Natural immunoglobulin M (IgM) antibodies have been shown to recognize post‐ischemic neoepitopes following reperfusion of tissues and to activate complement. Specifically, IgM antibodies and ...complement have been shown to drive hepatic ischemia reperfusion injury (IRI). Herein, we investigate the therapeutic effect of C2 scFv (single‐chain antibody construct with specificity of a natural IgM antibody) on hepatic IRI in C57BL/6 mice. Compared with PBS‐treated mice, C2 scFv‐treated mice displayed almost no necrotic areas, significant reduction in serum ALT, AST and LDH levels, and significantly reduced in the number of TUNEL positive cells. Moreover, C2 scFv‐treated mice exhibited a notable reduction in inflammatory cells after hepatic IRI than PBS‐treated mice. The serum IL‐6, IL‐1β, TNF‐α and MPC‐1 levels were also severely suppressed by C2 scFv. Interestingly, C2 scFv reconstituted hepatic inflammation and IRI in Rag1−/− mice. We found that C2 scFv promoted hepatic cell death and increased inflammatory cytokines and infiltration of inflammatory cells after hepatic IRI in Rag1−/− mice. In addition, IgM and complement 3d (C3d) were deposited in WT mice and in Rag1−/− mice reconstituted with C2 scFv, indicating that C2 scFv can affect IgM binding and complement activation and reconstitute hepatic IRI. C3d expression was significantly lower in C57BL/6 mice treated with C2 scFv compared to PBS, indicating that excessive exogenous C2 scFv inhibited complement activation. These data suggest that C2 scFv alleviates hepatic IRI by blocking complement activation, and treatment with C2 scFv may be a promising therapy for hepatic IRI.
Although it has been reported that deficiency of toll-like receptor 4 (TLR4) is associated with reduced atherosclerosis in atherosclerosis-prone mice and attenuated pro-inflammatory state in diabetic ...mice, it remains undetermined whether treatment with a TLR4 antagonist reduces atherosclerosis in nondiabetic or diabetic mice that have TLR4 expression. In this study, we determined the effect of Rhodobacter sphaeroides lipopolysaccharide (Rs-LPS), an established TLR4 antagonist, on early-stage atherosclerosis in nondiabetic and streptozotocin-induced diabetic apolipoprotein E-deficient (Apoe−/−) mice. Analysis of atherosclerotic lesions of both en face aortas and cross sections of aortic roots showed that administration of Rs-LPS in 14-week-old diabetic Apoe−/− mice for 10 weeks significantly reduced atherosclerotic lesions. Although atherosclerotic lesions in nondiabetic Apoe−/− mice appeared to be decreased by Rs-LPS treatment, the difference was not statistically significant. Metabolic study showed that Rs-LPS significantly lowered serum levels of cholesterol and triglycerides in nondiabetic mice but not in diabetic mice. Furthermore, immunohistochemistry studies showed that Rs-LPS inhibited the expression of interleukin 6 and matrix metalloproteinase-9 and reduced the content of monocytes and macrophages in atherosclerotic plaques. Taken together, this study demonstrated for the first time that TLR4 antagonist inhibited vascular inflammation and atherogenesis in diabetic Apoe−/− mice and lowered serum cholesterol and triglyceride levels in nondiabetic Apoe−/− mice.
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Background:Neutral sphingomyelinase 1 (NSMase1) catalyzes sphingomyelin to generate ceramide and mediates tumor cell apoptosis; however, the roles of NSMase1 in hepatocellular ...carcinoma (HCC) remain unclear. This study aims to evaluate the clinical value and prognostic significance of NSMase1 in HCC. Methods:A total of 142 patients who underwent radical hepatectomy were involved in this study. The expression of NSMase1 in HCC tissues and adjacent nontumorous liver tissues (ANLTs) was detected by quantitative real-time polymerase chain reaction and immunohistochemistry, and the association between NSMase1 expression and clinicopathologic features as well as prognosis of HCC patients was analyzed. Univariate and multivariate analyses were applied to identify independent prognostic factors. Results: NSMase1, at both mRNA and protein levels, was significantly decreased in HCC tissues compared to ANLTs. Low NSMase1 expression was associated with tumor size ( P= 0.029), TNM stage ( P= 0.040) and recurrence ( P= 0.006). Statistically, both the overall survival (OS) and disease-free survival (DFS) of low NSMase1 expression group were significantly shorter compared with high NSMase1 expression group ( p= 0.001; p= 0.001; respectively). Remarkably, the multivariate analysis showed that the low NSMase1 expression was an independent prognostic factor for OS (hazard ratio = 1.840; 95% confidence interval, 1.178-2.875, P= 0.007) and DFS (hazard ratio = 1.706; 95% confidence interval, 1.096-2.655, P= 0.018) in all enrolled HCC patients. Conclusions: NSMase1 down-regulation might actually serve as an independent prognostic factor for HCC patients. However, the roles of “NSMase1-ceramide” metabolic network in HCC deserve further studies.
Neutral sphingomyelinase 1 (NSMase1) mediates caspase‐3 activation and apoptosis. However, the role of NSMase1, especially exosome‐borne NSMase1 in hepatocellular carcinoma (HCC), remains unclear. We ...report that NSMase1, which converts sphingomyelin (SM) to ceramide, was significantly downregulated in HCC tissues. Low NSMase1 expression predicted poor long‐term survival of HCC patients. NSMase1 downregulation in HCC resulted in increased SM and reduced ceramide (Cer) that led to an increased SM/Cer ratio. Interestingly, NSMase1 and NSMase activity were also decreased in exosomes isolated from HCC tissues and cell lines. Furthermore, NSMase activity increased in exosomes isolated from the culture medium of L02 cells transfected with pEGFP‐C3‐NSMase1 (NSMase1‐Exo). NSMase1‐Exo suppressed HCC cell growth and induced apoptosis via reduction of the SM/Cer ratio. Thus, NSMase1 in exosomes inhibits HCC growth by decreasing the SM/Cer ratio.
Downregulation of NSMase1 in HCC tissues leads to NSMase1 decrease in exosomes secreted from HCC cells. These exosomes with low NSMase1 promote cancer cell proliferation by increasing SM/Cer ratio. Conversely, treating HCC cell with NSMase1‐enriched exosomes isolated from L02 cells transfected with NSMase1 plasmids results in cancer cell apoptosis due to the decrease of SM/Cer ratio.
Aldo‐keto reductase family 1 member B10 (AKR1B10) is a secretory protein overexpressed in hepatocellular carcinoma (HCC). We aimed to evaluate AKR1B10 as a serum marker for detection of HCC. Herein, ...we conducted a cohort study that consecutively enrolled 1,244 participants from three independent hospitals, including HCC, healthy controls (HCs), benign liver tumors (BLTs), chronic hepatitis B (CHB), and liver cirrhosis (LC). Serum AKR1B10 was tested by time‐resolved fluorescent assays. Data were plotted for receiver operating characteristic (ROC) curve analyses. Alpha‐fetoprotein (AFP) was analyzed for comparison. An exploratory discovery cohort demonstrated that serum AKR1B10 increased in patients with HCC (1,567.3 ± 292.6 pg/mL; n = 69) compared with HCs (85.7 ± 10.9 pg/mL; n = 66; P < 0.0001). A training cohort of 519 participants yielded an optimal diagnostic cutoff of serum AKR1B10 at 267.9 pg/mL. When ROC curve was plotted for HCC versus all controls (HC + BLT + CHB + LC), serum AKR1B10 had diagnostic parameters of the area under the curve (AUC) 0.896, sensitivity 72.7%, and specificity 95.7%, which were better than AFP with AUC 0.816, sensitivity 65.1%, and specificity 88.9%. Impressively, AKR1B10 showed promising diagnostic potential in early‐stage HCC and AFP‐negative HCC. Combination of AKR1B10 with AFP increased diagnostic accuracy for HCC compared with AKR1B10 or AFP alone. A validation cohort of 522 participants confirmed these findings. An independent cohort of 68 patients with HCC who were followed up showed that serum AKR1B10 dramatically decreased 1 day after operation and was nearly back to normal 3 days after operation. Conclusion: AKR1B10 is a potent serum marker for detection of HCC and early‐stage HCC, with better diagnostic performance than AFP.
AKR1B10 is involved in hepatocarcinogenesis via modulation of fatty acid and lipid synthesis. AKR1B10 inhibition results in apoptosis of tumor cells whose lipids, especially phospholipids, were ...decreased by over 50%, suggesting involvement of phospholipids like sphingosine-1-phosphate (S1P) in AKR1B10's oncogenic function. Using a co-culture system, we found that co-culture of QSG-7701 (human hepatocyte) with HepG2 (hepatoma cell line) increases QSG-7701's proliferation, in which AKR1B10-S1P signaling plays a pivotal role. Consistent with previous findings, AKR1B10 mRNA and protein levels were higher in primary hepatocellular carcinoma (PHC) tissues than in peri-tumor tissues. Interestingly, the level of S1P was also higher in PHC tissues than in peri-tumor tissues. After analyzing the correlation between AKR1B10 mRNA expression in PHC tissues and the clinical data, we found that AKR1B10 mRNA expression was associated with serum alpha-fetoprotein (AFP), tumor-node-metastasis (TNM) stage, and lymph node metastasis, but not with other clinicopathologic variables. A higher AKR1B10 mRNA expression level is related to a shorter DFS (disease free survival) and OS (overall survival), serving as an independent predictor of DFS and OS in PHC patients with surgical resection.
Premature trypsinogen activation is the early event of acute pancreatitis. Therefore, the studies on the processes of trypsinogen activation induced by compounds are important to understand mechanism ...underly acute pancreatitis under various conditions. Calcium overload in the early stage of acute pancreatitis was previously found to cause intracellular trypsinogen activation; however, treatment of acute pancreatitis using calcium channel blockers did not produced consistent results. Proteasome activity that could be inhibited by some calcium channel blocker has recently been reported to affect the development of acute pancreatitis; however, the associated mechanism were not fully understood. Here, the roles of nicardipine were investigated in trypsinogen activation in pancreatic acinar cells. The results showed that nicardipine could increase cathepsin B activity that caused trypsinogen activation, but higher concentration of nicardipine or prolonged treatment had an opposite effect. The effects of short time treatment of nicardipine at low concentration were studied here. Proteasome inhibition was observed under nicardipine treatment that contributed to the up-regulation in cytosolic calcium. Increased cytosolic calcium from ER induced by nicardipine resulted in the release and activation of cathepsin B. Meanwhile, calcium chelator inhibited cathepsin B as well as trypsinogen activation. Consistently, proteasome activator protected acinar cells from injury induced by nicardipine. Moreover, proteasome inhibition caused by nicardipine depended on CaMKII. In conclusion, CaMKII down-regulation/proteasome inhibition/cytosolic calcium up-regulation/cathepsin B activation/trypsinogen activation axis was present in pancreatic acinar cells injury under nicardipine treatment.
Polymer hydrogels are generally insufficient biomechanics, strong resistance to cell adhesion, and weak bioactivity which limits their application in bone tissue engineering considerably. In order to ...develop a bone tissue engineering material with both good mechanical properties, osteogenic and angiogenic activity. Nanofibers carrying DNA plasmid (pNF) are introduced to gelatin methacryloyl (GelMA) and thiolated chitosan (TCS) system for preparing a novel GelMA/TCS/pNF composite hydrogel with dual network structure. By characterization of the compressive measurements, the resulting composite scaffold shows greatly enhanced mechanical strength (0.53 MPa) and is not damaged after 20 cycles of compression. And the fabricated composite scaffold displays sustained release of bone morphogenetic protein‐2 that can induce osteogenic differentiation and angiopoietin‐1 that promotes vascularization. The cell experiment shows that this system can significantly promote MC3T3‐E1 cell attachment, proliferation, as well as osteogenic‐related and angiogenic‐related genes expression of MC3T3‐E1 cells. Moreover, the in vivo results show that the composite scaffold with activated gene fibers can significantly promote osteogenesis and vascularization leading to favorable capacity of bone regeneration, meaning that the resulting biomimetic composite hydrogel scaffolds are excellent candidates for bone repair materials.
Herein, a multifunctional hydrogel‐fiber composite with activated gene (ANG and BMP‐2)‐based gelatin methacryloyl and thiolated chitosan is successfully prepared via photopolymerization and in situ soaking process, which integrates with good mechanical properties, improved cytocompatibility, upregulated osteodifferentiation, and angiogenesis. Promising clinical application in bone regeneration is presented through enhancement of osteogenesis and vascularization capability.
The accumulation of amyloid beta (Aβ) plaques in the brain is a hallmark of Alzheimer’s disease (AD) pathology. Microglial activation-mediated neuroinflammation has been implicated in the ...pathogenesis of AD and the expression levels of interleukin-6 (IL-6) were increased in the brains of AD patients. However, the mechanisms by which IL-6 expression is regulated in human microglia are incompletely understood. Here, we show that Aβ
1-40
oligomers (Aβ
40
) dose-dependently stimulate IL-6 expression in HMC3 human microglial cells. Treatment with Aβ
40
promotes the transcription of IL-6 and tumor necrosis factor α (TNFα) mRNAs in both HMC3 and THP-1 cells. Mechanistic studies reveal that Aβ
40
-induced increase of IL-6 secretion is associated with the activation of p38 mitogen-activated protein kinase (p38 MAPK). Inhibition of p38 MAPK by BIRB 796 or SB202190 abrogates Aβ
40
-induced increase of IL-6 production. Through analyzing brain specimens, we found that the immunoreactivity for IL-6 and phosphorylated (the activated form) p38 MAPK was markedly higher in microglia of AD patients than in age-matched control subjects. Moreover, our studies identified the co-localization of IL-6 with phosphorylated p38 MAPK in microglia in the cortices of AD patients. Taken together, these results indicate that p38 MAPK is a major regulator of Aβ-induced IL-6 production in human microglia, which suggests that targeting p38 MAPK may represent a new approach to ameliorate Aβ accumulation-induced neuroinflammation in AD.
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