Celastrus orbiculatus extract reverses precancerous lesions of gastric cancer by inhibiting autophagy via regulating the PDCD4-ATG5 signaling pathway.

OBJECTIVES: Celastrus orbiculatus ethyl acetate extract (COE) is the main extract of the stem of the Chinese herbal C. orbiculatus, which has anti-tumor and anti-inflammatory biological effects. Our previous study showed that COE had a certain reversal effect on the precancerous lesions of gastric cancer (PLGC) in rats, but the exact mechanism of action remains elusive. We aimed to explore the therapeutic effects of COE on PLGC and the potential mechanisms. METHODS: The PLGC rat model was successfully constructed by N-methyl-N´-nitro-N-nitrosoguanidine (MNNG) multifactorial induction method. Then, COE was prepared to treat the PLGC rat model. Hematoxylin & eosin staining was used to observe gastric mucosal lesions in rats, AB-PAS and HID-AB staining were used to observe intestinal metaplasia. PDCD4-ATG5 signaling pathway was detected by immunohistochemistry (IHC) and reverse transcription polymerase chain reaction (RT-PCR) in vivo, and autophagy level was detected by IHC, transmission electron microscopy, and RT-PCR in vivo. Besides, the PLGC (MC) cell model was successfully constructed by treating GES-1 cells with MNNG. Then, the morphology, proliferation, and apoptosis of MC cells, and the role of the PDCD4-ATG5 signaling pathway and autophagy in MC cells were evaluated by COE and after the overexpression of PDCD4 treatment. KEY FINDINGS: COE significantly improved gastric mucosal injury and cellular heteromorphism and retarded the progression of PLGC in rats. Further studies indicated COE not only inhibited the level of autophagy but also interfered with the PDCD4-ATG5 signaling pathway in vivo. On the other hand, COE treatment could effectively reverse MC cell damage, inhibit MC cell proliferation, and promote MC cell apoptosis. Furthermore, COE also promoted PDCD4 and inhibited ATG5 expression in vitro, and the inhibitory effect of COE on ATG5-mediated autophagy was further enhanced after the overexpression of PDCD4. CONCLUSIONS: The study revealed that COE could regulate the PDCD4-ATG5 signaling pathway to inhibit autophagy in gastric epithelial cells, which contributes to reversing the progression of PLGC.

Low-dose interleukin-2 can improve salivary secretion but not lymphocyte infiltration of salivary glands in a murine model of Sjögren's syndrome.

INTRODUCTION/AIM: Effects of low-dose interleukin-2 (IL-2) on the exocrine glandular glands of Sjögren's syndrome are unknown. The aim of this study was to investigate the effects of low-dose IL-2 on salivary gland structure and function in a murine model of Sjögren's syndrome. MATERIALS AND METHODS: Non-obese diabetic/Ltj (NOD) mice were used as the animal model of Sjögren's syndrome, and low-dose IL-2 or phosphate buffered saline was administered subcutaneously from 5 weeks of age, while ICR mice were used as controls. Some mice were sacrificed at 9 weeks of age, while the other mice that continued to receive treatment were sacrificed at 23 weeks. We determined the salivary flow rate of mice every 3 weeks during the intervention. After the mice were sacrificed, one submandibular gland was removed for pathological evaluation, while the other submandibular gland was used to measure the levels of 25 cytokines by Luminex technology. Cervical lymph nodes and spleens were examined by flow cytometry for the proportions of CD8+ T cells and Treg cells. RESULTS: The results showed that the salivary flow rate of NOD mice was slower than that of control-group mice, and there were more pathological changes in the submandibular gland. The levels of many cytokines in the submandibular gland were elevated. The proportion of CD8+ T cells in the cervical lymph nodes and spleens was increased; however, the proportion of Treg cells was decreased. After treatment with IL-2, the exocrine function of the salivary glands of mice was improved. IL-2 also promoted the proliferation of Treg cells in the cervical lymph nodes and spleens, but it did not alter the extent of lymphocyte infiltration in the submandibular gland. The levels of cytokines in the submandibular glands, as well as the proportion of CD8+ T cells in the cervical lymph nodes and spleens, were unchanged significantly after IL-2 treatment. CONCLUSION: Our results demonstrate that treatment with low-dose IL-2 improves the secretory function of the exocrine glands of mice with Sjögren's syndrome, but it does not reverse the structural damage of the exocrine glands.

Targeting EZH2 prevents the occurrence and mitigates the development of Sjögren's syndrome in mice.

OBJECTIVE: We analyzed RNA-SEQ data and found that EZH2 gene expression in salivary glands (SGs) of Sjögren's syndrome (SS) patients was up-regulated and correlated with pathological injury. In this study, we sought to determine if inhibiting EZH2 would ameliorate SS-like disease in NOD/Ltj (NOD) mice. METHODS: We analyzed RNA-SEQ data of SGs of patients with SS from data obtained from the GEO database to explore the correlation between EZH2 gene expression and the progression of SS. Inhibition of EZH2 in the NOD mice was achieved by intraperitoneal administration of GSK343 using both a preventative and a therapeutic model. The effects of GSK343 on SGs secretion and pathological damage, as well as the levels and functions of T cells, B cells, Myeloid-derived suppressor cells (MDSCs), and other immune cells were evaluated. RESULTS: The expression levels of the gene encoding EZH2 in the SGs of SS patients were significantly higher than the non-SS sicca patients, and the expression levels were positively correlated with the severity of the SGs pathological damage. GSK343 treatment significantly increased the salivary flow rate and pathological damage of the SGs in the NOD mice compared to the control mice. In addition, GSK343 significantly inhibited the number and pro-inflammatory-factor secretion of CD4+ and CD8+ T cells and inhibited the increase in the Th1/Th2 cell ratio caused by SS. RNA-SEQ data also showed that EZH2 inhibited several inflammatory pathways during the pathogenesis of SS. CONCLUSIONS: EZH2 expression was up-regulated in the submandibular gland tissue of SS patients.Inhibition of EZH2 alleviated SS-like disease in NOD mice, suggesting that EZH2 might be a potential target for the clinical treatment of SS.

Dangguibuxue decoction abolishes abnormal accumulation of erythroid progenitor cells induced by melanoma.

ETHNOPHARMACOLOGIC RELEVANCE: Dangguibuxue decoction (DGBX), is a well-known traditional Chinese medicine that contains two types of materials used to treat anemia. In this study, we aimed to explore the effect and mechanism of DGBX on abolishing erythroid progenitor cell (Ter119+CD71+) accumulation induced by melanoma. MATERIALS AND METHODS: B16/F10 melanoma cells were used to establish transplanted and metastatic melanoma models. DGBX or normal saline were administered intragastrically daily after the models were established. Tumor sizes and metastatic nodules were observed after tumor cell inoculation. To further test the function of DGBX on erythroid progenitor cell (EPC) accumulation and immunosuppressive abilities, the percentage of EPCs in the blood, and spleen were quantified with flow cytometry. The proportion of CD8+ T cells and related functional mediators, IFN-γ and TNF-α,were also quantified with flow cytometry. To further strengthen our in vivo observations, DGBX serum was prepared from the rats three days after DGBX was administered. Liquid chromatography-mass spectrometry was carried out to control the quality of the experiments. B16/F10 melanomacells were cultured with DGBX serum, and proliferation and apoptosis were observed with the CCK8 assay and AnnexinV/7AAD staining, respectively. EPCs were isolated from B16/F10-bearing mice and cultured under erythroid differentiation conditions. EPCs were treated with DGBX serum, and mature red cell proportions and cell denucleations were tested with flow cytometry and Giemsa staining of the cultured EPCs. Flow cytometry and qPCR were used to analyze the effects of DGBX on the expression of key molecules involved in erythroid development and to explore the mechanism by which DGBX relieves abnormal EPC accumulation. RESULTS: DGBX treatments significantly reduced B16 melanoma tumor sizes and metastatic nodules. Most importantly, our study strongly suggested that DGBX could alleviate anemia, and systematically enhance anti-tumor immune responses by reducing abnormal EPC accumulation. Moreover, DGBX serum treatments had no direct effect on tumor cell proliferation and apoptosis, but could promote EPCs to differentiate into mature red blood cells, in vitro. Mechanistically, at least in part, DGBX relieved abnormal EPC accumulation by altering the "master switch" transcription factors, Pu.1 and Gata-1. CONCLUSIONS: DGBX significantly alleviates abnormal tumor-induced EPC accumulation, inhibits B16 melanoma progression, and enhances anti-tumor immune responses.