[Effects and mechanism of pyrroloquinoline quinine on mitochondrial function and cell survival of rat bone marrow mesenchymal stem cells under oxidative stress].

Objective: To observe the effects of pyrroloquinoline quinine (PQQ) on the mitochondrial function and cell survival of rat bone marrow mesenchymal stem cells (BMSCs) under oxidative stress, and to explore its mechanism. Methods: BMSCs of rats were cultured in vitro with Dulbecco's minimum essential medium/F12 medium containing fetal bovine serum in the volume fraction of 10% (hereinafter referred to as normal medium). The rat BMSCs of third to fifth passages in logarithmic growth phase were selected for the following experiments. (1) The cells were divided into normal control group, normal control+ PQQ group, hydrogen peroxide (H(2)O(2)) alone group, and H(2)O(2)+ PQQ group. The cells in normal control group were cultured in normal medium for 24 hours; the cells in normal control+ PQQ group were cultured in normal medium containing 100 µmol/L PQQ for 24 hours; the cells in H(2)O(2) alone group were cultured in normal medium containing 200 µmol/L H(2)O(2) for 24 hours; the cells in H(2)O(2)+ PQQ group were pre-incubated with normal medium containing 100 µmol/L PQQ for 2 hours, and then with H(2)O(2) added to the concentration of 200 µmol/L and cultured for 24 hours. The cell morphology of each group was observed under the inverted phase contrast microscope, and the cell survival rate was detected by cell count kit 8 method. (2) Five batches of cells were collected, and the cells of each batch were divided into normal control group, H(2)O(2) alone group, and H(2)O(2)+ PQQ group. The cells in each group received the same treatment as that in the corresponding group of experiment (1). After 24 hours of culture, one batch of cells was collected for apoptosis detection by flow cytometry, and the apoptosis rate was calculated. One batch of cells was subjected to mitochondrial membrane potential assay and JC-1 fluorescent staining observation using the JC-1 mitochondrial membrane potential detection kit and the inverted phase contrast fluorescence microscope, respectively. One batch of cells was collected for mitochondrial morphology observation under the transmission electron microscope. One batch of cells was subjected to catalase (CAT) and superoxide dismutase (SOD) activity assay by CAT activity assay kit and SOD activity assay kit, respectively. One batch of cells was subjected to Western blotting for determination of protein level of Epac1, adenine monophosphate activated protein kinase (AMPK), phosphorylated AMPK, cysteinyl aspartate-specific proteinase 3 (caspase-3), and cleaved caspase-3, and the phosphorylation level of AMPK and cleaved caspase-3/caspase-3 ratio were calculated. Six replicates were measured in each group for each index except for morphological observation. Data were statistically analyzed with one-way analysis of variance and independent sample equal variance t test. Results: (1) After 24 hours of culture, compared with those in normal control group (the cell survival rate was set to 100.0%), there was an increase in cell vacuole and a decrease in cell number in H(2)O(2) alone group, and the cell survival rate was significantly reduced to (74.3±2.9)% (t=6.39, P<0.01). Compared with those in H(2)O(2) alone group, the cell morphology of H(2)O(2)+ PQQ group was significantly improved, and the cell survival rate was significantly increased to (116.9±4.2)% (t=6.92, P<0.01); the cell survival rate in normal control+ PQQ group was (101.2±1.1)%, close to that of control group (t=1.06, P>0.05). (2) After 24 hours of culture, compared with (13.6±1.0)% in normal control group, the apoptosis rate of cells in H(2)O(2) alone group was significantly increased to (37.1±2.0)% (t=10.57, P<0.01). Compared with that in H(2)O(2) alone group, the apoptosis rate of cells in H(2)O(2)+ PQQ group was significantly declined to (17.0±0.7)% (t=9.49, P<0.01). (3) After 24 hours of culture, compared with those in normal control group, the mitochondrial membrane potential of cells in H(2)O(2) alone group was depolarized, the JC-1 fluorescent dye mainly existed in the cytoplasm in the form of monomer, which emitted green fluorescence, and a significant decrease in mitochondrial membrane potential was shown (t=4.18, P<0.01). Compared with those in H(2)O(2) alone group, the mitochondrial membrane potential of cells in H(2)O(2)+ PQQ group was increased to normal level (t=4.43, P<0.01), and the JC-1 fluorescent dye accumulated in mitochondria following the polarized mitochondrial membrane potential and emitted red fluorescence. (4) After 24 hours of culture, compared with that in normal control group, the mitochondrial structure of cells in H(2)O(2) alone group was disordered, with disappeared mitochondrial cristae and decreased mitochondrial matrix density. Compared with that in H(2)O(2) alone group, the mitochondrial structure of cells in H(2)O(2)+ PQQ group was regular and intact, with clearly visible mitochondrial cristae and increased mitochondrial matrix density. (5) After 24 hours of culture, compared with those in normal control group, the CAT activity of cells in H(2)O(2) alone group was significantly increased (t=4.54, P<0.05), and the SOD activity was significantly decreased (t=3.93, P<0.05). Compared with those in H(2)O(2) alone group, the CAT activity of cells in H(2)O(2)+ PQQ group was obviously increased (t=8.65, P<0.01), while there was no significant change in the SOD activity (t=0.72, P>0.05). (6) After 24 hours of culture, compared with those in normal control group, the protein expression of Epac1 of cells in H(2)O(2) alone group was significantly decreased (t=4.67, P<0.01), while the AMPK phosphorylation level and the cleaved caspase-3/caspase-3 ratio were significantly increased (t=7.88, 3.62, P<0.01). Compared with those in H(2)O(2) alone group, the protein expression of Epac1 and the AMPK phosphorylation level of cells in H(2)O(2)+ PQQ group were both significantly increased (t=4.34, 16.37, P<0.01), while the cleaved caspase-3/caspase-3 ratio was significantly declined (t=3.17, P<0.05). Conclusions: Pretreatment with PQQ can improve the mitochondrial function, reduce cell apoptosis rate, and enhance cell survival rate of rat BMSCs under oxidative stress, which may be related to the up-regulation of Epac1 protein expression, activation of AMPK signaling pathway, and down-regulation of cleaved caspase-3 protein level.

[Effects of microRNA-34a on regulating silent information regulator 1 and influence of the factor on myocardial damage of rats with severe burns at early stage].

Objective: To explore the effects of microRNA-34a on regulating silent information regulator 1 (SIRT1) and influence of SIRT1 on myocardial damage of rats with severe burns at early stage. Methods: (1) Twenty-four Sprague-Dawley (SD) rats were divided into sham injury (SI) group, simple burns (SB) group and SIRT1 agonist (SA) group according to the random number table (the same grouping method below), with 8 rats in each group. Rats in groups SB and SA were inflicted with 30% total body surface area full-thickness scald (hereinafter referred to as burns) on the back, and rats in group SI were sham injuried on the back. Immediately after injury, rats in groups SI and SB were intraperitoneally injected with normal saline of 50 mL/kg, and rats in group SA were intraperitoneally injected with normal saline of 50 mL/kg and 1 mg/mL resveratrol of 50 mg/kg. At 6 h post injury, abdominal aortic blood was collected to make serum and myocardial tissue of rats was collected. (2) Myocardial cells of twelve neonatal SD rats were collected and divided into microRNA-34a mimic control (MMC) group, microRNA-34a mimic (MM) group, microRNA-34a inhibitor control (MIC) group, and microRNA-34a inhibitor (MI) group, which were respectively transfected with gene sequences of mimic control, mimic, inhibitor control, and inhibitor of microRNA-34a. The microRNA-34a expression level and protein expression level of SIRT1 in myocardial cells were respectively detected by real-time fluorescence quantitative reverse transcription polymerase chain reaction (RT-PCR) and Western blotting. Another batch of myocardial cells were divided into microRNA-34a inhibitor control+ burn serum (MCB) group, microRNA-34a inhibitor+ burn serum (MB) group, and microRNA-34a inhibitor+ burn serum + EX527 (MBE) group. Myocardial cells in group MCB were transfected with gene sequence of inhibitor control, and myocardial cells in the later groups were transfected with gene sequence of inhibitor of microRNA-34a. After transfection of 48 h, myocardial cells in group MBE were cultured in Dulbecco's modified Eagle's medium (DMEM) solution for 6 hours, with serum in group SB of volume fraction of 10% and final amount-of-substance concentration of 1 mol/L, and myocardial cells in the other 2 groups were cultured in DMEM solution with serum from rats of group SB of volume fraction of 10%. The protein expression levels of myocardial cells of SIRT1, cleaved-caspase-3, and Bax were detected by Western blotting. (3) Myocardial tissue from (1) was collected to detect expression levels of microRNA-34a and mRNA of SIRT1 in groups SI and SB by real-time fluorescence quantitative RT-PCR. Morphology of myocardial tissue of rats in groups SI, SB, and SA was observed with biological image navigator. The mRNA expression levels of interleukin 1ß (IL-1ß) and tumor necrosis factor (TNF-α) of rats in groups SI, SB, and SA were detected by real-time fluorescence quantitative RT-PCR. The expression levels of cleaved-caspase-3, and Bax of myocardial tissue of rats in groups SI, SB, and SA were detected by Western blotting. Data were processed with one-way analysis of variance and least-significant difference test. Results: (1) After transfection of 48 h, the expression level of microRNA-34a of myocardial cells in group MM was 4.67±0.92, significantly higher than 1.03±0.04 in group MMC (P<0.01); the protein expression level of SIRT1 of myocardial cells in group MM was 0.35±0.06, significantly lower than 1.12±0.11 in group MMC (P<0.01). After transfection of 48 h, the expression level of microRNA-34a of myocardial cells in group MI was 0.26±0.07, significantly lower than 1.33±0.07 in group MIC (P<0.01); the protein expression level of SIRT1 of myocardial cells in group MIC was 1.12±0.16, significantly lower than 1.74±0.34 in group MI (P<0.01). At 6 h after culture, compared with those in group MCB, the SIRT1 protein expression level of myocardial cells in group MB was significantly increased (P<0.05), while cleaved-caspase-3 and Bax protein expression levels of myocardial cells in group MB were significantly decreased (P<0.05). Compared with those in group MB, the SIRT1 protein expression level of myocardial cells in group MBE was with no significantly statistical difference (P>0.05), and cleaved-caspase-3 and Bax protein expression levels were significantly increased (P<0.05). (2) At 6 h post injury, compared with that in group SI, the microRNA-34a expression level of myocardial tissue in group SB was significantly increased (P<0.01), and the mRNA expression level of SIRT1 of myocardial tissue in group SB was significantly decreased (P<0.01). At 6 h post injury, myocardial cells in group SI arranged neatly with normal nucleus and no inflammatory cells infiltration; myocardial cells in group SB arranged disorderly, with no abnormal nucleus, and obvious inflammatory cells infiltration; myocardial cells in group SA arranged neatly, with normal nucleus and little inflammatory cells infiltration. At 6 h post injury, compared with those in group SB, the mRNA expression levels of IL-1ß and TNF-α, and the protein expression levels of cleaved-caspase-3 and Bax of myocardial tissue in groups SI and SA were significantly decreased (P<0.01). Conclusions: The microRNA-34a expression level of myocardial tissue of rats with severe burns at early stage increases, which decreases the expression level of SIRT1, and increases the expression levels of IL-1ß, TNF-α, cleaved-caspase-3 and Bax, leading to obvious myocardial damage. Activation of SIRT1 can alleviate myocardial damage of rats with severe burns at early stage through decreasing expression levels of IL-1ß, TNF-α, cleaved-caspase-3, and Bax.

[Effects of activating silent information regulator 1 on early kidney damage in rats with severe burn].

Objective: To investigate the effects of activating silent information regulator 1 (SIRT1) on the early kidney damage in rats with severe burn. Methods: Thirty healthy male SD rats were divided into sham injury group (SI), pure burn group (PB), and SIRT1 activator group (SA) according to the random number table, with 10 rats in each group. Rats in groups PB and SA were inflicted with 30% total body surface area full-thickness scald (hereinafter referred to as burn) on the back. Immediately after injury, rats in group PB were intraperitoneally injected with normal saline in the dosage of 50 mL/kg, and those in group SA with 1 mg/mL (final mass concentration) resveratrol in the dosage of 50 mL/kg. Rats in group SI were sham injured and intraperitoneally injected with normal saline in the dosage of 50 mL/kg immediately after injury. Kidney tissue and abdominal aorta blood of rats in the three groups were collected at 24 hours after injury. The morphology of kidney tissue was observed after HE staining. The serum content of creatinine and urea nitrogen was determined with enzyme-linked immunosorbent assay. Protein expressions of SIRT1, Bax, and Bcl-2 in kidney tissue were determined with Western blotting. mRNA expressions of tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1ß), and IL-10 in kidney tissue were determined with real-time fluorescent quantitative reverse transcription polymerase chain reaction. Data were processed with one-way analysis of variance and LSD-t test. Results: (1) In rats of group SI, structures of kidney tubules and glomeruli were intact. In rats of group PB, structures of kidney tubules were not clear with casts in them, and glomeruli showed pyknosis. In rats of group SA, structures of kidney tubules were relatively intact, and the pyknosis of glomeruli were slighter as compared with that of group PB with fewer glomeruli showing pyknosis. (2) The serum content of creatinine and urea nitrogen in rats of group PB was (67±14) µmol/L and (22.0±4.4) mmol/L, respectively, which was significantly higher than that of group SI [(28±7) µmol/L and (5.5±1.2) mmol/L respectively, with t values respectively 6.07 and 11.53, P values below 0.01]. The serum content of creatinine and urea nitrogen in rats of group SA was (39±9) µmol/L and (14.1±1.7) mmol/L, respectively, significantly lower than that of group PB (with t values respectively 4.09 and 4.17, P values below 0.01). (3) Compared with those of group SI, protein expressions of SIRT1 and Bcl-2 in kidney tissue of rats in group PB were significantly decreased (with t values respectively 16.32 and 19.58, P values below 0.01), while the protein expression of Bax was significantly increased (t=5.98, P<0.01). Compared with those of group PB, protein expressions of SIRT1 and Bcl-2 in kidney tissue of rats in group SA were significantly increased (with t values respectively 6.94 and 5.37, P values below 0.01), while the protein expression of Bax was significantly decreased (t=3.44, P<0.01). (4) mRNA expressions of TNF-α, IL-1ß, and IL-10 in kidney tissue of rats in group PB were 17.0±4.0, 2.27±0.59, and 2.5±0.9, respectively, significantly higher than those of group SI (1.0, 1.00, and 1.0, respectively, with t values from 3.27 to 8.93, P<0.05 or P<0.01). mRNA expressions of TNF-α and IL-1ß in kidney tissue of rats in group SA were 6.8±1.2 and 1.18±0.26, respectively, significantly lower than those of group PB (with t values respectively 4.59 and 4.32, P values below 0.01). mRNA expression of IL-10 in kidney tissue of rats in group SA was 5.0±1.0, significantly higher than that of group PB (t=5.51, P<0.01). Conclusions: Activating SIRT1 on early stage of severe burn in rats can decrease levels of creatinine and urea nitrogen, thus improving the kidney function. It can down-regulate the protein expression of Bax and up-regulate the protein expression of Bcl-2, thus reducing the apoptosis in kidney tissue. Meanwhile, it can inhibit expressions of TNF-α and IL-1ß and promote the expression of IL-10, thus alleviating the inflammatory response in kidney.

Identification of novel compound heterozygous RECQL4 mutations and prenatal diagnosis of Baller-Gerold syndrome: a case report.

Birth defects are structural and/or functional malformations present at birth that cause physical or mental disability and are important public health problems. Our study was aimed at genetic analysis and prenatal diagnosis of congenital anomalies to understand the cause of certain birth defects. Karyotypes and array-comparative genomic hybridization (aCGH) were performed on a pregnant woman, surrounding amniotic fluid, and her husband. A short-stature panel genetic test was conducted in accordance with the phenotype of the fetus. Following examination, it was determined that the karyotype and aCGH results were normal. The RECQL4 gene in the fetus showed compound heterozygous mutations, and each parent was found to be a carrier of one of the mutations. The two heterozygous mutations (c.2059-1G>C and c.2141_2142delAG) were detected in the RECQL4 (NM_004260) gene in the fetus; therefore, the fetus was predicted to have Baller-Gerold syndrome. These two mutations have not previously been reported. In addition, these results identified a 25% risk of the parents having a sec-ond conceptus with this congenital disease. Therefore, prenatal genetic diagnosis was highly recommended for future pregnancies.

[NF-kappaB modulates activation of the BMP-2 gene by trichostatin A].

In this study, we investigated trichostatin A (TSA), a histone deacetylase inhibitor, increased the Bone morphogenetic protein-2 (BMP-2) mRNA level in human osteoblasts line. Deletion analysis of the promoter region revealed that TSA-induced luciferase was regulated by the BMP-2 promoter spanning from -320 to-310. Electrophoresis mobility shift assay (EMSA) and Chromatin immunoprecipitation (CHIP) assay proved that this position was nuclear factor NF-kappaB responsive element. The results above demonstrated that acetylation plays a crucial role in BMP-2 expression and acetylation of NF-kappaB p65 and p50 subunits by TSA treatment may activate the BMP-2 promoter.

[Determination of activity of bone alkaline phosphatase in whole serum by high performance liquid chromatography].

An analytical method has been established for the determination of bone alkaline phosphatase (BALP) activity using high performance liquid chromatography(HPLC). The stainless steel column was 22 cm x 4.6 mm i.d. packed with totally porous, spherical silica particles. A solution of methanol and 0.01 mol/L CH3COOH(30/70, V/V) adjusted to pH 4.8 was employed as the mobile phase. The flow rate was 0.8 mL/min. Chromatography was performed with ultraviolet detector at 275 nm. Linear calibration curve for BALP was measured within the range of 8-200 mg/L with correlation coefficient of 0.996. The lowest detection limit was 3 ng. The HPLC method described is simple, rapid and reliable and suitable for clinical monitoring.

Prognostic markers in clinically localised prostate cancer.

The prognostic value of immunohistochemical staining of P53, BCL-2, p27kip1, PSA, AR and MIB-1 was compared with that of established prognostic variables (Gleason score, surgical margins, tumour volume) following radical prostatectomy. Five groups were selected: negative margins with stable serum PSA (n=11), negative margins with rising serum PSA (n=7), positive margins with stable serum PSA (n=7), positive margins with rising serum PSA (6) and patients with micrometastatic disease diagnosed in lymph nodes removed during radical prostatectomy (n=8). Gleason score and tumour volume were of prognostic significance and immunohistochemical staining for MIB-1 and BCL-2 showed added independent prognostic significance in multivariate analysis.