Tacrolimus inhibits insulin release and promotes apoptosis of Min6 cells through the inhibition of the PI3K/Akt/mTOR pathway.

As a calcineurin inhibitor, tacrolimus is commonly used as a first­line immunosuppressant in organ transplant recipients. Post­transplantation diabetes mellitus (PTDM) is a common complication following kidney transplantation and is associated with immunosuppressant drugs, such as tacrolimus. PTDM caused by tacrolimus may be related to its influence on insulin secretion and insulin resistance. However, the specific mechanism has not been fully elucidated. The aim of the present study was to investigate whether the PI3K/Akt/mTOR signaling pathway served an important role in the pathogenesis of PTDM induced by tacrolimus. In the present study, the Cell Counting Kit­8 assay was used to measure the effect of tacrolimus on the viability of Min6 mouse insulinoma cells. The effects of tacrolimus on the insulin secretion and the activity of caspase­3 of Min6 cells stimulated by glucose exposure were measured by ELISA. Superoxide dismutase (SOD) and malondialdehyde (MDA) levels were measured using WST­8 and thiobarbituric acid assays, respectively. The effects of tacrolimus on the mRNA expression levels of PI3K, Akt and mTOR were detected by reverse transcription­quantitative PCR (RT­qPCR), whereas the protein expression levels of PI3K, Akt, mTOR, phosphorylated (p)­AKT and p­mTOR in Min6 cells were assessed using western blotting. The present data indicated that, compared with the control group, 5, 25 and 50 ng/ml tacrolimus treatment could inhibit the insulin secretion of Min6 cells stimulated by glucose solution, and 50 ng/ml tacrolimus could notably decrease the stimulation index (P<0.05). Moreover, 50 ng/ml tacrolimus markedly increased the activity of caspase­3 by 175.1% (P<0.05), it also decreased the SOD activity (P<0.01) and increased MDA levels (P<0.05). The RT­qPCR results demonstrated that the mRNA expression levels of PI3K, Akt and mTOR were downregulated by 25 and 50 ng/ml tacrolimus (P<0.01). Furthermore, the western blotting results suggested that tacrolimus had no significant effects on the expression levels of total PI3K, Akt and mTOR proteins (P>0.05), but 25 and 50 ng/ml tacrolimus could significantly inhibit the expression levels of p­Akt and p­mTOR (P<0.01). In conclusion, tacrolimus decreased the activity and insulin secretion of pancreatic ß cells and induced the apoptosis of islet ß cells by inhibiting the mRNA expression levels of PI3K, Akt and mTOR and reducing the phosphorylation of Akt and mTOR proteins in the PI3K/Akt/mTOR signaling pathway, which may ultimately lead to the occurrence of diabetes mellitus, and may be considered as one of the specific mechanisms of PTDM caused by tacrolimus.

Exosomal circ_DLGAP4 promotes diabetic kidney disease progression by sponging miR-143 and targeting ERBB3/NF-κB/MMP-2 axis.

Diabetic kidney disease (DKD) is closely associated with the high risk of cardiovascular disease and mortality. Exosomal circRNAs can exert significant roles in the pathology of various diseases. Nevertheless, the role of exosomal circRNAs in DKD progression remains barely known. Circular RNA DLGAP4 has been reported to be in involved in acute ischemic stroke. In our study, we found exosomal circ_DLGAP4 was increased in the exosomes isolated from HG-treated mesangial cells (MCs), DKD patients, and DKD rat models compared with the corresponding normal subjects. Then, we observed that exo-circ_DLGAP4 significantly promoted proliferation and fibrosis of MCs cells. Moreover, to study the underlying mechanism of circ_DLGAP4 in regulating DKD, bioinformatics method was consulted and miR-143 was predicted as its target. The direct correlation between miR-143 and circ_DLGAP4 was validated in MCs. MCs proliferation and fibrosis were increased by circ_DLGAP4, which could be decreased by mimic-miR-143. Next, elevated expression of Erb-b2 receptor tyrosine kinase 3 (ERBB3) is involved in various diseases. However, the function of ERBB3 in DKD development remains poorly known. Next, ERBB3 was predicted as the downstream target for miR-143. It was displayed that circ_DLGAP4 promoted proliferation and fibrosis of MCs by sponging miR-143 and regulating ERBB3/NF-κB/MMP-2 axis. Meanwhile, the loss of exo-circ_DLGAP4 induced miR-143 and repressed ERBB3/NF-κB/MMP-2 expression in MCs. Subsequently, in vivo assays were performed and it was proved that overexpression of circ_DLGAP4 markedly promoted DKD progression in vivo via modulating miR-143/ERBB3/NF-κB/MMP-2. In conclusion, we indicated that exosomal circ_DLGAP4 could prove a novel insight for DKD development.

Loss of lncRNA MIAT ameliorates proliferation and fibrosis of diabetic nephropathy through reducing E2F3 expression.

Diabetic nephropathy (DN) is a serious kidney disease resulted from diabetes. Dys-regulated proliferation and extracellular matrix (ECM) accumulation in mesangial cells contribute to DN progression. In this study, we tested expression level of MIAT in DN patients and mesangial cells treated by high glucose (HG). Up-regulation of MIAT was observed in DN. Then, functional assays displayed that silence of MIAT by siRNA significantly repressed the proliferation and cycle progression in mesangial cells induced by HG. Meanwhile, we found that collagen IV, fibronectin and TGF-ß1 protein expression was obviously triggered by HG, which could be rescued by loss of MIAT. Then, further assessment indicated that MIAT served as sponge harbouring miR-147a. Moreover, miR-147a was decreased in DN, which exhibited an antagonistic effect of MIAT on modulating mesangial cell proliferation and fibrosis. Moreover, bioinformatics analysis displayed that E2F transcription factor 3 (E2F3) could act as direct target of miR-147a. We demonstrated that E2F3 was greatly increased in DN and the direct binding association between miR-147a and E2F3 was evidenced using luciferase reporter assay. In summary, our data explored the underlying mechanism of DN pathogenesis validated that MIAT induced mesangial cell proliferation and fibrosis via sponging miR-147a and regulating E2F3.

Circ-AKT3 inhibits the accumulation of extracellular matrix of mesangial cells in diabetic nephropathy via modulating miR-296-3p/E-cadherin signals.

Diabetic nephropathy is a leading cause of end-stage renal disease globally. The vital role of circular RNAs (circRNAs) has been reported in diabetic nephropathy progression, but the molecular mechanism linking diabetic nephropathy to circRNAs remains elusive. In this study, we investigated the significant function of circ-AKT3/miR-296-3p/E-cadherin regulatory network on the extracellular matrix accumulation in mesangial cells in diabetic nephropathy. The expression of circ-AKT3 and fibrosis-associated proteins, including fibronectin, collagen type I and collagen type IV, was assessed via RT-PCR and Western blot analysis in diabetic nephropathy animal model and mouse mesangial SV40-MES13 cells. Luciferase reporter assays were used to investigate interactions among E-cadherin, circ-AKT3 and miR-296-3p in mouse mesangial SV40-MES13 cells. Cell apoptosis was evaluated via flow cytometry. The level of circ-AKT3 was significantly lower in diabetic nephropathy mice model group and mouse mesangial SV40-MES13 cells treated with high-concentration (25 mmol/L) glucose. In addition, circ-AKT3 overexpression inhibited the level of fibrosis-associated protein, such as fibronectin, collagen type I and collagen type IV. Circ-AKT3 overexpression also inhibited the apoptosis of mouse mesangial SV40-MES13 cells treated with high glucose. Luciferase reporter assay and bioinformatics tools identified that circ-AKT3 could act as a sponge of miR-296-3p and E-cadherin was the miR-296-3p direct target. Moreover, circ-AKT3/miR-296-3p/E-cadherin modulated the extracellular matrix of mouse mesangial cells in high-concentration (25 mmol/L) glucose, inhibiting the synthesis of related extracellular matrix protein. In conclusion, circ-AKT3 inhibited the extracellular matrix accumulation in diabetic nephropathy mesangial cells through modulating miR-296-3p/E-cadherin signals, which might offer novel potential opportunities for clinical diagnosis targets and therapeutic biomarkers for diabetic nephropathy.

Dinitrosyliron Complex [(PMDTA)Fe(NO)2]: Intermediate for Nitric Oxide Monooxygenation Activity in Nonheme Iron Complex.

Despite a comprehensive study on the biosynthesis and function of nitric oxide, biological metabolism of nitric oxide, especially when its concentration exceeds the cytotoxic level, remains elusive. Oxidation of nitric oxide by O2 in aqueous solution has been known to yield NO2-. On the other hand, a biomimetic study on the metal-mediated conversion of NO to NO2-/NO3- via O2 reactivity disclosed a conceivable pathway for aerobic metabolism of NO. During the NO-to-NO3- conversion, transient formation of metal-bound peroxynitrite and subsequent release of •NO2 via O-O bond cleavage were evidenced by nitration of tyrosine residue or 2,4-di-tert-butylphenol (DTBP). However, the synthetic/catalytic/enzymatic cycle for conversion of nitric oxide into a nitrite pool is not reported. In this study, sequential reaction of the ferrous complex [(PMDTA)Fe(κ2-O,O'-NO2)(κ1-O-NO2)] (3; PMDTA = pentamethyldiethylenetriamine) with NO(g), KC8, and O2 established a synthetic cycle, complex 3 → {Fe(NO)2}9 DNIC [(PMDTA)Fe(NO)2][NO2] (4) → {Fe(NO)2}10 DNIC [(PMDTA)Fe(NO)2] (1) → [(PMDTA)(NO)Fe(κ2-O,N-ONOO)] (2) → complex 3, for the transformation of nitric oxide into nitrite. In contrast to the reported reactivity of metal-bound peroxynitrite toward nitration of DTBP, peroxynitrite-bound MNIC 2 lacks phenol nitration reactivity toward DTBP. Presumably, the [(PMDTA)Fe] core in {Fe(NO)}8 MNIC 2 provides a mononuclear template for intramolecular interaction between Fe-bound peroxynitrite and Fe-bound NO-, yielding Fe-bound nitrite stabilized in the form of complex 3. This [(PMDTA)Fe]-core-mediated concerted peroxynitrite homolytic O-O bond cleavage and combination of the O atom with Fe-bound NO- reveals a novel and effective pathway for NO-to-NO2- transformation. Regarding the reported assembly of the dinitrosyliron unit (DNIU) [Fe(NO)2] in the biological system, this synthetic cycle highlights DNIU as a potential intermediate for nitric oxide monooxygenation activity in a nonheme iron system.

Downregulation of XIST ameliorates acute kidney injury by sponging miR-142-5p and targeting PDCD4.

Acute kidney injury (AKI) is a common kidney disease that markedly affects public health. To date, the roles of long noncoding RNA XIST in AKI are poorly understood. Here, we investigated the biological functions of XIST in AKI. We observed that XIST expression increased in patients with AKI and HK-2 cells stimulated by CoCl2 . In addition, a rat AKI model induced by ischemia-reperfusion was established. Tumor necrosis factor-α, interleukin-6, and cyclooxygenase-2 messenger RNA expression were induced in vivo; moreover, XIST expression was upregulated. Knockdown of XIST significantly repressed CoCl2 -triggered injury in HK-2 cells. However, microRNA (miR)-142-5p, a downstream target of XIST, was downregulated in AKI. miR-142-5p was repressed by XIST and miR-142-5p could inhibit CoCl2 -induced injury in HK-2 cells. Moreover, PDCD4 expression was significantly increased in AKI. PDCD4 was predicted to be the target of miR-142-5p. Subsequently, loss of PDCD4 was able to retard injury in HK-2 cells exposed to CoCl2. Thus, we suggest that XIST regulates miR-142-5p and PDCD4, and it has the potential to function as a biomarker in therapeutic strategies for AKI.

Hepatic Arterial Infusion Chemotherapy Using Oxaliplatin Plus 5-Fluorouracil Versus Transarterial Chemoembolization/Embolization for the Treatment of Advanced Hepatocellular Carcinoma with Major Portal Vein Tumor Thrombosis.

PURPOSE: To compare the efficacy and safety of hepatic arterial infusion chemotherapy (HAIC) to transarterial chemoembolization/embolization (TACE/TAE) for the treatment of advanced hepatocellular carcinoma (HCC) with major portal vein tumor thrombosis (PVTT). MATERIALS AND METHODS: Forty-six patients with advanced HCC with major PVTT who underwent HAIC or TACE/TAE between April 2013 and April 2017 were included. In the HAIC group (n = 22), oxaliplatin (35-40 mg/m2 for 2 h) and 5-fluorouracil (600-800 mg/m2 for 22 h) on days 1-3 every 4 weeks were administered for a maximum of six serial courses. In the TACE/TAE group (n = 24), an emulsion of epirubicin (40-60 mg) and lipiodol was administered followed by particles (cTACE), or particles alone embolization (TAE). Overall survival (OS), tumor response according to mRECIST, progression-free survival (PFS), and adverse events were investigated. RESULTS: Median OS was 20.8 months in the HAIC group versus 4.0 months in the TACE/TAE group (P < 0.001; hazard ratio [HR], 0.17). The HAIC group showed higher tumor response rates than the TACE/TAE group (59.1% [13/22] vs. 22.7% [5/22]; P = 0.014) and a longer median PFS (9.6 vs. 1.5 months; P < 0.001; HR, 0.09). The Child-Pugh class (P = 0.007) and treatment method (P = 0.002) were independent risk factors of survival. The most frequent grade 3 or worse treatment-related adverse events were liver dysfunction (2 [9.1%] vs. 5 [20.8%]), hematological abnormalities (1 [4.5%] vs. 2 [8.3%]), and fever (1 [4.5%] vs. 4 [16.7%]). One treatment-related death due to acute liver failure occurred 3 days after TACE treatment. CONCLUSION: HAIC may significantly improve OS and provide better tumor control with mild side effects and preserved liver function in patients with advanced HCC with major PVTT compared to TACE/TAE treatment.

circCCT3 Modulates Vascular Endothelial Growth Factor A and Wnt Signaling to Enhance Colorectal Cancer Metastasis Through Sponging miR-613.

Colorectal cancer (CRC) has been suggested to be one of the leading cancer types all over the world. Till now, the molecular mechanism by which circCCT3 regulates CRC remains to be clarified. To detect mRNA and protein levels of various genes, Reverse Transcription-quantitative PCR and western blot were used in our study. Luciferase reporter assay was utilized to probe direct interaction between genes. We used transwell assay to assess the invasion ability of CRC cells. For apoptosis detection, immunofluorescence of CRC cells by Annexin V staining was performed. We carried out bioinformatic analysis to show higher expression of circCCT3 in human clinical CRC tumors. Low level of circCCT3 was closely associated with higher disease-free survival of CRC patients. Moreover, we found that circCCT3 was linked to advanced stage of CRC. miR-613 is the target of circCCT3 and responsible for circCCT3-modulated invasion and apoptosis of CRC cells. In addition, we identified WNT3 and vascular endothelial growth factor A (VEGFA) as downstream effectors of miR-613 in CRC cells. WNT3 and VEGFA overexpression resulted in partial rescue of miR-613-mediated phenotypes of CRC cells. In conclusion, we propose that circCCT3 contributes to CRC metastasis via miR-613/WNT3 or miR-613/VEGFA, promoting the development of therapeutical approaches for treating CRC.

Intrabullous Adhesion Pexia (IBAP) by Percutaneous Pulmonary Bulla Centesis: An Alternative for the Surgical Treatment of Giant Pulmonary Bulla (GPB).

Background and Objective: Most patients with giant pulmonary bulla (GPB) are treated by surgery; however, there is a subset for whom surgery is not a viable option, such as those with contraindications, or those unwilling to undergo operation. Therefore, an alternative minimally invasive method is desired for this subpopulation. The aim of this study was to explore an alternative procedure for treating GPB. Methods: This was a prospective, nonrandomized, single-arm, unblinded study evaluating the efficacy and safety of intrabulla adhesion pexia (IBAP) procedure in GPB patients. The study was conducted between December 2004 and April 2017. Results: There were 38 cases in 36 patients (33 males and 3 females) with the target GPB cavities varying in size (range, 10 cm × 7 cm × 5 cm to 15 cm × 8 cm × 30 cm (anteroposterior diameter × medial-lateral diameter × superoinferior diameter)). After IBAP treatment, the closure ratio of GPB in one month was 86.84% (33/38), while the dyspnea index significantly decreased from 4.11 ± 1.11 to 2.24 ± 1.15 (P < 0.01). In addition, the mean FEV1 (L) increased from 1.06 ± 0.73 to 1.57 ± 1.13 (P < 0.01), while RV (L) decreased from 2.77 ± 0.54 to 2.36 ± 0.38 (P < 0.01) and TLC (L) decreased from 6.46 ± 1.21 to 5.86 ± 1.08 (P < 0.01). Moreover, PaO2 (mmHg) increased from 52.18 ± 8.31 to 68.29 ± 12.34, while the 6 MWD increased by 129.36% from 131.58 ± 105.24 to 301.79 ± 197.90 (P < 0.01). Collectively, these data indicated significant improvement in pulmonary function and exercise tolerance after IBAP treatment. Furthermore, no deaths occurred during IBAP treatment, and no cases of aggravated GPB relapse were reported during the 12-month follow-up period. Conclusions: IBAP is a promising strategy for the treatment of GPB. Our findings demonstrated that IBAP had a noteworthy therapeutic effect, desirable safety, and ideal long-term efficacy for GPB.

Electrodeposited-film electrodes derived from a precursor dinitrosyl iron complex for electrocatalytic water splitting.

In artificial photosynthesis, water splitting plays an important role for the conversion and storage of renewable energy sources. Here, we report a study on the electrocatalytic properties of the electrodeposited-film electrodes derived from irreversible electro-reduction/-oxidation of a molecular dinitrosyl iron complex (DNIC) {Fe(NO)2}9 [(Me6tren)Fe(NO)2]+ (Me6tren = tris[2-(dimethylamino)ethyl]amine) for the hydrogen evolution reaction (HER) and the oxygen evolution reaction (OER) in alkaline solution, individually. For HER, the overpotential and Tafel slope for the electrodeposited-film cathode are lower than those of the equiv.-weight Pt/C electrode. The electrodeposited-film anode for the OER is stable for 139 h. Integration of the electrodeposited-film cathode and anode into a single electrode-pair device for electrocatalytic water splitting exhibits an onset voltage of 1.77 V, achieving a geometrical current density of 10 mA cm-2.

Peritoneum as the sole distant metastatic site of lung adenosquamous cell carcinoma: a case report.

BACKGROUND: Peritoneum metastasis of lung cancer is a rare event which, in addition to the peritoneum, usually involves multiple metastatic tissues. Here we report a case of a patient with lung adenosquamous cell carcinoma with the peritoneum as the sole distant metastatic site. CASE PRESENTATION: An 82-year-old Han Chinese man, in the teaching profession, was diagnosed with lung adenosquamous cell carcinoma in the upper lobe of his left lung with the involvement of ipsilateral hilar and mediastinal lymph nodes, and was initially staged as IIIa (cT2N2M0). Molecular testing identified a mutation at KRAS G12A. Due to his poor physical condition, our patient was given gamma knife radiotherapy with a total dose of 28.0 Gy. Two weeks later, our patient was diagnosed as peritoneal metastasis identified by using magnetic resonance imaging and confirmed with ascitic cytology and peritoneal histology. No other distant metastatic sites such as liver, brain, bone, paranephroi, and lungs were found. Subsequently, our patient received palliative intraperitoneal chemotherapy, and died within 2 months. CONCLUSIONS: Our patient represented a rare case of lung adenosquamous cell carcinoma harboring the KRAS G12A mutation, which metastasized distantly to the peritoneum only, and progressed rapidly.

[Effect of co-inhibition of MCT1 gene and NHE1 gene on proliferation and growth of human lung adenocarcinoma cells].

BACKGROUND & OBJECTIVE: The authors previously discovered that the intracellular pH values (pHi) of tumor cells could be decreased, which led tumor cells to acidify and die, when the expressions of the first subtype of monocarboxylate transporter (MCT1) gene and the first subtype of Na+/H+ exchanger (NHE1) gene in tumor cell membranes had been inhibited by each corresponding antisense gene respectively. However it was not clear whether there were co-effects on the pHi, proliferation, and growth of tumor cells, after two genes were inhibited at the same time. METHODS: pLXSN-MCT1 and pLXSN-NHE1, the corresponding antisense gene expression vectors of MCT1 and NHE1 genes, were introduced into human lung adenocarcinoma A549 cells at the same time with electroporation. The positive colonies were selected by G418. Using PCR and RT-PCR technique, it was confirmed that antisense genes of MCT1 and NHE1 genes integrated with A549 genomic DNA. The pHi and lactate content were detected with spectrophotometry. Cell growth cycle was measured with flow cytometer. The cells co-transfected antisense genes, cells transfected MCT1 antisense gene and A549 cells were respectively inoculated in nude mouse subcutaneous to observe the growth of transplantation tumors. RESULTS: Compared with A549 cells, the pHi of cells co-transfected antisense genes was remarkably decreased (P < 0.05), while lactate content was remarkably increased (P < 0.001). The cell cycle was blocked at G0-G1 period. And the transplantation tumors of nude mice inoculated co-transfected antisense gene-cells and inoculated MCT1-antisense-gene-transfected cells were significantly lighter and smaller than ones inoculated A549 cells (P < 0.001). CONCLUSION: MCT1 and NHE1 genes play important regulation roles in proliferation and growth of tumor cells, probably by affecting pHi.