation with CD4 T cells and the NLRP3 inflammasome. RESULTS: GQD increased the survival rate of TNBS-induced UC in BALB/c mice, and significantly improved their body weight, DAI score, colonic weight and index, spleen index, and histological characteristics. The intestinal barrier dysfunction was repaired after GQD administration through promoting the expression of tight junction proteins (Occludin and ZO-1). GQD restored the balance of Th2/Th1 and Tregs/Th17 cells immune response of colitis mice, primarily inhibiting the increase in Th2/Th1 ratio and their transcription factor production (GATA3 and T-bet). Morever, GQD changed the secretion of Th1-/Th2-/Th17-/Tregs-related cytokines (IL-2, IL-12, IL-5, IL-13, IL-6, IL-10, and IL-17A) and reduced the expressions of IL-1ß, IL-18. Transcriptome results suggested that GQD could also remodel the immune inflammatory response of colitis by inhibiting NOD-like receptor signaling pathway, and Western blot, immunohistochemistry and RT-PCR further revealed that GQD exerted anti-inflammatory effects by inhibiting the NLRP3 inflammasome, such as down-regulating the expression of NLRP3, Caspase-1 and IL-1ß. More interestingly, GQD regulated gut microbiota dysbiosis, suppressed the overgrowth of conditional pathogenic gut bacteria like Helicobacter, Proteobacteria, and Mucispirillum, while the probiotic gut microbiota, such as Lactobacillus, Muribaculaceae, Ruminiclostridium_6, Akkermansia, and Ruminococcaceae_unclassified were increased. We further confirmed that GQD-treated gut microbiota was sufficient to relieve TNBS-induced colitis by FMT, involving the modulation of Th2/Th1 and Tregs/Th17 balance, inhibition of NLRP3 inflammasome activation, and enhancement of colonic barrier function. CONCLUSIONS: GQD might alleviate TNBS-induced UC via regulating Th2/Th1 and Tregs/Th17 cells Balance, inhibiting NLRP3 inflammasome and reshaping gut microbiota, which may provide a novel strategy for patients with colitis.
Subject(s)
Colitis, Ulcerative , Colitis , Drugs, Chinese Herbal , Gastrointestinal Microbiome , Humans , Mice , Animals , Colitis, Ulcerative/chemically induced , Colitis, Ulcerative/drug therapy , Drugs, Chinese Herbal/adverse effects , Inflammasomes/metabolism , Interleukin-18/metabolism , Interleukin-18/pharmacology , Interleukin-18/therapeutic use , NLR Family, Pyrin Domain-Containing 3 Protein/metabolism , Th17 Cells , Occludin/metabolism , RNA, Ribosomal, 16S/metabolism , Mice, Inbred CBA , Colitis/drug therapy , Cytokines/metabolism , Trinitrobenzenes/metabolism , Trinitrobenzenes/pharmacology , Trinitrobenzenes/therapeutic use , Anti-Inflammatory Agents/pharmacology , Body Weight , Caspases/metabolism , Disease Models, Animal , ColonABSTRACT
BACKGROUND: Panax notoginseng saponins (PNS), the main active component of Panax notoginseng, can promote vascular microcirculation. PNS exhibits antitumor effects in various cancers. However, the molecular basis of the relationship between PNS and tumor blood vessels remains unclear. PURPOSE: To study the relationship between PNS inhibiting the growth and metastasis of breast cancer and promoting the normalization of blood vessels. METHODS: We performed laser speckle imaging of tumor microvessels and observed the effects of PNS on tumor growth and metastasis of MMTV-PyMT (FVB) spontaneous breast cancer in a transgenic mouse model. Immunohistochemical staining of Ki67 and CD31 was performed for tumors, scanning electron microscopy was used to observe tumor vascular morphology, and flow cytometry was used to detect tumor tissue immune microenvironment (TME). RNA-seq analysis was performed using the main vessels of the tumor tissues of the mice. HUVECs were cultured in tumor supernatant in vitro to simulate tumor microenvironment and verify the sequencing differential key genes. RESULTS: After treatment with PNS, we observed that tumor growth was suppressed, the blood perfusion of the systemic tumor microvessels in the mice increased, and the number of lung metastases decreased. Moreover, the vascular density of the primary tumor increased, and the vascular epidermis was smoother and flatter. Moreover, the number of tumor-associated macrophages in the tumor microenvironment was reduced, and the expression levels of IL-6, IL-10, and TNF-α were reduced in the tumor tissues. PNS downregulated the expression of multiple genes associated with tumor angiogenesis, migration, and adhesion. In vitro tubule formation experiments revealed that PNS promoted the formation and connection of tumor blood vessels and normalized the vessel morphology primarily by inhibiting EphA2 expression. In addition, PNS inhibited the expression of tumor vascular marker proteins and vascular migration adhesion-related proteins in vivo. CONCLUSION: In this study, we found that PNS promoted the generation and connection of tumor vascular endothelial cells, revealing the key role of EphA2 in endothelial cell adhesion and tumor blood vessel morphology. PNS can inhibit the proliferation and metastasis of breast cancer by inhibiting EphA2, improving the immune microenvironment of breast cancer and promoting the normalization of tumor blood vessels.