Cannabinoid receptor 2 alleviates sepsis-associated acute lung injury by modulating maturation of dendritic cells.

BACKGROUND: Dendritic cells (DCs) play a key role in a variety of inflammatory lung diseases, but their role in sepsis-associated acute lung injury (SA-ALI) is currently not been illuminated. Cannabinoid receptor 2 (CNR2) has been reported to regulate the DCs maturation. However, whether the CNR2 in DCs contributes to therapeutic therapy for SA-ALI remain unclear. In current study, the role of CNR2 on DCs maturation and inflammatory during SA-ALI is to explored. METHODS: First, the CNR2 level was analyzed in isolated Peripheral Blood Mononuclear Cells (PBMCs) and Bronchoalveolar Lavage Fluid (BALF) from patient with SA-ALI by qRT-PCR and flow cytometry. Subsequently, HU308, a specific agonist of CNR2, and SR144528, a specific antagonist of CNR2, were introduced to explore the function of CNR2 on DCs maturation and inflammatory during SA-ALI. Finally, CNR2 conditional knockout mice were generated to further confirm the function of DCs maturation and Inflammation during SA-ALI. RESULTS: First, we found that the expression of CNR2 on DCs was decreased in patient with SA-ALI. Besides, the result showed HU308 could decrease the maturation of DCs and the level of inflammatory cytokines, simultaneously reduce pulmonary pathological injury after LPS-induced sepsis in mice. In contrast of HU308, SR144528 exhibits opposite function of DCs maturate, inflammatory cytokines and lung pathological injury. Furthermore, comparing with SR144528 treatment, similar results were obtained in DCs specific CNR2 knockout mice after LPS treatment. CONCLUSION: CNR2 could alleviate SA-ALI by modulating maturation of DCs and inflammatory factors levels. Targeting CNR2 signaling specifically in DCs has therapeutic potential for the treatment of SA-ALI.

Exosomal miR-127-5p from BMSCs alleviated sepsis-related acute lung injury by inhibiting neutrophil extracellular trap formation.

Neutrophil extracellular traps (NETs) play an important role in sepsis-related acute lung injury (ALI). Bone marrow mesenchymal stem cells (BMSCs)-derived exosomes and miRNA are becoming promising agents for the treatment of ALI. The current study aimed to elucidate the mechanism by BMSCs-derived exosomes carrying miR-127-5p inhibiting to the formation of NETs in sepsis-related ALI. We successfully isolated exosomes from BMSCs and confirmed that miR-127-5p was enriched in the exosomes. ALI mice treated with BMSCs-derived exosomes histologically improved, and the release of NETs and inflammatory factors in lung tissue and peripheral blood of mice also decreased compared with LPS group, while the protective effect of exosomes was attenuated after the knockdown of miR-127-5p. Using dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay, we identified CD64 as a direct target of miR-127-5p. Meanwhile, BMSCs-derived exosomes can synergize with anti-CD64 mab in ALI mice to reduce tissue damage, inhibit the release of inflammatory factors and NETs formation. The synergistic effect of exosomes was attenuated when miR-127-5p was down-regulated. These findings suggest that exosomal miR-127-5p derived from BMSCs is a potential therapeutic agent for treatment of sepsis-induced ALI through reducing NETs formation by targeting CD64.

Upregulation of TRPM8 can promote the colon cancer liver metastasis through mediating Akt/GSK-3 signal pathway.

This study aims to clarify the function of transient receptor potential melastatin 8 (TRPM8) in colon cancer liver metastasis. First, TRPM8 expression was determined by Western blotting in colon cancer patients with/without liver metastasis. Second, colon cancer cells were grouped into Mock, siCON, and siTRPM8 groups. Then, a series of in vitro experiments were conducted. Last, CT26 cells were used to construct colon cancer liver metastasis models on mice in vivo, followed by comparison of liver metastasis and determination of AKT/glycogen synthase kinase-3ß (GSK-3ß) pathway. Consequently, TRPM8 was upregulated in both colon cancer patients with/without liver metastasis, especially in those with metastasis. Compared with Mock and siCON groups, cells in siTRPM8 group demonstrated significant decreases in clone numbers, cell invasion, and migration; and obvious downregulations of p-AKT/AKT, p-GSK3ß/GSK3ß, Snail, and Vimentin, with an upregulation of E-cadherin. For in vivo experiments, a sharp decrease was observed in metastatic liver of mice in siTRPM8 group, with significant downregulations of p-AKT/AKT, p-GSK3ß/GSK3ß, Snail, and Vimentin and an upregulation of E-cadherin, as compared with Mock and siCON groups. Thus, TRPM8 was upregulated in colon cancer patients with liver metastasis, and silencing TRPM8 may suppress the progression and epithelial-mesenchymal transition of colon cancer cells to block its liver metastasis possibly by inhibiting AKT/GSK-3ß pathway.

[Structural and functional changes in islet beta cells in severely scalded rats].

OBJECTIVE: To observe the structural and functional changes in islet beta cells in severely scalded rats, and to explore its relationship with dysfunction of glycometabolism. METHODS: Seventy-two Wistar rats were divided into scald (S) group and sham injury (SI) group according to the random number table, with 36 rats in each group. Rats in group S were inflicted with 50%TBSA full-thickness scald by a 12-s immersion of back and a 6-s immersion of abdomen in 94 °C hot water. Rats in group SI were sham injured through immersion of back and abdomen in 37 °C warm water. At post injury hour (PIH) 6 and on post injury day (PID) 3 and 7, plasma glucose level was measured for intraperitoneal glucose tolerance test (IPGTT) in 12 rats of each group, and the area under the curve (AUC) of plasma glucose level was calculated. After the IPGTT, pancreatic tissue was harvested and subjected to a double immunostaining for insulin and cell nuclei to determine the pancreatic insulin-positive area ratio, and the area and number of beta cells in the islets (referred to as "the three indicators in the islets"). Data were processed with the analysis of repeated measures and factorial designed analysis of variance, and LSD test was applied for paired comparison. RESULTS: (1) At PIH 6 and on PID 3, the overall plasma glucose levels of rats in group S before and after injection of glucose and at each time point were obviously higher than those of rats in group SI (with F values of main effects respectively 79.372 and 32.962, P values all below 0.001; with P values of paired comparison below 0.05 or 0.01). On PID 7, the overall plasma glucose levels in the two groups before and after injection of glucose and at each time point were close (with P values all above 0.05). (2) The overall AUC of plasma glucose levels of rats in group S was higher than that of rats in group SI (main effects: F = 337.87, P < 0.01). Compared with those of rats in group SI [(1019 ± 32), (1003 ± 72) mmol·min·L(-1)], the AUCs of plasma glucose levels of rats in group S were higher at PIH 6 and on PID 3 [(1501 ± 163), (1132 ± 67) mmol·min·L(-1), P values all below 0.001]. The AUCs of plasma glucose levels were close between two groups on PID 7 (P > 0.05). The AUCs of plasma glucose levels on PID 3 and 7 were both lower than that at PIH 6 in rats of group S (with P values all below 0.001). (3) The three indicators in the islets in rats of group S were all lower than those of rats in group SI (with F values of main effects respectively 135.17, 24.75 and 39.35, P values all below 0.01). There were no significant differences in the three indicators in the islets at PIH 6 between two groups (with P values all above 0.05). The three indicators in the islets of rats in group S on PID 3 and 7 [0.47 ± 0.05, 0.51 ± 0.07; (0.032 ± 0.008), (0.037 ± 0.008) mm(2); (303 ± 64), (341 ± 58) cells] were significantly lower than those of rats in group SI [0.63 ± 0.05, 0.64 ± 0.06; (0.043 ± 0.011), (0.044 ± 0.012) mm(2); (398 ± 112), (387 ± 90) cells; P < 0.05 or P < 0.01] and that at PIH 6 within group S (P < 0.05 or P < 0.01). CONCLUSIONS: The number of beta cells is reduced, and the insulin secretion function of beta cells is decreased in the scalded rats, and they may constitute the cause of dysfunction of glycometabolism, mainly manifested as hyperglycemia.

Growth inhibitory effect of 4-phenyl butyric acid on human gastric cancer cells is associated with cell cycle arrest.

AIM: To investigate the growth effects of 4-phenyl butyric acid (PBA) on human gastric carcinoma cells and their mechanisms. METHODS: Moderately-differentiated human gastric carcinoma SGC-7901 and lowly-differentiated MGC-803 cells were treated with 5, 10, 20, 40, and 60 µmol/L PBA for 1-4 d. Cell proliferation was detected using the MTT colorimetric assay. Cell cycle distributions were examined using flow cytometry. RESULTS: The proliferation of gastric carcinoma cells was inhibited by PBA in a dose- and time-dependent fashion. Flow cytometry showed that SGC-7901 cells treated with low concentrations of PBA were arrested at the G0/G1 phase, whereas cells treated with high concentrations of PBA were arrested at the G2/M phase. Although MGC-803 cells treated with low concentrations of PBA were also arrested at the G0/ G1 phase, cells treated with high concentrations of PBA were arrested at the S phase. CONCLUSION: The growth inhibitory effect of PBA on gastric cancer cells is associated with alteration of the cell cycle. For moderately-differentiated gastric cancer cells, the cell cycle was arrested at the G0 /G1 and G2/M phases. For lowly-differentiated gastric cancer cells, the cell cycle was arrested at the G0/G1 and S phases.

[Study on the correlation between membrane protein Flotillin-1 and PrPc endocytosis].

OBJECTIVE: To explore whether the membrane-associated protein Flotillin-1 has relationship with endocytosis of PrPc. METHODS: The expression of Flotillin-1 in different cell lines was detected with the method of Western Blot; the interaction between Flotillin-1 and PrPc in Cells which were treated with copper ions was observed using immunoprecipitation method. RESULTS: (1) Flotillin-1 was widely expressed in many cell lines without significant difference in the amounts of expression level; (2) Only in the appearance of copper ions, the protein complexes of PrPc and Flotillin-1 can be detected with the method of IP, which were related to copper ions concentration and processing time. CONCLUSION: The membrane-associated protein Flotillin-1 has the relationship with the endocytosis of PrPc.

[Mutant D178N prion protein converts spontaneously in RT-QuIC assay].

OBJECTIVE: To study the conversion of mutant D178N prion protein in RT-QuIC assay. METHODS: The D178N mutant prion PRNP was generated by the method of single site mutation. The mutant PRNP gene was inserted into plasmids of pET24. The full and N-truncated recombinant human prion proteins were expressed and purified. The fibril formations of these proteins were real-time monitored by the method of RT-QuIC. The ability to resist proteinase K (PK) of these fibrils was analyzed. RESULTS: We succeed to construct human PrP-D178N plamids. The N-truncated human prion protein with D178N (PrP90-231-D178N) can convert spontaneously in RT-QuIC, while full length of human prion D178N protein (PrP23-231-D178N) fails to convert spontaneously. The spontaneously generated fibril has been domenstrated it is partily PK-resistant. CONCLUSION: The N-terminal of prion protein (23-90) plays an important role for the D178N mutant protein spontaneously conversion, which provide the clues for study the pathogenesis of genetic CJD.