Beneficial Effects of Celastrol on Immune Balance by Modulating Gut Microbiota in Experimental Ulcerative Colitis Mice

Ulcerative colitis (UC) is a chronic inflammatory bowel disease caused by many factors including colonic inflammation and microbiota dysbiosis. Previous studies have indicated that celastrol (CSR) has strong anti-inflammatory and immune-inhibitory effects. Here, we investigated the effects of CSR on colonic inflammation and mucosal immunity in an experimental colitis model, and addressed the mechanism by which CSR exerts the protective effects. We characterized the therapeutic effects and the potential mechanism of CSR on treating UC using histological staining, intestinal permeability assay, cytokine assay, flow cytometry, fecal microbiota transplantation (FMT), 16S rRNA sequencing, untargeted metabolomics, and cell differentiation. CSR administration significantly ameliorated the dextran sodium sulfate (DSS)-induced colitis in mice, which was evidenced by the recovered body weight and colon length as well as the decreased disease activity index (DAI) score and intestinal permeability. Meanwhile, CSR down-regulated the production of pro-inflammatory cytokines and up-regulated the amount of anti-inflammatory mediators at both mRNA and protein levels, and improved the balances of Treg/Th1 and Treg/Th17 to maintain the colonic immune homeostasis. Notably, all the therapeutic effects were exerted in a gut microbiota-dependent manner. Furthermore, CSR treatment increased the gut microbiota diversity and changed the compositions of the gut microbiota and metabolites, which is probably associated with the gut microbiota-mediated protective effects. In conclusion, this study provides the strong evidence that CSR may be a promising therapeutic drug for UC.     

Recombinant human lecithin‐cholesterol acyltransferase Fc fusion: Analysis of N‐ and O‐linked glycans and identification and elimination of a xylose‐based O‐linked tetrasaccharide core in the linker region

Recombinant human lecithin‐cholesterol acyltransferase Fc fusion (huLCAT‐Fc) is a chimeric protein produced by fusing human Fc to the C‐terminus of the human enzyme via a linker sequence. The huLCAT‐Fc homodimer contains five N‐linked glycosylation sites per monomer. The heterogeneity and site‐specific distribution of the various glycans were examined using enzymatic digestion and LC‐MS/MS, followed by automatic processing. Almost all of the N‐linked glycans in human LCAT are fucosylated and sialylated. The predominant LCAT N‐linked glycoforms are biantennary glycans, followed by triantennary sugars, whereas the level of tetraantennary glycans is much lower. Glycans at the Fc N‐linked site exclusively contain typical asialobiantennary structures. HuLCAT‐Fc was also confirmed to have mucin‐type glycans attached at T₄₀₇ and S₄₀₉. When LCAT‐Fc fusions were constructed using a G‐S‐G‐G‐G‐G linker, an unexpected +632 Da xylose‐based glycosaminoglycan (GAG) tetrasaccharide core of Xyl‐Gal‐Gal‐GlcA was attached to S₄₁₈. Several minor intermediate species including Xyl, Xyl‐Gal, Xyl‐Gal‐Gal, and a phosphorylated GAG core were also present. The mucin‐type O‐linked glycans can be effectively released by sialidase and O‐glycanase; however, the GAG could only be removed and localized using chemical alkaline β‐elimination and targeted LC‐MS/MS. E₄₁₆ (the C‐terminus of LCAT) combined with the linker sequence is likely serving as a substrate for peptide O‐xylosyltransferase. HuLCAT‐Fc shares some homology with the proposed consensus site near the linker sequence, in particular, the residues underlined PPP E₄₁₆GS₄₁₈G G G GDK. GAG incorporation can be eliminated through engineering by shifting the linker Ser residue downstream in the linker sequence.     

Changes in levels of pancreatic endoplasmic reticulum proteins that function in translocation and maturation of secretory proteins in response to cholecystokinin

Two pathways operate to target newly-synthesised proteins to the endoplasmic reticulum. In one, the signal recognition particle attaches to the signal sequences of nascent chains on ribosomes and slows or stops translation until contact is made with the docking protein at the membrane. The second operates via molecular chaperons. The pathways converge at the level of a 43 kDa signal binding protein integrated into the membrane, where translocation through a proteinaceous pore is initiated. In the lumen, proteins fold and disulphide formation is catalysed by the enzyme protein disulphide isomerase. The heavy chain binding protein may attach to unassembled or unfolded proteins and prevent their exit from the ER to the Golgi. Cholecystokinin (CCK) treatment increases the biosynthesis and secretion of pancreatic proteins, increases the levels of PDI and the 43 kDa binding protein, and reduces levels of BiP. These proteins may be possible targets for genetic manipulation to improve processing of heterologous proteins from cultured mammalian cells.     

Indole-3-carbinol blocks platelet-derived growth factor-stimulated vascular smooth muscle cell function and reduces neointima formation in vivo

The purpose of this study was to determine the effect and associated cell signaling mechanisms of indole-3-carbinol (I3C) on platelet-derived growth factor (PDGF)-BB-induced proliferation and migration of cultured vascular smooth muscle cells (VSMCs) and neointima formation in a carotid injury model. Our data demonstrated that I3C inhibited PDGF-BB-induced proliferation of VSMCs in a dose-dependent manner without causing cell cytotoxicity, as assessed by 5-bromo-2′-deoxyuridine incorporation and WST-1 assays. Further studies revealed that the antiproliferative effect of I3C was caused by the arrest of cells in both the G0/G1 and S phases. Moreover, I3C treatment inhibited migration of VSMCs and partly reversed the expression of smooth-muscle-specific contractile markers. We also demonstrated that I3C-induced growth inhibition was associated with an inhibition of the expression of cyclin D1 and cyclin-dependent kinase 4/6, as well as an increase in p27^Kip1 levels in PDGF-stimulated VSMCs. These beneficial effects of I3C on VSMCs appeared to be at least partly mediated by the inhibition of Akt and the subsequent activation of glycogen synthase kinase (GSK) 3β. Furthermore, using a mouse carotid artery injury model, we found that treatment with 150 mg/kg I3C resulted in a significant reduction of the neointima/media ratio and cells positive for proliferating cell nuclear antigen. These results demonstrate that I3C can suppress the proliferation and migration of VSMCs and neointima hyperplasia after vascular injury via inhibition of the Akt/GSK3β pathway and suggest that this might be feasible as part of a therapeutic strategy for vascular proliferative diseases.     

Expression of honeybee prepromelittin as a fusion protein in Escherichia coli

Strategies for the expression of precursors of eukaryotic secreted proteins as part of fused proteins in Escherichia coli have been explored. A fusion protein with β-galactosidase at the N-terminal end and honeybee prepromelittin at the C-terminal end (β-gal-pM) was expressed in low amounts as a cleaved polypeptide, from which the promelittin portion had been removed. Inclusion in the induction culture of 10 mM MgCl₂ or 8.3% (v/v) ethanol, inhibitors of signal peptidase, gave rise to the full-length β-gal-pM fusion protein. The results suggest that a soluble recombinant fusion protein with a signal peptide in an internal location 660 residues from the N-terminus is recognized by the E. coli translocation apparatus in the inner membrane and by leader peptidase. High-level production (about 45% of total cellular proteins) of prepromelittin was achieved when it was part of a fusion protein at the C-terminus of a truncated insoluble polypeptide from bacteriophage gene 10. This fusion protein separated into inclusion bodies in an aggregated form. In contrast, attempts to express prepromelittin by itself or at the N-terminal end of a fusion with mouse dihydrofolate reductase (pM-DHFR) proved unsuccessful.     

定向激活HIF-1协同脂肪干细胞对糖尿病小鼠皮肤创伤修复的促进作用

目的探讨定向激活HIF-1协同脂肪干细胞（ASCs）对糖尿病小鼠损伤修复的影响及机制。 方法8周龄雄性SPF级C57BJ/L小鼠68只,行腹腔注射1%链脲佐菌素（STZ,60?mg/?kg）的柠檬酸-柠檬酸钠缓冲液,3 d后空腹血糖大于16.7 mmol/L的小鼠用于实验。在小鼠背部做直径为1 cm的圆形皮肤全层创口,将动物随机分为空载质粒（EV）组,CA5-HIF-1α转染（CA5）组,EV+saline组,CA5+saline组,EV+ASCs组,CA5+ASCs组。于给药后第0、3、7、10、14、17和21天,观察并记录创口面积大小,利用激光多普勒血流成像（LDPI）检测血流灌注情况,荧光定量PCR法检测HIF-1α及VEGF mRNA表达水平,HE染色检测血管密度,Western blot法检测VEGE蛋白表达水平。用Bonferroni或Tukey post hoc方差分析法进行统计学分析。 结果CA5-HIF-1α基因注射剂量为125 μg时,局部HIF-1α mRNA表达水平较高为10.40±0.22（F = 19.48,P = 0.025）,故选用125 μg用于后续实验。于给药后第3 ~ 14天,CA5组小鼠的伤口面积分别为7?828.92±294.28、7?285.97±118.24、4?050.41±301.97、1?292.35±101.14小于EV组9?062.00±225.75、8?534.42±189.35、5?634.59±198.06、2?308.15±245.36（F?=?41.37、32.16、27.29、25.16,P?=?0.028、0.034、0.038、0.042）。CA5组小鼠皮肤血流于第10、17天（253.06±8.34、250.59±10.13）均大于EV组（158.31±9.98、169.73±7.28）（F?=?21.53、26.08,P?=?0.038、0.032）。在治疗后3 ~ 14天,CA5+saline组（7?656.92±177.03、7?163.83±128.24、4?238.23±228.36、1?316.52±90.75）、EV+ASCs组(7?593.64±192.12、7?233.08±146.86、4?097.58±227.91)及CA5+ASCs组的伤口面积小于对照组（6?745.25±203.16、6?159.35±168.72、3?682.06±257.30）（F?=?39.58、44.09、34.67,P?=?0.031、0.028、0.037）,且CA5+ASCs组最小。本研究还发现CA5+saline组（262.05±9.34、248.45±11.13）、EV+ASCs组（215.33±10.75、185.82±10.47）及CA5+ASCs组（322.54±12.27、292.49±9.57）的小鼠皮肤血流于第10天和第17天均大于对照组（161.30±5.64、134.57±8.67）（F?=?29.15、17.38,P?=?0.026、0.034）,CA5+ASCs组血流增加幅度最大。此外,CA5+saline组、EV+ASCs组及CA5+ASCs组血管密度大于对照组,且CA5+ASCs组最高。CA5+saline组、EV+ASCs组及CA5+ASCs组VEGF mRNA及蛋白表达水平分别为2.03±0.14、2.16±0.13、3.41±0.18和1.75±0.12、1.82±0.06、2.96±0.14,高于对照组1.05±0.02和1.03±0.05（F?=?34.08、29.53,P?=?0.019、0.021）,且CA5+ASCs组最高。 结论定向激活HIF-1基因协调脂肪干细胞可促进糖尿病小鼠创伤修复,可能与其调控VEGF表达有关。