Xuanfu Daizhe Tang alleviates reflux esophagitis in rats by inhibiting the STAT1/TREM-1 pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Reflux esophagitis (RE) is a common chronic inflammatory disease of the esophageal mucosa with a high prevalence and recurrence rate, for which a satisfactory therapeutic strategy is still lacking. Chinese medicine has its characteristics and advantages in treating RE, and the clinical application of Xuanfu Daizhe Tang (XDT) in treating RE has achieved sound therapeutic effects. However, there needs to be more research on its mechanism of action. AIM OF THE STUDY: The present work aimed to investigate the mechanism of XDT action in RE through the Signal Transducer and Activator of Transcription 1 (STAT1)/Triggering Receptor Expressed on Myeloid cells-1 (TREM-1) pathway. MATERIALS AND METHODS: The main active components of XDT were analyzed by ultra-performance liquid chromatography-mass spectrometer (UPLC-MS). The effect of XDT on RE was evaluated in a rat model of RE induced by "Cardioplasty + pyloric ligation + Roux-en-Y esophagojejunostomy". Each administration group was treated by gavage. The degree of damage to the esophageal mucosa was evaluated by visual observation, and the Potential of Hydrogen (PH) method and Hematoxylin-eosin staining (HE) staining were performed. Serum levels of Interleukin-1ß (IL-1ß), Interleukin-6 (IL-6), Tumor Necrosis Factor alpha (TNF-α), and Inducible Nitric Oxide Synthase (iNOS) were measured by ELISA. Quantitative Real-time PCR (qPCR), Western Blot (WB), and Immunofluorescence (IF) methods were used to detect Claudin-4, Claudin-5, TREM-1, and p-STAT1 in esophageal tissues for studying the mechanism of action and signaling pathway of XDT. Immunohistochemistry (IHC) analysis was used to detect the expression of TREM-1 and CD68 in esophageal tissues. Flow Cytometry (FC) was used to detect the polarization of macrophages in the blood. After conducting preliminary experiments to verify our hypothesis, we performed molecular docking between the active component of XDT and STAT1 derived from rats and parallel experiments with STAT1 inhibitor. The selective increaser of STAT1 transcription (2-NP) group was used to validate the mechanism by which XDT acts. RESULTS: XDT alleviated esophageal injury and attenuated histopathological changes in RE rats. XDT also inhibited the inflammatory response and decreased serum IL-1ß, IL-6, TNF-α, and iNOS levels in RE rats. qPCR and WB results revealed that XDT inhibited the expression of Claudin-4, Claudin-5, TREM-1, and STAT1 in the esophageal mucosa of RE rats. IHC and FC results showed that XDT reduced TREM-1 levels in esophageal tissues and polarized macrophages toward M2. The molecular docking results showed that rat-derived STAT1 can strongly bind to Isochronogenic acid A in XDT. The parallel experimental results of STAT1 inhibitor showed that XDT has anti-inflammatory effects similar to STAT1 inhibitors. The 2-NP group confirmed that XDT exerts its therapeutic effect on reflux esophagitis through the STAT1/TREM-1 pathway, with STAT1 as the upstream protein. CONCLUSIONS: This study suggests that XDT may treat reflux esophagitis by modulating the STAT1/TREM-1 pathway.

Overexpression of miR-122 Impairs Intestinal Barrier Function and Aggravates Acute Pancreatitis by Downregulating Occludin Expression.

Acute pancreatitis (AP) causes intestinal barrier damage, resulting in systemic inflammatory response syndrome (SIRS) or multiple organ dysfunction syndrome (MODS), which are important factors affecting AP severity and mortality. Here, we studied the mechanism of miR-122 in regulating intestinal barrier function in AP. AP rat model was constructed via intraperitoneal injection of ketamine, and primary intestinal epithelial cells were isolated from rats for in vitro studies. HE staining was used to assess pathological alterations of pancreas and intestines tissues. Inflammatory factors were detected by ELISA assay. qRT-PCR and WB were used to detect the expressions of miR-122 and occluding, respectively. Then dual-luciferase reporter assay, intestinal permeability test, and cell permeability were performed in vivo and in vitro to probe the molecular mechanism of miR-122 in regulating intestinal barrier function in AP. The expression of miR-122 was upregulated in AP rats, while the expression of occludin was downregulated, and the intestinal permeability was increased in AP rats and primary intestinal epithelial cells isolated from rats. Inhibition of miR-122 regulated intestinal barrier function through mediating occludin expression. miR-122 regulated intestinal barrier function to affect AP through mediating occludin expression in vivo. These results provided evidence that miR-122 overexpression impaired intestinal barrier function via regulation of occludin expression, thus promoting AP progression.

Oleanolic acid regulates the Treg/Th17 imbalance in gastric cancer by targeting IL-6 with miR-98-5p.

BACKGROUND: Gastric cancer (GC) was a type of malignant tumor with a very high fatality rate. Oleanolic acid (OA) was a class of pentacyclic triterpenes which was proved to have anti-cancer activity. While the specific molecular mechanism of OA's role in inhibiting GC was not fully understood. This study aimed to explore how OA played an anti-cancer role in GC. METHODS: Expression of miR-98-5p was examined using qPCR, and expression levels of Treg/Th17-related factors were evaluated using qPCR and western blot. Flow cytometry was conducted to assess the proportion of Treg cells and Th17 cells. Besides, dual luciferase reporter assay was performed to verify that IL-6 was a target of miR-98-5p. RESULTS: Downregulation of miR-98-5p and upregulation of Treg/Th17-related factors were observed in GC tissues. What's more, the Treg/Th17 imbalance was found in PBMCs of GC patients. Overexpression of miR-98-5p promoted balance of Treg/Th17 cells via directly targeting IL-6 to downregulate expression of IL-6. Finally, OA could promote balance of Treg/Th17 cells by upregulating expression of miR-98-5p. DISCUSSION: All our results proved that OA could promote balance of Treg/Th17 cells in GC by targeting IL-6 with miR-98-5p, indicating a potential drug for treatment of GC.

Inhibition of NF-κB is required for oleanolic acid to downregulate PD-L1 by promoting DNA demethylation in gastric cancer cells.

Gastric cancer is one of the most common causes of cancer-related death worldwide. Immunotherapy via programmed cell death protein 1 (PD-1)/programmed cell death-ligand 1 (PD-L1) blockade has shown benefits for gastric cancer. Epigenetic DNA methylation critically regulates cancer immune checkpoints. We investigated how the natural compound oleanolic acid (OA) affected PD-L1 expression in gastric cancer cells. Interleukin-1ß (IL-1ß) at 20 ng/mL was used to stimulate human gastric cancer MKN-45 cells. IL-1ß significantly increased PD-L1 expression, which was abolished by OA. Next, OA-treated MKN-45 cells were co-cultured with activated and PD-1-overexpressing Jurkat T cells. OA restored IL-2 levels in the co-culture system and increased T cell killing toward MKN-45 cells. Overexpression of PD-L1 eliminated OA-enhanced T cell killing capacity; however, PD-1 blocking antibody abrogated the cytotoxicity of T cells. Moreover, OA abolished IL-1ß-increased DNA demethylase activity in MKN-45 cells. DNA methyltransferase inhibitor 5-azacytidine rescued OA-reduced PD-L1 expression; whereas DNA demethylation inhibitor gemcitabine inhibited PD-L1 expression, and, in combination with OA, provided more potent inhibitory effects. Furthermore, OA selectively reduced the expression of DNA demethylase TET3 in IL-1ß-treated MKN-45 cells, and overexpression of TET3 restored OA-reduced PD-L1 expression. Finally, OA disrupted nuclear factor κB (NF-κB) signaling IL-1ß-treated MKN-45 cells, and overexpression of NF-κB restored OA downregulation of TET3 and PD-L1. The cytotoxicity of T cells toward MKN-45 cells was also weakened by NF-κB overexpression. Altogether, OA blocked the IL-1ß/NF-κB/TET3 axis in gastric cancer cells, leading to DNA hypomethylation and downregulation of PD-L1. Our discoveries suggested OA as an epigenetic modulator for immunotherapy or an adjuvant therapy against gastric cancer.

Oleanolic acid reduces aerobic glycolysis-associated proliferation by inhibiting yes-associated protein in gastric cancer cells.

Gastric cancer represents a common malignancy of digestive tract with high incidence and mortality. Increasing evidence suggests that the growth of gastric tumor cells relies largely on aerobic glycolysis. Currently, many potential anti-cancer candidates are derived from natural products. Here, we evaluated the effects of oleanolic acid (OA), a triterpenoid component widely found in the plants of Oleaceae family, on aerobic glycolysis and proliferation in human MKN-45 and SGC-7901 gastric cancer cells. Our results demonstrated that OA reduced the viability and proliferation of gastric cancer cells and inhibited the expression of cyclin A and cyclin-dependent kinase 2. OA blocked glycolysis in these cells evidenced by decreases in the uptake and consumption of glucose, intracellular lactate levels and extracellular acidification rate. Glycolysis inhibitor 2-deoxy-d-glucose, similar to OA, suppressed gastric cancer cell proliferation. OA also decreased the expression and intracellular activities of glycolysis rate-limiting enzymes hexokinase 2 (HK2) and phosphofructokinase 1 (PFK1). Moreover, OA downregulated the expression of hypoxia inducible factor-1α (HIF-1α) and decreased its nuclear abundance. Upregulation of HIF-1α by deferoxamine rescued OA-inhibited HK2 and PFK1. Furthermore, OA reduced the nuclear abundance of yes-associated protein (YAP) in gastric tumor cells. YAP inhibitor verteporfin, similar to OA, downregulated the expression of HIF-1α and glycolytic enzymes in gastric cancer cells; whereas overexpression of YAP abrogated all these effects of OA. Collectively, inhibition of YAP was responsible for OA blockade of HIF-1α-mediated aerobic glycolysis and proliferation in human gastric tumor cells. OA could be developed as a promising candidate for gastric cancer treatment.

[Expression of CXCR7 in Acute Monocytic Leukemia and Its Effects on THP-1 Cell Functions].

OBJECTIVE: To study the expression of stromal cell derived factor-1α (SDF-1α) receptor CXCR7 in acute monocytic leukemia (AML-M5), and its effects on proliferation, apoptosis, invasion of acute monocytic leukemia cell line THP-1. METHODS: CXCR7 protein and mRNA expression levels in THP-1 cells and peripheral blood mononuclear cells (PBMNC) from the newly diagnosed AML-M5 patients and normal individuals were detected by flow cytometry, Western blot and RT-PCR respectively. CCK8, Annexin V/PI double staining and Transwell assay were used to observe the effects of CXCR7 on the proliferation, apoptosis, and invasion of THP-1 cells in vitro. RESULTS: The expression of CXCR7 on immature cell surface of the newly diagnosed AML-M5 patients was significantly higher than that in the control group (P<0.05). CXCR7 was also highly expressed on THP-1 cells surface. The CXCR7 protein and mRNA levels in THP-1 cells and PBMNC of AML-M5 patients were significantly higher than those in the control group (P<0.05). The THP-1 cell proliferation activity was higher in SDF-1α-treated group, but this activity could be inhibited by CXCR7 antibody (P<0.01). CXCR7 antibody did not affect THP-1 cell apoptosis (P>0.05). CXCR7 antibody could inhibit SDF-1α -induced THP-1 cell invasiveness (P<0.01). CONCLUSION: CXCR7 highly expresses in AML-M5 patients and THP-1 cells, and involves in cell proliferation and invasion. The blocking CXCR7 expression can reduce the risk of AML-M5 cell infiltration.

Increase of C3a is Associated with Hemorrhagic Propensity in Patients with Immune Thrombocytopenia.

BACKGROUND: Complement activation is critically involved in multiple autoimmune diseases. Immune thrombocytopenia (ITP) is a hemorrhagic condition with enhanced platelet clearance caused by antiplatelet autoantibodies. However, the roles of complements C3a, C5a, and soluble C5b-9 (sC5b-9) in the hemorrhage of ITP remain unknown. METHODS: Plasma C3a, C5a, and sC5b-9 levels in ITP patients were measured by enzyme-linked immunosorbent assay (ELISA). Antiplatelet autoantibodies (anti-GPIIb/IIIa and anti-GPIbα) were evaluated by modified monoclonal antibody immobilization of platelet antigen (MAIPA) assay. The severity of bleeding was assessed using the validated bleeding score for each ITP patient at onset. RESULTS: Levels of C3a, C5a, and sC5b-9 were significantly increased in active ITP patients, compared with those in controls (p < 0.001). However, levels of C3a, C5a, and sC5b-9 were not changed by treatment of HD-DXM. In addition, the C3a levels were correlated with the increase in bleeding scores from the patients with ITP (p < 0.05, r = 0.256). In contrast, neither platelet counts nor antiplatelet autoantibodies (anti-GPIIb/IIIa and anti-GPIbα) showed any correlation with levels of C3a, C5a, and sC5b-9. CONCLUSIONS: Levels of C3a, C5a, and sC5b-9 are increased in patients with ITP, suggesting a hyperactive complement system. Certain complement components, such as C3a, may contribute to hemorrhage of patients with ITP.