MicroRNAs involved in neoplastic transformation of liver cancer stem cells

<p>Abstract</p> <p>Background</p> <p>The existence of cancer stem cells in hepatocellular carcinoma (HCC) has been verified by characterizing side population (SP) cells based on efflux of Hoechst 33342 dye from stem cells. Recent advances in microRNA (miRNA) biology have revealed that miRNAs play an important role in embryonic development and tumorigenesis. However, it is still unclear which miRNAs participate in the neoplastic transformation of liver cancer stem cells (LCSCs) during hepatocarcinogenesis.</p> <p>Methods</p> <p>To identify the unique set of miRNAs differentially regulated in LCSCs, we applied SP sorting to primary cultures of F344 rat HCC cancer cells treated with diethylnitrosamine (DEN) and normal syngenic fetal liver cells, and the stem-like characteristics of SP cells were verified through detecting expression of CD90.1, AFP and CK-7. Global miRNA expression profiles of two groups of SP cells were screened through microarray platform.</p> <p>Results</p> <p>A total of 68 miRNAs, including miR-10b, miR-21, miR-470*, miR-34c-3p, and let-7i*, were identified as overexpressed in SP of HCC cells compared to fetal liver cells. Ten miRNAs were underexpressed, including miR-200a* and miR-148b*. These miRNAs were validated using stem-loop real-time reverse transcriptase polymerase chain reaction (RT-PCR).</p> <p>Conclusions</p> <p>Our results suggest that LCSCs may have a distinct miRNA expression fingerprint during hepatocarcinogenesis. Dissecting these relationships will provide a new understanding of the function of miRNA in the process of neoplastic transformation of LCSCs.</p

Astragaloside IV, a Novel Antioxidant, Prevents Glucose-Induced Podocyte Apoptosis In Vitro and In Vivo

Glucose-induced reactive oxygen species (ROS) production initiates podocyte apoptosis, which represents a novel early mechanism leading to diabetic nephropathy (DN). Here, we tested the hypothesis that Astragaloside IV(AS-IV) exerts antioxidant and antiapoptotic effects on podocytes under diabetic conditions. Apoptosis, albuminuria, ROS generation, caspase-3 activity and cleavage, as well as Bax and Bcl-2 mRNA and protein expression were measured in vitro and in vivo. Cultured podocytes were exposed to high glucose (HG) with 50, 100 and 200 µg/ml of AS-IV for 24 h. AS-IV significantly attenuated HG-induced podocyte apoptosis and ROS production. This antiapoptotic effect was associated with restoration of Bax and Bcl-2 expression, as well as inhibition of caspase-3 activation and overexpression. In streptozotocin (STZ)-induced diabetic rats, severe hyperglycemia and albuminuria were developed. Increased apoptosis, Bax expression, caspase-3 activity and cleavage while decreased Bcl-2 expression were detected in diabetic rats. However, pretreatment with AS-IV (2.5, 5, 10 mg·kg−1·d−1) for 14 weeks ameliorated podocyte apoptosis, caspase-3 activation, renal histopathology, podocyte foot process effacement, albuminuria and oxidative stress. Expression of Bax and Bcl-2 mRNA and protein in kidney cortex was partially restored by AS-IV pretreatment. These findings suggested AS-IV, a novel antioxidant, to prevent Glucose-Induced podocyte apoptosis partly through restoring the balance of Bax and Bcl-2 expression and inhibiting caspase-3 activation

Finite element analysis of rapid canine retraction through reducing resistance and distraction

Objective: The aims of this study were to compare different surgical approaches to rapid canine retraction by designing and selecting the most effective method of reducing resistance by a three-dimensional finite element analysis. Material and Methods: Three-dimensional finite element models of different approaches to rapid canine retraction by reducing resistance and distraction were established, including maxillary teeth, periodontal ligament, and alveolar. The models were designed to dissect the periodontal ligament, root, and alveolar separately. A 1.5 N force vector was loaded bilaterally to the center of the crown between first molar and canine, to retract the canine distally. The value of total deformation was used to assess the initial displacement of the canine and molar at the beginning of force loading. Stress intensity and force distribution were analyzed and evaluated by Ansys 13.0 through comparison of equivalent (von Mises) stress and maximum shear stress. Results: The maximum value of total deformation with the three kinds of models occurred in the distal part of the canine crown and gradually reduced from the crown to the apex of the canine; compared with the canines in model 3 and model 1, the canine in model 2 had the maximum value of displacement, up to 1.9812 mm. The lowest equivalent (von Mises) stress and the lowest maximum shear stress were concentrated mainly on the distal side of the canine root in model 2. The distribution of equivalent (von Mises) stress and maximum shear stress on the PDL of the canine in the three models was highly concentrated on the distal edge of the canine cervix. . Conclusions: Removal of the bone in the pathway of canine retraction results in low stress intensity for canine movement. Periodontal distraction aided by surgical undermining of the interseptal bone would reduce resistance and effectively accelerate the speed of canine retraction

The effect of capsaicin on expression patterns of CGRP in trigeminal ganglion and trigeminal nucleus caudalis following experimental tooth movement in rats

Objectives The aim of this study was to explore the effect of capsaicin on expression patterns of calcitonin gene-related peptide (CGRP) in the trigeminal ganglion (TG) and trigeminal subnucleus caudalis (Vc) following experimental tooth movement. Material and Methods Male Sprague-Dawley rats were used in this study and divided into small-dose capsaicin+force group, large-dose capsaicin+force group, saline+force group, and no force group. Closed coil springs were used to mimic orthodontic forces in all groups except for the no force group, in which springs were inactivated. Capsaicin and saline were injected into periodontal tissues. Rats were euthanized at 0 h, 12 h, 1 d, 3 d, 5 d, and 7 d following experimental tooth movement. Then, TG and Vc were obtained for immunohistochemical staining and western blotting against CGRP. Results Immunohistochemical results indicated that CGRP positive neurons were located in the TG, and CGRP immunoreactive fibers were distributed in the Vc. Immunohistochemical semiquantitative analysis and western blotting analysis demonstrated that CGRP expression levels both in TG and Vc were elevated at 12 h, 1 d, 3 d, 5 d, and 7 d in the saline + force group. However, both small-dose and large-dose capsaicin could decrease CGRP expression in TG and Vc at 1 d and 3 d following experimental tooth movement, as compared with the saline + force group. Conclusions These results suggest that capsaicin could regulate CGRP expression in TG and Vc following experimental tooth movement in rats

Magnolin Protects against Contrast-Induced Nephropathy in Rats via Antioxidation and Antiapoptosis

BACKGROUND: Magnolin is the major active ingredient of the herb Magnolia fargesii which has anti-inflammatory and antioxidative effects. Oxidative stress and apoptosis are involved in the pathogenesis of contrast-induced nephropathy (CIN). We hypothesize that Magnolin could protect against CIN through antioxidative and antiapoptotic properties. METHODS: To test whether Magnolin could attenuate CIN, oxidative stress and apoptosis, in vivo and in vitro, we utilized a rat model of ioversol-induced CIN and a cell model of oxidative stress in which HK2 cells were treated with H2O2. Rats were assigned to 4 groups (n = 6 per group): control group, ioversol group (ioversol-induced CIN), vehicle group (CIN rats pretreated with vehicle), and Magnolin group (CIN rats pretreated with 1 mg/kg Magnolin). RESULTS: The results showed that magnolin ameliorated the renal tubular necrosis, apoptosis, and the deterioration of renal function (P < 0.05). Furthermore, Magnolin reduced the renal oxidative stress, suppressed caspase-3 activity, and increased Bcl-2 expression in vivo and in vitro. CONCLUSION: Magnolin might protect CIN in rats through antioxidation and antiapoptosis

Renal protective effect and mechanism research of hypoxia inducible factor-1α inhibitor YC-1 in diabetic nephropathy mice

Objective·To investigate the effect of hypoxia inducible factor-1α (HIF-1α) inhibitor YC-1 on the progression of diabetic nephropathy (DN) in mice and the potential mechanism.Methods·Ten-week-old male db/db mice (DN model) and their nondiabetic wild-type (WT) littermates were divided into 4 groups (n=6) according to whether treated with YC-1 or not: WT group, WT+YC-1 group, DB group, and DB+YC-1 group. The treatment groups were intraperitoneally injected with YC-1 (20 mg·kg-1) once a day, while the non-treatment groups received the same volumes of DMSO injection. After a total of 8 weeks of intervention, blood glucose, body weight, and kidney weight of all mice were measured. Serum, urine and kidney tissue samples were harvested. Serum creatinine, urinary albumin-to-creatinine ratio (UACR), and urine neutropil gelatinase-associated lipocalin (NGAL) levels were detected. The kidneys were stained with ‎hemat‎oxyli‎n-eosin (H-E) and periodic acid-Schiff (PAS) to observe the pathological changes. Masson staining was used to detect fibrosis, collagen-Ⅰ was detected by immunohistochemistry, and α-smooth muscle actin (α-SMA) was detected by Western blotting. The expression of HIF-1α was detected by both Western blotting and immunohistochemistry. TUNEL staining and Western blotting for apoptosis-related proteins were used to observe the cell apoptosis level. Superoxide dismutase (SOD) activity and malondialdehyde (MDA) level were detected by the kits. Endoplasmic reticulum stress (ERS) markers, including immunoglobulin heavy chain binding protein (BiP, also known as GRP78), phospho-protein kinase R-like endoplasmic reticulum kinase (p-PERK), total PERK, phospho-eukaryotic initiation factor 2α (p-eIF2α), total eIF2α, activating transcription factor 4 (ATF4), and C/EBP homologous protein (CHOP), were determined by Western blotting.Results·Compared with the WT group, the DB group showed significant rise of blood glucose, loss of renal function, severe kidney histopathology injuries and kidney fibrosis, increase of renal HIF-1α expression, and aggravated oxidative stress and ERS. Whilst there were no significant changes in blood glucose, YC-1 treatment notably reduced kidney weight/body weight ratio, serum creatinine, UACR, and urine NGAL levels in db/db mice. YC-1 treatment ameliorated kidney histopathology injuries and kidney fibrosis, and decreased the expressions of collagen-Ⅰ and α-SMA. YC-1 treatment also reduced the number of TUNEL positive cells, the expression of HIF-1α and pro-apoptotic proteins including BAX and cleaved caspase-3, and MDA level in the kidneys of db/db mice, while promoting anti-apoptotic protein BCL-2 expression and SOD activity. The expressions of ERS markers GRP78, p-PERK, p-eIF2α, ATF4, and CHOP were likewise significantly decreased in DB+YC-1 group.Conclusion·HIF-1α inhibitor YC-1 inhibits oxidative stress and abnormal activation of ERS, improving cell apoptosis and fibrosis in the kidneys of DN mice, which would attenuate the aggravation of pathological damage and loss of kidney function

Ischemic Duration and Frequency Determines AKI-to-CKD Progression Monitored by Dynamic Changes of Tubular Biomarkers in IRI Mice

Ischemia reperfusion injury (IRI) is one of the most common causes of acute kidney injury (AKI). However, the pathogenesis and biomarkers predicting the progression of IRI-induced AKI to chronic kidney disease (CKD) remain unclear. A side-by-side comparison between different IRI animal models with variable ischemic duration and episodes was performed. The dynamic changes of KIM-1 and NGAL continuously from AKI to CKD phases were studied as well. Short-term duration of ischemia induced mild renal tubule-interstitial injury which was completely reversed at acute phase of kidney injury, while long-term duration of ischemia caused severe tubular damage, cell apoptosis and inflammatory infiltration at early disease stage, leading to permanent chronic kidney fibrosis at the late stage. Repeated attacks of moderate IRI accelerated the progression of AKI to CKD. Different from serum and urine levels of KIM-1 that increased at acute phase of IRI then declined gradually in chronic phase, NGAL increased continuously during AKI-to-CKD transition. Severity and frequency of ischemia injury determines the progression and outcome of ischemia-induced AKI. Inflammation, apoptosis and fibrogenesis likely participate in the progression of AKI to CKD. Both KIM-1 and NGAL enable noninvasive and early detection of AKI, but NGAL is associated better with the process of AKI-to-CKD progression

Novel Evolved Immunoglobulin (Ig)-Binding Molecules Enhance the Detection of IgM against Hepatitis C Virus

Detection of specific antibodies against hepatitis C virus (HCV) is the most widely available test for viral diagnosis and monitoring of HCV infections. However, narrowing the serologic window of anti-HCV detection by enhancing anti-HCV IgM detection has remained to be a problem. Herein, we used LD5, a novel evolved immunoglobulin-binding molecule (NEIBM) with a high affinity for IgM, to develop a new anti-HCV enzyme-linked immunosorbent assay (ELISA) using horseradish peroxidase-labeled LD5 (HRP-LD5) as the conjugated enzyme complex. The HRP-LD5 assay showed detection efficacy that is comparable with two kinds of domestic diagnostic kits and the Abbott 3.0 kit when tested against the national reference panel. Moreover, the HRP-LD5 assay showed a higher detection rate (55.9%, 95% confidence intervals (95% CI) 0.489, 0.629) than that of a domestic diagnostic ELISA kit (Chang Zheng) (53.3%, 95% CI 0.463, 0.603) in 195 hemodialysis patient serum samples. Five serum samples that were positive using the HRP-LD5 assay and negative with the conventional anti-HCV diagnostic ELISA kits were all positive for HCV RNA, and 4 of them had detectable antibodies when tested with the established anti-HCV IgM assay. An IgM confirmation study revealed the IgM reaction nature of these five serum samples. These results demonstrate that HRP-LD5 improved anti-HCV detection by enhancing the detection of anti-HCV IgM, which may have potential value for the early diagnosis and screening of hepatitis C and other infectious diseases